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RJPT - Formulation and Evaluation of Albumin Microspheres containing Stavudine
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Formulation and Evaluation of Albumin
Microspheres containing Stavudine
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Author(s): V. Swathi (search.aspx?key=V. Swathi), G. Velrajan (search.aspx?key=G. Velrajan), Balasubramaniam
(search.aspx?key=Balasubramaniam)
Email(s): swathivpharma@gmail.com (mailto:swathivpharma@gmail.com)
DOI: Not Available
Address: V. Swathi*, G. Velrajan, Balasubramaniam
Department of Pharmaceutics, Smt. Sarojini Ramulamma College of Pharmacy, Seshadrinagar, Mahabubnagar,
Andhra Pradesh- 509 001, India.
*Corresponding Author
Published In: Volume - 6,
Keywords:
Issue - 2, Year - 2013 (Issues.aspx?VID=6&IID=2)
Microspheres ()
Stavudine ()
egg albumin ()
gluteraldehyde ()
single emulsion technique. ()
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RJPT - Formulation and Evaluation of Albumin Microspheres containing Stavudine
Cite this article:
V. Swathi, G. Velrajan, Balasubramaniam. Formulation and Evaluation of Albumin
Microspheres containing Stavudine. Research J. Pharm. and Tech. 6(2): Feb. 2013; Page
174-177.
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Formulation and Evaluation of Albumin
Microspheres containing Stavudine
V. Swathi*, G. Velrajan, Balasubramaniam
Department of Pharmaceutics, Smt. Sarojini Ramulamma College of Pharmacy, Seshadrinagar, Mahabubnagar, Andhra
Pradesh- 509 001, India.
*Corresponding Author E-mail: swathivpharma@gmail.com
ABSTRACT:
Sustained release microspheres of Stavudine, the FDA- approved drug for the treatment of HIV infections, AIDS and AIDS- related
conditions administered either alone or in combination with other antiretro-viral agents. Stavudine is typically administered orally as
a capsule and an oral solution. The virustatic drug has a very short half-life (1.30h). Its dose is 40mg twice or thrice daily. However
patients receiving stavudine develop neuropathy and lactic acidosis. The side effects of stavudine are dose dependent and a reduction
of the total administered dose reduces the severity of the toxicity. Microencapsulation is a process whereby one can formulate
controlled/sustained action dose form for drugs having a short half-life. Microencapsulation improves the drug absorption and
minimizes side effects due to the localized build up of drugs against the gastrointestinal mucosa. The main objective of the present
study was to formulate sustained release microspheres of stavudine which will retard drug release from dosage form, reduces the
frequency of drug administration, minimizing the adverse effects thereby increasing the patient compliance. Sustained release
microspheres were prepared by single emulsion technique followed by heat denaturation and chemical cross linking with
gluteraldehyde using egg albumin as a release retarding agent. These microspheres were evaluated for yield, flow properties,
entrapment efficiency and in-vitro release kinetics. The results showed that yield, entrapment efficiency was influenced by polymer
concentration and stirring speed. Results of the in vitro study showed that the desired release rate was achieved by F6, F5
formulations releasing drug up to 12hrs. FT-IR results proved that there is no interaction between drug and polymer. SEM results of
optimized microspheres showed discrete, spherical microspheres.
KEYWORDS: Microspheres, Stavudine, egg albumin, gluteraldehyde, single emulsion technique
INTRODUCTION:
The term ‘microsphere’ describes a monolithic spherical structure with the drug or therapeutic agent distributed throughout the
matrix either as a molecular dispersion or as a dispersion of particles3. Several pharmaceutical formulation approaches may be
applied to the development of controlled release or sustained release formulations, polymeric microsphere is one of them2. Albumin
is a major plasma protein constituent, accounting for ~55% of the total protein in human plasma. It is having unique advantage that
after performing their function they degrade into non-toxic monomers9. Albumin microspheres have been extensively investigated in
6
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RJPT - Formulation and Evaluation of Albumin Microspheres containing Stavudine
controlled/sustained release systems as vehicles for the delivery of therapeutic agents6.
Albumin is used for preparation of microspheres because it is non-antigenic, biodegradable, and free from toxicity and readily
available3,8. GA is one of the most common dialdehydes used to crosslink bio-prostheses and proteins8. Stavudine is a nucleoside
reverse transcriptase inhibitors and used in the treatment of infection by retrovirus, primarily HIV7, 10. It is absorbed and eliminated
rapidly following oral administration producing peak plasma concentration within 1hr, elimination half life 1.5 hours following
single or multiple dose10. Sustained release oral delivery systems are designed to achieve therapeutically effective concentrations of
drug in the systemic circulation over an extended period of time, thus achieving better patient compliance, reduction of adverse
effects4, 7. Microencapsulation is a process whereby one can formulate controlled/sustained action dose form for drugs having a short
half-life2. In this study, sustained release microspheres were prepared by single emulsion technique followed by heat denaturation
and chemical cross linking with gluteraldehyde using egg albumin as a release retarding agent. The microspheres evaluated for yield,
flow properties, entrapment efficiency and in-vitro release of the microspheres. The results showed that yield, entrapment efficiency
were influenced by polymer concentration, stirring speed. From the results of the in vitro study shows that the desired release rate is
achieved by F6, F5 formulations releasing drug up to 12hrs.FT-IR results showed there is no interaction between drug and polymer.
SEM results of optimized microspheres showed discrete, spherical microspheres.
MATERIALS AND METHODS:
Materials:
Stavudine was a gift sample from Hetero pharmaceuticals HYD, Egg Albumin, gluteraldehyde, liquid paraffin obtained from
commercial sources. All other reagents and chemicals used are of analytical reagent grade.
Preparation method of sustained release microspheres:
A total of 6 formulations of stavudine were prepared as shown in Table I, using varying concentrations of egg albumin, span 80 and
gluteraldehyde. The principle of “Single Emulsion technique followed by heat denaturation and chemical cross-linking with
gluteraldehyde” was adopted. A weighed amount of Egg Albumin was dissolved in distilled water. Stavudine (40mg) was dissolved
in prepared Egg Albumin solution. One ml of this solution was added drop wise to preheated (60°C) liquid paraffin (40 ml)
containing 1% w/v of span 80 as an emulsifying agent while stirring the whole system at 2500 rpm for 1hr.
Table I: Composition of Stavudine loaded Egg Albumin microspheres:
Ingredients
Stavudine
Egg Albumin conc.(w/v)
Span 80(% w/v)
Vol. of Gluteraldehyde (ml)
Batch Code
F1
F2
40
40
10
10
1
1
0.5
1
F3
40
10
1
2
F4
40
20
1
0.5
F5
40
20
1
1
F6
40
20
1
2
The temperature was reduced to 40°C for hardening process with continuous stirring and was maintained for 25 min. The resulting
microspheres were stabilized using gluteraldehyde solution and stirred for a period of 15 min. Microspheres formed were then
separated from the oil phase by filtration and were washed with n-hexane to remove the excess oil. Finally, they were washed with
water and then dried at room temperature for 24 hours.
Evaluation of microspheres:
Yield of sustained microspheres4,1,6,8
The percent practical yield of microspheres was calculated from the amount of microspheres obtained divided by the total amount of
all non-volatile components.
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Actual weight of the microspheres
% Yield=----------------------------------------------X 100
Total weight of all non-volatile components
Drug entrapment efficiency (DEE) 4,1,9,10,5
40mg of Stavudine loaded microspheres was weighed and dissolved in minimal amount of pH 7.4 phosphate buffer. The volume was
adjusted to 100ml with buffer. The resulting solution was then suitably diluted and analyzed for drug content spectrophotometrically
at 266nm using pH 7.4 phosphate buffer as blank.
The amount of drug entrapped in the microspheres was calculated using the following formula.
Amount of drug actually present
DEE=----------------------------------------------X 100
Theoretical drug load expected
Flow properties1,5
Microspheres were poured gently through a glass funnel into a graduated cylinder cut exactly to 10ml mark. Excess microspheres
were removed using a spatula and the weight of the cylinder with pellets required for filling the cylinder volume was calculated. The
cylinder was then tapped from a height 2.0cm until the time when there was no more decrease in the volume.
The flow properties and packing properties were investigated by measuring the bulk density, tapped density, Carr’s compressibility
index, Hausner ratio.
— Bulk density(g/ml) = Mass/V0
— Tapped density (g/ml) =Mass/Vf
— Compressibility Index = 100(V0-Vf)/V0
— Hausner Ratio= V0/Vf
— Angle of Repose=Tanθ=Tan θ-1(h/r)
In vitro drug release study1,3
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RJPT - Formulation and Evaluation of Albumin Microspheres containing Stavudine
In vitro drug release studies were carried out for all formulations in USP type I dissolution test apparatus. Microspheres equivalent to
40 mg of stavudine were poured into dissolution medium containing 900 ml of pH 7.4 phosphate buffer maintained at 37 ± 0.5ºC at
a rotation speed of 50 rpm. 5 ml aliquots were withdrawn at a predetermined intervals and equal volume of dissolution medium was
replaced to maintain sink conditions. The necessary dilutions were made with pH 7.4 phosphate buffer and the solution was analysed
for the drug content spectrophotometrically using UV-Visible spectrophotometer at 266 nm against an appropriate blank. From this
cumulative percentage drug release was calculated and plotted against function of time to study the pattern of drug release. The
results are presented in figure.
Table II: Percentage yield,% Entrapment efficiency an Flow properties of stavudine microspheres
Formulation
code
% practical
yield± SD
%Entrapment
Efficiency± SD
Bulk Density
(gm/ml) ± SD
F1
F2
F3
F4
F5
F6
81.73±0.19
80.29±0.15
82.69±0.12
87.50±0.18
81.54±0.23
84.62±0.29
54.0±0.62
58.8±0.45
60.2±0.95
64.1±0.87
67.0±0.68
72.5±0.93
0.75±0.05
0.65±0.08
0.62±0.03
0.81±0.07
0.65±0.04
0.68±0.06
Tapped
Density
(gm/ml) ± SD
0.85±0.18
0.72±0.19
0.67±0.16
0.92±0.12
0.70±0.17
0.75±0.21
Compressibility
Index(%)± SD
Hausner
Ratio
Angle of Repose
(degrees) ± SD
11.76±0.73
9.7±0.64
7.7±0.91
12.5±0.87
8.32±0.78
9.3±0.89
1.13
1.10
1.08
1.14
1.09
1.10
28.2±1.45
27.6±1.21
27.8±1.69
27.4±1.14
26.5±1.58
25.4±1.86
Stability studies1,6
Accelerated stability studies were carried out to assess shelf-life of a product, but the conditions of storage are harsher. The purpose
of stability study is not only to characterize the degradation of a drug product but also to establish an expiration dating period or shelf
life of drug product. The samples of microspheres were kept at 40ºC and 75% RH for 3months in the stability chamber and the
samples were withdrawn and analyzed at a period of 0,1,2 and 3months. The samples were analyzed for physical appearance, particle
size, %entrapment efficiency and in vitro release studies.
RESULTS AND DISCUSSION:
The prepared sustained release microspheres were evaluated for various parameters such as yield, drug entrapment efficiency, flow
properties and in vitro drug release and results presented in Table II. The effect of preparation and process variables such as drug
polymer ratio, Concentration of Glutaraldehyde on yield, entrapment efficiency, flow properties, in-vitro release of Stavudine
sustained microspheres were also studied.
From Table II the effect of drug, polymer and gluteraldehyde concentration in different ratios on % yield of Stavudine microspheres
were 80.29% to 87.50%. The maximum 87.50% was obtained with F-4 and minimum 80.29% with F-2. The maximum entrapment
efficiency was found for F6 (72.5%) while it was lowest for F1 (54.0%). Formulations F1, F2, F3, F4, F5, F6 with different volumes
of polymer (10%, 20%), gluteraldehyde showed increase in entrapment efficiency. This demonstrates that an increase in the amount
of polymer and gluteraldehyde results in an increase in entrapment efficiency. Angle of repose, Hausner ratio, and Compressibility
index was determined to predict flowability. A higher Hausner ratio indicates greater cohesion between particles while a high
Compressibility index is indicative of the tendency to form bridges, Tan θ value between 25-30 shows excellent flow properties.
In Vitro Drug Release of Stavudine microspheres:
Fig 1 shows as the polymer and glutaraldehyde concentrations increase, a decrease in the release was observed. The formulations F1
to F6 showed their release around 12hrs, where the F1 showed 99% release in 12hrs, F6 showed slow release of 82.88% in 12hrs, the
comparisons of cumulative drug release of all formulations is shown in fig.1
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Fig 1: plot of cumulative percentage drug release vs time for formulations F1 to F6
FT-IR Studies:
FTIR study conducted on Shimadzu and reveals there was no interaction between drug and polymer as shown in fig 2.
Fig 2: FTIR spectra of optimised formulation (F6)
Scanning Electron Microscope:
SEM photograph of optimized Stavudine microspheres F-6 at 100´ magnification, showed discrete, spherical microspheres. The
presence of drug crystal on the surface of microspheres revealed that the microspheres were having some rough surface as shown in
Fig 3.
Fig 3: SEM photographs of Stavudine microspheres at 100x magnification
Stability studies:
During the Accelerated stability studies no significant change was observed. The Cumulative % drug release from the microspheres
was decreased from 83.09% to 82.88 %, over a period of 3 months at accelerated conditions.
Mathematical Model Fitting Of In Vitro Drug Release11
The kinetic analysis, of formulation F6 obeys Non-fickian diffusion and first-order release kinetics11 as evidenced form Table III.
Data obtained from in-vitro release studies were fitted to various kinetic equations to find the mechanism of drug release from the
formulation F6 polymeric microspheres. The kinetic models used were,
Mt / M∞ = ktn
Log [Mt / M∞] = log k + n (First order equation)
Log Qt = log Qo + kt / 2.303 (Higuchi equation)
Mt / M∞= kHt1/2 (Korsemeyer- peppas equation)
Where, Mt and M∞ are cumulative amounts of drug release at time t and infinite time.
Determining the correlation coefficient assessed fitness of the data into various kinetic models. The rate constants or respective
models were also calculated from slope.
Table III: Estimated Values of R2 (Correlation Coefficient) For Optimised Formulation F6.
Formulae
F6
ZeroOrder
R2
0. 9406
FirstOrder
R2
0. 9656
Higuchi
R2
Peppas
Model R2
0. 9245
0. 95
CONCLUSION:
The development of the Stavudine microspheres was to retard drug release from dosage form, reduces the frequency of drug
administration, and minimizing the adverse effects there by increasing the patient compliance from the sustained release
microspheres. In addition, these formulations reduce the need of frequent administration apart from enhance patient compliance. The
in vitro drug release was found to be non-fickian. The dissolution studies, stability studies showed satisfactory results. The drug
release was studied up to 12 hrs and results indicate that release from albumin loaded stavudine microspheres in ratio F6 was
maximum.
ACKNOWLEDGEMENT:
The study was supported by my parents V. Shyam Sunder Reddy and V. Sharadha and I thank our college management for providing
facilities for my research work.
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Development and Research. 3(2); April-June 2011; 211-216.
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Prashant Singh, S.K. Senthil Kumar, S. Parthiban, Peeyush, T. Tamizh Manil. Formulation and Evaluation of Ivabradine Hydrochloride Loaded Egg
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