Development and In-Vitro Characterization of Extended

I

Development and In-Vitro Characterization of

Extended Release Pellets of Itopride

Hydrochloride

Muhammad Iqbal Nasiri

B.Pharm., M.Phil.

DEPARTMENT OF PHARMACEUTICS

FACULTY OF PHARMACY

UNIVERSITY OF KARACHI

PAKISTAN

2016

II

Development and In-Vitro Characterization of

Extended Release Pellets of Itopride

Hydrochloride

Muhammad Iqbal Nasiri B.Pharm., M.Phil.

Thesis submitted for the partial fulfillment of the

degree of Doctor of Philosophy (Ph.D.) in

Pharmaceutics

Department of pharmaceutics

Faculty of pharmacy

University of Karachi

Karachi – 75270

Pakistan

III

IV

DEDICATED TO,

My dearest PARENTS, whom prayers, love, support and encouragement

gave me strength, to complete this task. They have been the foundation of

all what I am.

All my Brothers and Sister, for their payers, support and unconditional

encouragement.

My sons Muhammad Haseeb Nasiri, Ahsan Iqbal Nasiri, my daughters

Sadaf Fatima and Parisa Fatima, for their unconditional love and patience.

My loving wife, Kaniz Fatima, for their company with unconditional love,

care, support, patience and understanding.

V

ACKNOWLEDGEMENT

All praises are to ALMIGHTY ALLAH, the most beneficent and the most merciful, who

endowed me the right direction and courage to perform this research work successfully.

ALLAH always showered his blessing onto me, in every steps of my life especially during

this research project.

First of all, I would like to acknowledge my deepest gratitude to my research supervisor,

Dr. Rabia Ismail Yousuf, Assistant professor, Department of Pharmaceutics, Faculty of

Pharmacy, University of Karachi, who took great interest throughout this research work. She

provided her guidance and motivation during the study. I express my appreciation for her

kindness, encouragement, support and patience during the whole period of this research

project.

I wish to express my sincere gratitude to Prof. Dr. Muhammad Harris Shoaib, chairman,

Department of Pharmaceutics, Faculty of Pharmacy, University of Karachi, for providing

valuable information in the areas of this research work. I really appreciate his vision, energy,

patience, support and dedication to the research and research students.

I am also very thankful to Prof. Dr. Syed Baqir Shyum Naqvi, for his support, advice,

encouragement and sincere help whenever needed throughout my educational period and

especially during this research work.

I am thankful to Prof. Dr. Iqbal Azhar, Dean, Faculty of Pharmacy, University of Karachi,

Prof. Dr. Dilnawaz Shaikh and Prof. Dr. Azhar Hussain Dean Faculty of Pharmacy,

Hamdard University for their encouragement and support during this research work.

I am extremely thankful to my dear Uncle Dr. Muhammad Hassan for his moral support,

encouragement, best wishes and prayers not only to complete this task, but also at every

steps of my life especially the era i.e. from Intermediate to Pharmacy, I never forget it.

I want pay especial thanks to all my Teachers, Bosses and Colleagues Dr. Iyad Naeem, Dr.

Rehana saeed, Dr. Sabahat Jabeen, Kamran Ahmed, Ms. Faaiza Qazi and Mr. Fahad Siddiqui

from University of Karachi, Mr. Ejaz-Un-Nabi, Mr. Zafar Hussain and Mr. Mukaish Khan

from Abbott Laboratories Pakistan, Mr. Sohail Anwer, Kamran Zaheer, Humera sarwar and

VI

Liaquat Raza from Hamdard University Karachi, Mr. Tariq Ali and Hafiz Muhammad

Arshad from Dow University of Health Sciences, Mr. Ahsaan Ahmed from Jinnah Sindh

Medical University, and all others seniors and juniors researchers who gave me moral

support to complete this difficult task.

I am thankful to Uncle Akber Ali Nasiri, Hussain Nasiri, Uncle Abbas, Shaikh Raza Salihi,

Uncle Ismail, Zulfiqar Ali Haideri, Ali Kazim, Itikhar Hussain Nasiri, Imtiyaz Hussain

Nasiri, Ejaz Hussain Nasiri, Muhammad Hussain Abbasi, Muhammad Ali Maashafipa,

Sajjad, Gulzar, Ahmed, Yousuf, Shabbir Charagi, brother Abbas, shabbir, Ejaz, Ghulam

Murtaza, Askari, Abid Haideri, Yasra Ali, Alima Nizam, Dr. Ather, Sultan Ali, Shahid

Zehzad, Abbas Malik, Muhammad Ali zubdavi, Muhammad Tahir Aain, Mohsin Haideri,

Aslam Maqdar, who always have sincere feelings and prayers for my success.

I am also thankful for the cooperation and the assistance provided by the staffs of the

Department of Pharmaceutics, Faculty of Pharmacy, University of Karachi and Hamdard

University Karachi.

I would like to thanks my affectionate Parents, Brothers, Shaikh Munawar Hussian,

Mujammad. Arif, Tajjammul Hassan, Zeeshan Haider and sister, AzraBatool for their

prayers, help, support, and motivation that they provided during my entire educational

processes.

My deepest thanks to my lovely sons and daughters for their patience that made study

possible.

Last but not least, I am most grateful to my loving, caring, and passionate wife Kaniz

Fatima, whose encouragement, prayers and support helped me to overcome various hurdles

at each and every step of this research plan as well as during my whole career.

MUHAMMAD IQBAL NASIRI

VII

DEPARTMENT OF PHARMACEUTICS

FACULTY OF PHARMACY

UNIVERSITY OF KARACHI

KARACHI

CERTIFICATE

It is certified that Mr. Muhammad Iqbal Nasiri S/O Ghulam Muhammad Nasiri has

completed his research work entitled “Development and In-Vitro Characterization of

Extended Release Pellets of Itopride Hydrochloride”, under my supervision and

guidance in Department of Pharmaceutics, Faculty of Pharmacy, University of Karachi.

His research work is original and his dissertation is worthy of presentation to the BASR,

University of Karachi for the award of degree of Doctor of Philosophy (PhD) in

Pharmaceutics.

RESEARCH SUPERVISOR

Dr. Rabia Ismail Yousuf Assistant Professor

Department of Pharmaceutics

Faculty of Pharmacy

University of Karachi

VIII

TABLE OF CONTENTS

DEDICATION…………………………………………………….……………….…. IV

ACKNOWLEDGEMENT …………………………………………………………..…V

CERTIFICATE …………………………...……………………………..……….…. VII

TABLE OF CONTENTS ………………………………….….……..………….…. VIII

LIST OF TABLES ……………………………………………………………………XV

LIST OF FIGURES……………………………………………………………….XXVII

LIST OF SYMBOLSAND ABBREVIATIONS…………………………….……XXXI

ABSTRACT ………………………………………………………………….…. XXXIV

KHULASA……………………………………………………………………..……...XL

1. INTRODUCTION…………………………………………………...... 1

1.1. Extended release dosage forms…………….……..…….…………..……….…... 2

1.1.1. Classification of extended release (ER) dosage forms………..….…..…..….. 4

1.1.1.1. Single unit ER dosage form……………………..………….…....…...….. 4

1.1.1.1.1. Matrix systems………………………………………………….…….….4

1.1.1.1.2. Reservoir systems.……………………….………………...…...….….…5

1.1.1.2. Multi-unit ER dosage form………………………….…..……………..…...5

1.1.1.2.1. Matrix systems………………………………………………………...…6

1.1.1.2.2. Reservoir systems………………………...…...…………………........…6

1.1.2. Advantages and disadvantages of ER dosage forms………………………....7

1.1.3. Polymers used in extended release dosage forms……………………….........8

1.2. Formulation development and optimization……………………...….….….…...9

1.3. Characterization of extended release dosage forms…………………………....10

1.3.1. Dissolution testing for single unit system……………………………….…...…11

1.3.2. Dissolution testing for multiparticulate pellet system………………….…..…..11

1.3.3. Dissolution testing…………………………………………………..…….…....12

1.4. Factors influencing the bioavailability of extended release dosage

forms…………………………………....…………………………………………13

1.4.1. Physiological properties of gastrointestinal tract, effect of food, pH and emptying

time……………...…...……………………………………………………..…....13

1.4.2. Effect of physicochemical properties of drug……………………………...…...14

1.4.3. Effect of diseased conditions………………………………………..…..…..….15

1.5. Extended release pellets formulation……………………..….…….……..…….15

1.5.1. Pellets and pelletization technique…………………………...……..….………15

1.5.2. Methods of pelletization…………………………..……………..…….……..16

IX

1.5.2.1. Extrusion and spheronization…………………………………….…….17

1.6. Pharmacokinetics studies…………………...…...……...……………………….17

1.6.1. Area under the plasma concentration time curve…………..……………………18

1.6.2. Volume of distribution (Vd)……………………………………...…..…….…....18

1.6.3. Clearance (Cl)……………………………………………………..……...……..19

1.6.4. Half-life (t1/2) ………………………..……………………………..……………19

1.6.5. Area under the first moment time curve (AUMC)……………………...….........20

1.6.6. Mean residence time (MRT)…………………………………………………….20 1.6.7. Cmax and Tmax ……………………………............................................................20 1.6.8. Rate Constants…………………………………………………………………...21

1.7. Itopride hydrochloride………………..………...….…………………………….22

1.7.1. Physicochemical properties of Itopride HCl………………………………..…...22

1.7.2. Chemical structure………………………………………………..…...…...........23

1.7.3. Mechanism of action………………………………………………..…………...23

1.7.4. Therapeutic indications……………………………………………….……...….23

1.7.5. Dosage and administration………………...…………………………………….24

1.7.6. Pharmacokinetics of Itopride HCl……………………………………….………24

1.7.6.1. Absorption and distribution………………………………………..….….….24

1.7.6.2. Effect of food………………………………………………………..…....…24

1.7.6.3. Metabolism…………………………………………………….….…..….….25

1.7.6.4. Elimination and excretion……………………………………..…………….25

1.7.7. Drug Interactions…………………………………………………...…………....25

1.7.8. Safety and adverse effects…………………………………………............….…25

2. OBJECTIVES OF THE STUDY…………………………………….27

3. LITERATURE SURVEY……………………………………...…......29

3.1. Formulation development of extended release dosage forms………….……30

3.2. Pellets formulations by extrusion-spheronization techniques……….………33

3.3. Hydroxypropyl methylcellulose (HPMC) and ethylcellulose (EC) containing

formulations…………………………………………….....................................36

3.4. Eudragit and Kollicoat containing formulations……………..……………...40

3.5. Release kinetics evaluation of formulations……………………………..........43

X

3.6. Itopride hydrochloride containing formulations……….…………………….47

3.7. Analytical methods for itopride hydrochloride…………..…………...……...50

3.8. Pharmacokinetic studies of Itopride hydrochloride……………...….……....52

4. MATERIAL AND METHODS………………………………………56

4.1. Formulation development of extended release pellets………………...….….57

4.1.1. Materials………………………………………………………………….….57

4.1.2. Methods……………………………………………...………………….…...57

4.1.2.1. Preparation of pellet formulations…………………………………….….57

4.1.2.2. Extended release coating…………………………………………….…...58

4.1.3. Flow Properties of Pellets…………………..…………………………….....63

4.1.3.1. Bulk density……………………………..…………………………..……63

4.1.3.2. Tapped density…………………………………..………………...…...…63

4.1.3.3. Carr’s index………………………………………..…………….....…….63

4.1.3.4. Hausner ratio………………………………………..…………….….…...64

4.1.3.5. Angle of repose………………………………………..………….….…...64

4.1.4. Characterization of Itopride HCl Pellet Formulations……..……….…....65

4.1.4.1. Friability………………...……………………….……………………….65

4.1.4.2. Assessment of pellets surface morphology….……………………….......65

4.1.4.2.1. Sieve analysis……………………………………………...…………65

4.1.4.2.2. Image analysis…………………………………………………...…...65

4.1.4.2.3. Scanning electron microscopy (SEM)…………….………………….66 4.1.4.3. Fourier transform infrared spectroscopy (FTIR)……..……………….…66

4.1.4.4. Drug content analysis………………………………...…………….…….67

4.1.4.5. In-vitro drug release study……………………………………………......67

4.1.5. Drug release kinetic studies ……………………………………………......67

4.1.5.1. Model dependent methods……………………………………….…….…68

4.1.5.1.1. Zero-order kinetics…………………………………….……...…...…68

4.1.5.1.2. First order kinetics………………………………………………..….68

4.1.5.1.3. Higuchi model…………………………………..………….....……...69

4.1.5.1.4. Korsmeyer–Peppas model……………………………………....……69

4.1.5.1.5. Hixson – Crowell model…………………………………..….……...70

4.1.5.1.6. Baker–Lonsdale model…………………………………….……...….70

4.1.5.2. Model independent method…………………………………...….………71

XI

4.1.6. Stability Studies……………………………………………….…...…..…....71

4.1.6.1. Stability Studies of Itopride HCl Pellets……………………………….…72

4.1.7. Method development and validation for the analysis of Itopride HCl in

human plasma…………………………………………….….…...………....73

4.1.7.1. Equipment, software, chemical and glassware………………….………..73

4.1.7.2. Preparation of mobile phase…………………………………..….…...….74

4.1.7.3. Preparation of standard stock and working solutions………….……..….74

4.1.7.3.1. Chromatographic Conditions………………………………...…….….75

4.1.7.3.2. Sample preparation and determination of drug in human plasma…......75

4.1.7.3.3. Retention Time………………………………...……………………....75

4.1.7.3.4. Method Validation……………………………….………………...….75

4.1.7.3.4.1. Selectivity………………………………………………….….….76

4.1.7.3.4.2. Linearity…………………………………………………….…….76

4.1.7.3.4.3. Accuracy and precision……………………………….……...…..76

4.1.7.3.4.3.1. Intraday precision…………………………………...….….…..76

4.1.7.3.4.3.2. Interday precision……………………………………………...77

4.1.7.3.4.4. Limit of quantification (LOQ) and limit of detection (LOD……..77

4.1.7.3.4.5. Analytical recovery……………………….………………..….….77

4.1.7.3.4.6. Stability of the drug in plasma……………………………….......77

4.1.8. Pharmacokinetic studies of extended release Itopride HCl pellets….…...78

4.1.8.1. Ethics board approval…………………………………………………...…78

4.1.8.2. Study venue…………………………………………………...…………...78

4.1.8.3. Design of study………………………………………………….….….….78

4.1.8.4. Volunteers selection……………………………………………………….79

4.1.8.4.1. Criteria for inclusion……………………………………….….………79

4.1.8.4.2. Criteria for exclusion………………………………….……….………79

4.1.8.4.3. Declaration of consent………………………………….……….….….80

4.1.8.5. Blood sampling protocol………………………………….……….............80

4.1.8.5.1. Requirement……………………………………….….……………….80

4.1.8.5.2. Procedure………………………………………….…………….….….80

4.1.8.6. Determination of pharmacokinetic parameters………………………...….81

4.1.8.6.1. Compartmental parameters…………………….….……………….….81

4.1.8.6.2. Non - Compartmental parameters…………………….…………….…81

XII

4.1.8.7. Statistical analysis of the pharmacokinetic data…………………….…......81

4.1.8.7.1. Latin Square ANOVA for two formulations………….………..……...81

4.1.8.7.2. Two one-sided t test………………………………………………..….82

5. RESULTS………………………...……….…………………………...83

5.1. Formulation Development of Extended Release Itopride HCl

Pellets……………………………………………………………………….….84

5.2. Characterizations of ITP pellet formulations ………………………………84

5.3. Pharmacokinetic Studies of extended release ITP pellets…………………..86

6. DISCUSSION……………………………………………….…….….207

6.1. Physical and chemical evaluation of uncoated ITP pellet

formulations………………………………………………………………….210

6.2. Image analysis……………………………………………...………………...210 6.3. Scanning electron microscopy (SEM)……………………….……………...211

6.4. Fourier transform infrared spectroscopy (FTIR)…………….…….….…212

6.5. In vitro drug release studies…………………………………………...........212

6.5.1. Effect of HPMC viscosity grade and concentration on drug release……...213

6.5.2. Effect of ethyl cellulose concentrations…………………………….….….214

6.5.3. Effect of EC coating on pellet formulation……………………………….214

6.5.4. Effect of Eudragit RS/RL 100 coating on pellet formulations…………....215

6.5.5. Effect of Kollicoat SR 30D coating on pellet formulations………...….…215

6.5.6. Effect of dissolution medium on drug release…………………………….216

6.5.6.1. Ethylcellulose coated pellet formulations………………………….….216

6.5.6.2. Eudragit RS/RL 100 coated pellet formulations……………………....217

6.5.6.3. Kollicoat SR 30D coated pellet formulations………………………....218

6.6. Drug release kinetics studies……………………………………...…………219

6.6.1. Kinetics of EC coated pellets………………………………….….……...219

6.6.2. Kinetics of Eudragit RS/RL100 coated pellets…………………….….…220

6.6.3. Kinetics of Kollicoat SR 30D coated pellets………………………...…...220

6.7. Drug release mechanism………………………………………………....….221 6.8. Model independent method…………………………………………....…....222

6.9. Stability studies……………………………………………….….…….…….223

XIII

6.10. Bio-analytical method validation…………….….………………...…….….224

6.11. Pharmacokinetics analysis and comparative bioavailability study of ER

Itopride HCl encapsulated pellets and tablets……………………….....….227

6.11.1. Compartmental and non-compartmental analysis…………………...228

6.11.1.1. Cmax, Tmax, and AUC…………………………….………………...228

6.11.1.2. Volume of distribution and Clearance……………...……...…...….230

6.11.1.3. Half-lives and rate constants……………………..………………...230

6.11.1.4. AUMC and MRT………………………………………..…...…….232

6.11.2. Statistical analysis for establishing bioequivalence…………..……...232

6.11.2.1. Cmax, Tmax, AUC0-∞, AUClast and AUCtot…………………………....232

6.11.2.2. Analysis of other pharmacokinetic parameters…….……………....235

7. CONCLUSION………………………...….…………………………238

8. REFERENCES…………………………...….………………………241

APPENDIX – I……………………...……….……………………..……261

1. Statistical Analysis for Log Transformed Data…………………………………261

A) EC5-F11 (Pellets) Under Fed and Fasted Conditions; (A = Fasted and B =

Fed)…………………………………………………………………………….261

B) Ganaton OD (Tablet) Under Fed and Fasted Conditions; (C = Fasted and D =

Fed)…………………………………………………………………………………….292

2. Statistical Analysis for Non Log Transformed Data……………………..…….332

A) EC5-F11 (Pellets) Under Fed and Fasted Conditions; (A = Fasted and B =

Fed)………………………………………………………..……………..…….332

B) Ganaton OD (Tablet) Under Fed and Fasted Conditions; (C = Fasted and D

= Fed)…………………………………………………………………………..352

XIV

APPENDIX – II………………………………………………………….382

8.1. Software Generated Report of Stability Analysis (Minitab version 17.1.0)

…………………...……………..……...…...…………………...……………...…383

A. At Accelerated temperature (40 0C/75% RH) ……………………………...383

B. At Room Temperature (25 °C /60% RH) …….….…….…………….……..391

8.2. Publication form dissertation ……………….………...…………….…………398

8.3. Ethical Approval Letter………………………………………...……….……....399

XV

LIST OF TABLES

TABLE 1……………………………………………………………………………..…13

Gastrointestinal Tract: Physical Dimensions and Dynamics

TABLE 2……………………………………………………………………………..…60

Composition of Itopride hydrochloride pellets formulations (F1 – F16)

TABLE 3……………………………………………………………………………..…61

Composition of Itopride hydrochloride pellets formulations (F17 – F31).

TABLE 4……………………………………………………………………………..…62

Composition of Ethylcellulose coating solution

TABLE 5……………………………………………………………………………..…62

Composition of Eudragit® RS/RL100 coating solution

TABLE 6……………………………………………………………………………..…62

Composition of Kollicoat® SR 30 D coating dispersion

TABLE 7……………………………………………………………………………..…64

Flow properties of Hausner ratio, compressibility index and angle of repose (USP35-

NF30, 2012)

TABLE 8…………………………………………………………………………..……72

Storage conditions for stability studies (ICH)

TABLE 9……………………………………………………………………………..…88

Evaluation of Uncoated Pellets Formulations (F1 – F16)

TABLE 10…………………………………………………………………………...….89

Evaluation of Uncoated Pellets Formulations (F17 – F31)

TABLE 11…………………………………………………………………………...….90

Image Analysis of Pellet Formulations (F1 – F16), (n > 50)

TABLE 12…………………………………………………………………………..…..91

Image Analysis of Pellet Formulations (F17 – F31), (n > 50)

TABLE 13…………………………………………………………………………..…115

Drug Release Kinetics of EC (5% dispersion) Coated Formulations

XVI

TABLE 14…………………………………………………………………………..…116

Drug Release Kinetics of Eudragit RL/RS 100 (15% dispersion) Coated Formulations

TABLE 15…………………………………………………………………………..…117

Drug Release Kinetics of Kollicoat SR 30 D (50% Dispersion) Coated Formulations

TABLE 16……………………………………………………………….………….…118

Similarity factor (f2) evaluation of ITP pellet formulations with reference to EC5-F11

TABLE 17……………………………………………………………………….….…119

Stability studies and shelf life of EC5-F11at room temperature (25 °C/60% RH)

TABLE 18……………………………………………………………………….….…120

Stability studies and shelf life of EC5-F11at accelerated temperature (40 °C/75% RH)

TABLE 19……………………………………………………………………….….…121

Standard Calibration Curve (Linearity, Accuracy and Precision) in Mobile Phase

TABLE 20……………………………………………………………………….….…122

Standard Calibration Curve (Linearity, Accuracy and Precision) in Plasma

TABLE 21……………………………………………………………………….….…123

Back Calculated Concentration of Itopride HCl Standard Calibration Curve (Linearity,

Accuracy and Precision) in Plasma

TABLE 22……………………………………………………………………………..126

Intraday and Interday Accuracy and Precision of Itopride HCl in Plasma

TABLE 23…………………………..…………………………………………………127

Limit of quantification (LOQ) of Itopride HCl in Plasma

TABLE 24……………………………………………………………………………..128

Limit of Detection (LOD) of Itopride HCl in Plasma

TABLE 25…………………………………………………………………………..…129

Absolute Analytical Recovery

TABLE 26…………………………………………………………………………..…130

Freeze and Thaw stability of Itopride HCl

TABLE 27…………………………………………………………………………..…131

Itopride HCl Degradation in Freeze Thaw stability

TABLE 28…………………………………………………………………………..…132

Long Term stability of Itopride HCl in Plasma

TABLE 29………………………………………………..……………………………133

Itopride HCl Degradation in Long Term Stability

XVII

TABLE 30a………………………………………………………………..……….….136

Details of Volunteers Participated in Pharmacokinetic Studies of Itopride HCl 150 mg

Pellets (EC5-F11) Under Fed and Fasted States

TABLE 30b………………………………………………………………..……….….137

Details of Volunteers Participated in Pharmacokinetic Studies of Itopride HCl 150 mg

Tablet (Ganaton OD) Under Fed and Fasted States

TABLE 31…………………………………………………………………………..…138

Plasma Drug Concentration of Itopride HCl 150 mg pellet (EC5-F11) in 12 Healthy

Human Volunteers under Fed State

TABLE 32………………………………………………………………………..……139

Compartmental Analysis of Different Pharmacokinetic Parameters of Itopride HCl 150

mg pellet (EC5-F11) Under Fed State

TABLE 33…………………………………………………………………………..…140

Non-Compartmental Analysis of Different Pharmacokinetic Parameters of Itopride HCl

150 mg pellet (EC5-F11) Under Fed State

TALBE 34…………………………………………………………………………..…141

Plasma Drug Concentration of Itopride HCl 150 mg pellet (EC5-F11) in 12 Healthy

Human Volunteers under Fasted State

TABLE 35……………………………………………………………………..………142


mg pellet (EC5-F11) Under Fasted State

TABLE 36……………………………………………………………………….……143

Non- Compartmental Analysis of Different Pharmacokinetic Parameters of Itopride HCl

150 mg pellet (EC5-F11) Under Fasted State

TABLE 37………………………………………………………………………….…144

Plasma Drug Concentration of Itopride HCl 150 mg Tablet (Ganaton OD) In 12 Healthy

Human Volunteers under Fed State

TABLE 38…………………………………………………………………….………145


mg pellet (Ganaton OD) under Fed State

TABLE 39………………………………………………………………………….…146

Non Compartmental Analysis of Different Pharmacokinetic Parameters of Itopride HCl

150 mg pellet (Ganaton OD) under Fed State

XVIII

TABLE 40……………………………………………………………………….……147

Plasma Drug Concentration of Itopride HCl 150 mg Tablet (Ganaton OD) In 12 Healthy

Human Volunteers under Fasted State

TABLE 41…………………………………………………………………….….……148


mg pellet (Ganaton OD) under Fasted State

TABLE 42……………………………………………………………………..………149

Non-Compartmental Analysis of Different Pharmacokinetic Parameters of Itopride HCl

150 mg pellet (Ganaton OD) under Fasted State

TABLE 43………………………………………………………………………….….155

Mean Pharmacokinetic Log Transformed Parameters of Extended Release Itopride HCl

150 mg Encapsulated Pellets (EC5-F11) Versus Itopride HCl 150 mg Tablet (Ganaton

OD), with Geometric Mean Ratios at 90% CI

TABLE 44………………………………………………………………………….….156

Mean Pharmacokinetic Non-Log Transformed Parameters of Extended Release Itopride

HCl 150 mg Encapsulated Pellets (EC5-F11) Versus Itopride HCl 150 mg Tablet

(Ganaton OD), with Geometric Mean Ratios at 90% CI

TABLE 45…………………………………………………………………………......157

Comparative Bioavailability of Itopride HCl 150 mg Pellets (EC5-F11) To That of

Itopride HCl 150 mg Tablet (Ganaton OD)

APPENDIX – I………………………………………………...261

STATISTICAL ANALYSIS FOR LOG TRANSFORMED DATA: ……………..262

EC5-F11 (Pellets) Under Fed and Fasted Conditions; (A = Fasted and B = Fed)

TABLE 46………………………………………………………………………..……262

LATIN SQUARE DESIGN: ANOVA TABLE for Ka

TABLE 46…………………………………………………………………………..…263

LATIN SQUARE DESIGN: ANOVA TABLE for Kel

TABLE 48………………………………………………………………………….…264

LATIN SQUARE DESIGN: ANOVA TABLE for ALPHA (α)

XIX

TABLE 49………………………………………………………………………….…265

LATIN SQUARE DESIGN: ANOVA TABLE for BETA (β)

TABLE 50……………………………………………………………………….……266

LATIN SQUARE DESIGN: ANOVA TABLE for K12

TABLE 51………………………………………………………………………….…267


TABLE 52……………………………….……………………………………………268

LATIN SQUARE DESIGN: ANOVA TABLE for Cmax calc

TABLE 53…………………………………………….………………………………269

LATIN SQUARE DESIGN: ANOVA TABLE for Tmax calc

TABLE 54………………………………………………………………….…………270

LATIN SQUARE DESIGN: ANOVA TABLE for Cmax

TABLE 55……………………………………………………………………….……271

LATIN SQUARE DESIGN: ANOVA TABLE for AUC0-∞

TABLE 56……………………………………………………………………….……272

LATIN SQUARE DESIGN: ANOVA TABLE for Vc

TABLE 57……………………………………………………………………….……273

LATIN SQUARE DESIGN: ANOVA TABLE for Clearance (Cl)

TABLE 58………………………………………………………………………….…274

LATIN SQUARE DESIGN: ANOVA TABLE for T1/2ka

TABLE 59………………………………………………………………………….…275

LATIN SQUARE DESIGN: ANOVA TABLE for Tabs

TABLE 60……………………………………………………………………..………276

LATIN SQUARE DESIGN: ANOVA TABLE for T1/2α

TABLE 61………………………………………………………………….…………277

LATIN SQUARE DESIGN: ANOVA TABLE for T1/2β

TABLE 62……………………………………………………………………….……278

LATIN SQUARE DESIGN: ANOVA TABLE for T1/2kel

TABLE 63……………………………………………………………………….……279

LATIN SQUARE DESIGN: ANOVA TABLE for AUMC

TABLE 64………………………………………………………………………….…280

LATIN SQUARE DESIGN: ANOVA TABLE for MRT

TABLE 65…………………………………………………………………….………281

LATIN SQUARE DESIGN: ANOVA TABLE for Lz

XX

TABLE 66…………………………………………………………………….………282

LATIN SQUARE DESIGN: ANOVA TABLE for HVD

TABLE 67……………………………………………………………………….……283

LATIN SQUARE DESIGN: ANOVA TABLE for AUClast

TABLE 68……………………………………………………………………….……283

LATIN SQUARE DESIGN: ANOVA TABLE for AUCtotal

TABLE 69……………………………………………………………………….……284

LATIN SQUARE DESIGN: ANOVA TABLE for AUCextra

TABLE 70…………………………………………………………………….………285

LATIN SQUARE DESIGN: ANOVA TABLE for %AUCextra

TABLE 71……………………………………………………………………….……286

LATIN SQUARE DESIGN: ANOVA TABLE for AUMClast

TABLE 72……………………………………………………………………….……287

LATIN SQUARE DESIGN: ANOVA TABLE for AUMCtotal

TABLE 73……………………………………………………………………….……289

LATIN SQUARE DESIGN: ANOVA TABLE for AUMCextra

TABLE 74………………………………………………………………………….…290

LATIN SQUARE DESIGN: ANOVA TABLE for Vz

TABLE 75………………………………………………………………………….…291

LATIN SQUARE DESIGN: ANOVA TABLE for T1/2Lz

STATISTICAL TEST FOR LOG TRANSFORMED DATA: Ganaton OD (Tablet)

Under Fed and Fasted Conditions; (C = Fasted and D = Fed)……………………292

TABLE 76………………………………………………………………………….…292


TABLE 77…………………………………………………………………….………293


TABLE 78……………………………………………………………………….……294


TABLE 79…………………………………………………………………………….295


TABLE 80……………………………………………………………………….……296


XXI

TABLE 81…………………………………………………………………….………297


TABLE 82………………………………………………………………………….…298


TABLE 83………………………………………………………………………….…299


TABLE 84……………………………………………….……………………….…...300


TABLE 85……………………………………………………………….……………301


TABLE 86…………………………………………………………………….………302


TABLE 87……………………………………………………………………….……303


TABLE 88………………………………………………………………………….…304


TABLE 89……………………………………………………………………….……305


TABLE 90……………………………………………………………….……………306


TABLE 91………………………………………………………………….…………307


TABLE 92………………………………………………………………………….…308


TABLE 93……………………………………………………………………….……309


TABLE 94……………………………………………………………………….……310


TABLE 95………………………………………………………………………….…311


TABLE 96………………………………………………………………………….…312


TABLE 97…………………………………………………………………….………313


XXII

TABLE 98………………………………………………………………………….…314


TABLE 99………………………………………………………………………….…315


TABLE 100……………………………………………………………………….…...316


TABLE 101……………………………………………………………………….…...317


TABLE 102……………………………………………………………………………318


TABLE 103………………………………………………………………………........319


TABLE 104………………………………………………………………………........320


TABLE 105………………………………………………………………..……..........321


STATISTICAL ANALYSIS FOR NON-LOG TRANSFERMED DATA………...322

EC5-F11 (Pellets) Under Fed and Fasted Conditions; (A = Fasted and B = Fed)

TABLE 106………………………………………………………………………........322


TABLE 107…………………………………………………………………………....323


TABLE 108……………………………………………………………………………324


TABLE 109…………………………………………………………………….….…..325


TABLE 110…………………………………………………………………………....326


TABLE 111………………………………………………………………………...….327


TABLE 112………………………………………………………………………...….328


XXIII

TABLE 113………………………………………………………………………...….329


TABLE 114…………………………………………………………………………....330


TABLE 115…………………………………………………………………………....331


TABLE 116……………………………………………………………………..….….332


TABLE 117………………………………………………………………………...….333


TABEL 118………………………………………………………………...………….334


TABLE 119……………………………………………………………………………335


TABLE 120………………………………………………………..……………….….336


TABLE 121………………………………………………………………………...….337


TABLE 122……………………………………………………..………………….….338


TABLE 123……………………………………………………………………..….….339


TABLE 124…………………………………………………………………………....340


TABLE 125………………………………………………………………………...….341


TABLE 126……………………………………………………………………………342


TABLE 127……………………………………………………………………………343


TABLE 128……………………………………………………………………………344


TABLE 129……………………………………………………………………………345


XXIV

TABLE 130……………………………………………………………………………346


TABLE 131……………………………………………………………………………347


TABLE 132……………………………………………………………………………348


TABLE 133……………………………………………………………………………349


TABLE 134…………………………………………………………………………....350


TABLE 135……………………………………………………………………………351


STATISTICAL ANALYSIS FOR NON-LOG TRANSFERMED DATA: Ganaton

OD (Tablet) Under Fed and Fasted Conditions; (C = Fasted and D = Fed)………352

TABLE 136…………………………………………………………………..…….….352


TABLE 137……………………………………………………………………...…….353


TABLE 138…………………………………………………………..…………….….354


TABLE 139…………………………………………………………………..…….….355


TABLE 140………………………………………………………………………...….356


TABLE 141………………………………………………………………………...….357


TABLE 142…………………………………………………………………………....358


TABLE 143…………………………………………………………………………....359


TABLE 144……………………………………………………………………………360


XXV

TABLE 145……………………………………………………………………………361


TABLE 146……………………………………………………………………………362


TABLE 147……………………………………………………………………………363

LATIN SQUARE DESIGN: ANOVA TABLE for T1/2ka.

TABLE 148……………………………………………………………………………364


TABLE 149……………………………………………………………………………365


TABLE 150……………………………………………………………………………366


TABLE 151……………………………………………………………………………367


TABLE 152……………………………………………………………………………368


TABLE 153…………………………………………………………………………....369


TABLE 154……………………………………………………………………………370


TABLE 155……………………………………………………………………………371


TABLE 156……………………………………………………………………………372


TABLE 157……………………………………………………………………………373


TABLE 158……………………………………………………………………………374


TABLE 159……………………………………………………………………………375


TABLE 160……………………………………………………………………………376


TABLE 161……………………………………………………………………………377


XXVI

TABLE 162……………………………………………………………………………378


TABLE 163……………………………………………………………………………379


TABLE 164……………………………………………………………………………380


TABLE 165……………………………………………………………………………381


XXVII

LIST OF FIGURES

FIGURE 1………………………………………………………………………....….… 3

Plasma drug concentration of different dosage forms after administration

FIGURE 2……………………………………………………………………………..... 4

Drug release pattern from diffusion-controlled matrix tablets

FIGURE 3……………………………………………………………………..................5

Drug release pattern from diffusion-controlled reservoir system

FIGURE 4……………………………………………………………………..………..22

Structure of Itopride HCl

FIGRUE 5………………………………………………………………………………92

Stereo micrographs of (a) uncoated plain and (b) matrix ITP pellets

FIGURE 6……………………………………………………………………................93

Stereo micrographs of (a) ethylcellulose coated F1, (b) F6, (c) F11, (d) F16 and (e) F21,

Itopride pellet formulations

FIGURE 7………………………………………………..…………………………......94

Stereo micrographs of (a) Eudragit® RS/RL100 coated F1, (b) F6, (c) F11, (d) F16 and

(e) F21, Itopride pellet formulations

FIGRUE 8……………………………………………………………………..……..…95

Stereo micrographs of (a) Kollicoat® SR 30Dcoated F1, (b) F6, (c) F11, (d) F16 and (e)

F21, Itopride pellet formulations

FIGURE 9………………………………………………………………………...….....96

SEM surface images of EC coated (5% dispersion) (a) F1, (b) F6, (c) F11, (d) F16 and

(e) F21, Itopride pellet formulations

FIGURE 10……………………………………………………………....................…..97

SEM cross-sectional images of EC coated (5% dispersion) (a) F1, (b) F6, (c) F11, (d)

F16 and (e) F21, Itopride pellet formulations

FIGURE 11…………………………………………………………………….……….98

FTIR spectra of (a) Pure ITP, (b) ITP + HPMC (K4M), (c) ITP + HPMC (K15M), (d)

ITP + HPMC (K100M), and (e) ITP + EC (7cps).

XXVIII

FIGURE 12………………………………………………………….………………...100

In-vitro Drug Release Profile of Uncoated Plain Pellet Formulations (F1 – F5)

FIGRUE 13………………………………………………………………….………...101

In-vitro Drug Release Profile of Uncoated Matrix Pellet Formulations (a) F6 – F18 and

(b) F19 – F31in 0.1 N HCl at pH 1.2

FIGURE 14………………………………………………………………………..…..102

In-vitro Drug Release Profile of Pellets Formulation F1 coated with (a) 5%, (b) 10% and

(c) 15% ethylcellulose dispersion

FIGURE 15……………………………………………………………………………103

In-vitro Drug Release Profile of Pellets Formulation F6 coated with (a) 5%, (b) 10% and

(c) 15% ethylcellulose dispersion

FIGURE 16……………………………………………………………………………104

In-vitro Drug Release Profile of Pellets Formulation F11 coated with (a) 5%, (b) 10%

and (c) 15% ethylcellulose dispersion

FIGURE 17………………………………………………………………………..…..105



FIGRUE 18………………………………………………………………………...….106



FIGRUE 19……………………………………………………………………………107

In-vitro Drug Release Profile of Pellet Formulation F1 coated with (a) 5%, (b) 10% and

(c) 15% Eudragit RL/RS 100 (2:1, w/w) dispersion

FIGRUE 20………………………………………………………………………........108



FIGRUE 21…………………………………………………………………………....109



FIGURE 22…………………………………………………………………………....110



XXIX

FIGRUE 23……………………………………………………………………….…...111



FIGRUE 24………………………………………………………………………..…..112

In-vitro Drug Release Profile of Pellet Formulation F1 coated with 50% Kollicoat SR

30D dispersion

FIGURE 25……………………………………………………………………………112


30D dispersion

FIGRUE 26……………………………………………………………………………113


30D dispersion

FIGURE 27……………………………………………………………………………113


30D dispersion

FIGRUE 28……………………………………………………………………………114

Drug Release Profile of Pellet Formulation F21 coated with 50% Kollicoat SR 30D

dispersion

FIGRUE 29……………………………………………………………………………114

In-vitro Drug Release Profile of Ganaton OD 150 mg Tablet (Reference Product) at pH

1.2, 4.5 and 6.8

FIGRUE 30a………………………………………………………………….…….....124

Calibration curve of itopride HCl in plasma

FIGRUE 30b……………………………………………………………………....…..125

Calibration curve of itopride HCl in plasma

FIGURE 31…………………………………………………………………….…...…134

Chromatograms for Linearity Curve in Mobile Phase (Conc. Ranges from 0.05 – 2.0

µg/mL)

FIGURE 32……………………………………………………………………….…...135

Chromatograms for Linearity Curve in Plasma (Conc. Ranges from 0.05 – 2.0 µg/mL)

FIGURE 33……………………………………………………………………………150

Plasma Concentration Vs Time Profile Comparison of Itopride HCl 150 mg pellet (EC5-

F11) in 12 Healthy Volunteers Under Fasted and Fasted State

XXX

FIGURE 34…………………………………………………………………….…..….152

Plasma Concentration Vs Time Profile Comparison of Itopride HCl 150 mg Tablet

(Ganaton OD) in 12 Healthy Volunteers under Fed and Fasted State

FIGURE 35………………………………………………............................................154

Mean Plasma Concentration Vs Time Profile Comparison of Itopride HCL 150 mg

Pellets (EC5-F11) and Itopride HCL 150 mg Tablet (Ganaton OD) in Twelve Healthy

Volunteers Under Fed and Fasted State

CHROMATOGRAMS……………………………………………………………….158

FIGURE 36……………………………………………………………………………159

Chromatograms of Plasma Samples of Itopride HCl 150 mg Pellets (EC5-F11) in 12

Healthy Volunteers under Fed state

FIGURE 37……………………………………………………………………......….157

Chromatograms of Plasma Samples of Itopride HCl 150 mg Pellets (EC5-F11) in 12

Healthy Volunteers under Fasted State

FIGURE 38………………………………………………………………………...…183

Chromatograms of Plasma Samples of Itopride HCl 150 mg Tablet (Ganaton OD) in 12

Healthy Volunteers under Fed State

FIGURE 39………………………………………………………………...………....195

Chromatograms of Plasma Samples of Itopride HCl 150 mg Tablet (Ganaton OD) in 12

Healthy Volunteers under Fasted State

XXXI

LIST OF SYMBOLS AND ABBREVIATIONS

AR Aspect ratio

% AUCextra Percentage of AUC extra respect to AUC total

α Overall distribution rate constant

β Distribution rate constant

Lz orλz Elimination rate constant in non-compartmental analysis

ANOVA Analysis of variance

AUC0-∞ or AUC Area under the curve from zero to infinity

AUCextra Extraplotted area under the curve

AUClast AUC from t-0 to t last (last sampling time point)

AUCtotal AUClast + AUCextra

AUMCextra Extraplotted area under the moment curve

AUMClast AUMC from t-0 to t last

AUMCtotal AUMClast + AUMCextra

API Active Pharmaceutical Ingredient

CCD Central composite design

BE Bioequivalence

Cl Total clearance

CI confidence interval

Cmax Maximum plasma concentration

CR Controlled release

CYP3A4 Cytochrome P450 3A4

DSC Differential Scanning Calorimetry

EC Ethyl Cellulose

EMEA European Medicine Agency

XXXII

EPR Enhanced permeability and retention (in partitioning)

ER Extended release

f1 Dissimilarity Factor

f2 Similarity Factor

FTIR Fourier transform infrared, spectroscopy

GERD Gastroesophageal Reflux Disease

GI Gastro Intestinal

GIT Gastro Intestinal Tract

FDA Food and Drug Administration

HEC Hydroxyethylcellulose

HPLC High Performance Liquid Chromatography

HPMC Hydroxypropyl Methyl Cellulose

HPC Hydroxypropylcellulose

HVD Half Value Duration. Describe the time coverage of drug

concentration in the plasma between half of Cmax and Cmax.

This parameter is estimated as the last time the

concentration time curve falls below the half- Cmax

subtracted by the first time that it claims above half- Cmax.

ICH International conference on Harmonization

ITP Itopride HCl

K0 Zero order rate constant

K First order rate constant

KH Higuchi rate constant

K12 Elimination rate constant from central compartment to

peripheral compartment

K21 Elimination rate constant from peripheral compartment to

central compartment

Kel Elimination rate constant from central compartment

XXXIII

LOD Limit of Detection

LOQ Limit of Quantification

MC methylcellulose

MCC Microcrystalline cellulose

PG Propylene glycol

PK Pharmacokinetics

SEM Scanning electron microscopy

SR Sustained release

TEC Triethyl citrate

Tmax Time to reach maximum plasma concentration

T1/2 Half Life

T1/2a Absorption Half life

T1/2kel Elimination Half life

T1/2β Disposition Half life

T1/2α Distribution Half life

Tlag Lag time

Vc Apparent volume of central compartment

Vd Volume of distribution

WHO World Health Organization

XXXIV

ABSTRACT

Pharmaceutical research and development is backbone of the health care sector.

Pharmaceutical formulation scientists are striving all over the world to develop drug

delivery systems to achieve therapeutic goal along with better patient compliance.

Formulation development of novel dosage form is a continuous process and an integral

part of pharmaceutical manufacturing. Currently pharmaceutical manufacturers are

focusing on the development of extended release dosage forms in order to enhance

patients’ concordance and improve quality of life. The development of extended release

pellets is one of the approaches to control the rate of drug delivery for the desirable

period of time. In Pakistan more than 20 multinational pharmaceutical industries are

operated all over the country and the national pharmaceutical companies are exhibiting

tremendous growth since the last few decades. But there is limited no of pharmaceutical

companies involved in the manufacturing of controlled release systems like pellets, and

the application of extrusion-spheronization technique for the production of pellets has

still not been reported in this region. Therefore, in current study, an effort has been made

to prepare pellets of highly water soluble drug Itopride Hydrochloride by extrusion-

spheronization technique.

Itopride HCl is a prokinetic drug which is now consider as a drug of choice in

Gastroesophageal Reflux Disease (GERD) because of its dual action i.e. anti-

cholinesterase (AchE) activity and dopamine D2 receptor antagonistic activity, that’s

why recommended for the therapy of different gastrointestinal motility diseases. In the

present study, the extrusion–spheronization technique was used to develop extended

release Itopride HCl (ITP) coated pellets. Different polymers like hydroxypropyl

methylcellulose (HPMC K4M, K15M, and K100M) and ethyl cellulose (EC-7 cps) were

evaluated for the influence of concentration and viscosity grade on the release profile of

Itopride HCl. Since, the population pharmacokinetic data of this drug is not available, so

pharmacokinetic study was conducted on local healthy Pakistani subjects.

The plain pellet (without polymer) and matrix pellet formulations composed of

hydroxypropyl methylcellulose (10–50%) (HPMC K4M, K15M, and K100M), ethyl

cellulose (10–30%) (EC-7 cps) microcrystalline cellulose (10–30%) and a fixed quantity

XXXV

of lactose (10%). Total thirty-one formulations were prepared, i.e. formulations F1 – F5

(without polymers), F6 – F10 (HPMC K4M), F11 – F15 (HPMC K15M), F16 – F20

(HPMC K100M), F21 – F25 (EC 7 cps), F26 – F28 (30 – 50% HPMC K100M) and F29

– F31 (HPMC K100M + EC 7cps). Five pellet formulations one without polymer (F1)

and one from each polymer category (F6, F11, F16 and F21) were screened out for

coating using different levels (5–15%) of ethylcellulose (Premium 10 cps), Eudragit®

RS/RL100 (2:1, w/w), and 50% dispersion of Kollicoat® SR 30D. The initial burst

release of drug from matrix pellets was excellently controlled by coating with 5% ethyl

cellulose (10 cps) dispersion which extended the drug release up to desired period of

time i.e. 12 h.

The physical and chemical properties such angle of repose, bulk density, tapped density,

compressibility index, Hausner ratio, friability and drug content of uncoated ITP pellet

formulations were evaluated and the results found satisfactory. The surface morphology

and cross section of pellets were analyzed by using Scanning electron microscope. Both

uncoated and coated pellets were found to be spherical with aspect ratio closer to 1. The

selected coated formulations were also analyzed by using Fourier transform infrared

spectroscopy and no incompatibility between drug–excipients was found. The coated

pellet formulations were tested for multiple point dissolution release profiles in HCL

buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8). The DD Solver (an add-in

software for MS Excel) was used to analyze the dissolution profile data for drug release

kinetics. Higuchi model best describes the kinetics of all EC coated formulations (R2 =

0.922–0.999) and first order kinetics (R2 = 0.982–0.999). EC coated pellet (F1, F6, F16

and F21) followed Hixson-Crowell (R2 = 0.981–0.998) and Baker-Lonsdale model (R2 =

0.911–0.999). However, drug release from HPMC matrix EC coated pellets (F11)

followed zero-order kinetics (R2 = 0.988 – 0.991). The release exponent n for the EC

coated matrix formulations F11, F16 and F21 ranged from 0.459 to 0.828, indicating

non-Fickian diffusion mechanism (anomalous transport), whereas, for F1 and F6 the

value of release exponent n ranged from 0.298 to 0.438, showing Fickian diffusion

mechanism. The coefficients correlation (R2) values for all the 15% Eudragit RS/RL 100

coated formulations were ranged from 0.921 – 0.997, showing a good linearity. The

values of n for all the Eudragit RS/RL 100 coated formulations F1, F6, F11, F16 and F21

ranged from 0.177 to 0.431, showing Fickian diffusion mechanism. The coefficients

correlation (R2) values for all the Kollicoat SR 30D were 0.933 – 0.997, and the values

XXXVI

of n for F6, and F11, were 0.127 to 0.264, exhibiting Fickian type of drug release

mechanism, whereas, the n value for F1, F16 and F21, ranged from 0.918 to 1.596,

exhibiting Super Case-II Transport. EC5-F11 (5% EC coated Itopride HCl pellet)

qualified all pharmaceutical quality attributes, and followed zero order release kinetics,

therefore considered as a reference formulation for the calculation of similarity factor

(f2). Similarity (>50%) was found for 5% EC coated F6 formulation in phosphate buffer

(pH 4.5 & 6.8) and 5% EC coated F21 in phosphate buffer pH 4.5.

The stability studies of 5% EC coated ITP pellet formulations, F1, F6, F11, F16 and F21,

were conducted as per ICH guidelines to assess their physical appearance, and

percentage drug content. All the formulations were packed into amber glass bottles and

stored at room temperature (25 ± 2°C /60 ± 5% RH) for 12 months and at accelerated

temperature (40 ± 2°C /75 ± 5% RH) for 6 months. The data were analyzed by using

Minitab 17 and shelf-lives of all formulations were calculated. The observed shelf-lives

were found in the range of 17.6 to 23.7 months at room temperature and 11.8 to 16.27

months at accelerated temperature.

An HPLC method was modified and validated according to FDA guideline for the

estimation of Itopride HCl in human plasma for pharmacokinetic evaluation. HPLC

column Phenomenex - C18 (250 mm, 4.6 mm, 5 µm) was used for the isocratic elution

using mobile phase consisted of acetonitrile and 0.05 M KH2PO4 (pH 4.0) in the ratio of

70:30 % (v/v). A linear relationship was found in the concentration ranged from 0.05 to 2

µg/ml with mean coefficient of correlation (r2 = 0.9989). The Itopride HCl was detected

at 258 nm with a flow of 1 ml/min and the mean retention time was 7.28 minutes. All

validation parameters were within the acceptance limits.

For pharmacokinetic evaluation, EC5-F11 (5% EC coated F11 pellet formulation) was

selected and study was carried out on 12 healthy human volunteers under fed and fasted

conditions as per FDA guidelines. The study was designed as single centered, single

dose, open labelled, randomized, two treatments, two sequence, four periods crossover

fashion, with two weeks washout period. The reference product was Ganaton OD 150

mg tablet, a product of Abbott Laboratories Pakistan. Hamdard University Ethical

Review Board (HU-ERB) and Board of Advanced Studies and Research, University of

Karachi were approved the study protocol. Before the study, written consent from all

XXXVII

volunteers was obtained. A blood sample of 5 ml was taken at 0, 0.5, 1, 2, 4, 6, 8, 12, 24

and 48 hours and plasma was separated and frozen at -20 °C. The concentration of drug

in the plasma samples was estimated by using validated HPLC method. The data of both

test and reference products were analyzed through compartmental and non-

compartmental models using pharmacokinetic software, Kinetica® Version 5.1

(Thermoelectron Corp, USA). The mean Cmax calculated value of test product under fed

and fasted conditions were 0.647 ± 0.011 µg/mL and 0.567 ± 0.020 µg/mL, while, the

mean Cmax calculated value of reference product were 0.634 ± 0.031 μg/mL (fed) and

0.565 ± 0.001 μg/mL (fasted). Calculated mean Tmax of test product under fed and fasting

conditions were 7.282 ± 0.088 h and 5.825 ± 0.246 h, while that of reference product,

were 7.404 ± 0.120 h and 5.910 ± 0.063 h, respectively. The mean values of AUClast and

AUC0-∞ for test product were 13.258 ± 0.323 µg.h/mL (fed) and 13.445 ± 0.427 µg.h/mL

(fed) and 11.966 ± 0.678 µg.h/mL (fasted) and 9.608 ± 0.592 µg.h/mL (fasted).

Similarly, the mean values of AUClast and AUC0-∞ for reference product under fed

conditions were 12.335 ± 0.831 mg/L× h and 13.422 ± 0.558 mg/L× h, and, under fasted

state were 9.837 ± 0.302 mg/L×h and 9.543 ± 0.397 mg/L×h, sequentially. The

elimination half-life (T1/2Kel) and absorption half-life (T1/2Ka) of test product were 6.814 ±

0.380 h (fed) and 3.701 ± 0.251 h (fed), and 5.556 ± 0.403 h (fasted) and 2.764 ± 0.278 h

(fasted). Similarly, the elimination half-life (T1/2Kel) and absorption half-life (T1/2Ka) of

reference product under fed and fasted conditions were 6.648± 0.0.581 h and 4.010±

0.338, and 5.717± 0.465 h and 2.676 ± 0.106 h, respectively.

Compartmental analysis provided the mean volume of distribution (Vc) value for test

product under fed and fasting conditions as 109.637 ± 4.354 L and 125.306 ± 7.966 L,

whereas the mean clearance (Cl) values under same conditions were 15.664 ± 0.929L/h

and 11.167 ± 0.365 L/h, respectively. Similarly, for reference product the values of Vc

were found to be 107.029 L (fed) and 129.473 L (fasted), and the values of Cl were

11.194 L/h (fed) and 15.743 L/h (fasted).

The rate constants at different phases were also determined for test product like, the

mean absorption rate constant (Ka), overall distribution rate constant (α) and elimination

rate constant (Kel) and their fed vs fasted values were observed as Ka (0.188 ± 0.013 and

0.253 ± 0.025 hr-1), α (0.132 ± 0.006 and 0.182 ± 0.010 hr-1) and Kel (0.102 ± 0.006 and

0.125 ± 0.009 hr-1). Similarly, these rate constants values for reference product were Ka

XXXVIII

(0.174 ± 0.016 and 0.259 ± 0.011 hr-1), α (0.129 ± 0.007 and 0.177 ± 0.005 hr-1) and Kel

(0.105 ± 0.010 and 0.122 ± 0.009 hr-1).

Through non-compartmental analysis, the mean AUMC values of test product were

215.469 ± 13.663 mg/L.hr2 (fed)and 136.280 ± 23.866 mg/L.hr2 (fasted), whereas, the

mean residence time (MRT) values were 22.640 ± 0.484 h (fed), and 30.684 ± 3.344 h

(fasted). Similarly, the fed vs fasted mean AUMC values of the reference product were

227.559 ± 42.152 mg/L. hr2 and 131.376 ± 13.118 mg/L.hr2, and, MRT values were

26.245 ± 2.351 h (fed) and 22.158 ± 0.715 h (fasted).

The values of micro rate constants from central to peripheral K12, and from peripheral to

central compartments K21, were also calculated through compartmental analysis, under

fed and fasted conditions, and the mean values of K12 were 0.008 ± 0.004 hr-1 and 0.021

± 0.024 hr-1 and of K21 were 0.098 ± 0.006 hr-1 and 0.103 ± 0.103 hr-1, respectively.

Statistical analysis i.e. two-way analysis of variance (ANOVA) and two one-sided t test

were applied using Kinetica software, in order to observe the effect of fed and fasted

states on pharmacokinetic parameters of Itopride HCl extended release formulation. In

this regard the test product (EC5-F11) under fasted (A) condition was compared with the

fed (B) conditions. Similarly, the reference product (Ganaton OD Tablet) was also

analyzed without food (C) and with food (D). The analysis was done on both log and

non-log transformed data, where p> 0.05 was considered as non-significant. The values

of geometric mean rations at 90 % confidence interval limit (0.8 – 1.25) of Cmax

calculated, Cmax observed, Tmax,AUC0-∞, AUClast and AUCtot of the test product when

compared under fed and fasted conditions, were found to be 1.141 (1.122 – 1.160), 1.169

(1.153 – 1.186), 1.250 (1.219 – 1. 284), 1.401 (1.345– 1.460), 1.351(1.321– 1.380), and

1.348(1.317–1.379), respectively. While, for reference product, these values were Cmax

calculated 1.121 (1.092 – 1.151), Cmax observed 1.171 (1.158 – 1.184), Tmax, 1.253 (1.240

– 1.266), AUC0-∞1.406 (1.363 – 1.451), AUClast1.331 (1.298 – 1.365) and AUCtot1.318

(1.279 – 1.357). The results were further confirmed by Schuirmann‘s two one-sided t

test.

Non-log transformed data with Latin Square ANOVA was also applied for the test

product (EC5-F11) and reference product (Ganaton OD Tablet). The analysis revealed

XXXIX

no significance difference for test and reference products, under fed and fasted

conditions for Cmax calculated, Cmax observed and Tmax. There was no difference observed

in the outcomes of logarithmically transformed or non-transformed data.

On the basis of the current study, it can be concluded that the combinations of HPMC

(10% K15M matrix former), and EC, 10 cps (coating agent) with MCC and Lactose, can

be used effectively to control the release of Itopride HCl (highly water soluble drug), for

extended period of time, when formulated in pellet dosage form by extrusion and

spheronization technique. When the pharmacokinetic parameters of test (EC5-F11) were

compared with that of reference (Ganaton OD Tablet) product under fed and fasted

states, then both the products were found equivalent. The relative bioavailability of test

(EC5-F11) and reference (Ganaton OD) products was 100.171% under fed state and

100.681% under fasted state. The geometric mean value of Tmax, at 90% CI of test and

reference products were 1.250 (1.219 – 1. 284) and1.253 (1.240 – 1.266) respectively,

indicating that food had delayed the absorption of Itopride in both formulations, without

any significant change in oral bioavailability.

XL

XLI

XLII

XLIII

XLIV

XLV

1

1. INTRODUCTION

2

1.1. Extended release dosage forms:

With the passage of time, pharmaceutical scientists have designed advanced oral drug

delivery systems to achieve therapeutic target. In order to obtain desirable therapeutic

plasma drug concentration, the drug release should be appropriate from the delivery

system and this goal is more challenging for the modified release drug delivery systems.

In order to avert repeated administration of unit dosage forms (immediate release tablets

and capsules), extended release formulations have been produced to maintain the

therapeutic level of drug in the plasma and to avoid toxic concentration. Therefore, to

overcome fluctuation in plasma drug levels and to reduce frequency of administration,

various formulations have been designed to control the therapeutic plasma drug

concentrations over extended period of time (FDA, 1997). Extended release formulations

(also regarded as controlled release) of short half-life drugs are better option for the long

term clinical management of chronically ill patients (Samal, H.B. et al., 2011: Lachman,

L. et al., 1986).

There are many extended release formulations of various drugs available in market,

which are economical and exhibiting better patient compliance. (Wilson, B. et al.,

2011a). There are different methods of manufacturing modified release dosing units like

direct compression method, wet and dry granulation methods, pelletization technique and

even coating with a suitable polymer, can effectively control the release of several drugs

with different physico-chemical properties (Bose, A. et al., 2013). Several controlled

release dosage forms have been developed usingwater soluble and insoluble polymers,

membrane-controlled system, waxes and osmotic system. The release of water soluble

drugs can be control by using different combinations of hydrophobic and hydrophilic

polymers (Reza, M.S. et al., 2003). Whereas to produce an extended release drug

delivery system of hydrophobic drugs, the low solubility is the main issue to overcome,

that can be improved by applying combination of techniques, like using polymers and

solid dispersion method (Tanaka, N. et al., 2006). However, extended release matrix

tablets produced by employing various hydrophilic and hydrophobic polymers, are

considered as the simplest approach to design this drug releasing system (Shaikh, A.C. et

al., 2011). The release of drug through the barrier gel (surrounded the matrix) is not only

influenced by its physical and mechanical characteristic but also due to drug solubility.

3

The swelling of matrix, erosion, and diffusion of drug, measures the kinetics and the

drug release mechanism (Chavhan, S. et al., 2011). Currently formulations of controlled

release matrix tables have gained tremendous popularity due to several advantages like,

simple processing steps and a low cost of fabrication. Several statistical techniques, that

now have been used by formulation scientists for the development and optimization of

controlled release formulations, have reduced the cost and duration of formulation design

by minimizing the number of trial batches (Bose, A. et al., 2013; Reddy, K.R. et al.,

2003).

Drugs with narrow absorption window in intestine and stomach are not suitable

candidates for sustained release delivery systems. Because the transit time of these drugs

is short in these segments, and will pass away from the site of absorption. Hence it

moves towards non absorbing part of GIT and results in less bioavailability of these

drugs (Chavanpatil, M. et al., 2005). The multiunit controlled release dosage forms such

as pellets, granules, or micro particles, consolidated into tablets, or filled into capsules

(hard gelatin), have become more important in pharmaceutical markets than single unit

drug delivery system (tablets and capsule), because of advantages like less difficulty in

esophageal transport, and thus a better patient compliance(Varshosaz, J. et al., 2012b).

Extended release dosage forms can be prepared by encapsulation of controlled release

pellets or granules or by compressing them into tablets. Plasma drug concentration of

different dosage forms are shown in Figure 1(Gad, S.C., 2008a).

Figure 1: Plasma drug concentration of different dosage forms after administration

PL

AS

MA

DR

UG

CO

NC

EN

TR

AT

ION

TIME

IR dosage form IV Injection

Oral Overdose CR dosage form

4

1.1.1. Classification of extended release(ER)dosage forms:

ER dosage forms can be classified into two classes; like single unit (e.g. tablets) and

multi-unit (e.g. pellets) dosage forms. Both types can further be subdivided into two,

depending upon the design of dosage form such as matrix system and reservoir system

(Araya, R., 2011).

1.1.1.1. Single unit ER dosage form

Single unit dosage form includes tablets or capsules, which further subdivided into,

matrix and reservoir systems.

1.1.1.1.1. Matrix systems

Drug release from matrix system e.g. tablets, followed several mechanisms included

dissolution, diffusion, swelling and erosion. In such system, the release properties of

drug are not generally zero-order. Drug at the surface is released first and the interior

drug diffuses out later with the passage of time. The path length for diffusion ofdrug

increases with the passage of time so the release rates decrease(Hacer, Y., 2002).

Figure 2: Drug release pattern from diffusion-controlled matrix tablets

5

1.1.1.1.2. Reservoir systems

In reservoir systems, i.e. coated tablets, beads, and particles, microcapsules and osmotic

pump systems, the drug release rate depends on the membrane thickness, permeability

and surface area. The systems or devices are coated with a solution of water-insoluble

and water-soluble polymers, when exposed to aqueous media, the surrounded coating

acts as semipermeable membrane, through which the drug diffuses out in the media in a

rate-limiting manner (Hacer, Y., 2002).

Figure 3: Drug release pattern from diffusion-controlled reservoir system

1.1.1.2. Multi-unit ER dosage form

The multiunit or multiparticulate dosage forms like pellets, are encapsulated or

compressed into a tablet or filled into a sachet, for accurate delivery of the recommended

dose. Multiparticulate drug delivery system (DDS) are getting popularity than single unit

dosage form because of having several benefits like less irritation to the gastric mucosa

(reduced localized concentration), reduced concentration fluctuation in plasma,

minimized risk of dose dumping and bettered bioavailability (Varshosaz, J. et al., 2012a:

Hamedelniel, E.I.M., 2011).

The release rate from film-coated pellets is controlled by the film thickness and rate

controlling polymers’ composition. For film-coated pellets, the uniformity of polymer

thickness and polymer weight-gain (relatively non-specific measurement), can determine

6

the rate of drug release (Liu, Y. et al., 2012). This multiparticulate system is also

subdivided into, matrix system and reservoir system.

1.1.1.2.1. Matrix systems

The matrix system (multiparticulate type) can be prepared by different methods such as

extrusion/spheronization (He, H. et al., 2011; Hamedelniel, E.I.M., 2011; Dukic, A. et

al., 2007; Chatchawalsaisin, J. et al., 2005), spherical crystal agglomeration

(Kachrimanis, K. et al., 2000; Manish, M. et al., 2005; Paradkar, A. et al., 2002) and

melt-solidification (Maheshwari, M. et al., 2003; Paradkar, A.R. et al., 2003). Although,

the formation of multiparticulate matrix systems is comparatively easy than reservoir

systems, but due to increase surface area of pellet, their extent of retardation of drug

release is limited.

1.1.1.2.2. Reservoir systems

Reservoir type of particulate systems can be prepared by extrusion /spheronization,

layering, congealing, spray drying, followed by coating using different polymeric

materials, like, polymethacrylate, chitosan, gelatin, poly (vinyl alcohol), biodegradable

polymers, and cellulose-based polymers. However, ethyl cellulose is one of the ideal

polymer for coating controlled release drug delivery systems. The addition of plasticizers

makes the film uniform, continuous and smooth. But, films prepared without plasticizers

makes the film hard, rigid, which may break easily. However, the plasticizers’ quality

can be evaluated by investigating physicochemical properties (Regdon Jr, G. et al.,

2012).

Organic solvent or water can be used to prepare extended release coating solutions or

dispersions. However, water based coating dispersions needed high temperature to

drying, resulting increased processing time and high energy utilization. While, used of

organic coating solutions having environmental and toxicological concerns (Terebesi, I.

&Bodmeier, R., 2010).

7

Methacrylate copolymers and ammoniomethacrylate copolymers (Eudragit® RS) are also

employed over solid drug delivery systems to develop extended-release. Aqueous

colloidal polymer dispersions or aqueous micronized polymer dispersions were

formulated by using a variety of polymers. However, aqueous coatings require high

energy of evaporation, higher coating temperature and/or long processing time, which

may cause thermal degradation of heat sensitive materials. The drug may also be

deteriorated during coating process on contact with water as some drug are moisture-

sensitive (Pearnchob, N. &Bodmeier, R., 2003b).

1.1.2. Advantages and disadvantages of ER dosage forms:

Advantages of extended release dosage form includes, the level of drug in plasma is

maintained for prolong period, reduced the overnight dose requirement (Aulton, M.E.

&Taylor, K.M., 2013), reduced severity of side-effects(Alekseev, K.V. et al., 2012) and

localized GIT adverse effects caused by irritant drugs from immediate release

formulations (Moursy, N. et al., 2003), these all features(Alekseev, K.V. et al., 2012)

improves patient compliance and adherence to therapy (Aulton, M.E. &Taylor, K.M.G.,

2013; Moursy, N. et al., 2003).

Extended release dosage forms have several disadvantages or limitations. Different

physiological factors, like GIT pH, activities of enzyme, gastric transit time, intestinal

transit time, food and disease conditions, may affect the release profiles of drugs.

Moreover, per unit cost of an ER formulation is comparatively greater than that of

conventional, immediate release formulation because of higher cost of manufacturing

(Aulton, M.E. & Taylor, K.M.G., 2013; Aulton, M.E., 2002).The unpredictability in drug

release, poor in vitro/in vivo correlation, and erratic systemic availability, are the other

issues which may cause hindrance in designing controlled release drug delivery systems

(Gad, S.C., 2008a). Dose dumping and burst liberation of drug from the dosage forms,

are other limitations to formulate drug in to controlled release system (Siepmann, J. et

al., 2011; Ghosh, T.K. &Jasti, B.R., 2004; De Haan, P. &Lerk, C., 1984).

8

1.1.3. Polymers used in extended release dosage forms

Hydrophilic polymers such as HPMC are extensively used in the formulations to control

the drug release(Bose, A. et al., 2013). The liberation of drug from the device depends

upon the solubility of drug. The drug release and solubility is pH-dependent which is

change by the changing pH environment in the gastro-intestinal tract. (Bose, A. et al.,

2013; Pygall, Samuel R et al., 2009). HPMC or hypromellose, a hydrophilic polymers

swell when exposed to water or body fluid and make a gel layer around the system. This

gel layer basically limits the drug release and decides about the failure or success of the

system (Tritt-Goc, J. et al., 2005).

The hydrophobic polymers have gained much popularity in the pharmaceutical industry

due to its economic benefits. In the preparation of matrix systems containing

hydrophobic polymers, the drug is uniformely dispersed in the polymeric matrix

(Dahiya, S. et al., 2008), while in case of reservoir systems, the drug core is coated with

polymericmemberan which bascially control the drug release (Kucera, S.A. et al., 2008).

Ethylcellulose is widely used in pharmaceutical coating (Pearnchob, N. &Bodmeier, R.,

2003a), to control drug release from solid drug delivery system (Patra, C. et al.,

2007),because of its low cost, excellent mechanical strength and sufficient film forming

properties (Lai, H.L. et al., 2010). Since ethylcellulose is hydrophobic in nature, which

reduces the water entrance into the solid polymeric matrix, and thus controlling the drug

release (Siepmann, J. et al., 2011).

Eudragit®RL100/RLPO and RS100/RSPO (ammoniomethacrylate copolymers) are also

hydrophobic polymers mentioned in the USP monograph and these polymers are pH-

independent, make them suitable for controlled release dosage forms (Siepmann, J. et al.,

2011; Lachman, L. et al., 1986).

Biodegradable natural and synthetic polymers are also used in ER formulations. The

common natural polymers, or biopolymers include, chitosan (Songsurang, K. et al.,

2011; Steckel, H. &Mindermann-Nogly, F., 2004), cellulose(Regdon Jr, G. et al., 2012)

sodium alginate and gelatin (Chen, F.-M. et al., 2007; Işiklan, N., 2006). Synthetic

9

biodegradable polymers used in CR formulation include poly(ε-caprolactone) and

polyanhydrides (Siepmann, J. et al., 2011).

Kollicoat® SR is available in the form of an aqueous colloidal dispersion for sustained

release coatings. It is reported that as the coating level of Kollicoat® SR 30D increased

over the pellets, then the propranolol HCl release was decreased. Application of 10 to

15% w/w coating mass retarded the release of propranolol HCl up to 12 h (Pearnchob, N.

&Bodmeier, R., 2003a).

1.2. Formulation development and optimization

The objective of preformulation studies is to get information about the physicochemical

characteristics of drug substance and pharmaceutical excipients, so that a well design and

target oriented formulation can be obtained. Preformulation investigations are designed

to identify the properties such as flow characteristics, solubility, effects of pH,

crystallography, particle size analysis and distribution, and compatibility with the

selected excipients. These are the parameters necessary to be pre-evaluated as they have

great influence on design of formulation, manufacturing technique, and

biopharmaceutical and pharmacokinetic characteristics of the emerging formulation.

Thus, preformulation studies is required to be performed in the development process as

early as possible. The processes involved in formulation development, remain continued

all over the product development phase, so that the product/drug stability, bioavailability

and delivery to targeted side can be improved (Swarbrick, J., 2006).

The physicochemical properties of drug, types and functions of additives incorporated in

the formulations and drug – excipient incompatibility are the crucial factors which needs

to be investigated during formulation development. As per global International

Conference on Harmonization (ICH) guidelines, the excipients should be selected

cautiously in any formulation design, since the quality and sources greatly affect the

final product quality (Siepmann, J. et al., 2011).Product formulations must meet a

number of goals such as they must release the active ingredients in a manner to give the

desired therapeutic effects and must comply with the regulatory and compendial

specifications. There are a variety of statistical tools that can be used to optimize

formulations to achieve the best values of all the factors (Bateman, S.D. et al., 1996).

10

Optimization of controlled release dosage form, by statistical experimental design, is an

efficient means to quantify and evaluate the formulation variables on crucial responses

like rate of drug release. (Furlanetto, S. et al., 2006)

1.3. Characterization of extended release dosage forms

The pivotal parameter that significantly influence the therapeutic performance of

extended release dosage form is dissolution testing. Therefore, a validated dissolution

procedure is required to characterize the extended release dosage forms in a justified

way(Durig, T. &Fassihi, R., 2000). There are factors that can affect the rate of liberation

of drug from the dosage form, such as temperature, rate of agitation and the combination

of the dissolution media (pH, ionic strength existence of surfactants, digestive enzymes,

and/or bile salts ) (Garbacz, G. et al., 2008). Among the dissolution conditions, pH of the

medium is one of the utmost influential factors influencing the solubility and dissolution

rate of drug. The media containing HCl, acetate, citrate, and phosphates in the pH range

1–7.6 are often applied to evaluate the effect of pH on drug release (Corrigan, O.I. et al.,

2003). Such factors play an important role in the characterization of extended release

formulations. The liberation of drug from the system is greatly influence by the physico-

chemical nature and amount of drug in the device, like, polymorphic form, crystallinity,

particle size, and solubility (Costa, P. &Lobo, J.M.S., 2001b).

The content of the dissolution medium is a significant factor in the design of dissolution

test. Several in vitro dissolution media similar to human physiological conditions have

been attempted to predict the in-vivo dissolution of drugs(Corrigan, O.I. et al., 2003).

The combination of simulated GIT fluids is depend on the results of the analytical

evaluation of GIT contents(Lindahl, A. et al., 1997). Used of simulated gastrointestinal

fluids, which is also known as ‘bio relevant dissolution media’, have gotten more

attention in pharmacopeial monographs like USP (Garbacz, G. et al., 2008).

11

1.3.1. Dissolution testing for ER single unit system

Dissolution test is usually conducted highly standardized conditions. But, formulations

containing hydrophilic matrices like HPMC, make highly viscoid mass after complete

hydration that may cause difficulty in subjecting such formulations to pharmacopeial

dissolution test specifications. In order to avoid this trouble, a double ring mesh was

introduced in the vessel of USP apparatus II, that will form an area between double

meshes structure in which tablet unit can be placed and will expose the entire surface of

unit dose to the dissolution medium (Durig, T. &Fassihi, R., 2000).

Bose, A et al., performed dissolution test of matrix tablet (HPMC) containing Itopride

HCl, to investigate the drug release behavior from the matrix system. Test was conducted

using 900 mL 0.1N HCl for first 2 h and the rest of hours at pH 6.8 phosphate buffer.

The dissolution media were kept at a temperature of 37 0C, using USP type I (basket)

apparatus(Bose, A. et al., 2013).

The release of drug from the sustained release matrix tablets is affected by several

factors which includes, the method of granulation, type of excipients, ratio of drug and

polymer, drug solubility and dissolution medium’s pH. Wilson et al., conducted the

dissolution test of sustained release levofloxacin matrix tablets on dissolution test

apparatus (USP type II) whose stirring rate was 100 rpm. The acidic (pH 1.2) and

phosphate (pH 6.8) buffers which were maintained at 37°C ± 0.5°C was used as

dissolution medium (Wilson, B. et al., 2011b). In vitro dissolution test of matrix system

containing tramadol HCl has been investigated by Tewari et al. Different ratios of

HPMC and ethyl cellulose were used to develop tablets. The 900 mL of degassed

demineralized water was used as dissolution medium (kept at 37°C ± 0.5°C), and

dissolution was carried out on USP type II apparatus, rotating at 50 rpm(Tiwari, S.B. et

al., 2003).

1.3.2. Dissolution testing for multiparticulate ER pellet system

Multiple unit dosage forms appear to be more reliable as controlled release system due to

variability in gastric transit time. For pellets system the first dissolution apparatus used

was USP type III (reciprocating cylinder) (Joshi, A. et al., 2008).

12

Pellets are small particles which can dispersed over a large area of intestine and may be

capable for the local management of colon disorders. Ferrari and co researchers

performed the dissolution test of metronidazole coated pellets and chitosan to plausible

colonic drug delivery using Bio-Dis III reciprocating cylinder apparatus(Ferrari, P.C. et

al., 2013). The apparatus was set with a vibration rate of 8.0 dips per minute (dpm).

Different buffer solutions such as simulated gastric fluid (pH 1.2), acetate buffer (pH

4.5), and phosphate buffer (pH 4.5, 6.0, 6.8 and 7.2), were used as dissolution media,

maintained at 37 0C(Ferrari, P.C. et al., 2013; Han, X. et al., 2013).

Multiparticulate systems such as pellets have various benefits over single unit dosage

form like there is no risk of dose dumping associated with multiparticulate systems,

moreover, short gastric residence time make the system more suitable for quick

therapeutics response. USP basket type apparatus at 100rpm was also used for the

dissolution of sustained-release nicotinic acid pellets, in combination with immediate

release simvastatin(Zhao, X. et al., 2010).

1.3.3. Dissolution testing

Dissolution testing is a great quality control tool in the product development, to assess

the drug liberation behavior from the solid drug dosage forms. Initially this test was

developed for immediate release, then spreaded to controlled release solid drug delivery

systems and novel or special drug delivery systems. The goal of this test is to evaluate

the biopharmaceutical characteristics of the formulation and to ensure the quality of

dosage form within a defined set of specification criteria (Siewert, M. et al., 2003). It has

been identified that in-vitro dissolution is a significant element in the drug development

which can be used for the assessment of different products for bioequivalence. The drug

dissolution profiles are presented by different kinetics models in which f is a function of t

(time) with respect to the quantity of drug released from the dosage forms (Costa, P.

&Lobo, J.M.S., 2001b).

In vitro dissolution testing is conducted in a defined apparatus under precisely defined

conditions. This is due to the overall geometry of the apparatus which can affect the

hydrodynamics of the drug release media and finally influence the drug release from the

13

dosage forms. It has been shown historically that a slight change in the design of the

vessel can significantly affect the drug release. The dissolution testing can be used for

different purposes which includes, product research and developments, stability testing,

final quality control, process monitoring and prediction of biological performance (Wen,

H. &Park, K., 2011).

Jantratid, E. and coworkers evaluated in-vitro dissolution of modified release delivery

systemof diclofenac sodium using USP Apparatus III and IV and compared with USP

Apparatus I and II(Jantratid, E. et al., 2009).

Some studies adopted compendial approach in order to perform dissolution test using

media, described in the pharmacopeias (Jantratid, E. et al., 2009; Takka, S. et al., 2003),

whereas, some surfactants (synthetic)were incorporated to the compendial media (Rossi,

R.C. et al., 2007), but these conditions do not absolutely represent the GIT condition and

the interpretation of results can only be done on empirical basis.

1.4. Factors influencing the bioavailability of extended release dosage

forms:

1.4.1. Physiological properties of gastrointestinal tract, effect of food,

pH and emptying time

Physiological properties in different compartments of GIT are presented in Table 1.

*(Gad, S.C., 2008b)

14

The bioavailability of drug can potentially be influenced by complex GIT organ system

like stomach (capacity approx. 1.5 L in fed state and <50 mL under fasted conditions),

small intestine (4-5 m) and large intestine. Total small intestinal surface area is about 200

m2 in an adult, which made it a good site of drug absorption. Colon is the final part of the

gastrointestinal tract which is approximately 1.5 m(Aulton, M.E., 2002).

The food particularly fatty foods, delay in gastric emptying from 3 to 5 h and hence

results in delayed absorption of drugs (Welling, P.G., 1996). The transit time is about 8

– 12 h for most of the drugs and drug products and the maximum absorption half - life

should be about 3 – 4 h; contrarily, the system will move away from the possible

absorptive area before the completion of drug release. This is the reason that many

controlled release dosage forms have less bioavailability than immediate release dosage

forms due to incomplete and/or poor rate of drug release from the device (Anal, A.K.,

2008). Multiparticulate dosage forms such as pellets in the presence of food will move

toward intestine more slowly because it mix thoroughly with the food and then enter into

small intestine, this transit is greatly affected by the bulk and calories per weight of the

ingested food(Basit, A.W. et al., 2004). The fed/ fasted states of the stomach and the

dosage form greatly influence the gastric emptying time and intestinal transit time of

many pharmaceuticals. Nimmo et al., reported that solution as well as pellets having

diameter <2 mm leave the stomach rapidly, and the single dose units having diameters

>7 mm and if administered with heavy food can remain in the stomach for up to 10

hours. The transit time of drug in small intestine is about 3 hours (Nimmo, J. et al.,

1973).

1.4.2. Effect of physicochemical properties of drug

There are number of formulation and process factors which influence the drug release

from the device includes, physicochemical characteristics of the drug, type and quantity

of additives in the formulations, and the manufacturing process parameters (Furlanetto,

Sandra et al., 2006). These parameters could be the soluble or insoluble fillers ,

surfactants, pH adjusting agents, drug/diluent ratio as well as the drug solubility (Sousa,

J. et al., 2002). The physiochemical properties that can influence the dissolution rate and

thus bioavailability of drugs are the particle size, solubility, hydrophilicity, molecular

15

size, wettability and the form/states of the drug (i.e. salt or free form, crystalline or

amorphous) (Aulton, M.E., 2002; Dressman, J.B. et al., 1998).

1.4.3. Effect of diseased conditions

The absorption and ultimately the bioavailability of the orally administered drug is also

greatly influenced by gastric disease conditions. Local diseases can changethe pH of

stomach that can influence the stability, dissolution and/or absorption of the drug. The

bioavailability of the drug is also different in normal individuals than patients subjected

to gastric surgery(Nimmo, J. et al., 1973). Different diseases conditions like,

inflammation of ileum, impaired gall bladder and or liver function, can have

malabsorption of fat or bile salt which possibly causes serious implications on

bioavailability of lipophilic drugs. Intestinal transit time of patients suffering from

irritable bowel syndrome and motor disorders, also significantly affects drug

bioavailability. Similarly patients with active ulcerative colitis with faster colon transit

becomes rate limiting step for drugs’ availability in systemic circulation, (McConnell,

E.L. et al., 2008).

1.5. Extended release pellets formulation

1.5.1. Pellets and pelletization technique

Pellets are spherical particles which are encapsulated or compressed into tablets intended

for oral used. The size of pellets ranges normally between 100 – 1000 µm. Pellets are

produced either by an extrusion/spheronization technique, or by layering core pellets

with drug dispersion, or by a fluidized bed chamber. These pellets are coated using

different layers of polymers to control drug release as well as to provide protection of the

(Sibanc, R. et al., 2013). Pellets are geometrically defined agglomerate which can be

obtained by utilizing various materials in different processing conditions. Size of pellets

usually ranges from 500 – 1500 µm, intended for oral use(Sultana, S. et al., 2010).

Pelletization is a process of size enlargement, with a size range of 500 – 1500 µm and

10% intra-agglomerate porosity. The popularity of multiple unit extended release pellets

in single unit dosage forms (tablets or capsules) is increasing day by day because of their

16

ability to spread in the GIT uniformly, reduction in plasma level variability, decreased

risk of local irritation and dose dumping (Kojima, M.& Nakagami, H., 2002; Liu, Y. et

al., 2012; Rahman, M.A. et al., 2009).

There are many technical benefits associated with pellets, such as pellets have good

flowability, great strength, less friability, uniform particle size distribution, and

admirable quality for coating application. Pellets can be used for separation of

incompatible drugs and/ or excipients. Such ingredients can be converted into pellets and

administer in a single dose after encapsulation (Supriya, P. et al., 2012). Pellets can be

manufactured by using layering process but are not uniform in size and with low drug

loading capacity, therefore most of the formulation scientists prefer

extrusion/spheronization process to prepare uniform size pellets(Young, C.R. et al.,

2002).The advancement in the formation of pellets have led to the development of

different techniques like, hot-melt extrusion freeze pelletization and cryopelletization

(Rahman, M.A. et al., 2009). The discharge of drug from the extended release pellets is

primarily controlled by either by coating membrane, or a matrix, but the application of

coating technology has increased to regulate the drug release rate. Since the

manufacturing and in process control of matrix controlled release pellets are easy than

coated controlled release pellets, therefore matrix pellets are preferred (Kojima, M.&

Nakagami, H., 2002). Pellets formations have some limitations which includes;

processing is highly expensive due to the requirements of specialized equipment and

trained personnel. Quality production of pellets is very difficult, because the amount of

water added, critically determines the overall integrity of pellets, since inappropriate

timing and amount of adding pelletizing fluid (water), may cause over-wetting of pellet

(Supriya, P. et al., 2012).

1.5.2. Methods of pelletization

There are different techniques of pelletization like melt pelletization, globulation or

droplets formation, balling, cryopelletization and extrusion-spheronization etc. But, drug

layering or powder layering is the most extensively used technique in pharmaceutical

industry (Chien, Y., 1988).. Among these techniques extrusion – spheronization is the

most popular one, and this technique has been used in the present research work.

17

1.5.2.1. Extrusion and spheronization

It is multistage process, where dry mixing of ingredients is carried out to obtain a

uniform powder dispersion, and then wet massing using planetary mixer (Bashaiwoldu,

A.B. et al., 2004). These wet mass are then converted into extrudates of uniform

diameter by extrusion process using extruder. The extrudates are then broken into lesser

fragments having length equal to their diameter and then converted into spheres (Rowe,

R., 1985). Extruders are of different types like, Screw feed extruder, Gravity feed

extruder and Piston feed extruder (Ghebre-Selassie, I., 1989). The most important part of

spheronizer is the friction plate or a rotating disk (200 – 400 rpm) with a grooved surface

to increase the frictional forces. Spheronization of a extrudates commonly takes 2 – 10

minutes (Gamlen, M., 1985) but the speed and time of spheronization is validated during

experiment. The conversion of rods into spheres during spheronization process depends

on the moisture in the extrudates e.g. if the mass is very much dry, then the spheres

either may not be formed or may be dumbbell in shape (Nakahara, N., 1964). The room

temperature can be used to dry pellets (Hellen, L. et al., 1993; Hasznos, L. et al., 1992)

or the pellets can be dried in an oven by using temperature 40° ± 1°C for 12 hours or

they can be dried in microwave oven for 30 minutes (Bataille, B. et al., 1993). To

obtained desirable size distribution of pellets, screening may be required(Husson, I. et

al., 1992).

1.6. Pharmacokinetics studies

In a pharmacokinetic study, parameters such as rate constants, plasma peak drug

concentrations (Cmax), time to reach maximum plasma concentrations (Tmax), volume of

distribution (Vd), half-life (t1/2), area under the plasma concentration-time curve (AUC0-t,

AUC0-∞) are determined in order to asses ADME (absorption, distribution, metabolism

and elimination) of a drug (Yehia, Yoon, S. et al., 2014; S.A. et al., 2013; Cho, Kyung-

Jin et al., 2010; Shargel, L. et al., 2004; Boroujerdi, M., 2001; Rouge, N. et al., 1998).

These parameters can be analyzed by using the following formulae.

18

1.6.1. Area under the plasma concentration time curve

The measurement of rate and extent of drug (Bioavailability) can be determined by the help

of Area under the curve (AUC), that can be expressed as AUC0-t and AUC0-∞, Trapezoidal

method is the commonly reported method for the estimation of area under the curve

(Yehia, S. et al., 2012; Rouge, N. et al., 1998) and it can also be estimated by using the

following formulae (Shargel, L. et al., 2004; Boroujerdi, M., 2001).

𝐀𝐔𝐂∞ = 𝐀𝐔𝐂𝟎−𝒕 + 𝑪𝒍𝒂𝒔𝒕 𝒌𝒆𝒍⁄ (1)

AUC0-∞ can also be determined by the following equations;

𝐀𝐔𝐂∞ = 𝐀 𝛂⁄ + 𝐁 𝛃⁄ + 𝐂 𝐊⁄𝐚 (2)

𝐀𝐔𝐂∞ =𝑭×𝑫

𝐊𝟏𝟎 ×𝐕𝐝𝜷 (3)

AUC0-t can be calculated by using following equations

𝐀𝐔𝐂𝟎−𝒕 = 𝐀 𝐀𝒆−∝𝒕⁄ + 𝐁 𝐁𝒆−𝛃𝒕⁄ + 𝐂 𝐂𝒆−𝐊𝐚 𝒕⁄ (4)

𝐀𝐔𝐂𝟎−𝒕 = ∑𝒏

𝒊 = 𝟎(

𝒚𝒊+𝒚𝒊+𝟏

𝟐) (𝒙𝒊+𝟏 − 𝒙𝒊) (5)

1.6.2. Volume of distribution(Vd)

The volume which contains the total amount of drug taken at the same concentration and

observed in the blood plasma, is known as volume of distribution. The relation of plasma

concentration of drug to the amount of drug in the body is expressed by apparent volume

of distribution (Vd) (Shargel, L. et al., 2004).Volume of distribution in the central

compartment (Vc) and in elimination phase (Vdβ) can be calculated as;

𝐕𝐜 =𝐊𝐚×𝐅×𝐃(𝐊𝟐𝟏−𝐊𝐚)

−𝐂×(𝛃−𝐊𝐚)×(𝛂−𝐊𝐚) (6)

19

𝐕𝒅𝜷 =𝐊𝟏𝟎×𝐕𝐜

𝜷 (7)

Where F is the fraction of the dose absorbed.

1.6.3. Clearance (Cl)

Clearance can be defined as “the amount of drug removed from plasma per unit time”.

Clearance can be estimated without consideration of the compartment model and is often

calculated by a non-compartmental approach (Shargel, L. et al., 2004), using the

following equation.

𝐂𝒍 = 𝐕𝒅𝜷×𝜷 (8)

1.6.4. Half-life (t1/2)

Half-life (t1/2) is defined as “the time for drug concentration to reduce to half of its

initial concentration”(Dhillon, S. &Kostrzewski, A., 2006). There are different equations

used for the determination of drug half-lives in respective phases of absorption,

distribution and elimination;

𝒕𝟏/𝟐 𝜷 =𝟎.𝟔𝟗𝟑

𝜷 (9)

𝒕𝟏/𝟐 𝜶 =𝟎.𝟔𝟗𝟑

𝛂 (10)

𝒕𝟏/𝟐 𝑲𝒂=

𝟎.𝟔𝟗𝟑

𝑲𝒂 (11)

𝒕𝟏/𝟐 𝑲𝟏𝟐=

𝟎.𝟔𝟗𝟑

𝑲𝟏𝟐 (12)

20

𝒕𝟏/𝟐 𝑲𝟐𝟏=

𝟎.𝟔𝟗𝟑

𝑲𝟐𝟏 (13)

Equation 1 Half Life based on K21 value

𝒕𝟏/𝟐 𝑲𝟏𝟎=

𝟎.𝟔𝟗𝟑

𝑲𝟏𝟎 (14)

1.6.5. Area under the first moment time curve (AUMC)

AUMC is defined as “the area under the first-moment curve which is obtained from the

plot of the product of drug concentration in plasma and time versus time from zero to

infinity” (Sisinthy, S. et al., 2015; Bauer, L.A. & Gibaldi, M., 1983) and can be

calculated by the following formulae;

𝑨𝑼𝑴𝑪 = ∫ 𝒕×𝑪𝒑(∞)

𝟎𝒅𝒕 = ∑ (

(𝒕𝒏×𝑪𝒑𝒏)+(𝒕𝒏+𝟏)

𝟐× ∆𝒕)𝒕

𝟎 +𝑪𝒑𝒍𝒂𝒔𝒕

𝑲𝟐 + (𝒕𝒍𝒂𝒔𝒕 × 𝑪𝒑𝒍𝒂𝒔𝒕)

𝑲 (15)

𝑨𝑼𝑴𝑪 = [𝑨𝒌𝒂

𝒌𝒂− 𝜶(

𝟏

𝜶𝟐 − 𝟏

𝒌𝒂𝟐

)] + [𝑩𝒌𝒂

𝒌𝒂− 𝜷(

𝟏

𝜷𝟐 − 𝟏

𝒌𝒂𝟐

)] (16)

1.6.6. Mean residence time(MRT)

The MRT can be assessed by measuring the ability of the drugs to remain in the body. It

the average time spent by the amount of drug in the body before its elimination (Sisinthy,

S. et al., 2015)and it is measured by;

𝑴𝑹𝑻 =𝑨𝑼𝑪

𝑪𝒑𝟎 (17)

1.6.7. Cmax and Tmax

Cmax represents the maximum plasma concentration and Tmax represent to the time needed

to achieve maximum plasma drug concentration soon after administration (Yoon, S. et

al., 2014; Shargel, L. et al., 2004). Both can be obtained directly from the graph.

21

1.6.8. Rate constants

The rate constants measure the rate of transfer, at which drug enters into the body

through central compartment. It includes rate constants for absorption (Ka), for the

movement of drug from central to peripheral compartment (K12), from peripheral to

central compartment (K21), and for the elimination of drug from central compartment

(Kel) (Shoaib, M.H. et al., 2008; Shargel, L. et al., 2004;Ritschel, W.A., 1992).

𝜷 =𝒍𝒏𝑪𝟏−𝒍𝒏𝑪𝟐

𝒕𝟐−𝒕𝟏 (18)

Where β represent overall disposition rate constant and C1andC2 are the concentrations at

time t1and t2 respectively

𝜶 =𝒍𝒏𝑪𝟏 𝒅𝒊𝒇𝒇−𝒍𝒏𝑪𝟐 𝒅𝒊𝒇𝒇

𝒕𝟐−𝒕𝟏 (19)

Where, α is overall distribution rate constant, while ln C1diffand ln C2 diffare difference

between post absorptive phase plasma concentration at time t1 and t2 and back

extrapolated mono-exponential β slope (μg/ml).

𝒌𝒂 =𝒍𝒏𝑪𝒂𝟏−𝒍𝒏𝑪𝒂𝟐

𝒕𝟐−𝒕𝟏 (20)

In the above equation, ka represents the absorption rate constant, and Ca1 and Ca2are the

differences among actual concentration of plasma at time t1 and t2 and mono-exponential

phase slope (μg/ml).

𝑲𝟏𝟐 = (𝑨×𝑩×(𝜷−𝜶)𝟐

𝑪×(𝑨×𝜷+𝑩×𝜶)) (21)

Where K12 is distribution rate constant from central to peripheral compartment, A, B and

C are the intercepts of the extrapolated lines of distribution, disposition and absorption

correspondingly. Whereas α and β are the overall distribution and disposition rate

constant.

22

𝑲𝟐𝟏 = (𝑨×𝑩+(𝜷×𝜶)

𝑪) (22)

K21is distribution rate constant from peripheral compartment to the central compartment.

𝑲𝟏𝟎 =𝑪

𝑨× (𝜶 + 𝑩

𝜷⁄ − 𝑪𝑲𝜶

⁄ ) (23)

Overall elimination rate constant is K10 in the above equation.

1.7. Itopride hydrochloride (ITP)

Hokuriku Seiyaker Co. Limited, Japan, developed and marketed ITP(a novel prokinetic

agent) for the first time in 1995 (Ahmed, S. et al., 2013; Gupta, S. et al., 2004). Itopride

is best candidate for gastro-esophageal reflux disease which enhances gastrointestinal

motility (Ahmed, S. et al., 2013; Shah, Sanjay et al., 2012) and is used to relieve

gastrointestinal symptoms such as nausea, non-ulcer gastritis, epigastric discomfort,

diabetic gastroparesis and functional dyspepsia (Sisinthy, S.P. et al., 2015; Yoon, S. et

al., 2014; Yehia, S.A. et al., 2013; Parmar, H., 2011).

1.7.1. Physicochemical properties of Itopride HCl

Chemically Itopride is as N-[P-[2-[dimethyl amino] ethoxyl] benzyl] veratramide

hydrochloride and molecular formula is C20H26O4 HCl (Penumajji, S. &Bobbarala, V.,

2009; Gupta, S et al., 2004). Its molecular weight is 358.43144 g/mol. It is white,

odorless fine powder, with melting point of about 197 0C in DSC analysis (Shah, Sanjay

et al., 2012). It is highly water soluble drug (Sisinthy, S.P. et al., 2015; Yehia, S.A. et al.,

2013; Prajapati, B.G. et al., 2010b).

http://en.wikipedia.org/wiki/Gastrointestinal

http://en.wikipedia.org/wiki/Motility

23

1.7.2. Chemical structure

The chemical structure of Itopride hydrochloride is(Rasheed, S.H. et al., 2011; Singh,

S.S. et al., 2005).

Figure 4: Structure of Itopride HCl

1.7.3. Mechanism of action

Itopride HCl has dual pharmacological action like, anticholinesterase and dopamine D2

(5-HT4) receptor antagonistic activity (Gupta, K. R. et al., 2010). Dopamine D2

receptors and acetylcholine esterase are inhibited by ITP which in turn increases the

concentration of acetylcholine. Increase concentration of acetylcholine, enhances the

GIT motility, esophageal sphincter pressure, gastric emptying and stimulate the gastric

motility (Rasheed, S.H. et al., 2011). The contraction of smooth muscle is stimulated by

acetylcholine released from enteric nerve endings that bind with M3 receptors present

on the GIT smooth muscle layer (Tsubouchi, T. et al., 2003). The enzyme

anticholinesterase is inhibited by ITP, thus preventing the degradation of acetylcholine

(Bose, A. et al., 2009). Itopride is unique from other prokinetic agents available in the

market due to its dual mechanism of action (Gupta, S. et al., 2004).

1.7.4. Therapeutic Indications

Various clinical studies were conducted and it was observed that the therapeutic

treatment of disorders like, non-ulcer dyspepsia or chronic gastritis, diabetic

gastroparesis, reflux esophagitis and functional dyspepsia was found to be effective

when treated with ITP (Yoon, S. et al., 2014; Inoue, K. et al., 1999; Otsuba, T. et al.,

1998; Masayuki, N. et al., 1997; Tsubouchi, T. et al., 2003;). There are studies reported

24

that the efficacy of itopride is superior than metoclopramide and cisapride in patients of

NUD (Kamath, V.K. et al., 2003; Myoshi, A. et al., 1994). Another comparative study of

itopride and domperidone have found similar efficacy in the symptomatic management

of NUD (Sawant, P. et al., 2002).

1.7.5. Dosage and administration

Itopride hydrochloride is available as 50 mg IR and 150 mg SR dosage form e.g.

Ganaton® 50 mg and Ganaton® OD 150 mg tablet marketed by Abbott Laboratories

Pakistan. Itopride is given in 50 mg doses thrice a day for adult in empty stomach an

hour before meal (Gupta, S. et al., 2004). Itopride 50mg is given three times in a day in

combination with Proton pump inhibitor in order to obtain profound therapeutic

effect(Prajapati, B.G. et al., 2010a; Prajapati, B.G. et al., 2010b).

1.7.6. Pharmacokinetics of Itopride HCl

1.7.6.1. Absorption and distribution

When Itopride is given orally, it rapidly absorbs from the stomach and upper part of the

small intestine and widely distributes throughout the body (Karen, H.D. et al., 2012).

The Itopride has rapid onset of action as the plasma concentrations are achieved within

half an hour after oral administration (Yoon, S. et al., 2014; Yehia, S. et al., 2012; Gupta,

Seema et al., 2004), unlike cisapride and mosapride, which take about 60 min. to reach

plasma peak concentrations.

1.7.6.2. Effect of food

The absorption of drug is not effected by food but it has been reported that the rate of

absorption of Itopride from ER dosage form is delayed by presence of food (e.g. 4.4

hours versus 3.1 hours for Tmax with and without food, respectively)with changes in its oral

bioavailability(Yoon, S. et al., 2014).

25

1.7.6.3. Metabolism

The enzyme flavin mono-oxygenase (FMO) metabolizes Itopride to N-oxide metabolite

in the liver by the oxidation of tertiary amine N-dimethyl group (Singh, S.S. et al., 2005),

whereas, cisapride and mosapride are metabolized by the enzyme cytochrome P450

(CYP) (Yoon, S. et al., 2014). The biological half-life of Itopride is approximately 6

hours (Karen, H.D. et al., 2012), that makes itopride a good candidate for designing

extended release dosage form.

1.7.6.4. Elimination and excretion

It is mainly excreted through the kidneys unchanged and in the form of metabolites

(Yehia, S. et al., 2012).

1.7.7. Drug Interactions

Since Itopride is metabolized by the liver enzyme flavin monooxygenase (FMO) rather

than cytochrome P450 enzyme system, which is a different (rare) metabolic pathway,

reduces the risk of drug – drug interaction of itopride with other drugs significantly.

However, other drugs of the same class i.e. cisapride and mosapride citrate, have

potential of drug-drug interaction with cytochrome P450 enzyme inhibitors (Choi, H.Y.

et al., 2012; Mushiroda, T. et al., 2000).

1.7.8. Safety and adverse effects

Itopride HCl is well tolerated drug which has no considerable effects on central nervous

system and cardiovascular system. Whereas, extra pyramidal side effects and

hyperprolactinemia are seen in the cases of other antiemetic agents like metoclopramide

and domperidone. Some common adverse effects are usually observed in patients

receiving itopride hydrochloride such as diarrhea, headache, abdominal pain/discomfort

etc. (Choi, H.Y. et al., 2012; Gupta, S. et al., 2004).

26

Cisapride and mosapride are associated for prolongation of QT intervals leading to

cardiotoxicity, whereas, Itopride has no cardio toxic effects, as proved in some

preclinical and clinical studies (Gupta, S. et al., 2005; Kakuichi, M. et al., 1997). In

females, pregnancy related side effects of Itopride HCl has not been established and no

abnormalities were observed in animal studies(Gupta, S. et al., 2004).

27

2. OBJECTIVES OF THE STUDY

28

The objective of this research workwas,

1. To develop extended release matrix and coated pellets of itopride HCl (ITP) by

extrusion- spheronization technique using hydroxypropyl methylcellulose

(HPMC - K4M, K15M and K100M), ethyl cellulose (EC - 7cps), microcrystalline

cellulose and lactose.

2. To characterize the trial pellets formulations, on the basis of physicochemical

parameters such as angle of repose, bulk density, tapped density, Carr’s index,

Hauser’s ratio, particle size analysis, image analysis, SEM and FTIR

3. To determine drug content and dissolution profiles at different pH (1.2, 4.5 and

6.8) of extended release pellets formulations.

4. To evaluate the dissolution rate kinetics using by applying different kinetic

models such as First order, Zero order, Higuchi, Korsmeyer-Peppas, Hixson

Crowell, and Baker Lonsdale etc.

5. To apply model independent approach in order to evaluate similarity factor by

comparing, trial extended release pellet formulations’ dissolution profiles with

that of the optimized formulation.

6. To conduct the stability studies of optimized extended release ITP pellets

formulations as per ICH guidelines under accelerated condition as well as general

condition (room temperature).

7. To develop and validate the analytical method for the analysis of itopride HCl

from human plasma using HPLC as per FDA guidelines.

8. Pharmacokinetic study of newly best (optimized) developed extended release

pellet formulation in Pakistani population using both compartmental and non-

compartmental models.

9. To perform comparative bioavailability studies of developed ER ITP pellets (150

mg) formulation with the Reference product of Itopride HCl, under fasted and fed

conditions.

10. To statistically analyze the pharmacokinetic parameters of both test and reference

products using Latin Square Two Formulations Analysis of Variance (ANOVA).

29

3. LITERATURE SURVEY

30

3.1. Formulation development of extended release dosage forms

In the areas of formulation development, controlled release dosage form is one of the

important dosage from which provides continuous therapeutic efficacy. These dosage

forms reducing dosing frequency, by greatly improving the patient compliance.

Sisinthy, S. et al., formulated a CR layered matrix tablets of Itopride HCl containing

polyethylene oxide release retardant. On the basis of the dissolution profile of the

developed formulations, one of the best formulation was selected for in vivo study in

healthy human subjects. It was concluded on the basis of in-vitro/vivo studies, that

polyethylene oxide based layered matrix tablets exhibited controlled release of Itopride

hydrochloride (Sisinthy, S. et al., 2015).

Wei Y.-m. et al., applied solid dispersion method to produce a sustained release dosage

form, for less water-soluble drug (nifedipine) to promote the solubility. Polyvinyl

pyrrolidone and stearic acid were used to establish the dosage form but the release of

nifedipine could not control. To control the release, the system was coated with

ethylcellulose of different viscosity grade like EC10, 45 and 100cp, which extended the

dissolution rate from 16 to 20 h. The drug release mechanism was diffusion followed

with erosion. The oral absorption of the drug was very rapid in rabbits, showing

improved oral bioavailability (Wei, Y.-m. et al., 2013).

Remya, P. et al., used wruster process method for the development of venlafaxine HCl

sustained release pellets using HPMC (E6), Ethylcellulose (7cps), and MCC (101). The

dissolution studies were carried out and results showed that formulations controlled the

release of drug up to 20 hours. The optimized formulation exhibited good disperse ability

of drug freely in the gastrointestinal tract, for obtaining more bioavailability (Remya, P.

et al., 2012).

Gupta, N.V. et al., developed olanzapine matrix pellets for controlled release drug

delivery by pelletization technique. For this purpose, sodium alginate, sodium lauryl

sulphate glyceryl palmito-stearate, and microcrystalline cellulose were used. Scanning

electron microscope photographs confirmed the sphericity of the pellets. The

compatibility between drug-polymers was confirmed by differential scanning

31

colorimetric and furrier transformed infrared spectroscopic studies. The result showed

that the formulation F5 controlled the drug release for more than 24 h and drug release

mechanism followed was Fickian diffusion (Gupta, N.V. et al., 2011)

Zurao, P.G., developed hot melt matrix pellets of diltiazem HCl by extrusion-

spheronization technique using low melting waxes. Dissolution profiles of the matrix

pellets were quit similar with the dissolution pattern mentioned in USP. This similarity in

the result, was without the application of a polymeric membrane (Zurao, P.G., 2010).

Larsson, M. et al., investigated the influence of co-ingested alcohol (ethanol) on the

liberation of drug from controlled release formulations, because of the concerns of

regulatory authorities of possible dose dumping. Influence of the water permeability of

hydroxypropyl cellulose and ethylcellulose coated formulations in the existence of

ethanol in the dissolution medium was also studied. It was observed that higher

concentration of ethanol, enhanced the water permeability of the films with low

hydroxypropyl cellulose content due to swelling. However, the water permissibility

decreased for films with higher hydroxypropyl cellulose content, possibly because of the

swelling of the ethylcellulose, blocking the pores (Larsson, M. et al., 2010).

Kranz, H. et al., investigated the release pattern of low/poor aqueous soluble drug

(theophylline) from microcrystalline cellulose and carrageenan-based pellets. Drug

release mechanism followed by microcrystalline cellulose based pellets was pure

diffusion. The drug diffusivity was significantly regulated by addition of small quantity

of polyethylene glycol 6000 (pore former) and croscarmellose sodium (disintegrant). The

drug release periods of microcrystalline cellulose based pellets ranged from few minutes

to certain hours. Inversely, high doses of drug were released within few minutes from

carrageenan-based pellets as pellets were rapidly disintegrated upon contact with

aqueous media (Kranz, H. et al., 2009).

Dey, N. et al., reviewed an article to focus the recent trends of pharmaceutical research

of multiparticulate controlled or delayed release drug delivery systems. This system

having low risk of dose dumping, short gastric residence time and flexibility in drug

release patterns. The release of drug from microparticles depends upon the carrier used

32

and the loaded drug quantity. Therefore, oral controlled and delayed release formulations

can be designed by multiparticulate drug delivery systems (Dey, N. et al., 2008).

Eskandari, S. et al., developed nonpareil ER indomethacin pellets by using

centrifugation or powder layering method. The pellets were prepared using sugar,

Avicel PH 101 and lactose with drug powder, treated with HPC-L binder solution. The

prepared pellets were coated with Eudragit NE 30 D and the drug was evaluated by

HPLC method. The dissolution profile achieved from pellets was compared with

Indocid®75 Capsule. Dissolution release data revealed that by increasing the quantity of

Eudragit NE 30D, Opadray and Sodium dodecyl sulfate in coating solution, the drug

release rate was greatly controlled (Eskandari, S. et al., 2007).

Reddy, K.R. et al., developed SR nicorandil matrix tablets using ethylcellulose, Eudragit

RL-100, Eudragit RS-100, polyvinylpyrrolidone, HPMC, sodium CMC, and sodium

alginate. The dissolution profiles were determined and the results revealed that

formulation containing drug to HPMC ratio 1:4 using ethanol as granulating agent,

controlled the drug release up to 24 hours. The drug release mechanism followed was

diffusion controlled. However, formulation containing drug to HPMC ratio 1:4, using

4% ethylcellulose dispersion (wt./vol.) as granulating agent, showed satisfactory drug

release initially and the maintenance release pattern was identical with the theoretical

release data, followed diffusion coupled with erosion drug release mechanism (Reddy,

K.R. et al., 2003).

Sanchez-Lafuente, C. et al.,had studied the didanosine release from matrix tablets and

its associated process and formulation variables by applying the statistical experimental

design. Direct compress technique was used to prepare tablets using blends of Eudragit

RS-PM and Ethocel-100. The percentage drug released, dissolution efficiency and 10

percent of the drug dissolved in specified period of time, were the responses while tablet

compression force, drug content and polymers and their particle size ratio were

considered as independent variables. It was demonstrated that the obtained experimental

values of the optimized formulation and the models reliability in the extended-release

formulation of matrix tablets, highly agreed with the predicted values (Sanchez-

Lafuente, C. et al., 2002).

33

Vergote. G.J. et al., developed CR ketoprofen pellet formulation (nanocrystal and

microcrystal) using mixture of microcrystalline wax, waxy maltodextrin and drum dried

corn starch. The pellets were prepared by melt pelletization technique. Dissolution

profiles of nanocrystalline ketoprofen loaded wax based pellets were compared with

microcrystalline ketoprofen loaded wax based pellets and commercially available

sustained release product. It was observed that by varying the concentration of drum

dried corn starch, the drug release rate was increased. Drug dissolution rate was

increased by adding surfactants such as sodium lauryl sulphate. Complete release of

nanocrystalline ketoprofen was observed from the matrix pellet formulations, however,

both the techniques showed sustained release (Vergote, G.J. et al., 2001).

3.2. Pellets formulations by extrusion-spheronization techniques

Singh, G. et al., formulated and evaluated sustained release pellets of furosemide by

extrusion/spheronization technique. A ratio of 1:3 of drug to polymer was used to design

the formulation and optimized using 32 central composite methodology. Two-tailed

paired t test and one-way ANOVA were used to statistically analyze the in-vitro

dissolution data. Dissolution data demonstrated that furosemide SR pellets exhibited

great SR characteristics and significant (P≤0.05) difference as compared to pure drug

and commercial product (Singh, G. et al., 2012).

Pai, R. & Kohli, K., formulated Sertraline hydrochloride matrix pellets by

extrusion/spheronization method using 10, 20 and 30 % (w/w) of sodium alginate and

calcium chloride solution. Direct compression approach was used to compressed pellets

into tablets and drug content uniformity was evaluated. The drug was released

completely from pellets containing alginate within 4 hours, while pellets treated with

calcium chloride showed the desired release profile (Pai, R. &Kohli, K., 2011).

Akhgari, A.et al., prepared acacia and tragacanth based ibuprofen and theophylline

pellets by extrusion-spheronization method using 32 factorial design. Different

proportion of acacia and tragacanth and different concentrations of drug were used and

the effect of factors on response were analyzed by linear regression. Pellets containing

acacia alone showed greater mechanical strength and rapid drug release rate as compared

34

to tragacanth based pellets. Whereas, addition of small quantity of tragacanth to

ibuprofen based matrix pellets formulation exhibited delay in drug release. (Akhgari, A.

et al., 2011).

Pana, K. et al., prepared matrix sustained release pellets of poorly soluble drug

nimodipine (NMD). The nimodipine was coground to improve the dissolution with

excipients such as chitosan (matrix former), MCC, mannitol, sodium dodecyl sulphate,

crospovidone and croscarmellose sodium. Extrusion-spheronization technique was used

to convert the coground mixtures into pellets. Croscarmellose sodium was found the

most effective and crospovidone was the least effective in bettering the release of drug

from both coground mixture and pellets. So, using this technique, sustained release

matrix pellets could be obtained (Pana, K. et al., 2009).

Sriamornsak, P. et al., prepared theophylline pellets using sodium alginate by

extrusion/spheronization. It was observed that the shape and size of pellets was

influenced by the types and quantity of sodium alginate and calcium salts. 75–85% drug

was released from most of pellet formulations within 1 hour. However, drug release

profile was changed by addition of calcium salts, depending upon the solubility of the

calcium salts used. The drug release mechanism followed was Higuchi and Korsmeyer–

Peppas equations (Sriamornsak, P. et al., 2008).

Dukic, A. et al., developed modified starch based theophylline anhydrous pellets by

extrusion/spheronization. The surface response design was used for the optimization of

the formulations. Sorbitol, binder, and water level were used as formulation variables

whereas, the spheronization speed was the only process variable. Among these variables

sorbitol showed significant influence on properties of pellets and consistency of wet

mass which was confirmed by NMR and Mixer torque rheometry. Within 20 mins, all

formulations showed complete drug release, indicating the higher potential of modified

starch in development of formulations by extrusion/spheronization (Dukic, A. et al.,

2007).

Almeida, P.S .et al., developed and evaluated starch-dextrin based pellets using

extrusion–spheronization for more suitable release properties. The quantity of wetting

agent (optimized) was characterized by torque-rheometry. The results demonstrated that

35

that mixtures of corn or wheat starch and 20% white dextrin produced good quality

pellets with better size and shape distributions (Almeida, P.S. et al., 2005).

Steckel, H. & Mindermann-Nogly, F., successfully prepared chitosan pellets by

extrusion/spheronization method. The formulations consisting of microcrystalline

cellulose in concentrations from 0 to 70% were extruded using different ratio of water

and diluted acetic acid solution. The obtained beads were investigated for assessment of

morphological and mechanical properties. The mass fraction of chitosan could be

increased to 100% within the pellets when granulation was carried out using dilute acetic

acid (Steckel, H. &Mindermann-Nogly, F., 2004).

Tomer, G. et al., produced pellets formulations by extrusion and spheronization

technology. The formulations consisted of five drug-models, 4-parahydroxybenzoic acid,

methyl, propyl and butyl paraben and propyl gallate, were mixed in different ratio with

microcrystalline cellulose (MCC) and various levels of water. Combined analysis

showed that determination of pellet size distribution was most significantly influenced by

two factors i.e. type of model drug and the levels of water. Propyl gallate (PG) was the

most consistent material which produced very highly quality pellets with constant

median diameter, shape factor, size range and roundness (Tomer, G. et al., 2002).

Santos, H. et al., used chitosan, cellulose microcrystalline, and povidone to prepare

diclofenac sodium loaded pellets by extrusion/spheronization. The influence of different

concentration of chitosan, type of fillers and composition of the liquid binders on

physical properties of pellets were studied by using three-factor factorial design.

Analysis of variance showed that the surface roughness of pellets was significantly

increased as chitosan load increased and pellets containing tribasic calcium phosphate

exhibited smoother surface as compared to other fillers (Santos, H. et al., 2002).

Lustig-Gustafsson, C.et al., evaluated the effect of drug solubility and water content on

the formation of microcrystalline based pellets by extrusion and spheronization. It was

found that the force requires to extrude the wet mass via the ram extruder was decrease

as the amount of water added increased. The water level needed to produce the best

quality pellets was found to reduce as a linear function of the natural logarithm of the

drug water solubility (Lustig-Gustafsson, C. et al., 1999).

36

3.3. Hydroxypropyl methylcellulose (HPMC) and ethylcellulose (EC)

containing formulations

Verma, N.K., prepared ethylcellulose (EC) coated microcapsules for controlled release

of paracetamol by an emulsion-solvent evaporation method. Different proportions of

core and coat were used for microcapsules and evaluated for size distribution, drug

entrapment and release kinetics. The drug release was controlled from the ethylcellulose

coated microcapsules, depending upon the core: coat ratio, size of microcapsules and

wall thickness, followed by non-fickian diffusion. It was also noted that formulation

prepared using cyclohexane as solvent showed good release rates (Verma, N.K., 2013).

Hosseini, A. et al., developed and optimized extended release ethylcellulose (EC)

coated propranolol hydrochloride pellets to be compressed into rapidly disintegrating

tablets. The coated pellets were converted into cushion layered with tablet excipients

using MCC, lactose or sorbitol, to avoid segregation problems and to prevent the

integrity of the EC coating brittleness during compression. In short, without changes in

the release profile of EC coated pellets, MCC based cushion layer facilitated segregation-

free compression into fast disintegrating tablets. It was also proved when compared to

the blends of coated pellets and powder excipients compressed conventionally (Hosseini,

A. et al., 2013).

Elias, N.M. et al., designed sustained release pellets based on HPMC & ethylcellulose to

evaluate the release profile of diltiazem hydrochloride. The pellets were prepared by

applying drug binder solution on neutral pellets which is then followed by spraying 6%

coating solution of HPMC and EC in different ratios. The in vitro dissolution profiles

revealed that ethylcellulose retarded highest release rate of the drug than HPMC and the

retarding ability reduces with decreased in concentration (Elias, N.M. et al., 2012).

Chowdary, K.P.R. et al., prepared controlled release microcapsules of pioglitazone

using ethyl cellulose (EC) and ethylene vinyl acetate copolymer (EVA) by an

emulsification solvent evaporation method. The release of drug from the microcapsules

were extended over 24 hours followed by non-fickian diffusion mechanism, depending

upon the wall thickness, size and core: coat ratio of both polymers. The in-vitro

37

dissolution results demonstrated that EVA based microcapsules were found more

suitable for the design of CR formulations as compared to EC based microcapsules,

because the later one were more permeable (Chowdary, K.P.R. et al., 2012).

RegdonJr, G. et al., investigated the influence of concentration of plasticizer (triethyl

citrate) and thermal stability of ethylcellulose (EC-10 and EC-45 cps) coating used for

modified release dosage form. Thermo-sensitive genic male sterile (TG–MS)

examinations was used to study the effect of storage time by regulating the changes in

the thermos-analytical parameters. It was observed after mass spectroscopy and TG–MS

examinations that coating composed of EC-45 with 5% triethyl citrate was better for

making MR as compare to EC 10 and the films was stable until about 200oC (Regdon Jr,

G. et al., 2012).

Parmar, H. et al., designed mucoadhesive microparticle of Itopride HCl to retain the

drug in the stomach and to prolong the residence time at absorption site, thereby

prolonging the drug release. Sodium alginate, carbopol 934 and HPMC K15M were used

in the formulations by orifice ionic gelation method. The efficiency of entrapment was in

the range of 41.32 to 81.68 %. The in vitro results showed that carbopol 934 containing

microparticle sustained the drug release and had greater mucoadhesive strength than that

of HPMC K15 (Parmar, H. et al., 2011).

Aguilar-de-Leyva, A. et al., used different ratios of hydroxypropylmethyl cellulose and

microcrystalline cellulose in the preparation of matrix pellets with the help percolation

theory to investigate the release pattern of clozapine. Pellets were produced using

different particle size fraction of clozapine. It was noted that the percolation threshold

has a significant effect on the system. Based on percolation theory, it was possible to

demonstrate the release pattern of clozapine from matrix pellets (Aguilar-de-Leyva, A. et

al., 2011).

Chowdary, K.P.R.& Dana, S.B., prepared ethylcellulose (EC) coated diclofenac

microcapsules by emulsion solvent evaporation technique, to investigate the controlled

release property of EC as a coating polymer. Ethylcellulose was found as an effective CR

polymer and extended the release over 12 – 16 hours followed by non-fickian diffusion

(Chowdary, K.P.R. &Dana, S.B., 2011).

38

Siddique, S. et al., developed sustained release encapsulated metoprolol tartrate matrix

coated granules. Matrix pellets were prepared utilizing various level of HPMC (K100M)

and ethyl cellulose and then coated with various ratio of Eudragit® RL/RS. The capsules

extended the release of drug up to 12 h, following Korsemeyer–Peppas kinetic model.

The optimized formulation was evaluated for bio-absorption using rabbit model which

revealed that in-vivo drug release was prolonged up to 10 h. A close correlation

(R2=0.9434) was established between the in vitro drug release and in vivo absorption

(Siddique, S. et al., 2010).

Zhao, X. et al., prepared sustained-release nicotinic acid (NA) pellets by extrusion–

spheronization which was then coated with EC and polyvinyl pyrrolidone K30 in

different ratios. For immediate release, a milled suspension of simvastatin (SIM) was

layered over coated pellets. The pellets formulations were compared with marketed

brands (NER/S; SIMCOR, Abbott) and the results were found very similar in different

media (Zhao, X. et al., 2010).

Duarte, A.R. et al., prepared ethyl/methyl cellulose blends to develop controlled release

microspheres using the supercritical antisolvent (SAS) technique. In this technique,

methyl cellulose was added to enhance the solubility of less soluble drug molecule.

Different operational conditions were evaluated to investigate the effects of temperature,

pressure, carbon dioxide and liquid flow on particle size distribution. Mixture of

dichloromethane and dimethyl sulfoxide (4:1 ratio) were used to precipitate the

microspheres with process conditions of 40 ◦C and 80 bar (Duarte, A.R. et al., 2006).

Sadeghi, F. et al., investigated the effects of different coating levels of Surelease®

(ethylcellulose aqueous dispersion) on the release of metoclopramide HCl and diclofenac

sodium pellets. It has been observed that the release of drug from the dosage from was

reduced when coating level of polymer was increased. However, the release of

diclofenac sodium was comparatively faster than metoclopramide hydrochloride. But

this effect was markedly increased with metoclopramide hydrochloride after curing. This

differences in the release behaviour of the two drugs were may be due the presence of

anionic ammonium oleate in the Surelease® which may have interacted with the cationic

metoclopramide and a high molecular size complex was made to diffuse slowly via the

39

film. The coating process of pellets may also affected by decreasing the volume of air

flow rate and removal of ammonia in the coater (Sadeghi, F. et al., 2003).

Law, M.F.L. & Deasy, P.B., used HPMC, hydroxypropyl cellulose, sodium

carboxymethyl cellulose, or polyvinyl pyrrolidone combined microcrystalline cellulose

to aid extrusion–spheronization, either by spray-drying or physical mixing. Twenty

percent of this mixture was mixed with eighty percent of lactose using water as binding

agent to produce pellets by extrusion–spheronization. It was noted that excipients

combined by spray-dry, produced pellets with better sphericity and higher yield

compared with the physical mixed excipient. SEM, DSC and XRD analysis, showed that

disintegration of the component of MCC into smaller crystallites was prominent in the

spray-drying excipients with the hydrophilic polymer. Formulations containing

hydroxypropyl cellulose or polyvinyl pyrrolidone produced highly spherical and

maximum yield pellets among other hydrophilic polymers because of the least adhesive

strength (Law, M.F.L. &Deasy, P.B., 1998)

Chatlapalli, R.R. & Rohera, B.D., were characterized the physical properties of HPMC

and hydroxyethyl cellulose (HEC) to investigate their use as pelletization aid. Three

placebo pellets were prepared using HPMC, HEC and MCC by extrusion and

spheronisation method. Isopropyl alcohol was used as wetting agent and prepared pellets

were evaluated for their physical characteristics. The findings indicated that HPMC can

be a great choice as a pelletization aid when the formulations excipients are required to

be completely water soluble. HPMC can also use to achieved modified release dosage

form as well as in pellet systems having water-labile drugs, and/or where aqueous

system cannot be employ as wet massing agent (Chatlapalli, R.R. &Rohera, B.D., 1998).

Yuen, K.H. et al., developed sustained release pellets of theophylline by extrusion

spheronisation method. The pellets formulations were coated using fluidized bed coating

technique with ethylcellulose aqueous dispersion. The drug release was found

unsatisfactory from pellets coated with ethylcellulose alone, but the release profile was

found very much satisfactory by addition of high viscosity grade of methylcellulose. The

in-vitro drug release was linear, pH independent, and was stable after thermal treatment

of one year (Yuen, K.H. et al., 1993).

40

3.4. Eudragit and Kollicoat Containing Formulations

Ferrari, P.C. et al., developed chitosan based metronidazole pellets for colonic drug

delivery. Kollicoat® SR 30 D was used as an inner layer and Kollicoat® MAE 30 DP as

an outer enteric layer to coat these pellets using fluidized-bed coater. Bio-Dis dissolution

results showed that the drug release, intestinal permeation and swelling was influenced

by chitosan and coating composition. The mechanism of drug release was diffusion and

erosion controlled simultaneously confirmed by Weibull equation. It was concluded that

chitosan based pellets exhibited as a great system of colonic drug delivery and coating

film was the main drug release controlling factor whereas chitosan was controlling the

drug intestinal permeation (Ferrari, P.C. et al., 2013).

Asnani, A.J. & Parashar, V.V., developed a multiparticulate system of theophylline by

powder layering technique in which nonpareil seeds were used as core material. Drug

was sprinkled over the nonpareil seeds using PVP K-30 in IPA as binder. The pellets

were coated with different proportion of Eudragit®: RL (3:1, 2:1) and ethyl cellulose and

then collected on the basis of percentage coating (5%, 10%, 15% and 20%). The in-vitro

dissolution results demonstrated that 15% ethyl cellulose and 15% & 20% Eudragit® RS:

RL (3:1) coated pellets retarded the drug release up to 12 hours while in at same

percentage of coating with Eudragit®RS: RL (2:1) was unable to retard. Higuchi and

Korsmeyer – Peppas were the best fitted models followed by all the formulations.

Spherical and porous pellets were produced, confirmed by SEM study (Asnani, A.J.

&Parashar, V.V., 2013).

Bolcskei, E. et al., produced matrix pellets by extrusion–spheronization technique using

Eudragit NE 30D. The formulation was developed by factorial design to determine the

critical control points of the process. Water Quantity, spheronization speed, dosing speed

and duration of spheronization were the factors. The hardness, significant factors and

aspect ratio were determined (Bolcskei, E. et al., 2012).

Liu, Y. et al., used extrusion/spheronization method in the development of sustained

release venlafaxine hydrochloride pellets. Eudragit® NE30D and ethylcellulose (10cps)

were used to coat the pellets formulation. The oral relative bioavailability of pellets

formulations was compared (fasted state) with a reference SR capsules (Effexor® XR), in

41

six healthy beagle dogs. The in-vivo results showed that pellets formulations had better

bioavailability as compared reference product. The oral absorption and in-vivo drug

release was strongly influenced by micro-structure of film and roughness of surface

caused by coating techniques (Liu, Y. et al., 2012).

Sadeghi, F. et al., produced ibuprofen pellets by extrusion-spheronization using high

concentration (30.4 or 38%) of Eudragit RL with the help of polyethylene glycol

(PEG400) as plasticizer. It was noticed that with addition of 1 or 3% PEG400 the mean

dissolution time (MDT) increased but not in the case of pellets with 5% PEG400. In

short, PEG400 change behavior of pellets from brittle to plastic which may be

meaningful when pellets are supposed to be converted into tablets (Sadeghi, F. et al.,

2011).

Andreazza, I.F. & Ferraz, H.G., prepared pellets of ascorbic acid via extrusion and

spheronization method. These pellets were coated with different level of Kollicoat SR 30

D dispersion (5.07; 8.26 and 10.35%) using fluid bed coater. After in-vitro dissolution

studies of both pellets formulations and commercial product, it was found that pellets

coated with 5.07% of polymer showed comparable dissolution profile to that of

commercial brand (Andreazza, I.F. &Ferraz, H.G., 2011).

Sultana, S. et al., used extrusion and spheronization technique to prepare sustained

release salbutamol sulphate pellets. Pellets were coated by applying aqueous dispersion

of Eudragit RS® 30 D and Kollicoat SR® 30 D of different levels (5%, 10%, 15%, 20%

& 25 % (w/w)). In-vitro evaluation data showed that drug release mechanism followed

was diffusion controlled or non-Fickian transport. It was noted that the MDT was high

with Eudragit® RS 30D as compare to Kollicoat® SR 30D and this values increased

with the increasing concentration of polymers (Sultana, S. et al., 2010).

Ahmed, I. et al., developed ambroxol hydrochloride pellets containing hydroxypropyl

methylcellulose, MCC, lactose, and maize starch by Extrusion-Spheronization technique.

These pellets formulations were coated with Eudragit RL 30D and Eudragit RS 30D

dispersion in various ratios (1:1, 1:1.5, 1:2, 1:2.5 and 1:3). The in vitro dissolution data

exhibited that by the increasing concentration of Eudragit RS 30 D, the drug release was

decreased, followed by first order and Higuchi release kinetics models. The combination

42

of these two polymers showed significant (p<0.05) effects on drug release, analyzed by

one-way ANOVA (Ahmed, I. et al., 2008).

Ghaffari, A. et al., prepared theophylline pellets using microcrystalline cellulose by the

extrusion-spheronization method. Mixture of pectin/chitosan/Eudragit® RS aqueous

dispersions was used as coating agent using a fluidized-bed coater. The formulations

were evaluated for coating mass gain and quantity of pectin/chitosan complex (5, 10, 15

and 20%, m/m). It was observed that the drug release rate and pattern of some

formulations exhibited bimodal and burst release, which was accelerated by the presence

of pectinolytic enzymes in the colonic medium. Formulations with 15 or 20% (m/m) of

pectin-chitosan and 20% (m/m) of coating mass gain showed better release rate and

surface erosion was observed with exposure to pectinolytic enzymes (Ghaffari, A. et al.,

2006).

Sawicki, W. & Łunio, R., prepared pellets of verapamil hydrochloride by wet

granulation using microcrystalline cellulose and sodium hydrocarbonate. Kollicoat SR

30 D was used to coat the pellets, followed by compression to produce tablets to prolong

the stomach residence time and to control the drug release. The effect of 10%

concentration of three plasticizers such triethyl citrate, propylene glycol and dibuthyl

sebecate were also examined. It was found that coated pellets having same thickness

using different plasticizer showed different drug release pattern. The effect of flotation

and film thickness was also defined before compression. The influence of compression

force on friability and hardness and flotation and pellet aggregation were also evaluated

for the best formulation (Sawicki, W. &Łunio, R., 2005).

Shao, Z.J. et al., investigated the influence of Kollicoat SR 30D on the release of

diphenhydramine hydrochloride from nonpareil-based systems. The influence of types

and concentration of plasticizers on drug release rate were also evaluated. The drug

loaded beads were coated in fluid bed wurster coater using plasticizers such as propylene

glycol (10% wt/wt), triethyl citrate (2.5%), and dibutylsebacute (2.5%), talc, and red #30

lake dye. It was found that the types of plasticizer play a vital role in drug release

mechanisms i.e. propylene glycol plasticized coating retarded the drug release as the

coating level increases. Formulations plasticized with dibutylsebacate showed slowest

dissolution, followed by propylene glycol and triethyl citrate and unplasticized

43

formulations revealed fastest dissolution. Significant difference between cured and

uncured dissolution profiles of all four formulations were observed after stability studies

at accelerated. In conclusion, controlling drug release from such system, is significantly

influence by the types and concentration of plasticizers and curing conditions (Shao, Z.J.

et al., 2002).

3.5. Release kinetics evaluation of formulations

Rao, P. S. et al., formulated Itopride HCl SR pellets using ethyl cellulose N50 by

solution/suspension layer technique. The pellets formulations were evaluated for

physicochemical, micrometrics properties as well as dissolution studies. Comparative

dissolution characterization of all formulations indicated that the optimized formulation

released 96.46% drug in 12 h by following Higuchi model exhibiting diffusion controlled

release mechanism (Rao, P.S. et al., 2014).

Dandag, P. et al., developed sustained release Itopride hydrochloride floating beads for

gastroretentive delivery by emulsion gelation methods using methoxy pectin and sodium

alginate as sustained release polymers and sunflower oils as floating agent. The influence

of polymers types and level, in-vitro release, buoyancy, entrapment efficiency, and

release kinetics were studied. The optimized formulation was compared with marketed

brand and stability study performed for 90days. The in-vivo floating study conducted on

rabbits and X-ray examination exhibited that the beads were floating up to 10 h in

stomach. In vitro release showed that the formulations followed zero order and fitted

Korsmeyer-peppas model i.e. non-Fickian release, indicating diffusion and swelling

(Dandag, P. et al., 2013).

Asok, G. et al., by using direct compression technique, developed sustained release

tablets of itopride hydrochloride. HPMC K4M, HPMC K15M, Carbopol 971P, Metolose

60SH-50, Gaur gum, and Xanthan gum in different ratio (1:1) and levels (15%, 25%,

35%) were used. The physical parameters, % assay and in-vitro drug release of all

formulations were also be studies. The formulation, F8 followed first order kinetic and

Korsmeyer-Peppas models, showing non-Fickian release (Asok, G. et al., 2013).

44

Jose, S. et al., developed chitosan based multiparticulate microspheres for colon targeted

delivery of ondansetron for irritable bowel syndrome by emulsion cross linking method.

The influence of drug-polymer proportion, emulsifier concentration and chitosan on drug

entrapment efficiency were investigated. By using solvent evaporation technique, the

initial in-vitro burst drug release was controlled by microencapsulation of optimized

chitosan microspheres using Eudragit S-100. The optimized formulation was fitted into

different kinetic data and drug release mechanism followed was Korsmeyer-Peppas

model (Jose, S. et al., 2010).

Pygall, S. R. et al., studied the drug release mechanism in sodium citrate buffered

formulation by incorporating different agents into the HPMC matrices, to protect the acid

sensitive drugs. The formulations were prepared using 39% HPMC 2910 and 2208 as

matrices former, 10% felbinac, dextrose and sodium citrate. The impact of sodium citrate

on gel layer formation, pH of internal layer and drug release mechanism was evaluated.

In-vitro dissolution results demonstrated that with the increase concentration of citrate in

the gel layer of HPMC 2910 matrices, suppressed the particle swelling, interfere with

diffusion barrier integrity thereby enhancing the immediate release and decreasing the

drug solubility and extended release. Whereas, HPMC 2208 resisted this osmotic-

mediated effects (Pygall, S. R. et al., 2009).

Belgamwar, V. et al., prepared oral mucoadhesive Metoprolol tartrate microspheres by

ionic gelation technique using HPMC (K4M, K15M, K100M, and E50LV), Carbopol

(971P, 974P) and Polycarbophil. The liberation of drug from the system was controlled

by cross linked sodium alginate /calcium chloride. The prepared microspheres were

characterized for physical properties, swelling index, drug release kinetic and greater

mucoadhesive properties. It was observed that the mucoadhesive properties of HPMC

was greater than carbopol and polycarbophil. The drug release was controlled to a period

of 12 hours, followed by non-Fickian drug release kinetics (Belgamwar, V. et al., 2009).

Fursule, R. et al., prepared floating amoxicillin trihydrate beads for gastroretentive drug

delivery using gel forming sodium alginate and oil as floating agent. The spheres were

characterized for surface morphology, diameter, encapsulation efficiency, and percentage

buoyancy of floating drug. To evaluate the drug release mechanism, beads formulations

were fitted into different kinetics models. Formulations followed Higuchi model showing

45

that the release is by diffusion mechanism with n = 0.5, indicating Fickian diffusion

(Fursule, R. et al., 2009).

Muschert, S. et al., elucidated the release of diltiazem HCl from aqueous ethylcellulose

dispersion coated pellets consisting of poly(vinyl alcohol)-poly(ethylene glycol)

copolymer, microcrystalline cellulose or sugar. Experimental results of all formulations

showed that the release of drug from ethylcellulose coated pellets was controlled by drug

diffusion and the diffusion coefficient was determined with thin films in the

macromolecular networks and used to predict successfully the release rate from coated

pellets (Muschert, S. et al., 2009).

Varshosaz, J. et al., developed bioadhesive floating ciprofloxacin tablets to enhance the

drug residence time in the proximal areas of the GIT. HPMC, Sodium carboxymethyl

cellulose, polymetacrylic acid, polyacrylic acid, citric acid, and sodium bicarbonate were

incorporated to produce the effervescent tablets. The polymers types exhibited no effects

on floating lag time but, 5% effervescent base in the tablets showed longer lag time than

10%. However, increasing concentration of Sodium carboxymethyl cellulose enhanced

mucoadhesion and all formulations followed Higuchi model, non-Fickian release

mechanism (Varshosaz, J. et al., 2006).

Varshosaz, J. et al., prepared sustained release tramadol HCl matrix tablets by direct

compression method. Various proportion of Xanthan gum: HPMC, Xanthan gum: Guar

gum and mixture of three polymers (Guar gum, Xanthan gum and HPMC) as well as

alone, were used in different formulations. The polymers hydration rate and drug release

rate of all formulations were evaluated. The mean dissolution time of formulations

containing only Xanthan gum was highest, following a zero-order model and the release

mechanisms were swelling, diffusion, and erosion. The drug release was not controlled

in formulations containing Guar gum alone while combinations of natural gums with

HPMC could retard drug release. However, formulations containing pure HPMC and

H8G2 were found similar to reference brand (Topalgic-LP) indicating same similarity

factor ( f2) values (Varshosaz, J. et al., 2006).

Chavanpatil, M. D. et al., developed sustained release ofloxacin gastroretentive delivery

system using psyllium husk, HPMC K100M as retarding agents and crosspovidone as a

46

swelling agent. The floating, swelling, bioadhesive properties and in-vitro dissolution

profile of the optimized formulations were evaluated. The drug release mechanism was

observed to be non-Fickian type, followed by Higuchi kinetic model. The release of drug

from developed formulation was similar when compared with marketed brand (Zanocin

OD), according to similarity factor (f2). The combination of two polymers showed

significant (P < 0.005) bioadhesive property than HPMC K100M and psyllium husk

alone (Chavanpatil, M.D. et al., 2006).

Zimm, K.R.et al., prepared acetaminophen (10%) based pellets consisted of

microcrystalline cellulose (90%) by extrusion/ spheronization technology. The release of

drug from the multiparticulate pellets system was evaluated with the help of Higuchi

square root of time and cubic equations to explain the drug release from a single planar

system and spherical pellets, respectively. The data were statistically compared and the

results described that use the Higuchi cubic equation is better than square root because

cubic equation is more sensitive to change in particle size (Zimm, K.R. et al., 1996).

Tapia, C.et al., prepared diclofenac sodium based spheres by extrusion and

spheronisation using various concentration of chitosan solution. The drug release was

retarded up to 30 minutes to 6 hours in the formulations containing chitosan as compare

to formulations without chitosan. Thus a hydrophilic gel was used to avoid coating. All

the formulations followed first order release kinetics and when plotted as a function of

the square root of time a straight line is formed. The mechanism of drug release followed

was non-Fickian diffusion type. Dissolution tester speed did not effect on the drug

release rate confirming diffusion process was controlled within spheres rather than

diffusion layer. (Tapia, C. et al., 1993).

47

3.6. Itopride Hydrochloride containing formulations

Rao, M.R. & Shelar, S.U., developed controlled release oral floating in situ gel of

itopride hydrochloride using gellan gum, calcium carbonate and HPMC K100M. The

formulations were optimized by applying 32 factorial designs. The influence of various

concentration and viscosity of gellan gum and HPMC K100M on drug release was

investigated. The floating lag time, duration, pH, drug content, gel strength, in-vitro drug

release and gelling capacity were also investigated. The results showed that the

formulations controlled the drug release, and the release mechanism followed was

Korsmeyer-Peppas model. The in vivo result showed that Tmax of gel was higher when

compare with plain drug however, the AUC0-12 h was 90% higher than plain drug (Rao,

M.R. &Shelar, S.U., 2015).

Biswal, B. & Karna, N., prepared sustained released pellets of Itopride hydrochloride by

Fluidized bed technique. The pellets formulations were optimized in terms of sphericity

and percentage drug release using microcrystalline cellulose, hypromellose 15 cps and

ethyl cellulose 50 cps. The pellets were checked for physicochemical properties, aspect

ratio, sizing, density, in-vitro drug release and binder’s concentration. With respect to

types of polymer and %age coating levels on the pellets, the release of drug was non-

linear. Therefore, an attempt was made to compress these multi-unit particulate system

into tablets and then investigated for in-vitro drug release, %age drug content, weight

variation, thickness, hardness and disintegration time (Biswal, B. &Karna, N., 2014).

Bose, A. et al., prepared SR Itopride HCl matrix tablets. The formulations were

optimized by response surface methodology. The formulations consisted of different

combination of HPMC K100M, polyvinyl pyrolidine (PVP) and lactose. The in vitro

dissolution study demonstrated that combination of HPMC K100M, PVP and lactose

controlled the release of drug for more than 12 h, best expressed by Higuchi model

(Bose, A. et al., 2013).

Ahmed, S. et al., investigated the compatibility of itopride HCl with some

pharmaceutical excipients used in specific dosage form. As the drug-excipient or

excipient-excipient interaction, is one of the most important factor to study in the field of

48

science and research technology. Differential scanning calorimetry and FTIR were used

for this purpose. Excipients tested were, hydroxypropyl methylcellulose, sodium carboxy

methylcellulose, Eudragit RSpm, Carbopol 934p, ethylcellulose, microcrystalline

cellulose, magnesium stearate, citric acid, sodium bicarbonate, and mixtures of these

with the drug (1:1 w/w). Thermal analysis and FTIR confirmed that all the excipients

evaluated were compatible with the drug (Ahmed, S. et al., 2013).

Soni, S., developed and evaluated the ethyl cellulose N-14 and N-20 based itopride HCL

pellets for controlled release. The formulated pellets were characterized for

micromeritics properties, hardness and dissolution rate. The superiority of the pellets

formulated with different grades of ethylcellulose was established after the comparative

evaluation of the above parameters (Soni, S., 2013).

Karen, H.D. et al., formulated floating gastro-retentive tablet of Itopride HCl by an

effervescent approach to enhance the gastric residence time using HPMC (K100M,

K15M, K4M) and sodium bicarbonate as effervescent agent by direct compression

method and evaluated for physical and chemical characterization (Karen, H.D. et al.,

2012).

Gandhi, P.A.et al., developed Itopride hydrochloride matrix tablets by using Central

composite design. HPMC K15M (X1) and K100M (X2) were used in the formulation as

matrix former. The X1 and X2 were chosen as independent variables whereas, the

release of drug at different time intervals was as dependent variables. In-vitro dissolution

data were fitted to different kinetics models and ANOVA, regression analysis and

response surface plot were performed for dependent variables. The optimized

formulation (FB7) showed same dissolution profile as innovator by similarity factor

(f2=83.86) and difference factor (f1=3.65). The optimized formulation followed zero

order kinetics (R2=0.9825) with non-Fickian release mechanism (n=0.5377) (Gandhi,

P.A. et al., 2012).

Santhosh, K.M. et al., formulated hydrodynamic balanced tablet of Itopride

hydrochloride for controlled release and to restrain the drug release to the stomach. The

formulations were composed of HPMC of different grades, Eudragit PSPO and gas

generating agent, sodium bicarbonate, and direct compression technique. The swelling of

49

the tablet was increased by increasing viscosity of the polymers. The buoyancy time of

all the formulations was ranged from 8-12h. Formulation (F12) containing Drug: HPMC

E15 in 1:2 ratios exhibited good controlled release of 74.26% over a duration of 12 h

(Santhosh, K.M. et al., 2010).

Hiremath, D. et al., designed extended release matrix tablet of Itopride hydrochloride

using HMPM K100 and gum karaya alone and in combination to investigate their

controlled release behaviour of highly water soluble drug, prepared by direct

compression. FTIR and DSC confirmed the compatibility of drugs with excipients. In-

vitro dissolution studies revealed that increased concentration of polymers decrease the

release rate and most of the formulations retarded the drug release up to 24 h, following

zero order kinetics and showed non- Fickian (anomalous) release with n values in

between 0.5-0.89 (Hiremath, D. et al., 2010).

Chandira, R.M. et al., developed gastroretentive floating tablet of Itopride

hydrochloride employing an effervescent approach, to enhance the gastric residence

time. The tablets were produced by direct compression method, using HPMC (K100M,

K15M), and Carbopol 934 P. Gas generating agents used were sodium bicarbonate and

citric acid. All the formulations were evaluated for physical and chemical

characterizations. It was found after dissolution studies that formulation containing drug,

HMPC K100m (125 mg), HPMC K15M (40 mg), and Carbopol (40 mg) sustained the

release of drug for 24 h, followed by non‐fickian diffusion mechanism. No prominent

changes was noted in the optimized formulation after stability studies at accelerated

temperature of 400oC/75% RH (Chandira, R.M. et al., 2010).

Prajapati, B.G. et al., formulated the sustained release Itopride hydrochloride tablet and

investigated the release pattern of drug from the formulations based on HPMC K4M,

HPMC K100M, ethyl cellulose and pregelatinized starch. The concentration of polymers

was optimized on the hypothetical release profile of trial and error based. The optimized

formulation (F11) showed similar dissolution profile to the hypothetical release profile

and the similarity factor (f2) value was 80.25. After the application of mathematical

models it was found that optimized formulation followed Higuchi model and regression

coefficient was 0.9962 (Prajapati, B.G. et al., 2010a).

50

Prajapati, G.G. et al., developed itopride hydrochloride matrix tablet for sustained

release using 32 factorial designs to optimize the polymers concentrations. The

formulations composed of HPMC K4M and K100M individually and in combination

with different ratios. In-vitro dissolution studies of the optimized (F019) and reference

product were conducted and the drug release at 2 h, 6 h, and10 h were noted. It was

found that both the test formulation and reference product were similar, indicated by

similarity factor (f2 = 86.04) (Prajapati, B.G. et al., 2010b).

Chhipa, P. et al., formulated sustained release pellets of Itopride hydrochloride and

coated with different coating levels of ethyl cellulose and Kollicoat SR 30 D. The

formulated pellets were evaluated for physical and chemical parameters. The release of

drug from the system was extremely influenced by the types of polymers and levels of

coating and 10 % (w/w) EC coated pellets showed a good release profiles. The optimized

formulation was compared with commercial Ganaton OD capsules and dissolution data

of the both formulations were identical, with the value for the similarity factor (f2 = 77.6)

(Chhipa, P. et al., 2009).

3.7. Analytical methods for Itopride hydrochloride

Rasheed, S.H.et al., developed RP-HPLC method for the quantitative determination of

Rabeprazole sodium and Itopride hydrochloride. Phenomenex C18 column and mobile

phase of ammonium acetate buffer and methanol (20:80v/v) were used and the drugs

were detected at 286 nm in 10 minutes. The method was validated according to ICH

guidelines for linearity, accuracy, precision, limit of quantitation and limit of detection.

Linearity results of RP and IH were in ranges of 37.5-375 μg/mL and 5-50 μg/mL,

correlation coefficients were 0.9997 and 0.9995, and accuracy were 98.6-100.7 and

99.42 -100.81 respectively (Rasheed, S.H. et al., 2011).

Zate, S.U. et al., developed itopride hydrochloride estimation method using UV-visible

spectrophotometry at 258nm using 0.1N HCl medium. The method was validated for

linearity, specificity, accuracy and precision. Regression analysis indicated good

correlation in the concentration ranges from 5–100 µg/ml and percentage recovery was

96.80 to 106.84 with standard deviation of 0.293 to 3.98% (Zate, S.U. et al., 2010).

51

Umamaheswari, D. et al., designed RP-HPLC method for simultaneous determination

of Itopride hydrochloride and Rabeprazole sodium using UV detection at 268nm.

Different proportion of methanol: water: acetonitrile (50:40:10) were used as mobile

phase. The retention time were 2100 and 6700 for itopride hydrochloride and rabeprazole

sodium respectively. The method has been validated (Umamaheswari, D. et al., 2010).

Gupta, K.R. et al., developed three UV methods for the estimation of Itopride

hydrochloride at different wavelength. Method A at 258.0 nm, Method B at 247nm and

Method C used AUC at 262.0-254.0 nm. All the three methods obeyed Beer- Lambert’s

law in concentration ranges from 5–50 μg/mL. The method has been validated by

recovery studies and results found satisfactory (Gupta, K.R. et al., 2010).

Sharma, R.et al., developed RP-HPLC technique for simultaneous quantification of

Rabeprazole sodium and Itopride HCl using UV detection at 266 nm. Linearity of

Rabeprazole sodium and Itopride HCl were found in the concentration ranges from 2–16

μg/mL and 5–55 μg/mL with a correlation coefficient of 0.9992 and 0.9996 respectively.

Recovery studies has been used for the validation of the method (Sharma, R. et al.,

2010).

Ma, J. et al., developed RP-HPLC plasma method for estimation of Itopride

hydrochloride using fluorescence detection. Hypersil BDS C18 column was used.

Isocratic mobile phase consisted of ammonium acetate and methanol (30:70, v/v) and

moxifloxacin used as internal standard. The excitation and emission wavelengths was set

at 304 and 344 nm, sequentially. The technique was validated in the concentration range

of 5–1000.0 ng/ml, LLOQ was 5 ng/ml and the percentage extractive recovery of

Itopride was > 80.77% from plasma. The intra and interday accuracy of the drug were

greater than 82.94% with a precision of 2.81–4.37% and 82.91% with a precision of

6.89–9.54%, correspondingly. The successfully developed method was applied in the

bioequivalence studies of itopride hydrochloride in human healthy volunteers (Ma, J. et

al., 2009).

Bose, A. et al., used LC-MS for the simultaneous estimation of Itopride HCl and

Domperidone in human plasma. Both the drugs were extracted from the plasma using

ethyl acetate and borax solution (saturated). The mobile phase consisted of water–

52

methanol (2:98, v/v) and 0.5% formic acid. The linearity in human plasma for Itopride

HCl was in the concentration range of 3.33–500 ng/mL and for domperidone 3.33–100

ng/mL. The itopride HCl and domperidone were measured by using the precursor to

product ion transitions of m/z 359.1–72.3 and 426.0–147.2 respectively (Bose, A. et al.,

2009).

Choragudi, S.F. & Settaluri, V.S., used spectrophotometer for the determination of

Itopride hydrochloride with UV-Visible detection at 520 nm. The method was based on

the formation of blue colored chromogen which was formed by reacting the drug,

sodium carbonate and Folinciocaltaeu reagent. The analytical results were validated

statistically as well as by recovery studies (Choragudi, S.F. &Settaluri, V.S., 2008).

3.8. Pharmacokinetic Studies of Itopride Hydrochloride

Rao, M. R. et al., performed an in-vivo studies of CR floating oral in situ gel of Itopride

hydrochloride in male Wistar rats of weighing 230–330 g. Six rats were employed in the

study and they were divided into three groups. First group was as negative control, pure

itopride solution was administered to second group and the optimized formulation was

given to third group (10mg/kg of animal weight). The analysis was carried out using

HPLC with UV detection at 288 nm. The Tmax and Cmax of the test formulation were

found to be 6 h and 0.163 µg/mL respectively. The AUC0-12 h of the test formulation was

found 90% higher than pure drug (Rao, M.R. et al., 2014).

Yehia, S. A. et al., performed the pharmacokinetic evaluation of optimized Itopride

hydrochloride microspheres formulation in human healthy volunteers. The study used

was two-way cross over using six healthy human subjects, with two weeks washing

period. The sampling was carried out up to 48 hours and analyzed using HPLC method.

The Cmax, Tmax, AUC0-∞, AUC0-48 and MRT were 1624 ± 168 ng/mL, 6 ±2, 85835 ± 6116

ng.h/mL, 29728 ± 761 ng.h/mL, 108 ± 9 h respectively. The optimized formulation was

compared with reference standard (Ganaton tablet) and the relative bioavailability of

itopride was 317.9 %. (Yehia, S.A. et al., 2013).

53

Sisinthy, S. et al., conducted pharmacokinetic studies of best formulation (IMP3L2) on

human subjects. The study performed was single dose, two periods, cross over and eight

healthy human volunteers were participated. Blood sampling were done over a period of

30 h and analyzed by RP-HPLC method. The mean Cmax, Tmax,T1/2,, Ka, Ke, AUC0-∞, and

MRT were found to be 164.5±6.09 ng/mL, 8 h, 10.47±0.873 h-1, 0.049±0.005 h-1,

0.067±0.005 h-1,3521.89±112.601 h-1, and 14.19±0.669 ng/mL/h, respectively (Sisinthy,

S. et al., 2013).

Yehia, S. et al., performed pharmacokinetics studies of the optimized SR tablet of

Itopride hydrochloride in human healthy subjects in comparison to the Ganaton® 50mg

tablet (Abbott) as a reference product. The comparison was carried out using cross over

study on six healthy male adults with two weeks wash out period. The pharmacokinetic

parameters such as Cmax, Tmax, AUC0– ∞, and MRT were estimated using HPLC method.

The analysis indicated that Cmax and AUC0– ∞ of the test formulation were higher and the

relative bioavailability of itopride was found to be 243% when compared to Ganaton® 50

mg tablet (Yehia, S. et al., 2012).

Choi, H.Y. et al., compared the bioavailability and tolerability of Revaprazan and

Itopride combination therapy in human healthy male Korean subjects. The study

consisted of multiple-dose, randomized, sequenced, crossover on 30 healthy volunteers

with one-week washout period. Sampling was done for 24 hours and analysis was carried

out using HPLC coupled with LC/MS-MS. The drug tolerability including vital signs,

clinical chemistry testing and interview were conducted throughout the experiment and

total 15 mild adverse effects were reported in 8 volunteers with no clinically significant

difference b/w the groups. The geometric mean ratios (90% CI) of Cmax,ss, and AUC,ss

with revaprazan and itopride were 0.92 (0.84 –1.00) & 0.96 (0.89 –1.03), and 1.07 (0.96

–1.20) & 1.12 (1.06 –1.18), respectively. It was concluded that monotherapy of

revaprazan and in combination with itopride in healthy Korean male volunteers were

well tolerated (Choi, H.Y. et al., 2012).

Cho, K. et al., performed the pharmacokinetics and bioequivalence study of two

different formulations of Itopride HCl (50 mg) in human volunteers. Twenty-eight

healthy male human subjects were participated a randomized, 2-sequence, crossover

design study with washout period of one-week. The blood samples were drawn up to 24

54

h. The plasma drug concentrations were determined by using HPLC method coupled

with fluorescence detector. The LOD of Itopride HCl was 5 ng/ml and no endogenous

substances interfered with analysis. The mean Cmax, Tmax, T1/2, AUC0–24h, and AUC0– ∞,

were 303.72 ng/ml, 0.75 h, 2.95 h, 865.28 ng/ml/h, 873.04 ng/ml/h, sequentially, for the

test formulation and 268.01 ng/ml, 0.78 h, 2.83 h, 833.00 ng/ml/h, and 830.97 ng/ml/h,

respectively, for the reference products. The AUC0– ∞, and Cmax were log-transformed and

tested by ANOVA. The 90% CI of AUC0– ∞, and Cmax were 100.57%–109.56% and

105.46%– 121.18%, respectively. The results indicated that both formulations were

found to be bioequivalent (Cho, K. et al., 2010).

Sahoo, B.K. et al., performed a pharmacokinetics study of Rabeprazole and Itopride

HCl. Twelve healthy Indian male subjects were participated in the bioequivalence study

of test and reference formulations, in a randomized, single-dose, two-treatment, two-

period, crossover study, with a washout period of one-week. Analysis of both drugs were

performed by UV detection HPLC method. The logarithmically transformed analysis of

both test sand reference formulations indicated that no significant differences (P > 0.05)

between AUC0– ∞, and Cmax values observed. The 90% confidence interval of the both

parameters were within the bioequivalence limits (0.8–1.25). The relative bioavailability

of both test and reference formulations were 98.24 and 93.65%, respectively (Sahoo,

B.K. et al., 2009).

Penumajji, S. & Bobbarala, V., conducted pharmacokinetic and bioequivalence study

of Rabiplus-XT (test) and Rabium Plus (reference) SR capsules. Both the formulations

contained Rabeprazole sodium 20mg + Itopride HCl 150 mg. A single dose, randomized,

two-way, crossover study was performed on 12 healthy human volunteers to compare the

pharmacokinetic parameters of test with reference product. Blood samples were drawn

up to 48 h and the plasma levels were determined by HPLC. The pharmacokinetics

parameters like, Cmax, Tmax, AUC0–48h, T1/2, and MRT were computed which suggested

that both the formulations were found to be bioequivalent (Penumajji, S. &Bobbarala,

V., 2009).

Kang, Y. et al., performed pharmacokinetics study of SR tablets of Itopride

hydrochloride and compared with dispersible tablets of Itopride hydrochloride using

healthy dogs. Both the formulations were given separately to the six healthy dogs, a

55

single dose, two periods, cross over study. Plasma drug concentration was determined

by HPLC method. The Cmax, Tmax, t1/2, AUC0-∞ of the SR tablet were 548.6±54.2 μg/L,

6.7±1.2 h, 6.0±0.6 h and 6.6±0.5 mg/L/h, respectively and Cmax, Tmax, t1/2, AUC0-∞ of

dispersible tablets were 798.1±42.5 μg/L, 1.83±0.25 h, 5.72±0.26 h and 7.0±0.6 mg/L/h

respectively (Kang, Y. et al., 2008).

Wang, B.-J. et al., compared the pharmacokinetics and bioavailability of Itopride

hydrochloride dispersion tablets (100 mg) with a reference tablet (100 mg). Eighteen

healthy male human volunteers were divided into two groups one for dispersion and

other for tablet, in a single dose, two-period, crossover study, divided randomly into 2

groups. The blood samples were drawn up to 24 h and assayed by HPLC. The Cmax, Tmax,

t1/2, AUC0-24h, AUC0-∞, of the dispersion tablet were 601.78±159.29 ng/ml, 0.78±0.35 h,

15.0±3.41 h, 2721.6± 577.92 ng/ml/h, and 4860.60± 804.80 ng/ml/h, respectively and the

corresponding parameters of the tablet were 611.22±166.32 ng/ml, 0.78±0.35 h,

14.6±3.13 h, 2632.3±614.27 ng/ml/h and 4675.90±645.00 ng/ml/h, respectively. The

relative bioavailability of the dispersion tablet was 103.87±6.31% when compared with

reference tablet. The results of ANOVA and two one-side test showed that both

formulations were bioequivalent (Wang, B.-J. et al., 2003).

56

4. MATERIAL AND METHODS

57

4.1. Formulation development of extended release itopride hydrochloride

pellets

4.1.1. Materials

Itopride Hydrochloride (ITP) was kindly gifted by Abbott Laboratories (Pakistan)

Limited.

Methocel (Hydroxypropyl methylcellulose/ HPMC-K4M, K15M and K100M)

(Colorcon, Kent, England).

Ethocel (Ethyl cellulose; EC-7 cps and 10 cps) (Colorcon, Kent, England). (Avicel

PH101, (FMC Corporation, USA)

Lactose monohydrate (Sigma-Aldrich Laborchemikalien, GmbH, Germany)

Eudragit® RS-100 (Colorcon, Kent, England).

Eudragit® RL-100(Colorcon, Kent, England).

Kollicoat SR 30D (BASF, Ludwigshafen, Germany)

Triacetin USP/FCC, (Colorcon, Kent, England).

Triethyl citrate (TEC) (Merck, l Co., Greensboro, NC)

Propylene glycol (BASF, Ludwigshafen, Germany)

Talc (BDH Laboratories Suppliers, Poole, England)

4.1.2. Method

4.1.2.1. Preparation of pellet formulations

Thirty-one (31) formulations of Itopride Hydrochloride (ITP) plain (without polymers)

and matrix pellets were designed manually, containing microcrystalline cellulose (MCC;

Avicel PH101), Hydroxypropyl methylcellulose (HPMC-K4M, K15M, K100M), Ethyl

cellulose (EC-7cps) and Lactose DC. The amount of ITP HCL in each pellets

formulation was kept constant at 150 mg as available in market. The plain pellets

formulations (F1 – F5), composed of MCC and lactose in the concentration ranges from

10–40%. The quantity of different concentrations and viscosity grades polymers used in

the matrix pellets formulations (F6 – F31) ranges from 10–30% (HPMC K4M, K15 and

EC-7cps) and 10 – 50% (HPMC- K100), while amount of lactose DC in matrix pellets

58

formulations (F6 – F31) was also kept constant at 10%. Composition of all the pellets

formulations are listed in Table 2 and Table 3.

The quantities of drug and excipients were accurately weighted, mixed and wet massed

in a planetary mixer. For HMPC matrix pellets, isopropyl alcohol (IPA) was used as

granulating fluid while distilled water for plain pellets and EC matrix pellets. However,

to achieve the spherical pellets, the quantity of granulating fluid was adjusted. The wet

mass was then passed through laboratory scale mini screw extruder (Caleva Process

Solution Ltd, Model. M.S.E, Dorset, UK) with 1 mm screen, operated at a speed of 55–

65 rpm. The collected extrudates were broken down into small pieces manually with a

length equal to their diameter and then transferred into the multi-bowl bench top

spheronizer (Caleva Process Solution Ltd, Model M.B.S 120, Dorset, UK) fitted with a

crosshatched plate with grooves running at right angle to one another. The speed of

spheronizer was adjusted at 600 to 800 rpm and operated for 10 min. Details of the used

spheronizer is given in manufacturer’s website (Caleva, P.S.). The plain and EC matrix

pellets were dried for 8 h while HPMC matrix pellets were for 4 h at 40oC in hot air

oven. The dried pellets were then passed through 18–24 mesh screen for size analysis.

All the pellets formulations were evaluated for in vitro drug release, to investigate the

influence of concentration and viscosity grades of the above-mentioned polymers on

release profile of Itopride HCl.

4.1.2.2. Extended release coating

Five formulations were selected for extended release coating from thirty-one trial pellet

formulations. F1 (10% lactose without polymers) was selected on the basis of good

sphericity and physical appearance, whereas F6 (10% HPMC K4M), F11 (10% HPMC

K15M), F16 (10% HPMC K100M) and F21 (10% Ethylcellulose 7 cps) were the

formulations containing lowest concentration of each viscosity grades of polymers

(HPMC and EC). The selected formulations were coated using three different types of

polymers such as Ethylcellulose (EC), Eudragit® RS/RL100 (2:1, w/w), and Kollicoat®

SR 30D. The coating levels of EC and Eudragit® RS/RL100 (2:1, w/w) were 5, 10, and

15% whereas Kollicoat® SR 30D was 50% dispersion as available. The aims of coating

were, to overcome the initial burst release of drug from the pellets and to extend the

59

release up to 12 h, as well as to investigate the controlled release behavior of these

polymers. The compositions of coating solution of EC, Eudragit® RS/RL100 and

Kollicoat® SR 30Dare given in Table 4, 5 and 6, respectively.

Accurately weighted quantity of EC (polymer), triacetin (plasticizer), and talc

(antitacking agent) were dispersed in a mixture of isopropyl alcohol (IPA) and water.

The coating dispersion were mixed in homogenizer for 10 min, filtered through a nylon

cloth before coating and kept on agitation during the entire process of coating.

The Eudragit®RS/RL100 were powdered into mortar and pestle and then the accurately

weighed quantity was added slowly into 50% of the diluents mixture and mixed for 30

min. in homogenizer. In a separate container, the talc (antitacking agent) and triethyl

citrate (plasticizer), were added in the remaining diluent mixture and stirred for

15min.using high shear mixer. Both the mixtures were mixed while stirring with a

conventional stirrer and then passed through a 0.5 mm sieve. During the coating process,

the solution was kept on agitation.

The accurately weighed quantity of Kollicoat® SR30D dispersion and propylene glycol

were transferred in a suitable container containing 35 ml water and mixed for 10 min.

using conventional stirrer. Talc was added in the remaining quantity of water using

separate container and mixed for 5 min. with stirrer. Both the mixtures were mixed while

stirring and then homogenized for 10 minutes.

Pellets were coated in a conventional coating pan (Erweka). The coating parameters were

as follows:

Batch size = 50 gram pellets, inlet temperature = 60–65oC, pan speed = 40 r/min, spray

pressure = 40 psi, spray rate = 1 mL/min, and atomizing nozzle diameter of spray gun =

1 mm. After the application of the polymers dispersion, the obtained weight gained by

pellets was determined. Finally, the coated pellets were dried at 40oC for 2 h in an oven

and evaluated for in vitro drug release.

60

TABLE 2

Composition of Itopride hydrochloride pellets formulations

Formulation

code

Drug

(ITP)

MCC

pH-

101

Lactose

DC

HPMC

K4M

HPMC

K15M

HPMC

K100M

EC

7cps

Drug

(ITP)

MCC

pH-

101

Lactose

DC

HPMC

K4M

HPMC

K15M

HPMC

K100M

EC

7cps

Total

weight

per

capsule

(%) (%) (%) (%) (%) (%) (%) (mg) (mg) (mg) (mg) (mg) (mg) (mg) (mg/Cap)

F1 60 30 10 - - - - 150 75 25 - - - - 250

F2 60 20 20 - - - - 150 50 50 - - - - 250

F3 60 10 30 - - - - 150 25 75 - - - - 250

F4 60 - 40 - - - - 150 - 100 - - - - 250

F5 60 40 - - - - - 150 100 - - - - - 250

F6 50 30 10 10 - - - 150 90 30 30 - - - 300

F7 50 25 10 15 - - - 150 75 30 45 - - - 300

F8 50 20 10 20 - - - 150 60 30 60 - - - 300

F9 50 15 10 25 - - - 150 45 30 75 - - - 300

F10 50 10 10 30 - - - 150 30 30 90 - - - 300

F11 50 30 10 - 10 - - 150 90 30 - 30 - - 300

F12 50 25 10 - 15 - - 150 75 30 - 45 - - 300

F13 50 20 10 - 20 - - 150 60 30 - 60 - - 300

F14 50 15 10 - 25 - - 150 45 30 - 75 - - 300

F15 50 10 10 - 30 - - 150 30 30 - 90 - - 300

61

TABLE 3

Composition of Itopride hydrochloride pellets formulations

Formulation

code

Drug

(ITP)

MCC

pH-

101

Lactose

DC

HPMC

K4M

HPMC

K15M

HPMC

K100M

EC

7cps

Drug

(ITP)

MCC

pH-

101

Lactose

DC

HPMC

K4M

HPMC

K15M

HPMC

K100M

EC

7cps

Total

weight

per

capsule

(%) (%) (%) (%) (%) (%) (%) (mg) (mg) (mg) (mg) (mg) (mg) (mg) (mg/Cap)

F16 50 30 10 - - 10 - 150 90 30 - - 30 - 300

F17 50 25 10 - - 15 - 150 75 30 - - 45 - 300

F18 50 20 10 - - 20 - 150 60 30 - - 60 - 300

F19 50 15 10 - - 25 - 150 45 30 - - 75 - 300

F20 50 10 10 - - 30 - 150 30 30 - - 90 - 300

F21 50 30 10 - - - 10 150 90 30 - - - 30 300

F22 50 25 10 - - - 15 150 75 30 - - - 45 300

F23 50 20 10 - - - 20 150 60 30 - - - 60 300

F24 50 15 10 - - - 25 150 45 30 - - - 75 300

F25 50 10 10 - - - 30 150 30 30 - - - 90 300

F26 37.5 22.5 10 - - 30 - 150 90 40 - - 120 - 400

F27 37.5 12.5 10 - - 40 - 150 50 40 - - 160 - 400

F28 37.5 2.5 10 - - 50 - 150 10 40 - - 200 - 400

F29 37.5 32.5 10 - - 10 10 150 130 40 - - 40 40 400

F30 37.5 22.5 10 - - 15 15 150 90 40 - - 60 60 400

F31 37.5 12.5 10 - - 20 20 150 50 40 - - 80 80 400

62

TABLE 4

Composition of Ethylcellulose coating solution

Compositions Quantity

(5%)

Quantity

(10%)

Quantity

(15%)

Ethyl Cellulose (EC-10cps) 5.0g 10 g 15 g

Triacetin USP/FCC 1.0g 1.5 g 2 g

Talc 1.0g 1.5 g 2 g

Isopropyl Alcohol 90 ml 90 ml 90 ml

Water 10ml 10 ml 10 ml

TABLE 5

Composition of Eudragit® RS/RL100 coating solution


(5%)

Quantity

(10%)

Quantity

(15%)

Eudragit RS 100/RL 100 (2:1) 5 g 10 g 15 g

Triethyl citrate (TEC) 0.5 g 1 g 1.5 g

Talc 2.5 g 5 g 7.5 g

Isopropyl Alcohol (IPA) 90 ml 90 ml 90 ml

Water (diluent) 10 ml 10 ml 10 ml

TABLE 6

Composition of Kollicoat® SR 30 D coating dispersion


(50% dispersion)

Kollicoat SR 30D 50.0 g

Propylene glycol 1.5 g

Talc 5 g

Water 43.5g

63

4.1.3. Flow Properties of Pellets

Flow properties of the drug loaded pellets formulations were determined using the procedure

mentioned in US Pharmacopeia (USP35-NF30, 2013).

4.1.3.1. Bulk density

Bulk densities were calculated using 10 g of pellets in measuring cylinder and the

volume was noted in ml. Bulk density of pellets was determined by using the following

formula.

𝑩𝒖𝒍𝒌 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 =𝑴

𝑽o

(24)

Where,

M is the mass of pellets in g and Vo is the initial volume of pellets in ml.

4.1.3.2. Tapped density

The Tapped densities were measured by tapping the pellets till constant volume, using

the following formula.

𝑻𝒂𝒑𝒑𝒆𝒅 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 = 𝑴

𝑽𝒇 (25)

Where,

M is the mass of pellets in g and Vf is the final volume of pellets after tapping in ml.

4.1.3.3. Carr’s index

The Compressibility/ Carr’s Index of pellets were determined by using the following

formula

𝑪𝒂𝒓𝒓′𝒔 𝑰𝒏𝒅𝒆𝒙 = (𝑽𝒐 − 𝑽𝒇

𝑽𝒐 ) ×𝟏𝟎𝟎 (26)

Where,

Vo is initial volume and Vf is the final volume of pellets

64

4.1.3.4. Hausner ratio

It is the ratio between the two volumes and was calculate by the following formula

𝑯𝒂𝒖𝒔𝒏𝒆𝒓 𝒓𝒂𝒕𝒊𝒐 = 𝑽𝒐

𝑽𝒇 (27)

Where, Vo is initial volume and Vf is final tapped volume of pellets

4.1.3.5. Angle of repose

The angles of repose of pellets sample can be determined by measuring the height of

cone and was calculated by using the following equation;

𝒕𝒂𝒏(∝) = 𝒉𝒆𝒊𝒈𝒉𝒕

𝟎.𝟓 𝒃𝒂𝒔𝒆 (28)

Where,∝ is the angle of repose.

TABLE 7: Flow properties of Hausner ratio, compressibility index and angle of

repose (USP35-NF30, 2012)

Flow Properties Hausner ratio Compressibility Index (%) Angle of repose (degrees)

Excellent 1.00–1.11 <10 25–30

Good 1.12–1.18 11–15 31–35

Fair 1.19–1.25 16–20 36–40

Passable 1.26–1.34 21–25 41–45

Poor 1.35–1.45 26–31 46–55

Very poor 1.46–1.59 32–37 56–65

Very, very poor >1.60 >38 >66

65

4.1.4. Characterization of Itopride HCl pellet formulations:

Itopride HCl loaded pellets were evaluated for various pharmaceutical quality analysis,

as described below.

4.1.4.1. Friability

A sample of 10 g uncoated and coated pellets were placed in the friabilator (Erweka

GmbH D-63150, Husenstamm, Germany) to calculate the friability. The pellets were

subjected to falling shocks at a rotational speed of 25 rpm for 10 min. Fines were

removed by sieving through a 250 µm mesh. The fraction above 250 µm was used to

calculate the friability by following formula (Dukic-Ott, A. et al., 2007).

𝑭𝒓𝒊𝒂𝒃𝒊𝒍𝒊𝒕𝒚 (%) = (𝑰𝒏𝒊𝒕𝒊𝒂𝒍 𝑾𝒆𝒊𝒈𝒉𝒕−𝑭𝒊𝒏𝒂𝒍 𝑾𝒆𝒊𝒈𝒉𝒕)

𝑰𝒏𝒊𝒕𝒊𝒂𝒍 𝑾𝒆𝒊𝒈𝒉𝒕 ×𝟏𝟎𝟎 (29)

The Acceptance criteria: % friability less than 1% is considered acceptable.

4.1.4.2. Assessment of pellets surface morphology

4.1.4.2.1. Sieve analysis

Sieve analysis of pellets formulations were performed using series of standard sieves

(1700, 1400, 1180, 1000, 710, 500 and 250 μm) and agitated for 10 min on sieve shaker

(Erweka, Germany). The size of pellets in the range of 710 – 1400μm were considered

appropriate pellets and subjected for further analysis (Law, M.F.L. &Deasy, P.B., 1998;

Wiwattanapatapee, R. et al., 2004).

4.1.4.2.2. Image analysis

Image analysis of the pellets were carried out using stereomicroscope (Am Scope

Digital, LED-1444A, USA) at x10 magnification by spreading over a flat surface and

66

digital images of each formulation (n ≥ 50) were taken. Feret diameter, aspect ratio and

sphericity (shape factor) of these images were measured by using image analysis

software (NIH Image J 1.47v, USA).The average caliper distance of 36 measurements

around the particle employing a 5° angle of rotation is referred as Feret diameter,

whereas sphericity is the degree of roundness of each particle. The ratio of length to

width of each particle is known as Aspect ratio. The aspect ratio and sphericity were

calculated using the following equations:

𝑨𝒔𝒑𝒆𝒄𝒕 𝒓𝒂𝒕𝒊𝒐 (𝑨𝑹) = 𝒅𝒎𝒂𝒙 𝒅𝒎𝒊𝒏⁄ (30)

𝑺𝒑𝒉𝒆𝒓𝒊𝒄𝒊𝒕𝒚 (𝑺𝒉𝒂𝒑𝒆 𝑭𝒂𝒄𝒕𝒐𝒓) = 𝟒𝝅𝑨 𝑷𝟐⁄ (31)

Where, A is the area, P is the perimeter, dmax is the longest and dmin is the shortest Feret

diameters. The calculated value of ‘‘1’’ indicates a particle that is perfectly

round.(Akhgari, A. et al., 2011; Gupta, N.V. et al., 2011; Fursule, R. et al., 2009;

Sriamornsak, P. et al., 2008).

4.1.4.2.3. Scanning electron microscopy (SEM)

The surface morphology and cross section of successful pellets formulations (EC coated

only), controlling the drug release for 12 hours, were examined by using scanning

electron microscope (JSM-6380A, Jeol, Japan). With the help of double-sided tape, the

pellets were mounted on brass stub. The stubs were then coated with gold film by Auto

Coater (JFC-1500, Jeol, Japan) up to 300°A. The sample was then placed in a sample

chamber and scanning was performed under different magnifications ranging from

3,500x to 11,000x at 10 kV to 16kV voltage.

4.1.4.3. Fourier transform infrared spectroscopy (FTIR)

The possible interaction between drug and excipients was determined by Fourier

transform infrared spectrophotometer (Nicolet-6700, Thermo ScientificTM, USA).

Infrared spectra of pure drug and successful formulations were recorded (OMNICTMTM

Specta Software) over the wave numbers ranging from 4500 to 1000 cm-1.

67

4.1.4.4. Drug content analysis

The ITP HCl loaded pellets were evaluated for drug content. Mortar and pestle was used

to crushed the accurately weighed ITP pellets (300 and 400 mg, with respect to

formulations), and transferred to a 100 mL volumetric flask. Then, 50 mL of 0.1 N

hydrochloric acid (HCl) solution was added to the flask and sonicated (Digital Ultrasonic

Cleaner—Supersonic X3, Germany) for 10 min. The same solution was employed to

make up the volume. The sample was analyzed after filtration and appropriate dilution to

25 µg/mL, using spectrophotometer (Shimadzu UV 1800, Japan) with UV detection at

258 nm(Rao, P.S. et al., 2014: Chhipa, P. et al., 2009).

4.1.4.5. In-vitro drug release study

Accurately weighed quantity of uncoated and coated ITP pellets (n = 6) were added to

the USP dissolution apparatus – II (Erweka D-63150, Germany) for in-vitro drug release

studies and operated for a period of 12 h. The dissolution media used were 900 mL of

hydrochloric acid (pH 1.2) and phosphate buffer (pH 4.5 and 6.8), maintained at

temperature 37 ± 0.5°C. The paddle speed kept at 50 rpm. The 5 ml of aliquots were

drawn at regular time intervals of 1, 2, 3, 4, 6, 8, 10, and 12 h and which replaced with 5

ml of fresh medium maintained at same temperature. The collected samples were filtered

and then analyzed after appropriate dilution, using UV Spectrophotometer(Shimadzu UV

1800, Japan) at 258 nm(Shah, S. et al., 2012: Chhipa, P. et al., 2009).

The marketed Itopride Hydrochloride 150 mg sustained release (24 hours) tablet brand,

was also evaluated for in-vitro drug release profile in the same media. The all release

studies were performed in triplicate and the amount of drug released from the samples

was calculated in percentage. The mean percentage drug release with standard deviation

values vs time were plotted.

4.1.5. Drug release kinetic studies

Dissolution of drug means transfer of drug from its solid phase to the surrounding

medium and solubility is a thermodynamic property of a drug and medium while the

68

dissolution rate is a kinetic property (Siepmann, J. et al., 2011). The dissolution data can

be compared and performed by using model dependent and independent methods.

4.1.5.1. Model dependent methods

Model dependent methods are used when dissolution comparison of different

formulations are needed. Different theories of kinetic models have been proposed for

explaining dissolution of drug from immediate or controlled release dosage forms

(Yuksel, N. et al., 2000). The in-vitro release data were fitted to various kinetics models

such as Zero order, First order, Higuchi, Korsmeyer-Peppas, Hixson-Crowell, and Baker

-Lonsdale, to interpret drug release rate from matrix-coated pellets using MS Excel (DD

Solver). The release rate of drug can also be calculated by the following equations.

4.1.5.1.1. Zero-order kinetics

The model describes the dissolution and slow release of drug from the device that do not

separate. Mathematically it can be express as:

𝑸𝒕 = 𝑲𝒐𝒕(32)

Where Qt is the amount of drug dissolved in time t, K0is zero order release constant and t

is time in hours. This model can be used to explain the release of drug from the

transdermal systems, matrix /coated tablets and osmotic systems (Costa, P. &Lobo,

J.M.S., 2001a; Lachman, L. et al., 1986).

4.1.5.1.2. First order kinetics

Gibaldi, M.& Feldman, S., first time proposed this model (Gibaldi, M. & Feldman, S.,

1967). This model can be expressed as:

𝑳𝒐𝒈𝑸𝒕 = 𝒍𝒐𝒈 𝑸𝒐 − 𝑲𝒕

𝟐.𝟑𝟎𝟑 (33)

69

Where, Qt is the concentration of drug released at time t, Q0 is the initial concentration of

drug in the solution, tis the time and K is the first order release constant. This model can

be used to demonstrate the dissolution of drug from the porous matrices containing

water-soluble drugs (Dash, S. et al., 2010; Lachman, L. et al., 1986).

4.1.5.1.3. Higuchi model

The first mathematical kinetic model suggested by Higuchi, T. in 1961 and 1963 to

evaluate the release of drug from the matrices (Higuchi, T., 1961; Higuchi, T., 1963).

This model explain that the amount of drug release from the matrix system is directly

proportional to the square root of the time of exposure to the dissolution medium(Dash,

S. et al., 2010).

It can be expressed as:

𝑸 = 𝒌𝑯. 𝒕𝟏/𝟐 (34)

Where, kH, is the Higuchi release rate constant, and t is the time in hours.

4.1.5.1.4. Korsmeyer–Peppas model

This model was derived by Korsmeyer et al. in 1983 in which the first 60% in vitro drug

release data were fitted into the model to described the fraction of drug release from a

non-swelling or insoluble polymer matrix system. To study the release kinetics, in-vitro

dissolution data were plotted as log cumulative percentage drug release versus log time.

Korsmeyer–Peppas is mathematically calculated using following equation:

𝑴𝒕

𝑴∞ = 𝑲𝒕𝒏 (35)

Where, Mt /M∞ is the fraction of drug released at time t, K is the release rate constant, n

is the release exponent, and t is the time in hours (Dash, S. et al., 2010; Korsmeyer,

R.W. et al., 1983).

70

Then, Peppas, N., in 1985 used the value of exponent ‘n’ to characterize the drug release

mechanism from the dosage form. If n = 0.5 it indicates Fickian diffusion, if

0.45<n<0.89 indicates non-Fickian diffusion or anomalous diffusion, if n = 0.89

indicates case-II transport and if n > 0.89 indicates super case - II transport(Peppas, N.,

1985).

4.1.5.1.5. Hixson – Crowell model

Hixson, A. and Crowell J., explained that the particles’ regular area is proportional to the

cube root of its volume(Hixson, A. &Crowell, J., 1931). It is mathematically written

given in following equation:

𝑸𝒐𝟏/𝟑

− 𝑸𝒕𝟏/𝟑

= 𝑲𝐇𝐂 ×𝒕 (36)

Where Q0 is the initial amount of drug in the dosage form, Qt is the remaining amount of

drug in the dosage format time t, KHC is the Hixson - Cowell release constant

incorporating the surface – volume relation and tis the time in hours.

4.1.5.1.6. Baker–Lonsdale model

This model was developed from the Higuchi by Baker and Lonsdale in 1974.It described

the drug release from spherical matrix or microspheres. Mathematically expressed in the

following equation:

𝒇𝟏 = 𝟑

𝟐[𝟏 − (𝟏 −

𝑴𝒕

𝑴∞) ]

𝑴𝒕

𝑴∞ = 𝒌𝒕 (37)

Where, Mt is the amount of drug release at time t, M∞ is the amount of drug released at

an infinite time, K is the release constant which corresponds to the slope. The data

obtained from in vitro drug release studies were plotted as[d (Mt /M∞)] / dt with respect to

the root of time inverse (Dash, S. et al., 2010).

71

4.1.5.2. Model independent method

Model-independent method can further be divided into ratio tests and pair-wise

procedures. The ratio tests describe the relations between the reference and test product

i.e. ratio between percentage dissolved drug with respect to time or ratio between mean

dissolution time. Whereas the pair-wise procedures include;

(i) Differential factor (f1) which determines the percentage error between two

curves at different times and both the formulations would be considered similar if

the valve of f1 is less than50.

(ii) Similarity factor (f2) which measures the differences between the test and

reference products at different times and the two formulations would be similar if

the valve of f2 is more than 50 i.e. 50-100. It can be calculated by this equation:

𝒇𝟐 = 𝟓𝟎×𝒍𝒐𝒈 {[𝟏 + (𝟏

𝑵) ∑(𝑹𝒊 − 𝑻𝒊)

𝟐]−𝟎.𝟓

} ×𝟏𝟎𝟎(38)

Where Ri is the percent dissolved of reference drug, Ti is the percent dissolved of test

drug at each time point and N is the number of samples(Costa, P. &Lobo, J.M.S., 2001a).

4.1.6. Stability studies

Stability studies is an important part of product development (ICH, 2003b). The aim of

stability study is to evaluate the quality of a drug substance/ product that changes with

the passage of time under the influence of temperature, humidity and light(WHO, 2009).

On the basis of stability study, we can recommend the storage conditions, retests periods,

and shelf lives of the product to be established. The shelf lives of the product is

determined on the basis of the assay, using International Conference on Harmonization

(ICH) guidelines and Pharmacopoeias stability indicating methods(Gupta, K.R. et al.,

2010).

72

The formulations are subjected for a period of 6 months in humidity chamber at the

accelerated temperature as per ICH guidelines (40°C±2°C and 75%±5% RH). The

samples are taken out at different time intervals and investigated for physical appearance,

percentage content and dissolution profiles (Sisinthy, S. et al., 2015).

TABLE 8: Storage conditions for stability studies (ICH)

Intended

storage

conditions

Stability Study Storage conditions

Minimum time

required for

submission

Room

Temperature

Long term

25 °C ± 2 °C / 60% ± 5% RH or

30 °C ± 2 °C / 65% ± 5% RH or

30 °C ± 2 °C / 75% ± 5% RH

12 months

Intermediate 30 °C ± 2 °C / 65% ± 5% RH 6 months

Accelerated 40 °C ± 2 °C / 75% ± 5% RH 6 months

Refrigerator

Long term 5° C ± 3 °C RH 12 months

Accelerated

25 °C ± 2 °C / 60% ± 5% RH or

30 °C ± 2 °C / 65% ± 5% RH or

30 °C ± 2 °C / 75% ± 5% RH

6 months

Freezer Long term -20 °C ± 5 °C 12 months

(WHO, 2009).

4.1.6.1. Stability studies of Itopride HCl pellets

Stability studies of the selected coated formulations (F1, F6, F11, F16 and F21) were

conducted to assess their physical appearance, and drug content. The samples were

subjected to storage at room temperature (25 °C± 2 °C/ 60% ± 5% RH) and at

accelerated temperature (40± 2 °C / 75% ± 5%RH). Samples subjected to room

temperature were analyzed at 0 and 12 months while samples subjected to accelerated

temperature were analyzed at 0, 3 and 6 months as per ICH guideline (Q1E evaluation

for stability data). The shelf-life of the coated pellets formulations were also calculated

using software Minitab (version 17.1.0.), data analysis tool for drug stability studies.

73

4.1.7. Method development and validation for the analysis of itopride HCl

in human plasma

High performance liquid chromatography method was developed and validated for the

determination of Itopride in the human plasma. The reported method (Yehia, S. et al.,

2012)for the determination of Itopride, was modified and validated according to FDA

guidelines(FDA, 2001).

4.1.7.1. Equipment, software, chemical and glassware

1. Analytical balance (Sartorius, Germany)

2. High Performance Liquid Chromatography Pump (LC-10 AT VP, Shimadzu

Corp., Kyoto, Japan)

3. UV Detector (SPD-10A VP, Shimadzu Corp., Kyoto, Japan)

4. Communication Bus Module (CBM 102, Shimadzu Corp., Kyoto, Japan)

5. HPLC Column (Phenomenex -18, 4.6 x 250 mm, 5µm)

6. Guard column C18, 4.0 x 2 mm

7. HPLC Software Class GC 10, (Shimadzu Corp., Kyoto, Japan)

8. Ultra-Sonic bath (Clifton, Nickel Electro Ltd. Somerset, England)

9. Centrifuge machine (Hereues, Osterode, Germany)

10. Vortex mixer (Whirl mixer, England)

11. Filtration assembly (Sartorius, Gorringen, Germany)

12. Swinney Filter assembly (Millipore, England)

13. Microliter syringe (Hamilton, Switzerland)

14. Micropippette (Mettler Toledo, Scwerzenbach, England)

15. pH meter (Mettler Toledo, Scwerzenbach, England)

16. Distillation assembly (Hamilton Laboratories, Kent, England)

74

17. Water Deionizer (Elga, Highwycombe, England)

18. Membrane filters 0.45 μ pore size, 47 mm diameter (Schleicher &Schuell, Dasel,

Germany)

19. Swinney Filters (Millipore, England)

20. Vacuum pump (China)

21. Methanol, HPLC grade (Merck, Darmstadt, Germany)

22. Acetonitrile, HPLC grade (Merck, Darmstadt, Germany)

23. Potassium Dihydrogen Phosphate (Merck Millipore, Germany)

24. Ortho-Phosphoric Acid (Merck Millipore, Germany)

25. Sodium hydroxide (Merck, Darmstadt, Germany)

26. Diethyl ether (BDH Chemicals Ltd., Poole, England)

27. Moxifloxacin (Abbot Laboratories Pakistan)

28. Glassware i.e. Beakers, Funnel, Volumetric Flask, Graduated cylinder, Pipettes,

(Pyrex, England)

4.1.7.2. Preparation of mobile phase

Mobile Phase consisted of acetonitrile and Potassium di Hydrogen Phosphate (KH2PO4)

buffer (0.05 M) adjusted at pH 4.0 with ortho-phosphoric acid (1 M) in a ratio of 70:30%

(v/v). The mobile phase was filtered through membrane filter (0.45μm) using filtration

assembly and sonicated to degas before use.

4.1.7.3. Preparation of standard stock and working solutions

The standard stock solution of Itopride HCl and moxifloxacin (internal standard) of 100

μg/ml were prepared in mobile phase by dissolving accurately weighed quantity of both

drugs. From this standard stock solution of Itopride, serial dilutions of 0.05, 0.1, 0.2, 0.4,

75

0.6, 0.8, 1 and 2 μg/ml were prepared in mobile phase and plasma. 50 μl moxifloxacin

was added to each concentration as internal standard.

4.1.7.3.1. Chromatographic conditions

The HPLC system consisted of isocratic pump, UV-Vis detector, C18 guard column and

C18 column (Phenomenex C-18, 4.6 x 250 mm, 5µm) was adjusted at a flow rate of 1

ml/min. The detection wavelength of Itopride was set at 258 nm.

4.1.7.3.2. Sample preparation and determination of drug in human

plasma

2 ml of diethyl ether was added as deproteinating agent, to 1 ml thawed volunteer

plasma sample for extraction.

The mixture was then mixed for 2 minutes using vortex mixer.

Centrifuged at 3000 rpm for 10 minutes.

The supernatant organic layer was drawn using micropipette and transferred into

another set of tubes.

The solvent was evaporated in a stream of Nitrogen at 40oC to dryness.

The residue was reconstituted with 350 µl of mobile phase.

100 µl was injected into the HPLC system using microliter syringe.

4.1.7.3.3. Retention Time

Retention time of Itopride HCl was found 7.28 min and 8.56 min for IS (moxifloxacin).

4.1.7.3.4. Method Validation

The method was validated as per FDA guidelines(FDA, 2001) in terms of selectivity,

linearity, accuracy, precision and stability.

76

4.1.7.3.4.1. Selectivity

Six blank plasma samples were injected and then compared with the samples containing

Itopride HCl in the concentrations of 2, 0.4 and 0.05 ug/ml, in order to establish

selectivity.

4.1.7.3.4.2. Linearity

Linearity was determined by preparing a serial dilution of drug in plasma in the

concentration range of 0.05-2 ug/ml in triplicate. The calibration curves were constructed

and co-efficient of correlation (r2) was determined. Back calculation of all concentrations

by calibration curves was done and standard deviation (SD), % CV (coefficient of

variance) and % accuracy were calculated.

4.1.7.3.4.3. Accuracy and precision

“The degree of closeness of the test concentrations of the analyte with its true

concentrations is termed as accuracy". For the estimation of accuracy, five samples of

each concentration of Itopride HCl(0.05, 0.1, 0.2, 0.6, 0.8, 1 & 2 µg/ml) were analyzed

in mobile phase and plasma and observed for the deviation from the mean of the trues

values i.e. relative standard deviation should not be more than 15% (Table 19 and 21).

Whereas, the degree of agreement among individual test results and how individual

results are scattered from the mean value is referred as precision. The developed method

was also validated for intraday and interday precision.

4.1.7.3.4.3.1. Intraday precision

For the determination of intraday precision, four different concentrations (0.05, 0.4, 0.8

& 2 µg/ml) of itopride were evaluated at different time in a day by the proposed method.

The drug concentration in plasma was calculated by standard calibration curves. For this

five concentration SD, % CV and % accuracy were calculated.

77

4.1.7.3.4.3.2. Interday precision

For interday precision, five samples of the above mentioned concentrations were

analyzed for three consecutive days at different and same times. To calculate the

concentration of Itopride an average of two calibration curves was used and % accuracy,

SD, and % RSD were determined.

4.1.7.3.4.4. Limit of quantification (LOQ) and limit of detection (LOD)

Limit of quantification (LOQ) and limit of detection (LOD) were determined by

analyzing five samples of different concentrations i.e. 0.03, 0.05, 0.1, 0.2, 0.6, 0.8, 1 & 2

µg/ml. The LOQ was found to be 0.05 ug/ml, whereas the detection was carried out till

0.03 ug/ml. Concentrations were determined by back calculation and SD, % CV, %

accuracy and precision were also determined. The chromatograms of the lowest

concentrations of Itopride were observed, and signal to noise ratios were determined for

LOQ and LOD

4.1.7.3.4.5. Analytical recovery

The absolute analytical recovery was determined by comparing the data of drug spiked in

plasma and in mobile phase. Five samples of three different concentrations (0.2, 0.6 &

2µg/ml) were evaluated for the calculation of % recovery.

4.1.7.3.4.6. Stability of the drug in plasma

Freeze and thaw short and long term stability studies of Itopride in plasma were

conducted, for which two concentrations were selected i.e. 0.1 µg/ml and 2 µg/ml, and

20 samples of each concentration were analyzed. Initially five fresh samples of each

concentration were evaluated and other remaining samples were stored at -20 °C for 24

hours. Next day, further five samples (cycle 1) of each concentration were thawed and

investigated while remaining samples were kept frozen for next 24 hours. The procedure

was repeated for cycle 2 and 3. The samples of cycle 1, 2 and 3, were compared with that

of fresh samples in order to observe any degradation. `

78

For the long term stability study, 20 samples of each low and high concentrations in

plasma were prepared and five freshly prepared samples were analyzed and the

remaining were stored at -20 °C. Five samples of each concentration were tested after 2,

3 and 6 weeks of storage, and the chromatograms were compared with that of fresh

samples of same concentrations.

4.1.8. Pharmacokinetic Studies of Extended Release Itopride HCl Pellets

The extended release Itopride pellets formulation EC5-F11, containing 10% HPMC-

K15M was chosen for pharmacokinetic studies in healthy human volunteers due to its

best physicochemical properties and zero order release kinetics.

4.1.8.1. Ethical board approval

The protocol was approved by Hamdard University Ethical Review Board (HU-ERB)

and the Board of Advanced Studies and Research (BASR), University of Karachi,

Karachi.

4.1.8.2. Study venue

The study was conducted at Department of Pharmaceutics, Faculty of Pharmacy,

University of Karachi, under the supervision of principal investigator and physician.

4.1.8.3. Design of study

The design of study was a single centered, single dose, randomized; crossover, design to

compare and evaluate the pharmacokinetics of extended release Itopride pellets (150mg,

encapsulated) and Ganaton OD (once daily) tablet (150 mg) – Abbott Pakistan as

reference brand in 12 healthy human volunteers. The pharmacokinetic parameters of

both test and reference products were determined and compared under fasting and fed

conditions. Plasma samples from volunteers were drawn at regular time interval till 48

79

hours and the samples were analyzed for Itopride concentration using validated HPLC

method.

4.1.8.4. Volunteers selection

Twelve (12) healthy human subjects of age 18-27 years, were selected for the study. All

the volunteers were medically examined for their blood test, blood pressure, weights,

heights and habitual history (non-smokers and non-addicted).

4.1.8.4.1. Criteria for inclusion

Age limit of healthy volunteers was between 18-27 years.

With weight ranges between ±10 percent of the ideal body weight.

All physical and medical examinations were within normal limits.

Allergy history was also analyzed and not reported.

4.1.8.4.2. Criteria for exclusion

Volunteers whose heights and weight were not in normal range.

Failure of diagnostic tests in normal and clinical conditions.

Any type of acute infection within two weeks prior to study.

Addiction of drug or alcohol or smokers having more than 10 cigarettes per day.

Special diet users i.e., spicy, vegetarian, rich diet.

Person who cannot leave the grape fruit and beverages at least two days before and

till the end of study.

Subjects having any chronic disease which may alter the pharmacokinetics of drug.

Subjects participated in any other pharmacokinetic studies within three months

before the present studies.

Any hospitalization history within three months before studies.

Subjects donated blood of 500 ml, 750 ml, 1000 ml, 1250 ml, 1500 ml, 2000 ml and

2500 ml, within 14, 30, 90, 120, 180, 270 days and within a year respectively.

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4.1.8.4.3. Declaration of consent

All the volunteers were informed in writing about the consequences and possible

outcomes of study. A consent declaration form was prepared both in English and Urdu

(local language), after the approval from Hamdard University Ethics Review Board(HU-

ERB) and BASR University of Karachi and signed consent collected before study.

4.1.8.5. Blood Sampling Protocol

4.1.8.5.1. Requirement

IV cannula with stopper (Master IV Catheter, Korea)

Syringes10 cc, disposable (Becton Dickenson, USA)

Centrifuge heparinized tubes (USP specification)

Alcohol swabs and surgical cottons

Ethanol analytical grade (Merck, Darmstadt, Germany)

Different sizes of Microliter pipettes (Hamilton, Switzerland) with their tips.

Refrigerator, with a chiller cabinet having temperature of -20°C, (LG Electronics,

Korea)

4.1.8.5.2. Procedure

In order to observe the effect of fed and fasted conditions, the study period was

divided in to four phases, and was randomized between EC5-F11(encapsulated

150mg pellets) and Ganaton OD (150mg Tablet) under Fed and Fasted states (Table

30a and 30b).

The extended release encapsulated Itopride HCl 150 mg pellets (EC5-F11) and 150

mg tablet (Ganaton OD) were administered orally to selected volunteers with 240 ml

(8 fluid ounce) of plain water in fed and fasted conditions (FDA, 2002).

The subjects were kept fasted for at least 10 hours before drug administration and, no

beverages/soft drinks were allowed at least 4 hours after dosing (FDA, 2002).

81

The subjects started recommended meal 30 minutes before the administration of drug

products, however, the doses were administered 30 minutes after the start of the food

(FDA, 2002).

Two weeks of washed out period was provided between each phase of study.

The sampling time was 0, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h.

5 ml blood sample was drawn from each volunteer.

Plasma was separated after centrifugation at 4000 rpm for 10 minutes and was kept

frozen at -20 °C till analysis.

4.1.8.6. Determination of pharmacokinetic parameters

Compartmental and non-compartmental pharmacokinetics parameters of EC5-F11

extended release Itopride HCl 150 mg pellets and Ganaton OD 150 mg tablet, were

calculated using pharmacokinetics software Kinetica version 5.1 (Thermoelectron,

USA).

4.1.8.6.1. Compartmental parameters

Two-compartmental model was applied on the basis of time verses concentrations data

and the parameters determined were Cmax, Tmax (observed and calculated) Ka, A, B, Ke,

alpha, beta, K12, K21, AUC0-∞, AUC0-t., Vc, Cl, Kel, T1/2ka, T1/2α, Tabs, T1/2kel.

4.1.8.6.2. Non - Compartmental parameters

Non-compartmental parameters calculated were, λz, Vz, Vss, HVD, AUClast,

AUCextrapolated, AUCtotal, % AUCextrapolated, AUMClast, AUMCextrapolated, AUMC, AUMCtotal

and MRT.

4.1.8.7. Statistical analysis of the pharmacokinetic data

For the evaluation of pharmacokinetic parameters, Two-way ANOVA and Two-one-sided t

test were used. Analysis was performed for both non transformed and log transformed data

as per FDA guidelines(www.fda.gov).

82

4.1.8.7.1. Latin square ANOVA for two formulations

Latin square ANOVA (two-way) was applied using software Kinetica version 5.1

(Thermoelectron corp, USA) for the evaluation of all the pharmacokinetics parameters.

The study was open labelled, single dose, randomized, two treatments, two sequence,

four period randomized cross over study.

4.1.8.7.2. Two one-sided t test

Kinetica version 5.1 (Thermoelectron corp, USA) was used for the measurement of Two

unilateral t-tests.

In the table the lower t and the upper t was concluded on the basis of the following rules.

1. If t (lower) ≥ t and t (upper) ≥ t then the data can conclude equivalent

2. If t (lower) ≤ t and t (upper) ≤ t then the data cannot conclude equivalent

83

5. RESULTS

84

RESULTS

In current study, extrusion-spheronization technique was used to formulate Itopride

Hydrochloride in extended release dosage form. Several trial formulations were designed

with the addition of different types of polymers in various concentrations, which were

then subjected to pharmaceutical quality evaluation with the objective of optimization.

The optimum formulation’s pharmacokinetics under fed and fasted conditions, was also

determined and comparative bioavailability study was conducted, in order to compare

pharmacokinetic parameters of the optimized formulation with that of marketed one.

5.1. Formulation development of extended release Itopride HCl pellets

Itopride HCl (ITP) plain (without polymer) and matrix pellets (hydrophilic and

hydrophobic polymers) were developed by using MCC (sphericity enhancer) and lactose

(filler). Different percentage ranges (10-50%) and viscosity grades of HPMC K4M (4000

cps), K15M (15000 cps), and K100M (100000 cps). Similarly, EC (7 cps) was used in

different ranges (10-30%) to extend the drug release rate. Thirty-one pellet formulations

were prepared by extrusion-spheronization method and their compositions are given in

Table 2 and 3. Five formulations were selected for coating i.e. F1 (30% MCC and 10%

Lactose, without polymer), F6 (HPMC K4M, 4000cps), F11 (HPMC K15M, 15000cps),

F16 (HPMC K100M, 100000cps), and F21 (EC Premium 7cps). These five selected

pellet formulations were coated with different levels (5-15%) of ethylcellulose (Premium

10 cps) coating dispersion and Eudragit® RS/RL100 (2:1, w/w), whereas, Kollicoat® SR

30D was applied as 50% dispersion, to controlled the initial burst release of drug and to

control the rate of drug release up to 12 h. Compositions of EC (10 cps), Eudragit®

RS/RL100 and Kollicoat SR 30D coating dispersions are given in Table 4, 5 and 6,

respectively.

5.2. Characterizations of ITP pellet formulations

The pre-formulation determination of flow and compression characteristics of trial

batches, such as angle of repose, bulk density, tapped density, compressibility index,

Hausner ratio, friability and percentage drug content of uncoated ITP pellet formulations

85

are given in Table 9 and 10. The surface morphology of uncoated and coated pellets

formulations were evaluated by using Stereomicroscope. Images taken by

Stereomicroscope of uncoated plain and matrix pellets are given in Figure 5a and 5b.

Images of ethylcellulose coated F1, F6, F11, F16 and F21 formulations are shown in

Figure 6a, b, c, d and e, respectively. Images of Eudragit® RS/RL100 coated F1, F6, F11,

F16 and F21 formulations and Kollicoat® SR 30D coated F1, F6, F11, F16 and F21

formulations are given in Figure 7a, b, c, d and e, and 8a, b, c, d and e, sequentially.

Table 11 and 12 shows Feret diameter, aspect ratio (AR) and sphericity/shape factor of

the ITP pellets. For analysis, more than 50 pellets (n>50) from each formulation were

subjected to measurement. The best selected 5% EC coated pellet formulations were also

characterized for their morphology, surface properties and cross-sectional studies using

SEM. The morphology and surface characteristics of 5% EC coated pellet formulations

F1, F6, F11, F16, and F21 are shown in Figure 9a, b, c, d and e, while the cross-sectional

pictures of these selected coated pellet formulations are expressed as Figure 10a, b, c, d

and e, respectively. These five selected formulations were also analyzed to detect any

possible interaction between pure drug (ITP) and polymers used in the formulations

using FTIR spectroscopy. The recorded infrared spectrums of pure ITP, HPMC (K4M) +

ITP, HPMC (K15M) + ITP, HPMC (K100M) + ITP, and EC (7 cps) + ITP, indicated that

there was no drug-polymers interaction occurred, as shown in Figure 11a, b, c, d and e

correspondingly.

Multiple point dissolution studies of both uncoated and coated pellets formulations were

performed. In-vitro dissolution profiles of uncoated plain (without polymers) and matrix

pellets formulations of ITP containing MCC, lactose, HPMC (K4M, K15M and K100M)

and EC (7cps) in 0.1 N HCl (pH 1.2) are shown in Figure12 and 13. The in-vitro

dissolution profiles of all coated pellet formulations in different media such as HCl

buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8), are exhibited in Figure 14 to

Figure 28. The dissolution profile of Ganaton OD 150 mg Tablet was also evaluated in

the above mentioned three pH, and result is given in Figure 29.

Different kinetic models, i.e., Zero order, First order, Higuchi, Hixson–Crowell, and

Baker–Lonsdale were applied to interpret the release kinetics from the EC (5%

dispersion), Eudragit RL/RS (15% dispersion) and Kollicoat SR 30D (50% dispersion)

coated pellets formulations (F1, F6, F11, F16 and F21) using DD Solver (a Microsoft

86

Excel based software). Table 13 to Table 15 exhibits the release kinetic data of all the

selected coated formulations. Formulation F11 coated with 5% dispersion of EC (coded

as EC5-F11) was selected as best formulation because it was best fitted to Higuchi,

Korsmeyer–Peppas, zero order and Hixson Crowell kinetics models. Similarity factor

(f2) was calculated using DD-solver. The percentage drug release at different time

interval was determined for F1, F6, F16 and F21, coated with 5% EC, 15% Eudragit

RL/RS and 50% Kollicoat SR 30 D dispersion, separately, and were compared with

EC5-F11, selected as reference formulation (on the basis of drug release). Table 16

shows the similarity factor (f2) of ITP pellet formulations with EC5-F11 (reference).

The selected 5% EC coated ITP pellet formulations F1, F6, F11, F16 and F21 were

subjected to stability studies to determine their physical appearance and percentage drug

content. All the formulations filled in rubber-capped amber glass bottles, were stored at

25 ○C/60% RH (under normal ambient conditions) and 40 oC/75% RH (under stress

condition) for 12 months according to ICH guidelines. The percentage content was

determined at the end of 3, 6, and 12 months and results are given in Table 17 and 18.

The stability data were assessed by using software Minitab (version 17.1.0.) to calculate

the shelf-life of the coated pellet formulations. The stability analysis report and graphs

generated by Minitab (version 17.1.0.) is also included as separately in Appendix 9.1.

5.3. Pharmacokinetic studies of extended release ITP pellets

High performance liquid chromatographic (HPLC) method was developed for the

determination of Itopride HCl and validated for selectivity, linearity, accuracy, precision,

and stability according to FDA guidelines. The standard calibration curves both in

mobile phase and plasma for Itopride with back calculated concentrations are given in

Table 19 to 21 and presented in Figure30a and 30b. Figure 31 and 32 shows the

chromatograms of standard calibration curves both in mobile phase and in plasma,

respectively. The method was also validated for intraday and interday accuracy and

precision, limit of quantification (LOQ), limit of detection (LOD), analytical recovery,

Freeze and Thaw stability of the drug in plasma and long term stability of the drug in

plasma, are shown in table 22 to 29.

87

Comparative pharmacokinetic studies of EC5-F11 pellets (150 mg) with Ganaton OD

tablet (150 mg), were conducted on 12 healthy human volunteers according to FDA

criteria. The details of these subjects and the sequence in which the EC5-F11 and

Ganaton OD tablet was administered under fed and fasted state is presented in table 30a

and 30b. EC5-F11 was selected for pharmacokinetic study on the basis of their

physicochemical properties and zero order kinetic release. Table 31, 32, 33 gives details

about the plasma drug concentration profiles under fed state, compartmental and non-

compartmental pharmacokinetic analysis of EC5-F11, whereas table 37, 38, and 39 for

Ganaton OD tablet, respectively. Similarly, table 34, 35 and 36 shows details about the

plasma drug concentration profiles under fasted condition, compartmental and non-

compartmental pharmacokinetic analysis of the EC5-F11, whereas table 40, 41 and 42

for Ganaton OD tablet, respectively. The plasma drug concentration vs time plot of EC5-

F11 and Ganaton OD in individual subject under fed and fasted states are exhibited in

figure 33 and 34, respectively. The mean plasma concentration vs time plot of the both

EC5-F11 and Ganaton OD tablet is given in figure 35.

Table 163 and 164 exhibit details about the mean pharmacokinetic log and non-log

transformed parameters of ER Itopride HCl 150 mg encapsulated pellets (EC5-F11)

versus Itopride HCl 150 mg tablet (Ganaton OD), with geometric mean ratios at 90%

confidence interval (CI). Table 165 shows the relative bioavailability of the EC5-F11

(test product) when compared to Ganaton OD (reference product).

Chromatograms of different volunteers both in fed and fasting states are shown in figure

36 to 39. The pharmacokinetic analysis reports generated by pharmacokinetic software

“Kinetica version 5.1” are shown in table 46 to 105 for logarithmically transformed data

of both fasted and fed states. Similarly, table 106 to 165 shows non-logarithmically

transformed data of both fasted and fed states. Each table contains the results of

ANOVA, geometric mean ratio, confidence interval and two one-sided t-test of

logarithmically and non-logarithmically transferred data.

88

TABLE 9

Evaluation of uncoated pellets formulations (F1 – F16)

Formulations

code

Physical Evaluation Chemical Evaluation

Bulk Density

(g/cm3)

Tapped Density

(g/ cm3)

Carr’s Index

(%) Hausner Ratio

Angle of Repose

(θ0)

Friability

(%)

Drug content Analysis

(%)

F1 0.655 0.771 15.05 1.18 24.43 0.61 101.16

F2 0.667 0.784 14.92 1.17 25.55 0.55 98.93

F3 0.649 0.766 15.27 1.18 26.76 0.57 99.36

F4 0.673 0.795 15.35 1.18 25.91 0.49 97.89

F5 0.672 0.802 16.21 1.19 26.11 0.52 100.25

F6 0.670 0.768 12.76 1.14 26.81 0.47 102.08

F7 0.680 0.778 12.60 1.14 27.78 0.48 99.11

F8 0.681 0.773 11.90 1.13 25.62 0.45 98.86

F9 0.659 0.765 13.86 1.16 25.21 0.44 97.56

F10 0.673 0.759 11.33 1.12 28.16 0.38 101.72

F11 0.678 0.781 13.19 1.15 26.64 0.42 99.67

F12 0.657 0.788 16.62 1.20 28.45 0.39 100.08

F13 0.667 0.765 12.81 1.14 25.51 0.43 98.78

F14 0.705 0.806 12.53 1.14 27.49 0.35 96.12

F15 0.652 0.793 17.78 1.21 28.72 0.42 100.14

F16 0.695 0.814 14.62 1.17 25.23 0.32 99.89

89

TABLE 10

Evaluation of uncoated pellets formulations (F17 – F31)

Formulations

code

Physical Evaluation Chemical

Evaluation

Bulk Density

(g/cm3)

Tapped

Density

(g/ cm3)

Carr’s Index

(%) Hausner Ratio

Angle of Repose

(θ0) Friability

(%)

Drug content

Analysis (%)

F17 0.683 0.778 12.21 1.14 28.33 0.34 99.56

F18 0.674 0.785 14.14 1.16 27.18 0.28 102.4

F19 0.665 0.810 17.90 1.22 26.67 0.41 100.21

F20 0.702 0.801 12.36 1.14 28.64 0.26 98.77

F21 0.687 0.828 17.03 1.20 26.54 0.45 99.87

F22 0.679 0.789 13.94 1.16 29.22 0.39 98.89

F23 0.669 0.776 13.79 1.16 24.42 0.36 100.63

F24 0.704 0.797 11.67 1.13 26.72 0.44 97.75

F25 0.656 0.749 12.42 1.14 26.56 0.33 98.57

F26 0.710 0.799 11.14 1.13 27.89 0.31 99.97

F27 0.708 0.820 13.66 1.16 28.65 0.29 100.22

F28 0.699 0.811 13.81 1.16 29.31 0.36 101.33

F29 0.677 0.788 11.29 1.16 29.55 0.30 96.99

F30 0.705 0.799 11.76 1.13 27.89 0.28 98.43

F31 0.711 0.802 11.35 1.13 28.11 0.27 97.37

90

TABLE 11

Image analysis of pellet formulations (F1 – F16),(n > 50)

Formulations

code

Area

(A)

Perimeter

(P)

Feret diameter

(mm) Aspect ratio

Sphericity/

Shape factor

F1 377139 2263.74 727.27 1.06 0.99

F2 91420 1106.21 368.27 1.09 0.92

F3 17460 501.48 155.80 1.03 0.97

F4 414636 2335.15 770.27 1.10 0.91

F5 311826 2253.85 678.60 1.11 0.90

F6 497732 2580.06 833.39 1.01 0.99

F7 437306 2531.03 811.61 1.17 0.86

F8 377139 2263.74 727.27 1.06 0.94

F9 78759 1167.05 359.84 1.22 0.82

F10 97466 1161.86 381.08 1.17 0.86

F11 103305 1285.13 416.04 1.33 0.75

F12 311826 2253.85 678.60 1.11 0.90

F13 76267 1092.01 331.20 1.13 0.89

F14 322780 2216.96 690.81 1.11 0.9

F15 95579 1176.62 385.02 1.25 0.80

F16 94213 1194.93 411.81 1.09 0.92

91

TABLE 12

Image analysis of pellet formulations (F17 – F31),(n > 50)

Formulations

code

Area

(A)

Perimeter

(P)

Feret diameter

(mm) Aspect ratio

Sphericity/

Shape factor

F17 329764 2163.04 709.48 1.11 0.90

F18 17753 513.64 165.85 1.10 0.91

F19 379640 2266.84 745.85 1.11 0.90

F20 417434 2412.64 786.05 1.12 0.89

F21 467816 2613.66 816.28 1.08 0.93

F22 337799 2236.12 702.28 1.08 0.93

F23 343189 2221.44 707.14 1.09 0.92

F24 428359 2450.42 787.55 1.14 0.88

F25 333887 2119.19 690.16 1.11 0.91

F26 130788 1365.73 452.99 1.25 0.80

F27 107093 1351.88 399.81 1.15 0.87

F28 364907 2244.95 745.38 1.16 0.86

F29 114341 1281.78 422.30 1.26 0.80

F30 136780 1409.64 470.11 1.25 0.79

F31 72728 1072.25 351.27 1.35 0.74

92

(a)

(b)

FIGURE 5: Stereo micrographs of (a) uncoated plain and (b) matrix ITP pellets

93

(a) (b)

(c) (d)

(e)

FIGURE 6: Stereo micrographs of (a) Ethylcellulose coated F1, (b) F6, (c) F11, (d)

F16 and (e) F21, Itopride pellet formulations

94

(a) (b)

(c) (d)

(e)

FIGURE 7: Stereo micrographs of (a) Eudragit® RS/RL100 coated F1, (b) F6, (c)

F11, (d) F16 and (e) F21, Itopride pellet formulations

95

(a) (b)

(c) (d)

(e)

FIGURE 8: Stereo micrographs of (a) Kollicoat® SR 30Dcoated F1, (b) F6, (c) F11,

(d) F16 and (e) F21, Itopride pellet formulations

96

(a) (b)

(c) (d)

(e)

FIGURE 9: SEM surface images of EC coated (5% dispersion) (a) F1, (b) F6, (c)

F11, (d) F16 and (e) F21, Itopride pellet formulations

97

(a) (b)

(b) (d)

(e)

FIGURE 10: SEM cross-sectional images of EC coated (5% dispersion) (a) F1, (b)

F6, (c) F11, (d) F16 and (e) F21, Itopride pellet formulations

98

(a) (b)

(c) (d)

FIGURE 11: FTIR spectra of (a) Pure ITP, (b) ITP + HPMC (K4M), (c) ITP + HPMC (K15M), (d) ITP + HPMC (K100M), and (e)

ITP + EC (7cps).

99

Continued…...

(e)

FIGURE 11: FTIR spectra of (a) Pure ITP, (b) ITP + HPMC (K4M), (c) ITP + HPMC (K15M), (d) ITP + HPMC (K100M), and (e)

ITP + EC (7cps).

100

FIGURE 12: In-vitro drug release profile of uncoated plain pellet formulations (F1 – F5)

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60

Per

centa

ge

Dru

g R

elea

se

Time (min.)

Plain Pellets (without polymers) in 0.1 N HCL at pH-1.2 (n = 6)

F1

F2

F3

F4

F5

101

(a) (b)

FIGURE 13: In-vitro Drug release profile of uncoated matrix pellet formulations (a) F6 – F18 and (b) F19 – F31, in 0.1 N HCl at pH

1.2

20

30

40

50

60

70

80

90

100

0 2 4 6

Dru

g R

elea

se %

Time (h)

Drug Release from Matrix Pellets (0.1 N HCL

pH-1.2, (n = 6))F6

F7

F8

F9

F10

F11

F12

F13

F14

F15

F16

F17

F1820

30

40

50

60

70

80

90

100

0 2 4 6

Dru

g R

elea

se %

Time (h)

Drug Release from Matrix Pellets (0.1 N HCL pH-

1.2, (n = 6))

F19

F20

F21

F22

F23

F24

F25

F26

F27

F28

F29

F30

F31

102

(a) (b)

(c)

FIGURE 14: In-vitrodrug release profile of pellets formulation F1 coated with (a) 5%, (b) 10% and (c) 15% Ethylcellulose

dispersion

0

20

40

60

80

100

0 2 4 6 8 10 12

Per

centa

ge

Dru

g R

elea

se

Time (h)

F1= Coated with 5% EC dispersion, (n = 6)

(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 2 4 6 8 10 12

Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 2 4 6 8 10 12

Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

103

(a) (b)

(c)


dispersion

0

20

40

60

80

100

-3 2 7 12

Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 2 4 6 8 10 12

Per

centa

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Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 2 4 6 8 10 12

Per

cen

tage

Dru

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elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

104

(a) ( b)

(c)


dispersion

0

20

40

60

80

100

-3 2 7 12

Per

centa

ge

Dru

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elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 2 4 6 8 10 12

Per

centa

ge

Dru

g R

elea

se

Time (h)

F11 = Coated with 10% EC dispersion, (n = 6)

(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 2 4 6 8 10 12Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

105

(a) (b)

(c)


dispersion

0

20

40

60

80

100

0 2 4 6 8 10 12Per

centa

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Dru

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Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

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60

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0 2 4 6 8 10 12Per

centa

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Dru

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Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

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100

0 2 4 6 8 10 12

Per

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(pH-1.2)

(pH-4.5)

(pH-6.8)

106

(a) (b)

(c)


dispersion

0

20

40

60

80

100

0 2 4 6 8 10 12Per

centa

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Dru

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Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

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100

0 2 4 6 8 10 12Per

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Time (h)


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(pH-4.5)

(pH-6.8)

0

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Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

107

(a) (b)

(c)

FIGURE 19: In-vitrodrug release profile of pellet formulation F1 coated with (a) 5%, (b) 10% and (c) 15% Eudragit RL/RS 100

(2:1, w/w) dispersion

0

20

40

60

80

100

0 2 4 6Per

centa

ge

Dru

g R

elea

se

Time (h)

F1 = Coated with 5% Eudragit RL/RS 100, (n = 6)

(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 2 4 6Per

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se

Time (h)

F1= Coated with 15% Eudragit RL/RS 100, (n = 6)

(pH-1.2)

(pH-4.5)

(pH-6.8)

108

(a) (b)

(c)

FIGURE 20: In-vitro drug release profile of pellet formulation F6 coated with (a) 5%, (b) 10% and (c) 15% Eudragit RL/RS 100


0

20

40

60

80

100

0 2 4 6Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 2 4 6Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

109

(a) (b)

(c)



0

20

40

60

80

100

0 2 4 6Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 2 4 6Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

110

(a) (b)

(c)



0

20

40

60

80

100

0 2 4 6Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 1 2 3 4 5 6Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

111

(a) (b)

(c)



0

20

40

60

80

100

0 1 2 3 4 5 6Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)0

20

40

60

80

100

0 2 4 6Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8

Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

112

FIGURE 24: In-vitro drug release profile of pellet formulation F1 coated with 50%

Kollicoat SR 30D dispersion



0

20

40

60

80

100

0 2 4 6

Per

cen

tage

Dru

g R

elea

se

Time (h)

F1 = Coated with 50% Kollicoat SR 30D Dispersion, (n = 6)

(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 2 4 6

Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

113





0

20

40

60

80

100

0 1 2 3 4 5 6

Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

20

40

60

80

100

0 2 4 6

Per

centa

ge

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

114



FIGURE 29: In-vitro drug release profile of Ganaton OD 150 mg tablet (Reference

Product) at pH 1.2, 4.5 and 6.8

0

20

40

60

80

100

0 1 2 3 4 5 6

Per

cen

tage

Dru

g R

elea

se

Time (h)


(pH-1.2)

(pH-4.5)

(pH-6.8)

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 25

Dru

g R

elea

se .%

Time (h)

Ganaton OD 150 mg Tablet, (n = 6)

pH 1.2

pH 4.5

pH

6.8

115

TABLE 13

Drug release kinetics of EC (5% dispersion) coated formulations

Formulations

code First Order Zero Order Higuchi Korsmeyer-peppas Hixson-Crowell Baker-Lonsdale

r2 k1(hr-1) r2 k(hr-1) r2 kH(hr-1/2) r2 n kkp(hr–n) r2 kHC(hr-1/3) r2 kbl(hr-1)

0.1 N HCl pH 1.2

F1 0.997 0.303 0.847 6.085 0.939 30.894 0.992 0.314 47.919 0.990 0.069 0.999 0.038

F6 0.999 0.265 0.865 6.197 0.955 30.267 0.984 0.353 42.661 0.991 0.062 0.993 0.033

F11 0.965 0.175 0.990 7.907 0.973 28.642 0.998 0.755 15.390 0.985 0.047 0.897 0.026

F16 0.996 0.247 0.906 7.252 0.987 30.566 0.988 0.459 33.368 0.997 0.061 0.955 0.034

F21 0.989 0.191 0.961 6.517 0.999 28.239 0.998 0.500 28.231 0.997 0.047 0.971 0.023

Phosphate buffer pH 4.5

F1 0.993 0.322 0.803 6.597 0.922 31.872 0.971 0.337 45.534 0.981 0.077 0.989 0.038

F6 0.996 0.234 0.895 7.037 0.987 29.904 0.992 0.438 34.035 0.986 0.057 0.967 0.031

F11 0.966 0.169 0.991 8.311 0.965 26.664 0.996 0.828 12.998 0.988 0.046 0.892 0.025

F16 0.988 0.231 0.922 8.546 0.989 31.305 0.988 0.472 33.184 0.997 0.061 0.978 0.030

F21 0.982 0.206 0.960 7.847 0.980 30.077 0.994 0.603 23.155 0.996 0.053 0.956 0.027


F1 0.994 0.348 0.756 6.129 0.877 31.531 0.956 0.298 49.896 0.982 0.085 0.986 0.042

F6 0.995 0.249 0.897 6.697 0.979 30.403 0.990 0.407 37.501 0.992 0.059 0.911 0.034

F11 0.967 0.181 0.988 7.961 0.975 29.011 0.998 0.738 16.218 0.988 0.048 0.895 0.027

F16 0.993 0.226 0.931 7.552 0.994 29.727 0.993 0.516 28.754 0.998 0.057 0.967 0.030

F21 0.990 0.204 0.946 6.588 0.996 28.817 0.997 0.468 31.011 0.995 0.050 0.918 0.020

116

TABLE 14

Drugrelease kinetics of Eudragit RL/RS 100 (15% dispersion) coated formulations

Formulations

code

First Order Zero Order Higuchi Korsmeyer-peppas Hixson-Crowell Baker-Lonsdale

r2 k1(hr-1) r2 k(hr-1) r2 kH(hr-

1/2) r2 n kkp(hr–n) r2

kHC(hr-

1/3) r2 kbl(hr-1)

0.1 N HCl pH 1.2

F1 0.959 0.324 0.942 5.082 0.966 29.450 0.975 0.283 54.201 0.982 0.063 0.977 0.044

F6 0.958 0.338 0.866 5.341 0.906 30.179 0.921 0.255 58.300 0.966 0.069 0.955 0.047

F11 0.981 0.365 0.910 5.501 0.949 30.775 0.984 0.239 61.260 0.996 0.073 0.993 0.050

F16 0.982 0.434 0.972 7.362 0.953 35.177 0.979 0.254 62.622 0.995 0.088 0.976 0.048

F21 0.962 0.398 0.950 6.942 0.952 34.815 0.932 0.431 21.505 0.965 0.076 0.966 0.052


F1 0.981 0.343 0.946 4.831 0.961 29.148 0.990 0.230 62.192 0.996 0.068 0.995 0.048

F6 0.947 0.317 0.941 5.738 0.966 30.840 0.964 0.345 47.199 0.976 0.065 0.964 0.044

F11 0.971 0.393 0.828 4.868 0.874 29.424 0.943 0.180 69.300 0.980 0.077 0.975 0.053

F16 0.925 0.339 0.976 7.312 0.981 34.004 0.973 0.356 29.188 0.961 0.071 0.944 0.049

F21 0.934 0.415 0.943 8.085 0.970 36.756 0.967 0.352 52.535 0.970 0.092 0.959 0.063


F1 0.986 0.429 0.834 5.177 0.889 30.413 0.947 0.177 70.186 0.988 0.086 0.983 0.059

F6 0.981 0.411 0.839 5.732 0.894 31.679 0.961 0.209 66.085 0.991 0.084 0.987 0.057

F11 0.974 0.405 0.926 7.064 0.959 34.444 0.972 0.275 59.609 0.992 0.083 0.984 0.058

F16 0.973 0.411 0.966 7.983 0.986 36.044 0.997 0.317 55.384 0.993 0.085 0.988 0.058

F21 0.959 0.320 0.955 5.743 0.979 30.855 0.986 0.313 51.143 0.981 0.065 0.978 0.045

117

TABLE 15

Drug release kinetics of Kollicoat SR 30 D (50% Dispersion) coated formulations

Formulations

code

First Order Zero Order Higuchi Korsmeyer-peppas Hixson-Crowell Baker-Lonsdale

r2 k1(hr-1) r2 k(hr-1) r2 kH(hr-

1/2) r2 n kkp(hr–n) r2

kHC(hr-

1/3) r2 kbl(hr-1)

0.1 N HCl pH 1.2

F1 0.822 0.449 0.964 6.932 0.946 34.261 0.937 1.512 1.028 0.871 0.081 0.870 0.060

F6 0.941 0.6333 0.986 5.783 0.992 32.479 0.993 0.261 62.929 0.976 0.099 0.983 0.073

F11 0.963 0.645 0.976 8.149 0.989 37.783 0.997 0.259 66.826 0.990 0.113 0.990 0.082

F16 0.979 0.633 0.960 8.847 0.978 39.018 0.996 0.918 72.348 0.993 0.114 0.994 0.083

F21 0.877 0.545 0.996 7.823 0.992 36.661 0.994 1.384 1.990 0.925 0.098 0.926 0.072


F1 0.805 0.485 0.966 6.631 0.950 33.887 0.942 1.482 1.071 0.861 0.085 0.864 0.063

F6 0.913 0.606 0.957 5.324 0.963 31.232 0.933 0.264 61.301 0.951 0.094 0.956 0.069

F11 0.991 0.612 0.984 8.149 0.922 33.775 0.961 0.127 82.332 0.986 0.101 0.983 0.074

F16 0.873 0.510 0.985 9.287 0.974 38.993 0.975 1.596 1.195 0.920 0.099 0.910 0.071

F21 0.904 0.413 0.953 5.841 0.951 31.663 0.906 1.039 5.050 0.925 0.073 0.926 0.054


F1 0.835 0.577 0.978 6.717 0.970 34.539 0.957 1.317 2.054 0.893 0.097 0.901 0.072

F6 0.973 0.553 0.963 6.367 0.975 33.603 0.978 0.207 70.845 0.990 0.093 0.990 0.068

F11 0.969 0.612 0.925 6.975 0.941 35.153 0.962 0.139 81.021 0.970 0.102 0.973 0.075

F16 0.884 0.595 0.995 8.789 0.990 38.774 0.990 1.134 5.622 0.933 0.108 0.932 0.079

F21 0.902 0.437 0.993 7.171 0.994 34.628 0.988 0.924 24.219 0.976 0.082 0.971 0.059

118

TABLE 16

Similarity factor (f2) evaluation of ITP pellet formulations with reference to EC5-F11

Comparison f2 Dissolution Profile

0.1 N HCl pH 4.5 pH 6.8

5% EC Coated Formulations

F1 31.98 30.13 31.92 Dissimilar

F6 35.41 39.48 40.81 Dissimilar

F16 43.81 50.02 52.7 Dissimilar in HCl and similar in phosphate buffer

F21 47.59 50.32 47.03 Similar in phosphate (pH 4.5) and dissimilar HCl and

phosphate(6.8)

15% Eudragit RS/RL 100 Coated Formulations

F1 17.06 13.97 16.91 Dissimilar

F6 17.78 16.12 17.71 Dissimilar

F16 16.92 14.27 17.19 Dissimilar

F21 18.71 16.16 18.8 Dissimilar

50% Kollicoat SR 30D Coated Formulations

F1 11.73 9.32 10.91 Dissimilar

F6 10.15 8.24 11.35 Dissimilar

F16 11.93 10.71 12.01 Dissimilar

F21 11.55 9.87 12.63 Dissimilar

119

TABLE 17

Stability studies and shelf life of 5% EC coated pellets formulations at room temperature (25 ± 2°C/60 ± 5% RH)

Study Period Test

5% EC coated pellets formulations

F1 F6 F11 F16 F21

0 Month

Physical appearance No change No change No change No change No change

Drug Content (%)

101.16 100.25 99.67 97.28 99.87

6 Months


Drug Content (%)

99.21 98.94 98.55 95.76 96.37

12 Months


Drug Content (%)

97.87 97.69 97.37 94.68 93.72

Shelf Life (Months) 17.60 22.33 23.70 22.59 19.23

120

TABLE 18

Stability studies and shelf life of 5% EC coated pellets formulations at accelerated temperature (40 ± 2 °C/75 ± 5% RH)

Study Period Test

5% EC coated pellets formulations

F1 F6 F11 F16 F21

0 Month


Drug Content (%)

101.16 100.25 99.67 97.28 99.87

3 Months


Drug Content (%)

98.09 99.32 98.56 96.11 97.67

6 Months


Drug Content (%)

96.72 97.89 97.11 95.18 96.23

Shelf Life (Months) 11.18 16.03 16.27 15.41 15.90

121

TABLE 19

Standard calibration curve (Linearity, accuracy and precision) in mobile phase

Concentration (µg/ml)

Back Calculated Concentration of Itopride HCl (µg/ml) Mean

Conc. SD

% CV as Precision

Accuracy (%)

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

2 1.981 1.938 2.106 2.022 1.908 1.991 0.077 3.889 99.55

1 1.012 0.907 1.109 1.088 0.962 1.016 0.085 8.332 101.56

0.8 0.83 0.821 0.708 0.843 0.766 0.794 0.056 7.074 99.20

0.6 0.62 0.582 0.607 0.612 0.583 0.601 0.017 2.886 100.13

0.4 0.393 0.418 0.379 0.409 0.394 0.399 0.015 3.807 99.65

0.2 0.21 0.189 0.167 0.175 0.206 0.189 0.019 9.910 94.70

0.1 0.101 0.082 0.092 0.077 0.117 0.094 0.016 16.981 93.80

0.05 0.051 0.048 0.047 0.046 0.045 0.047 0.002 4.857 94.80

r2 = 0.9998

122

TABLE 20

Standard calibration curve (Linearity, accuracy and precision) in plasma

Concentration (µg/ml) Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Mean SD %CV

2 190988 191231 191061 190165 191456 190980 490.007 0.257

1 99235 99342 99351 99243 99272 99289 54.711 0.055

0.8 78294 78135 78376 78356 78196 78271 103.561 0.132

0.6 57898 57913 58063 57524 57876 57855 199.047 0.344

0.4 35491 36123 35143 35211 35365 35467 391.097 1.103

0.2 18082 18985 18126 18168 18116 18295 386.717 2.114

0.1 8221 8381 8131 8278 8087 8220 117.149 1.425

0.05 3535 3496 3527 3532 3493 3517 20.403 0.580

R2 0.9991 0.9993 0.9990 0.9990 0.9992 0.9991 - -

123

TABLE 21

Back calculated concentration of Itopride HCl standard calibration curve (Linearity, accuracy and precision) in plasma


Back Calculated Concentration of Itopride HCl (µg/ml)

Mean SD %CV Assay (%)


2 1.975 1.978 1.976 1.967 1.980 1.977 0.005 0.254 98.83

1 1.036 1.037 1.037 1.036 1.036 1.036 0.001 0.054 103.65

0.8 0.821 0.820 0.822 0.822 0.820 0.821 0.001 0.129 102.63

0.6 0.612 0.613 0.614 0.609 0.612 0.613 0.002 0.333 102.16

0.4 0.383 0.389 0.379 0.380 0.382 0.384 0.004 1.043 95.96

0.2 0.196 0.205 0.196 0.197 0.196 0.199 0.004 2.004 99.56

0.1 0.094 0.096 0.093 0.095 0.093 0.094 0.001 1.281 94.35

0.05 0.046 0.045 0.046 0.046 0.045 0.046 0.000 0.462 91.19

124

FIGURE 30a: Calibration curve of ItoprideHCl in plasma

y = 96934x - 933.37

R² = 0.9991

0

50000

100000

150000

200000

0 0.5 1 1.5 2 2.5

PE

AK

AR

EA

CONC. (µg/ml)

Sample 1 Caliberation Curve

Sample 1

Linear (Sample 1)

y = 96839x - 639.2

R² = 0.9993

0

50000

100000

150000

200000

0 0.5 1 1.5 2 2.5

PE

AK

AR

EA

CONC. (µg/ml)


Sample 2

Linear (Sample 2)

y = 97026x - 987.98

R² = 0.999

0

50000

100000

150000

200000

0 0.5 1 1.5 2 2.5

PE

AK

AR

EA

CONC. (µg/ml)


Sample 3

Linear (Sample 3)

y = 96559x - 850.34

R² = 0.999

0

50000

100000

150000

200000

0 0.5 1 1.5 2 2.5

PE

AK

AR

EA

CONC. (µg/ml)


Sample 4

Linear (Sample 4)

125

FIGURE 30b: Calibration curve of Itopride HCl in plasma

y = 97192x - 1084.7

R² = 0.9992

0

50000

100000

150000

200000

0 0.5 1 1.5 2 2.5

PE

AK

AR

EA

CONC. (µg/ml)


Sample 5

Linear (Sample 5)

y = 96910x - 899.12

R² = 0.9991

0

50000

100000

150000

200000

0 0.5 1 1.5 2 2.5

PE

AK

AR

EA

CONC. (µg/ml)

Caliberation Curve of Mean Value

Mean

Linear (Mean)

126

TABLE 22

Intraday and interday accuracy and precision of Itopride HCl in plasma

Selected concentrations in validated method (µg/ml)

0.05μg/ml 0.4μg/ml 0.8μg/ml 2μg/ml

Intraday

Mean (n=5) 0.049 0.391 0.808 1.983

Recovery (%) 98.340 97.737 101.048 99.168

SD 0.002 0.008 0.011 0.011

% CV 4.835 2.141 1.401 0.544

Interday

Mean (n=5) 0.049 0.374 0.800 2.014

Recovery (%) 94.919 96.691 100.751 101.761

SD 0.003 0.012 0.013 0.052

% CV 5.871 3.180 1.677 2.596

127

TABLE 23

Limit of quantification (LOQ) of Itopride HCl in plasma





2 1.985 2.102 1.969 2.014 1.998 2.019 0.052 2.589 100.95

1 1.024 1.017 1.026 1.038 1.016 1.022 0.009 0.859 102.22

0.8 0.813 0.810 0.801 0.807 0.799 0.808 0.006 0.731 101.00

0.6 0.602 0.611 0.613 0.609 0.602 0.609 0.005 0.831 101.49

0.4 0.393 0.361 0.374 0.376 0.367 0.376 0.012 3.164 94.05

0.2 0.194 0.186 0.188 0.190 0.194 0.189 0.004 1.991 94.63

0.1 0.097 0.097 0.095 0.097 0.095 0.097 0.001 1.096 96.59

0.05 0.045 0.051 0.047 0.052 0.050 0.048 0.003 6.033 95.93

128

TABLE 24

Limit of detection (LOD) of Itopride HCl in plasma





0.05 0.051 0.046 0.052 0.052 0.048 0.050 0.003 5.349 99.96

0.03 0.021 0.017 0.021 0.022 0.019 0.020 0.002 10.169 65.56

0.015 0.007 0.005 0.009 0.008 0.011 0.007 0.002 31.944 46.67

0.008 - - - - - - - - -

129

TABLE 25

Absolute analytical recovery

Concentration (µg/ml) Peak Area of Itopride HCl in Plasma

Mean SD %CV Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

2 191088 192231 192061 190165 191356 191380.2 829.160 0.433

0.6 57978 57813 58163 57524 57986 57892.8 240.486 0.415

0.2 18082 18985 18126 18168 18116 18295.4 386.717 2.114

Concentration (µg/ml) Peak Area of Itopride HCl in Mobile Phase

Mean SD %CV Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

2 195877 193921 196051 194324 195231 195080.8 937.505 0.481

0.6 58367 58432 58652 59123 58432 58601.2 311.032 0.531

0.2 19776 19675 19754 19812 19793 19762 53.127 0.269

Mean Peak Area

Concentration (µg/ml) Mean Peak Area in Plasma

Mean Peak Area in Mobile phase

% Recovery

2 191380.2 195080.8 98.1030

0.6 57892.8 58601.2 98.7912

0.2 18295.4 19762 92.5787

130

TABLE 26

Freeze and thaw stability of Itopride HCl

Sample No Low concentration (0.1 µg/ml) High concentration (2µg/ml)

Fresh Sample FT Cycle 1 FT Cycle 2 FT Cycle 3 Fresh Sample FT Cycle 1 FT Cycle 2 FT Cycle 3

1 0.108 0.104 0.098 0.096 1.985 1.945 1.935 1.915

2 0.094 0.093 0.094 0.099 1.992 2.010 1.987 2.026

3 0.097 0.097 0.101 0.092 2.037 1.978 2.046 1.946

4 0.102 0.092 0.094 0.098 1.971 2.043 1.997 2.042

5 0.098 0.102 0.097 0.096 2.013 1.917 1.870 1.930

Mean 0.100 0.098 0.097 0.096 2.000 1.979 1.967 1.972

SD 0.005 0.005 0.003 0.003 0.026 0.050 0.067 0.058

% CV 5.415 5.451 3.047 2.789 1.291 2.534 3.408 2.947

% Accuracy 99.80 97.60 96.80 96.20 99.98 98.93 98.35 98.59

*FT Cycle = Freeze-thaw cycle

131

TABLE 27

Itopride HCl degradation in freeze thaw stability

Sample No

% Degradation in Low concentration (0.1 µg/ml) % Degradation in High concentration (2µg/ml)

FT Cycle 1 FT Cycle 2 FT Cycle 3 FT Cycle 1 FT Cycle 2 FT Cycle 3

1 3.704 9.259 11.111 2.015 2.519 3.526

2 1.064 0.000 -5.319 -0.904 0.251 -1.707

3 0.000 -4.124 5.155 2.896 -0.442 4.467

4 9.804 7.843 3.922 -3.653 -1.319 -3.602

5 -4.082 1.020 2.041 4.769 7.104 4.123

Mean 2.098 2.800 3.382 1.025 1.623 1.362

Gross Mean Degradation (%) 2.760 1.337

*FT Cycle = Freeze-thaw cycle

132

TABLE 28

Long term stability of Itopride HCl in plasma

Sample No

Low concentration (0.1 µg/ml) High concentration (2 µg/ml)

Fresh Sample After 2 Weeks After 3 Weeks After 6 Weeks Fresh Sample After 2 Weeks After 3 Weeks After 6 Weeks

at -20 °C

at -20 °C

1 0.098 0.099 0.093 0.087 1.991 1.978 1.899 1.931

2 0.106 0.097 0.107 0.098 1.989 1.954 1.975 1.911

3 0.094 0.109 0.091 0.096 1.987 2.046 1.896 2.006

4 0.110 0.091 0.097 0.090 2.071 1.943 1.992 1.832

5 0.093 0.094 0.090 0.097 2.011 1.925 1.847 1.812

Mean 0.100 0.098 0.096 0.094 2.010 1.969 1.922 1.898

SD 0.007 0.007 0.007 0.005 0.036 0.047 0.060 0.079

% CV 7.482 6.996 7.232 5.157 1.768 2.388 3.137 4.137

% Accuracy 100.20 98.00 95.60 93.60 100.49 98.46 96.09 94.92

http://www.rapidtables.com/convert/temperature/fahrenheit-to-celsius.htm


133

Table 29

Itopride HCl degradation in long term stability

Sample No

% Degradation in Low concentration (0.1 µg/ml) % Degradation in High concentration (2 µg/ml)

After 2 Weeks After 3 Weeks After 6 Weeks After 2 Weeks After 3 Weeks After 6 Weeks

1 -1.020 5.102 11.224 0.653 4.621 3.014

2 8.491 -0.943 7.547 1.760 0.704 3.922

3 -15.957 3.191 -2.128 -2.969 4.580 -0.956

4 17.273 11.818 18.182 6.181 3.815 11.540

5 -1.075 3.226 -4.301 4.276 8.155 9.896

Mean 1.542 4.479 6.105 1.980 4.375 5.483

Gross Mean Degradation (%) 4.041 3.946

134

FIGURE 31: Chromatograms for linearity curve in mobile phase(Conc. ranges from 0.05 – 2.0 µg/mL)

135

FIGURE 32: Chromatograms for linearity curve in plasma (Conc. ranges from 0.05 – 2.0 µg/mL)

136

TABLE 30a

Details of volunteers participated in pharmacokinetic studies of Itopride HCl 150 mg pellets (EC5-F11) under fed and fasted states

S. No Volunteer

Code Sequence

Age (Yr)

Weight (Kg)

Height (ft.in)

Blood Pressure (mmHg)


Phase I Phase II

1 V1 AB 22 72 5'9" 120/70 105/60

2 V2 BA 21 69 5'6" 120/75 120/80

3 V3 AB 24 70 5'8" 115/65 105/65

4 V4 BA 23 69 5'7" 110/70 117/80

5 V5 AB 23 65 5'5" 115/75 115/75

6 V6 BA 25 70 5'7" 115/65 118/75

7 V7 AB 21 71 5'9" 120/70 120/70

8 V8 BA 22 78 5'10" 120/80 115/80

9 V9 AB 23 66 5'7" 115/75 110/65

10 V10 BA 24 73 5'9" 125/85 120/80

11 V11 AB 21 72 5'8" 120/80 120/70

12 V12 BA 25 67 5'6" 115/70 115/75

A = EC5-F11 (Fasted)

B = EC5-F11 (Fed)

137

TABLE 30b

Details of volunteers participated in pharmacokinetic studies of Itopride HCl 150 mg tablet (Ganaton OD) under fed and fasted states

S. No Volunteer

Code Sequence

Age (Yr)

Weight (Kg)

Height (ft.in)



Phase III Phase IV

1 V1 CD 23 74 5'8" 113/72 115/72

2 V2 DC 24 70 5'7" 122/72 120/75

3 V3 CD 25 71 5'7" 120/70 115/70

4 V4 DC 21 72 5'8" 115/72 118/68

5 V5 CD 22 66 5'6" 120/80 118/72

6 V6 DC 24 72 5'6" 112/65 115/69

7 V7 CD 25 72 5'8" 114/72 113/68

8 V8 DC 22 77 5'11" 110/75 114/72

9 V9 CD 22 67 5'8" 118/72 117/68

10 V10 DC 25 78 5'7" 120/80 118/70

11 V11 CD 22 74 5'9" 118/70 122/72

12 V12 DC 25 68 5'7" 113/65 118/74

C = Ganaton OD (Fasted)

D = Ganaton OD (Fed)

138

TABLE 31

Plasma drug concentration of Itopride HCl 150 mg pellet(EC5-F11)in 12 healthy human volunteers under fed state

Time (hr)

Concentration (µg/ml) Mean SD

V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12

0.5 0.106 0.109 0.097 0.089 0.093 0.091 0.104 0.098 0.107 0.110 0.093 0.114 0.101 0.008

1 0.182 0.183 0.176 0.166 0.151 0.157 0.166 0.163 0.183 0.184 0.159 0.184 0.171 0.012

2 0.289 0.291 0.287 0.281 0.277 0.303 0.315 0.296 0.291 0.293 0.305 0.300 0.294 0.011

4 0.481 0.482 0.478 0.476 0.462 0.456 0.473 0.463 0.482 0.484 0.459 0.490 0.474 0.011

6 0.678 0.681 0.677 0.671 0.667 0.654 0.664 0.655 0.679 0.685 0.657 0.687 0.671 0.012

8 0.789 0.791 0.786 0.768 0.781 0.747 0.764 0.737 0.791 0.792 0.750 0.784 0.773 0.020

12 0.475 0.477 0.474 0.466 0.468 0.465 0.472 0.463 0.476 0.478 0.468 0.495 0.473 0.009

24 0.212 0.214 0.211 0.208 0.202 0.198 0.206 0.205 0.213 0.217 0.201 0.225 0.209 0.008

48 0.082 0.082 0.080 0.086 0.084 0.075 0.085 0.074 0.083 0.084 0.077 0.084 0.081 0.004

139

TABLE 32

Compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg pellet(EC5-F11) under fed state

Table 32:

Volunteer

code Ka Kel α β K12 K21

Cmax

(calc)

Tmax

(calc) AUC Vc Cl T1/2(ka) Tabs T1/2 α T1/2 β T1/2Kel

hr-1 hr-1 hr-1 hr-1 hr-1 hr-1 μg/mL hr mg/L×h L L/h hr hr hr hr hr

V1 0.184 0.102 0.129 0.078 0.006 0.098 0.653 7.321 13.552 108.176 11.068 3.767 18.835 5.392 8.870 6.775

V2 0.184 0.102 0.128 0.078 0.006 0.098 0.655 7.329 13.625 107.975 11.009 3.771 18.856 5.406 8.850 6.798

V3 0.186 0.102 0.129 0.078 0.006 0.099 0.651 7.323 13.477 108.862 11.130 3.734 18.669 5.391 8.830 6.780

V4 0.212 0.095 0.142 0.059 0.018 0.089 0.649 7.079 13.783 114.372 10.883 3.265 16.324 4.870 11.672 7.285

V5 0.205 0.100 0.141 0.065 0.014 0.091 0.650 7.182 13.414 111.550 11.182 3.384 16.918 4.928 10.683 6.915

V6 0.161 0.121 0.143 0.097 0.004 0.114 0.631 7.351 12.518 98.772 11.983 4.316 21.579 4.857 7.121 5.713

V7 0.193 0.098 0.132 0.069 0.010 0.093 0.641 7.198 13.549 112.584 11.071 3.599 17.996 5.235 10.063 7.049

V8 0.191 0.100 0.127 0.080 0.006 0.101 0.626 7.275 12.961 115.605 11.573 3.637 18.183 5.445 8.699 6.924

V9 0.184 0.102 0.129 0.077 0.007 0.097 0.654 7.316 13.631 108.124 11.005 3.759 18.793 5.368 9.025 6.810

V10 0.185 0.101 0.130 0.075 0.008 0.097 0.657 7.318 13.801 107.976 10.869 3.748 18.739 5.318 9.265 6.886

V11 0.188 0.103 0.128 0.080 0.006 0.100 0.632 7.277 12.982 112.654 11.555 3.694 18.472 5.398 8.691 6.758

V12 0.185 0.098 0.127 0.077 0.006 0.100 0.661 7.398 14.051 109.000 10.676 3.740 18.701 5.441 8.978 7.077

Mean 0.188 0.102 0.132 0.076 0.008 0.098 0.647 7.281 13.445 109.637 11.167 3.701 18.505 5.254 9.229 6.814

SD 0.013 0.006 0.006 0.009 0.004 0.006 0.011 0.088 0.427 4.354 0.365 0.252 1.262 0.230 1.141 0.380

%CV 6.653 6.330 4.572 12.222 50.133 6.545 1.748 1.202 3.178 3.971 3.268 6.821 6.821 4.380 12.367 5.581

Min 0.161 0.095 0.127 0.059 0.004 0.089 0.626 7.079 12.518 98.772 10.676 3.265 16.324 4.857 7.121 5.713

Max. 0.212 0.121 0.143 0.097 0.018 0.114 0.661 7.398 14.051 115.605 11.983 4.316 21.579 5.445 11.672 7.285

140

TABLE 33

Non-compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg pellet(EC5-F11) under fedstate

Table 33:

Volunteer

code AUMC MRT Lz AUClast AUCextra AUCtotal AUMClast AUMCextra AUMCtotal HVD T1/2 Lz

mg/L×(h)2 h L/hr mg/L×h mg/L×h mg/L×h mg/L×(h)2 mg/L×(h)2 mg/L×(h)2 h h

V1 214.469 22.628 0.047 13.4019 1.727 15.129 223.056 119.28 342.335 11.663 8.870

V2 216.026 22.580 0.048 13.4685 1.721 15.19 224.211 118.771 342.982 11.711 8.850

V3 212.589 22.393 0.048 13.3159 1.662 14.978 221.096 114.306 335.402 11.669 8.830

V4 238.700 23.465 0.045 13.2238 1.890 15.114 222.363 132.299 354.662 11.823 11.672

V5 220.254 23.190 0.046 13.0857 1.821 14.907 218.775 126.918 345.693 11.359 10.683

V6 184.928 21.992 0.049 12.758 1.524 14.282 209.985 104.099 314.084 12.158 7.121

V7 223.295 23.191 0.046 13.2428 1.844 15.087 221.333 128.549 349.882 12.214 10.063

V8 204.555 21.879 0.050 12.8316 1.488 14.320 211.945 101.364 313.309 12.494 8.699

V9 217.059 22.740 0.047 13.4564 1.759 15.215 224.312 121.679 345.991 11.671 9.025

V10 222.522 22.822 0.047 13.5791 1.785 15.365 227.004 123.644 350.649 11.781 9.265

V11 203.047 22.212 0.049 12.8897 1.582 14.472 213.024 108.419 321.443 12.237 8.691

V12 228.181 22.592 0.048 13.8407 1.746 15.587 232 120.143 352.143 12.582 8.978

Mean 215.469 22.640 0.048 13.258 1.713 14.970 220.759 118.289 339.048 11.947 9.229

SD 13.663 0.484 0.001 0.323 0.126 0.411 6.437 9.639 14.755 0.379 1.141

%CV 6.341 2.138 2.707 2.438 7.372 2.742 2.916 8.148 4.352 3.174 12.367

Min 184.928 21.879 0.045 12.758 1.488 14.282 209.985 101.364 313.309 11.359 7.121

Max. 238.700 23.465 0.050 13.841 1.890 15.587 232.000 132.299 354.662 12.582 11.672

141

TABLE 34

Plasma drug concentration of Itopride HCl 150 mg pellet(EC5-F11) in 12 healthy human volunteers under fasted state

Time

(hr)


V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12

0.5 0.099 0.094 0.089 0.096 0.107 0.097 0.095 0.095 0.096 0.102 0.101 0.099 0.098 0.005

1 0.151 0.165 0.188 0.143 0.164 0.168 0.166 0.159 0.166 0.172 0.174 0.155 0.164 0.012

2 0.298 0.371 0.362 0.265 0.267 0.261 0.271 0.286 0.291 0.297 0.296 0.293 0.297 0.035

4 0.442 0.513 0.522 0.427 0.429 0.421 0.427 0.431 0.442 0.461 0.462 0.452 0.452 0.033

6 0.662 0.692 0.689 0.623 0.658 0.652 0.676 0.678 0.651 0.684 0.686 0.678 0.669 0.020

8 0.598 0.602 0.613 0.545 0.549 0.556 0.574 0.568 0.549 0.577 0.575 0.577 0.574 0.022

12 0.311 0.312 0.297 0.312 0.307 0.318 0.297 0.292 0.292 0.305 0.313 0.305 0.305 0.009

24 0.192 0.147 0.131 0.192 0.197 0.167 0.197 0.199 0.198 0.202 0.198 0.202 0.185 0.024

48 0.065 0.058 0.062 0.061 0.059 0.058 0.068 0.059 0.060 0.062 0.060 0.062 0.061 0.003

142

TABLE 35

Compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg pellet (EC5-F11) under fasted state

Volunteer


Cmax

(calc)

Tmax



V1 0.278 0.110 0.189 0.074 0.026 0.128 0.555 5.927 9.841 139.112 15.242 2.492 12.458 3.668 9.340 6.326

V2 0.308 0.113 0.188 0.059 0.036 0.098 0.612 5.304 10.549 126.119 14.220 2.253 11.264 3.690 11.817 6.148

V3 0.221 0.139 0.202 0.037 0.046 0.054 0.589 5.399 10.436 103.283 14.373 3.139 15.696 3.432 18.655 4.981

V4 0.248 0.132 0.174 0.084 0.016 0.110 0.560 5.933 9.011 126.068 16.646 2.800 13.998 3.974 8.297 5.250

V5 0.248 0.128 0.174 0.083 0.016 0.113 0.571 5.944 9.354 124.862 16.036 2.793 13.963 3.982 8.330 5.397

V6 0.206 0.115 0.199 0.067 0.035 0.116 0.532 6.236 10.524 123.588 14.253 3.361 16.805 3.485 10.338 6.010

V7 0.254 0.129 0.176 0.076 0.019 0.103 0.564 5.878 9.179 126.248 16.343 2.731 13.656 3.942 9.123 5.355

V8 0.251 0.130 0.176 0.076 0.019 0.102 0.561 5.864 9.129 125.922 16.432 2.758 13.791 3.944 9.179 5.312

V9 0.256 0.124 0.175 0.073 0.021 0.104 0.571 5.883 9.582 126.542 15.655 2.707 13.533 3.958 9.458 5.603

V10 0.254 0.131 0.177 0.076 0.019 0.102 0.561 5.850 9.072 126.345 16.534 2.732 13.661 3.922 9.167 5.297

V11 0.255 0.128 0.174 0.073 0.020 0.100 0.556 5.846 9.104 128.732 16.476 2.715 13.577 3.980 9.437 5.416

V12 0.258 0.124 0.176 0.073 0.021 0.103 0.570 5.841 9.517 126.855 15.761 2.690 13.448 3.949 9.527 5.579

Mean 0.253 0.125 0.182 0.071 0.024 0.103 0.567 5.825 9.608 125.306 15.664 2.764 13.821 3.827 10.222 5.556

SD 0.025 0.009 0.010 0.012 0.009 0.018 0.020 0.246 0.592 7.966 0.929 0.278 1.389 0.203 2.809 0.403

%CV 9.929 6.963 5.606 17.596 38.745 17.075 3.453 4.227 6.156 6.357 5.932 10.047 10.047 5.307 27.483 7.259

Min 0.206 0.110 0.174 0.037 0.016 0.054 0.532 5.304 9.011 103.283 14.220 2.253 11.264 3.432 8.297 4.981

Max. 0.308 0.139 0.202 0.084 0.046 0.128 0.612 6.236 10.549 139.112 16.646 3.361 16.805 3.982 18.655 6.326

143

TABLE 36

Non-compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg pellet (EC5-F11) under fasted state

Volunteer



V1 143.167 28.030 0.035 12.786 2.759 15.545 225.508 210.209 435.716 10.544 9.340

V2 162.328 28.954 0.034 12.912 2.969 15.881 229.810 230.000 459.810 9.931 11.817

V3 186.282 28.242 0.035 13.074 2.838 15.912 232.051 217.330 449.382 10.108 18.655

V4 114.270 33.876 0.029 11.493 3.691 15.185 210.553 303.842 514.395 9.502 8.297

V5 120.896 32.897 0.030 11.259 3.440 14.699 203.592 279.960 483.552 9.286 8.330

V6 169.882 31.250 0.032 11.525 3.157 14.682 208.556 250.262 458.818 10.244 10.338

V7 120.232 32.898 0.029 11.623 3.539 15.162 206.018 292.781 498.799 8.527 9.123

V8 119.394 31.526 0.030 11.674 3.274 14.948 205.356 265.890 471.246 8.771 9.179

V9 130.513 31.917 0.030 11.767 3.385 15.152 206.766 276.827 483.593 8.759 9.458

V10 118.233 35.058 0.027 12.228 4.189 16.418 219.841 355.728 575.570 8.633 9.167

V11 120.796 30.963 0.031 12.263 3.289 15.552 217.737 263.786 481.524 8.642 9.437

V12 129.367 22.604 0.045 10.991 1.379 12.369 182.745 96.855 279.599 8.758 9.527

Mean 136.280 30.684 0.032 11.966 3.159 15.125 212.378 253.622 466.000 9.309 10.222

SD 23.866 3.344 0.005 0.678 0.682 1.010 13.626 63.471 68.978 0.731 2.809

%CV 17.513 10.899 14.750 5.669 21.591 6.679 6.416 25.026 14.802 7.849 27.483

Min 114.270 22.604 0.027 10.991 1.379 12.369 182.745 96.855 279.599 8.527 8.297

Max. 186.282 35.058 0.045 13.074 4.189 16.418 232.051 355.728 575.570 10.544 18.655

144

TABLE 37

Plasma drug concentration of Itopride HCl150 mg tablet(Ganaton OD) In 12 healthy human volunteers under fed state

Time

(hr)


V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12

0.5 0.109 0.112 0.107 0.102 0.103 0.115 0.097 0.101 0.106 0.105 0.103 0.102 0.105 0.005

1 0.179 0.182 0.186 0.179 0.171 0.185 0.141 0.147 0.162 0.161 0.157 0.146 0.166 0.016

2 0.291 0.296 0.291 0.283 0.295 0.301 0.243 0.246 0.255 0.252 0.246 0.255 0.271 0.023

4 0.476 0.482 0.485 0.473 0.475 0.491 0.418 0.422 0.421 0.419 0.409 0.424 0.450 0.033

6 0.687 0.691 0.687 0.669 0.658 0.688 0.636 0.639 0.638 0.636 0.631 0.647 0.659 0.024

8 0.771 0.782 0.779 0.767 0.761 0.785 0.747 0.737 0.763 0.761 0.757 0.741 0.763 0.015

12 0.481 0.494 0.488 0.479 0.482 0.496 0.403 0.406 0.424 0.421 0.419 0.408 0.450 0.039

24 0.221 0.227 0.218 0.214 0.221 0.226 0.179 0.183 0.202 0.206 0.201 0.187 0.207 0.017

48 0.071 0.072 0.073 0.074 0.082 0.085 0.071 0.073 0.074 0.065 0.063 0.074 0.073 0.006

145

TABLE 38

Compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg tablet (Ganaton OD) under fed state

Volunteer


Cmax

(calc)

Tmax



V1 0.157 0.118 0.140 0.093 0.005 0.110 0.653 7.462 13.295 95.626 11.283 4.405 22.027 4.946 7.456 5.875

V2 0.185 0.099 0.118 0.088 0.002 0.105 0.660 7.459 13.812 109.907 10.860 3.756 18.780 5.864 7.904 7.015

V3 0.184 0.101 0.120 0.088 0.002 0.104 0.657 7.386 13.553 109.101 11.068 3.760 18.802 5.760 7.873 6.833

V4 0.184 0.101 0.121 0.086 0.003 0.103 0.643 7.417 13.336 111.492 11.247 3.766 18.828 5.743 8.020 6.871

V5 0.186 0.097 0.129 0.077 0.007 0.102 0.641 7.425 13.683 112.793 10.963 3.727 18.636 5.356 9.026 7.132

V6 0.186 0.097 0.128 0.076 0.007 0.100 0.662 7.396 14.125 109.018 10.620 3.733 18.664 5.419 9.112 7.116

V7 0.195 0.096 0.134 0.066 0.012 0.092 0.646 7.178 13.842 112.453 10.837 3.562 17.809 5.189 10.458 7.193

V8 0.172 0.099 0.130 0.020 0.025 0.026 0.575 7.216 13.465 112.997 11.140 4.034 20.172 5.340 34.770 7.031

V9 0.160 0.108 0.139 0.036 0.021 0.047 0.600 7.361 13.446 102.841 11.156 4.342 21.712 4.972 19.077 6.390

V10 0.155 0.118 0.138 0.071 0.008 0.083 0.606 7.498 12.553 101.192 11.949 4.484 22.421 5.038 9.786 5.870

V11 0.146 0.125 0.130 0.113 0.000 0.117 0.602 7.634 12.158 98.431 12.338 4.763 23.817 5.331 6.159 5.530

V12 0.183 0.100 0.125 0.082 0.005 0.102 0.658 7.415 13.800 108.493 10.869 3.790 18.952 5.543 8.492 6.919

Mean 0.174 0.105 0.129 0.075 0.008 0.091 0.634 7.404 13.422 107.029 11.194 4.010 20.052 5.375 11.511 6.648

SD 0.016 0.010 0.007 0.025 0.008 0.027 0.030 0.120 0.558 5.973 0.490 0.388 1.938 0.309 8.016 0.581

%CV 9.123 9.479 5.735 33.466 94.958 29.876 4.682 1.617 4.160 5.581 4.374 9.665 9.665 5.752 69.640 8.733

Min 0.146 0.096 0.118 0.020 0.000 0.026 0.575 7.178 12.158 95.626 10.620 3.562 17.809 4.946 6.159 5.530

Max. 0.195 0.125 0.140 0.113 0.025 0.117 0.662 7.634 14.125 112.997 12.338 4.763 23.817 5.864 34.770 7.193

146

TABLE 39

Non-compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg tablet (Ganaton OD) under fed state

Volunteer



V1 201.973 27.553 0.041 13.063 2.970 16.033 226.403 215.355 441.758 12.071 7.456

V2 217.522 28.022 0.036 13.729 3.069 16.798 239.277 231.443 470.720 12.841 7.904

V3 210.307 28.241 0.040 13.072 3.168 16.240 226.987 231.655 458.642 12.315 7.873

V4 208.280 30.558 0.033 13.672 3.721 17.393 240.527 290.958 531.485 12.233 8.020

V5 223.661 22.343 0.051 12.398 1.684 14.082 200.936 113.688 314.624 11.236 9.026

V6 230.767 25.499 0.046 11.600 2.268 13.867 195.174 158.425 353.599 10.490 9.112

V7 232.926 24.511 0.047 11.699 1.924 13.623 200.912 133.000 333.912 11.492 10.458

V8 344.546 26.962 0.039 11.674 2.386 14.060 203.052 176.036 379.088 11.427 34.770

V9 262.784 26.736 0.039 11.616 2.334 13.950 201.358 171.592 372.950 11.492 19.077

V10 197.566 24.909 0.047 11.546 1.987 13.533 199.182 137.902 337.085 10.989 9.786

V11 180.936 26.789 0.039 11.965 2.417 14.381 207.406 177.860 385.266 11.218 6.159

V12 219.437 22.822 0.046 11.983 1.603 13.586 198.394 111.661 310.055 10.116 8.492

Mean 227.559 26.245 0.042 12.335 2.461 14.795 211.634 179.131 390.765 11.493 11.511

SD 42.152 2.351 0.005 0.831 0.649 1.401 16.735 54.333 69.866 0.779 8.016

%CV 18.524 8.958 12.650 6.735 26.361 9.472 7.907 30.331 17.879 6.779 69.640

Min 180.936 22.343 0.033 11.546 1.603 13.533 195.174 111.661 310.055 10.116 6.159

Max. 344.546 30.558 0.051 13.729 3.721 17.393 240.527 290.958 531.485 12.841 34.770

147

TABLE 40

Plasma drug concentration of itopride HCl 150 mg tablet (Ganaton OD) In 12 healthy human volunteers under fasted state

Time (hr)


Mean SD

V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12

0.5 0.101 0.102 0.107 0.105 0.104 0.103 0.104 0.109 0.104 0.104 0.105 0.103 0.104 0.002

1 0.122 0.121 0.125 0.131 0.132 0.133 0.133 0.136 0.135 0.132 0.134 0.136 0.131 0.005

2 0.282 0.281 0.289 0.281 0.282 0.283 0.285 0.281 0.283 0.285 0.283 0.286 0.283 0.002

4 0.435 0.436 0.434 0.434 0.435 0.436 0.436 0.433 0.434 0.433 0.431 0.436 0.434 0.002

6 0.652 0.651 0.651 0.655 0.656 0.664 0.652 0.661 0.658 0.657 0.655 0.655 0.656 0.004

8 0.609 0.608 0.607 0.603 0.604 0.591 0.603 0.602 0.601 0.597 0.599 0.602 0.602 0.005

12 0.308 0.307 0.318 0.302 0.305 0.300 0.304 0.297 0.302 0.299 0.301 0.302 0.304 0.005

24 0.161 0.16 0.166 0.127 0.126 0.131 0.129 0.126 0.127 0.127 0.126 0.127 0.136 0.016

48 0.062 0.061 0.057 0.061 0.062 0.057 0.061 0.06 0.057 0.065 0.061 0.06 0.060 0.002

148

TABLE 41

Compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg tablet (Ganaton OD) under fasted state

Volunteer


Cmax

(calc)

Tmax



V1 0.275 0.108 0.184 0.070 0.027 0.119 0.564 5.972 10.225 136.404 14.670 2.523 12.613 3.770 9.945 6.445

V2 0.275 0.108 0.184 0.070 0.026 0.120 0.564 5.965 10.158 136.270 14.767 2.522 12.612 3.764 9.834 6.396

V3 0.277 0.106 0.187 0.083 0.018 0.145 0.565 6.056 10.055 140.244 14.917 2.501 12.503 3.698 8.397 6.517

V4 0.248 0.132 0.175 0.084 0.016 0.112 0.565 5.925 9.118 125.020 16.451 2.793 13.967 3.960 8.253 5.267

V5 0.253 0.126 0.174 0.072 0.021 0.099 0.566 5.902 9.416 126.458 15.930 2.737 13.684 3.993 9.668 5.503

V6 0.258 0.126 0.178 0.077 0.020 0.108 0.565 5.854 9.289 127.718 16.148 2.688 13.438 3.902 8.990 5.482

V7 0.256 0.124 0.175 0.072 0.021 0.101 0.565 5.887 9.457 127.668 15.862 2.710 13.552 3.967 9.660 5.579

V8 0.252 0.129 0.175 0.074 0.020 0.101 0.565 5.871 9.273 125.658 16.176 2.750 13.748 3.954 9.331 5.384

V9 0.248 0.132 0.175 0.084 0.016 0.112 0.566 5.912 9.125 124.977 16.438 2.793 13.967 3.962 8.263 5.270

V10 0.262 0.120 0.174 0.060 0.027 0.087 0.563 5.821 9.712 129.053 15.445 2.644 13.220 3.974 11.579 5.792

V11 0.254 0.126 0.174 0.072 0.020 0.100 0.562 5.884 9.340 127.258 16.060 2.733 13.664 3.979 9.570 5.492

V12 0.254 0.126 0.174 0.074 0.020 0.102 0.565 5.873 9.344 126.949 16.053 2.724 13.621 3.980 9.350 5.481

Mean 0.259 0.122 0.177 0.074 0.021 0.109 0.565 5.910 9.543 129.473 15.743 2.676 13.382 3.909 9.403 5.717

SD 0.011 0.009 0.005 0.007 0.004 0.015 0.001 0.063 0.397 5.150 0.636 0.106 0.528 0.103 0.914 0.465

%CV 4.054 7.624 2.714 9.273 18.911 13.559 0.200 1.066 4.157 3.978 4.043 3.943 3.943 2.637 9.719 8.127

Min 0.248 0.106 0.174 0.060 0.016 0.087 0.562 5.821 9.118 124.977 14.670 2.501 12.503 3.698 8.253 5.267

Max. 0.277 0.132 0.187 0.084 0.027 0.145 0.566 6.056 10.225 140.244 16.451 2.793 13.967 3.993 11.579 6.517

149

Table 42

Non-compartmental analysis of different pharmacokinetic parameters of Itopride HCl 150 mg tablet (Ganaton OD) under fasted state

Volunteer



V1 153.724 22.723 0.044 10.323 1.414 11.737 166.576 100.124 266.700 9.092 9.945

V2 151.424 22.543 0.044 10.278 1.380 11.657 165.365 97.423 262.788 9.083 9.834

V3 142.612 21.515 0.047 10.399 1.205 11.604 166.314 83.341 249.655 9.352 8.397

V4 115.784 22.778 0.042 9.678 1.437 11.115 150.351 102.828 253.179 8.923 8.253

V5 127.456 20.684 0.056 9.705 1.106 10.811 150.838 72.779 223.616 8.973 9.668

V6 123.036 21.895 0.044 9.664 1.281 10.945 149.351 90.282 239.633 8.762 8.990

V7 129.175 22.710 0.043 9.733 1.429 11.161 151.447 102.028 253.475 9.049 9.660

V8 122.849 22.643 0.042 9.622 1.412 11.034 148.850 100.992 249.842 8.744 9.331

V9 115.938 21.852 0.044 9.621 1.282 10.903 147.899 90.353 238.252 8.893 8.263

V10 143.636 21.204 0.055 9.710 1.188 10.898 152.353 78.732 231.085 8.868 11.579

V11 125.975 22.821 0.042 9.642 1.441 11.083 149.693 103.235 252.929 8.908 9.570

V12 124.900 22.523 0.043 9.674 1.397 11.071 149.755 99.606 249.362 8.977 9.350

Mean 131.376 22.158 0.046 9.837 1.331 11.168 154.066 93.477 247.543 8.969 9.403

SD 13.118 0.715 0.005 0.302 0.115 0.318 7.345 10.349 12.434 0.164 0.914

%CV 9.985 3.228 10.419 3.069 8.630 2.845 4.767 11.071 5.023 1.832 9.719

Min 115.784 20.684 0.042 9.621 1.106 10.811 147.899 72.779 223.616 8.744 8.253

Max. 153.724 22.821 0.056 10.399 1.441 11.737 166.576 103.235 266.700 9.352 11.579

150

FIGURE 33

Plasma concentration versus time profile comparison of Itopride HCl 150 mg

pellet(EC5-F11) in 12 healthy volunteers under fasted and fasted state

0

0.2

0.4

0.6

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V1)

Fed State

(V1)

0

0.2

0.4

0.6

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted State

(V2)

Fed State

(V2)

0

0.2

0.4

0.6

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L

Time (h)

ER ITP Pellet

Fasted state

(V3)

Fed state (V3)

0

0.2

0.4

0.6

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted State

(V4)

Fed state

(V4)

0

0.2

0.4

0.6

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V5)

Fed state

(V5)

0

0.2

0.4

0.6

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V6)Fed state

(V6)

151

Continued…

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V7)Fed state

(V7)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V8)Fed state

(V8)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V9)Fed state

(V9)

00.10.20.30.40.50.60.70.8

0 20 40 60Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted

state

(V10)

Fed state

(V10)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V11)

Fed state

(V11)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

ER ITP Pellet

Fasted state

(V12)

Fed state

(V12)

152

FIGURE 34

Plasma concentration versus time profile comparison of Itopride HCl 150 mg tablet

(Ganaton OD) in 12 healthy volunteers under fed and fasted state

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted

state (V1)Fed State

(V1)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted State

(V2)

Fed State

(V2)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted

state (V3)

Fed state

(V3)

0

0.2

0.4

0.6

0.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted State

(V4)

Fed state

(V4)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V5)

Fed state (V5)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V6)

Fed state

(V6)

153

Continued…..

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V7)

Fed state

(V7)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V8)

Fed state (V8)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V9)

Fed state

(V9)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V10)

Fed state

(V10)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V11)

Fed state

(V11)

00.10.20.30.40.50.60.70.8

0 20 40 60

Pla

sma

conce

ntr

atio

n o

f

Ito

pri

de

HC

l (µ

g/m

L)

Time (h)

Ganaton OD Tablet

Fasted state

(V12)

Fed state

(V12)

154

Figure 35

Mean plasma concentration versus time profile comparison of Itopride HCl 150 mg

pellets (EC5-F11) and Itopride HCl 150 mg tablet (Ganaton OD) in 12 healthy

volunteers under fed and fasted state

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

0 10 20 30 40 50Ito

pri

de

HC

l P

lasm

a C

on

cen

trat

ion

(µ

g/m

L)

Time (h)

Mean Plot of ER ITP Pellet

Fasted State

Fed State

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

0 10 20 30 40 50

Ito

pri

de

HC

l P

lasm

a C

once

ntr

atio

n (

µg/m

L

Time (h)

Mean Plot of Ganaton OD Tablet

Fasted State

Fed State

155

TABLE 43

Mean pharmacokinetic log transformed parameters of extended release Itopride HCl 150 mg encapsulated pellets (EC5-F11) versus

Itopride HCl 150 mg tablet (Ganaton OD), with geometric mean ratios at 90% CI

Parameters

Fed State Fasted State

ER Pellets

(EC5-F11)

ER Tablet

(Ganaton OD) Geomean Ratio (CI)

ER Pellets

(EC5-F11)

ER Tablet


Cmax,µg/mL 0.647 0.634 1.0215 (0.9974 – 1.0463) 0.567 0.565 1.0034 (0.9848 – 1.0223)

Tmax,h 7.281 7.404 0.98343 (0.9736 – 0.9933) 5.825 5.910 0.9848 (0.958 – 1.0125)

AUC0-t,mg/L×h 13.258 12.335 1.0133 (0.9847 – 1.0428) 11.966 9.837 0.9986 (0.9844 – 1.0129)

AUC0-∞,mg/L×h 13.445 13.422 1.0223 (0.9912 – 1.0543) 9.608 9.543 0.9994 (0.9816 – 1.0176)

AUMC, mg/L×(h)2 215.469 227.559 0.9577 (0.8654 – 1.060) 136.280 131.376 1.0286 (0.9486 – 1.1154)

AUMCtotal, mg/L×(h)2 339.048 390.765 1.0470 (0.9981 – 1.0983) 466.000 247.543 0.9981 (0.9525 – 1.0460)

Cl, L/h 11.167 11.194 0.9979 (0.96891 – 1.0278) 15.664 15.743 0.9941 (0.9669 – 1.022)

Vc,L 109.637 107.029 1.0251 (0.9930 – 1.0582) 125.306 129.473 0.9665 (0.9207 – 1.0146)

T1/2Kel, h 6.814 6.648 1.0272 (0.9689 – 1.089) 5.556 5.717 0.9723 (0.9273 – 1.0194)

MRT, h 22.640 26.245 1.0242 (0.9980 – 1.0511) 30.684 22.158 0.9987 (0.9669 – 1.0315)

156

TABLE 44

Mean pharmacokinetic non-log transformed parameters of extended release Itopride HCl 150 mg encapsulated pellets (EC5-F11) versus

Itopride HCl 150 mg tablet (Ganaton OD), with geometric mean ratios at 90% CI

Parameters

Fed State Fasted State

ER Pellets

(EC5-F11)

ER Tablet


ER Pellets

(EC5-F11)

ER Tablet


Cmax,µg/mL 0.647 0.634 1.0206 (0.9971 – 1.0442) 0.567 0.565 1.0039 (0.985 – 1.0229)

Tmax,h 7.281 7.404 0.9833 (0.9734 – 0.9932) 5.825 5.910 0.9856 (0.9588 – 1.0125)

AUC0-t,mg/L×h 13.258 12.335 1.0124 (0.9840 – 1.0409) 11.966 9.837 0.9987 (0.9844 – 1.0130)

AUC0-∞,mg/L×h 13.445 13.422 1.0214 (0.9906 – 1.0521) 9.608 9.543 0.9995 (0.9817 – 1.0174)

AUMC, mg/L×(h)2 215.469 227.559 0.9468 (0.8352 – 1.0585) 136.280 131.376 1.0373 (0.9475 – 1.1271)

AUMCtotal, mg/L×(h)2 339.048 390.765 1.0453 (0.9974 – 1.0933) 466.000 247.543 0.9977 (0.953 – 1.0425)

Cl, L/h 11.167 11.194 1.0024 (0.9724 – 1.0325) 15.664 15.743 0.9949 (0.9678 – 1.0221)

Vc,L 109.637 107.029 1.0243 (0.9933 – 1.0554) 125.306 129.473 0.9678 (0.9222 – 1.0134)

T1/2Kel, h 6.814 6.648 1.0250 (0.9690 – 1.0810) 5.556 5.717 0.9717 (0.9246 – 1.0188)

MRT, h 22.640 26.245 1.0236 (0.9974 – 1.0498) 30.684 22.158 0.9984 (0.9670 – 1.0299)

157

TABLE 45

Comparative bioavailability of Itopride HCl 150 mg pellets (EC5-F11) to that of Itopride HCl 150 mg tablet (Ganaton OD)

Conditions Formula %age Relative

bioavailability

Fed state

% Relative bioavailability =𝑀𝑒𝑎𝑛 [𝐴𝑈𝐶]𝐴

𝑀𝑒𝑎𝑛 [𝐴𝑈𝐶]𝐵

Where,

A = test product (ER coated ITP pellet)

B = reference product (Ganaton OD tablet)

% Relative bioavailability =13.445

13.422

100.171

Fasting state

% Relative bioavailability =9.608

9.543

100.681

158

5.5 Chromatograms

159

FIGURE 36

Chromatograms of plasma samples of Itopride HCl 150 mg Pellets (EC5-F11) in 12 healthy volunteers under fed state

VOLUNTEER 1 (V1)

160

VOLUNTEER 2 (V2)

161

VOLUNTEER 3 (V3)

162

VOLUNTEER 4 (V4)

163

VOLUNTEER 5 (V5)

164

VOLUNTEER 6 (V6)

165

VOLUNTEER 7 (V7)

166

VOLUNTEER 8 (V8)

167

VOLUNTEER 9 (V9)

168

VOLUNTEER 10 (V10)

169

VOLUNTEER 11 (V11)

170

VOLUNTEER 12 (V12)

171

FIGURE 37

Chromatograms of plasma samples of Itopride HCl 150 mg pellets (EC5-F11) in 12 healthy volunteers under fasted state

VOLUNTEER 1 (V1)

172

VOLUNTEER 2 (V2)

173

VOLUNTEER 3 (V3)

174

VOLUNTEER 4 (V4)

175

VOLUNTEER 5 (V5)

176

VOLUNTEER 6 (V6)

177

VOLUNTEER 7 (V7)

178

VOLUNTEER 8 (V8)

179

VOLUNTEER 9 (V9)

180

VOLUNTEER 10 (V10)

181

VOLUNTEER 11 (V11)

182

VOLUNTEER 12 (V12)

183

FIGURE 38

Chromatograms of plasma samples of itopride HCl 150 mg tablet (Ganaton OD) in 12 healthy volunteers under fed state

VOLUNTEER 1 (V1)

184

VOLUNTEER 2 (V2)

185

VOLUNTEER 3 (V3)

186

VOLUNTEER 4 (V4)

187

VOLUNTEER 5 (V5)

188

VOLUNTEER 6 (V6)

189

VOLUNTEER 7 (V7)

190

VOLUNTEER 8 (V8)

191

VOLUNTEER 9 (V9)

192

VOLUNTEER 10 (V10)

193

VOLUNTEER 11 (V11)

194

VOLUNTEER 12 (V12)

195

FIGURE 39

Chromatograms of plasma samples of Itopride HCl 150 mg tablet (Ganaton OD) in 12 healthy volunteers under fasted state

VOLUNTEER 1 (V1)

196

VOLUNTEER 2 (V2)

197

VOLUNTEER 3 (V3)

198

VOLUNTEER 4 (V4)

199

VOLUNTEER 5 (V5)

200

VOLUNTEER 6 (V6)

201

VOLUNTEER 7 (V7)

202

VOLUNTEER 8 (V8)

203

VOLUNTEER 9 (V9)

204

VOLUNTEER 10 (V10)

205

VOLUNTEER 11 (V11)

206

VOLUNTEER 12 (V12)

207

6. DISCUSSION

208

DISCUSSION

There are more than 800 large volume pharmaceutical formulations producing

companies in Pakistan, catering more than 90% of the national pharmaceutical products

need. Since the last decades, national pharmaceutical industry has been growing, but still

there is only few pharmaceutical manufacturing units producing specialized dosage

forms, especially there are less companies involved in manufacturing of controlled

release drug delivery system. The primary goal of the controlled release system is to

deliver drug at a constant rate to obtain zero order release kinetics (Sisinthy, S. et al.,

2015). In order to obtain a cost effective, efficient extended release system, type of

excipients and method of manufacturing should be selected wisely. Pelletization can be

used effectively to formulate extended release dosage form. The size of pharmaceutical

pellets ranges from 0.2 to 1.5 mm (Asnani, A.J. &Parashar, V.V., 2013) and to control

the drug release for extended period of time by formulating matrix pellets, is a very

difficult task because of their greater surface area.

The objective of the present study was to prepare extended release matrix pellets of ITP

by extrusion and spheronization technique as it produces pellets with high sphericity,

narrow particle size distribution, compact structure, and low hygroscopicity. The

influence of different concentrations and viscosity grades of polymers on to the release

of highly water soluble drug (ITP) was also investigated. The matrix pellets were

developed using various polymers of different viscosity grades, like HPMC K4M (4000

cps), K15M (15000 cps), K100M (100000 cps) and EC (7cps) as mentioned in table 2

and 3. To obtain controlled release system, use of hydrophilic and hydrophobic

polymers are very common (Rao, M.R. &Shelar, S.U., 2015) like among hydrophilic

polymers, HPMC of different viscosity (K4M- 4000cps, K15 M- 15000 cps and K100M

– 100000 cps) and copolymers of acrylic and methacrylic acids such as Eudragit RL,

RS, and NE, have been used widely by several researchers as matrix formers for

extended release system (Alekseev, K.V. et al., 2012). Comparatively the controlled

release pellets can be developed easily by applying coating solution or dispersion of

different polymers like ethyl cellulose and Eudragit® RL:RS, to the surface of

pellets(Asnani, A.J. &Parashar, V.V., 2013; Gamlen, M., 1985; Goodman, G., 1985).

Kollicoat SR30D dispersion (consisted of polyvinyl acetate stabilized with povidone and

209

sodium lauryl sulfate) is also used as coating material for the formation of pH-

independent controlled release devices (Alekseev, K.V. et al., 2012). Ethyl cellulose; a

hydrophobic polymer which is non-toxic and inert, has been used extensively as a

coating material for tablets and granules as well as matrix forming agent for sustained

release dosage forms (Desai, J. et al., 2006; Songsurang, K. et al., 2011). Coating with a

hydrophobic polymer such as ethyl cellulose (EC) over drug-loaded pellets, offers a

reliable method of regulating the drug release. Drug release from the coated pellets

occurs either through diffusion process and/or pores created by dissolution of soluble

components incorporated into the coat(Yuen, K.H. et al., 1993).Therefore, in present

study five different formulations (F1, F6, F11, F16 and F21) consisted of lowest

percentage (10%) of each viscosity grade polymers, were selected for coating to control

the initial fast release of highly water soluble drug from the matrix pellets. The coating

was performed using EC (10 cps), Eudragit RL/RS 100 (2:1 w/w) and Kollicoat SR 30D

dispersion (table 4, 5 and 6), to extended drug release up to the desired time period.

The physicochemical properties of pellet formulations were evaluated by image analysis,

scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR)

and drug content analysis. Moreover, in-vitro drug release rates were determined in

different dissolution media (HCl pH 1.2, Phosphate buffer pH 4.5and 6.8) at 37 ± 0.5○C.

The drug release profiles were studied by applying different kinetic models. Stability

studies of 5% EC coated pellet formulations were also conducted at room temperature

(25 ○C/60% RH) and at accelerated temperature (40 ○C/75% RH) for period of 12

months as per ICH guidelines and data were analyzed using Minitab v 17.1.0.

The pharmacokinetic evaluation of optimized ITP pellet formulation EC5-F11was

performed to assess the in-vivo performance and then compared with reference brand

Ganaton OD tablet (Abbott, Pakistan). For the estimation of Itopride HCl in plasma, an

HPLC method was developed and validated.

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6.1. Physical and chemical evaluation of uncoated ITP pellet

formulations

ITP pellet formulations were evaluated for bulk density, tapped density, compressibility

index, Hausner ratio, angle of repose, and friability and all the results of these parameters

are given in Table 9 and 10. The values of Hausner ratio (1.12 – 1.22) indicated that flow

properties were good and passable, whereas values of angle of repose (24.42 – 29.55)

exhibited excellent flow characteristic. The percentage friability (0.26 – 0.61%) was

found within the prescribed limit (< 1%) and no considerable difference was observed

between plain (without polymer), EC (7 cps) and HPMC of different viscosity grade

(K4M, K15M, and K100M) pellets. The percent content of ITP in each formulation (F1 –

F31) was found within the prescribed limit in the range of 96.12 – 102.4% showing

uniformity of drug content, as given in Table 9 and 10. Gupta, N. V. et al., reported

good flow property of Olanzapine matrix pellets, through the determination of angle of

repose, tapped density, bulk density and Carr’s index, and the friability was also

observed within the Pharmacopeial limit (Gupta, N.V. et al., 2011).Rao, P. S. et al., also

reported the values of angle of repose of Itopride pellets composed of Ethylcellulose

N50, i.e.in between 22.78 to 33.82 showing good flow behavior, while the drug content

in each formulation was ranged from 99.16 – 99.59%(Rao, P.S. et al., 2014).

6.2. Image analysis

Stereomicroscope was used to take images of uncoated and coated pellets (Figure 5, 6, 7

and 8) and these images were analyzed to calculate different physical parameters. The

results of the microscopy showed that nearly all pellet formulations were spherical in

shape. Table 11 and 12 shows the Feret diameter, aspect ratio (AR) and sphericity/shape

factor of the ITP pellets prepared by extrusion/spheronization method. For analysis, more

than 50pellets (n>50) from each formulation were subjected to measurement. The effect

of type and concentrations of polymer on Feret diameter of pellets was found negligible.

The value of sphericity or shape factor was found in the range of 0.75–0.99, and that of

aspect ratio in the range of 1.01–1.33(closer to 1), indicating that the pellets are spherical

in shape. Cespi et al., in 2007, Dukic et al., in 2007, Thommes & Kleinebudde, in 2006,

and Chatlapalli & Rohera, in 1998also used Stereomicroscope for image analysis of

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pellets and parameters analyzed were roundness/sphericity and radius ratio /aspect ratio.

Dukic, A.et al., reported the aspect ratio values of Theophylline anhydrous starch based

pellets 1.11–1.25, whereas the shape factor ranged from 0.45–0.58. Cespi, M.et al.,

explained that for a sphere the radius ratio is 1 and if the value deviates, the sphericity

reduces accordingly with the surface irregularity. Cespi, M. et al., worked on

viscoelasticity of pellets and reported the value of roundness as 1.076–1.093, while,

values of radius ratio ranged from 1.071to1.135.Thommes&Kleinebudde, determined the

ratio between the maximum Feret diameter and the Feret diameter perpendicular to the

maximum Feret diameter and reported the mean Feret diameter as 1.08–1.46 (Cespi, M.

et al., 2007; Dukic, A. et al., 2007; Thommes, M. &Kleinebudde, P., 2006: Chatlapalli,

R.R. &Rohera, B.D., 1998).

Podczeck, F.et al., demonstrated that pharmaceutical pellets should have an aspect ratio

less than 1.1(Podczeck, F. et al., 1999). In the present study, the sphericity of pellets was

influenced by varying the amount of granulating liquid. Chopra, S.et al. also observed

that the consistency of the formulation ingredients greatly influenced the shape and

surface properties of pellets(Chopra, S. et al., 2013).

6.3. Scanning electron microscopy (SEM)

The morphology and surface properties of EC coated (5% dispersion) pellet formulations

F1, F6, F11, F16, and F21 are shown in Figure 9a, 9b, 9c, 9d and 9e, sequentially, which

revealed that the pellets coated with EC were smooth and spherical in shape exhibited

smooth film coating. The cross-sectional picture of coated pellet formulations at higher

magnifications showed that the particles formed good matrix structure by coalescence as

shown in Figure 10a, 10b, 10c, 10d and 10e, of formulations F1, F6, F11, F16 and F21,

respectively. Rao, P. S. et al., and Shah, Sanjay et al., also conducted morphological

studies of Itopride Hydrochloride pellets using SEM at different magnifications, to

evaluate sphericity of pellets (Rao, P.S. et al., 2014; Shah, Sanjay et al., 2012).

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6.4. Fourier transform infrared spectroscopy (FTIR)

The drug-polymers compatibility studies of EC coated pellet formulations were

conducted to detect any possible interaction between pure drug (ITP) and excipients used

in the formulations, using FTIR spectroscopy. The recorded infrared spectra of pure ITP

and coated pellets formulations indicated that no drug-polymers interaction occurred.

The characteristic peaks of the pure ITP at3278.60 cm-1, 3225.75 cm-1(NH asymmetric

and symmetric str.), 2937.30 cm-1(C – H str. of aromatic nucleus), 2618.56 cm-1 (C – H

str. of methyl group), 1649.05 cm-1(C = O bending), 1577.91, 1544.46, 1508.80 cm-1 (C

= C aromatic str.),1226.78 cm-1 (C – N aromatic str.) 1012.13 cm-1 (C – O aromatic str.),

respectively are shown in Figure 11a. FTIR spectrums of HPMC with ITP are shown in

Figure11b, 11c, and 11d. The peak at 3278.60 to 3225.75 cm-1was due to OH vibrational

stretching. The symmetric stretching mode for methyl and hydroxypropyl group was

found to be 2941.60 cm-1. The maximum of the absorption band due to hydroxyl group

of EC, (3277.21cm-1) was detected. Figure 11e shows similar behavior for the ITP/EC

mixture. All these results indicated that no chemical interaction occurred between ITP

and polymers used in formulations. Ahmed, S. et al., conducted compatibility study of

Itopride HCl with HPMC, ethylcellulose and microcrystalline cellulose using FTIR and

no drug-excipients interaction was reported (Ahmed, S. et al., 2013). Rao, P. S. et

al.,also perfomed compatiblity study of Itopride HCl with HPMC and EC and the

spectra indicated that the drug was compatible with the excipients used (Rao, P.S. et al.,

2014).

6.5. In vitro drug release studies

It was found that plain pellet (without polymer) formulations (F1 – F5) released more

than 90% drug within 30 minutes and all matrix uncoated pellet formulations (F6 – F31)

released drug within 6 hours. All uncoated matrix pellet formulations of ITP containing

HPMC (F6 – F20) exhibited swelling except formulations containing EC (F21 –F25),

however, none of the formulation disintegrated during the entire dissolution period.

The in-vitro drug release profile of reference product (Ganaton OD 150 mg Tablet,

Abbott Pakistan), was also evaluated in HCl buffer (pH 1.2) and phosphate buffer (pH

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4.5 and 6.8), shown in Figure 29, 18%, 41%, 60%, 92% and > 97% Itopride HCl was

released at 1, 3, 6, 12 and 16 h, respectively.

6.5.1. Effect of HPMC viscosity grade and concentration on drug release

In the present study, the influence of three viscosity grades of HPMC polymers, i.e.,

K4M (4000cps), K15M (15000cps), and K100M (100000cps) on ITP release were

evaluated. Figure 13a shows 90% drug released by F-6 (10%K4M), 85% by F-11 (10%

K15M) and 90% by F-16 (10% K100M) at 1 h, whereas the drug release of F-7, F-12

and F-17 is also given in the same figure 13a, showing 73% drug release at 1 h for F-7,

93% for F-12, and 85% for F-17 containing 15% K4M, K15M, and K100M,

respectively. The comparison of drug release of F-8, F-13, and F-18 is also shown in

Figure 13a, indicating 85% drug released at 1 h for F-8, 70% for F-13 and 75% for F-18

containing 20% K4M, K15M, and K100M, correspondingly. The drug release

comparison of F-9, F-14 and F-19 is given in figure 13a and 13b, revealing 75% drug

released at 1 h for F-9 and F-14, while 73% for F-19 containing 25% K4M, K15M and

K100M. The drug release comparison of F-10 (30% K4M), F-15 (30% K15M), and F-20

(30% K100M) indicated 90%, 66%, and 56% drug release at 1 h. The drug release

comparison of F-26 (30% K100M), F-27 (40%, K100M) and F-28 (50%, K100M) is

shown in figure 13b, showing 75% drug release for F-26, 72% for F-27 and 68% for F-

27. HPMC was used in viscosity range of 4000– 100,000 cps in formulations F-6 to F-20

and F-26 to F-31(Table 2 and 3). Cumulative % ITP release vs time up to 12 h showed

inverse relationship for formulations containing F-6 to F-20 and F-26 to F-31. Qazi. F.et

al., also observed a similar trend for HPMC concentration and viscosity grade, and

reported that the viscous gel layer of HPMC increased both the diffusion path length as

well as resistance to diffusion(Qazi, F. et al., 2013). Jamzad, S. and Fassihi, R., also

employed HPMC of different viscosity grades in different ratio for glipizide CR matrix

formulation (Jamzad, S. &Fassihi, R., 2006). However, in the current study maximum

viscosity grade (K100M; 100,000 cps) and concentration (50%) failed to control the

release of ITP up to 12 h. This fast release of highly water soluble drug was due to the

rapid diffusion of dissolved drug through the hydrophilic gel network.

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6.5.2. Effect of ethyl cellulose concentration

Formulations F-21 to F-25 were composed of EC(Premium 7 cps) in the concentration

range of 10–30%. F-21 (10%), F-22 (15%), F-23 (20%), F-24 (25%) and F-25 (30%)

exhibited 90% drug release within 3 h for F-21, F-22 and within 4 h for F-23, F-24 and

F-25, respectively. Formulation F-29 (10% HPMC - K100M and 10% EC – 7cps), F-30

(15% HPMC - K100M and 15% EC – 7cps) and F-31 (20% HPMC K100M and 20% EC

– 7cps) showed more than 70% drug release within 1 h and more than 90% in 3 h

(Figure13b). Like HPMC, EC also failed to retard the ITP release up to the desired time

period.

6.5.3. Effect of EC coating on pellets formulations

The burst release of drug revealed that matrix uncoated pellet formulations containing

HPMC and EC alone or in combination, failed to control the drug release up to the

targeted time period even at a higher viscosity grade and concentration of polymer, i.e.,

50% HPMC and 30% EC. Therefore, five formulations F1 (without polymer), F6, F11,

F16 and F21 containing 10% HPMC K4M, K15M, K100M, and EC (7 cps), were

selected for coating in order to overcome the initial burst release through formation of

thin film around the plain and matrix pellets. These selected pellet formulations were

coated with different levels (5, 10 and 15%) of ethylcellulose (Premium 10 cps) coating

dispersion (Table 4). Therefore, EC dispersions was prepared in solvent comprised of

IPA and water in a ratio of 9:1 (Table 4) providing a viscosity of 77 cp. Viscosity grade

is determined by the molecular weight of the ethyl cellulose polymer chain. In the

current work, EC (10 cps) was selected because a higher molecular weight film forms a

greater number of entanglements with reduced free volume, resulting in a decrease in

drug transport through polymer layer. This coating dispersion formed strong film with

good adhesion around the cores containing 10% polymer. Thus, release of ITP was

successfully extended up to 12 h by the application f 5% EC coating on matrix pellets.

Whereas, application of 10% EC coating dispersion, excessively controlled the drug

release for longer time and released up to 64 – 70% in 12 h. Similarly, application of

15% EC coating dispersion further reduced the drug release rate i.e. up to 25 – 33% in 12

h. This may be due to formation of water insoluble ethyl cellulose film causing decreased

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permeability in water as the film thickness increased. Elias, N.M.et al., demonstrated that

higher viscosity grades of ethyl cellulose (10 cps) tend to produce stronger and more

durable films, thus used in drug microencapsulation. They also used IPA to prepare

coating dispersion of EC (Elias, N.M. et al., 2012).Dow Chemical, and Regdon Jr, G.et

al., tested that EC is practically insoluble in water but dissolves in aliphatic alcohols like

isopropyl alcohol (IPA) (Dow Chemical, 2013; Regdon Jr, G. et al., 2012). Pearnchob,

N. and Bodmeier, R., also used ethylcellulose micronized particles for pellets coating

and triethyl citrate as plasticizer (Pearnchob, N. &Bodmeier, R., 2003a).

6.5.4. Effect of Eudragit RS/RL 100coating on pellet formulations

The selected five (F1, F6, F11, H16, and F21) pellet formulations were also coated with

different levels (5, 10 and 15%) of Eudragit® RS/RL100 (2:1, w/w) coating dispersion.

Eudragit RS/RL 100 (2:1) dispersions was prepared in solvent consisted of IPA and

water in a ratio of 9:1 (Table 5). The influence of the levels of Eudragit®RS/RL100 on

the release properties of Itopride HCl was determined from their dissolution profiles,

which are given in Figure 19 – 23. Application of 5, 10 and 15% coating dispersions,

were unable to control the initial burst release and did not control the drug release up to

the targeted time period of 12 h. Almost 57 – 77% drug was released within in 1h and

more than 95% drug release within 6 h from the mentioned coated formulations. Kucera,

S.A.et al., has used a blend of Eudragit®RS/RL 30D (95:5) with 15% TEC as a

plasticizer to control the drug release, which extended the release up to 12 h (Kucera,

S.A. et al., 2008). El-Malah, Y. & Nazzal, S., used Eudragit®RS 30 as a release

controlling film because of its ability to form a continuous film upon spraying (El-

Malah, Y. &Nazzal, S., 2008). Pearnchob, N. and Bodmeier, R., used Eudragit RS 30 in

the coating of pellets for extended release, showing that drug release extended up to 6 h

(Pearnchob, N. &Bodmeier, R., 2003b).

6.5.5. Effect of Kollicoat SR 30D coating on pellet formulations

The above mentioned five formulations were also coated with 50% dispersion of

Kollicoat® SR 30D. This polymer was also unable to control the initial fast release and

approximately 76% drug was released within 1h. The coating levels could not be

increased as the dispersion of Kollicoat® SR 30D was water based and it was very

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difficult to coat pellets (high levels) in a simple conventional coating pan. Andreazza,

I.F. & Ferraz, H.G.,, in 2011 and Shao, Z.J. et al., in 2002 used Kollicoat® SR 30D in

the coating of highly water soluble containing pellets and demonstrated that polymer

effectively controlled the drug release from the systems. The rate of release of drug may

be controlled if the level of the coating layer is increased. Kollicoat SR 30D is an

aqueous dispersion of polyvinyl acetate (pH-independent polymer) which can precisely

delay the drug release from dosage, irrespective of the type of dosage form (BASF,

2012).

6.5.6. Effect of dissolution medium on drug release

Effect of dissolution medium on drug release of ethylcellulose, Eudragit RS/RL 100 and

Kollicoat SR 30D coated pellet formulations were also evaluated.

6.5.6.1. Ethylcellulose coated pellet formulations

The in vitro drug release profiles of F1, F6, F11, F16 and F21, (each coated with 5%,

10%, and 15% of EC)-, in HCl buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8) are

shown in Figure14a, 14b, and 14c, to Figure 18a, 18b, and 18c, respectively. The drug

release profile of 5% EC coated F1 indicated that 19%, 60%, 80%, and 96%, whereas,

F6 showed that 17%,48%, 73%, and 98% Itopride HCl was released at 1, 3, 6, and 12 h,

respectively. However, it was observed that the drug release rate was more controlled in

F11 i.e., 8% (1h), 26% (3h), 52% (6h), and >94%(12h). Pellet formulations F16 and

F21showed 14%, 38%, 70% and >96%, and 20%, 45%, 66%, and >95% drug release at

same time points.

The drug release profile of 10% EC coated F1 showed that 8%, 16%, 29%, and 65%,

while F6 indicated that 11%, 19%, 32% and 66% Itopride HCl was released at 1, 3, 6,

and 12 h, respectively. However, the drug release rate in F11 was observed as 7% (1h),

15%(3h), 31%(6h) and 64%(12h). For the same time intervals of 1, 3, 6 and 12hpellet

formulation F16 released 12%, 22%, 40% and 68% drug and F21 released the drug 10%,

23%, 35% and 64%.

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The percentage drug release of 15% EC coated F1 was0.9%, 2%, 8% and 25%, while, F6

showed 1%, 2%, 10%, and 27% Itopride HCl release at the same time intervals. Pellet

formulation F11 and F16 also exhibited excessive control on drug release i.e. 0.8%, 2%,

9%, and 25%, and 1%, 3%, 11% and 29% at 1, 3, 6 and 12 h, sequentially. However, in

case of F21 only 1%, 3%, 10% and 32% drug was released. The results revealed that the

release of ITP from EC-coated pellets was not significantly influenced by the pH of the

dissolution media. However, the release rate was found slightly low at pH 4.5. Muschert,

S. et al., also observed pH-independent release profile from ethyl cellulose-coated pellets

(Muschert, S. et al., 2009). Furthermore, a pH-independent release rate was also found in

ethyl cellulose controlled release matrix pellets(Kojima, M.&Nakagami, H., 2002).

Whereas Liu, Y.et al., reported difference in release profile in different dissolution

media, for ethyl cellulose (10 cps)-coated pellets, especially great difference was

observed at pH 4.5 (Liu, Y. et al., 2012).

6.5.6.2. Eudragit RS/RL 100 coated pellet formulations

The in vitro drug release profiles of F1, F6, F11, F16 and F21, each coated with 5%,

10%, and 15% of Eudragit RS/RL 100 (2:1, w/w), in HCl buffer (pH 1.2) and phosphate

buffer (pH 4.5 and 6.8) are shown in Figure19a, 19b, and 19c, and exhibited by Figure

23a, 23b, and 23c, respectively. The drug release profile of 5% Eudragit RS/RL 100

coated F1 indicated 79%, 91%, and >97% drug release, whereas, F6 showed 77%, 95%,

and >97% Itopride HCl release at 1, 3, and 6 h, respectively. However, the percentage

drug release of F11 was 77%(1h), 96%(3h), and >97%(6h). Pellet formulations F16 and

F21 showed 80%, 96%, and 99%, and 83%, 98%, and 99% drug release at same time

intervals.

The drug release of 10% Eudragit RS/RL 100 (2:1, w/w)coated F1 showed 68%, 82%,

and 97%, F6 indicated 71%, 93%, and 96% Itopride HCl release at 1, 3, and 6 h,

sequentially. However, the drug release from F11 was 74%, 93%, and >97%, F16 was

75%, 96%, and 99% and F21 was70%, 91%, and >96% at 1, 3, and 6 h, respectively.

The pellet formulation F1 coated with 15% Eudragit RS/RL 100 (2:1, w/w) exhibited

60%, 77%, and >98%, while, F6 formulation coated with the same concentration of

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polymer, showed57%, 73%, and > 95% drug release at 1, 3, and 8 h, respectively. Pellet

formulation F11 and F16 exhibited 62%, 78%, and >96%, and 58%, 76%, and >98%

drug release at 1, 3, and 8 h, correspondingly. However, in case of F21,56%, 74%, and

99% drug was released at same time interval.

The results indicated that the release of ITP from Eudragit RS/RL 100 coated pellets was

independent of the dissolution media pH. However, all the three coating levels were

unable to control the initial burst released, unlike EC coated pellets. Ahmed, I.et al.,

reported a difference in dissolution profile of Eudragit RS/RL 30 D (2:1) coated

Ambroxol Hydrochloride pellets and also indicated that the mean dissolution time values

increased with the increase in concentration of Eudragit RS 30 D., which may be due to

its lower permeability (Ahmed, I. et al., 2008). Pearnchob, N. and Bodmeier, R., also

applied Eudragit RS 30 to formulate extended release pellets of Propranolol

Hydrochloride, showing different dissolution profile (Pearnchob, N. &Bodmeier, R.,

2003b).

6.5.6.3. Kollicoat SR 30D coated pellet formulations

The release profiles of pellet formulations F1, F6, F11, F16 and F21 each coated with

Kollicoat SR 30D (50% dispersion) in HCl buffer (pH 1.2) and phosphate buffer (pH 4.5

and 6.8) are shown in Figure 24–28. The drug release profile of F1 exhibited 76%, 87%,

and 96%, whereas, F6 showed 77%, 93%, and 99% drug release at 1, 3, and 6 h,

respectively. However, the drug release of F11 was 73%, 91%, and 98%.Pellet

formulations F16 and F21 showed 70%, 88%, and >95%, and 73%, 87%, and >97% drug

release at same time points consecutively. Contrarily, according to the findings reported

by Andreazza, I.F. & Ferraz, H.G., in 2011 and Shao, Z.J.et al., in 2002, who formulated

extended release pellets of highly water soluble drugs i.e. Ascorbic acid and

Diphenhydramine Hydrochloride, e the initial release was comparatively better

controlled in pellets coated with the same polymer. Dashevsky, A.et al., coated

Verapamil Hydrochloride (a water soluble drug), pellets with Kollicoat SR 30D for

extended release, then applied top coating with Kollicoat MAE 30 DP as enteric

polymer. In 0.1 N HCl, both the polymers were insoluble and the drug release was

decreased with rise in amount of polymers, while in phosphate buffer (pH 6.8), the

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release was found better, since the polymer was soluble at this pH,. (Dashevsky, A. et

al., 2004).

6.6. Drug release kinetics studies

Different kinetic models, i.e., First order, Zero order, Higuchi, Hixson–Crowell, and

Baker–Lonsdale were applied to interpret the release kinetics of Itopride from the plain

and matrix-coated pellets (F1, F6, F11, F16 and F21), using DD Solver, an add-in

program for Microsoft excel. Zhang, Y.et al., also used DD Solver for the dissolution

data profile comparison and modeling (Zhang, Y. et al., 2010).

6.6.1. Kinetics of EC coated pellets

Table 13 shows the details of release kinetic data of 5% EC coated plain and matrix

formulations in HCl buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8). EC coated

formulation F1 (without polymer), Matrix coated formulation F6 (HPMC K4M), F16

(HPMC K100M) and F21 (Premium EC, 7cps) showed linear relationship when First

order kinetic model was applied (R2 = 0.982 – 0.999). These formulations exhibited

concentration-dependent drug release in HCl buffer (pH 1.2) and phosphate buffer (pH

4.5 and 6.8). However, EC matrix-coated formulation F11 (HPMC K15M) showed

concentration-independent drug release as highest linearity was observed when Zero

order model was applied (R2 = 0.988 – 0.991).The in vitro release of all coated pellet

formulations was best explained by the Higuchi kinetic model with coefficient of

correlation (R2) values in HCl buffer pH 1.2 ranges from 0.939 – 0.999, in phosphate

buffer pH 4.5 from 0.922 – 0.989, and pH 6.8 from 0.877 – 0.996. The values of

coefficient of correlation indicate that drug diffuses at a comparatively slower rate with

the increase in diffusion distance. When Hixson Crowell model was applied, all

formulations presented linearity (R2 = 0.981– 0.998) in all three pH values, indicating

that dissolution changes as a function of time progressively, as the change in surface area

and diameter occurs. Similarly, the coefficient of correlation obtained when the Baker

and Lonsdale model was plotted was in the range of 0.911–0.999 except for F11 (R2 =

0.892 – 0.897). Rao, P.S.et al., formulated sustained release pellets of Itopride HCl by

EC coating and reported a highest linearity values (R2= 0.94 – 0.98) with first order

release kinetic, followed by Higuchi model (Rao, P.S. et al., 2014). Chowdary, K.P.R.

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and Dana, S.B., also analyzed EC coated microcapsules of diclofenac according to first

order, zero order, Higuchi and Peppas kinetics models. The correlation coefficient (R2)

values in the first order model were higher when compared to the zero order model in all

the formulations (Chowdary, K.P.R. &Dana, S.B., 2011).

6.6.2. Kinetics of Eudragit RS/RL100 Coated Pellets

Table 14 shows the release kinetic data of 15% Eudragit RS/RL 100 coated plain and

matrix formulations in HCl buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8).

Eudragit RS/RL 100 coated formulations F1, F6, F11, F16 and F21 were best fitted to

First order kinetic model(R2 = 0.925 – 0.986), indicating concentration-dependent drug

release in HCl buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8). Formulation F16

showed concentration-independent drug release as showed compliance with Zero order

kinetics (R2 = 0.966 – 0.976) and other formulations exhibited poor fit to zero order

kinetics. All the Eudragit coated pellet formulations was also best explained by Higuchi

kinetics model with linearity values at all three pH, and ranges from 0.874 – 0.986.

When Hixson Crowell model was applied, all formulations presented a good linearity (R2

= 0.961– 0.996), A linear relationship was also found when the Baker-Lonsdale model

was plotted (R2 = 0.944–0.995). Ahmed, I.et al., also explained a poor fit to zero order

kinetics at lower concentration of Eudragit RS 30D, when Ambroxol Hydrochloride

pellets were subjected to release kinetic analysis (Ahmed, I. et al., 2008). Vasilevska,, K.

et al., coated diltiazem pellets with Eudragit RS/RL 100 (70:30) for controlled release

purpose and applied first order (R2 = 0.9966), Higuchi (R2 = 0.9875), Hixson Crowell

(R2 = 0.9949) and Weibull function (R2 = 0.9967), showing no significant differences

between the correlation coefficients values (Vasilevska, K. et al., 1992).

6.6.3. Kinetics of Kollicoat SR 30D Coated Pellets

The release kinetic data of Kollicoat SR 30D coated plain and matrix formulations, in

HCl buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8) are presented in Table 15.

Kollicoat SR 30D coated pellet formulations F6 and F11 followed first order with

coefficient of correlation values between 0.913 – 0.991, while formulation F1, F16 and

F21 were poorly fitted to first order (R2= 0.805 – 0.904). Whereas all the coated

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formulations were best fitted to zero order kinetics (R2= 0.957 – 0.996), followed by

Higuchi kinetics model (R2= 0.922 – 0.994), then Hixson Crowell model (R2= 0.920 –

0.993), except formulation F1 (R2= 0.861 – 0.893). A linear relationship was also found

when the Baker-Lonsdale model was plotted (R2 = 0.910 – 0.994), except for F1 (R2 =

0.864 – 0.901). Li-Fang, F.et al., used Korsmeyer–Peppas model to analyze drug release

mechanism from dosage forms and the release data indicated that the correlation

coefficient (r2) was greater than 0.99 in all cases(Li-Fang, F. et al., 2009).

6.7. Drug release mechanism

The drug release mechanism was determined by plotting first 60% of the in vitro release

data in the Korsmeyer–Peppas model. The correlation coefficients for all the 5% EC

coated formulations were high (R2 = 0.956 –0.998) enough to evaluate the drug release

behavior, in HCl buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8).The release

exponent (n) and kinetic rate constant (k) are presented in Table 13. The release

exponent n for the EC coated matrix formulations F11, F16 and F21 ranged from 0.459

to 0.828, indicating non-Fickian diffusion mechanism (anomalous transport), whereas,

for F1 and F6 the value of release exponent n ranged from 0.298 to 0.438, showing

Fickian diffusion mechanism. A similar release mechanism was found by Liu, Y.et al.,

who reported the value of n between 0.45 and 0.89 for ethyl cellulose coated pellets (Liu,

Y. et al., 2012). Chowdary, K.P.R.& Dana, S.B., analyzed the release data of EC coated

diclofenac microcapsules according to Peppas equation, and the release exponent (n) was

ranged from 0.4740–0.867 for all the formulation s exhibiting that the release

mechanism was non Fickian in nature (Chowdary, K.P.R. &Dana, S.B., 2011). Rao, P. S.

et al., reported the values of release exponent (n)for different formulations between

0.513–0.589 showing non- Fickian diffusion(Rao, P.S. et al., 2014). The release

exponent (n) was a function of polymer used and the physico-chemical property of the

drug molecule itself(Bose, A. et al., 2013). When a film-coated pellet comes into contact

with dissolution medium, it absorbs the medium and swells. The drug present inside the

polymer coat dissolves in the medium which permeates through the polymer coat. The

polymer coat swells until an equilibrium is established between the elastic strength of the

polymer and the hydration that promotes diffusion. Permeation of dissolution medium

continues until the core is saturated due to the difference in osmotic pressure and

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diffusion control transport through the film coat in both directions. Permeability of the

film coat is further increased by the expansion of the polymer network due to swelling

induced by the medium influx (Liu, Y. et al., 2012).

The coefficients of correlation (R2) values for all the 15% Eudragit RS/RL 100 coated

pellet formulations were ranged from 0.921–0.997, showing a good linearity, in HCl

buffer (pH 1.2) and phosphate buffer (pH 4.5 and 6.8). The release exponent (n) and

kinetic rate constant (k) are given in Table 14. The values of ‘n’ for all the Eudragit

RS/RL 100 coated formulations were 0.177 – 0.283 (F1), 0.209 – 0.345 (F6), 0.180 –

0.275 (F11), 0.254 – 0.356 (F16) and 0.313 – 0.431 (F21), indicating Fickian diffusion

mechanism. Ghaffari, A.et al. ,reported the value of n between 0.385 and 0.402 for

Eudragit RS 30D/RL 100 coated Theophylline pellets, showing Fickian diffusion

mechanism (Ghaffari, A. et al., 2006). Thriveni, M.et al., reported that Tizanidine

Hydrochloride pellets coated with a mixture of HPMC E-5, Ethyl cellulose N-50 and

Eudragit L-100, followed zero order release kinetics and exhibited non-Fickian case-II

diffusion, (Thriveni, M. et al., 2013).

The coefficients correlation (R2) values for all the Kollicoat SR 30D coated formulations

were ranged from 0.933 –0.997, showing a good linearity, in HCl buffer (pH 1.2) and

phosphate buffer (pH 4.5 and 6.8). Table 15 shows the release exponent (n) and kinetic

rate constant (k).The values of n for Kollicoat SR 30 D coated formulations F6, and F11,

ranged from 0.127 to 0.264, indicating Fickian diffusion mechanism, whereas, the n

value for F1, F16 and F21, ranged from 0.918 to 1.596, proving Super Case-II Transport.

Li-Fang, F.et al., reported the release exponent(n) values ranged from 0.95 to 1.33 for all

chitosan/Kollicoat SR30D coated tablets of theophylline, indicating ‘Super case II’

transport, since the values of exponent was greater than 0.89(Li-Fang, F. et al., 2009).

6.8. Model independent method

Similarity test (f2) was conducted for selected formulations with reference to EC5-F11

(5% EC coated pellets, containing 10% HPMC-K15M). The reason of selection of EC5-

F11 as reference, was its good compliance with the all pharmaceutical quality attributes.

Formulation F16 coated with 5% EC, demonstrated similarity in dissolution profiles with

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EC5-F11, in phosphate buffer pH 4.5 (f2 = 50.02), and 6.8 (f2 = 52.70), whereas F21

showed similarity in phosphate buffer pH 4.5 (f2 = 50.32), only. Formulation F1 and F6

were dissimilar with EC5-F11 (Table 16). However, 15% Eudragit RS/RL100 coated and

50% Kollicoat SR 30D dispersion coated formulations F1, F6 F16 and F21 showed

dissimilarity in dissolution profile with EC5-F11 (Table 16). Shoaib, M.H.et al., have

used similarity factor (f2) for dissolution profile comparison of different drugs and

provided a detailed description of model independent and dependent approaches (Shoaib,

M.H. et al., 2010).

6.9. Stability studies

5% EC coated ITP pellet formulations, F1, F6, F11, F16 and F21, were subjected to

stability studies to assess their physical appearance, and drug content. All the

formulations, in rubber-capped amber glass bottles were stored at 25 ± 2°C/60 ±5% RH

for 12 months and at 40 ± 2°C/75 ±5% RH for 6 months as per ICH guidelines(ICH,

2003b; Rao, P.S. et al., 2014; Singh, B.N., 2007).The percentage drug content and

physical appearance were evaluated at 0, 3, and 6 months for formulations stored at

accelerated temperature (Table 17) while for formulations stored at room temperature,

evaluated at 0, 6 and 12 months (Table 18). The results showed no significant change in

physical appearance, and drug content and found within the ICH specification at both

conditions. Barrier coating made with ethyl cellulose in organic solvent remained stable

overtime(Dow Chemical, 2013).The stability data were analyzed by using software

Minitab (version 17.1.0), to determine the shelf-life of the pellet formulations. The

results indicated that the pellets formulations with lower acceptance limit of90% of label

claim could provide a shelf-life of17.6 (F1), 22.33 (F6), 23.70(F11), 22.59 (F16) and

19.23 months (F21), when stored at room temperature. Similarly, a shelf-life of

formulations, stored at accelerated temperature, were also evaluated, and found to be

11.18, 16.03, 16.27, 15.41 and 15.90 months for formulations F1, F6, F11, F16 and F21,

sequentially. Thriveni, M.et al., also conducted stability study of SR pellets formulation

of Tizandine HCl containing HPMC, EC and Eudragit, as per ICH guidelines under

accelerated (40±2°C/75±5% RH), intermediate (30±2°C/65±5% RH) and long term

(25±2°C/60±5% RH) conditions and observed no change in drug content, disintegration

time and dissolution profiles(Thriveni, M. et al., 2013). Mutalik, S. et al., also

conducted accelerated stability study (40°C ± 2 °C/ 75 ± 5% RH) for six months

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according to ICH guidelines, for HPMC K4M containing Aceclofenac SR tablets and

found no significant change in drug content, color and other physical parameters

(Mutalik, S. et al., 2007). In current work the stability analysis report (output) was

generated by Minitab 17 (See Appendix – II).

6.10. Bio-analytical method validation

There are various HPLC methods that have been developed and reported by different

researchers for the estimation of Itopride HCl in various biological matrices. Some of the

reported HPLC methods have been developed using UV detector(Sisinthy, S. et al.,

2015; Yehia, S.A. et al., 2013: Penumajji, S. & Bobbarala, V., 2009)and some methods

with fluorescence detection have been reported (Cho, K. et al., 2010; Ma, J. et al.,

2009). Lee, H. W.et al., evaluated Itopride HCl in human plasma by using liquid

chromatography coupled to tandem mass spectrometric detection(ESI-MS/MS)(Lee,

H.W. et al., 2007). Bose, A. et al., also developed simultaneous determination of Itopride

HCl and Domperidone in human plasma by HPLC coupled to LC-MS detection (Bose,

A. et al., 2009).

The HPLC method used in the present study is a modification of the method by Yehia, S.

A. et al.(Yehia, S. et al., 2012). Liquid-liquid extraction technique was used in the

extraction of Itopride HCl from the biological fluid using diethyl ether. Sisinthy, S. et

al., also used diethyl ether for the extraction of Itopride HCl in plasma (Sisinthy, S. et

al., 2015).The HPLC system (LC-10 AT VP), UV Detector (SPD-10A VP),

Communication Bus Module (CBM 102), and HPLC column Phenomenex -C18 (250

mm, 4.6 mm, 5 µm), along with guard column, were used in this proposed method. The

mobile phase used was modified and consisted of acetonitrile and 0.05 M KH2PO4 (pH

4.0) in the ratio of 70:30 % (v/v). The pH of buffer was adjusted using 1.0 M ortho-

phosphoric acid. For the analysis, the system was run at a flow rate of 1 ml/min and the

detection wavelength was carried out at 258 nm. The mean retention time of Itopride

HCl and internal standard (Moxifloxacin) was found 7.28 and 8.56 minutes, respectively.

The plasma samples were treated with diethyl ether for the extraction of Itopride HCl,

before injecting in to HPLC system.

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The method was validated according to FDA guidelines for Bio-analytical Method

Validation (FDA, 2001), and the method was found to be linear, accurate, precise and

sensitive with good stability results. The linearity showed in a concentration ranged from

0.05 µg/ml to 2µg/ml with mean coefficient of correlation (r2) of 0.9998 in mobile phase

and 0.9991 in plasma (Table 19, 20 & 21 and Figure 30a & 30b).Accuracy in mobile

phase and plasma were found ranged from 93.80 to 101.56% and 91.19 to 103.65 %,

respectively. The intraday accuracy and precision on four different concentrations 0.05,

0.4, 0.8 and 2 µg/ml was found to be 98.340, 97.737, 101.048 and 99.168%, and

percentage CV values were 4.835, 2.141, 1.401 and 0.555%, respectively (Table 22).

The interday accuracy and precision values of four different concentrations 0.05, 0.4, 0.8

and 2 µg/ml on three different consecutive days were 94.919, 96.691, 100.751, and

101.761%, and percentage CV values were, 5.871, 3.180, 1.677 and 2.596%,

respectively (Table 22). The lower limit of quantification (LOQ) was 0.05 µg/ml,

validated by spiking five samples of concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1

and 2µg/ml in plasma and results of accuracy were found in the range 94.05 – 102.22%

(Table 23). Five samples of each concentration 0.05, 0.03, 0.015 and 0.008µg/ml, were

spiked in plasma for the lower limit of detection (LOD). It was found that concentration

0.008 µg/ml was not detected while concentration 0.015, 0.03 and 0.05µg/ml was

detected with an accuracy of 46.67, 65.56 and 99.96%, respectively (Table 24). The

mean % recovery values of each three concentrations 0.2, 0.6 and 2µg/ml, were 92.579,

98.791 and 98.103%, respectively(Table 25).Freeze and thaw stability were determined

by spiking five samples of 0.1 µg/ml (low concentration) and five samples of 2 µg/ml

(high concentration), run freshly and for three freeze thaw (FT) cycles (Table 26).The

%CV values for fresh samples of low and high concentrations were found as5.415 and

1.291%, while, % accuracy of low and high concentration were found as 99.80 and

99.98%, respectively. The % CV and % accuracy values for FT cycle 1, 2 & 3 of low

concentration (0.1 µg/ml) were found to be 5.451, 3.047, 2.789 and 97.60, 96.80,

96.20%, sequentially. The % CV and % accuracy values for FT cycle 1, 2 & 3 of high

concentration (2 µg/ml) were found to be 2.534, 3.408, 2.947 and 9.93, 98.35, 98.59%,

respectively (Table 26). Itopride HCl degradation in freeze thaw cycles was also

estimated with five samples of low (0.1 µg/ml) and high concentration (2 µg/ml). The

gross mean degradation in low concentration (0.1 µg/ml) was found 2.760% while in

high concentration (2 µg/ml) it was 1.337% (Table 27). Long term stability was

determined by study of fresh sample of same concentration during their storage for two,

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three and six weeks at -20 °C. The % CV for fresh sample of low and high

concentrations was 7.482 and 1.786, and % accuracy values were 100.20 and 100.49%,

respectively. The %CV in low concentration (0.1 µg/ml) after two, three and six weeks

was 6.996, 7.232 and 5.157%, and % accuracy was 98.00, 95.60 and 93.60%,

respectively. For high concentration (2 µg/ml) % CV after two, three and six weeks were

2.388, 3.137, and 4.137% and% accuracy were 98.46, 96.09 and 94.92%, respectively

(Table 28). The gross mean degradation of Itopride HCl in long term stability in low (0.1

µg/ml) and high (2 µg/ml) concentration were found to be 4.042 % and 3.946%,

respectively (Table 29).

There are several researchers, who have used costly, complex and/or time consuming

methods for the detection of Itopride in human plasma, but the proposed method is

comparatively less time consuming and is cost effective. Choi, et al., developed and

validated method for HPLC coupled with the API 5000, MS/MS detector for

simultaneous determination of Revaprazan 200mg and Itopride 150 mg in plasma using

Hypersil gold column (150 x 2.1 mm, 5 µm) and mobile phase composed of 10 mM

ammoniumacetate: acetonitrile (20:80 v/v, containing 0.1% formic acid). (Choi, H.Y. et

al., 2012). Rasheed, S.H.et al., developed and validated RP-HPLC method for the

simultaneous determination of Rabeprazole sodium and Itopride HCl in a tablet dosage

form, using Phenomenex C-18 column (250 mm × 4.6 mm, 5 μm). The mobile phase

consisted of mixture of 50 mM ammonium acetate: methanol (20:80v/v), pH 4.5 adjusted

with acetic acid, and set at a flow rate of 1.3 mL/min with PDA detection at 286 nm. The

% accuracy of Rabeprazole and Itopride were in the range of 98 – 100% and 99 – 100%.

(Rasheed, S.H. et al., 2011). Umamaheswari, D.et al., also developed and validated RP-

HPLC method for the quantitative determination of Itopride HCl and Rabeprazole

sodium. The chromatographic conditions were as, HPLC column Phenomenex Luna C-

18 (250 x 4.6mm, 5μ), mobile phase Methanol: Water (80:20), flow rate of 1ml/min,

injection volume 20 µl, and detection wavelength 268 nm (Umamaheswari, D. et al.,

2010).


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6.11. Pharmacokinetics analysis and comparative bioavailability study of

ER Itopride HCl encapsulated pellets and tablets:

In the present study, the pharmacokinetics of the developed and optimized extended

release ITP pellet formulation EC5-F11 was determined in 12 healthy male volunteers

coded as, V1–V12. The EC5-F11 was selected on the basis of its best physicochemical

characteristics, release kinetics and highest shelf-life (16.27 months at accelerated

temperature and 23.70 months at ambient temperature). The brand Ganaton OD Tablet

(150 mg) manufactured by Abbot Laboratories, was selected for comparative

bioavailability. The study was conducted in the Department of Pharmaceutics, Faculty of

Pharmacy, University of Karachi, after approval from the Hamdard University Ethical

Review Board (ERB-16-09) and Board of Advanced Studies University of Karachi. The

volunteers were screened initially before the study (Table 30). The study design was

single center, single dose, open labeled, randomized, two treatments, two sequence, four

periods, crossover study with the washout period of two weeks. The optimized

formulation, EC5-F11 was selected for comparative bioavailability study according to

FDA guidelines (FDA, 2003). The EC5-F11 (encapsulated pellets) and reference product

(Ganaton OD Tablet) were taken orally with 250 ml of water in fasted (overnight fasting

of 10 hours) and fed conditions and the blood sample of 5 ml was collected at time 0,

0.5, 1, 2, 4, 6, 8, 12, 24 and 48 hours. The design of sampling time was based on

reported elimination half-lives. The EC5-F11 (Pellets, Test Product) under fasted

condition was coded as “A” and under fed condition it was coded as “B”. Similarly, the

Ganaton OD (Tablet, Reference Product) under fasted state was coded as “C” and under

fed state it was coded as “D”. The plasma drug concentration of each volunteer was

analyzed by validated HPLC method (Table 19–29, Figure 33–35). The

pharmacokinetics parameters of the both products were determined by using Kinetica

(Ver 5.1, Thermoelectron Corp., USA), using Macro-extravascular method two

compartmental and non-compartmental models (Table 32 – 42).

Comparative bioavailability parameters of EC5-F11 (test) and Ganaton OD (reference)

such as Cmax, Tmax, and AUC0-∞ were compared using two-way analysis of variance

(ANOVA). Schirmann’s two one-sided t test was also used for this comparative analysis

and significant difference was considered at p< 0.05. The two formulations were

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considered to have similar bioavailability if 90% confidence interval (CI) for the

reference and test falls between 0.80 to 1.25, after logarithmic and non-logarithmic

transformation of pharmacokinetics parameters (Cmax, Tmax, and AUC0-∞.).

6.11.1. Compartmental and non-compartmental analysis

The plasma drug concentration vs time profile was pharmacokinetically analyzed for

both compartmental and non-compartmental parameters by using Kinetica®, version 5.1,

Thermoelectron Corp., USA (Presented in Table 32, 33, 35, 36, 38, 39, 41and 42). The

data was fitted into oral two compartmental model based on the graph (Figure 33 – 35),

in which extrapolated disposition line was below the distribution nose. Gannu, R. et al.,

Brunner, M.et al.,and Pellock, J.M.et al.,have been used Kinetica in the calculation of

pharmacokinetic parameters of different drugs (Gannu, R. et al., 2007; Brunner, M. et

al., 2003; Pellock, J.M. et al., 2001). Jia, J.et al.,and Shoaib, M.H.et al.,used

compartmental model (Jia, J. et al., 2011; Shoaib, M.H. et al., 2008) and Yoon, S.et al.,

Cho, K. et al.,Ma, J. et al.,Sahoo et al., Lee, H. W. et al.,and Singh, S. S. et al.,used non-

compartmental model for analysis of different drugs (Yoon, S. et al., 2014; Cho, K. et

al., 2010; Ma, J. et al., 2009; Sahoo, B.K. et al., 2009; Lee, H.W. et al., 2007; Singh,

S.S. et al., 2005).

6.11.1.1. Cmax, Tmax, and AUC

The mean Cmax calculated of EC5-F11 under fed condition was 0.647 ± 0.011 μg/mL and

under fasted condition was 0.567 ± 0.020 μg/mL, whereas, the mean Cmax calculated, of

Ganaton OD Tablet under fed state was 0.634 ± 0.031μg/mL, and under fasted condition,

was 0.565 ± 0.001 μg/mL. Calculated mean Tmax of EC5-F11 under fed and fasting

conditions were 7.282 ± 0.088 h, and 5.825 ± 0.246 h, while that of Ganaton OD Tablet,

was 7.404 ± 0.120 h (fed) and5.910 ± 0.063 h (fasting) respectively, (Table 32, 35, 38,

41 and Figure 33). Penumajji, S. & Bobbarala, V., conducted bioequivalence study of

Itopride HCl 150 mg SR Capsules under fasted conditions and reported mean Cmax values

of 0.763 ± 0.021 µg/mL for test and 0.731 ± 0.017 µg/mL for reference products. The

peak concentration were achieved at (Tmax), 7.25± 0.13 for test and 7.17 ± 0.16 h

(Penumajji, S. &Bobbarala, V., 2009). Another pharmacokinetic study of Itopride HCl

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100 mg dispersion tablets was reported by Wang, et al, in 2003, on 8 healthy male

Chinese volunteers, in which the Cmax and Tmax, values were 0.602 ± 0.159 µg/mL and

0.78 ± 0.35 h, after fitted into two compartmental model (Wang, B.-J. et al., 2003).

Sahoo, B.K. et al., also performed pharmacokinetics and bioequivalence study of fixed

dose combination of Rabeprazole (20 mg) and Itopride (150 mg) after oral

administration in a randomized, single-dose, two-period, two-treatment crossover

fashion. The study was conducted under 10 hours fasting and the Cmax and Tmax, values

found was 1.136 ± 0.078 µg/mL and 2.12 ± 0.43 h through non-compartmental method

(Sahoo, B.K. et al., 2009). The mean reported Cmax value by Yehia, S. et al., for 100 mg

SR tablet, was 2.553 ± 0.630 µg/mL and Tmax, was 6 ± 0.04 h (Yehia, S. et al., 2012).

Sisinthy, S. et al., also reported the mean Cmax and Tmax, values of 75 mg Itopride CR

tablet i.e. 0.197 ± 8.48 µg/mL and 5 h respectively (Sisinthy, S. et al., 2015). Yehia, S.

A. et al.,in 2013 conducted another study and reported mean Cmax and Tmax, values for

100 mg Itopride SR tablet as 1.624 ± 0.168 µg/mL and 6± 2 h (Yehia, S.A. et al., 2013).

Yoon, S. et al., reported the mean Cmax and Tmax, values for 150 mg Itopride ER tablet,

administered to 24 Korean volunteers, under fed condition, and the values were 0.426 ±

0.159 µg/mL and 4.4 ± 2 h, whereas under fasting condition the values were

comparatively lesser i.e. 0.244 ± 0.094 µg/mL and 3.1 ± 3 h sequentially (Yoon, S. et al.,

2014).

In the present work, AUC0-∞for EC5-F11 under fed state was 13.445 ± 0.427 mg/L× h,

and under fasted state was 9.608 ± 0.592 mg/L×h, while AUC0-∞for Ganaton OD under

fed state was 13.422 ± 0.558 mg/L× h and under fasted state the value was 9.543 ± 0.397

mg/L×h through compartmental analysis (Table 32, 35, 38 and 41). AUC0-t of EC5-F11

under fed and fasted states were 13.258 ± 0.323 mg/L× h, and 11.966 ± 0.678 mg/L×h,

while that of Ganaton OD, 12.335 ± 0.831 mg/L× h (fed) and 9.837 ± 0.302 mg/L×h

(fasting)respectively, obtained through non-compartmental analysis (Table 33, 36, 39

and 42). Whereas, when non compartmental analysis was performed, AUCtot ofEC5-F11

under fed and fasted conditions were 15.125 ± 1.010 mg/L×h, and 14.970 ± 0.411 mg/L×

h, and that of Ganaton OD, 11.168 ± 0.318 mg/L×h (fed) and 14.795 ± 1.401 mg/L× h

(fasting), consecutively (Table 33, 36, 39 and 42). Sahoo, B.K.et al., in 2009 reported

AUC0-tvalue for 150 mg Itopride ER tablet as 6.213 ± 0.431 µg.h/mL, whereas, its values

reported by Penumajji, et al., in 2009 and Yehia, S. A.et al., in 2012 and 2013, were

10.463 ± 0.332 µg.h/mL, 22.755 ± 0.697 µg.h/mL and 29.728 ± 0.761 µg.h/mL,

230

respectively. However, Choi, H.Y.et al., in 2012 reported 0.565 µg.h/mL AUC0-t in 27

Korean volunteers (Choi, H.Y. et al., 2012).

In the present study, it was observed that the AUC values were nearly same when

compared with published data but some reported values were found considerably

different. The results revealed that Cmax was increased when EC5-F11 was given under

fed state by 14% (0.647 ± 0.011 versus 0.567 ± 0.020 μg/mL, with and without food,

respectively) and the time taken to reach peak concentration was delayed by 1.46 h

(7.281 h versus 5.825 h, with and without food, respectively), similarly, AUC0–t was

increased by 11% under fed state (fed; 13.258 ± 0.323 versus fasted, 11.966 ± 0.678

μg/mL).Welling, demonstrated that food may delay gastric emptying, and then after drug

absorption (Welling, P.G., 1977). Change in gastric emptying, might have contributed to

delay the time to reach the peak concentration of Itopride. Moreover, Itopride HCl can

change intestinal motility, which may further alter the absorption rate of ER Itopride

HCl.

6.11.1.2. Volume of distribution and Clearance

The mean volume of distribution (Vc) values of EC5-F11through compartmental analysis

under fed and fasting conditions were found 109.637 ± 4.354 L and 125.306 ± 7.966 L,

whereas the mean clearance (Cl) values under same conditions were 15.664 ±

0.929L/hand 11.167 ± 0.365 L/h, respectively (Table 32, 38 and Figure 33). Zhao, R.-s

.et al., reported the clearance of 100 mg Itopride HCl in 10 healthy Chinese volunteers as

42.2±8.4 L (Zhao, R.-s. et al., 2005). Whereas, Vunnam, R.R.et al., calculated the mean

volume of distribution and clearance in healthy rats and reported as 497.65 mL and

11.58±0.042 mL/h, respectively (Vunnam, R.R. et al., 2015).

6.11.1.3. Half-lives and rate constants

In the present study, the mean disposition rate constant (β) under fed and fasted

conditions were calculated as 0.076 ± 0.009 hr-1and 0.071 ± 0.012 hr-1from

compartmental analysis, while, terminal phase rate constant (Lz) was calculated as 0.048

± 0.001 hr-1and 0.032 ± 0.005 hr-1through non-compartmental analysis, respectively.

231

Under fed and fasted conditions, the mean absorption rate constant (Ka), overall

distribution rate constant (α) and elimination rate constant (Kel) were observed as Ka

(0.188 ± 0.013and 0.253 ± 0.025hr-1), α (0.132 ± 0.006and 0.182 ± 0.010hr-1) and Kel

(0.102 ± 0.006and 0.125 ± 0.009 hr-1). The elimination rate constant (Kel) for 150 mg

SR tablet was reported by Sahoo, B. K. et al., in 2009 as 0.198 ±0.02hr-1, which is closer

to the present study. Sisinthy, SP, et al., in 2015 reported the absorption rate constant

(Ka) as 0.078±0.007hr-1and elimination rate constant (Kel) as 0.08±0.008hr-1for100 mg

Itopride HCl tablet. Another study conducted by Singh, S. S. et al., in 2005 for 50 mg

tablet and reported terminal phase rate constant (Lz) as 2.72 ± 0.63hr-1.

The micro rate constants values from central compartment to peripheral K12, and from

peripheral to central compartment K21, were also calculated through compartmental

analysis (Table 32). Under fed and fasted conditions, the mean values of K12were

observed as 0.008 ± 0.004 hr-1and 0.021 ± 0.024 hr-1while K21 were estimated as 0.098 ±

0.006 hr-1 and 0.103 ± 0.103 hr-1, respectively.

The absorption half-life (T1/2Ka), distribution half-life (T 1/2 α), disposition half-life (T1/2β)

and elimination half-life (T1/2Kel) were determined from compartmental analysis while

terminal phase half-life (T1/2Lz) was calculated by non-compartmental analysis. Under

fed and fasted conditions, the mean absorption half-lives (T1/2Ka) were observed as 3.701

± 0.251 h and 2.764 ± 0.278 h, the mean values of distribution half-life (T1/2α) were

5.254 ± 0.230 h and 3.827 ± 0.203 h, disposition half-life (T1/2β) 9.299 ± 1.141 h and

10.222 ± 2.809 h and elimination half-life (T1/2Kel) were 6.814 ± 0.380 h, and 5.556 ±

0.403 respectively. Whereas, the half-life of terminal phase (T1/2Lz) were calculated to be

9.229 ± 1.141 h (fed) and 10.222 ± 2.809 h (fasted). The elimination half-life (T½)

reported by Sisinthy, S.et al., in 2015 was 8.19±0.861 h, Yoon, et al., in 2014 was 7.4 ±

3.2 h (fasted), and 5.9 ± 1.1 h (fed) under fasting condition. Another study conducted

under fasting condition, by Bose A. et al., in 2009 and Ma, J.et al., in 2009, and their

reported T½value were similar i.e. 4.60 ± 1.32 h, and4.26 ± 0.68 h for 50 mg tablet.

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6.11.1.4. AUMC and MRT

Under fed and fasted conditions, the mean AUMC values of EC5-F11were found to be

215.469 ± 13.663 mg/L.hr2and 136.280 ± 23.866 mg/L.hr2, the mean values of AUMClast

were 220.759 ± 6.437mg/L.hr2and 213.378± 13.626 mg/L.hr2, whereas, the mean

AUMCtotal values were 339.048 ± 14.755 and 466.000 ± 68.978 mg/L.hr2, respectively.

Under fed and fasted conditions, the mean residence time (MRT) values of Itopride HCl

were found to be 22.640 ± 0.484 h, and 30.684 ± 3.344 h. Sisinthy, S.P. et al., in 2015

determined MRT value of 11.91±0.513 h for 50 mg Itopride HCl tablet (Sisinthy, S. et

al., 2015), while Yehia, SAet al., in 2012 reported 48.60 ± 42.4 h for 100 mg Itopride

HCl tablet (Yehia, S. et al., 2012). Yehia, S. A. et al., conducted another study in 2013

for 100 mg Itopride HCl tablet and reported the mean MRT value as 108 ± 9 h.

6.11.2. Statistical Analysis for Establishing Bioequivalence

6.11.2.1. Cmax, Tmax, AUC0-∞ , AUClast and AUCtot.

There are few researchers who have applied Latin square two-way analysis of variance

(ANOVA) test to compare the pharmacokinetics parameters of different drugs

administered under fed and fasting conditions like Yoon, S. et al., and Wright, et al.,

(Wright, C.W. et al., 2009; Yoon, S. et al., 2014).

In the present study, two-way analysis of variance (ANOVA) test was also applied using

Kinetica software for analysis of the test product (EC5-F11) under fasted (A) and fed (B)

conditions and the reference product (Ganaton OD Tablet) under fasted (C) and fed (D)

conditions. Latin Square option was used for analysis on conventional two treatments,

two sequence, four periods, randomized cross over study. As per FDA guidelines

different parameters like subject effect nested, sequence effect, and period effect were

determined by Latin Square ANOVA (Kinetica, User manual). After Log transformation,

the data obtained under fed and fasting conditions, was analyzed for test product (EC5-

F11), and the geometric mean values of Cmax calculated as0.566 ± 1.035 (fast) and 0.646

± 1.017 (fed) with geometric mean ratio of 1.141. The ANOVA effect of period,

sequence, and subject was not found significant. Two one-sided t test further supports the

233

ANOVA results (see Appendix-I, Table 52). Geometric means values of Cmax observed

for test product under fasted and fed states were 0.661 ± 1.02 and 0.773 ± 1.02, with

geometric ratio of 1.169. The period, subject and sequence effect were also observed

insignificant. The lower and upper Schuirmann’s two one-sided t test values were 8.597

and 48.875 (see Appendix-I, Table 54). The 90% CI for Cmax calculated was 1.122 –

1.160 and that for Cmax observed was 1.153 – 1.186, which were observed within the

bioequivalence range of 0.8 – 1.25.

The geometric mean values for Tmax under fasted and fed states were 5.821 ± 1.044 and

7.280 ± 1.012 h, respectively, and the geometric ratio was 1.250, with 90% CI limit of

1.219 – 1. 284. The subject, and sequence effects were found insignificant. The lower

and upper two one-sided t tests were -0.044 and 31.402 for Tmax calculated (see

Appendix-I, Table 53).

The geometric mean values of AUC0-∞under fasting and fed conditions were found to be

9.592 ± 1.062mg/L×h and 13.439 ± 1.033mg/L×h, with a geometric mean ratio of 1.401.

The 90% confidence interval was 1.345– 1.460, and the lower and upper two one-sided t

test values were -5.039 and 24.748 (see Appendix-I, Table 55). Similarly, the geometric

mean values of AUClast were determined and found to be 9.814 ± 1.035 (fasted) and

13.254 ± 1.025 (fed), with a geometric mean ratio of 1.351. The 90% confidence

interval limit was 1.321– 1.380, with the lower and upper two one-sided t test values of -

6.409 and 43.381 (see Appendix-I, Table 67). The geometric mean values of AUCtot

under fasting and fed conditions were found to be 11.043± 1.037 and 14.883 ± 1.028,

and geometric mean ratio was calculated to be 1.348. The 90% CI was 1.317–1.379 and

the upper and lower two one-sided t test values were -5.912 and 40.972 (see Appendix-I,

Table 68). Through ANOVA, the effect of subject sequence, and period of all these

parameters were observed insignificant. The results showed that the 90% CI values of

AUC0-∞, AUClast, AUCtot and Tmax fell out of the bioequivalence limit of 0.8 – 1.25.

Schuirmann’s two one-sided t test further confirmed the results.

For the reference product (Ganaton OD Tablet), two-way analysis of variance was also

applied with and without logarithmic transformation. The Log transformed data analysis,

exhibited the geometric mean values of Cmax calculated under fasted and fed conditions

as 0.564 ± 1.002 and 0.633 ± 1.048 with geometric mean ratio of 1.121. The effect of

234

period, sequence, and subject were not observed significant. Two one-sided t test further

supports the ANOVA findings (see Appendix-I, Table 82). Geometric mean values of

Cmax observed for reference product were 0.655 ± 1.006 (fast) and 0.767 ± 1.017(fed),

and geometric ratio was 1.171. The period, subject and sequence effect were also found t

insignificant. The lower and upper Schuirmann’s two one-sided t test values were 10.646

and 61.962 (see Appendix-I, Table 95). Similarly, 90% CI limit of 1.092 – 1.151 was

computed for Cmax calculated and 1.158 – 1.184for Cmax observed, the values were within

the prescribed limits of 0.8 – 1.25.

For Tmax under fasted and fed states the geometric mean values were 5.910 ± 1.010 and

7.403 ± 1.016 h, respectively. The geometric mean ratio was 1.253, at 90% CI values of

1.240 – 1.266. The subject, and sequence effect were found also not significant. The

lower and upper two one-sided t tests were determined to be -0.372 and 79.195 (see

Appendix-I, Table 83).

The geometric mean values of AUC0-∞ were found to be 9.535 ± 1.042 mg/L×h and

13.411 ± 1.044 mg/L×h, under fasting and fed states correspondingly, and the geometric

mean ratio was calculated to be 1.406. The 90% confidence interval was 1.363 – 1.451,

and the upper and lower two one-sided t test values were -6.811 and 32.582 (see

Appendix-I, Table 84).The geometric mean values of AUClast under fasting and fed

conditions were found to be 9.828 ± 1.031 and 13.080 ± 1.052, with a geometric mean

ratio of 1.331 (90 %CI; 1.298 – 1.365). The two one-sided t test values were -4.518

(lower) and 36.680 (upper) (see Appendix-I, Table 97). Similarly, the geometric mean

values of AUCtot under fasting and fed conditions were calculated as 11.049 ± 1.034 and

14.559 ± 1.053, with a geometric mean ratio of 1.318 (90 %CI; 1.279 – 1.357). The

lower and upper two one-sided t test values were -3.224 and 30.543 (see Appendix-I,

Table 99). The ANOVA results revealed that, the effects of subject sequence, and period

of all these parameters were insignificant. The results showed that the 90% CI values of

AUC0-∞, AUClast, AUCtot and Tmax were outside the bioequivalence limit of 0.8 – 1.25,that

was also showing compliance with Schuirmann’s two one-sided t test.

235

6.11.2.2. Analysis of other pharmacokinetic parameters

In addition to Cmax, Tmax, AUClast, and AUC0-∞, other pharmacokinetic parameters were

also estimated for both test (EC5-F11) and reference (Ganaton OD Tablet) formulations

under fed and fasting conditions, with and without log transformation. There was no

significant period, subject and sequence effect observed for the other pharmacokinetics

parameters (Ka, Kel, α, β, K12, K21, Lz, T1/2ka, T1/2kel, T1/2kα, T1/2β, and Cl) of both test

(EC5-F11) and reference (Ganaton OD Tablet) products. The values of geometric mean

ratio at 90% CI limit, of different rate constants were found to be, Ka0.7450 (0.7049 –

0.7873), Kel0.8147 (0.7669 – 0.8654), α 0.7281 (0.7011 – 0.7561), β 1.0873 (0.9292 –

1.2724), K120.3190 (0.2190 – 0.4645), K21 0.9717 (0.8612 – 1.0965) and Lz 1.059

(1.0162 – 1.1043) for test product when fasted and fed states were compared. While, for

reference product, these values were observed as Ka 0.6696(0.6345 – 0.7067), Kel 0.8607

(0.8045 – 0.9205), α, 0.7280 (0.7011 – 0.7559), β, 0.9299 (0.7087 – 1.2203), K12,

0.2484(0.1353 – 0.4561), K21, 0.7865(0.6207 – 0.9967) and Lz, 1.082 (1.023 – 1.1453).

The results revealed thatK21, and Lzof test product showed equivalence under fed and

fasted conditions, whereas, Ka, α, β, Kel, K12, and K21, exhibited nonequivalence.

Similarly, for reference product only Kel was concluded as equivalent (see Appendix-I,

Table 46 – 51, 65, 76 –81, 94). The half-lives values of corresponding rate constants, i.e.

T1/2ka, T1/2α, T1/2β, T1/2kel, and T1/2Lzof both test and reference products were successfully

calculated and their geometric mean ratio and the CI limit were found to be 1.3422

(1.2701 to 1.4186), 1.3734 (1.3225 to 1.4262), 0.9196 (0.7859 to 1.0761), 1.2274

(1.1555 to 1.3038), and 0.9196 (0.7859 to 1.0761) respectively. While for reference

product the geometric mean ratio and CI limits of these half-lives were 1.4932 (1.4151 to

1.5758), 1.3735 (1.3229 to 1.4262), 1.0753 (0.8194 to 1.411), 1.1617 (1.086 to 1.242),

and 1.0753 (0.8194 to 1.411), correspondingly. The geometric mean ratio values of

T1/2βand T1/2kel, of reference product were found within the acceptance limit of 0.8 to

1.25 and therefore concluded equivalent for both fed and fasted conditions. Whereas,

T1/2ka, and T1/2α were observed nonequivalent. Contrarily, these half-lives were found

non-equivalent under fed and fasted conditions for test products. The results were further

endorsed by two one-sided t test for all the parameters. Geometric mean ratio (90% CI)

of Vc of test and reference products were found to be consecutively 0.876 (0.841 to

0.912) and 0.826 (0.794 to 0.858), when compared with and without food. In case of

236

clearance, the geometric mean ratio values (90% CI) of test and reference

formulationwere0.7137 (0.685 to 0.743) and 0.7109 (0.689 to 0.733), respectively.

For AUMC, AUMClast AUMCtotal and AUMCextra of test product, the geometric mean

ratio values (90% CI) were 1.598 (1.4297 - 1.7873), 1.448 (1.4058 - 1.4933), 1.394

(1.3439 - 1.4478), and 1.305 (1.2063 - 1.4139), while for reference product the values

were 1.716 (1.524 - 1.934), 1.413 (1.3678 - 1.4612), 1.329 (1.2577 - 1.4059) and 1.181

(1.0459 - 1.3332), respectively. The geometric mean ratio (90% CI) of Tabs, and Vz of

test product were 1.3422 (1.2701 - 1.4186), and 0.7004 (0.67172 - 0.73041), while for

reference product the values were 1.4932 (1.415 - 1.5758), 1.2986 (1.2339 - 1.3667 and

0.7011 (0.66642 - 0.73778), respectively. The geometric mean ratio and CI limit values

of MRT were 1.03499 (1.014 - 1.056) for test product, and 1.0092 (0.9769 - 1.0426) for

reference product.

Non-log transformed data with Latin Square ANOVA was also applied for the test

product (EC5-F11) and reference products (Ganaton OD Tablet) The analysis revealed

that no significance difference was observed for test and reference products, under fed

and fasted conditions for Cmax calculated, Cmax observed and Tmax There was no

difference observed in the outcomes of logarithmically transformed or non-transformed

data (see Appendix-I, Table 106 – 165).

However, upon comparison of pharmacokinetic parameters of test (EC5-F11) and

reference (Ganaton OD Tablet) product with and without food, both the products were

found bioequivalent. Table 43 and 44 shows comparative mean pharmacokinetic log and

non-log transformed parameters of test product (EC5-F11) versus reference product

(Ganaton OD), with geometric mean ratios at 90% CI, exhibiting no considerable

differences between the two formulations under fed and fasted conditions.

According to current study, the relative bioavailability of test (EC5-F11) and reference

(Ganaton OD) products, was 100.171% under fed state and 100.681% under fasted state

(Table 45). Therefore, it can be concluded that food delayed the absorption of Itopride in

both formulations, without any significant change in oral bioavailability. Yoon, S. et al.,

in 2014 examined the effect of food on the ER Itopride HCl formulation and reported

AUC0–t and Cmax, and within the prescribed limits of 0.80 to 1.25. Choi, et al., in 2012

237

also conducted bioequivalence analysis of Itopride (150 mg), at 90% CIs using log-

transformed data, their reported Cmax, and AUC, were also observed within the range

(Choi, H.Y. et al., 2012).

238

7. CONCLUSION

239

CONCLUSION

Following conclusion can be made in the present research work on the basis of the

observations and findings.

1. The plain pellets, ER matrix pellets of HPMC (K4M, K15M and K100M) as well

as matrix pellets of ethyl cellulose were prepared by extrusion and

spheronization technique.

2. The ER matrix pellets failed to control the release of ITP 150mg, for 12 h even at

a higher concentration (50%).

3. To overcome the burst release of highly water soluble drug ITP, different

polymers such as ethylcellulose (10 cps), Eudragit® RS/RL 100 and Kollicoat SR

30D were used at different levels (5 – 15%).

4. The initial fast release of ITP was effectively controlled for up to 12 h by the

application of 5% EC dispersion on 10% matrix pellets of K4M, K15M, K100M,

and EC (7 cps).

5. Drug release kinetics was studied using different kinetic models such as Zero

order, first order, Higuchi, Korsmeyer-Peppas, Hixson-Crowell and Baker

Lonsdale and it was found that extended release EC5-F11formulation of ITP

followed zero order release kinetics. All coated matrix pellets were also best

explained by the Higuchi kinetic model. Drug release mechanism for all coated

formulations was non-Fickian diffusion (anomalous transport).

6. FTIR spectra of the pure drug and coated formulations indicated absence of

interaction among excipients, coating polymers, and pure drug.

7. Shelf lives of ER coated ITP pellet formulations were calculated by using

Minitab 17 after accelerated and room temperature stability studies and the self-

lives were found up to 23 months.

240

8. Pharmacokinetics study of optimized extended release ITP coated formulation

(EC5-F11) was successfully evaluated in local healthy male volunteers under

fasting and fed conditions for compartmental and non-compartmental parameters

and compared with other reported literature.

9. On the basis of analysis of variance results it can be concluded that food delayed

the absorption of drug without effecting bioavailability.

10. The pharmacokinetic parameters of the optimized formulation (EC5-F11) was

compared with marketed reference brand (Ganaton OD Tablet, Abbott

Laboratories Pakistan), and both the products were found equivalent.

11. Thus, ethyl cellulose was found to be an excellent rate controlling agent for

highly water soluble drug ITP. This study has established that extended release

pellet formulation of ITP can be a good oral alternative formulation for the

treatment of gastrointestinal symptoms such as nausea, vomiting, heartburn,

epigastric discomfort, non-ulcer dyspepsia, gastro-esophageal reflux disease, and

gastritis.

241

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261

APPENDIX – I

262

3. STATISTICAL ANALYSIS FOR LOG TRANSFORMED

DATA

A) EC5-F11 (Pellets) Under Fed and Fasted Conditions; (A = Fasted

and B = Fed)

TABLE 46


LATIN SQUARE with Log (neperian) option

SOURCE D.F SS MS F p

Period 1 0.000941313 0.000941313 0.168617 0.69 NS

Subject(Seq) 10 0.101281 0.0101281 1.81425 0.1808 NS

Formulation 1 0.51988 0.51988 93.1261 2.201e-006 ***

Sequence 1 0.000502833 0.000502833 0.0900723 0.7702 NS

Error 10 0.0558254 0.00558254

Total 23 0.678431

--------------------------------------------------------------------------------

N Mean SD SEM GeoMean Geo SD

Formulation (Fasted) =

A 12 -1.37869 0.0994844 0.0287187 0.251909 1.1046

Formulation (Fed) = B 12 -1.67305 0.0672054 0.0194005 0.187674 1.06952

--------------------------------------------------------------------------------

Root Mean Square Error = 0.0747164; CV = -0.0489665

phi = 6.82371

Power of the test = 1

1 - (Power of the test) = 2.419e-011

Minimum detectable difference = 0.294358

--------------------------------------------------------------------------------

BIOEQUIVALENCE TESTS FOR: Level A and level B

Reference Confidence Interval: [ 0.8, 1.25]

Geomean Ratio (Fasted/Fed) = 0.74501

90% standard confidence interval

(around the ratio: [Fasted form]/ [Fed form]) = [ 0.70494, 0.78736]

t(0.05 - 10df) = 1.8125

Cannot conclude equivalence.

--------------------------------------------------------------------------------

TWO ONE-SIDED T-TESTS FOR

Level A and level B

Lower: t(10df) = -2.3347

Upper: t(10df) = 16.966

t(0.05 - 10df) = 1.8125

Cannot conclude equivalence

263

TABLE 47




Period 1 0.00124902 0.00124902 0.187578 0.6741 NS

Subject(Seq) 10 0.0265222 0.00265222 0.398309 0.9187 NS

Formulation 1 0.251956 0.251956 37.8386 0.0001081 ***

Sequence 1 1.79251e-005 1.79251e-005 0.00269198 0.9596 NS

Error 10 0.066587 0.0066587

Total 23 0.346332

--------------------------------------------------------------------------------


Formulation (Fasted)

= A

12 -2.07904 0.071099 0.0205245 0.12505 1.07369

Formulation (Fed) =

B

12 -2.28397 0.0593683 0.0171381 0.101879 1.06117

-------------------------------------------------------------------------------


phi = 4.34963

Power of the test = 0.999794

1 - (Power of the test) = 0.000206439


-------------------------------------------------------------------------------

BIOEQUIVALENCE TESTS FOR:

Level A and level B




(around the ratio: [Fasted form]/[Fed form])=[ 0.76698, 0.86542]

t(0.05 - 10df) = 1.8125


-------------------------------- ------------------------------------------------

TWO ONE-SIDED T-TESTS FOR:

Level A and level B

Lower: t(10df) = 0.547

Upper: t(10df) = 12.85

t(0.05 - 10df) = 1.8125


264

TABLE 48




Period 1 0.000256499 0.000256499 0.098575 0.76 NS

Subject(Seq) 10 0.0283093 0.00283093 1.08795 0.4483 NS

Formulation 1 0.60403 0.60403 232.135 3.009e-008 ***

Sequence 1 0.000222112 0.000222112 0.08536 0.7761 NS

Error 10 0.0260207 0.00260207

Total 23 0.658838

--------------------------------------------------------------------------------



= A

12 -1.70734 0.0545738 0.0157541 0.181348 1.05609

Formulation (Fed) =

B

12 -2.02462 0.0447694 0.0129238 0.132043 1.04579

--------------------------------------------------------------------------------


phi = 10.7735

The power of the test is near to 1

1 - (Power of the test) is near to 0


--------------------------------------------------------------------------------


Level A and level B




(around the ratio: [Fasted form]/[fed form])=[ 0.70115, 0.75613]

t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -4.5208

Upper: t(10df) = 25.951

t(0.05 - 10df) = 1.8125


265

TABLE 49




Period 1 0.00105046 0.00105046 0.0232959 0.8817 NS

Subject(Seq) 10 0.220495 0.0220495 0.488987 0.8626 NS

Formulation 1 0.0420867 0.0420867 0.933346 0.3568 NS

Sequence 1 0.0145268 0.0145268 0.322157 0.5828 NS

Error 10 0.450923 0.0450923

Total 23 0.729082

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 -2.66572 0.21797

3

0.062923

4

0.0695492 1.24355

Formulation (Fed) =

B

12 -2.58197 0.12223

7

0.035286

8

0.075625 1.13002

--------------------------------------------------------------------------------


phi = 0.683135


1 - (Power of the test) = 0.858542


--------------------------------------------------------------------------------


Level A and level B




(around the ratio: [fasted form]/[fed form])=[ 0.92925, 1.2724]

t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 1.6079

Upper: t(10df) = 3.5401

t(0.05 - 10df) = 1.8125


266

TABLE 50




Period 1 0.0148736 0.0148736 0.0576813 0.8151 NS

Subject(Seq) 10 0.586005 0.0586005 0.227259 0.9859 NS

Formulation 1 7.83218 7.83218 30.374 0.0002577 ***

Sequence 1 0.0121922 0.0121922 0.0472825 0.8322 NS

Error 10 2.57858 0.257858

Total 23 11.0238

--------------------------------------------------------------------------------



= A

12 -3.76816 0.342294 0.0988118 0.0230946 1.40817

Formulation (Fed) =

B

12 -4.91068 0.415915 0.120064 0.0073674

7

1.51576

--------------------------------------------------------------------------------


phi = 3.89705


1 - (Power of the test) = 0.00149227


--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -4.4349

Upper: t(10df) = 6.5877

t(0.05 - 10df) = 1.8125


267

TABLE 51




Period 1 0.00267611 0.00267611 0.100533 0.7577 NS

Subject(Seq) 10 0.247103 0.0247103 0.928289 0.5457 NS

Formulation 1 0.00491335 0.00491335 0.184579 0.6766 NS

Sequence 1 0.0195077 0.0195077 0.732842 0.412 NS

Error 10 0.266192 0.0266192

Total 23 0.540393

--------------------------------------------------------------------------------



= A

12 -2.29401 0.211245 0.0609811 0.100861 1.23521

Formulation (Fed) =

B

12 -2.32263 0.0636839 0.018384 0.0980159 1.06576

--------------------------------------------------------------------------------


phi = 0.303792




--------------------------------------------------------------------------------

BIOEQUIVALENCE TESTS FOR

Level A and level B





t(0.05 - 10df) = 1.8125

Can conclude equivalence

--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 2.9205

Upper: t(10df) = 3.7798

t(0.05 - 10df) = 1.8125


268

TABLE 52




Period 1 1.01155e-005 1.01155e-005 0.0215802 0.8861 NS

Subject(Seq) 10 0.0115109 0.00115109 2.45571 0.08632 NS

Formulation 1 0.105058 0.105058 224.128 3.562e-008 ***

Sequence 1 2.90958e-005 2.90958e-005 0.0620724 0.8083 NS

Error 10 0.00468739 0.000468739

Total 23 0.121295

--------------------------------------------------------------------------------



= A

1

2 -0.568261 0.0341615 0.00986159 0.56651 1.03475

Formulation (Fed) =

B

1

2 -0.435937 0.017582 0.00507548 0.646658 1.01774

--------------------------------------------------------------------------------


phi = 10.586




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 10.275

Upper: t(10df) = 40.217

t(0.05 - 10df) = 1.8125


269

TABLE 53




Period 1 8.28593e-007 8.28593e-007 0.0006818 0.9797 NS

Subject(Seq) 10 0.00988439 0.000988439 0.813327 0.6249 NS

Formulation 1 0.300435 0.300435 247.21 2.222e-008 ***

Sequence 1 6.65142e-005 6.65142e-005 0.0547305 0.8197 NS

Error 10 0.012153 0.0012153

Total 23 0.32254

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 1.76139 0.043165

2

0.0124607 5.82051 1.04411

Formulation (Fed) =

B

12 1.98516 0.012095

1

0.0034915

6

7.28018 1.01217

--------------------------------------------------------------------------------

Root Mean Square Error = 0.0348612; CV = 0.0186098

phi = 11.1178




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -0.043931

Upper: t(10df) = 31.402

t(0.05 - 10df) = 1.8125


270

TABLE 54




Period 1 0.000101955 0.000101955 0.2818 0.6071 NS

Subject(Seq) 10 0.00887973 0.000887973 2.45434 0.08645 NS

Formulation 1 0.146744 0.146744 405.596 2.005e-009 ***

Sequence 1 0.000158034 0.000158034 0.436803 0.5236 NS

Error 10 0.00361797 0.000361797

Total 23 0.159501

--------------------------------------------------------------------------------

N Mean SD SEM GeoMean Geo

SD

Formulation

(Fasted) = A

12 -

0.413731

0.022499

1

0.0064949

3

0.661179 1.0227

5

Formulation (Fed) =

B

12 -

0.257343

0.025565

2

0.0073800

5

0.773103 1.0258

9

--------------------------------------------------------------------------------


phi = 14.2407




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125

Can conclude equivalence.

--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 8.5967

Upper: t(10df) = 48.875

t(0.05 - 10df) = 1.8125


271

TABLE 55




Period 1 1.49092e-005 1.49092e-005 0.00484592 0.9459 NS

Subject(Seq) 10 0.0208235 0.00208235 0.676822 0.7258 NS

Formulation 1 0.682441 0.682441 221.813 3.744e-008 ***

Sequence 1 3.88879e-005 3.88879e-005 0.0126397 0.9127 NS

Error 10 0.0307665 0.00307665

Total 23 0.734085

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 2.26091 0.060470

2

0.0174562 9.59182 1.06234

Formulation (Fed) =

B

12 2.59816 0.032221

8

0.0093016

4

13.439 1.03275

--------------------------------------------------------------------------------


phi = 10.5312




-------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -5.0392

Upper: t(10df) = 24.748

t(0.05 - 10df) = 1.8125


272

TABLE 56




Period 1 0.000990975 0.000990975 0.324295 0.5816 NS

Subject(Seq) 10 0.0362348 0.00362348 1.18578 0.3964 NS

Formulation 1 0.105076 0.105076 34.386 0.0001586 ***

Sequence 1 4.004e-006 4.004e-006 0.0013103 0.9718 NS

Error 10 0.0305578 0.00305578

Total 23 0.172864

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 4.82877 0.067177

9

0.019392

6

125.057 1.06949

Formulation (Fed) =

B

12 4.69643 0.040615

8

0.011724

8

109.556 1.04145

--------------------------------------------------------------------------------


phi = 4.14644


1 - (Power of the test) = 0.000520032


--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 4.0238

Upper: t(10df) = 15.752

t(0.05 - 10df) = 1.8125


273

TABLE 57




Period 1 1.49141e-005 1.49141e-005 0.0048475 0.9459 NS

Subject(Seq) 10 0.0208227 0.00208227 0.676798 0.7258 NS

Formulation 1 0.682444 0.682444 221.814 3.744e-008 ***

Sequence 1 3.88982e-005 3.88982e-005 0.012643 0.9127 NS

Error 10 0.0307665 0.00307665

Total 23 0.734087

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 2.74973 0.060469

2

0.017456 15.6383 1.06233

Formulation (Fed) =

B

12 2.41247 0.032222

6

0.0093018

5

11.1615 1.03275

--------------------------------------------------------------------------------


phi = 10.5312




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -5.0392

Upper: t(10df) = 24.748

t(0.05 - 10df) = 1.8125


274

TABLE 58




Period 1 0.000941404 0.000941404 0.16863 0.69 NS

Subject(Seq) 10 0.101282 0.0101282 1.81422 0.1809 NS

Formulation 1 0.519885 0.519885 93.1246 2.201e-006 ***

Sequence 1 0.000502859 0.000502859 0.0900749 0.7702 NS

Error 10 0.0558267 0.00558267

Total 23 0.678437

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 1.01218 0.099485

4

0.028719 2.75158 1.1046

Formulation (Fed) =

B

12 1.30653 0.067205

3

0.019400

5

3.69335 1.06952

--------------------------------------------------------------------------------


phi = 6.82366




--------------------------------------------------------------------------------


Level A and level B




(around the ratio: [fasted form]/ [fed form]) = [ 1.2701, 1.4186]

t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -2.3347

Upper: t(10df) = 16.966

t(0.05 - 10df) = 1.8125


275

TABLE 59




Period 1 0.000941377 0.000941377 0.168623 0.69 NS

Subject(Seq) 10 0.101281 0.0101281 1.81418 0.1809 NS

Formulation 1 0.519884 0.519884 93.1238 2.201e-006 ***

Sequence 1 0.00050292 0.00050292 0.0900852 0.7702 NS

Error 10 0.0558272 0.00558272

Total 23 0.678436

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 2.62161 0.099484

6

0.028718

7

13.7579 1.1046

Formulation (Fed) =

B

12 2.91597 0.067206 0.019400

7

18.4668 1.06952

--------------------------------------------------------------------------------


phi = 6.82363




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -2.3347

Upper: t(10df) = 16.965

t(0.05 - 10df) = 1.8125


276

TABLE 60




Period 1 0.000256556 0.000256556 0.0985946 0.76 NS

Subject(Seq) 10 0.0283091 0.00283091 1.08792 0.4483 NS

Formulation 1 0.604037 0.604037 232.132 3.009e-008 ***

Sequence 1 0.000222228 0.000222228 0.0854023 0.7761 NS

Error 10 0.0260213 0.00260213

Total 23 0.658846

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 1.34082 0.054574

1

0.015754

2

3.82218 1.05609

Formulation (Fed) =

B

12 1.65811 0.044769

6

0.012923

9

5.24939 1.04579

--------------------------------------------------------------------------------


phi = 10.7734




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -4.5208

Upper: t(10df) = 25.951

t(0.05 - 10df) = 1.8125


277

TABLE 61




Period 1 0.00105038 0.00105038 0.0232941 0.8817 NS

Subject(Seq) 10 0.220494 0.0220494 0.488987 0.8626 NS

Formulation 1 0.0420862 0.0420862 0.93334 0.3568 NS

Sequence 1 0.014527 0.014527 0.322163 0.5828 NS

Error 10 0.45092 0.045092

Total 23 0.729078

--------------------------------------------------------------------------------



= A

12 2.29921 0.217972 0.062923

2

9.96628 1.24355

Formulation (Fed) =

B

12 2.21546 0.122237 0.035286

8

9.16558 1.13002

--------------------------------------------------------------------------------


phi = 0.683132




-------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 1.6079

Upper: t(10df) = 3.5401

t(0.05 - 10df) = 1.8125


278

TABLE 62




Period 1 0.00124898 0.00124898 0.187568 0.6741 NS

Subject(Seq) 10 0.0265228 0.00265228 0.39831 0.9187 NS

Formulation 1 0.251954 0.251954 37.8376 0.0001081 ***

Sequence 1 1.79234e-005 1.79234e-005 0.00269168 0.9596 NS

Error 10 0.0665883 0.00665883

Total 23 0.346332

--------------------------------------------------------------------------------



= A

12 1.71253 0.0710998 0.0205247 5.54298 1.07369

Formulation (Fed) =

B

12 1.91745 0.0593687 0.0171383 6.80361 1.06117

--------------------------------------------------------------------------------


phi = 4.34958


1 - (Power of the test) = 0.000206496


--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 0.54702

Upper: t(10df) = 12.849

t(0.05 - 10df) = 1.8125


279

TABLE 63




Period 1 0.000161948 0.000161948 0.00711692 0.9344 NS

Subject(Seq) 10 0.113694 0.0113694 0.499638 0.8554 NS

Formulation 1 1.32012 1.32012 58.0136 1.805e-005 ***

Sequence 1 0.000140213 0.000140213 0.00616177 0.939 NS

Error 10 0.227553 0.0227553

Total 23 1.66167

--------------------------------------------------------------------------------



= A

12 4.90185 0.163811 0.0472883 134.539 1.17799

Formulation (Fed) =

B

12 5.37092 0.064929 0.0187434 215.06 1.06708

--------------------------------------------------------------------------------


phi = 5.3858




-------------------------------------------------------------------------------


Level A and level B




(around the ratio: [fasted form]/ [fed form]) = [ 1.4297, 1.7873]

t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -3.9933

Upper: t(10df) = 11.24

t(0.05 - 10df) = 1.8125


280

TABLE 64




Period 1 0.000530742 0.000530742 0.715611 0.4174 NS

Subject(Seq) 10 0.0103184 0.00103184 1.39125 0.3057 NS

Formulation 1 0.00709841 0.00709841 9.57094 0.01137 ***

Sequence 1 4.99637e-005 4.99637e-005 0.0673671 0.8005 NS

Error 10 0.00741663 0.000741663

Total 23 0.0254141

--------------------------------------------------------------------------------



= A

12 3.07379 0.0352869 0.0101864 21.6238 1.03592

Formulation (Fed) =

B

12 3.10819 0.0204915 0.0059154 22.3805 1.0207

--------------------------------------------------------------------------------


phi = 2.18757




--------------------------------------------------------------------------------


Level A and level B




(around the ratio:[fasted form]/[fed form])=[ 1.0143, 1.0561]

t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 16.977

Upper: t(10df) = 23.164

t(0.05 - 10df) = 1.8125


281

TABLE 65




Period 1 0.00270923 0.00270923 0.857993 0.3761 NS

Subject(Seq) 10 0.0459559 0.00459559 1.45539 0.2819 NS

Formulation 1 0.0199304 0.0199304 6.31181 0.03079 ***

Sequence 1 0.000500521 0.000500521 0.158512 0.6989 NS

Error 10 0.0315763 0.00315763

Total 23 0.100672

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 -

3.10341

0.081284

2

0.0234647 0.044896 1.08468

Formulation (Fed) =

B

12 -

3.04577

0.027075 0.0078158

7

0.047559

6

1.02744

--------------------------------------------------------------------------------


phi = 1.77649




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 7.2147

Upper: t(10df) = 12.239

t(0.05 - 10df) = 1.8125


282

TABLE 66




Period 1 0.000771393 0.000771393 0.667784 0.4329 NS

Subject(Seq) 10 0.00520453 0.000520453 0.450548 0.8877 NS

Formulation 1 0.494446 0.494446 428.035 1.541e-009 ***

Sequence 1 0.000993229 0.000993229 0.859824 0.3756 NS

Error 10 0.0115515 0.00115515

Total 23 0.512966

--------------------------------------------------------------------------------



= A

12 2.19293 0.0261623 0.0075524 8.96143 1.02651

Formulation (Fed) =

B

12 2.48 0.0316107 0.00912522 11.9412 1.03212

--------------------------------------------------------------------------------


phi = 14.6293




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -4.607

Upper: t(10df) = 36.771

t(0.05 - 10df) = 1.8125


283

TABLE 67




Period 1 1.23519e-005 1.23519e-005 0.0141365 0.9077 NS

Subject(Seq) 10 0.0109386 0.00109386 1.2519 0.3646 NS

Formulation 1 0.541757 0.541757 620.028 2.496e-010 ***

Sequence 1 2.71581e-005 2.71581e-005 0.0310818 0.8636 NS

Error 10 0.00873761 0.000873761

Total 23 0.561472

--------------------------------------------------------------------------------



= A

12 9.19158 0.0346022 0.00998878 9.81419 1.03521

Formulation (Fed) =

B

12 9.49207 0.0243933 0.00704174 13.2542 1.02469

--------------------------------------------------------------------------------


phi = 17.6072




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -6.4092

Upper: t(10df) = 43.391

t(0.05 - 10df) = 1.8125


284

TABLE 68




Period 1 1.71975e-005 1.71975e-005 0.0176891 0.8968 NS

Subject(Seq) 10 0.013564 0.0013564 1.39517 0.3042 NS

Formulation 1 0.534274 0.534274 549.545 4.52e-010 ***

Sequence 1 4.13926e-005 4.13926e-005 0.0425757 0.8407 NS

Error 10 0.00972211 0.000972211

Total 23 0.557619

--------------------------------------------------------------------------------



= A

12 9.30959 0.036856 0.0106394 11.0434 1.03754

Formulation (Fed) =

B

12 9.60799 0.0276384 0.00797852 14.8832 1.02802

--------------------------------------------------------------------------------


phi = 16.5763




--------------------------------------------------------------------------------


Level A and level B




(around the ratio: [fasted form]/[fed form]) = [ 1.317, 1.3792]

t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -5.9125

Upper: t(10df) = 40.972

t(0.05 - 10df) = 1.8125


285

TABLE 69




Period 1 0.00581868 0.00581868 0.689515 0.4257 NS

Subject(Seq) 10 0.14119 0.014119 1.67311 0.2149 NS

Formulation 1 0.488873 0.488873 57.9316 1.817e-005 ***

Sequence 1 0.000559846 0.000559846 0.066342 0.802 NS

Error 10 0.084388 0.0084388

Total 23 0.72083

--------------------------------------------------------------------------------



= A

12 7.10877 0.124825 0.0360338 12.2265 1.13295

Formulation (Fed) =

B

12 7.39422 0.0742008 0.0214199 16.2656 1.07702

-------------------------------------------------------------------------------


phi = 5.38199




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -1.6612

Upper: t(10df) = 13.561

t(0.05 - 10df) = 1.8125


286

TABLE 70




Period 1 0.00520338 0.00520338 0.885718 0.3688 NS

Subject(Seq) 10 0.0917918 0.00917918 1.56248 0.2465 NS

Formulation 1 0.00100775 0.00100775 0.17154 0.6875 NS

Sequence 1 0.000296639 0.000296639 0.0504938 0.8267 NS

Error 10 0.0587476 0.00587476

Total 23 0.157047

--------------------------------------------------------------------------------



= A

12 2.40436 0.106 0.0305997 11.0713 1.11182

Formulation (Fed) =

B

12 2.3914 0.0543074 0.0156772 10.9288 1.05581

--------------------------------------------------------------------------------


phi = 0.292865




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 6.7171

Upper: t(10df) = 7.5454

t(0.05 - 10df) = 1.8125


287

TABLE 71




Period 1 3.02083e-006 3.02083e-006 0.00181465 0.9669 NS

Subject(Seq) 10 0.0137876 0.00137876 0.828238 0.6143 NS

Formulation 1 0.825069 0.825069 495.629 7.508e-010 ***

Sequence 1 6.35996e-005 6.35996e-005 0.0382051 0.8489 NS

Error 10 0.0166469 0.00166469

Total 23 0.85557

--------------------------------------------------------------------------------



= A

12 11.9336 0.0437726 0.0126361 152.300 1.04474

Formulation (Fed) =

B

12 12.3044 0.0292709 0.00844978 220.672 1.0297

--------------------------------------------------------------------------------


phi = 15.7421




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -8.8662

Upper: t(10df) = 35.659

t(0.05 - 10df) = 1.8125


288

TABLE 72




Period 1 0.000738873 0.000738873 0.292127 0.6007 NS

Subject(Seq) 10 0.0361003 0.00361003 1.42729 0.2921 NS

Formulation 1 0.664532 0.664532 262.735 1.656e-008 ***

Sequence 1 0.000182322 0.000182322 0.0720843 0.7938 NS

Error 10 0.0252929 0.00252929

Total 23 0.726847

--------------------------------------------------------------------------------



= A

12 12.3834 0.0612354 0.0176771 238.800 1.06315

Formulation (Fed) =

B

12 12.7162 0.0437627 0.0126332 333.094 1.04473

-------------------------------------------------------------------------------


phi = 11.4616




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -5.3408

Upper: t(10df) = 27.077

t(0.05 - 10df) = 1.8125


289

TABLE 73




Period 1 0.00834137 0.00834137 0.724532 0.4146 NS

Subject(Seq) 10 0.192216 0.0192216 1.66959 0.2158 NS

Formulation 1 0.42762 0.42762 37.1431 0.0001165 ***

Sequence 1 0.000887664 0.000887664 0.0771025 0.7869 NS

Error 10 0.115128 0.0115128

Total 23 0.744192

--------------------------------------------------------------------------------



= A

12 11.3618 0.148505 0.0428696 85.9752 1.1601

Formulation (Fed) =

B

12 11.6288 0.0820103 0.0236743 112.283 1.08547

--------------------------------------------------------------------------------


phi = 4.30947


1 - (Power of the test) = 0.000248953


--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -1.0004

Upper: t(10df) = 11.189

t(0.05 - 10df) = 1.8125


290

TABLE 74




Period 1 0.00229491 0.00229491 0.716284 0.4172 NS

Subject(Seq) 10 0.0313288 0.00313288 0.977828 0.5138 NS

Formulation 1 0.76058 0.76058 237.39 2.702e-008 ***

Sequence 1 0.000253894 0.000253894 0.079245 0.7841 NS

Error 10 0.0320392 0.00320392

Total 23 0.826496

--------------------------------------------------------------------------------



= A

12 -1.19555 0.0731345 0.0211121 0.302539 1.07588

Formulation (Fed) =

B

12 -1.55158 0.0253727 0.00732448 0.211912 1.0257

-------------------------------------------------------------------------------


phi = 10.8947




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = -5.751

Upper: t(10df) = 25.064

t(0.05 - 10df) = 1.8125


291

TABLE 75




Period 1 0.00105038 0.00105038 0.0232941 0.8817 NS

Subject(Seq) 10 0.220494 0.0220494 0.488987 0.8626 NS

Formulation 1 0.0420862 0.0420862 0.93334 0.3568 NS

Sequence 1 0.014527 0.014527 0.322163 0.5828 NS

Error 10 0.45092 0.045092

Total 23 0.729078

--------------------------------------------------------------------------------



= A

12 2.29921 0.217972 0.0629232 9.96628 1.24355

Formulation (Fed) =

B

12 2.21546 0.122237 0.0352868 9.16558 1.13002

-------------------------------------------------------------------------------


phi = 0.683132




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level A and level B

Lower: t(10df) = 1.6079

Upper: t(10df) = 3.5401

t(0.05 - 10df) = 1.8125


292

B) Ganaton OD (Tablet) Under Fed and Fasted Conditions;(C = Fasted

and D = Fed)

TABLE 76




Period 1 0.00319772 0.00319772 0.604725 0.4548 NS

Subject(Seq) 10 0.0574029 0.00574029 1.08555 0.4496 NS

Formulation 1 0.964674 0.964674 182.431 9.536e-008 ***

Sequence 1 0.00134342 0.00134342 0.254056 0.6252 NS

Error 10 0.052879 0.0052879

Total 23 1.0795

-------------------------------------------------------------------------------



= C

12 -1.35028 0.0399899 0.0115441 0.259168 1.0408

Formulation (Fed) =

D

12 -1.75125 0.0940173 0.0271405 0.173556 1.09858

--------------------------------------------------------------------------------


phi = 9.55067




--------------------------------------------------------------------------------

BIOEQUIVALENCE TESTS FOR:Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------

TWO ONE-SIDED T-TESTS FOR: Level C and level D

Lower: t(10df) = -5.9901

Upper: t(10df) = 21.023

t(0.05 - 10df) = 1.8125


293

TABLE 77




Period 1 0.00898698 0.00898698 1.09122 0.3208 NS

Subject(Seq) 10 0.0675536 0.00675536 0.820249 0.6199 NS

Formulation 1 0.134914 0.134914 16.3815 0.002333 ***

Sequence 1 0.000702443 0.000702443 0.085292 0.7762 NS

Error 10 0.0823575 0.00823575

Total 23 0.294514

--------------------------------------------------------------------------------



= C

12 -2.10712 0.0787986 0.0227472 0.121587 1.08199

Formulation (Fed) =

D

12 -2.25708 0.0911039 0.0262994 0.104656 1.09538

--------------------------------------------------------------------------------


phi = 2.86195




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 1.9755

Upper: t(10df) = 10.07

t(0.05 - 10df) = 1.8125


294

TABLE 78




Period 1 0.00194574 0.00194574 0.754295 0.4055 NS

Subject(Seq) 10 0.0126002 0.00126002 0.488465 0.863 NS

Formulation 1 0.604512 0.604512 234.348 2.875e-008 ***

Sequence 1 0.00383719 0.00383719 1.48755 0.2506 NS

Error 10 0.0257954 0.00257954

Total 23 0.64869

--------------------------------------------------------------------------------



= C

12 -1.72936 0.0267593 0.00772475 0.177399 1.02712

Formulation (Fed) =

D

12 -2.04677 0.0574471 0.0165835 0.129151 1.05913

--------------------------------------------------------------------------------


phi = 10.8247




-------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -4.5465

Upper: t(10df) = 26.07

t(0.05 - 10df) = 1.8125


295

TABLE 79




Period 1 0.024259 0.024259 0.179876 0.6805 NS

Subject(Seq) 10 1.17376 0.117376 0.870321 0.5848 NS

Formulation 1 0.0316219 0.0316219 0.23447 0.6387 NS

Sequence 1 0.0522317 0.0522317 0.387287 0.5477 NS

Error 10 1.34865 0.134865

Total 23 2.63053

--------------------------------------------------------------------------------



= C

12 -2.60341 0.0947203 0.0273434 0.0740207 1.09935

Formulation (Fed) =

D

12 -2.67601 0.476752 0.137626 0.0688374 1.61083

--------------------------------------------------------------------------------


phi = 0.342396




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


-------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 1.0041

Upper: t(10df) = 1.9726

t(0.05 - 10df) = 1.8125


296

TABLE 80




Period 1 0.0554996 0.0554996 0.0823077 0.7801 NS

Subject(Seq) 10 6.13924 0.613924 0.910468 0.5575 NS

Formulation 1 11.6379 11.6379 17.2594 0.001966 ***

Sequence 1 0.115277 0.115277 0.170959 0.688 NS

Error 10 6.74294 0.674294

Total 23 24.6908

--------------------------------------------------------------------------------



= C

12 -3.87913 0.186253 0.0537667 0.0206688 1.20473

Formulation (Fed) =

D

12 -5.27184 1.07329 0.309831 0.00513413 2.92497

--------------------------------------------------------------------------------


phi = 2.93763




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -3.4888

Upper: t(10df) = 4.8201

t(0.05 - 10df) = 1.8125


297

TABLE 81




Period 1 0.0110386 0.0110386 0.107777 0.7495 NS

Subject(Seq) 10 1.21152 0.121152 1.18288 0.3979 NS

Formulation 1 0.345771 0.345771 3.37597 0.09601 NS

Sequence 1 0.0696877 0.0696877 0.680405 0.4287 NS

Error 10 1.02421 0.102421

Total 23 2.66223

--------------------------------------------------------------------------------



= C

12 -2.22564 0.128885 0.037206 0.107998 1.13756

Formulation (Fed) =

D

12 -2.4657 0.440426 0.12714 0.0849494 1.55337

--------------------------------------------------------------------------------


phi = 1.29923




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -0.12947

Upper: t(10df) = 3.5453

t(0.05 - 10df) = 1.8125


298

TABLE 82




Period 1 5.4328e-007 5.4328e-007 0.000440654 0.9837 NS

Subject(Seq) 10 0.0127678 0.00127678 1.03559 0.4785 NS

Formulation 1 0.0785245 0.0785245 63.6912 1.203e-005 ***

Sequence 1 7.76067e-009 7.76067e-009 6.29467e-006 0.998 NS

Error 10 0.0123289 0.00123289

Total 23 0.103622

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 -

0.571676

0.00200371 0.00057842 0.564579 1.00201

Formulation (Fed) =

D

12 -

0.457275

0.0477237 0.0137767 0.633006 1.04888

--------------------------------------------------------------------------------


phi = 5.64319




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 7.586

Upper: t(10df) = 23.547

t(0.05 - 10df) = 1.8125


299

TABLE 83




Period 1 8.76752e-005 8.76752e-005 0.455822 0.5149 NS

Subject(Seq) 10 0.00198409 0.000198409 1.03152 0.4809 NS

Formulation 1 0.304432 0.304432 1582.74 2.407e-012 ***

Sequence 1 0.000135249 0.000135249 0.703157 0.4213 NS

Error 10 0.00192345 0.000192345

Total 23 0.308563

--------------------------------------------------------------------------------



= C

12 1.77661 0.0106145 0.00306415 5.90979 1.01067

Formulation (Fed) =

D

12 2.00186 0.016212 0.00468 7.40283 1.01634

--------------------------------------------------------------------------------


phi = 28.1313




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -0.37248

Upper: t(10df) = 79.195

t(0.05 - 10df) = 1.8125


300

TABLE 84




Period 1 0.00105878 0.00105878 0.588428 0.4608 NS

Subject(Seq) 10 0.0194542 0.00194542 1.08118 0.4521 NS

Formulation 1 0.698061 0.698061 387.953 2.492e-009 ***

Sequence 1 3.5191e-006 3.5191e-006 0.00195577 0.9656 NS

Error 10 0.0179934 0.00179934

Total 23 0.736571

--------------------------------------------------------------------------------



= C

12 2.255 0.0410064 0.0118375 9.53525 1.04186

Formulation (Fed) =

D

12 2.59609 0.0426541 0.0123132 13.4112 1.04358

--------------------------------------------------------------------------------


phi = 13.9276




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -6.811

Upper: t(10df) = 32.582

t(0.05 - 10df) = 1.8125


301

TABLE 85




Period 1 0.00387628 0.00387628 1.39156 0.2654 NS

Subject(Seq) 10 0.0202034 0.00202034 0.72529 0.6894 NS

Formulation 1 0.21921 0.21921 78.6948 4.711e-006 ***

Sequence 1 0.000606403 0.000606403 0.217695 0.6508 NS

Error 10 0.0278557 0.00278557

Total 23 0.271751

--------------------------------------------------------------------------------



= C

12 4.86277 0.0389875 0.0112547 129.382 1.03976

Formulation (Fed) =

D

12 4.67163 0.0570658 0.0164735 106.871 1.05873

--------------------------------------------------------------------------------


phi = 6.27275




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 1.4853

Upper: t(10df) = 19.227

t(0.05 - 10df) = 1.8125


302

TABLE 86




Period 1 0.00105894 0.00105894 0.588469 0.4607 NS

Subject(Seq) 10 0.0194549 0.00194549 1.08114 0.4521 NS

Formulation 1 0.698056 0.698056 387.922 2.493e-009 ***

Sequence 1 3.53937e-006 3.53937e-006 0.00196689 0.9655 NS

Error 10 0.0179948 0.00179948

Total 23 0.736568

--------------------------------------------------------------------------------



= C

12 2.75564 0.0410076 0.0118379 15.7311 1.04186

Formulation (Fed) =

D

12 2.41455 0.0426554 0.0123136 11.1847 1.04358

--------------------------------------------------------------------------------


phi = 13.927




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -6.8107

Upper: t(10df) = 32.581

t(0.05 - 10df) = 1.8125


303

TABLE 87




Period 1 0.00319762 0.00319762 0.604706 0.4548 NS

Subject(Seq) 10 0.0574034 0.00574034 1.08556 0.4496 NS

Formulation 1 0.964675 0.964675 182.43 9.536e-008 ***

Sequence 1 0.00134345 0.00134345 0.254061 0.6251 NS

Error 10 0.052879 0.0052879

Total 23 1.0795

--------------------------------------------------------------------------------



= C

12 0.983767 0.0399903 0.0115442 2.67451 1.0408

Formulation (Fed) =

D

12 1.38474 0.0940174 0.0271405 3.99379 1.09858

-------------------------------------------------------------------------------


phi = 9.55067




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -5.9901

Upper: t(10df) = 21.023

t(0.05 - 10df) = 1.8125


304

TABLE 88




Period 1 0.00319787 0.00319787 0.604771 0.4548 NS

Subject(Seq) 10 0.0574039 0.00574039 1.0856 0.4496 NS

Formulation 1 0.964679 0.964679 182.437 9.534e-008 ***

Sequence 1 0.0013435 0.0013435 0.254078 0.6251 NS

Error 10 0.0528774 0.00528774

Total 23 1.0795

--------------------------------------------------------------------------------



= C

12 2.5932 0.0399884 0.0115437 13.3726 1.0408

Formulation (Fed) =

D

12 2.99418 0.0940178 0.0271406 19.9689 1.09858

--------------------------------------------------------------------------------


phi = 9.55083




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -5.9903

Upper: t(10df) = 21.024

t(0.05 - 10df) = 1.8125


305

TABLE 89




Period 1 0.00194616 0.00194616 0.754495 0.4054 NS

Subject(Seq) 10 0.0126001 0.00126001 0.488485 0.863 NS

Formulation 1 0.60451 0.60451 234.359 2.874e-008 ***

Sequence 1 0.003837 0.003837 1.48754 0.2506 NS

Error 10 0.0257942 0.00257942

Total 23 0.648688

--------------------------------------------------------------------------------



= C

12 1.36284 0.0267597 0.00772485 3.90728 1.02712

Formulation (Fed) =

D

12 1.68026 0.0574461 0.0165833 5.36693 1.05913

--------------------------------------------------------------------------------


phi = 10.8249




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -4.5466

Upper: t(10df) = 26.071

t(0.05 - 10df) = 1.8125


306

TABLE 90




Period 1 0.0242592 0.0242592 0.179877 0.6805 NS

Subject(Seq) 10 1.17377 0.117377 0.870321 0.5848 NS

Formulation 1 0.0316221 0.0316221 0.234471 0.6387 NS

Sequence 1 0.0522313 0.0522313 0.387283 0.5477 NS

Error 10 1.34866 0.134866

Total 23 2.63054

--------------------------------------------------------------------------------



= C

12 2.2369 0.0947198 0.0273432 9.36424 1.09935

Formulation (Fed) =

D

12 2.30949 0.476753 0.137627 10.0693 1.61084

--------------------------------------------------------------------------------


phi = 0.342397




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 1.0041

Upper: t(10df) = 1.9726

t(0.05 - 10df) = 1.8125


307

TABLE 91




Period 1 0.00898755 0.00898755 1.0913 0.3208 NS

Subject(Seq) 10 0.0675525 0.00675525 0.820247 0.6199 NS

Formulation 1 0.13491 0.13491 16.3812 0.002333 ***

Sequence 1 0.000702411 0.000702411 0.0852892 0.7762 NS

Error 10 0.0823563 0.00823563

Total 23 0.294508

--------------------------------------------------------------------------------



= C

12 1.74061 0.0787987 0.0227472 5.70084 1.08199

Formulation (Fed) =

D

12 1.89056 0.091103 0.0262992 6.6231 1.09538

--------------------------------------------------------------------------------


phi = 2.86192




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 1.9756

Upper: t(10df) = 10.07

t(0.05 - 10df) = 1.8125


308

TABLE 92




Period 1 0.010344 0.010344 0.399217 0.5417 NS

Subject(Seq) 10 0.128095 0.0128095 0.494369 0.859 NS

Formulation 1 1.75262 1.75262 67.6407 9.234e-006 ***

Sequence 1 0.00354297 0.00354297 0.136738 0.7193 NS

Error 10 0.259107 0.0259107

Total 23 2.15371

--------------------------------------------------------------------------------



= C

12 4.87362 0.097803 0.0282333 130.793 1.10275

Formulation (Fed) =

D

12 5.41408 0.164004 0.0473437 224.546 1.17822

--------------------------------------------------------------------------------


phi = 5.81553




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -4.8288

Upper: t(10df) = 11.62

t(0.05 - 10df) = 1.8125


309

TABLE 93




Period 1 2.8112e-005 2.8112e-005 0.0145593 0.9063 NS

Subject(Seq) 10 0.0173259 0.00173259 0.897312 0.5663 NS

Formulation 1 0.000504624 0.000504624 0.261346 0.6203 NS

Sequence 1 5.96601e-005 5.96601e-005 0.0308981 0.864 NS

Error 10 0.0193086 0.00193086

Total 23 0.0372269

--------------------------------------------------------------------------------



= C

12 3.07509 0.0410174 0.0118407 21.6519 1.04187

Formulation (Fed) =

D

12 3.08426 0.0406935 0.0117472 21.8514 1.04153

--------------------------------------------------------------------------------


phi = 0.361487




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 11.928

Upper: t(10df) = 12.95

t(0.05 - 10df) = 1.8125


310

TABLE 94




Period 1 6.43888e-005 6.43888e-005 0.0110582 0.9183 NS

Subject(Seq) 10 0.0757503 0.00757503 1.30094 0.3427 NS

Formulation 1 0.0376108 0.0376108 6.4593 0.02929 ***

Sequence 1 0.000381074 0.000381074 0.0654459 0.8033 NS

Error 10 0.0582273 0.00582273

Total 23 0.172034

--------------------------------------------------------------------------------



= C

12 -3.08702 0.0954195 0.0275452 0.0456376 1.10012

Formulation (Fed) =

D

12 -3.00785 0.0558158 0.0161126 0.0493978 1.0574

--------------------------------------------------------------------------------


phi = 1.79712




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 4.6215

Upper: t(10df) = 9.7045

t(0.05 - 10df) = 1.8125


311

TABLE 95




Period 1 5.82385e-006 5.82385e-006 0.0256924 0.8758 NS

Subject(Seq) 10 0.00146793 0.000146793 0.647589 0.7478 NS

Formulation 1 0.149226 0.149226 658.323 1.857e-010 ***

Sequence 1 8.80834e-005 8.80834e-005 0.388587 0.547 NS

Error 10 0.00226676 0.000226676

Total 23 0.153055

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 -

0.422247

0.00610468 0.00176227 0.655572 1.00612

Formulation (Fed) =

D

12 -

0.264541

0.0176292 0.0050891 0.767558 1.01779

--------------------------------------------------------------------------------


phi = 18.1428




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 10.646

Upper: t(10df) = 61.962

t(0.05 - 10df) = 1.8125


312

TABLE 96




Period 1 0.000697527 0.000697527 0.146266 0.7101 NS

Subject(Seq) 10 0.0675211 0.00675211 1.41587 0.2963 NS

Formulation 1 0.409702 0.409702 85.9114 3.173e-006 ***

Sequence 1 0.000222482 0.000222482 0.0466529 0.8333 NS

Error 10 0.0476889 0.00476889

Total 23 0.525832

--------------------------------------------------------------------------------



= C

12 2.19358 0.0182007 0.00525408 8.96724 1.01837

Formulation (Fed) =

D

12 2.45489 0.101124 0.0291919 11.6451 1.10641

--------------------------------------------------------------------------------


phi = 6.55406




-------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -1.3538

Upper: t(10df) = 17.184

t(0.05 - 10df) = 1.8125


313

TABLE 97




Period 1 0.000814959 0.000814959 0.705404 0.4206 NS

Subject(Seq) 10 0.0256812 0.00256812 2.22288 0.1119 NS

Formulation 1 0.490231 0.490231 424.329 1.608e-009 ***

Sequence 1 4.25953e-005 4.25953e-005 0.0368692 0.8516 NS

Error 10 0.0115531 0.00115531

Total 23 0.528323

--------------------------------------------------------------------------------



= C

12 9.19298 0.0305645 0.00882322 9.82787 1.03104

Formulation (Fed) =

D

12 9.47882 0.0502862 0.0145164 13.0797 1.05157

--------------------------------------------------------------------------------


phi = 14.5659




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -4.5183

Upper: t(10df) = 36.68

t(0.05 - 10df) = 1.8125


314

TABLE 98




Period 1 0.000471996 0.000471996 0.0228826 0.8828 NS

Subject(Seq) 10 0.203999 0.0203999 0.988993 0.5068 NS

Formulation 1 0.218843 0.218843 10.6096 0.008614 ***

Sequence 1 0.000935425 0.000935425 0.0453498 0.8356 NS

Error 10 0.206269 0.0206269

Total 23 0.630518

--------------------------------------------------------------------------------



= C

12 7.10119 0.135013 0.038975 12.134 1.14455

Formulation (Fed) =

D

12 7.29217 0.138551 0.0399962 14.6875 1.14861

--------------------------------------------------------------------------------


phi = 2.30322




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 0.54854

Upper: t(10df) = 7.063

t(0.05 - 10df) = 1.8125


315

TABLE 99




Period 1 0.000793229 0.000793229 0.495413 0.4976 NS

Subject(Seq) 10 0.025213 0.0025213 1.57468 0.2428 NS

Formulation 1 0.456398 0.456398 285.044 1.117e-008 ***

Sequence 1 9.08345e-006 9.08345e-006 0.00567308 0.9414 NS

Error 10 0.0160115 0.00160115

Total 23 0.498424

--------------------------------------------------------------------------------



= C

12 9.31013 0.0334667 0.009661 11.0494 1.03403

Formulation (Fed) =

D

12 9.58594 0.0519673 0.0150017 14.5586 1.05334

--------------------------------------------------------------------------------


phi = 11.9383




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -3.2235

Upper: t(10df) = 30.543

t(0.05 - 10df) = 1.8125


316

TABLE 100




Period 1 4.14602e-005 4.14602e-005 0.0030857 0.9568 NS

Subject(Seq) 10 0.162185 0.0162185 1.20707 0.3859 NS

Formulation 1 0.0431669 0.0431669 3.21272 0.1033 NS

Sequence 1 0.00112904 0.00112904 0.0840292 0.7778 NS

Error 10 0.134362 0.0134362

Total 23 0.340885

--------------------------------------------------------------------------------



= C

12 2.39622 0.117468 0.03391 10.9816 1.12465

Formulation (Fed) =

D

12 2.3114 0.115181 0.0332498 10.0886 1.12208

--------------------------------------------------------------------------------


phi = 1.26742




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 2.923

Upper: t(10df) = 6.5078

t(0.05 - 10df) = 1.8125


317

TABLE 101




Period 1 0.00157984 0.00157984 0.794316 0.3937 NS

Subject(Seq) 10 0.0337533 0.00337533 1.69706 0.2087 NS

Formulation 1 0.719279 0.719279 361.641 3.511e-009 ***

Sequence 1 9.93077e-005 9.93077e-005 0.0499301 0.8277 NS

Error 10 0.0198893 0.00198893

Total 23 0.774601

--------------------------------------------------------------------------------



= C

12 11.9428 0.0475587 0.013729 153.700 1.04871

Formulation (Fed) =

D

12 12.289 0.0526062 0.0151861 217.291 1.05401

--------------------------------------------------------------------------------


phi = 13.4469




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -6.7608

Upper: t(10df) = 31.273

t(0.05 - 10df) = 1.8125


318

TABLE 102




Period 1 0.00111999 0.00111999 0.197807 0.666 NS

Subject(Seq) 10 0.0521763 0.00521763 0.921511 0.5501 NS

Formulation 1 0.487255 0.487255 86.0565 3.149e-006 ***

Sequence 1 2.21544e-005 2.21544e-005 0.00391279 0.9514 NS

Error 10 0.0566203 0.00566203

Total 23 0.597194

--------------------------------------------------------------------------------



= C

12 12.3852 0.0672281 0.0194071 239.241 1.06954

Formulation (Fed) =

D

12 12.6702 0.073992 0.0213596 318.125 1.0768

--------------------------------------------------------------------------------


phi = 6.55959




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -2.0127

Upper: t(10df) = 16.541

t(0.05 - 10df) = 1.8125


319

TABLE 103




Period 1 0.000377127 0.000377127 0.0140202 0.9081 NS

Subject(Seq) 10 0.279773 0.0279773 1.04009 0.4758 NS

Formulation 1 0.165837 0.165837 6.16524 0.03238 ***

Sequence 1 0.00132444 0.00132444 0.0492378 0.8289 NS

Error 10 0.268987 0.0268987

Total 23 0.716299

--------------------------------------------------------------------------------



= C

12 11.3493 0.162567 0.0469292 849.062 1.17653

Formulation (Fed) =

D

12 11.5156 0.153668 0.0443601 1002.63 1.1661

--------------------------------------------------------------------------------


phi = 1.75574




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 0.84969

Upper: t(10df) = 5.8157

t(0.05 - 10df) = 1.8125


320

TABLE 104




Period 1 0.00130964 0.00130964 0.277131 0.6101 NS

Subject(Seq) 10 0.0877941 0.00877941 1.8578 0.1716 NS

Formulation 1 0.756041 0.756041 159.985 1.778e-007 ***

Sequence 1 0.000507997 0.000507997 0.107497 0.7498 NS

Error 10 0.047257 0.0047257

Total 23 0.89291

--------------------------------------------------------------------------------



= C

12 -1.21248 0.0877018 0.0253173 0.29746 1.09166

Formulation (Fed) =

D

12 -1.56745 0.0689276 0.0198977 0.208576 1.07136

--------------------------------------------------------------------------------


phi = 8.94385




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = -4.6974

Upper: t(10df) = 20.6

t(0.05 - 10df) = 1.8125


321

TABLE 105




Period 1 0.0242592 0.0242592 0.179877 0.6805 NS

Subject(Seq) 10 1.17377 0.117377 0.870321 0.5848 NS

Formulation 1 0.0316221 0.0316221 0.234471 0.6387 NS

Sequence 1 0.0522313 0.0522313 0.387283 0.5477 NS

Error 10 1.34866 0.134866

Total 23 2.63054

--------------------------------------------------------------------------------



= C

12 2.2369 0.0947198 0.0273432 9.36424 1.09935

Formulation (Fed) =

D

12 2.30949 0.476753 0.137627 10.0693 1.61084

--------------------------------------------------------------------------------


phi = 0.342397




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


--------------------------------------------------------------------------------


Level C and level D

Lower: t(10df) = 1.0041

Upper: t(10df) = 1.9726

t(0.05 - 10df) = 1.8125


322

4. STATISTICAL ANALYSIS FOR NON LOG

TRANSFORMED DATA

A) EC5-F11 (Pellets) Under Fed and Fasted Conditions; (A = Fasted

and B = Fed)

TABLE 106




Period 1 4.64733e-005 4.64733e-005 0.132799 0.7231 NS

Subject(Seq) 10 0.00511609 0.000511609 1.46194 0.2796 NS

Formulation 1 0.025342 0.025342 72.4156 6.827e-006 ***

Sequence 1 3.91476e-006 3.91476e-006 0.0111865 0.9179 NS

Error 10 0.00349953 0.000349953

Total 23 0.034008

--------------------------------------------------------------------------------



= A

12 0.25305 0.0251248 0.0072529 0.251909 1.1046

Formulation (Fed) =

B

12 0.18806 0.0125125 0.0036120

6

0.187674 1.06952

--------------------------------------------------------------------------------


phi = 6.01729




-------------------------------------------------------------------------------


Level A and level B


Mean Ratio (Fasted/Fed) = 0.743174



t(0.05 - 10df) = 1.8125


323

TABLE 107




Period 1 2.06304e-005 2.06304e-005 0.229343 0.6423 NS

Subject(Seq) 10 0.000376214 3.76214e-005 0.418228 0.9073 NS

Formulation 1 0.00325297 0.00325297 36.1625 0.0001297 ***

Sequence 1 3.55875e-007 3.55875e-007 0.00395618 0.9511 NS

Error 10 0.000899542 8.99542e-005

Total 23 0.00454971

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 0.125336 0.00872652 0.00251913 0.12505 1.07369

Formulation (Fed) =

B

12 0.102051 0.00646015 0.00186489 0.101879 1.06117

-------------------------------------------------------------------------------


phi = 4.2522


1 - (Power of the test) = 0.00032386


--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


324

TABLE 108


LATIN SQUARE DESIGN: ANOVA TABLE for ALPHA


Period 1 5.53824e-006 5.53824e-006 0.0756522 0.7889 NS

Subject(Seq) 10 0.000800507 8.00507e-005 1.09349 0.4452 NS

Formulation 1 0.0146623 0.0146623 200.286 6.107e-008 ***

Sequence 1 3.87287e-006 3.87287e-006 0.0529033 0.8227 NS

Error 10 0.000732066 7.32066e-005

Total 23 0.0162043

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 0.1816 0.0101813 0.00293909 0.181348 1.05609

Formulation (Fed) =

B

12 0.132167 0.00604328 0.00174454 0.132043 1.04579

--------------------------------------------------------------------------------


phi = 10.0072




-------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


325

TABLE 109



Period 1 6.50631e-007 6.50631e-007 0.00346694 0.9542 NS

Subject(Seq) 10 0.00073304 7.3304e-005 0.390606 0.9229 NS

Formulation 1 0.000166901 0.000166901 0.889346 0.3679 NS

Sequence 1 5.28642e-005 5.28642e-005 0.281691 0.6072 NS

Error 10 0.00187667 0.000187667

Total 23 0.00283013

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 0.0708688 0.0124702 0.00359984 0.0695492 1.24355

Formulation (Fed)

= B

12 0.0761429 0.00930618 0.00268646 0.075625 1.13002

--------------------------------------------------------------------------------


phi = 0.666838




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


326

TABLE 110



Period 1 1.10729e-008 1.10729e-008 0.000137772 0.9909 NS

Subject(Seq) 10 0.000364873 3.64873e-005 0.453984 0.8855 NS

Formulation 1 0.00161814 0.00161814 20.1332 0.001166 ***

Sequence 1 3.81909e-007 3.81909e-007 0.00475181 0.9464 NS

Error 10 0.000803713 8.03713e-005

Total 23 0.00278711

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 0.0244791 0.00948452 0.00273795 0.0230946 1.40817

Formulation

(Fed) = B

12 0.00805688 0.00403915 0.001166 0.00736747 1.51576

--------------------------------------------------------------------------------


phi = 3.17279




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


327

TABLE 111



Period 1 1.6287e-006 1.6287e-006 0.00863444 0.9278 NS

Subject(Seq) 10 0.00183675 0.000183675 0.973741 0.5164 NS

Formulation 1 0.000119 0.000119 0.630869 0.4455 NS

Sequence 1 0.000109284 0.000109284 0.579363 0.4641 NS

Error 10 0.00188628 0.000188628

Total 23 0.00395294

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 0.102655 0.017528 0.00505991 0.100861 1.23521

Formulation (Fed)

= B

12 0.0982014 0.00642715 0.00185536 0.0980159 1.06576

--------------------------------------------------------------------------------


phi = 0.561636




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


328

TABLE 112



Period 1 1.87209e-006 1.87209e-006 0.0119799 0.915 NS

Subject(Seq) 10 0.00404827 0.000404827 2.59056 0.07462 NS

Formulation 1 0.0383374 0.0383374 245.328 2.306e-008 ***

Sequence 1 6.71301e-006 6.71301e-006 0.0429578 0.84 NS

Error 10 0.0015627 0.00015627

Total 23 0.0439569

--------------------------------------------------------------------------------



= A

12 0.566815 0.0195725 0.00565009 0.56651 1.03475

Formulation (Fed) =

B

12 0.64675 0.0113043 0.00326326 0.646658 1.01774

--------------------------------------------------------------------------------


phi = 11.0754




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


329

TABLE 113



Period 1 1.3515e-005 1.3515e-005 0.000322382 0.986 NS

Subject(Seq) 10 0.32858 0.032858 0.783782 0.6463 NS

Formulation 1 12.7067 12.7067 303.101 8.29e-009 ***

Sequence 1 0.00331938 0.00331938 0.0791791 0.7841 NS

Error 10 0.419224 0.0419224

Total 23 13.4578

--------------------------------------------------------------------------------



= A

12 5.82541 0.246223 0.0710784 5.82051 1.04411

Formulation (Fed) =

B

12 7.28067 0.0875187 0.0252645 7.28018 1.01217

--------------------------------------------------------------------------------


phi = 12.3106




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


330

TABLE 114



Period 1 6.01667e-005 6.01667e-005 0.312095 0.5887 NS

Subject(Seq) 10 0.00464117 0.000464117 2.40745 0.09101 NS

Formulation 1 0.075264 0.075264 390.407 2.416e-009 ***

Sequence 1 8.81667e-005 8.81667e-005 0.457336 0.5142 NS

Error 10 0.00192783 0.000192783

Total 23 0.0819813

--------------------------------------------------------------------------------



= A

12 0.661333 0.0150534 0.00434555 0.661179 1.02275

Formulation (Fed) =

B

12 0.773333 0.0195975 0.0056573 0.773103 1.02589

--------------------------------------------------------------------------------


phi = 13.9715




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


331

TABLE 115



Period 1 0.00123855 0.00123855 0.00346029 0.9543 NS

Subject(Seq) 10 2.2683 0.22683 0.633722 0.7582 NS

Formulation 1 88.3484 88.3484 246.83 2.239e-008 ***

Sequence 1 0.00805237 0.00805237 0.0224969 0.8838 NS

Error 10 3.57932 0.357932

Total 23 94.2053

--------------------------------------------------------------------------------



= A

12 9.6081 0.591503 0.170752 9.59182 1.06234

Formulation (Fed) =

B

12 13.4454 0.427283 0.123346 13.439 1.03275

--------------------------------------------------------------------------------


phi = 11.1092




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


332

TABLE 116



Period 1 8.60212 8.60212 0.207043 0.6588 NS

Subject(Seq) 10 482.244 48.2244 1.1607 0.4092 NS

Formulation 1 1473.08 1473.08 35.4552 0.0001404 ***

Sequence 1 0.189464 0.189464 0.00456016 0.9475 NS

Error 10 415.476 41.5476

Total 23 2379.59

--------------------------------------------------------------------------------



= A

12 125.306 7.96595 2.29957 125.057 1.06949

Formulation (Fed) = B 12 109.637 4.3536 1.25678 109.556 1.04145

-------------------------------------------------------------------------------


phi = 4.21042


1 - (Power of the test) = 0.000391238


--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


333

TABLE 117



Period 1 0.00282968 0.00282968 0.00447053 0.948 NS

Subject(Seq) 10 4.62631 0.462631 0.730898 0.6853 NS

Formulation 1 121.358 121.358 191.73 7.523e-008 ***

Sequence 1 0.00366548 0.00366548 0.00579099 0.9408 NS

Error 10 6.32963 0.632963

Total 23 132.32

--------------------------------------------------------------------------------



= A

12 15.6642 0.929217 0.268242 15.6383 1.06233

Formulation (Fed) =

B

12 11.1669 0.364886 0.105334 11.1615 1.03275

--------------------------------------------------------------------------------


phi = 9.79106




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


334

TABLE 118



Period 1 0.0114023 0.0114023 0.244655 0.6315 NS

Subject(Seq) 10 1.05901 0.105901 2.27228 0.1058 NS

Formulation 1 5.26699 5.26699 113.012 9.049e-007 ***

Sequence 1 0.012928 0.012928 0.277391 0.6099 NS

Error 10 0.466055 0.0466055

Total 23 6.81638

--------------------------------------------------------------------------------



= A

12 2.76415 0.277705 0.0801666 2.75158 1.1046

Formulation (Fed) =

B

12 3.70107 0.252456 0.0728777 3.69335 1.06952

--------------------------------------------------------------------------------


phi = 7.51705




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


335

TABLE 119



Period 1 0.285057 0.285057 0.24465 0.6316 NS

Subject(Seq) 10 26.475 2.6475 2.27222 0.1058 NS

Formulation 1 131.675 131.675 113.01 9.05e-007 ***

Sequence 1 0.323222 0.323222 0.277406 0.6099 NS

Error 10 11.6516 1.16516

Total 23 170.41

--------------------------------------------------------------------------------



= A

12 13.8207 1.38852 0.400831 13.7579 1.1046


--------------------------------------------------------------------------------


phi = 7.51698




-------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


336

TABLE 120



Period 1 0.00591827 0.00591827 0.119669 0.7366 NS

Subject(Seq) 10 0.530083 0.0530083 1.07184 0.4574 NS

Formulation 1 12.2151 12.2151 246.994 2.232e-008 ***

Sequence 1 0.00582754 0.00582754 0.117834 0.7385 NS

Error 10 0.494553 0.0494553

Total 23 13.2515

--------------------------------------------------------------------------------



= A

12 3.82731 0.203131 0.0586387 3.82218 1.05609

Formulation (Fed) =

B

12 5.25414 0.230119 0.0664295 5.24939 1.04579

-------------------------------------------------------------------------------


phi = 11.1129




-------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


337

TABLE 121



Period 1 0.821148 0.821148 0.135163 0.7208 NS

Subject(Seq) 10 37.1699 3.71699 0.611824 0.7746 NS

Formulation 1 5.92144 5.92144 0.974681 0.3468 NS

Sequence 1 2.40374 2.40374 0.395661 0.5434 NS

Error 10 60.7526 6.07526

Total 23 107.069

--------------------------------------------------------------------------------



= A

12 10.2223 2.80937 0.810997 9.96628 1.24355


--------------------------------------------------------------------------------


phi = 0.698098




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


338

TABLE 122



Period 1 0.035269 0.035269 0.146103 0.7103 NS

Subject(Seq) 10 0.93029 0.093029 0.385377 0.9257 NS

Formulation 1 9.49754 9.49754 39.344 9.232e-005 ***

Sequence 1 0.000546356 0.000546356 0.00226331 0.963 NS

Error 10 2.41397 0.241397

Total 23 12.8776

--------------------------------------------------------------------------------



= A

12 5.55601 0.403295 0.116421 5.54298 1.07369

Formulation (Fed) =

B

12 6.81416 0.380306 0.109785 6.80361 1.06117

--------------------------------------------------------------------------------


phi = 4.43531


1 - (Power of the test) = 0.000137398


--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


339

TABLE 123



Period 1 6.62656 6.62656 0.0112817 0.9175 NS

Subject(Seq) 10 2437.91 243.791 0.415055 0.9092 NS

Formulation 1 37625.1 37625.1 64.057 1.173e-005 ***

Sequence 1 0.739557 0.739557 0.0012591 0.9724 NS

Error 10 5873.7 587.37

Total 23 45944.1

--------------------------------------------------------------------------------



= A

12 136.28 23.8661 6.88955 134.539 1.17799


--------------------------------------------------------------------------------


phi = 5.65937




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


340

TABLE 124



Period 1 0.230104 0.230104 0.683175 0.4278 NS

Subject(Seq) 10 4.65587 0.465587 1.38232 0.3092 NS

Formulation 1 3.36571 3.36571 9.99272 0.01014 ***

Sequence 1 0.016907 0.016907 0.0501967 0.8272 NS

Error 10 3.36816 0.336816

Total 23 11.6367

--------------------------------------------------------------------------------



= A

12 21.6358 0.735955 0.212452 21.6238 1.03592

Formulation (Fed) =

B

12 22.3848 0.458567 0.132377 22.3805 1.0207

--------------------------------------------------------------------------------


phi = 2.23525




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


341

TABLE 125



Period 1 7.11041e-006 7.11041e-006 0.950097 0.3527 NS

Subject(Seq) 10 0.00010968 1.0968e-005 1.46555 0.2784 NS

Formulation 1 3.85718e-005 3.85718e-005 5.15399 0.04655 ***

Sequence 1 1.50946e-006 1.50946e-006 0.201696 0.6629 NS

Error 10 7.48387e-005 7.48387e-006

Total 23 0.00023171

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 0.0450401 0.00398747 0.00115108 0.044896 1.08468

Formulation (Fed)

= B

12 0.0475756 0.00128766 0.000371715 0.0475596 1.02744

--------------------------------------------------------------------------------


phi = 1.6053




-------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


342

TABLE 126



Period 1 0.112138 0.112138 0.850224 0.3782 NS

Subject(Seq) 10 0.627813 0.0627813 0.476005 0.8713 NS

Formulation 1 53.3702 53.3702 404.651 2.028e-009 ***

Sequence 1 0.140436 0.140436 1.06478 0.3264 NS

Error 10 1.31892 0.131892

Total 23 55.5695

--------------------------------------------------------------------------------



= A

12 8.96426 0.236887 0.0683835 8.96143 1.02651

Formulation (Fed) =

B

12 11.9467 0.379238 0.109477 11.9412 1.03212

-------------------------------------------------------------------------------


phi = 14.2241




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


343

TABLE 127



Period 1 2454.91 2454.91 0.0228657 0.8828 NS

Subject(Seq) 10 1.35173e+006 135173 1.25903 0.3614 NS

Formulation 1 7.09292e+007 7.09292e+007 660.653 1.825e-010 ***

Sequence 1 5886.59 5886.59 0.0548293 0.8196 NS

Error 10 1.07362e+006 107362

Total 23 7.33629e+007

--------------------------------------------------------------------------------



= A

12 9819.6 341.755 98.6563 9814.19 1.03521


--------------------------------------------------------------------------------


phi = 18.1749




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


344

TABLE 128



Period 1 6692.93 6692.93 0.517634 0.4883 NS

Subject(Seq) 10 216292 21629.2 1.67281 0.215 NS

Formulation 1 959763 959763 74.2285 6.116e-006 ***

Sequence 1 27.7501 27.7501 0.0021462 0.964 NS

Error 10 129298 12929.8

Total 23 1.31207e+006

--------------------------------------------------------------------------------



= A

12 1230.65 134.443 38.8102 1222.65 1.13295


--------------------------------------------------------------------------------


phi = 6.09215




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


345

TABLE 129



Period 1 1040.17 1040.17 0.00684447 0.9357 NS

Subject(Seq) 10 2.11596e+006 211596 1.39234 0.3053 NS

Formulation 1 8.83907e+007 8.83907e+007 581.626 3.419e-010 ***

Sequence 1 6720.11 6720.11 0.0442195 0.8377 NS

Error 10 1.51972e+006 151972

Total 23 9.20341e+007

--------------------------------------------------------------------------------



= A

12 11050.2 405.311 117.003 11043.4 1.03754


--------------------------------------------------------------------------------


phi = 17.0532




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


346

TABLE 130



Period 1 0.479437 0.479437 0.760091 0.4037 NS

Subject(Seq) 10 9.68599 0.968599 1.5356 0.2549 NS

Formulation 1 0.197189 0.197189 0.31262 0.5884 NS

Sequence 1 0.0134692 0.0134692 0.0213538 0.8867 NS

Error 10 6.30762 0.630762

Total 23 16.6837

--------------------------------------------------------------------------------



= A

12 11.1248 1.07027 0.308961 11.0713 1.11182

Formulation (Fed) =

B

12 10.9436 0.594386 0.171585 10.9288 1.05581

--------------------------------------------------------------------------------


phi = 0.395361




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


347

TABLE 131



Period 1 349692 349692 0.00658651 0.9369 NS

Subject(Seq) 10 4.23819e+008 4.23819e+007 0.798269 0.6357 NS

Formulation 1 2.80087e+010 2.80087e+010 527.548 5.526e-010 ***

Sequence 1 3.32047e+006 3.32047e+006 0.0625416 0.8076 NS

Error 10 5.30922e+008 5.30922e+007

Total 23 2.89671e+010

--------------------------------------------------------------------------------



A

12 152435 6760.08 1951.47 152300 1.04474

Formulation (Fed) = B 12 220759 6436.57 1858.08 220672 1.0297

--------------------------------------------------------------------------------


phi = 16.2411




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


348

TABLE 132



Period 1 3.61131e+007 3.61131e+007 0.199723 0.6645 NS

Subject(Seq) 10 2.51262e+009 2.51262e+008 1.3896 0.3063 NS

Formulation 1 5.32281e+010 5.32281e+010 294.377 9.552e-009 ***

Sequence 1 5.06002e+006 5.06002e+006 0.0279843 0.8705 NS

Error 10 1.80816e+009 1.80816e+008

Total 23 5.759e+010

--------------------------------------------------------------------------------



A

12 239195 13835.2 3993.87 238800 1.06315


--------------------------------------------------------------------------------


phi = 12.1321




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


349

TABLE 133



Period 1 4.35792e+007 4.35792e+007 0.533857 0.4818 NS

Subject(Seq) 10 1.37e+009 1.37e+008 1.67828 0.2135 NS

Formulation 1 4.0138e+009 4.0138e+009 49.17 3.662e-005 ***

Sequence 1 183400 183400 0.0022467 0.9631 NS

Error 10 8.1631e+008 8.1631e+007

Total 23 6.24387e+009

--------------------------------------------------------------------------------



= A

12 86759.7 11020.6 3181.38 85975.2 1.1601


--------------------------------------------------------------------------------


phi = 4.95833




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


350

TABLE 134



Period 1 0.000164065 0.000164065 0.630604 0.4456 NS

Subject(Seq) 10 0.0025778 0.00025778 0.990809 0.5057 NS

Formulation 1 0.0500037 0.0500037 192.195 7.437e-008 ***

Sequence 1 3.76351e-005 3.76351e-005 0.144655 0.7117 NS

Error 10 0.00260171 0.000260171

Total 23 0.055385

--------------------------------------------------------------------------------


Formulation

(Fasted) = A

12 0.303265 0.0214587 0.0061946 0.302539 1.07588

Formulation (Fed) =

B

12 0.211974 0.00535949 0.00154715 0.211912 1.0257

--------------------------------------------------------------------------------


phi = 9.80294




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


351

TABLE 135



Period 1 0.821148 0.821148 0.135163 0.7208 NS

Subject(Seq) 10 37.1699 3.71699 0.611824 0.7746 NS

Formulation 1 5.92144 5.92144 0.974681 0.3468 NS

Sequence 1 2.40374 2.40374 0.395661 0.5434 NS

Error 10 60.7526 6.07526

Total 23 107.069

--------------------------------------------------------------------------------



= A

12 10.2223 2.80937 0.810997 9.96628 1.24355


--------------------------------------------------------------------------------


phi = 0.698098




--------------------------------------------------------------------------------


Level A and level B





t(0.05 - 10df) = 1.8125


352

B) Ganaton OD (Tablet) Under Fed and Fasted Conditions; (C =

Fasted and D = Fed)

TABLE 136



Period 1 0.00010162 0.00010162 0.551859 0.4746 NS

Subject(Seq) 10 0.00203094 0.000203094 1.10293 0.44 NS

Formulation 1 0.0434649 0.0434649 236.041 2.777e-008 ***

Sequence 1 2.19479e-005 2.19479e-005 0.11919 0.7371 NS

Error 10 0.00184141 0.000184141

Total 23 0.0474608

--------------------------------------------------------------------------------



= C

12 0.25936 0.0105147 0.00303534 0.259168 1.0408

Formulation (Fed) =

D

12 0.174247 0.0158967 0.00458899 0.173556 1.09858

--------------------------------------------------------------------------------


phi = 10.8637




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


353

TABLE 137



Period 1 0.000113851 0.000113851 1.07961 0.3233 NS

Subject(Seq) 10 0.000865563 8.65563e-005 0.820778 0.6196 NS

Formulation 1 0.00170565 0.00170565 16.174 0.002432 ***

Sequence 1 7.52718e-006 7.52718e-006 0.0713772 0.7948 NS

Error 10 0.00105456 0.000105456

Total 23 0.00374715

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 0.121926 0.00929521 0.0026833 0.121587 1.08199

Formulation (Fed) =

D

12 0.105066 0.00995944 0.00287504 0.104656 1.09538

--------------------------------------------------------------------------------


phi = 2.84376




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


354

TABLE 138



Period 1 2.73408e-005 2.73408e-005 0.517353 0.4884 NS

Subject(Seq) 10 0.000232194 2.32194e-005 0.439365 0.8946 NS

Formulation 1 0.0138879 0.0138879 262.792 1.655e-008 ***

Sequence 1 7.24329e-005 7.24329e-005 1.3706 0.2689 NS

Error 10 0.000528475 5.28475e-005

Total 23 0.0147484

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 0.177457 0.00481697 0.00139054 0.177399 1.02712

Formulation (Fed) =

D

12 0.129347 0.00741747 0.00214124 0.129151 1.05913

--------------------------------------------------------------------------------


phi = 11.4628




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


355

TABLE 139



Period 1 6.08437e-005 6.08437e-005 0.155828 0.7013 NS

Subject(Seq) 10 0.00325581 0.000325581 0.833854 0.6103 NS

Formulation 1 5.69615e-007 5.69615e-007 0.00145886 0.9703 NS

Sequence 1 0.000162906 0.000162906 0.417224 0.5329 NS

Error 10 0.00390453 0.000390453

Total 23 0.00738466

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 0.074321 0.00689156 0.00198942 0.0740207 1.09935

Formulation (Fed)

= D

12 0.0746291 0.0249757 0.00720987 0.0688374 1.61083

--------------------------------------------------------------------------------


phi = 0.0270079




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


356

TABLE 140



Period 1 5.29951e-007 5.29951e-007 0.0108702 0.919 NS

Subject(Seq) 10 0.00031268 3.1268e-005 0.641362 0.7525 NS

Formulation 1 0.00101905 0.00101905 20.9025 0.001023 ***

Sequence 1 2.90278e-006 2.90278e-006 0.0595411 0.8122 NS

Error 10 0.000487525 4.87525e-005

Total 23 0.00182268

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 0.0210028 0.00397178 0.00114655 0.0206688 1.20473

Formulation

(Fed) = D

12 0.00797044 0.00756855 0.00218485 0.00513413 2.92497

--------------------------------------------------------------------------------


phi = 3.23284




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


357

TABLE 141



Period 1 2.6614e-006 2.6614e-006 0.00627127 0.9384 NS

Subject(Seq) 10 0.00585992 0.000585992 1.38082 0.3097 NS

Formulation 1 0.00192461 0.00192461 4.5351 0.05906 NS

Sequence 1 0.000409426 0.000409426 0.964762 0.3492 NS

Error 10 0.0042438 0.00042438

Total 23 0.0124404

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 0.10885 0.0147584 0.00426039 0.107998 1.13756

Formulation (Fed) =

D

12 0.0909398 0.0271693 0.00784311 0.0849494 1.55337

--------------------------------------------------------------------------------


phi = 1.50584




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


358

TABLE 142



Period 1 9.588e-007 9.588e-007 0.0020105 0.9651 NS

Subject(Seq) 10 0.00492583 0.000492583 1.03289 0.4801 NS

Formulation 1 0.0286315 0.0286315 60.0373 1.556e-005 ***

Sequence 1 3.7475e-007 3.7475e-007 0.000785812 0.9782 NS

Error 10 0.00476895 0.000476895

Total 23 0.0383276

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 0.56458 0.00113027 0.00032628 0.564579 1.00201

Formulation (Fed) =

D

12 0.633659 0.0296679 0.0085644 0.633006 1.04888

--------------------------------------------------------------------------------


phi = 5.47893




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


359

TABLE 143



Period 1 0.0028026 0.0028026 0.292615 0.6004 NS

Subject(Seq) 10 0.0977259 0.00977259 1.02034 0.4876 NS

Formulation 1 13.3855 13.3855 1397.56 4.467e-012 ***

Sequence 1 0.00510154 0.00510154 0.532644 0.4822 NS

Error 10 0.0957777 0.00957777

Total 23 13.5869

--------------------------------------------------------------------------------



= C

12 5.9101 0.0630246 0.0181936 5.90979 1.01067

Formulation (Fed) =

D

12 7.40372 0.11974 0.034566 7.40283 1.01634

--------------------------------------------------------------------------------


phi = 26.4345




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


360

TABLE 144



Period 1 0.141453 0.141453 0.585159 0.462 NS

Subject(Seq) 10 2.59546 0.259546 1.07368 0.4564 NS

Formulation 1 90.3028 90.3028 373.561 2.997e-009 ***

Sequence 1 0.00632061 0.00632061 0.0261469 0.8748 NS

Error 10 2.41735 0.241735

Total 23 95.4634

--------------------------------------------------------------------------------



= C

12 9.54267 0.3967 0.114518 9.53525 1.04186

Formulation (Fed) =

D

12 13.4222 0.558366 0.161187 13.4112 1.04358

--------------------------------------------------------------------------------


phi = 13.6668




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


361

TABLE 145



Period 1 48.7222 48.7222 1.3269 0.2762 NS

Subject(Seq) 10 264.48 26.448 0.720286 0.6932 NS

Formulation 1 3022.52 3022.52 82.3155 3.849e-006 ***

Sequence 1 3.77111 3.77111 0.102703 0.7552 NS

Error 10 367.188 36.7188

Total 23 3706.68

--------------------------------------------------------------------------------



= C

12 129.473 5.14986 1.48664 129.382 1.03976

Formulation (Fed) = D 12 107.029 5.97289 1.72423 106.871 1.05873

--------------------------------------------------------------------------------


phi = 6.41543




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


362

TABLE 146



Period 1 0.187974 0.187974 0.566531 0.469 NS

Subject(Seq) 10 3.58626 0.358626 1.08086 0.4523 NS

Formulation 1 124.155 124.155 374.188 2.973e-009 ***

Sequence 1 0.00179574 0.00179574 0.00541214 0.9428 NS

Error 10 3.31798 0.331798

Total 23 131.249

--------------------------------------------------------------------------------



= C

12 15.7431 0.636489 0.183738 15.7311 1.04186

Formulation (Fed) =

D

12 11.1942 0.489686 0.14136 11.1847 1.04358

--------------------------------------------------------------------------------


phi = 13.6782




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


363

TABLE 147



Period 1 0.0524068 0.0524068 0.639482 0.4425 NS

Subject(Seq) 10 0.871264 0.0871264 1.06314 0.4624 NS

Formulation 1 10.6749 10.6749 130.258 4.675e-007 ***

Sequence 1 0.0317117 0.0317117 0.386956 0.5478 NS

Error 10 0.819519 0.0819519

Total 23 12.4498

--------------------------------------------------------------------------------



= C

12 2.67645 0.105526 0.0304626 2.67451 1.0408

Formulation (Fed) =

D

12 4.0103 0.387581 0.111885 3.99379 1.09858

--------------------------------------------------------------------------------


phi = 8.07024




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


364

TABLE 148



Period 1 1.31026 1.31026 0.639537 0.4425 NS

Subject(Seq) 10 21.7819 2.17819 1.06317 0.4624 NS

Formulation 1 266.873 266.873 130.26 4.675e-007 ***

Sequence 1 0.79283 0.79283 0.386979 0.5478 NS

Error 10 20.4877 2.04877

Total 23 311.245

--------------------------------------------------------------------------------



= C

12 13.3823 0.527604 0.152306 13.3726 1.0408

Formulation (Fed) =

D

12 20.0515 1.93791 0.559428 19.9689 1.09858

--------------------------------------------------------------------------------


phi = 8.07032




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


365

TABLE 149



Period 1 0.0628449 0.0628449 0.976514 0.3464 NS

Subject(Seq) 10 0.3609 0.03609 0.560783 0.8122 NS

Formulation 1 12.9039 12.9039 200.506 6.075e-008 ***

Sequence 1 0.101016 0.101016 1.56963 0.2388 NS

Error 10 0.643564 0.0643564

Total 23 14.0722

--------------------------------------------------------------------------------



= C

12 3.90855 0.103083 0.0297576 3.90728 1.02712

Formulation (Fed) =

D

12 5.37506 0.309168 0.0892493 5.36693 1.05913

--------------------------------------------------------------------------------


phi = 10.0127




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


366

TABLE 150



Period 1 10.9347 10.9347 0.305528 0.5926 NS

Subject(Seq) 10 330.775 33.0775 0.924219 0.5484 NS

Formulation 1 26.6505 26.6505 0.744643 0.4084 NS

Sequence 1 16.4477 16.4477 0.459566 0.5132 NS

Error 10 357.897 35.7897

Total 23 742.705

--------------------------------------------------------------------------------



= C

12 9.40344 0.913951 0.263835 9.36424 1.09935

Formulation (Fed) =

D

12 11.511 8.01627 2.3141 10.0693 1.61084

--------------------------------------------------------------------------------


phi = 0.610181




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


367

TABLE 151



Period 1 0.34369 0.34369 1.09614 0.3198 NS

Subject(Seq) 10 2.57198 0.257198 0.820289 0.6199 NS

Formulation 1 5.19177 5.19177 16.5583 0.002253 ***

Sequence 1 0.0313009 0.0313009 0.0998288 0.7585 NS

Error 10 3.13546 0.313546

Total 23 11.2742

--------------------------------------------------------------------------------



= C

12 5.71743 0.464655 0.134134 5.70084 1.08199

Formulation (Fed) =

D

12 6.64764 0.580555 0.167592 6.6231 1.09538

--------------------------------------------------------------------------------


phi = 2.87735




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


368

TABLE 152



Period 1 587.882 587.882 0.473133 0.5072 NS

Subject(Seq) 10 8069.94 806.994 0.649477 0.7464 NS

Formulation 1 55507 55507 44.6726 5.474e-005 ***

Sequence 1 354.817 354.817 0.28556 0.6048 NS

Error 10 12425.3 1242.53

Total 23 76945

--------------------------------------------------------------------------------



= C

12 131.376 13.1181 3.78688 130.793 1.10275


--------------------------------------------------------------------------------


phi = 4.72613




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


369

TABLE 153



Period 1 0.0165638 0.0165638 0.0184647 0.8946 NS

Subject(Seq) 10 8.15555 0.815555 0.909152 0.5584 NS

Formulation 1 0.239261 0.239261 0.266719 0.6168 NS

Sequence 1 0.0254606 0.0254606 0.0283826 0.8696 NS

Error 10 8.9705 0.89705

Total 23 17.4073

--------------------------------------------------------------------------------



= C

12 21.6684 0.869843 0.251102 21.6519 1.04187

Formulation (Fed) =

D

12 21.8681 0.896721 0.258861 21.8514 1.04153

--------------------------------------------------------------------------------


phi = 0.365184




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


370

TABLE 154



Period 1 1.72127e-007 1.72127e-007 0.0121079 0.9146 NS

Subject(Seq) 10 0.000178238 1.78238e-005 1.25378 0.3638 NS

Formulation 1 7.90952e-005 7.90952e-005 5.56379 0.04004 ***

Sequence 1 9.28857e-007 9.28857e-007 0.0653385 0.8034 NS

Error 10 0.000142161 1.42161e-005

Total 23 0.000400595

--------------------------------------------------------------------------------


Formulation

(Fasted) = C

12 0.045837 0.00466892 0.0013478 0.0456376 1.10012

Formulation

(Fed) = D

12 0.0494677 0.00272553 0.000786792 0.0493978 1.0574

--------------------------------------------------------------------------------


phi = 1.6679




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


371

TABLE 155



Period 1 1.04167e-006 1.04167e-006 0.00828418 0.9293 NS

Subject(Seq) 10 0.00086175 8.6175e-005 0.685334 0.7194 NS

Formulation 1 0.075376 0.075376 599.452 2.947e-010 ***

Sequence 1 4.5375e-005 4.5375e-005 0.360859 0.5614 NS

Error 10 0.00125742 0.000125742

Total 23 0.0775416

--------------------------------------------------------------------------------



= C

12 0.655583 0.0040104 0.0011577 0.655572 1.00612

Formulation (Fed) =

D

12 0.767667 0.0134457 0.00388145 0.767558 1.01779

--------------------------------------------------------------------------------


phi = 17.3126




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


372

TABLE 156



Period 1 0.0613647 0.0613647 0.0978204 0.7609 NS

Subject(Seq) 10 8.27394 0.827394 1.31894 0.335 NS

Formulation 1 44.7164 44.7164 71.2817 7.323e-006 ***

Sequence 1 0.0154113 0.0154113 0.0245669 0.8786 NS

Error 10 6.2732 0.62732

Total 23 59.3404

--------------------------------------------------------------------------------



= C

12 8.9686 0.164345 0.0474424 8.96724 1.01837

Formulation (Fed) =

D

12 11.6986 1.14124 0.329449 11.6451 1.10641

--------------------------------------------------------------------------------


phi = 5.97




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


373

TABLE 157



Period 1 105332 105332 0.561457 0.4709 NS

Subject(Seq) 10 3.68275e+006 368275 1.96303 0.1513 NS

Formulation 1 6.38685e+007 6.38685e+007 340.441 4.713e-009 ***

Sequence 1 699.408 699.408 0.00372808 0.9525 NS

Error 10 1.87605e+006 187605

Total 23 6.95333e+007

--------------------------------------------------------------------------------



= C

12 9832.12 305.434 88.1711 9827.87 1.03104


--------------------------------------------------------------------------------


phi = 13.0469




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


374

TABLE 158



Period 1 1228.5 1228.5 0.0344965 0.8564 NS

Subject(Seq) 10 356874 35687.4 1.00211 0.4987 NS

Formulation 1 401434 401434 11.2724 0.007274 ***

Sequence 1 1190.2 1190.2 0.033421 0.8586 NS

Error 10 356123 35612.3

Total 23 1.11685e+006

--------------------------------------------------------------------------------



= C

12 1223.04 152.173 43.9286 1213.4 1.14455


--------------------------------------------------------------------------------


phi = 2.37406




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


375

TABLE 159



Period 1 129287 129287 0.422658 0.5303 NS

Subject(Seq) 10 4.49411e+006 449411 1.46919 0.2771 NS

Formulation 1 7.4397e+007 7.4397e+007 243.215 2.404e-008 ***

Sequence 1 65.0104 65.0104 0.000212529 0.9887 NS

Error 10 3.0589e+006 305890

Total 23 8.20793e+007

--------------------------------------------------------------------------------



= C

12 11055.2 374.315 108.055 11049.4 1.03403


--------------------------------------------------------------------------------


phi = 11.0276




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


376

TABLE 160



Period 1 0.00142188 0.00142188 0.00102975 0.975 NS

Subject(Seq) 10 17.7436 1.77436 1.28502 0.3497 NS

Formulation 1 4.83087 4.83087 3.49861 0.09094 NS

Sequence 1 0.100944 0.100944 0.0731057 0.7924 NS

Error 10 13.808 1.3808

Total 23 36.4848

--------------------------------------------------------------------------------



= C

12 11.0481 1.21001 0.349299 10.9816 1.12465

Formulation (Fed) =

D

12 10.1508 1.18891 0.343209 10.0886 1.12208

--------------------------------------------------------------------------------


phi = 1.32261




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


377

TABLE 161



Period 1 5.16325e+007 5.16325e+007 0.682005 0.4282 NS

Subject(Seq) 10 1.2113e+009 1.2113e+008 1.59998 0.2353 NS

Formulation 1 2.43482e+010 2.43482e+010 321.611 6.216e-009 ***

Sequence 1 423473 423473 0.00559357 0.9419 NS

Error 10 7.5707e+008 7.5707e+007

Total 23 2.63686e+010

--------------------------------------------------------------------------------



C

12 153862 7490.08 2162.2 153700 1.04871

Formulation (Fed) = D 12 217564 11294.9 3260.54 217291 1.05401

--------------------------------------------------------------------------------


phi = 12.6809




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


378

TABLE 162



Period 1 9.05205e+007 9.05205e+007 0.198986 0.665 NS

Subject(Seq) 10 4.10679e+009 4.10679e+008 0.902774 0.5627 NS

Formulation 1 3.76222e+010 3.76222e+010 82.7029 3.768e-006 ***

Sequence 1 4.335e+006 4.335e+006 0.0095294 0.9242 NS

Error 10 4.54908e+009 4.54908e+008

Total 23 4.63729e+010

--------------------------------------------------------------------------------



C

12 239732 15870.3 4581.37 239241 1.06954


--------------------------------------------------------------------------------


phi = 6.43051




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


379

TABLE 163



Period 1 5.42184e+006 5.42184e+006 0.0254733 0.8764 NS

Subject(Seq) 10 2.26807e+009 2.26807e+008 1.0656 0.461 NS

Formulation 1 1.43834e+009 1.43834e+009 6.75771 0.02651 ***

Sequence 1 7.46783e+006 7.46783e+006 0.0350859 0.8552 NS

Error 10 2.12844e+009 2.12844e+008

Total 23 5.84774e+009

--------------------------------------------------------------------------------



= C

12 85870.7 12585.9 3633.22 84906.2 1.17653


--------------------------------------------------------------------------------


phi = 1.83817



Minimum detectable difference = 15483

--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


380

TABLE 164



Period 1 9.0373e-005 9.0373e-005 0.276377 0.6105 NS

Subject(Seq) 10 0.0059552 0.00059552 1.82121 0.1793 NS

Formulation 1 0.0480039 0.0480039 146.805 2.668e-007 ***

Sequence 1 5.43305e-005 5.43305e-005 0.166153 0.6921 NS

Error 10 0.00326992 0.000326992

Total 23 0.0573737

--------------------------------------------------------------------------------



= C

12 0.298484 0.0251966 0.00727363 0.29746 1.09166

Formulation (Fed) =

D

12 0.209038 0.0147287 0.0042518 0.208576 1.07136

--------------------------------------------------------------------------------


phi = 8.56751




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


381

TABLE 165



Period 1 10.9347 10.9347 0.305528 0.5926 NS

Subject(Seq) 10 330.775 33.0775 0.924219 0.5484 NS

Formulation 1 26.6505 26.6505 0.744643 0.4084 NS

Sequence 1 16.4477 16.4477 0.459566 0.5132 NS

Error 10 357.897 35.7897

Total 23 742.705

--------------------------------------------------------------------------------



= C

12 9.40344 0.913951 0.263835 9.36424 1.09935

Formulation (Fed) =

D

12 11.511 8.01627 2.3141 10.0693 1.61084

--------------------------------------------------------------------------------


phi = 0.610181




--------------------------------------------------------------------------------


Level C and level D





t(0.05 - 10df) = 1.8125


382

APPENDIX – II

383

1. Software Generated Report (Output) of Stability Analysis (Minitab

version 17.1.0)

A) At Accelerated temperature (40 0C/75% RH)

Formulation F1

384

Stability Study: % Assay versus Months

Model Summary

S R-sq R-sq(adj) R-sq(pred)

0.285774 98.64% 97.28% 81.62%

Coefficients

Term Coef SECoef T-Value P-Value VIF

Constant 100.043 0.261 383.49 0.002

Months -0.5733 0.0674 -8.51 0.074 1.00

Regression Equation

% Assay = 100.043 - 0.5733 Months

Shelf Life Estimation

Lower spec limit = 90 Upper spec limit = 110

Shelf life = time period in which you can be 95% confident that at least 50% of response

iswithin spec limits

Shelf life = 11.1821

385

Formulation F6


Model Summary


0.204124 98.53% 97.05% 80.10%

Coefficients


Constant 100.333 0.186 538.45 0.001

Months -0.3933 0.0481 -8.18 0.077 1.00

386

Regression Equation

% Assay = 100.333 - 0.3933 Months






Formulation F11

387


Model Summary


0.138804 99.42% 98.83% 92.11%

Coefficients


Constant 99.727 0.127 787.04 0.001

Months -0.4267 0.0327 -13.04 0.049 1.00

Regression Equation

% Assay = 99.727 - 0.4267 Months






Formulation F16

388


Model Summary


0.0979796 99.57% 99.13% 94.15%

Coefficients


Constant 97.2400 0.0894 1087.18 0.001

Months -0.3500 0.0231 -15.16 0.042 1.00

Regression Equation

% Assay = 97.2400 - 0.3500 Months






389

Formulation F21

Stability Study: % Assay versus Months (F21)

Model Summary


0.0979796 99.73% 99.45% 96.29%

Coefficients


Constant 98.9100 0.0894 1105.85 0.001

Months -0.4400 0.0231 -19.05 0.033 1.00

390

Regression Equation

% Assay = 98.9100 - 0.4400 Months






391

B) At Room Temperature (25 °C /60% RH)

Formulation F1

Stability Study: % Assay versus Months F1 (room temp)

Model Summary


0.159217 99.04% 98.08% 87.07%

Coefficients


Constant 100.225 0.145 689.57 0.001

Months -0.3817 0.0375 -10.17 0.062 1.00

http://www.thefreedictionary.com/%C2%B0C

392

Regression Equation

% Assay = 100.225 - 0.3817 Months






FormulationsF6

393


Model Summary


0.0244949 99.98% 99.96% 99.75%

Coefficients


Constant 100.240 0.022 4482.87 0.000

Months -0.42667 0.00577 -73.90 0.009 1.00

Regression Equation

% Assay = 100.240 - 0.42667 Months






Formulation F11

394


Model Summary


0.0204124 99.98% 99.97% 99.79%

Coefficients


Constant 99.6783 0.0186 5349.30 0.000

Months -0.38167 0.00481 -79.33 0.008 1.00

Regression Equation

% Assay = 99.6783 - 0.38167 Months




is within spec limits


395

Formulation F16

Stability Study: % Assay versus Months (F16) room temp

Model Summary


0.179629 99.05% 98.11% 87.23%

Coefficients


Constant 97.207 0.164 592.80 0.001

Months -0.2167 0.0212 -10.23 0.062 1.00

Regression Equation

396

% Assay = 97.207 - 0.2167 Months






Formulation F21

397


Model Summary


0.0612372 99.97% 99.94% 99.62%

Coefficients


Constant 98.8950 0.0559 1769.09 0.000

Months -0.42917 0.00722 -59.47 0.011 1.00

Regression Equation

% Assay = 98.8950 - 0.42917 Months




is within spec limits


398

2. Publicationfrom dissertation

Muhammad Iqbal Nasiri, Rabia Ismail Yousuf, Muhammad Harris Shoaib,

Muhammad Fayyaz, FaaizaQazi, Kamran Ahmed. Investigation on release of

highly water soluble drug from matrixcoated pellets prepared by extrusion–

spheronization technique. Journal of Coating Technology and Research, 13 (2)

333–344, 2016.DOI 10.1007/s11998-015-9749-1

399

3. Ethical Review Board Approval Letter

Documents

Development and In-Vitro Characterization of Extended