Experimental Work - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/9100/6/06_chapter 4... · Experimental work School of Pharmacy & Technology Management, SVKM’S NMIMS, Mumbai

Experimental Work

Chapter 4

Experimental work

School of Pharmacy & Technology Management, SVKM’S NMIMS, Mumbai 74

4.1 MATERIALS AND METHODS

Table 4.1: List of chemicals and solvents

S. No. Drugs Source

1. Diltiazem HCl Lupin Pharmaceutical Ltd., India

2. Verapamil Triveni Chemicals, Gujrat, India

______________________________________________________________________________

S. No. Chemicals Source

1. Acrylic acid anhydrous Fluka

2. Poly(vinyl alcohol) S.A Pharmchem Pvt. Ltd, Dupont

3. Poly (vinyl pyrrolidone) USP Signet Chemical Corporation, BASF

4. Azoisobutyronitrile Mody Chemi - Pharma Pvt. Ltd.

5. Potassium persulphate SD Fine chemicals

6. HPMC K15M USP Colorcon Asia Pvt. Ltd

7. Potassium bromide for IR Merck India Ltd.

8. Magnesium stearate SD Fine chemicals

9. Talc Qualigens

10. Glycerine Qualigens

11. Povidone K-30 USP Signet Chemical Corporation, BASF

12. Silica bag Tyvel silica bags

13. Sodium hydroxide Loba Chemicals

14. Pharmactose DCL 21 McLeod’s Pharm. Ltd.

______________________________________________________________________________

S. No. Solvents Source

1. Ethanol GR SD Fine chemicals

2. Ethyl methyl ketone for synthesis Qualigens

3. Ethyl acetate extrapure SD Fine chemicals

4. Dichloromethane GR SD fine Chemicals

5. Methanol AR SD Fine chemicals

6. Dimethyl formamide GR SD Fine chemicals

7. Glacial acetic acid GR SD Fine chemicals

8. Hexane AR Qualigens

9. Isopropyl alcohol AR SD Fine chemicals

10. Benzoyl chloride for syn Spectrochem

11. Glacial acetic acid AR Qualigens

12. Dichloromethane AR SD Fine chemicals

http://www.sulekhab2b.com/viewoffer/company/16306/triveni-chemicals.htm

http://www.tradeindia.com/Seller-99342-278568-839-CATALOGS/Industrial-Chemicals/MODY-CHEMI-PHARMA-PVT-LTD-.html

Experimental work


Table 4.2 List of equipments

S. No. Equipments Manufacturer, Model

1. FTIR Perkin Elmer, Spectrun RX I

2. IR Pellet compression machine Kimaya Engineering KP

3. Electronic weighing balance Shimadzu, AUX 220

4. Automated sample applicator Camag, Linomat

5. Bioadhesion test analyzer Modified double balance

6. Centrifuge Eltec, TC 650 D

7. Tablet Compression machine Karanavati

8. Deep freezer Sanyo, MDF-U333

9. Densitometer Camag

10. Development chamber (HPTLC) Camag

11. Digital pH meter Labindia, PICO+

12. Dissolution test apparatus Electrolab, TDT-08L

13. Franz diffusion cell Lab glass

14. Hardness tester Labline

15. Orbital Shaker Scigenics Biotech, Orbitek

16. Hot air oven Meta Lab, 6318

17. Refrigerator Toshiba, GR-M53UD-IND

18. Sampling cannula In-house

19. Sonicator Toschcon, SN-2

20. Stability chamber Thermolab

21. UV spectroscopy Perkin-Elmer, Lambda 25

22. Vernier calliper Mitotoyo, Japan

23. Cyclomixer Remi, CM 101

24. Melting apparatus Veego VMP-III

25. Vaccum oven Scientico, EVO-01R

_______________________________________________________________________

Experimental work


4.2 Analytical Method development

4.2.1 UV Spectrophotometry (For assay)

UV spectroscopic analysis was employed to determine the drug content and the concentration of

drug in aliquots obtained after dissolution studies.

Preparation of standard curves of DLZ

Standard curve of DLZ in distilled water

DLZ (100 mg) was accurately weighed and transferred to 100 ml volumetric flask. The drug was

dissolved in distilled water and volume was made up to 100 ml with distilled water. The stock

solution was further diluted with distilled water to obtain concentration ranging from 2-12 µg/ml.

The absorbance of each solution was recorded at 237 nm using UV-visible spectrophotometer in

1 cm path length quartz cuvette. Distilled water was taken for blank determinations. The

absorbance values were plotted against concentration (µg/ml) to obtain standard calibration plot

of DLZ. The data was treated statistically using linear least square regression method. The

above procedure was repeated six times.

Accuracy

Accuracy of analytical method is the closeness of test results obtained by the method to the true

value. For determining of DLZ concentration, standard solution was prepared. The above

solutions were subjected to UV absorbance at 237 nm and compared for its accuracy. The

method is said to be accurate for estimation if it gives < 2.0 % RSD at each concentration level.

RSD= SD * 100/ Mean

Precision

The precision is agreement between the results obtained when determinations are conducted.

Precision provides an indication of random errors and is generally subdivided into to cases:

repeatability and reproducibility, which are determined by calculating RSD of inter-day and

intra-day of DLZ. RSD should be < 2.0 % for the method in both inter-day and intra-day

determinations.

Experimental work


Linearity

The linearity of test procedure is defined as its ability (within a given range) to produce the

results which are directly proportional to concentration of analyte in the sample. Linearity of

light absorption determination should be examined to ensure that Beer’s law operates over the

range of interest. For evaluation of linearity of the UV method of DLZ, the standard solution was

prepared at 2, 4, 6, 8, 10 and 12 µg/ml concentration (n=3) and observation were taken at 237nm

in UV spectrophotometer. The method is said to be linear for estimation of DLZ if it is linear

over 2-12 µg/ml range. Least square regression method was used to determine the regression

coefficient, r and the equation y = mx + c for the best fitting line.

Experimental work


4.2.2 Development and Validation of Diltiazem using HPTLC (For bioassay)

Method development and optimization:

Stationary phase

Type: Silica gel 60F 254 precoated HPTLC plates (E. Merck, Germany) were used.

Pre-washing of plates: The plates were pre-washed in methanol for removing any impurities

picked up during storage in the laboratory environment.

Preparation of standard solution

10 mg of working standard of Diltiazem was accurately weighed and dissolved in 10 ml.

methanol to give a stock solution of 1 mg/ml (solution A)

Preparation of plasma sample

Plasma sample (480µL) was placed in glass tube followed by addition of 10 µL of Diltiazem (1

mg/mL, solution A) and 10 µL of Verapamil (500 µg/mL) as internal standard solution. 100 µL

of 0.2 N NaOH as added to the sample, the mixture was vortexed for 2 minutes, then 2 mL n-

hexane with 5% IPA as added. The solution was vortexed for 5 minutes and then centrifuged at

4000 rpm for 10 minutes. The organic phase was separated and dried. The residue was

reconstituted with 200 µL of methanol.

Table 4.3: Optimized Chromatographic Conditions

Application mode CAMAG LINOMATV using 25 µL

syringe

Development mode CAMAG twin trough chamber

Application band width 6 nm

Developing solvent Ethanol: DCM: Acetic acid :Water

(12:10:0.5:1)

Chamber saturation time 10 min

Developing distance 80 mm

Developing time 20 min

Scanner CAMAG TLC Scanner III

Integrator WIN CATS V 1.2.2

Scanning wavelength 240 nm (UV)

Experimental work


Once the chromatographic conditions were set, validation of analytical method was done for

standard solutions.

The HPTLC method developed was validated for following parameters:

Sensitivity

The sensitivity of the method was determined in terms of LOD, LOQ, linearity range and

correlation coefficient. Diltiazem was extracted from working stock solution (1 µg/ml) and

spotted on TLC plate in the range of 40-280 ng. The LOD was calculated as three times the noise

level and LOQ was calculated as ten times the noise level. A graph was obtained by plotting the

area under the peak of DLZ against the amount of DLZ (ng) to determine the linearity range and

correlation coefficient.

Selectivity and specificity

The selectivity of the assay was determined in relation to interferences from endogenous

substances and other commonly used antihypertensive drugs in human plasma.

Precision and accuracy

Different amount of Diltiazem covering the low, medium and higher ranges of the calibration

curve were spotted on the TLC plate. These spots were analyzed (n = 3) by using above

described HPTLC method. Precision was expressed as the % coefficient of variation (% CV) and

accuracy was expressed as a percentage [(observed concentration/theoretical concentration) ×

100)].

Reproducibility

The intra day reproducibility was evaluated by analyzing the amount of Diltiazem spotted on

TLC plate covering low, medium and higher ranges of calibration curve in replicates (n = 3). The

inter day reproducibility was evaluated analyzing the same amount of analyte over period of three

days (n = 3).

Experimental work


Recovery study

Recovery of Diltiazem was determined by spiking Diltiazem in human plasma to obtain three

different concentrations covering the low (40 ng/spot), medium (160 ng/spot) and higher (280

ng/spot) ranges of the calibration curve. The samples were then extracted and analyzed as

described earlier. The recovery was calculated by comparing the resultant peak areas with those

obtained from pure standards in methanol at the same concentrations.

Robustness

To study the robustness of the method, small deliberate variations in the mobile phase

composition (± 2%), chamber saturation period (± 10%), development distance (± 10) and time

for drying after development to scanning (00, 60 and 120 min) were carried out. One factor was

varied while keeping other constants, % RSD as considered to assess the robustness of the

method.

Experimental work


4.3 Polymer Synthesis

4.3.1.1 Synthesis of Poly acrylic acid (PAA)-Poly (vinyl pyrrolidone) (PVP)

interpolymer complex:

Under the lab conditions, complexes were synthesized by template polymerization of acrylic acid

in the presence of PVP as a template. The molar ratio used to prepare PVP/PAA interpolymer

complex was 1:1. To prepare the complexes, acrylic acid and PVP were dissolved in 90 ml of

ethanol and the solution was purged with nitrogen gas for 15-20 min to remove dissolved oxygen.

The polymerization was carried with AIBN (0.5 mmol) as an initiator at 60 0C for 15 h. The

supernatant was removed and the resulting precipitate was dissolved in dimethylformamide

(DMF) at 800

C. The dissolved solution was poured into an excess of cold ethyl acetate with

vigorous stirring. The resulting precipitate was dried in a vacuum oven at 60 0C for 24 (Chun et

al, 2002).

Fig. 4.1: Schematic representation of acrylic acid-PVP interpolymer complex formation

4.3.1.2 Characterization:

Experimental work


FTIR spectroscopy

PVP and AAPVP complex were subjected to Fourier Transform Infra-red spectroscopy in the

range of 4000- 400 cm-1

. FTIR spectrophotometer (Perkin-Elmer), USA) was used for the study.

DSC Analysis

Differential Scanning Calorimeter (Diamond TG/DTA, Perkin Elmer, USA) was used for

studying the thermal behaviour of the PVP and AAPVP complex. The melting studies were

performed in the temperature range of 50–300°C at the heating rate of 10°C/min in nitrogen

atmosphere.

4.3.1.3 Acute oral toxicity study

Previous reports in literature indicated that the polymer complex composed of AA and PVP in

1:1 mole ratio has adhesive properties higher than that of carbopol 971 P NF (Chun et al, 2002).

However, there have been no previous reports about the toxicity of this polymer complex to be

used in pharmaceutical formulation. So it was necessary to access its safety issue in our desired

buccal formulation. OECD guideline No.425 (Acute oral Toxicity-Acute Toxic class method)

was undertaken as a test procedure to ascertain the acute oral toxicity, occurred after the

administration of the test substance. The test procedure in this OECD guideline is of value of

minimizing the number of animals required to estimate the acute oral toxicity. The acute

intravenous toxicity study was conducted using preliminary limit dose test of the Up-and-Down

Procedure statistical program– AOT 425StatPgm (2001). In addition to the estimation of LD50

and confidence intervals, the test allows the observation of signs of toxicity.

Female Wistar rats (150-180 g) were individually identified and allowed to acclimate to the

laboratory conditions for 7 days before the start of the study. Only one rat received a dose at a

particular time. First animal received a dose of 55 mg/kg. Animal was observed for 3 hours after

oral administration for any toxicity signs, survival or death. If the first animal died or appeared

moribund, the second animal received a lower dose (175mg/kg). The dose progression or

Experimental work


reduction factor was 3.2 times of the previous dose. If no mortality was observed in the first

animal then the second animal received a higher dose (175 mg/kg, 550 mg/kg and 2000 mg/kg).

Dosing of the next animal was continued depending on the outcome of the previously dosed

animal for a fixed time interval (3 hour).

The test was stopped when one of the stopping criteria was met:

a) 5 reversals occur in any 6 consecutive animals tested.

b) 3 consecutive animals died at one dose level.

Survived animals were observed for long-term outcomes for a period of 14 days. The LD50

values were calculated using AOT 425 software (Environmental Protection Agency, USA) based

on the short term (3 hour) and long term outcome (14 days).

4.3.1.4 Buccal irritation test

Wistar rat was anaesthetised for 6 hours and tablet was placed in buccal cavity. After 6 hours,

tablet was removed and the animal was sacrificed. Buccal mucosa was taken for histological

study to see any adverse effect on the oral mucosa part.

Experimental work


4.3.2.1 Synthesis of Poly acrylic acid -Poly vinyl alcohol) (PVA) interpolymer

complex:

Under the lab conditions, complexes were synthesized by template polymerization of acrylic acid

in the presence of PVA. 15 ml of AA aqueous solution (6.7%) was mixed with 15 ml of the PVA

aqueous solution (4.1%). The monomer molar ratio used to prepare PVA/PAA interpolymer

complex was 1:1. Resultant solution was purged with nitrogen gas for 15-20 min to remove

dissolved oxygen. The polymerization was carried out with KPS (Potassium Persulphate)

(60mM), as an initiator, at 60˚C for 2 hours (Chun et al 2004).

CH2

H

OH

O + CH2 CH

n

CH2 CH

n

OH

Water, Potassium persulphateStirred at 60 0C for 3 hours

OHOH

CH2 CH

m

O

n

Acrylic acid Poly (vinyl alcohol)

Interpolymer complex

Nitrogen atmosphere

Fig. 4.2: Schematic representation of acrylic acid-PVA interpolymer complex formation

Experimental work


4.3.2.2 Characterization:

FTIR spectroscopy

PVA and AAPVA complex were subjected to Fourier Transform Infra-red spectroscopy in the

range of 4000- 400 cm-1


DSC Analysis


studying the thermal behaviour of PVA and AAPVA complex. The melting studies were

performed in the temperature range of 50–300°C at the heating rate of 10°C/min in nitrogen

atmosphere.

4.3.2.3 Acute oral toxicity study

Previous reports in literature indicated that the adhesive force of polymer complex composed of

AA and PVA was stronger than that of commercial Carbopol 971 P (Oh et al, 2004). However,

there have been no previous reports about the toxicity of this polymer complex to be used in

pharmaceutical formulation. So it was necessary to access its safety issue in our desired buccal

formulation. OECD guideline No.425 (Acute oral Toxicity-Acute Toxic class method) was

undertaken as a test procedure to ascertain the acute oral toxicity as mentioned in section 4.3.1.2.

4.3.2.4 Buccal irritation test

The rat was anaesthetised for 6 hours and patch was placed in buccal cavity. After 6 hours patch

was removed and the animal was sacrificed. Buccal mucosa was taken for histological study to

see any adverse effect on the oral mucosa part.

Experimental work


4.3.3 Synthesis of Acrylic acid - HPMC interpolymer complex

4.3.3.1a Synthesis of Acryloyl Chloride (ACl)

A mixture of 21.6 gm (0.3 moles) of acrylic acid (AA), 84.4 gm (0.6 moles) of benzoyl chloride,

and 0.05 gm of hydroquinone was distilled at a fairly rapid rate through a distilling column. The

distillate was collected in a receiver containing half a gram of hydroquinone, immersed in ice.

When the temperature at the top of the column, which remained between 60-70oC for most of the

distillation, had reached 85oC the distillation was discontinued. The crude product, weighing

between 21-22 gm, was then redistilled through the same column and the fraction boiling at 72-

74oC at 740 mm was collected. The yield of the final product was 18-19 gm or 68-72%.

4.3.3.1b Esterification reaction

2.7 gm of HPMC was dissolved in 100 ml of water with constant stirring so as to avoid lump

formation. 30 ml of MEK was added to the aqueous solution of HPMC (Solution-A) and was

kept in an ice-bath with constant stirring so as to maintain a temperature of 0-5 oC. 0.5 ml of ACl

was mixed with 30 ml of MEK (Solution-B). Solution-B was added drop wise to the solution-A,

kept on stirring in an ice-bath and was stirred for 3 hrs. The reaction mixture was subsequently

transferred to a petri-dish and was kept for drying at room temperature for 48 hrs. The film, so

obtained, was repeatedly washed with rectified spirit to wash off the unwanted free acrylic acid,

if any. The yield of the final product was 80%.

HPMC Acrylic acid HPMC acrylate+Benzoyl chloride

0-5 C

Ethyl methyl ketone

Fig. 4.3: Schematic representation of acrylic acid-HPMC

Experimental work


4.3.3.2 Characterization of Acrylic acid - HPMC interpolymer complex

The newly formed complex was analysed for solubility and melting point determination.

FTIR spectroscopy

HPMC and HPMC acrylate were subjected to Fourier Transform Infra-red spectroscopy in the

range of 4000- 400 cm-1


DSC Analysis


studying the thermal behaviour of the HPMC and the developed film. The temperature and

energy scales were calibrated as per the standard protocols supplied by the manufacturer. The

melting studies were performed in the temperature range of 50–300°C at the heating rate of

10°C/min in nitrogen atmosphere.

XRD analysis

HPMC and the esterified product were subjected to X-ray diffraction (XRD-PW 1700, Philips,

USA) using CuKα radiation generated at 40KV and 40 mA; the range of diffraction angle 2θ was

10.00-60.00o.

4.3.3.3 Toxicity studies

4.3.3.3a Hemocompatibility study

The complex formed was subjected to hemocompatibility test. The haemocompatibility test of

HPMC and HPMCAA were done as per the reported literatures with necessary modifications (Pal

et al, 2007; Mishra et al, 2008; Pal et al, 2008). This test aims at determining the % haemolysis

of the RBCs in the presence of the samples. The % haemolysis may be mathematically defined

as:

Experimental work


% 100Test Negative

Positive Negative

A AHaemolysis x

A A

(Equation 4.1)

where,

Atest Absorbance for test samples;

Anegative Absorbance for negative control;

Apositive Absorbance for positive control

In short, 5 ml of citrated blood was collected from a pathological laboratory and was

subsequently diluted to 20 ml with normal saline. For the preparation of the positive control, 0.5

ml of the diluted blood was transferred to a 15 ml falcon tube with the subsequent addition of the

0.5 ml of 0.01N hydrochloric acid. Thereafter, the volume was made up to 10 ml with normal

saline. Hydrochloric acid is a corrosive liquid and leads to the disruption of RBC membrane

thereby causing haemolysis. The negative control was prepared in the similar manner where the

hydrochloric acid was replaced with normal saline. For test samples, solutions (10 %, 20 %, 40

% and 80%) of the HPMCAA were prepared in normal saline. 0.5 ml of the solution was diluted

to 1 ml with normal saline, which was further diluted to 10 ml with normal saline. The samples

(positive control, negative control and test samples), so obtained, were incubated at 37 oC for 1 hr

and were subsequently centrifuged at 3000 rpm for 10 min. The supernatant was analyzed

spectrophotometrically at 545 nm. % haemolysis was calculated as per eq.4.1. If the %

haemolysis ≤5%, the test material is considered as highly hemocompatible, if the % haemolysis

in the range of 5-10%, the test material is considered as hemocompatible and if the % haemolysis

is ≥20%, the test material is considered as non-hemocompatible.

4.3.3.3b Acute oral toxicity study

OECD guideline No.425 (Acute oral Toxicity-Acute Toxic class method) was undertaken as a

test procedure to ascertain the acute oral toxicity as mentioned in section 4.3.1.2.

4.3.3.3c 28 days repeated Dose Toxicity (Sub-acute Oral Toxicity)

OECD guideline No. 407 (Sub-Acute Toxic class method) was undertaken as a test procedure.

Experimental work


Test animals:

i. Test Species: Rat (Rattus norvegicus) of either sex

ii. Strain: Wistar

iii. Total Number of Animals Used: 60

iv. Weight before study: 150-180g

Acclimatization Period:

7 days prior to commencement of dosing.

Animal Identification:

The animals were identified using picric acid. Appropriate labels were attached to the cages

indicating the study number, test substance code, group number and sex, dose, and cage number.

Environment Conditions:

Temperature: Maximum : 27 ºC, Minimum : 25ºC

Mean Relative Humidity: 70 %

Photoperiod: The photoperiod was 12 hours artificial light and 12

hours darkness

Housing:

i. Caging: Polypropylene rat cages covered with stainless steel grid top

were used. Clean rice husk was used as the bedding

material.

ii. Water Bottle: Each cage was supplied with propylene water bottles

(Capacity –250ml) with a stainless steel nozzle.

iii. Housing: 5 animals of 100 – 250 Grams of same sex per cage.

Experimental work


Diet and Water:

i. Diet: Rodent pellet diet (manufactured by ‘Nav Maharashtra

Chakan Mills Ltd.’) was provided ad libitum.

ii. Water: Drinking water filtered through ‘Aqua guard’ water filter

system was provided ad libitum.

Table 4.4: Allocation of Wistar Rats in 28 days oral toxicity study

Test Group Dose to Animal (mg/kg) Male Female

Control Vehicle (Distilled Water) 3 3

Low 100 mg/kg 3 3

Medium 200 mg/kg 3 3

High 500 mg/kg 3 3

Satellite Control Vehicle (Distilled Water) 3 3

Satellite (High) 500 mg/kg 3 3

Experimental Procedure:

Adult Wistar rats of either sex weighing between 150 to 180 g.

The animals are divided into following groups:

Group 1 Control / Vehicle Group (Distilled Water).

Group 2 HPMCAA (100 mg/kg)



Group 5 Satellite group (Distilled Water)

Group 6 Satellite group HPMCAA (500 mg/kg)

Each group contains 3 male and 3 female. Each cage houses 3 animals of the same sex either

male or female. Cages are labelled and the animals are marked for identification.

Experimental work


Route of Administration

Vehicle or drug solution was administered by oral route using a stainless steel feeding needle.

Maximum permissible volume to be administered was limited to 2ml/100 gm body weight in case

of aqueous solution. In case of doses where more volume of solution was required, total volume

of solution was divided into 2 or 3 equal fractions and administered after interval of 30 minutes.

The time of drug administered was kept same throughout the study.

Observations:

General health of the animals in terms of body weight, behavior, skin, hair, and food

consumption was carefully noted. Bedding material of each cage replaced regularly.

Food Consumption

Before administration of drug, the animals were weighed using a calibrated balance. For

recording the food consumption 150 g of standard rat food pellets (purchased from Amrut

laboratory manufactured by Nav Maharashtra Chakan Oil mills Ltd., Sangli) was kept in feed

tray of the animals. The unconsumed pellets was weighed and replaced with fresh 150 g of

pellets in each tray every day. The time of filling the food tray is noted down and kept constant

throughout the study. The animal’s had access to Aquaguard filtered water and food ad libitum.

Haematology

Before start of study and at the end of study period i.e. 28 days the animals were anaesthetized

(anaesthetic ether) and blood sample was collected. In case of satellite control and satellite 500

mg/kg group the blood sample was collected before the start of study and after one month of 28

days of study have been completed. The blood samples were collected in two separate bottles,

marked individually (animal number, group, haematology). EDTA (anti-coagulant) was used in

blood samples for heamatological parameters.

Histopathology

At the end of study period i.e. 28 days the animals were anaesthetized (anaesthetic ether) 24

hours after the last dose. In case of satellite control and satellite 500mg/kg group were killed by

anaesthetic ether 28 days of treatment.

Experimental work


The abdomen of rat was cut opened using stainless steel scissors and forceps in a pre disinfected

stainless steel necropsy table. The organs were collected very carefully and washed in sterile

phosphate buffered saline and weighed individually using Mettler balance. Care was taken by

marking all the organs as per their Group no. and Animal Id Code. The organs after weighing

were transferred into individually marked (with indelible ink) glass bottles containing 10%

formalin.

Experimental work


4.4. Preformulation studies

Preformulation testing is the first step in the rational development of dosage form. It can be

defined as an investigation of physical and chemical properties of a drug substance alone and

when combined with excipients. The overall objective of preformulation studies is to generate

information useful to the formulator in developing stable and bioavailable dosage forms, which

can be mass-produced.

4.4.1 API Characterization

Diltiazem HCl was received from Lupin Pharmaceutical Ltd, Pune, India. Following tests were

carried out which could result in essential information for formulation work.

Diltiazem has been authenticated by using techniques like TLC, FTIR, DSC and determining

melting point.

4.4.1.1 Organoleptic properties

Received drug was observed for general physical properties such as nature, colour and odour.

The solubility of the drug was determined in water as per IP.

4.4.1.2 Thin layer chromatography

It was carried out using silica gel GF 254 as the coating substance and ethanol: dichloromethane:

acetic acid: water in ratio of 12:10:1:05v/v/v/v was used as mobile phase. Development chamber

was saturated for 10 minutes and spotted plate was developed in mobile phase. Retention factor

was calculated as equation:

Rf = Distance travelled by solute front/ Distance travelled by solvent front (Equation 4.2)

4.4.1.3 Melting point

Melting point of DLZ was determined using digital melting point apparatus. Average of three

reading was calculated (n=3).

4.4.1.4 FTIR studies

Experimental work


FTIR studies were used for unique structural information extracted from the spectral data. Drug

was mixed with potassium bromide dried overnight at 1100C in ratio of 1:100. Mixture was

triturated for uniform mixing of drug. Mixture was pelletized in IR compression machine to

develop a thin film which was subjected to FTIR spectrophotometer in the range of 600-3600 cm-

1.

4.4.1.5 DSC studies

Differential scanning calorimetry (DSC) is one of the most widely used thermal analysis

technique for the characterization of pharmaceutical solids. Thermal events such as melting

point, recrystallization and glass transition temperature were measured. DSC studies of DLZ

were performed by 10 0C/min heating rate in the temperature range of 40-250

0C in inert

atmosphere.

4.4.2 Drug excipient compatibility studies

Drug- excipient compatibility study was carried out by placing drug alone and drug along with

individual excipients in certain ratio (tentative ratio of formulation) in stoppard vials at 40°C/

75%RH for two months. Samples were physically observed every weak and degradation was

examined by FTIR spectroscopy at the end of studies. Quantities of the excipients taken are

mentioned in Table 4.5. Physical observation of sample was done for any color change, lumps

formation or any other visual changes.

Table 4.5: Composition of samples for compatibility studies

S.No. Excipients Drug : Excipients ratio

1. Drug ---

2. AAPVP 1:4

3. PVP K-30 1:2

4. Magnesium stearate 1:0.25

5. Talc 1:0.25

6. DC Lactose 1:5

7. HPMCAA 1:3

8. AAPVP 1:4

Experimental work


4.5 Formulation Studies

Tablets have been the most commonly investigated dosage form for buccal drug delivery to date.

However, buccal patch may be preferred over adhesive tablet in terms of flexibility and comfort.

It was planned to prepare both formulations, i.e. tablet and patch, using the mucoadhesive

polymers synthesized.

AAPVP interpolymer complex was found to be insoluble in most of the commonly used solvents.

Hence, was not a suitable candidate for buccal patch formulation as formulation of patch requires

the solution of drug and excipents in a common solvent. Therefore, AAPVP interpolymer

complex was employed in buccal tablets formulation.

On contrary, AAPVA polymer complex did not give powder but was found to form flexible patch

after getting dried, due to the presence of PVA. Hence, it was not a suitable candidate for tablet

formulation. So it was used in buccal patch formulation.

Tablets were prepared using HPMC acrylate at different concentration ranging from 20-70% and

its affect on drug release was studied.

The literature documents that the dose of DLZ can be reduced up to 80% via buccal delivery,

owing to the avoidance of a hepatic first-pass effect (Ahuja et al, 1995). Therefore,

buccoadhesive formulations containing the lowest dose of DLZ, i.e., 30 mg, were worked upon in

the current study. Another work carried out on buccal tablets and patches of DLZ corroborates

the use of this dose level (Saisivam et al, 2000; Shayeda et al, 2009). There are reports

indicating that the buccoadhesives have been studied for drug release up to 2 days (Han et al,

1999; Guo JH, 1994). However, as a buccoadhesive tablet, film, or patch is unlikely to remain on

the buccal mucosa for such long a time, drug release in the present study was investigated only

up to 6 hr.

http://journal.pda.org/search?author1=Shayeda&sortspec=date&submit=Submit

Experimental work


4.5.1 Preparation of Mucoadhesive Buccal Tablet

Direct compression technique was employed for the formulation. In this technique, first

medicated layer was formed and backing layer blend was placed on medicated layer and

compressed to get bilayered tablet. Drug, polymers and excipients were properly mixed to get a

uniform blend and compressed. Similarly, blend of protective layer containing ethyl cellulose

was compressed on the previously compressed medicated tablet or loose compact to get bilayered

tablet. Colourant was incorporated in backing layer in order discriminate the two layers of the

tablet.

Since the polymer compex was a novel one, no range was known for its application. Initially

trial batches were formulated using the polymer complex AAPVP in range of 20-80% keeping all

the other variables constants and its effect on the dissolution attributes was evaluated.

Experimental

Trial 1:

Table 4.6: Composition of tablets for the effect of AAPVP on dissolution profile

It was observed that at low concentration (20%) of AAPVP, 70% of drug was released in first

hour leading to dose dumping phenomenon. While only 30% of drug was released after 6 hours

when the complex was high concentration (80%). 50-60% of drug as released in the formulation

containing 40-60 % of AAPVP, therefore this range was selected for further studies. Moreever, it

was found in the literature that starch, PVP K- 30 is added in the formulation as release modifier

to enhance the extent and release rate of drug. PVP K-30 was selected to enhance the extent of

drug release. AAPVP was selected in two concentrations, 40 % and 60%. For each

Batch Code Ta Tb Tc Td

Drug (DLZ) (mg) 30 30 30 30

AA-PVP (%) 20 40 60 80

Talc (%) 1 1 1 1

Mag. Stearate (%) 1 1 1 1

Lactose (DC) qs qs qs qs

Backing layer

Total (mg) 180 180 180 180

Experimental work


concentration, PVP K-30 was used in 5, 10, 20 and 30% concentration to study its effect on the

dissolution profile.

Trial 2:

Table 4.7: Composition of tablets for the effect of PVP K-30 on dissolution profile

Dissolution studies indicated that there was no significant change in the release profile when PVP

K-30 as used in 5% concentration. At 30 % concentration, although more than 90 % drug was

released but more than 50% of drug was released within1 hour. 70-92% drug release was

obtained when PVP K-30 was used in range of 10-20 %.

4.5.1.1 Experimental Design

It is desirable to develop an acceptable pharmaceutical formulation in shortest possible time

using minimum man power and raw materials. Traditionally pharmaceutical formulation is

developed by changing one variable at a time. This method is time consuming and requires a lot

of imaginative efforts. Moreover, it may be difficult to evolve an ideal formulation using this

classical technique since the joint effects of independent variables are not considered. It is

therefore very essential to understand the complexity of pharmaceutical formulations by using

established tools such as factorial design.

Factorial designs are used in experiment where effect of different factors or condition on

experimental results is to be elucidated. Factorial designs are designs of choice for simultaneous

determinations of effects of several factors and their interactions. A 32 randomized full factorial

design was utilized in the present study. In this design two factors were evaluated, each at three

Batch Code Te Tf Tg Th Ti Tj Tk Tl

Drug (DLZ) (mg) 30 30 30 30 30 30 30 30

AA-PVP (%) 40 40 40 40 60 60 60 60

PVP K-30 5 10 20 30 5 10 20 30

Talc (%) 1 1 1 1 1 1 1 1

Mag. Stearate (%) 1 1 1 1 1 1 1 1

Lactose (DC) qs qs qs qs qs qs qs qs

Backing layer

Total (mg) 180 180 180 180 180 180 180 180

Experimental work


levels, and experimental trials were carried out at all nine possible combinations. The factors

were selected based on preliminary study described as above.

A three-level, two-factor experimental design, as shown in Table 4.8, describes the proportion in

which the independent variables AAPVP, (X1), and PVP K-30 (X2), were used in the formulation

of mucoadhesive patches. The levels of the two factors were selected on the basis of the

preliminary studies carried out before implementing the experimental design. All other

formulation and processing variables were kept invariant throughout the study. The response

variables tested includes: bioadhesion force and percentage release after 6 hours. The quantity of

AAPVP was varied at three levels: 45, 52.5 and 60 % (w/w), whereas the quantity of PVP K-30

was varied at the levels of 12, 16 and 20% (w/w). All nine formulations were prepared in three

sets and analyzed individually for percentage release after 6 hours, bioadhesion force and other

patch properties. Checkpoint batch was also prepared to prove the validity of the evolved

mathematical model. The linear regression model was derived from Enter Method using SPSS

version 11.5, statistical software. Significant terms were chosen at 95% confidence interval

(p<0.05) for final equations. In addition, contour and surface plots were obtained by Statistica-6,

to represent the effect of the independent variables graphically.

Table 4.8: Factor Combinations as per the Chosen Experimental Design

Formulation code AAPVP (X1) (%) PVP K-30(X2) (%)

T1 45 12

T2 45 16

T3 45 20

T4 52.5 12

T5 52.5 16

T6 52.5 20

T7 60 12

T8 60 16

T9 60 20

Experimental work


Table 4.9: Composition of buccal tablets (DLZ-AAPVP)

4.5.1.2 Tablet Evaluation

4.5.1.2a Technological parameters

The diameter and thickness of the formulated tablets were measured using Vernier Callipers.

Total weight of the tablets was determined using digital balance.

4.5.1.2b Assay

Twenty tablets were taken and powdered; powder equivalent to one tablet was taken and

dissolved in 100ml of water. The solution was filtered through a membrane filter and the

absorbance was measured, after suitable dilution, spectrophotometrically at 237 nm.

4.5.1.2c Surface pH Study

The surface pH of the buccal tablets was determined in order to investigate the possibility of any

side effects in vivo. As an acidic or alkaline pH may irritate the buccal mucosa, the surface pH

was maintained as close to neutral as possible. The method adopted by Bottenberg was used to

determine the surface pH of tablets (Bottenberg et al, 1991). A combined glass electrode was

used for this purpose. The tablet was allowed to swell by keeping it in contact with 1 mL of

distilled water for 2 hours at room temperature. The pH was identified by bringing the electrode

Batch Code T1 T2 T3 T4 T5 T6 T7 T8 T9

Medicated layer

Drug (DLZ) (mg) 30 30 30 30 30 30 30 30 30

AA-PVP (%) 45 45 45 52.5 52.5 52.5 60 60 60

PVP K 30 (%) 12 16 20 12 16 20 12 16 20

Talc (%) 1 1 1 1 1 1 1 1 1

Mag. Stearate (%) 1 1 1 1 1 1 1 1 1

Lactose (DC) qs qs qs qs qs qs qs qs qs

Backing layer

Ethyl cellulose

Coarse powder: fine powder 4:1(mg) 40 40 40 40 40 40 40 40 40

Total weight (mg) 220 220 220 220 220 220 220 220 220

Experimental work


into contact with the tablet surface and allowing the surface to equilibrate for 1 minute. The

experiments were performed in triplicate, and average values were reported.

4.5.1.2d Swelling Study

The swelling properties of the tablets were evaluated using swelling index by determination of %

swelling (Parodi et al, 1996). Buccal tablets were weighed individually (designated as W1) and

placed separately in 2% agar gel plates, incubated at 37°C ± 1°C, and examined for any physical

changes. At regular 1-hour time intervals until 6 hours, tablets were removed from the gel plates

and excess surface water was removed carefully using the filter paper. The swollen tablets were

then reweighed (W2) and the swelling index (SI) were calculated using the following formula:

SI = [(W 2 − W 1)/ W 1] × 100

The experiments were performed in triplicate, and average values were reported.

4.5.1.2e Ex vivo Mucoadhesive Strength

A modified balance method was used for determining the ex vivo buccoadhesive strength (Gupta

et al, 1992). Sheep buccal mucosa was obtained from a local slaughterhouse and used within 2

hours of slaughter. The mucosal membrane was separated by removing underlying fat and loose

tissues. The membrane was washed with distilled water and then with phosphate buffer pH 6.8 at

37°C. The mucosa was cut into pieces and washed with phosphate buffer pH 6.8. A piece of

buccal mucosa was tied to a lower Teflon block which was placed in a beaker filled with

phosphate buffer pH 6.6 touching mucosal surface at 37°C ± 1°C. The buccal tablet was stuck to

the upper Teflon block with cyanoacrylate glue. The two sides of the balance were made equal

before the study, by keeping a weight on the right-hand pan. A weight of 5 g was removed from

the right-hand pan, which lowered the pan along with the tablet over the mucosa. The balance

was kept in this position for 5 minutes contact time. The water (equivalent to weight) was added

slowly with a burette (100 drops/min) to the right-hand pan until the tablet detached from the

mucosal surface. This detachment force gave the buccoadhesive strength of the buccal tablet in

grams. Force of adhesion and bond strength parameters were calculated.

Experimental work


4.5.1.2f. Ex vivo Residence Time

The Ex vivo mucoadhesion time was studied (n = 3) after application of tablets on freshly cut

sheep buccal mucosa. The fresh sheep buccal mucosa was fixed in the inner side of a beaker,

about 2.5 cm from the bottom, with cyanoacrylate glue. One side of each tablet was wetted with

1 drop of phosphate buffer (pH 6.6) and pasted to the sheep buccal mucosa by applying a light

force with a fingertip for 30 seconds. The beaker was filled with 200 mL of phosphate buffer

(pH 6.6) and was kept at 37°C ± 1°C. After 2 minutes, a 50-rpm stirring rate was applied to

simulate the buccal cavity environment, and tablet adhesion was monitored for 6 hours. The time

required for the tablet to detach from the sheep buccal mucosa was recorded as the mucoadhesion

time (Han et al, 1999).

4.5.1.2g Stability study of optimized formulation in human saliva

The stability studies of optimized tablets were performed in 5mL of natural human saliva kept at

room temperature for 6 hours. Tablets were physically examined for changes in shape at regular

time intervals of 0, 1, 2, 3, and 6 hours.

Experimental work


4.5.2 Preparation of Mucoadhesive Buccal Patch

AAPVA polymer complex did not give powder but was found to form flexible patch after getting

dried, due to the presence of PVA. Hence, it was not a suitable candidate for tablet formulation.

So it was used in buccal patch formulation. Solvent casting technique was used for the

preparation of patches (Anders R and Merkle HP, 1989). The dried patches were checked for any

imperfections or air bubbles and cut into circulars pieces of diameter 14 mm diameter using

cutter. Patches were meant to release the drug from only one side; therefore an impermeable

backing membrane (Tegaderm TM

adhesive surgical dressing, 3M Health Care, Germany) was

placed on the other side of the patch.

Trial 1:

Amount of plasticizer greatly affects the film properties. Glycerine and Propylene glycol were

used in concentration ranging from 5 -50% w/w.

It was observed that at low concentration of plasticizer (0-20% w/w for glycerine and 0-40% w/w

for propylene glycol) film formed was brittle and difficult to remove. At high concentration

(≥40% w/w for glycerine and ≥60 %w/w for propylene glycol), although the film was flexible but

was found to be oily. 30% w/w for glycerine and 50 %w/w for propylene glycol were found to be

ideal for getting smooth and flexible film. Glycerine at 30% w/w concentration was used further

in all the buccal film formulation. The film formed was smooth, homogenous and flexible.

Trial 2:

Since the polymer compex was a novel one, no range was known for its application. Initially

trial batches were formulated using the polymer complex AAPVA in range of 25-100 mg keeping

all the other variables constants and its effect on the dissolution attributes was evaluated.

Experimental work


Table 4.10: Composition of patches to study the effect of AAPVA on dissolution profile

It was observed that at low concentration (25mg) of AAPVA, more than 70% of drug was

released in first hour leading to dose dumping phenomenon. Whereas only a small fraction of

drug was released after 6 hours when the complex was used at high concentration (100 mg).50-

60% of drug as released in the formulation containing 50-75 mg of AAPVA. This range was

selected for further study. Hoever, it was found in the literature that PVP K- 30 is added in the

formulation to enhance the extent and release rate of drug. PVP K-30 was selected to enhance the

extent of drug release. AAPVA was selected in two concentrations, 50 and 75 mg. For each

concentration, PVP K-30 was used in 5, 10, 25 and 40 mg concentrations to study its effect on

the dissolution profile.

Trial 3: PVP K-30 was used in concentration of 5-40 mg keeping AAPVP at two levels (50 and

75 mg)

Table 4.11: Composition of patches for the effect of PVP K-30 on dissolution profile

No significant change as observed in the release profile when PVP K-30 was used in

concentration of 5 mg. At 40 mg concentration, although more than 90 % drug was released but

more than 50% of drug was release within1 hour. 70-92% drug release was obtained when PVP

K-30 was used in range of 10-40 mg.

Batch Code Pa Pb Pc Pd

Drug (DLZ) (mg) 30 30 30 30

AA-PVA (mg) 25 50 75 100

Glycerine (%w /w) 30 30 30 30

Water qs qs qs qs

Batch Code Pe Pf Pg Ph Pi Pj Pk Pl

Drug (DLZ) (mg) 30 30 30 30 30 30 30 30

AA-PVA (mg) 50 50 50 50 75 75 75 75

PVP K-30 (mg) 5 10 25 40 5 10 25 40

Glycerine (% w/w) 30 30 30 30 30 30 30 30

Water qs qs qs qs qs qs qs qs

Experimental work


4.5.2.1 Experimental Design:

A 32 randomized full factorial design was utilized in the present study as described in section

4.5.1.1. A three-level, two-factor experimental design, as shown in Table 4.12, describes the

proportion in which the independent variables AAPVA, (X1), and PVP K-30 (X2), were used in

the formulation of mucoadhesive patches. The response variables tested includes: bioadhesion

force (BF) and percentage release after 6 hours (Rel 6 hr). The quantity of AAPVP was varied at

three levels: 40, 60 and 80 mg, whereas the quantity of PVP K-30 was varied at the levels of 10,

20 and 30 mg. All nine formulations were prepared in three sets and analyzed individually for

percentage release after 6 hours, bioadhesion force and other patch properties.

Table 4.12: Factorial Combinations as per the Chosen Experimental Design

Formulation code AAPVA (X1) (mg) PVP K-30(X2) (mg)

P1 40 10

P2 40 20

P3 40 30

P4 60 10

P5 60 20

P6 60 30

P7 80 10

P8 80 20

P9 80 30

TABLE 4.13: Composition of buccal patch (DLZ-AAPVA)

Batch Code P1 P2 P3 P4 P5 P6 P7 P8 P9

Medicated layer

Drug (DLZ) (mg) 30 30 30 30 30 30 30 30 30

AA-PVA (mg) 40 40 40 60 60 60 80 80 80

PVP K 30 (mg) 10 20 30 10 20 30 10 20 30

Glycerin (%) 30 30 30 30 30 30 30 30 30

Backing layer

Tegaderm TM

adhesive surgical dressing

Experimental work


4.5.2.2 Patch Evaluation

4.5.2.2a Patch thickness

The thickness of each patch was measured at five different locations (center and four corners)

using a micrometer screw gauge and a mean value of five locations was used as a film thickness.

4.5.2.2b Folding endurance

The folding endurance of patches was determined by repeatedly folding the patch at the same

place till it broke or was folded up to 200 times without breaking (Khurana et al, 2000).

The

experiments were performed in triplicate, and average values were reported.

4.5.2.2c Content uniformity

Drug content uniformity was determined by dissolving the patch by homogenization in 100 mL

of an isotonic phosphate buffer (pH 6.6) under occasional shaking and then followed the

procedure given in section 4.5.1.2b.

4.5.2.2d Surface pH study

Surface pH was determined using combined glass electrode as mentioned in section 4.5.1.2c.

4.5.2.2e Swelling study

The swelling properties of the patches were evaluated using swelling index by determination of

% swelling as mentioned in section 4.5.1.2d.

4.5.2.2f Ex vivo Mucoadhesive Strength

Ex vivo Bioadhesive force was determined using in house modified double pan balance method

as mentioned in section 4.5.1.2 e.

Experimental work


4.5.2.2g Ex vivo Residence Time

Ex vivo Residence Time was determined using the beaker method as mentioned in section

4.5.1.2f.

4.5.2.2h In vitro Drug Release

Dissolution study was conducted similar to that mentioned in section 4.5.1.2g.

4.5.2.2i Stability of optimized formulation in human saliva

The stability studies of optimized patches were performed in 5mL of natural human as mentioned

in section 4.5.1.2i.

4.5.2.2j Mechanical Parameters of Buccal Patches:

Mechanical properties of the films were evaluated using a microprocessor based advanced force

guaze (Ultra Test, Mecmesin, UK) equipped with a 25 kg load cell. Film strip with dimensions

60 x 10 mm and free from air bubbles or physical imperfections were held between two clamps

positioned at a distance of 3 cm. During measurement, the top clamp at a rate of 2 mm/s pulled

the strips to a distance till the film broke. The force and elongation were measured when the film

broke. The mechanical properties were calculated according to the following formulae (peh,

1999). Measurements were run in four replicates for each formulation.

The following equations were used to calculate the mechanical properties of the films:

Experimental work


The tensile testing gives an indication of the strength and elasticity of the film, reflected by the

parameters: tensile strength (TS), elastic modulus (EM) and elongation at break (E/B). A soft

and weak polymer is characterized by a low TS, EM and E/B; a hard and brittle polymer is

defined by a moderate TS, high EM and low E/B; a soft and tough polymer is characterized by a

moderate TS, low EM and high E/B; whereas a hard and tough polymer is characterized by a

high TS, EM and E/B (Aulton et al, 1981). Another parameter, Strain has been used as an

indicator of the overall mechanical quality of the film (Rowe, 1983). A high strain value

indicates that the film is strong and elastic. Hence, it is suggested that a suitable buccal film

should have a relatively high TS, E/B and Strain but a low EM.

http://www.ualberta.ca/~csps/JPPS2(2)/K.Peh/polymeric.htm#TS

http://www.ualberta.ca/~csps/JPPS2(2)/K.Peh/polymeric.htm#EM

http://www.ualberta.ca/~csps/JPPS2(2)/K.Peh/polymeric.htm#E

Experimental work


4.5.3 Formulation Studies

Tablets were prepared using HPMC acrylate at different concentration and its affect on drug

release was studied.

Preparation of Mucoadhesive Buccal Tablet

Direct compression technique was employed for the formulation. Physical blend of drug,

polymers and excipients were properly mixed and passed through 20 mesh screen, then

compressed in tablet compression machine (Karanawati, Mumbai) using 8 mm flat faced punch.

Since the polymer complex was a novel one, no range was known for its application. Initially

trial batches were formulated using the polymer complex HPMCAA in range of 20-70% keeping

all the other variables constant and its effect on the dissolution attributes was evaluated.

Experimental

Table 4.14: Composition of tablets proposed to study the effect of HPMCAA

Batch Code F1 F2 F3 F4

Drug (DLZ) (mg) 30 30 30 30

HPMCAA (%) 20 40 60 70

Talc (%) 1 1 1 1

Mag. Stearate (%) 1 1 1 1

Lactose (DC) qs qs qs qs

Total (mg) 120 120 120 120

Experimental work


4.6 Stability studies

Stability studies of both optimized formulations (T13 and P 13) were conducted as per ICH

guidelines. Packing material was selected based on high polymer composition of formulation.

Tablets were packed in 30cc thick walled HDPE bottles with CRC caps. Each bottle containing

30 tablets and 2 g of silica bags were kept for real time (25°C/ 60% RH) and accelerated (40°C/

75% RH) stability study. Patches were kept in aluminium foil and subjected to above mentioned

stability conditions. Samples at each time point were observed for physical change and were

analyzed for dissolution, bioadhesive strength and total drug content.

Experimental work


4.7 In vivo studies

In vivo experiments were performed after approval of the protocol (CPSEA/SPTM/78/2010) from

Institutional Animals Ethics Committee. As per the protocol, optimized formulations were tested

in Albino rabbits for bioavailability studies. Rabbits were obtained from Haffkine Institute,

Mumbai. Studies were conducted in four rabbits in the weight range of 3 to 4 kgs. After

habitation of ten days they were exposed to three samples (Diltiazem solution, optimized buccal

formulations T13 & P 13) in serial one after other with wash out period of seven days (more than

seven half lives).

Figure 4.4: Rabbit in restrainer Figure 4.5: Feeding of oral solution to rabbit

Figure 4.6: Withdrawal of blood Figure 4.7: Buccal adhesive tablet

sample from rabbit ear vein applied at mucosa of rabbit

Experimental work


Rabbits were fasted for 24 h before drug administration and anaesthetised with pentobarbital (25

mg/kg). For oral administration, drug in aqueous solution in dose of 10mg/kg was administered

by a oral feeding needle (Figure 4.5) to all four rabbits and blood samples were withdrawn from

marginal ear vein (Figure 4.6) using insulin syringe at 0.5, 1, 1.5, 2, 3, 4, 5 and 6 hours. For the

blood withdrawal, rabbits were restrained in restrainer (Figure 4.4) and 1 mL of blood was

withdrawn from marginal ear vein. Withdrawn blood was mixed gently with a drop of heparin

solution (Inhep®) and centrifuged for 5 minutes at 5,000 rpm at 10°C. Centrifugation gives

sedimentation of blood component and supernatant plasma. Plasma was separated and processed

as mentioned earlier. Extracted drug was quantified in validated HPTLC method as mentioned in

section 4.2.2.2.

After a wash out period of seven days, optimized formulation (buccal tablet, T13) was positioned

just above the incisor tooth (on either side of the mouth) and held firmly in place with a finger

over the lip for 30 seconds to ensure adhesion (Figure 4.7). Blood sample were withdrawn and

treated as mentioned in earlier paragraph.

Third study sample of optimized formulation (buccal patch P 13) was also treated as that

mentioned in earlier paragraph for T13.

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Experimental Work - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/9100/6/06_chapter 4... · Experimental work School of Pharmacy & Technology Management, SVKM’S NMIMS, Mumbai