23

2.1 INTRODUCTION TO SOLID DISPERSION:

The enhancement of oral bioavailability of poorly water-soluble drugs remains

one of the most challenging aspects of drug development. Although salt formation,

co-solubilization and particle size reduction have commonly been used to increase

dissolution rate and thereby oral absorption and bioavailability of such drugs[10]

, there

are practical limitations of these techniques. The salt formation technique is not

feasible for neutral compounds and also the synthesis of appropriate salt forms of

drugs that are weakly acidic or weakly basic may often not be practical[14]

. Even when

salts can be prepared, an increased dissolution rate in the gastrointestinal tract may not

be achieved in many cases because of the reconversion of salts into aggregates of their

respective acid or base forms[13]

. The solubilization of drugs in organic solvents or in

aqueous media by the use of surfactants and cosolvents leads to liquid formulations

that are usually undesirable from the viewpoints of patient acceptability and

commercialization. Although particle size reduction is commonly used to increase

dissolution rate, there is a practical limit to size reduction achieved by commonly used

methods as controlled crystallization, grinding, pearl milling etc. The use of very fine

powders in a dosage form may also be problematic because of handling difficulties

and poor wettability due to charge development[12]

.

In 1961, Sekiguchi and Obi[96]

developed a practical method whereby most of the

limitations with the bioavailability enhancement of poorly water-soluble drugs can be

overcome, which was termed as „Solid Dispersion‟[52]

.

From conventional capsules and tablets, the dissolution rate is limited by the size

of the primary particles formed after disintegration of dosage forms. In this case, an

average particle size of 5 µm is usually the lower limit, although higher particle sizes

are preferred for ease of handling, formulation and manufacturing. On the other hand,

24

if a solid dispersion or a solid solution is used, a portion of the drug dissolves

immediately to saturate the gastrointestinal fluid and the excess drug precipitates out

as fine colloidal particles or oily globules of submicron size. Hence, due to promising

increase in the bioavailability of poorly water-soluble drugs, solid dispersion has

become one of the most active areas of research in the pharmaceutical field[49, 97]

.

2.1.1 DEFINITION AND TYPES OF SOLID DISPERSIONS:

2.1.1.1 Definition:

Solid dispersion technology is the science of dispersing one or more active

ingredients in an inert matrix in the solid stage to achieve an increased dissolution rate

or sustained release of drug, altered solid state properties and improved stability.

2.1.1.2 Types of Solid Dispersions:

A) Simple Eutectic Mixture:

Eutectic mixture of a sparingly water-soluble drug and a highly water-soluble

carrier may be regarded thermodynamically as an intimately blended physical mixture

of its two crystalline components (Fig. 2.1). These systems are usually prepared by

melt fusion method. When the eutectic mixture is exposed to water, the soluble carrier

dissolves leaving the drug in a microcrystalline state which gets solubilized rapidly.

The increase in surface area is mainly responsible for increased rate of dissolution[98]

.

Fig. 2.1: Hypothetical Phase Diagram of Eutectic Mixture

25

B) Solid Solutions:

Solid solutions consist of a solid solute dissolved in a solid solvent. These systems

are generally prepared by solvent evaporation or co-precipitation method, whereby

guest solute and carrier are dissolved in a common volatile solvent such as alcohol.

The solvent is allowed to evaporate, preferably by flash evaporation. As a result, a

mixed crystal containing amorphous drug in crystalline carrier is formed because the

two components crystallize together in a homogenous single phase system. Such

dispersions are also known as „Co-precipitates‟ or „Co-evaporates‟. This system

would be expected to yield much higher rates of dissolution than simple eutectic

systems. Because, the basic difference between solid solution and eutectic mixture is

that the drug is precipitated out in an amorphous form in solid dispersion/solution

while it is in crystalline form in eutectics[99, 100]

.

Solid solution can generally be classified according to the extent of miscibility

between the two components or the crystalline structure of the solid solution[101]

.

(i) Continuous solid solutions

(ii) Discontinuous solid solution

(iii) Substitutional solid solution

(iv) Interstitial solid solution

i) Continuous Solid Solutions:

In this system, the two components are miscible or soluble at solid state in all

proportions (Fig. 2.2). No established solid solution of this kind has been shown to

exhibit faster dissolution properties, although it is theoretically possible. It is obvious

that a faster dissolution rate would be obtained if the drug were present as a minor

component. However, the presence of a small amount of the soluble carrier in the

26

crystalline lattice of the poorly soluble drugs may also produce a dissolution rate

faster than the pure compound with similar particle size.

Fig. 2.2: Hypothetical Phase Diagram of Continuous Solid Solution

ii) Discontinuous Solid Solution:

In this system (Fig. 2.3), in contrast to the continuous solid solution, there is only

a limited solubility of a solute in a solid solvent. Each component is capable of

dissolving the other component to a certain degree above the eutectic temperature.

However, as the temperature is lowered, the solid solution regions become narrower.

The free energy of stable and limited solid solutions is also lower than that of pure

solvent.

Fig. 2.3: Hypothetical Phase Diagram of Discontinuous Solid Solution

27

iii) Substitutional Solid Solution:

As shown in Fig. 2.4, in this type of solid solution, the solute molecule substitutes

for the solvent molecules in the crystal lattice of the solid solvent. It can form a

continuous or discontinuous solid solution. The size and steric factors of the solute

molecules play a decisive role in the formation of solid solution. The size of the solute

and the solvent molecule should be as close as possible.

Fig. 2.4: Substitutional Solid Solution

iv) Interstitial Solid Solution:

The solute (guest) molecule occupies the interstitial space of the solvent (host)

lattice (Fig. 2.5). It usually forms only a discontinuous (limited) solid solution. The

size of the solute is critical in order to fit into the interstices. It was found that the

apparent diameter of the solute molecules should be less than that of the solvent in

order to obtain an extensive interstitial solid solution of metals.

Fig. 2.5: Interstitial Solid Solution

28

C) Glass Solution:

A glass solution is a homogenous system in which a glassy or a vitreous carrier

solubilizes drug molecules in its matrix[102]

. PVP dissolved in organic solvents

undergoes a transition to a glassy state upon evaporation of the solvent[103]

. The glassy

or vitreous state is usually obtained by an abrupt quenching of the melt. It is

characterized by transparency and brittleness below the glass transition temperature

(Tg). On heating, it softens progressively without a sharp melting point.

D) Compound or Complex Formation:

This system is characterized by complexation of two components in a binary

system during solid dispersion preparation. The availability of drug from complex or

compound depends on the solubility, association constant and intrinsic absorption rate

of complex. Rate of dissolution and gastrointestinal absorption can be increased by

the formation of a soluble complex with low association constant[104]

.

E) Amorphous Precipitation:

Amorphous precipitation occurs when drug precipitates as an amorphous form in

the inert carrier. The higher energy state of the drug in this system generally produces

much greater dissolution rates than the corresponding crystalline forms of the drug. It

is postulated that a drug with high super cooling property has more tendency to

solidify as an amorphous form in the presence of a carrier. Hence, amorphous

precipitation is rarely observed[105]

.

Fig. 2.6: Amorphous Precipitation

29

2.1.2 MECHANISM OF DISSOLUTION RATE ENHANCEMENT:

Corrigan[106]

reviewed the understanding of the mechanism of release from solid

dispersion. The increase in drug dissolution rate from solid dispersion system can be

attributed to a number of factors like particle size, crystalline or polymorphic forms

and wettability of drug etc. It is very difficult to show experimentally that any one

particular factor is more important than another. The main reasons postulated for the

observed improvements in dissolution from these systems are as follows[52]

:

a) Reduction of Particle Size:

In case of glass solution, solid solution and amorphous dispersions, particle size is

reduced. This may result in enhanced dissolution rate due to increase in the surface

area. Similarly, it has been suggested that the presentation of particles to dissolution

medium as physically separate entities may reduce aggregation.

b) Solubilization Effect:

The carrier material, as it dissolves, may have a solubilization effect on the drug.

Enhancement in solubility and dissolution rate of poorly soluble drugs is related to the

ability of carrier matrix to improve local drug solubility as well as wettability[107]

.

c) Wettability and Dispersibility:

The carrier material may also have an enhancing effect on the wettability and

dispersibility of the drug due to the surfactant action reducing the interfacial tension

between hydrophobic drug particle and aqueous solvent phase, increasing the

effective surface area exposed to the dissolution medium. This also retards

agglomeration or aggregation of the particles, which can slow down the dissolution.

d) Conversion of Polymorphic Nature of Solute:

Energy required to transfer a molecule from crystal lattice of a purely crystalline

solid is greater than that required for non-crystalline (amorphous) solid. Hence

30

amorphous state of a substance shows higher dissolution rates. But the amorphous

solids also demonstrate lack of physical stability due to natural tendency to form

crystals. Thus formation of metastable dispersions with reduced lattice energy would

result in faster dissolution rate and comparatively acceptable stability.

2.1.3 SELECTION OF CARRIER:

One of the most important steps in the formulation and development of solid

dispersion for various applications is selection of carrier. The properties of carrier

have a major influence on dissolution characteristics of the drug. A material should

possess following characteristics to be suitable carrier for increasing dissolution[108]

:

i. Freely water-soluble with intrinsic rapid dissolution properties

ii. Non-toxic nature and pharmacologically inertness

iii. Thermal stability preferably with low melting point especially for melt method

iv. Solubility in a variety of solvents and should pass through a vitreous state upon

solvent evaporation for the solvent method

v. Ability to increase the aqueous solubility of the drug

vi. Chemical compatibility and not forming a strongly bonded complex with drug.

2.1.4 POLYMERS USED IN SOLID DISPERSIONS:

A variety of polymers is offered as carriers for formulation of solid dispersion.

Table 2.1 represents various categories and examples of carriers. Some polymers used

in solid dispersions are as follows:

A) Polyethylene Glycols (PEG):

The term polyethylene glycols refer to compounds that are obtained by reacting

ethylene glycol with ethylene oxide. PEGs with molecular weight more than 300,000

are commonly termed as polyethylene oxides.

31

B) Polyvinyl Pyrrolidone (PVP):

PVPs have molecular weights ranging from 10,000 to 700,000. It is soluble in

solvents like water, ethanol, chloroform and isopropyl alcohol. PVP is not suitable for

preparation of solid dispersions prepared by melt method because it melts at a very

high temperature above 275ºC, where it gets decomposed.

C) Polymers and Surface Active Agent Combinations:

The addition of surfactants to dissolution medium lowers the interfacial tension

between drug and dissolution medium and promotes the wetting of the drug thereby

they enhance the solubility and dissolution of drug. Ternary dispersion systems have

higher dissolution rates than binary dispersion systems[109]

.

D) Cyclodextrins:

Cyclodextrins are primarily used to enhance solubility, chemical protection, taste

masking and improved handling by the conversion of liquids into solids by

entrapment of hydrophobic solute in hydrophilic cavity of CD[38-41]

. Advantages of

CD include increasing the stability of the drug, release profile during gastrointestinal

transit through modification of drug release site and time profile, decreasing local

tissue irritation and masking unpleasant taste.

E) Phospholipids:

Phospholipids are major structural components of cell membranes.

Phosphotidylcholine was first isolated from egg yolk and brain. In phosphatidyl

ethanolamine and phosphatidyl serine, the choline moiety is replaced by ethanolamine

and serine respectively. Other phospholipids that occur in tissues include

phosphotidyl ethanolamide, phosphotidyl serine and phosphotidyl glycerol. Naturally

occuring lecithins contain both a saturated fatty acid and unsaturated fatty acids with

some exceptions[72]

.

32

Table 2.1: Materials used as carrier for solid dispersion

Sr. No. Category Examples

1 Sugars

Dextrose, Sucrose, Galactose, Sorbitol,

Maltose, Xylitol, Mannitol[67]

, Lactose[64]

2 Acids Citric acid, Succinic Acid[68]

3 Polymeric materials

PVP[56]

, PEG[58]

, Celluloses like HPMC[60]

,

HEC, HPC, Pectin, Galactomannan, CDs[38]

4 Insoluble/ enteric polymer HPMC[60]

, Phthalate, Eudragits[71]

5 Surfactants

Polyoxyethylene stearate, Renex,

Poloxamers[63]

, texafor, Deoxycholic acid,

Tweens, Spans[65]

6 Miscellaneous

Pentaerythritol, Pentaerythrityl tetra acetate,

Urea[62]

, Urethane, Hydroxy alkyl xanthins

2.1.5 METHODS OF PREPARATION OF SOLID DISPERSIONS:

A) Fusion Process:

The fusion process is technically less difficult method of preparing dispersions

provided the drug and carrier are miscible in the molten state. Drug and carrier

mixture of eutectic composition is molten at temperature above its eutectic

temperature. Then molten mass is solidified on an ice bath and pulverized to a

powder. Since a super saturation of the drug can be obtained by quenching the melt

rapidly (when solute molecules are arrested in solvent matrix by instantaneous

solidification), rapid congealing is favoured. The solidification is often performed on

stainless steel plates to facilitate rapid heat loss. A modification of the process

involves spray congealing from a modified spray drier onto cold metal surfaces.

33

Decomposition should be avoided during fusion but is often dependent on

composition and affected by fusion time, temperature and rate of cooling. Therefore,

to maintain drug content and physicochemical stability of formulation at an

acceptable level, fusion must be effected at a temperature only just in excess of that

which completely melts both drug and carrier.

B) Solvent Evaporation Process:

Solid dispersion prepared by solvent removal process was termed by Bates et

al.[110]

as „Coprecipitates‟. But these systems should more correctly, be designated as

„Coevaporate‟, a term that has been recently adopted.

The solvent evaporation process uses organic solvents, the agent to intimately mix

the drug and carrier molecules and was initially used by Tachibana and Nakamura[111]

,

where, chloroform was used to co-dissolve β-carotene and PVP to form Co-evaporate.

The choice of solvent and its removal rate are critical parameters affecting the

quality of the solid dispersion. Since the chosen carriers are generally hydrophilic and

the drugs are hydrophobic, the selection of a common solvent is difficult and its

complete removal, necessitated by its toxic nature, is imperative. Vacuum evaporation

may be used for solvent removal at low temperature and also at a controlled rate.

More rapid removal of the solvent may be accomplished by freeze-drying. The

difficulties in selecting a common solvent to both drug and carrier may be overcome

by using an azeotropic mixture of solvent in water.

C) Fusion Solvent Method:

This method consists of dissolving the drug in a suitable solvent and incorporating

the solution directly in the melt of carrier. If the carrier is capable of holding a certain

proportion of liquid yet maintains its solid properties and if the liquid is innocuous,

34

then the need for solvent removal is eliminated. This method is particularly useful for

drugs that have high melting points or they are thermo-labile.

D) Supercritical Fluid Process:

Supercritical CO2 is a good solvent for water-insoluble as well as water-soluble

compounds under suitable conditions of temperature and pressure. Therefore, it has

potential as an alternative for conventional organic solvents used in solvent based

processes for forming solid dispersions due to its favorable properties of being non-

toxic and inexpensive. The process consists of the following steps[27, 28]

:

i. Charging the bioactive material and suitable polymer into the autoclave.

ii. Addition of supercritical CO2 under precise conditions of temperature and

pressure, that causes polymer to swell

iii. Mechanical stirring in the autoclave

iv. Rapid depressurization of the autoclave vessel through a computer controlled

orifice to obtain desired particle size.

The temperature condition used in this process is fairly mild (35-75°C), which

allows handling of heat sensitive biomolecules, such as enzymes and proteins.

2.1.6 ADVANTAGES AND DISADVANTAGES OF SOLID DISPERSIONS:

The advantages of solid dispersion include the rapid dissolution rates that result in

increased bioavailability and a reduction in pre-systemic metabolism. The latter

advantage may occur due to saturation of the enzyme responsible for

biotransformation of the drug or inhibition of the enzyme by the carrier, as in the case

of morphine-tristearin dispersion[112]

. Both can lead to the need for lower doses of the

drug. Other advantages include transformation of the liquid form of the drug into a

solid form (e.g. clofibrate and benzoyl benzoate can be incorporated into PEG 6000 to

give a solid, avoiding polymorphic changes and thereby bioavailability problems[113]

)

35

and protection of certain drugs by PEGs against decomposition by saliva to allow

buccal absorption.

The disadvantages of solid dispersion are related mainly to stability issue. Several

systems have shown changes in crystallinity and a decrease in dissolution rate with

aging[114, 115]

. Moisture and temperature have a more prominent deteriorating effect on

solid dispersions than on physical mixtures. Some solid dispersion may not lend them

to easy handling because of tackiness.

Fig. 2.7: Pharmaceutical Applications of Solid Dispersion

36

2.1.7 FUTURE PROSPECTS:

Despite many advantages of solid dispersion, issues related to preparation,

reproducibility, formulation, scale up and stability has limited its use in commercial

dosage forms for poorly water-soluble drugs. Successful development of solid

dispersion systems for preclinical, clinical and commercial use has been feasible in

recent years due to the availability of surface active and self-emulsifying carriers with

relatively low melting points. The preparation of dosage forms involves the

solubilization of drug in melted carriers and the filling of the hot solutions into hard

gelatin capsules because of the simplicity of manufacturing and scale-up processes,

the physicochemical properties and as a result, the bioavailability of solid dispersions

are not expected to change significantly during the scale-up. For this reason, the

popularity of the solid dispersion system to solve difficult bioavailability issues of

poorly water-soluble drugs will grow rapidly. As the dosage form can be developed

and prepared using small amount of drug substance in early stages of the drug

development process, the system might have an advantage over such other commonly

used bioavailability enhancement techniques such as micronization and soft gelatin

encapsulation.

One major focus of the future research will be the identification of new surface

active and self-emulsifying carriers for solid dispersion. Only a small number of such

carriers are currently available for oral use. Some carriers that are used only for

topical applications of drug may be qualified for oral use by conducting appropriate

toxicological testing. One limitation in the development of solid dispersion systems is

inadequate drug solubility in carrier, so a wider choice of carriers will increase the

success of dosage form development.

37

Research should also be directed towards identification and synthesis of new

possibilities of vehicles or excipients that would retard or prevent crystallization of

drugs from super-saturated systems. Attention must be given to any physiological,

pharmacological and toxicological effects of carriers. Many of the surface active and

self-emulsifying carriers are lipoidal in nature, so potential roles of such carriers on

drug absorption, especially on their inhibitory effects on CYP-3 based drug

metabolism and p-glycoprotein mediated drug efflux will require careful

consideration.

In addition to bioavailability enhancement, much recent efforts and advances in

the research on solid dispersion systems are directed towards the development of

extended release dosage forms.

Physical and chemical stability of both drug and carrier in a solid dispersion are

major developmental issues, so future research needs to be directed to address various

stability issues. The semisolid and waxy nature of solid dispersions poses unique

stability problem that might not be seen in other types of solid dosage forms.

Predictive methods are necessary for the investigation of any potential drug

crystallization and its impact on dissolution and bioavailability. Also possible drug-

carrier interactions must also be investigated.

38

2.2 REVIEW OF LITERATURE:

Sekiguchi and Obi[96]

in 1961 first demonstrated the unique approach of solid

dispersion to reduce the particle size and increase dissolution and absorption rate.

They proposed the formation of eutectic mixture of poorly soluble drug such as

sulfathiazole with physiologically inert, easily water-soluble carrier such as urea. The

eutectic mixture was prepared by melting the physical mixture of drug and carrier,

followed by a rapid solidification process. Upon exposure to aqueous fluid, the active

drug was expected to be released into the fluids as fine, dispersed particles because of

the fine dispersion of the drug in the solid eutectic mixture and the rapid dissolution

of the soluble matrix.

Goldberg et al. [99, 100, 107]

in a series of reports in 1965-66, presented a detailed

experimental and theoretical discussion on advantages of solid solution over the

eutectic mixture.

Tachibana and Nakamaru[111]

reported a novel method for preparing aqueous

colloidal dispersions of β-carotene by using water-soluble polymers such as polyvinyl

pyrrolidone. They dissolved the drug and the carrier in a common solvent and then

evaporated the solvent completely. A colloidal dispersion was obtained when the co-

precipitate was exposed to water.

Chiou and Riegelman[52]

advocated the application of glass solution to increase

dissolution rate. They used PEG 6000 as a dispersion carrier. It is demonstrated that

the pharmaceutical technique of solid dispersion can play an important role in

increasing dissolution, absorption and therapeutic efficacy of drugs in future.

Therefore, a thorough understanding of its fast release principles, methods of

preparation, selection of suitable carriers, determination of physical properties,

limitations and disadvantages is essential in its practical and effective applications.

39

Duncan et al. [103]

discussed the nature of glassy state with particular emphasis on

the molecular processes associated with glass transitional behavior and the use of

thermal methods for characterizing the glass transition temperature. 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 were outlined, with discussion given regarding the

problems associated with physical and chemical stability. Also, the principles of

freeze drying were described, including discussion of the relevance of glass

transitional behavior to product stability.

Xiaolin et al. [116]

studied hydrogen bonding patterns and strength in a series of

structurally related compounds. Seven 1, 4-dihydropyridine calcium channel blockers

were evaluated. They found that H-bonding patterns (acceptor group) varied between

the crystalline compounds, but were remarkably consistent in the amorphous

compounds. Thus the acceptor group in the amorphous phase is not necessarily the

same as in the crystalline counterpart.

Makoto Otsuka et al. [117]

studied effect of humidity on the physicochemical

properties of amorphous forms of cimetidine using differential scanning calorimetry,

isothermal micro-calorimetry and X-ray diffraction analysis. They suggested that the

crystallization process consists of an initial stage of the nuclei formation and a final

stage of crystal growth.

Urbanetz et al. [118]

investigated improvement in the storage stability of

nimodipine by preventing recrystallization. The first approach in order to prevent

recrystallization was the development of thermodynamically stable solid solutions by

using solvents added to enhance the solubility of nimodipine in the carrier material.

The second approach was to enhance storage stability by the addition of

40

recrystallization inhibitors to super-saturated solid solutions, thereby delaying the

transformation of the metastable super-saturated system to the thermodynamically

stable state. Stabilization by solubility enhancement was only successful at drug

loadings not exceeding 10% (w/w) using polyethylene glycol 300 as solubility

enhancing additive, while for second approach povidone K17 effectively prevents

recrystallization in solid solutions containing 20% (w/w) of nimodipine during storage

at 25°C over silica gel.

Paradkar et al. [119]

emphasized on stability aspects of formulated solid dispersion

of anti-inflammatory drug, valdecoxib with hydrophilic carriers selected PVP K30

and HPC by spray-drying method. The evaluation of SD system suggests that the drug

was transformed into its amorphous form to elicit increased dissolution rate. During

stability testing, saturation solubility of spray-dried valdecoxib dropped drastically

within 15 min. While, there was gradual decrease in saturation solubility and

dissolution rate of solid dispersion, over the period of 3 months, showing

comparatively enhanced stability.

Paradkar et al. [120]

, in another study, prepared solid dispersions of glibenclamide

and polyglycolized glycerides (Gelucire®) with the aid of silicon dioxide (Aerosil®

200) as an adsorbent by spray-drying technique. The study demonstrated the high

potential of spray-drying technique for obtaining stable free flowing SDs. Moreover

in vivo study in Swiss Albino mice also justified the improvement in the therapeutic

efficacy of amorphous drug in SDs over pure drug. SDs also showed improved

stability which could be due to the hydrogen bonding between the drug and the

carriers and the adsorption on the surface of amorphous silicon dioxide.

Ozeki et al. [121]

applied solid dispersion approach for controlled release of

phenacitin by the formation of an inter-polymer complex between methyl cellulose

41

and carbopol using 6 different molecular weights of methyl cellulose. The effect of

the ratio of polymer and molecular weight of methyl cellulose on the phenacitin

release was studied. The results of the study also clarify the mechanism of drug

release from the granules with help of semi-empirical mathematical model.

Seo et al. [122]

formulated solid dispersions of diazepam by melt agglomeration

method for improving the dissolution rate. Lactose monohydrate was melt

agglomerated with polyethylene glycol (PEG) 3000 and Gelucire® 50/13 as meltable

binders in a high shear mixer. The binders were added either as a mixture of melted

binder and diazepam by a pump-on procedure or by a melt-in procedure of solid

binder particles. Different drug concentrations, maximum manufacturing temperatures

and cooling rates were investigated. It was found that it is possible to increase the

dissolution rate of diazepam by melt agglomeration. A higher dissolution rate was

obtained with a lower drug concentration.

Chen et al. [123]

prepared solid dispersion of a model drug ABT- 963 with pluronic

by cooling the hot melt of the drug in the carrier. Results showed that, ABT-963

formed a eutectic mixture with Pluronic F68. Both the drug and poloxamer were

crystalline in the solid dispersion with a wide range of composition of each

component. The solid dispersion substantially increased the in vitro dissolution rate of

ABT-963 which was attributed to enhanced hydration of drug in a viscous micro-

environment formed by immediate release of poloxamer. Dosing of the dispersion to

fasted dogs resulted in a significant increase in oral bioavailability. These results

suggested that poloxamer is a promising material for developing solid dispersion.

Gines et al. [124]

, in 1995, studied thermal behavior of Gelucire® 53/10-cinnarizine

binary systems. It was noted from the analytical thermal techniques employed like

42

DSC and hot stage microscopy (HSM), that the molten Gelucire was able to dissolve

the crystals of cinnarizine.

Literature survey was also done for profiling of selected drug candidate-

Gliclazide (GLZ) for formulation of solid dispersion.

Glowka et al. [125]

evaluated bioavailability of gliclazide from some formulations

including conventional gliclazide tablet formulation as well as sustained release

formulation. It demonstrates poor dissolution rate of gliclazide.

Ozkan et al. [126]

prepared inclusion complexes of GLZ with β-CD using two

methods viz. neutralization and recrystallization. The study showed the inadequacy of

dissolution rate of gliclazide and emphasized on the need of solubility enhancement.

Cham et al. [127]

formulated inclusion complex of GLZ and β-CD by liquid/liquid

extraction method and neutralization method. The solid complex by liquid/liquid

extraction demonstrated a faster release profile attributed to decreased particle size

and wettability of hydrophobic GLZ.

Vijayalakshmi et al.[128]

attempted the solubility enhancement of GLZ by

inclusion complex with HP-β-CD and incorporated the solubility enhanced drug in a

matrix forming polymer (sodium carboxymethyl cellulose) for designing oral

controlled release tablets. The in vivo study was conducted on Newzealand rabbits.

The bioavailability obtained from the standardized solubility enhanced GLZ tablets

was greater than that of the tablets containing untreated gliclazide.

Abou-Auda et al. [129]

studied effect of β-CD complexation on solubility,

bioavailability as well as pharmacodynamic activity of GLZ. The prepared binary

system showed increased dissolution rate which can be correlated with

pharmacokinetic as well as hypoglycemic study in Beagle Dogs.

43

2.3 OBJECTIVES:

Solubility of a drug plays a very important role in dissolution and hence

absorption of drug which ultimately affects its bioavailability. Poorly soluble drugs

particularly of BCS Class II represent a problem for their scarce availability.

Gliclazide (GLZ) is a second generation hypoglycemic sulfonylurea oral

hypoglycemic agent used in the treatment of non insulin dependent diabetes mellitus

(NIDDM). Due to its short-term action, GLZ has been considered suitable for

diabetic patients with renal impairment and for elderly patients those have reduced

renal function and follow a sulphonyl urea treatment which may increase the risk of

hypoglycemia[130]

.

The remarkable recede in the therapeutic application and efficacy of gliclazide as

immediate release oral dosage forms is its very low aqueous solubility and inter-

individual variability in its bioavailability mainly because of its hydrophobic

molecular nature and crystalline characteristics[126-129, 131]

.

Hence, considering the factors affecting solubility and bioavailability, attempts

have been made to apply the principles of solid dispersion techniques to enhance the

dissolution and oral bioavailability of gliclazide with following objectives:

1. Formulation of solid dispersion for the improvement of solubility and

dissolution characteristics of gliclazide

2. Characterization and confirmation of amorphous dispersion

3. Characterization of solubility, dissolution rate and stability

4. In vivo evaluation of bioavailability

44

2.4 PLAN OF WORK:

1. Literature survey

2. Procurement of materials

3. Experimental

A. Drug authentication

B. Compatibility study

C. Calibration curve of gliclazide

D. Phase solubility study

E. Formulation of solid dispersion.

F. Evaluation and characterization of solid dispersion:

a. Drug content

b. Interaction study

c. Thermal study

d. Assessment of crystallinity

e. In vitro dissolution study

f. In vivo pharmacodynamic study

4. Stability study of optimized formulation

5. Compilation of data

45

2.5 DRUG PROFILE:

GLICLAZIDE[132-135]

Gliclazide (GLZ) is a second generation sulphonylurea, oral hypoglycemic agent

used in the treatment of non-insulin-dependant diabetes mellitus (NIDDM or Type-II

diabetes mellitus). GLZ improves defective insulin secretion and may reverse insulin

resistance observed in patients with NIDDM. These actions are reflected in blood

glucose level which is maintained during short and long-term administration and is

comparable with that achieved with other sulphonylurea agents.

Chemical Name:

N-[[(hexahydrocyclopenta[c]pyrrol-2(1H)-yl)amino]carbonyl]-4-methyl benzene)

sulfonamide

Chemical Structure:

Fig. 2.8: Chemical Structure of Gliclazide

Molecular Formula: C15H21N3O3S

Molecular Weight: 323.4

Description: Gliclazide is a white or almost white crystalline powder.

Solubility:

It is practically insoluble in water, freely soluble in methylene chloride, sparingly

soluble in acetone and slightly soluble in alcohol.

46

Pharmacodynamics:

Gliclazide reduces blood glucose levels in patients with NIDDM by correcting

both defective insulin secretion and peripheral insulin resistance. Unstimulated and

stimulated insulin secretions from pancreatic ß-cells are increased following the

administration of GLZ, with both first and second phases of secretion being affected.

GLZ binds to the ß-cell sulfonyl urea receptor. This binding subsequently blocks the

ATP-sensitive potassium channels. The binding results in channel closure leading to a

decreased K+ efflux and depolarization of the beta cells. This opens voltage sensitive

calcium channels in the ß-cell resulting in calmodulin activation, which in turn, leads

to exocytosis of secretory granules containing insulin[131,

132]

.

In addition, GLZ increases the sensitivity of ß-cells to glucose. It may have extra

pancreatic effect which restores peripheral insulin sensitivity such as decreasing

hepatic glucose production, increasing glucose clearance and skeletal muscle

glycogen synthesis activity. These effects do not appear to be mediated by effect on

insulin receptor number, affinity or function. There is some evidence that GLZ also

improves defective hematological activity in patients with NIDDM [136]

.

Pharmacokinetics:

Oral absorption of GLZ is similar in patients and healthy volunteers, but there is

inter-subject variation in time to reach peak plasma concentrations (Tmax)131

. Age

related differences in plasma peak concentrations (Cmax) and Tmax have been observed.

A single oral dose of 40 to 120 mg of gliclazide results in a Cmax of 2.2-8 µg/ml

within 2 to 8 hr. Tmax and Cmax are increased after repeated gliclazide administration.

The variability in absorption of GLZ could be related to its early dissolution in the

stomach leading to more variability in the absorption in the intestine[136]

. This process

resulted in low and variable bioavailability of the conventional dosage forms.

47

Steady state concentration is achieved after two days of administration of 40 to

120 mg of GLZ. It has low volume of distribution (13 to 24 L) in both patients and

healthy volunteers due to its high protein binding affinity (85 to 97%)[136]

. The

elimination half-life (t1/2) is about 8.1 to 20.5 hr in healthy volunteers and patients

after administration of a dose of 40 to 120 mg orally.

Moreover, its plasma clearance is 0.78 L/hr (13 ml/min). It is extensively

metabolized to 7 metabolites, which are excreted in urine. Therefore, renal

insufficiency has no effect on pharmacokinetic of GLZ.

Dosage and Administration:

GLZ is administered in doses ranging from 40 to 320 mg/day as tablets once to

three times daily. Recently, modified release formulations containing 20 mg or 30 mg

of GLZ have been developed to obtain a better predictable drug release[137, 138]

.

Indication:

Gradually accumulating evidence suggests that GLZ may be useful in NIDDM

patients. GLZ is an effective agent for the treatment of metabolic disorder associated

with NIDDM and may have the added advantage of potentially slowing the

progression of diabetic retinopathy, due to its hematological actions and that addition

to insulin therapy enables insulin dosage to be reduced[139]

. These actions, together

with its good tolerability and low incidence of hypoglycemia, allow GLZ to be well

placed within the array of oral hypoglycemic agents available for control of NIDDM.

Contraindication:

It is contraindicated in the conditions like insulin-dependent diabetes mellitus,

diabetic coma, pre-coma and extreme imbalance with tendency to acidosis, hepatic or

renal failure, surgical stress or acute infection.

48

Drug Interactions:

Glycemic control of GLZ may be reduced by diuretics, barbiturates, phenytoin,

rifampicin, corticosteroids, estrogens, estroprogestogens and pure progestogens. The

hypoglycemic action may be potentiated by salicylates, phenylbutazone,

sulphonamides, beta-blockers, clofibric acid, vitamin K antagonist, allopurinol,

theophylline, caffeine and monoamine oxidase inhibitors. Concomitant administration

of miconazole, perhexiline or cimetidine may result in hypoglycemia. Concomitant

administration of gliclazide with agents that increase blood glucose levels should not

be considered without careful monitoring of blood glucose levels to avoid

hyperglycemia.

Adverse Reactions:

Gastrointestinal disturbances:

Nausea, diarrhoea, gastric pain and vomiting.

Dermatological effects:

Rash, pruritus, urticaria, erythema and flushing.

Miscellaneous:

Headache and dizziness. GLZ appears to be associated with a low incidence of

hypoglycemia. It may have the potential to produce adverse cardiovascular effects.

However it has been an established agent for the treatment of NIDDM for a number

of years without any adverse cardiovascular effects.

49

2.6 POLYMER PROFILE:

POLOXAMER[140]

Poloxamers are nonionic polyoxyethylene- polyoxypropylene copolymers used

primarily in pharmaceutical formulations as emulsifying or solubilizing agents.

Synonyms:

Lutrol, Monolan, Pluronic, poloxalkol, poloxamera, polyethylene- propylene

glycol copolymer, polyoxyethylene– polyoxypropylene copolymer, Supronic and

Synperonic.

Chemical Name:

α-Hydro-ω-hydroxypoly(oxyethylene)-poly(oxypropylene)-poly-(oxyethylene) block

copolymer.

Chemical Structure:

Fig. 2.9: Chemical Structure of Poloxamer Monomer

Empirical Formula: HO(C2H4O)a(C3H6O)b(C2H4O)aH.

Molecular Weight: 1000 to > 16000

Description:

Poloxamers generally occur as white, waxy, free-flowing prilled granules or as

cast solids. They are practically odorless and tasteless. At room temperature,

poloxamer occurs as a colorless liquid.

50

Chemical Properties[141-143]

:

Poloxamers are nonionic polyoxyethylene- polyoxypropylene copolymers. The

polyoxyethylene segment is hydrophilic while the polyoxypropylene segment is

hydrophobic. All grades of poloxamers are chemically similar in composition,

differing only in the relative amounts of propylene and ethylene oxides added during

manufacturing. Their physical and surface-active properties vary over a wide range

and a number of different types are commercially available.

The nonproprietary name „poloxamer‟ is followed by a number. The first two

digits of which, when multiplied by 100, correspond to the approximate average

molecular weight of the polyoxypropylene portion of the copolymer and the third

digit, when multiplied by 10, corresponds to the percentage by weight of the

polyoxyethylene portion.

Similarly, with many of the trade names used for poloxamers, e.g. Pluronic F68

(BASF Corp.), the first digit arbitrarily represents the molecular weight of the

polyoxypropylene portion and the second digit represents the percent weight of the

oxyethylene portion. The letters „L‟, „P‟ and „F‟, stand for the physical form of the

poloxamer- liquid, paste or flakes respectively[143]

.

Typical Properties:

Acidity/alkalinity: pH = 5.0-7.4 for a 2.5% w/v aqueous solution.

Density: 1.06 g/cm3 at 25°C

Flash point: 260°C

Flowability: Solid poloxamers are free flowing.

HLB value: 0.5-30; 29 for poloxamer 188.

Melting point: 16°C for poloxamer 124, 52-57°C for poloxamer 188, 498°C

for poloxamer 237, 57°C for poloxamer 338, 52-57°C for poloxamer 407

51

Moisture Content:

Poloxamers generally contain less than 0.5% w/w water and are hygroscopic only

at relative humidity greater than 80%.

Solubility:

Poloxamers are very soluble in water and alcohol, practically insoluble in light

petroleum (50-70°C). Poloxamers are more soluble in cold water than hot water.

Functional Category:

Dispersing agent, emulsifier, solubilizing agent, tablet lubricant, wetting agent.

Applications in Pharmaceutical Technology[144]

:

Poloxamers are used as emulsifying agents in intravenous fat emulsions and as

solubilizing and stabilizing agents to maintain the clarity of elixirs and syrups.

Poloxamers may also be used as wetting agents, in ointments, suppository bases and

gels and as tablet binder and coating material.

Poloxamer 188 (Pluronic F68) has also been used as an emulsifying agent for

fluorocarbons used as artificial blood substitutes and in the preparation of solid

dispersion systems. Therapeutically, poloxamer 188 is administered orally as a

wetting agent and stool lubricant in the treatment of constipation. It is usually used in

combination with a laxative such as Danthron.

Poloxamers may also be used therapeutically as wetting agents in eye drop

formulations, in the treatment of kidney stones and as skin wound cleansers.

Poloxamer in the form of its hydrogel is used as lens refilling material for injectable

intraocular lens. In this, air vinyl was used as in vitro model for checking transparency

of the hydrogel[145]

.

Poloxamer demonstrates a thermoreversible behavior in which sol-gel conversion

is observed on increase in the temperature[146]

. Marked increase in gel strength was

52

found after addition of mucoadhesive polymer. Hence, this combination was found to

be used as in situ gelling and mucoadhesive drug delivery for enhancing ocular

bioavailability[147]

. The combination of bioadhesive polymer and poloxamer has been

successful in enhancement of bioavailability from various other routes of

administration like nasal absorption[148]

, vaginal[149]

, rectal[150]

and buccal

application[151]

. The mechanism of in situ gelling is also used for injectable

formulations like intramuscular and intravenous[152]

.

As poloxamer is a non ionic surfactant, it shows solubilization of poorly water-

soluble drugs when used in solid dispersion[123]

. It acts by creating a micellar micro-

environment around the drug particle enhancing the dissolution rate.

Stability and Storage Conditions:

Poloxamers are stable materials. Aqueous solutions of poloxamers are stable in

the presence of acids, alkalis and metal ions. However, aqueous solutions support

mold growth. The bulk material should be stored in a well-closed container in a cool,

dry place due to its hygroscopicity.

Incompatibilities:

Depending on the relative concentrations, poloxamer 188 is incompatible with

phenols and parabens.

Safety[153]

:

Poloxamers are used in a variety of oral, parenteral and topical pharmaceutical

formulations. These are generally regarded as nontoxic and nonirritant materials.

Poloxamers are not metabolized in the body.

Animal toxicity studies, with dogs and rabbits, have shown poloxamers to be

nonirritating and non-sensitizing when applied in 5% w/v and 10% w/v concentration

to the eyes, gums and skin.

53

2.7 EXPERIMENTAL:

2.7.1 Materials:

Gliclazide was a generous gift from Lupin Research Park, Pune, India. Pluronic

F68 and Pluronic F127 were kindly supplied as gift samples by BASF, Mumbai, India

All other chemicals and solvents were of analytical reagent grade.

2.7.2 Drug Authentication:

2.7.2.1 Melting Point:

Primary authentication of GLZ was done by melting point determination. Melting

point was checked by conventional capillary method and reported uncorrected.

2.7.2.2 FTIR Spectroscopy:

FTIR Spectroscopy of GLZ was done by using FTIR Spectrophotometer

(Schimadzu FTIR 8400S, Japan). The samples were scanned over wave number

region of 4000 to 400 cm-1

at resolution of 4 cm-1

. Samples were prepared using KBr

(spectroscopic grade) disks with hydraulic pellet press at pressure of 7-10 tons.

2.7.2.3 UV Spectroscopy:

A solution of 100 µg/ml concentration of GLZ in 0.1N hydrochloric acid was

prepared for determination of λmax. The sample was scanned on Double beam UV-VIS

spectrophotometer (Systronics -Double Beam Spectrophotometer- 2201).

2.7.2.4 Calibration Curve of Gliclazide:

Calibration curve of absorbance vs. concentration of GLZ was plotted in 0.1N

hydrochloric acid. The solutions of different concentrations (0-20 µg/ml) were

prepared from stock solution of 100 µg/ml concentration in triplicates. The

absorbances of solutions were read spectrophotometrically at 228.8 nm.

54

2.7.2.5 Solubility Study:

Absolute solubility of GLZ was carried out by the method reported by Higuchi

and Connor[154]

in distilled water and 0.1N HCl. Excess of GLZ was added to 10 ml

study fluid in a screw capped vial. Samples were shaken on rotary shaker at constant

speed at 25°C±2

°C for 48 hr. The saturated solutions after equilibration for 24 hr were

filtered through a membrane filter having pore size of 0.45 µm. Filtrates were suitably

diluted and estimated spectrophotometrically for GLZ content at 228.8 nm.

2.7.3 Compatibility Study:

The drug and excipients in different ratios were equally distributed in glass

ampoules. They were kept at 37°C, 45

°C, 60

°C and room temperature of 25

°C. The

samples were analyzed for its physical appearance, UV scanning to examine the

compatibility. Possibility of interaction was also studied by FTIR spectroscopy with

1:1 physical mixture of drug and excipients.

2.7.4 Phase Solubility Study:

The phase solubility analysis for GLZ was done by Higuchi-Connor‟s method154

with two grades of poloxamer viz. Pluronic F68 and Pluronic F127. Excess amount of

GLZ was added to screw-capped vials containing 10 ml of aqueous solutions of

Pluronic F68 and Pluronic F127 with varying concentrations (5% to 30%)[123]

. Vials

were shaken with rotary shaker for 48 hr at a controlled temperature at 25ºC±2ºC.

Supernatant was centrifuged after equilibration period for 24 hr. Aliquots were

analyzed by UV- spectrophotometry at 228.8 nm.

55

2.7.5 FORMULATION OF SOLID DISPERSION:

Different formulations of solid dispersions of GLZ were prepared with two grades

of poloxamers as carrier by two methods, viz. solvent evaporation (SE) and melt

fusion (MF) method[49, 51, 52, 58, 97]

. Corresponding physical mixtures (PM) were

prepared for comparative evaluation and determination of effect of methods of

preparation. The compositions of the formulations are shown in Table 2.2.

2.7.5.1 Solvent Evaporation Method:

Accurately weighed quantities of drug and carrier were homogenously dissolved

in sufficient volume of chloroform as a common volatile solvent. The solution was

allowed to evaporate at ambient temperature and the resulting solid mass was then

dried in a desiccator. Solid dispersions were sieved through sieve number 60 to

confirm the size uniformity.

2.7.5.2 Melt Fusion Method:

Accurately weighed amount of carrier was placed on a hot plate and molten, with

constant stirring, maintaining the critical temperature just below 70°C. An accurately

weighed amount of GLZ was incorporated into the molten carrier with stirring to

ensure homogeneity. The mixture was heated until a clear homogeneous melt was

obtained. It was cooled in an ice-bath, allowed to solidify and sieved through sieve

number 60.

2.7.5.3 Physical Mixture:

Physical mixtures were prepared by simple mixing of two components. The

appropriate amounts of drug and carrier were blended with minimum stirring pressure

in a mortar and pestle to form physical mixture. The mixture was passed through

sieve number 60 to obtain uniform size distribution.

56

Table 2.2: Formulation of Solid Dispersion

Method

Carrier

Solvent Evaporation Melt Dispersion Physical Mixture

Batch Ratio Batch Ratio Batch Ratio

Pluronic F68

GES 1 1:1 GEM 1 1:1 GEP 1 1:1

GES 2 1:2 GEM 2 1:2 GEP 2 1:2

GES 3 1:3 GEM 3 1:3 GEP 3 1:3

GES 4 1:4 GEM 4 1:4 GEP 4 1:4

Pluronic F127

GFS 1 1:1 GFM 1 1:1 GFP 1 1:1

GFS 2 1:2 GFM 2 1:2 GFP 2 1:2

GFS 3 1:4 GFM 3 1:4 GFP 3 1:4

GFS 4 1:9 GFM 4 1:9 GFP 4 1:9

2.7.6. EVALUATION OF SOLID DISPERSIONS:

2.7.6.1 Drug Content:

Solid dispersions and physical mixtures equivalent to 10 mg of GLZ were

weighed accurately and dissolved in 50 ml of 0.1N HCl and stirred well with

magnetic stirrer. The drug content was determined at 228.8 nm by UV

spectrophotometric analysis after suitable dilutions.

2.7.6.2 Thin Layer Chromatography:

Thin Layer Chromatography (TLC) was employed for primary assessment of

drug-polymer incompatibility and degradation[155]

. The study was carried out using

chromatographic grade silica gel-G stationary phase and benzene: methanol (9:1) as

optimized mobile phase. The sample solutions of GLZ, poloxamers and formulations

were prepared in methanol and spotted on stationary phase. Development of

chromatograph was performed in a glass TLC chamber previously saturated with

57

mobile phase. Visualization of spots was done in saturated Iodine chamber. Number

and positions of spots were observed to assess integrity of samples and any possibility

of degradation during formulation. Comparison of Rf values was done. All the

readings were taken in triplicate.

2.7.6.3 Fourier Transform Infra-Red Spectroscopy:

Assessment of chemical interaction and compatibility between drug and

excipients in physical mixtures as well as solid dispersions was done by FTIR

spectroscopy. FTIR spectra of GLZ, carriers, physical mixtures and solid dispersions

were recorded using a FTIR Spectrophotometer (Schimadzu FTIR spectrophotometer

8400S, Japan). Samples were prepared using KBr (spectroscopic grade) disks by

means of hydraulic pellet press at a pressure of 7-10 tons. The samples were scanned

from 4000 to 400 cm–1

at resolution of 4 cm-1

.

2.7.6.4 Thermal Analysis:

DSC serves as useful tool to assess the chemical compatibility as well as the

degree of crystallinity of excipients and drugs. The thermal behavior of the GLZ,

Poloxamers and formulations (1:1) were determined using a differential scanning

calorimeter (DSC SDT2960, TA Instruments Inc., USA) for identifying the chemical

compatibility of drug and polymers. Samples (3-5 mg) were accurately weighed into

50 mg aluminium pans and hermetically sealed. The samples were heated from 100 to

300°C at a rate of 10°C/min under a dry nitrogen gas purge.

2.7.6.5 X-Ray Diffractometry:

Degree of crystallinity is one of the factors influencing the solubility and

dissolution rate. Hence, crystalline nature of substances and extent of its conversion to

amorphous form was studied by X-Ray Diffractometry (XRD). XRD patterns were

studied using Philips PW 3710 X-Ray diffractometer. Samples were irradiated with

58

Cr- radiation of wavelength 2.289A° and analyzed between 5-40° (2θ). XRD patterns

were determined for GLZ, poloxamers and all formulations.

2.7.6.6 In vitro Dissolution Study:

The in vitro dissolution study was performed using USP-XXIV Type-II paddle

dissolution test apparatus (Electrolab TDT- 06P, India). The samples equivalent to 40

mg GLZ were placed in dissolution vessel containing 900 ml 0.1 N hydrochloric acid

as dissolution medium (pH 1.2) maintained at 37°C±0.5°C and stirred at 100 rpm. 5

ml aliquot samples were collected periodically and replaced with a fresh dissolution

medium to maintain sink conditions. After filtration through 0.45 µm membrane

filter, GLZ was estimated spectrophotometrically at 228.8 nm with suitable dilutions.

The dissolution profiles are represented as mean of three sets of readings[126]

.

Statistical analysis of dissolution data[156]

:

The drug release from solid dispersion was analyzed by applying various

mathematical models such as zero order, first order, Hixson-Crowell, Korsmeyer-

Peppas and Matrix models. Calculations were done for determination of rate constants

R2 values and release exponent (n-value). Also the dissolution profiles were compared

by calculating mean dissolution time (MDT), % dissolution efficiency (%DE) at

different time intervals. All the results were expressed as mean values ± standard

deviation. All data analyses were performed using PCP Disso V3 software.

2.7.6.7 In vivo Pharmacodynamic Study: [43, 157]

In vivo pharmacodynamic study of gliclazide solid dispersion was performed

on both healthy and diabetic animals. Male Wistar rats weighing 150-200 g were

obtained from National Toxicological Center Pune, India. They were housed in

polypropylene cages with husk bedding, renewed every 48 hr under 12:12 hr light-

dark cycle at around 25°C±2°C. They were fed with commercial pellet rat chow and

59

given water ad libitum. The experiment was carried out according to the guidelines of

the Committee for the Purpose of Control and Supervision of Experiments on

Animals (CPCSEA), New Delhi, India and the Institutional Animal Ethical

Committee (IAEC) approved protocol of this study (IAEC/2010-11/2A) at MVP

College of Pharmacy, Nasik, Maharashtra.

Induction of Diabetes in Rats by Alloxan:

Chemically induced (alloxan) diabetes in animals was given by Frerichs and

Creutzfeldt[158]

. The compound turned out to be specifically cytotoxic to pancreatic β-

cells. Triphasic time course is observed: an initial rise of glucose which is followed by

a decrease, probably due to depletion of islets from insulin, again followed by a

sustained increase of blood glucose. The alloxan-induced rat model represents an

acquired form of hyper-insulinemia, insulin resistance, hyper-triglyceridemia and

consequently dementia.

Dosing & Measurement of Blood Glucose Levels:

Six groups of Wistar rats were allowed free access to standard laboratory diet and

drinking fluid. Each group consisted of five wistar rats of either sex. One group was

assigned as „Control group‟ while, animals from group II, III and IV were fasted

overnight and were injected with freshly prepared alloxan solution intravenously at a

dose of 75 mg/kg body weight to induce diabetes. Group II was kept as diabetic

animals without treatment. Administration of drug and formulation was done in form

of suspension. Group V and VI were assigned as normal rats treated with GLZ and

formulation respectively. Groups III and V were given pure gliclazide at a dose of 8.3

mg/kg and group IV and VI were given gliclazide formulation (Batch GEM 3) at a

dose of 24.9 mg/kg (Table 2.3). Blood samples were collected through retro-orbital

plexus under ether anesthesia for determination of plasma glucose level on 0, 7, 14,

60

21 and 28th days, while at 0, 1, 2, 4, 6 and 24 hr for normal rats. The blood glucose

levels were determined using the glucose measuring biochemical kit (Span

Diagnostics Ltd., Surat, Gujarat). The instrument was self-calibrated, and the samples

were allowed to dry before the results were read to avoid contaminating the lens.

Statistical significance was tested by applying two-way ANOVA. The results

showing p< 0.05 were considered as significant.

Table 2.3: In vivo pharmacodynamic Study

Group Description Dose

I Control Group-Normal 0.5ml/100 g, p.o

II Control Group-Untreated Diabetic Alloxan: 75 mg/kg, i.v.

III Diabetic rats treated with GLZ 8.3 mg/kg p.o. for 21days

IV Diabetic rats treated with GLZ SD 24.9 mg/kg p.o. for 21days

V Diabetic rats treated with GLZ 8.3 mg/kg p.o.

VI Diabetic rats treated with GLZ SD 24.9 mg/kg p.o.

2.7.6.8 Stability Study:

The stability of optimized formulation was monitored up to 3 months at

accelerated stability conditions of temperature and relative humidity (30±2°C, RH

65±5% and 40±2°C, RH 75±5%) according to guidance of ICH guidelines for

stability[159]

. Samples were withdrawn after each time interval and evaluated for effect

of aging on drug content and dissolution rate.

61

2.8 RESULTS & DISCUSSION:

2.8.1 Authentication of Drug:

2.8.1.1 Melting point:

Melting point of GLZ was found to be in a range of 178 to 1810C. The reported

value is 180-182°C[133, 134]

. Thus, the melting point of GLZ sample complies with the

standard reported value.

2.8.1.2 UV Spectra Analysis:

The UV spectrum of GLZ in 0.1N HCl showed that the λmax was found to be at

228.8 nm which was in accordance with the previously reported values[126-128]

.

2.8.1.3 FTIR Spectroscopy:

Fig. 2.10 depicts FTIR spectrum of GLZ sample. It shows an intense peak at

3272.54 cm-1

corresponding to N-H stretch; 1712.24 cm-1

attributed to carbonyl

functionality. Sulphonyl group in pure GLZ can be characterized by strong symmetric

stretching peak at 1163.27 cm-1

and anti-symmetric vibration peak at 1348.98 cm-1

.

The observation of characteristic peaks of GLZ in FTIR spectra of sample

authenticates the sample as pure GLZ.

Fig. 2.10: FTIR Spectrum of Gliclazide

62

2.8.1.4 Solubility Study:

The solubility of GLZ in distilled water is found to be 57.607±3.677 µg/ml; while

the solubility in 0.1N HCl was 82.43±0.67 µg/ml.

2.8.2 Compatibility Study:

2.8.2.1 Compatibility Study by UV spectroscopy:

Compatibility study shows that the physical appearance of the mixture remains

unaffected on storage in different temperature condition. Compatibility was also

examined by using UV spectrophotometry at initial, second week and fourth week.

The scanning values were found in the range of 228.8 nm (Table 2.4).

Table 2.4: Compatibility Study by UV spectroscopy

Drug +

Excipient

λ max (nm) λ max at 37oC (nm) λ max at 45

oC (nm)

Initial

2nd

week

4th

week

Initial

2nd

week

4th

week

Initial

2nd

week

4th

week

GLZ+

Pluronic F68

228.9 228.8 228.8 228.6 228.7 228.8 228.8 228.8 228.6

GLZ+

Pluronic F127

229 228.8 228.8 228.8 228.8 229 228.8 228.6 228.4

2.8.2.2 Compatibility Study by FTIR Spectroscopy:

Compatibility study was carried out by using FTIR spectroscopy. Drug and

excipients with equal proportions showed all the characteristic peaks of their

respective functional groups. As shown in Fig. 2.11 and Fig. 2.12, there is no

significant shift observed in the positions of featured peaks of GLZ as well as

poloxamers. Hence, it can be considered that the drug and poloxamers are chemically

compatible and can be togetherly incorporated together in the formulation.

63

Fig. 2.11: Compatibility Study of GLZ and Pluronic F127 by FTIR spectroscopy

Fig. 2.12: Compatibility Study of GLZ and Pluronic F68 by FTIR spectroscopy

64

2.8.3 Standard Calibration Curve of Gliclazide:

The standard calibration curve of UV absorption vs. concentration of GLZ at

228.8 nm showed very good linearity characterized by good coefficient of correlation

(R2= 0.9999) over the concentration range of 0-20 µg/ml. Thus it was found to obey

Beer- Lambert‟s law over this range. The line equation of standard calibration curve

for estimation of GLZ in 0.1 N HCl can be given by equation 2.1.

Y = 0.0481X - 0.0015 (2.1)

2.8.4 Phase Solubility Study:

Poloxamers are water-soluble non-ionic surface active agents, which have been

widely used in pharmaceutical applications as emulsifier and solubilizing agents. As

stated in preformulation study, the intrinsic solubility of GLZ was 1.781 X 10-4

M/ml,

which is in accordance with the reviewed literature[158]

. At 25°C, aqueous solubility of

GLZ was found to be increasing with increased poloxamer concentration as the carrier

concentration increased both grades of poloxamer at the tested concentrations with AL

type of solubility phase diagram[154[

. Phase solubility curves with Pluronic F68 and

Pluronic F127 are shown in Fig. 2.13 and Fig. 2.14 respectively.

The significant enhancement in the solubility of GLZ might be attributed to the

surfactant effect due to polymeric phase which creates a favorable environment

around drug particles reducing the interfacial tension and enhancing wettability of the

hydrophobic drug. Thus, the use of poloxamer for designing solid dispersion for

solubility enhancement of GLZ appears to be a promising approach.

65

Fig. 2.13: Phase Solubility of GLZ in Pluronic F68

Fig. 2.14: Phase Solubility of GLZ in Pluronic F127

50

60

70

80

90

0 5 10 15

Sa

tura

tio

n s

olu

bil

ity

(µ

g/m

l)

Concentration of Pluronic F 68 (%w/v)

50

60

70

80

90

0 5 10 15 20 25

Satu

rati

on

solu

bil

ity

(µ

g/m

l)

Concentration of Pluronic F 127 (%w/v)

66

2.8.5 CHARACTERIZATION OF SOLID DISPERSIONS:

2.8.5.1 Thin Layer Chromatography:

The affinity shown by sample towards stationary and mobile phases in

chromatographic method is a function of polarity of the molecule and thereby,

chemical structure. Thus chromatographic techniques can be used as assessment tool

for determination of chemical interactions between drug and excipients.

As observed with Silica gel G as stationary phase and methanol:water as

optimized mobile phase. TLC showed a good resolution to distinctly separate the two

components. Poloxamers showed maximum affinity towards the stationary phase

showing no movement with development of mobile phase. Thus its Rf value was zero.

GLZ demonstrated a good development and showed a spot at 0.3 Rf value.

Physical mixtures and solvent evaporation batches showed two distinctly separate

spots at Rf equal to 0 and 0.3 corresponding to poloxamer and GLZ respectively,

suggesting no chemical interaction and degradation in PM and SE.

However, in case of the melt fusion batches prepared at about 80°C, spots were

observed at Rf equal to 0 & 0.3 of poloxamer and drug respectively, with additional

third spot at a different Rf (0.6). Thus developed chromatograms with a different

number and position of spots suggest the possibility of thermal decomposition at 80°C

operating temperature. But such spot was absent in melt fusions prepared at

temperature just below 70°C. Therefore, further temperature for solid dispersion by

melt dispersion method was critically controlled just below 70°C.

67

2.8.5.2 Drug Content:

Drug contents in prepared SDs were found to be ranging between 97.06 to 100.8

% (w/w) which complying the pharmacopoeial standards[133]

(Table 2.5). The assay

was found to be decreased in formulation batches by fusion method at 80°C,

supporting TLC results which suggest degradation, while batches prepared at 70°C

show acceptable drug content.

Table 2.5: Drug Content of Solid Dispersions.

Batch Assay Batch Assay Batch Assay

GEP 1 100.44±1.67 GEM 1 99.14±0.65 GFS 1 97.06±1.99

GEP 2 100.62±0.91 GEM 2 98.53±3.01 GFS 2 97.84±3.55

GEP 3 100.01±1.19 GEM 3 97.67±1.13 GFS 3 99.57±3.97

GEP 4 100.62±1.28 GEM 4 97.15±2.06 GFS 4 98.36±2.47

GES 1 99.49±0.78 GFP 1 99.92±1.69 GFM 1 98.01±2.09

GES 2 97.49±1.05 GFP 2 98.10±1.84 GFM 2 98.45±0.78

GES 3 98.97±0.52 GFP 3 99.49±1.58 GFM 3 98.27±3.46

GES 4 99.84±1.17 GFP 4 99.92±3.53 GFM 4 100.36±1.73

2.8.5.3 FTIR Spectra Analysis:

FTIR spectra of GLZ and solid dispersions are shown in Fig. 2.15-2.20. GLZ

showed intense peaks at 3272.54 cm-1

, corresponding to N-H stretch, 1712.24 cm-1

attributed to carbonyl functionality. Peaks at 1163.27 cm-1

and 1348.98 cm-1

correspond to sulphonyl group of symmetric stretching and anti-symmetric vibration

respectively. All the characteristic bands of GLZ remained unaffected in physical

mixtures as well as solid dispersions. Thus FTIR study suggested absence of

incompatibility. Also the prepared formulations were found chemically stable.

68

Fig. 2.15: FTIR Spectra of PMs: GEP 1, GEP 2, GEP 3 & GEP 4

Fig. 2.16: FTIR Spectra of SDs: GES 1, GES 2, GES 3 & GES 4

69

Fig. 2.17: FTIR Spectra of SDs: GEM 1, GEM 2, GEM 3 & GEM 4

Fig. 2.18: FTIR Spectra of PMs: GFP 1, GFP 2, GFP 3 & GFP 4

70

Fig. 2.19: FTIR Spectra of SDs: GFS 1, GFS 2, GFS 3 & GFS 4

Fig. 2.20: FTIR Spectra of SDs: GFM 1, GFM 2, GFM 3 & GFM 4

71

2.8.5.4 X-Ray Diffraction Study of Solid Dispersions:

Crystallinity is one of the most important properties of drug molecule, which

significantly influence the solubility and dissolution rate of solute. Crystalline

structure is considered as a comparatively stable structure due to the phenomenon of

the stabilization of energy by forming the crystal bonds. Thus due to less energy state,

its solubility is poor compared to the amorphous structure which represents a higher

energy state. Hence, crystallinity of pure GLZ and dispersions with poloxamers was

studied by X-Ray Diffractometry.

The degree of crystallinity of any substance can be assessed by the peak numbers

and the intensity of peak in XRD. More the number and intensity of peak, greater is

the crystallinity.

From XRD of GLZ (Fig. 2.21), it is evident that drug exhibits microcrystalline

nature. The XRD spectra of plain GLZ showed sharp and intense peaks of

crystallinity at a diffraction angle of 2θ at 10.67, 15.005, 16.805, 17.1, 18.195, 20.97

and 22.21 indicate the presence of crystalline drug.

Pluronic F68 demonstrated peaks at 2θ at 19.165, 23.225 and 38.405 (Fig. 2.22),

while Pluronic F127 showed peaks at 2θ at 19.305 and 23.365 (Fig. 2.23). The

physical mixtures showed all characteristics peaks of GLZ.

For the comparison of disorderness of formulations the term relative degree of

crystallinity (RDC) can be used. RDC can be calculated by equation 2.2[160]

. The

highest peak of drug was found to be at 10.67 and was selected for calculating RDC.

RDC =Highest peak intensity of formulation

Highest peak intensity of drug (2.2)

Fig. 2.24-2.29 show the XRDs of different formulations. The XRD patterns of the

formulations exhibited reduction in both number and intensity of peaks compared to

72

GLZ alone. This indicates that the crystallinity of GLZ is reduced by solid dispersion

approach which ultimately results in enhanced dissolution rates.

From the values of RDC (Table 2.6), it was observed that the reduction in degree

of crystallinity was observed more significant in the solid dispersions compared to

physical mixtures. Hence it can be concluded that the amorphization of drug is

dependent on method of preparation of solid dispersion. Melt fusion technique was

found to be more significantly useful in reduction of crystalline characteristics (RDC

ranging from 0.07 to 0.316) than solvent evaporation technique (RDC ranging from

0.093 to 1.1653). This might be attributed to molecular dispersion of amorphous drug

in carrier melt in the process of melt fusion. Subsequent solidification of melt

maintains the dispersed microstructure of the fusion. On the contrary, solvent

evaporation technique includes evaporation of volatile solvent followed by

recrystallization, leading to comparatively more crystalline character of drug than

melt fusion.

In SDs prepared by both MF and SE method shows gradual decrease in the peak

intensity with increased carrier content. Hence it can be noted that the decrease in the

drug crystallinity is a function of concentration of carrier in the system. It may be

attributed to the tendency of carrier molecules to keep the molecules of drug discrete

and away from each other thus inhibiting the crystalline bond formation. Hence

availability of more amount of carrier gives opportunity to make drug molecule as

more discrete entity leaving the drug molecule in an amorphous state with high

surface free energy which is responsible for faster dissolution process. From the

comparison between two grades of poloxamer, Pluronic F68 has produced more

significant effect on GLZ crystallinity than Pluronic F127, which can be observed

from the peak intensities from different solid dispersions and PMs.

73

Table 2.6: Relative Degree of Crystallinity by XRD study

Sample

Related Main peak

Sample

Related Main peak

Angle

2θ

Peak

intensity

(Count)

RDC Angle

2θ

Peak

intensity

(Count)

RDC

GLZ 10.670 1225 1.0000

GEP 1 10.465 250 0.1377 GFP 1 10.510 339 0.1868

GEP 2 10.555 538 0.2964 GFP 2 10.510 310 0.1708

GEP 3 10.560 185 0.1019 GFP 3 10.595 293 0.1614

GEP 4 10.555 269 0.1482 GFP 4 10.595 204 0.1124

GES 1 10.635 1781 0.9813 GFS 1 10.855 1018 0.5609

GES 2 10.770 169 0.0931 GFS 2 10.355 650 0.3581

GES 3 10.495 259 0.1427 GFS 3 10.570 2116 1.1658

GES 4 10.78 185 0.1019 GFS 4 10.640 416 0.2292

GEM 1 10.615 151 0.0832 GFM 1 10.70 620 0.3416

GEM 2 10.625 160 0.0881 GFM 2 10.645 272 0.1499

GEM 3 10.550 142 0.0782 GFM 3 10.620 272 0.1499

GEM 4 10.670 139 0.0766 GFM 4 10.610 196 0.108

Fig. 2.21: XRD Spectrum of Gliclazide

74

Fig. 2.22: XRD Spectrum of Pluronic F68

Fig. 2.23: XRD Spectrum of Pluronic F127

75

Fig. 2.24: XRD Spectra of PMs- GEP 1, GEP 2, GEP 3 & GEP 4

Fig. 2.25: XRD Spectra of SDs- GES 1, GES 2, GES 3 & GES 4

76

Fig. 2.26: XRD Spectra of SDs- GEM 1, GEM 2, GEM 3 & GEM 4

Fig. 2.27: XRD Spectra of PMs- GFP 1, GFP 2, GFP 3 & GFP 4

77

Fig. 2.28: XRD Spectra of SDs- GFS 1, GFS 2, GFS 3 & GFS 4

Fig. 2.29: XRD Spectra of SDs- GFM 1, GFM 2, GFM 3 & GFM 4

78

2.8.5.5 Thermal Analysis:

Thermal study can be utilized to assess the purity as well as the crystallinity of a

substance. A sharp endothermic peak in DSC denotes the absence of impurity as well

as highly crystalline character of a substance. DSC can also be applied to examine the

chemical compatibility of components in a mixture.

Fig. 2.30 and Fig. 2.31 depict the thermal behavior of pure drug and

corresponding drug carrier system. Thermogram of GLZ shows a sharp endothermic

peak at 173.84°C (ΔH= -158.53 J/g) corresponding to its melting, indicating its

crystalline nature. A remarkable difference was observed between thermograms of

GLZ and all solid dispersion batches. The solid dispersions as well as physical

mixtures showed the melting endotherm of GLZ almost at the same position which

indicates the chemical compatibility between drug and excipients.

The melting peaks in the drug-carrier systems demonstrated reduction in

intensities. This finding suggests the crystalline drug is converted to amorphous state.

The melt fusion of GLZ and Pluronic F127 showed additional peaks above 230°C

suggesting the possibility of thermal decomposition at higher temperature.

79

Fig. 2.30: DSC Thermogram of GLZ & Pluronic F68 Formulations

Fig. 2.31: DSC Thermogram of GLZ & Pluronic F127 Formulations

80

2.8.5.6 In vitro Dissolution Study:

Figures 2.32-2.37 and Table 2.7-2.8 render the comparison of dissolution behavior

of GLZ alone with solid dispersions by solvent evaporation and melt fusion methods

as well as corresponding physical mixtures.

It was observed that dissolution of GLZ alone was very slow and incomplete till

120 min. According to the obtained results the only 35.61±0.24% of GLZ is dissolved

after 2 hr. Percent drug dissolved at 10 min, 30 min and 60 min (Q10, Q30 and Q60

respectively) were 1.25±0.26, 11.06±0.94 and 25.24±1.37 respectively. Hence it is

evident that the drug is having poor dissolution rate and there is a need for dissolution

enhancement of GLZ.

From the dissolution profiles of solid dispersions, physical mixtures and drug, it

can be observed that the physical mixtures also showed enhanced dissolution rate.

Q10, Q30 and Q60 values with GEP 1 were found to be 11.29±0.36, 16.88±0.52 and

23.84±0.43 respectively and with GFP 1 were 10.94±0.4, 21.54±0.12 and 30.25±0.23

respectively which shows better dissolution rates compared to GLZ alone. The

improved dissolution without any application of solid dispersion technique might be

attributed to the surfactant effect of poloxamer which creates a polymeric micellar

micro-environment around drug particle. Thus increased wettability of drug causes

increased rate of mass transfer from the surface of drug particles. This result can be

correlated with findings of the phase solubility study which shows the solubilization

effect of poloxamer (Fig. 2.13-2.14). Still the drug dissolution from physical mixtures

was incomplete. The percent drug release from GEP 1 and GFP 1 in 120 min. was

40.92±0.19 and 42.15±1.67 respectively. Hence it can be concluded that only

solubilization action of poloxamer is not sufficient for the dissolution rate

enhancement of GLZ.

81

From Fig 2.32-2.37 and Table 2.7 & 2.8, it is evident that solid dispersions

exhibited faster as well as complete dissolution than the drug as well as physical

mixtures. The rate of dissolution is influenced by the factors like carrier

concentration, method of preparation of solid dispersion and grade of the poloxamer

which are discussed below in detail.

Effect of Poloxamer Concentration on Dissolution of GLZ:

From Fig. 2.32-2.37 and Table 2.7, it is seen that dissolution efficiency at 60 min

(%DE60min) with GES formulations with increasing carrier concentration were 31.53,

34.86, 42.00 and 42.75 respectively. The mean dissolution time (MDT) was found

decreasing with increasing poloxamer concentration. This is observed with all solid

dispersions as well as physical mixtures.

The carrier concentration imparts a significant effect on degree of crystallinity as

proven by XRD study and thus, on dissolution characteristics of drug. It is believed

that the carrier used in the solid dispersion system keeps the drug molecules away

from each other and prevents the crystal growth. Thus the crystalline drug is

converted to amorphous form. Also, poloxamer additionally generates the micellar

micro-environment around drug particles. Hence increased carrier concentration

results in higher dissolution rates.

82

Fig. 2.32: Dissolution Profile of PMs with Pluronic F68

Fig. 2.33: Dissolution Profile of PMs with Pluronic F127

0

10

20

30

40

50

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GEP1

GLZ

GEP2

GEP3

GEP 4

0

10

20

30

40

50

60

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GFP 1

GFP 2

GFP 3

GFP 4

83

Fig. 2.34: Dissolution Profile of SDs by solvent evaporation with Pluronic F68

Fig. 2.35: Dissolution Profile of SDs by solvent evaporation with Pluronic F127

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GES 1

GES 3

GES 4

GES 2

0

10

20

30

40

50

60

70

80

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GFS 1

GFS 2

GFS 3

GFS 4

84

Fig. 2.36: Dissolution Profile of SDs by Melt Fusion with Pluronic F68

Fig. 2.37: Dissolution Profile of SDs by Melt Fusion with Pluronic F127

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GEM1

GEM2

GEM3

GEM4

0

10

20

30

40

50

60

70

80

90

100

110

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GFM 1

GFM 2

GFM 3

GFM 4

85

Table 2.7: Mean Dissolution Time & Dissolution Efficiency at 15 min and 30 min

Batch % DE MDT

15 min 30 min 15 min 30 min

GLZ 1.45 4.56 9.92 17.63

GEP 1 8.48 11.77 5.41 9.08

GEP 2 10.24 14.38 6.17 8.02

GEP 3 12.13 17.49 6.46 8.48

GEP 4 16.65 22.29 5.72 6.88

GES 1 18.62 25.44 5.47 8.32

GES 2 21.85 28.75 5.40 6.79

GES 3 28.53 34.85 3.72 6.45

GES 4 29.73 36.16 3.88 5.94

GEM 1 56.45 72.56 5.25 5.94

GEM 2 52.67 65.55 4.40 6.11

GEM 3 64.98 77.35 3.84 4.82

GEM 4 72.57 84.71 3.67 3.97

GFP 1 8.57 13.70 7.04 10.92

GFP 2 9.95 15.04 6.54 10.07

GFP 3 10.12 15.29 6.41 10.25

GFP 4 16.56 23.55 6.28 8.32

GFS 1 14.78 19.59 3.80 9.76

GFS 2 18.98 26.03 4.48 10.03

GFS 3 23.90 32.45 5.33 8.34

GFS 4 19.51 24.65 3.98 7.60

GFM 1 58.39 73.39 4.90 5.56

GFM 2 58.29 74.62 5.13 6.02

GFM 3 61.64 77.85 4.77 6.11

GFM 4 64.89 80.40 4.62 5.40

86

Effect of Method of Preparation on Dissolution of GLZ:

Figures 2.38-2.41 show the comparison of dissolution from solid dispersions with

same concentration of poloxamer but different methods of preparation. It is clear that

melt fusion method was found to be the most successful for dissolution rate

enhancement of GLZ followed by solvent evaporation method and physical mixtures.

This can be attributed to the molecular dispersions with higher surface free energy

resulting in the pull of insoluble but discrete drug molecules into the bulk of the

solvent as dissolved entity. Additionally the solubilization effect of poloxamer

micelles prevents re-aggregation of drug molecules. Solvent evaporation method

showed faster dissolution than physical mixtures mainly due to presence of

amorphous drug in the crystalline carrier.

The result can also be correlated to results of XRD study which elaborates the

decrease in the degree of crystallinity of GLZ. As previously stated, XRD study

shows that melt fusion method showed highest degree of amorphization, while,

physical mixtures display minimum influence on the crystalline character of drug.

87

Fig. 2.38: Dissolution Profile of SDs by Different Methods with Pluronic F68

(1:1)

Fig. 2.39: Dissolution Profile of SDs by Different Methods with Pluronic F127

(1:1)

0

10

20

30

40

50

60

70

80

90

100

110

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GEP1

GES1

GEM1

0

10

20

30

40

50

60

70

80

90

100

110

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GFP1

GFS1

GFM1

88

Fig. 2.40: Dissolution Profile of SDs by Different Methods with Pluronic F68

(1:4)

Fig. 2.41: Dissolution Profile of SDs by Different Methods with Pluronic F127

(1:4)

0

10

20

30

40

50

60

70

80

90

100

110

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GEP4

GES4

GEM4

0

10

20

30

40

50

60

70

80

90

100

110

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GFP4

GFS4

GFM4

89

Effect of Grade of Poloxamer on Dissolution of GLZ:

Fig. 2.42-2.44 illustrate the dissolution profiles of solid dispersions with same

ratio of drug to carrier as well as same method of preparation, but with different

grades of poloxamer.

It can be concluded that in comparison with Pluronic F68, solid dispersions with

Pluronic F127 initially slower but complete dissolution of GLZ. In case of physical

mixtures and solvent evaporation batches, formulations with Pluronic F127 as carrier

initially showed slower release rate. The release constant „K‟ of GES 1 is 11.646 with

Korsemeyer-Peppas as the best fit dissolution model, while K-value of GFS 1 is 7.015

with matrix as best fit model. In case of physical mixture GEP 1 and GFP 1, K-values

were found to be 3.005 and 3.7952. But the amount released after 120 min was

significantly more with Pluronic F127 than Pluronic F68.

But in case of melt fusion approach (Fig. 2.44), both the carriers demonstrated a

similar drug release profile. The similarity of dissolution profiles can be shown by the

percent dissolution efficiency of GEM 1 and GFM 1 at 60 min, which were 84.10 and

84.49. This might be attributed to the molecularly dissolved melt of drug in the melt

of Pluronic irrespective of the molecular weight and grade of poloxamer. Thus the

molecularly dispersed drug in the polymeric crust dissolves immediately in the

solvent. Also, XRD study reveals that the drug in melt fusion with both carriers is

completed to its amorphous form. Hence due to the amorphization of drug in carrier,

dissolution profile of the melts was not completely dependent on the solubilization by

surfactant action of poloxamer and hence, similar with both grades of Pluronic.

90

Fig. 2.42: Dissolution Profile of SDs by Physical Mixture (1:1)

Fig. 2.43: Dissolution Profile of SDs by Solvent evaporation (1:1)

0

10

20

30

40

50

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GEP1

GFP1

0

10

20

30

40

50

60

70

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GES1

GFS1

91

Fig. 2.44: Dissolution Profile of SDs by melt fusion (1:1)

Statistical analysis of dissolution profiles:

Model dependent methods

Although model independent methods are simple and easy to apply, they lack

scientific justification. Different models of dissolution profile comparison were used

(Table 2.7). The results of these models indicate most of the solid dispersion

formulations followed Matrix and Peppas model as “best fit model” depending on R2

& K- values obtained from model fitting.

0

10

20

30

40

50

60

70

80

90

100

110

0 20 40 60 80 100 120

% C

um

ula

tiv

e R

elea

se

Time (Min.)

GLZ

GEM1

GFM1

92

Table 2.8: Model dependent analysis of dissolution of Solid dispersions

Model Zero Order 1st Order Matrix Peppas Hix-Crowell

Best Fit Batch R

2 K R

2 K R

2 K R

2 K R

2 K

GLZ 0.9771 0.3466 0.9816 -0.0042 0.9339 3.1031 0.9811 0.1174 0.9805 -0.0013 1st Order

GEP 1 0.4490 0.5358 0.7293 -0.0072 0.9331 5.1547 0.9872 11.6464 0.6589 -0.0022 Peppas

GEP 2 0.6967 0.6527 0.8996 -0.0098 0.9614 6.1952 0.9751 12.6893 0.8528 -0.0028 Peppas

GEP 3 0.3163 0.7285 0.8030 -0.0115 0.9143 7.0262 0.9868 19.4650 0.7039 -0.0033 Peppas

GEP 4 0.2747 0.7414 0.8027 -0.0118 0.9016 7.1539 0.9665 20.9410 0.6981 -0.0033 Peppas

GES 1 - 1.1862 - - 0.6102 11.8236 0.8638 44.7757 0.8817 -0.0123 --

GES 2 - 1.1596 - - 0.7320 11.4427 0.9827 42.8611 0.9423 -0.0109 --

GES 3 - 1.1902 - - - 11.9533 0.9578 62.8888 0.7912 -0.0120 --

GES 4 - 1.2047 - - - 12.2060 0.9005 76.6677 0.6650 -0.0130 --

GEM 1 0.9319 0.4048 0.9613 -0.0050 0.9798 3.7330 0.9859 3.0054 0.9534 -0.0016 Peppas

GEM 2 0.8902 0.4413 0.9421 -0.0056 0.9910 4.1133 0.9892 3.9958 0.9276 -0.0017 Matrix

GEM 3 0.7924 0.4550 0.8862 -0.0058 0.9909 4.3012 0.9892 5.4294 0.8592 -0.0018 Matrix

93

Model Zero Order 1st Order Matrix Peppas Hix-Crowell

Best Fit Batch R

2 K R

2 K R

2 K R

2 K R

2 K

GEM 4 0.4701 0.4817 0.6945 -0.0062 0.9381 4.6426 0.9860 9.9144 0.6336 -0.0019 Peppas

GFP 1 0.8642 0.5702 0.9544 -0.0080 0.9930 5.3334 0.9779 7.0148 0.9320 -0.0024 Matrix

GFP 2 0.7544 0.6736 0.9190 -0.0102 0.9856 6.3860 0.9925 9.9450 0.8779 -0.0029 Peppas

GFP 3 0.6028 0.7289 0.8763 -0.0115 0.9605 6.9784 0.9936 13.7720 0.8115 -0.0033 Peppas

GFP 4 0.7928 0.6344 0.9257 -0.0095 0.9680 5.9589 0.9651 10.7937 0.8990 -0.0027 Matrix

GFS 1 - 1.2037 - - 0.6056 11.991 0.8684 46.6527 0.8705 -0.0134 -

GFS 2 - 1.2006 - - 0.5647 11.9968 0.8600 47.8908 0.8210 -0.0134 -

GFS 3 - 1.1992 - - 0.4280 12.0421 0.8425 54.4490 0.7173 -0.0127 -

GFS 4 - 1.2000 - - 0.2479 12.0953 0.8059 59.9468 0.6105 -0.0127 -

GFM 1 0.8923 0.4069 0.9428 -0.0050 0.9961 3.7952 0.9918 3.1181 0.9283 -0.0016 Matrix

GFM 2 0.8715 0.4239 0.9342 -0.0053 0.9969 3.9674 0.9906 3.8949 0.9164 -0.0016 Matrix

GFM 3 0.8780 0.4352 0.9401 -0.0055 0.9970 4.0682 0.9926 3.9807 0.9227 -0.0017 Matrix

GFM 4 0.6437 0.5200 0.8217 -0.0069 0.9606 4.9642 0.9812 9.2137 0.7734 -0.0021 Peppas

94

2.8.5.7 In vivo Pharmacodynamic Study

The best batch was selected based on drug crystallinity and drug release. In vivo

pharmacodynamic study reveals that in diabetic animals, GLZ alone had reduced the

blood glucose level to 153.4±3.17 mg/ml on 7th

day (Fig. 2.45), while SD reduced

blood glucose level to 131.2±3.17 mg/ml. While in case of normal rats, the decrease

in glucose levels could be observed 1 h after administration (Figure 2.46-2.47). This

effect was gradually enhanced 6 h. The decrease in glucose levels reflects an increase

of GLZ in the blood levels as a result of the drug‟s dissolution and absorption.

Kahn and Shechter[161]

have suggested that a 25 % reduction in blood glucose

levels is considered as a significant hypoglycemic effect. Formulations containing

GLZ showed the lowest glucose level After applying the t-test analysis for the data

obtained from percentage decrease in blood glucose concentration (Fig. 2.47),

significant difference was observed between the untreated GLZ powder and the

formulation (p = 0.0285). This significant decrease was attributed to the improvement

of the bioavailability of GLZ solid dispersion.

Fig. 2.45: In vivo Pharmacodynamic Study of SD in Diabetic rats

100

130

160

190

220

250

280

0 7 14 21 28

Glu

cose

Lev

el m

d/d

l

Time in Days

CONTRO

L

DIABETI

C

GLZ

95

Fig. 2.46: In vivo pharmacodynamic evaluation of SD in Normal rats

Fig. 2.47: % decrease in Blood Glucose Level of Normal and Diabetic Rats

50

60

70

80

90

100

110

120

0 4 8 12 16 20 24

Blo

od

glu

cose

lev

el (

mg

/dl)

Time (Hr)

GLZ GLZ SD

0

10

20

30

40

50

60

0 4 8 12 16 20 24

% D

ecre

ase

in

blo

od

glu

cose

lev

el

Time (Hr)

GLZ

GLZ SD

96

2.8.5.8 Stability Study:

The solid dispersion GEM 3 and GES 3 were selected for stability study. The drug

contents observations of all formulations after 3 months were not significantly

affected by accelerated stability conditions; indicating chemical stability over a period

of 3 months. The drug contents of GEM 3 and GES 3 before and after exposure of

accelerated conditions are illustrated in table 2.9.

The accelerated conditions were found to affect on the dissolution profile of

solid dispersions systems, causing decrease in dissolution rate (Fig. 2.48), which

might be due to crystallization of GLZ upon aging. The effect was more significant

with melt dispersion.

Fig. 2.48: Effect of Aging on Dissolution of Solid dispersion

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

% D

rug

Rel

ea

se

Time in min.

GEM-3 stability

GEM-3

GES-3

GES-3 stability

97

Table 2.9: Drug Content- Stability Study

Batch Storage Condition

Drug content (%w/w)

0 month 1 month 2 months 3 months

GES 1

30±2°C, RH 65±5% 98.97±0.52 98.45±0.52 97.84±0.84 98.27±0.98

40±2°C, RH 75±5% 99.84±1.17 98.45±1.96 99.05±0.65 97.75±0.79

GEM 1

30±2°C, RH 65±5% 99.05±0.54 98.19±0.69 99.75±1.04 99.75±1.19

40±2°C, RH 75±5% 98.88±1.50 99.66±1.47 98.53±2.60 97.23±0.65

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2.9 CONCLUSION:

The aim of present investigation was to increase dissolution rate of gliclazide by

application of the approach of solid dispersion. In present work, preformulation study

was carried out to authenticate drug and to profile its solubility and phase solubility

study. Hence, from preformulation study it was concluded that the drug was authentic

based on the data obtained in FTIR spectrum and UV analysis.

The solubility of GLZ in distilled water is found to be 57.607±3.677 µg/ml; while

it was 82.43±0.67 µg/ml in 0.1N HCl. Dissolution of GLZ alone was very slow and

incomplete up to 120 min. According to the obtained results, only 35.61±0.24 % of

drug was dissolved after 2 hr. Hence, as the intrinsic solubility as well as rate of drug

dissolution is poor, there is strong need to enhance its solubility and dissolution.

Phase solubility study indicates that solubility of GLZ increased with poloxamer

concentration. The improvement in solubility can be attributed to effect of poloxamer.

Hence, poloxamer has ability to increase dissolution rate and can be considered as a

suitable carrier for formulation of solid dispersion.

Solid dispersions were formulated by solvent evaporation and melt fusion

techniques. For the comparative study, corresponding physical mixtures were also

prepared. Solid dispersions were characterized for chemical stability by Differential

Scanning Calorimetry, Fourier Transform Infra-Red Spectroscopy and Thin Layer

Chromatography and found to be stable. Degree of crystallinity was evaluated by X-

ray Diffractometry and DSC. GLZ was found to be highly crystalline. The

crystallinity of GLZ was reduced with increase in poloxamer concentration. The

amorphization was found more significant with melt fusion method. Drug release rate

was profiled in 0.1 N HCl and found to be dependent on method of preparation with

following order: GLZ< Physical Mixtures < Solvent evaporation < Melt fusion. The

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enhancement of drug dissolution rate was also found to be poloxamer concentration

dependent. Increased carrier concentration results in higher dissolution rates.

In vivo pharmacodynamic study in normal as well as diabetic animals reveals that

the solid dispersion was significantly effective to reduce the blood glucose level

compared to drug alone. This suggests improved bioavailability of drug due to

enhanced dissolution rate.

Stability study was performed at 30°C±2°C, RH 65%±5% and 40°C±2°C, RH

75%±5%. It was found that the drug content is not affected by stability conditions

upto 3 months suggesting good chemical stability on exposure of accelerated

conditions. But the drug release rate was decreased upon aging. Hence the stability

study show the lack of physical stability with hampered dissolution attributed to the

tendency of re-aggregation of molecularly dispersed drug to convert the amorphous

drug to crystalline form.

Finally it can be concluded that, binary solid dispersion system containing GLZ

and Pluronic with aid of melt dispersion technique was efficient to form amorphous

dispersion of GLZ. Thus, this may be potential application for formulation research in

improvement of dissolution rate of GLZ.