“FORMULATION AND EVALUATION OF A LYOPHILIZED DOSAGE FORM”

SYNOPSIS FOR M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA

BYRAHUL SANGAMKER

I- M.PHARMUNDER THE GUIDANCE OF

Mr. S. J. SHANKARDEPARTMENT OF PHARMACEUTICAL TECHNOLOGY

PES COLLEGE OF PHARMACYBENGALURU-560050

(2011-13)

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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BENGALURU

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the candidate and address PRESENT ADDRESSRAHUL SANGAMKERI- M. PHARM PHARMACEUTICAL TECHNOLOGYPES COLLEGE OF PHARMACYHANUMANTHANAGARBENGALURU-560 050rahulsangamkers383@gmail.com

PERMANENT ADDRESS:S/O Sangameshwar Rao6-3-29, Sangareddy AP 502001

2. Name of the institution PES COLLEGE OF PHARMACYHANUMANTHANAGARB.S.K.1st STAGEBENGALURU:-560 050

3. Course of the study MASTER OF PHARMACY(PHARMACEUTICAL TECHNOLOGY)

4. Date of Admission 30 JULY 2011

5. Title of the topic:

“FORMULATION AND EVALUATION OF A LYOPHILIZED DOSAGE FORM”

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6. Brief resume of the intended work:

6.1 Need for the study:

Lyophilization is a process more commonly known as freee drying. The word is derived from Greek, and means “made solvent loving”. Lyophilization is commonly used in pharmaceutical and biotechnology industries to improve the stability of formulations.2 The active pharmaceutical ingredient and accompanying excipients are first stabilized in a solvent (usually water), and the solution is rendered sterile by filtering through 0.2μm or equivalent sterilizing grade filters. The sterilized solution is filled into vials, and then loaded into a lyophilizer where the solution is frozen, and subsequently heated at a very low pressure to sublime the solvent and remove it from the formulation. Once the water is removed, the product vials are sealed under vacuum or an inert gas head space (i.e N2, Ar). The resulting highly porous cake has low moisture content and can be stored over extended periods of time at designated storage conditions until its intended use. Over the past few decades, the investigation of the fundamental physical phenomenon occurring in each step of freeze drying had led to producing a stable and elegant freeze-dried pharmaceutical dosage form.1

The relative importance of freeze drying in pharmaceutical science will continue to expand with the development of the next generation of therapeutic agents from discovery research through clinical trials, FDA approval, and market introduction. Many of the new products are chemically or physically unstable in aqueous solution and depend on the maintenance of the proper structure for the biological activity. Formulation and manufacture of injectable dosage forms of these agents present a challenge to the pharmaceutical scientist, and freeze drying will be necessary tool for the development of these products.2

The freeze-drying essentially consists of freezing stage, primary drying stage and secondary drying stage.3by means of lyophilization:

1. Stability of the compound is achieved and maintained.3

2. Enhanced stability of a dry powder.1

3. Removal of water without excessive heating of the product.4. Enhanced product stability in a dry state.5. Rapid and easy dissolution of reconstituted product.

There are many new parenteral products, including anti-infectives, anticancer drugs and biotechnology derived products manufactured as lyophilized products.

In this present study an attempt is made to reduce lyophilization cycle time of the lyophilized drug to improve its stability and reduce the time of manufacturing process.

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6.2 Review of the literature:

M. Glavas-Dodov et al studied the effects of lyophilisation on the stability

of liposomes containing 5-FU. Multilamellar liposomes containing 5-FU

were prepared by modified lipid film hydration method and were lyophilized

with or without saccharose as cryoprotectant. The effect of lyophilisation on

the stability of liposomes was evaluated by comparing the vesicle size,

encapsulation effeiciency and drug release rate before and after

lyophilisation. The process of lyophilisation, without cryoprotectant resulted

in particle size increase and significant content leakage. By the addition of

saccharose, the lipid bilayers become more stable and less permeable to the

encapsulated drug, saccharose imparted 5-FU retention of about 80% after

lyophilization/rehydration. Freeze-drying did not affect the particle size of

liposomes containing saccharose as cryoprotectant. The drug release profiles

of rehydrated liposomes followed Higuchi’s square root model. This research

has revealed that liposomes loaded with 5-FU and stabilized with a proper

cryoprotectant under optimized and well-controlled lyophilization conditions

could be suitable drug carriers in anticancer therapy.4

Kim S et al studied effective polymeric dispersants for vacuum, convection

and freeze drying of drug nanosuspensions and reported that only a small

amount of polymeric dispersants such as carrageenan, gelatin, and alginic

acid between 0.5 and 3 wt.% in various drug nanosuspensions can provide

sufficient redispersibility in vacuum, convection, and freeze drying. In

vacuum and freeze drying of naproxen nanosuspensions, the addition of only

0.5 wt.% carrageenan resulted in the formation of redispersable

nanoparticulate powders. The amounts of polymeric dispersants required for

redispersibility was lowest for carrageenan and highest for gelatin. The

specific interactions between the dispersants and steric stabilizers (or drugs),

in addition to viscosity increase during drying, appeared to effectively

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prevent irreversible particle aggregation.5

S.C. Tsinontides et al. framed Freeze drying—principles and practice for

successful scale-up to manufacturing. In this manuscript they described the

principal approach and tools utilized to successfully transfer the

lyophilization process of a labile pharmaceutical product from pilot plant to

manufacturing. Based on pilot plant data, the lyophilization cycle was tested

during limited scale-up trials in manufacturing to identify parameter set-

point values and test process parameter ranges. The limited data from

manufacturing were then used in a single vial mathematical model to

determine manufacturing lyophilizer heat transfer coefficients, and

subsequently evaluate the cycle robustness at scale-up operating conditions.

The lyophilisation cycle was then successfully demonstrated at target

parameter set-point values.6

Abdelwahed W et al. studied the impact of annealing on the freeze drying

process of nanocapsules. They investigated the impact of annealing on

primary and secondary drying characteristics and on nanocapsules (NC)

properties. Nanocapsules were prepared from poly-caprolactone (PCL)

biodegradable polymer and stabilized by polyvinyl alcohol (PVA), and then

freeze-dried with two cryoprotectants: sucrose and poly vinyl pyrrolidone

(PVP). Freeze-dried nanocapsules were characterized by size measurement

and transmission electron microscopy after reconstitution. The effect of

annealing on the kinetics of sublimation, on the mass transfer resistance and

on the porosity of the freeze-dried product has been studied in the case of

PVP. Finally, the effect of annealing on the kinetic of secondary drying was

studied and the results were coupled with the isotherm of sorption. Results

showed that PCL nanocapsules could be freeze-dried without any

modification of their properties in presence of the two cryoprotectants used.

Annealing of nanocapsules suspensions could accelerate the sublimation rate

without any modification of nanocapsules size in the case of the two studied

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cryoprotectants. Such improvement could be explained by the increase of ice

crystals size after annealing and by the diminution of mass transfer

resistance by the dried layer.7

Kadoya S et al. studied the physical properties and protein-stabilizing

effects of sugar alcohols in frozen aqueous solutions and freeze-dried solids.

Various frozen sugar alcohol solutions showed a glass transition of the

maximally freeze-concentrated phase at temperatures (Tg_s) that depended

largely on the solute molecular weights. Some oligosaccharide-derived sugar

alcohols (e.g., maltitol, lactitol, maltotriitol) formed glass-state amorphous

cake-structure freeze-dried solids. Microscopic observation of frozen

maltitol and lactitol solutions under vacuum (FDM) indicated onset of

physical collapse at temperatures (Tc) several degrees higher than their

Tg_s. Freeze-drying of pentitols (e.g., xylitol) and hexitols (e.g., sorbitol,

mannitol) resulted in collapsed or crystallized solids. The glass forming

sugar alcohols prevented activity loss of a model protein (LDH: lactate

dehydrogenase) during freeze-drying and subsequent storage at 50 ◦C. They

also protected bovine serum albumin (BSA) from lyophilization-induced

secondary structure perturbation. The glass-forming sugar alcohols showed

lower susceptibility to Maillard reaction with co-lyophilized l-lysine

compared to reducing and non-reducing disaccharides during storage at

elevated temperature.8

Guan T et al prepared the nimodipine-loaded nanoliposomes for injection

and evaluate their characteristics after lyophilization. Nimodipine-loaded

nanoliposomes were prepared by the emulsion-ultrasonic method with

sodium cholesterol sulfate (SCS) as the regulator and then lyophilized by

adding different cryoprotectants. SCS was used as a blender of regulator and

surfactant and helped to prepare smaller liposomes due to the steric

hindrance of the sulfate group. The results showed that nimodipine-loaded

nanoliposomes with a 20:1 of egg yolk lecithin PL-100M vs. SCS ratio had a

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particle size of 86.8±42.007 nm, a zeta potential of −13.94mV and an

entrapment efficiency (EE) of 94.34% and could be stored for 12 days at 25

°C. Because of the good bulking effect of mannitol and the preservative

effect of trehalose, they were used to obtain suitable lyophilized

nanoliposomes. The lyophiles containing 10% mannitol and 20% trehalose

had a good appearance and a slightly altered particle size after rehydration.

In addition, the lyophilized products were characterized by differential

scanning calorimetry, X-ray diffraction and scanning electron microscopy,

which confirmed the morphous state of trehalose, mannitol and the mixture.

Trehalose could inhibit mannitol crystallization to some extent. The drug

release from nanoliposomes before and after lyophilization in pH 7.4

phosphate buffer containing 30% ethanol was also examined and both

profiles were found to fit the Viswanathan equation.9

Schwarz C et al. carried out experiments on Freeze-drying of drug-free and

drug-loaded solid lipid nanoparticles (SLN). Solid lipid nanoparticles (SLN)

of a quality acceptable for i.v. administration were freeze-dried. Dynasan

112 and Compritol ATO 888 were used as lipid matrices for the SLN,

stabilisers were Lipoid S 75 and poloxamer 188, respectively. To study the

protective effect of various types and concentrations of cryoprotectants (e.g.

carbohydrates), freeze-thaw cycles were carried out as a pre-test. The sugar

trehalose proved to be most effective in preventing particle growth during

freezing and thawing and also in the freeze-drying process. Changes in

particle size distribution during lyophilisation could be minimised by

optimising the parameters of the lyophilisation process, i.e. freezing velocity

and redispersion method. Lyophilised drug-free SLN could be reconstituted

in a quality considered suitable for i.v. injection with regard to the size

distribution. Loading with model drugs (tetracaine, etomidate) impairs the

quality of reconstituted SLN. However, the lyophilisate quality is sufficient

for formulations less critical to limited particle growth, e.g. freeze-dried

SLN for oral administration.10

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Jonge J.D et al. stated that Inulin sugar glasses preserve the structural

integrity and biological activity of influenza virosomes during freeze-drying

and storage. Here they presented a procedure to generate influenza

virosomes as a stable dry-powder formulation by freezedrying

(lyophilization) using an amorphous inulin matrix as a stabilizer. In the

presence of inulin the structural integrity and fusogenic activity of virosomes

were fully preserved during freeze-drying. For example, the immunological

properties of the virosomes, i.e. the HA potency in vitro and the

immunogenic potential in vivo, were maintained during lyophilization in the

presence of inulin. In addition, compared to virosomes dispersed in buffer,

inulin-formulated virosomes showed substantially prolonged preservation of

the HA potency upon storage. Also the capacity of virosomes to mediate

cellular delivery of macromolecules was maintained during lyophilization in

the presence of inulin and upon subsequent storage. Specifically, when

dispersed in buffer, virosomes with encapsulated plasmid DNA lost their

transfection activity completely within 6weeks, whereas their transfection

activity was fully preserved for at least 12 weeks after incorporation in an

inulin matrix. Thus, in the presence of inulin as a stabilizing agent, the shelf-

life of influenza virosomes with and without encapsulated macromolecules

was considerably prolonged. Formulation of influenza virosomes as a dry-

powder is advantageous for storage and transport and offers the possibility to

develop needle-free dosage forms, e.g. for oral, nasal, pulmonal, or dermal

delivery.11

Xiang J et al. investigated the effects of different freeze–drying factors on

the rate of sublimation. The experiments were carried out in a custom-built

freeze–drying microbalance to accurately monitor the sample temperature

and control the chamber pressure. Twenty-four experiments were conducted

based on a full factorial design by changing four factors: freezing rate (fast

freezing or slow freezing), chamber temperature (35, 0, or −35 ◦C), chamber

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pressure (30 or 1000mTorr), and the presence or absence of an annealing

process. Lactate dehydrogenase (LDH), a tetrameric protein, was selected as

a model protein for this study. The statistical analysis of the experimental

results revealed that chamber temperature, analogous to the shelf

temperature, in this experiment system, had the greatest impact on the

sublimation rate. High chamber temperature resulted in high sublimation

rate, regardless of the chamber pressure and thermal history of the sample.

Chamber pressure was an important factor affecting the sublimation rate. In

addition, both chamber temperature and chamber pressure had significant

impact on sample temperature during freeze–drying. Annealing the samples

was the most critical step to preserve good freeze–dried cake structure.12

Alfadhel M et al. worked on Lyophilized inserts for nasal administration

harboring bacteriophage selective for Staphylococcus aureus. Here authors

showed that lyophilization of bacteriophages in 1 ml of a viscous solution of

1–2% (w/v) hydroxypropyl methylcellulose (HPMC) with/without the

addition of 1% (w/v) mannitol, contained in Eppendorf tubes, yields nasal

inserts composed of a highly porous leaflet-like matrix. Fluorescently

labeled bacteriophage were observed to be homogenously distributed

throughout the wafers of the dried matrix. The bacteriophage titer fell 10-

fold following lyophilization to 108 pfu per insert, then falling a further 100-

to 1000-fold over 6 to 12 months storage at 4 ◦C. This compares well with a

total dose of 6 × 105 pfu in 0.2 ml liquid applied into the ear during a recent

clinical trial in humans. The residual water content of the lyophilized inserts

was reduced upon the addition of mannitol to HPMC, but this did not have

any correlation to the lytic activity. Mannitol underwent a transition from its

amorphous to crystalline state during exposure of the inserts to increasing

relative humidities (as would be experienced in the nose), although this

transition was suppressed by higher HPMC concentrations and the presence

of buffer containing gelatin and bacteriophages. Our results therefore

suggest that lyophilized inserts harboring bacteriophage selective for S.

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aureus may be a novel means for the eradication of MRSA resident in the

nose.13

Puapermpoonsiri U et al. stabilized the baceteriophage during freeze

drying. Lyophilization is an established technique for the storage of

bacteriophage, but there is little consensus regarding drying cycles, additives

and moisture content specific to phage. Here, the addition of sucrose or

poly(ethylene glycol) 6000 yielded stable freeze-dried cakes only from high

concentrations (0.5Mand 5%, respectively), with addition of bacteriophage

otherwise causing collapse. Gelatin, which is added to storage media (a

solution of salts), played no role in maintaining bacteriophage stability

following lyophilization. A secondary drying cycle was most important for

maintaining bacteriophage activity. The addition of high concentrations of

PEG 6000 or sucrose generally caused a more rapid fall in bacteriophage

stability, over the first 7–14 d, but thereafter residual activities for all phage

formulations converged. There was no distinct change in the glass transition

temperatures (Tg) measured for the formulations containing the same

additive. Imaging of cakes containing fluorescently labeled bacteriophage

did not show gross aggregation or phase separation of bacteriophage during

lyophilization. However, the moisture content of the cake did correlate with

lytic activity, irrespective of the formulation, with a 4–6% moisture content

proving optimal. We propose that residual moisture is followed during

lyophilization of bacteriophage from minimal concentrations of bulking

agent.14

Kasper J.C et al. studied the Physico-chemical fundamentals, freezing

methods and consequences on process performance and quality attributes of

biopharmaceuticals involved in the freezing step in lyophilisation.

Lyophilization is a common, but cost-intensive, drying process to achieve

protein formulations with longterm stability. In the past, typical process

optimization has focused on the drying steps and the freezing step was rather

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ignored. However, the freezing step is an equally important step in

lyophilization, as it impacts both process performance and product quality.

While simple in concept, the freezing step is presumably the most complex

step in lyophilization. Therefore, in order to get a more comprehensive

understanding of the processes that occur during freezing, the physico-

chemical fundamentals of freezing are first summarized. The available

techniques that can be used to manipulate or directly control the freezing

process in lyophilization are also reviewed. In addition, the consequences of

the freezing step on quality attributes, such as sample morphology, physical

state of the product, residual moisture content, reconstitution time, and

performance of the primary and secondary drying phase, are discussed. A

special focus is given to the impact of the freezing process on protein

stability. This review aims to provide the reader with an awareness of not

only the importance but also the complexity of the freezing step in

lyophilization and its impact on quality attributes of biopharmaceuticals and

process performance. With a deeper understanding of freezing and the

possibility to directly control or at least manipulate the freezing behavior,

more efficient lyophilization cycles can be developed, and the quality and

stability of lyophilized biopharmaceuticals can be improved.15

Zhao D et al. developed lyophilized submicron emulsion for Cheliensisin A

(GC-51) which is a potent anti tumor drug with poor water solubility and

chemical instability for improving the therapeutic index of the drug. The

resultant lyophilized GC-51 submicron emulsion was much more stable than

its solution, which can be stored for years without significant change on

physicochemical properties .And its solubility was increased. The 50%

inhibitory concentration IC50 values were calculated from growth curves by

MTT assay on various tumor cell lines. Compared with the IC50 of GC-51

crude drug, that of lyophilized GC-51 submicron emulsion decreased. In the

time-dependent assay of tumor cell viability, lyophilized GC-51 submicron

emulsion exhibited significantly lower inhibition rate in the initial action

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times, but increased gradually afterwards. That means lyophilized submicron

emulsion as the vector for GC-51 had some protective and delayed release

effect. Compared with crude drug, the lyophilized GC-51 submicron

emulsion showed a significantly higher antitumor efficiency both in vivo and

in vitro, suggesting a potential application in tumor chemotherapy.16

Craig D.Q.M et al. studied the relevance of the amorphous state to

pharmaceutical dosage forms like glassy drugs and freeze dried systems.

Many pharmaceuticals, either by accident or design, may exist in a total or

partially amorphous state. Consequently, it is essential to have an

understanding of the physic chemical principles underpinning the behavior

of such systems. In this discussion, the nature of the glassy state was

described, with particular emphasis on the molecular processes associated

with glass transitional behaviour and the use of thermal methods for

characterising the glass transition temperature, Tg. The practicalities of such

measurements, the significance of the accompanying relaxation endotherm

and plasticization effects are considered. The advantages and difficulties

associated with the use of amorphous drugs will be outlined, with discussion

given regarding the problems associated with physical and chemical

stability. Finally, the principles of freeze drying will be described, including

discussion of the relevance of glass transitional behaviour to product

stability.17

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6.3 Main objectives of the study:

• Preformulation studies including IR, DSC and compatibility studies of drug.• To have several initial formulations prepared with suitable excipients and

comparing formulatons. • Determining the maximum allowable temperature permitted during freezing and

secondary drying i.e kmowing eutectic point, glass transition and/or collapse temperatures.

• Determining appropriate process parameters i.e rate of freezing, set point temperature during three phases, need for annealing, pressure during primary drying and pressure during secondary drying.

• Optimize the formulations. • Stability studies of the optimized formulation.

7. 7.1 Source of data

The data will be obtained from the literature survey and internet source.

The data will be obtained from the experimental work, which includes formulation and optimization of lyophilized, scale up techniques and stability studies of optimized formulation.

7.2 Method of collection of data (including sampling procedures if any)

The pharmacological details of the drug will be collected from various standard

books, journals and other sources like research literature databases such as Medline,

Pubmed, Science direct, etc.

Experimental data will be collected from the designed formulation and then

subjecting the formulation to different evaluation techniques and stability studies

obtained from Strides Arcolabs FDA approved facility in Bengaluru.

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8. LIST OF REFERENCES :

1. Guide to inspections of lyophilization of parenterals, office of regulatory affairs U.S.food and drug administration. Available at: http://www.fda.gov/ICECI/Inspections/InspectionGuides/ucm074909.htm.

2. www.wisegeek.com/what-is-lyophilization.htm

3. Akers MJ. Parenteralsprepations. In; Gennaro AR. Remington Editor: The Science and Practice of Pharmacy. 21st ed. Vol-1, Pennsylvania: Mack publishing Company; 1995; p.828-31.

4. Dodav MG, Kumbaradzi EF, Goracinova K, Simonosla M, Calis S, Jolevska S et al. The effects of lyophilization on the stability of liposomes containing 5-FU. Int J Pharm. 2005;292:79-86.

5. Kim S, Lee J. Effective polymeric depressants for vacuum, convention and freeze drying of nano suspensions. Int J Pharm. 2010;397:218-24.

6. Tsinontides SC, Rajniak P, Hualee WA, Placele J, Reynolds SD. Freeze drying, principles and practice for successful scale-up, manufacturing. Int J Pharm. 2004;280:1-6.

7. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: Formulation, process and storage considerations. Int J Pharm. 2005;309:178-88.

8. Kadoya S, Fujii K, Izutsu KI, Yonemochi E, Terada K, Yomota C et al. Freeze-drying of proteins with glass-forming oligosaccharide-derived sugar alcohols. Int J Pharm. 2010;389:107-13.

9. Guan T, Miao Y, Xu L, Yang S, Wang J,He H et al. Injectable nimodipine-loaded nanoliposomes: Preparation, lyophilization and characteristics. Int J Pharm. 2010;410:180-87.

10. Schwarz C, Mehnert W. Freeze-drying of drug-free and drug-loaded solid lipid nanoparticles (SLN). Int J Pharm. 1997;157:171-79.

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11. Jonge JD, Amorji JP, Hinrichs WLJ, Wilschut J, Huckriede A, Frijlink HW. Inulin sugar glasses preserve the structural integrity and biological activity of influenza virosomes during freeze-drying and storage. Eur J Pharm Sci. 2007;32:33-44.

12. Xiang J, Hey JM, Liedtke V, Wang DQ. Investigation of freeze-drying sublimation rates using freeze-drying microbalance technique. Int J Pharm. 2004;279:95-105.

13. Alfadel M, Puapermpoonsiri U, Ford SJ, McInnes FJ, Vander Walle CF. Lyophilized inserts for nasal administration harboring bacteriophage selective for Staphylococcus aureus: In vitro evaluation. Int J Pharm. 2011;416:280-87.

14. Puapermpoonsiri U, Ford SJ, Vander Walle CF. Stabilization of bacteriophage during freeze drying. Int J Pharm. 2010;389:168-175.

15. Kasper JC, Friess W. The freezing step in lyophilization: Physico-chemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals. Eur J Pharm Biopharm. 2011;78:248-63.

16. Dong Z, Tao G, Yao F, Yu N, Li-Li H, Jie L, et al. Lyophilized Cheliensisin A submicron emulsion for intravenous injection: Characterization, in vitro and in vivo antitumor effect. Int J Pharm. 2008;357:139-47.

17. Craig DQM, Royall PG, Kett VL, Hopton ML. The relevance of the amorphous state to pharmaceutical dosage forms: glassy drugs and freeze dried systems. Int J Pharm. 1999;179:179-207.

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12.

Signature of the candidate: (RAHUL SANGAMKER )

Remarks of the guide: Recommended

Name And Designation of: 11.1 Guide Dr. S. J. SHANKAR Asst. Professor and Head, Department of Pharmaceutical Technology, P.E.S College of Pharmacy, Banglore-50. 11.2 Signature

11.3 Co-Guide SHIVRAJ B. KATAGERI, Asst. Vice President-FDD, Strides Acrolabs Limited, Bilekahalli, Bannerghatta Road, Bangalore - 560 076. Ph: 66580290 Mobile: 9740011640 shivraj.katageri@strides.com 11.4 Signature

11.5 Head of the department Dr.S.J.Shankar Head of Department, Department of Pharmaceutical Technology, P.E.S College of Pharmacy, Banglore-50.

11.6 Signature

12.1 Remarks of the Principal Prof. Dr. S. Mohan Principal and director P.E.S College of Pharmacy, Bangalore-50.

12.2 Signature

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