Download 23 2.1 INTRODUCTION TO SOLID DISPERSION: The enhancement

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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,
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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
2.1.1.1
DEFINITION AND TYPES OF SOLID DISPERSIONS:
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
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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
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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
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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
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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
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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
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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.
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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].
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Table 2.1: Materials used as carrier for solid dispersion
Sr. No.
Category
1
Sugars
2
Acids
Examples
Dextrose, Sucrose, Galactose, Sorbitol,
Maltose, Xylitol, Mannitol[67], Lactose[64]
Citric acid, Succinic Acid[68]
PVP[56], PEG[58] , Celluloses like HPMC[60],
3
Polymeric materials
4
Insoluble/ enteric polymer
HEC, HPC, Pectin, Galactomannan, CDs[38]
HPMC[60], Phthalate, Eudragits[71]
Polyoxyethylene stearate, Renex,
5
Surfactants
Poloxamers[63], texafor, Deoxycholic acid,
Tweens, Spans[65]
Pentaerythritol, Pentaerythrityl tetra acetate,
6
Miscellaneous
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.
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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,
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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 nontoxic 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])
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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
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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.
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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 drugcarrier interactions must also be investigated.
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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 coprecipitate 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.
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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
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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
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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 microenvironment 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
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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 candidateGliclazide (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.
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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 interindividual 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
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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
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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 watersoluble drugs when used in solid dispersion[123]. It acts by creating a micellar microenvironment 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
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
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
Pluronic F68
Pluronic F127
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, KorsmeyerPeppas 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 lightdark 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 βcells. 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
2.7.6.8
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.
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
λ max (nm)
λ max at 37oC (nm)
λ max at 45oC (nm)
2nd
2nd
4th
week
week
Drug +
2nd
Excipient
4th
Initial
4th
Initial
week
week
228.9
228.8
228.8
229
228.8
228.8
Initial
week
week
228.6
228.7
228.8
228.8
228.8
228.6
228.8
228.8
229
228.8
228.6
228.4
GLZ+
Pluronic F68
GLZ+
Pluronic F127
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
2.8.4
(2.1)
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
Saturation solubility (µg/ml)
90
80
70
60
50
0
5
10
15
Concentration of Pluronic F 68 (%w/v)
Fig. 2.13: Phase Solubility of GLZ in Pluronic F68
Saturation solubility (µg/ml)
90
80
70
60
50
0
5
10
15
20
25
Concentration of Pluronic F 127 (%w/v)
Fig. 2.14: Phase Solubility of GLZ in Pluronic F127
66
2.8.5
2.8.5.1
CHARACTERIZATION OF SOLID DISPERSIONS:
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.
2.8.5.3
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
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
Related Main peak
Sample
Angle
Peak
2θ
intensity
RDC
Related Main peak
Sample
Angle
Peak
2θ
intensity
(Count)
RDC
(Count)
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
50
% Cumulative Release
40
30
GEP1
GLZ
20
GEP2
GEP3
10
GEP 4
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.32: Dissolution Profile of PMs with Pluronic F68
60
% Cumulative Release
50
40
30
GLZ
GFP 1
20
GFP 2
10
GFP 3
GFP 4
0
0
20
40
60
80
100
Time (Min.)
Fig. 2.33: Dissolution Profile of PMs with Pluronic F127
120
83
80
70
% Cumulative Release
60
50
40
30
GLZ
GES 1
20
GES 3
10
GES 4
GES 2
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.34: Dissolution Profile of SDs by solvent evaporation with Pluronic F68
80
GLZ
70
GFS 1
% Cumulative Release
GFS 2
60
GFS 3
GFS 4
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.35: Dissolution Profile of SDs by solvent evaporation with Pluronic F127
84
100
% Cumulative Release
90
GLZ
80
GEM1
70
GEM2
60
GEM3
50
GEM4
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.36: Dissolution Profile of SDs by Melt Fusion with Pluronic F68
110
GLZ
% Cumulative Release
100
90
GFM 1
80
GFM 2
70
GFM 3
60
GFM 4
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.37: Dissolution Profile of SDs by Melt Fusion with Pluronic F127
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
110
GLZ
100
GEP1
90
GES1
% Cumulative Release
80
GEM1
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.38: Dissolution Profile of SDs by Different Methods with Pluronic F68
(1:1)
% Cumulative Release
110
GLZ
100
GFP1
90
GFS1
80
GFM1
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.39: Dissolution Profile of SDs by Different Methods with Pluronic F127
(1:1)
88
GLZ
110
GEP4
100
GES4
% Cumulative Release
90
GEM4
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.40: Dissolution Profile of SDs by Different Methods with Pluronic F68
(1:4)
GLZ
110
GFP4
100
GFS4
% Cumulative Release
90
GFM4
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.41: Dissolution Profile of SDs by Different Methods with Pluronic F127
(1:4)
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
50
% Cumulative Release
40
30
GLZ
20
GEP1
GFP1
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.42: Dissolution Profile of SDs by Physical Mixture (1:1)
70
% Cumulative Release
60
50
40
30
GLZ
GES1
20
GFS1
10
0
0
20
40
60
80
100
120
Time (Min.)
Fig. 2.43: Dissolution Profile of SDs by Solvent evaporation (1:1)
91
110
GLZ
100
GEM1
GFM1
% Cumulative Release
90
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (Min.)
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.
92
Table 2.8: Model dependent analysis of dissolution of Solid dispersions
1st Order
Model
Zero Order
Matrix
Peppas
Hix-Crowell
Batch
R2
K
R2
K
R2
K
R2
K
R2
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
Best Fit
93
1st Order
Model
Zero Order
Matrix
Peppas
Hix-Crowell
Batch
R2
K
R2
K
R2
K
R2
K
R2
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
Best Fit
94
2.8.5.7
In vivo Pharmacodynamic Study
In vivo
The best batch was selected based on drug crystallinity and drug release.
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.
Glucose Level md/dl
280
CONTRO
L
DIABETI
C
GLZ
250
220
190
160
130
100
0
7
14
Time in Days
21
28
Fig. 2.45: In vivo Pharmacodynamic Study of SD in Diabetic rats
95
Blood glucose level (mg/dl)
120
110
100
90
GLZ
80
GLZ SD
70
60
50
0
4
8
12
Time (Hr)
16
20
24
Fig. 2.46: In vivo pharmacodynamic evaluation of SD in Normal rats
% Decrease in blood glucose level
60
50
40
30
20
GLZ
GLZ SD
10
0
0
4
8
12
Time (Hr)
16
20
24
Fig. 2.47: % decrease in Blood Glucose Level of Normal and Diabetic Rats
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.
GEM-3 stability
120
GEM-3
% Drug Release
100
GES-3
GES-3 stability
80
60
40
20
0
0
15
30
45
60
75
90
105
120
Time in min.
Fig. 2.48: Effect of Aging on Dissolution of Solid dispersion
97
Table 2.9: Drug Content- Stability Study
Drug content (%w/w)
Batch
Storage Condition
0 month
1 month
2 months
3 months
30±2°C, RH 65±5% 98.97±0.52 98.45±0.52 97.84±0.84 98.27±0.98
GES 1
40±2°C, RH 75±5% 99.84±1.17 98.45±1.96 99.05±0.65 97.75±0.79
30±2°C, RH 65±5% 99.05±0.54 98.19±0.69 99.75±1.04 99.75±1.19
GEM 1
40±2°C, RH 75±5% 98.88±1.50 99.66±1.47 98.53±2.60 97.23±0.65
98
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 Xray 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
99
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.