Formulation, Characterization and In-vitro Evaluation of Abacavir Sulphate Loaded Microspheres

 

N. Chandarsekaran¹*, M. Balamurugan²

¹The Erode College of Pharmacy, Erode, Tamilnadu, India

²Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur Campus, No.1, Jalan Menara Gading, 56000 Kuala Lumpur, Malaysia

*Corresponding Author E-mail:- cnchandrasekaran00@gmail.com

 

The aim of the present investigation was to formulate, characterize and evaluate Abacavir Sulphate (AS) loaded microspheres.  Microspheres were prepared by emulsion – solvent evaporation method, using Ethyl cellulose, HPMC K4M, Eudragit RSPO and Eudragit L 100 in different combinations and proportions, in order to achieve better controlled release. The entrapment efficiency of all the formulations was found to be in the range of 79.45±7.71 to 89.79± 5.47 %. The encapsulation efficiency was also found to be dependent on nature of polymer used in the formulation. Scanning electron microscopic analysis confirmed the formation of discrete, spherical, free flowing microspheres with a size range of 25.69±10.55mm to 37.93±26.39mm. All the formulations (F1-F8) exhibited anomalous diffusion mechanism and followed zero order kinetics. The formulation coded as F2 prepared with HPMC K4M was selected as best formulation showed 94 % of drug release at the end of 24 hours with best Physico- chemical properties. Therefore such a design can be used for the controlled delivery of AS in the form of microspheres for the treatment of HIV. Stability of the optimized formulation revealed promising results when stored under refrigeration and room temperature.

 

 

 

INTRODUCTION:

The present research work deals with the formulation, characterization and evaluation of Abacavir Sulphate (AS) loaded microspheres by using various combinations of polymers. AS is an antiretroviral drug which acts by inhibiting Nucleoside Reverse Transcriptase. It is widely used in the treatment of HIV as single drug or in combination with other NRTI’s. The oral bioavailability of AS is 83 %, and it is having very short biological half-life of 1.54 ± 0.63 hours,¹ requires frequent dosing. It causes an increased risk of gastrointestinal adverse events, neurological complaints and unique hypersensitivity syndrome. Consequently, the conventional dosage form has several drawbacks. To overcome the problems associated with AS and to improve its bioavailability, minimize the dosing frequency and adverse reactions it was decided to select this drug for the formulation of the microspheres.

 

MATERIALS AND METHODS

Materials

Abacavir sulphate was received as a gift sample from Apotex Laboratories. Bangalore, India. HPMC K4M, Colorcon Asia Pvt. Ltd. Mumbai, India. Eudragit RSPO, Eudragit L100, Ajantha Pharma Mumbai. Ethyl cellulose, SD Fine chemicals Ltd, Mumbai, India. All other ingredients used in the study were of laboratory and analytical grades.

 

Methods

Investigation of physicochemical compatibility of drug and polymers

Fourier Transform Infrared Spectrophotometer (FTIR) ^{2, 3}

Compatibility study of drug with the excipients was determined by FTIR Spectroscopy (Perkin Elmer Spectrum- RX1 FTIR). In the 1: 100 ratio (sample to KBr), the pellets were prepared at high compaction pressure by using KBr; then the prepared pellets were examined and the spectra of drug and other constituents in the formulations were compared with the original spectra.

 

 

Table 1: Composition of various formulations of AS microspheres.

	Abacavir Sulphate (mg)	EC (mg)	HPMC K4M (mg)	Eudragit RSPO (mg)	Eudragit L100 (mg)
F1	500	1000	-	-	-
F2	500	800	200	-	-
F3	500	800	-	200	-
F4	500	800	-	-	200
F5	500	800	100	100	-
F6	500	800	-	100	100
F7	500	800	100	-	100
F8	500	800	66.66	66.66	66.66

Weighed quantity of AS and polymer HPMC K4M were then dispersed in the above polymeric phase; and the whole contents were stirred for 2 hours. Then it was emulsified with the 100 ml of liquid paraffin with continuous stirring at 1000rpm in a magnetic stirrer. The stirring was continued for 2 hours to ensure complete evaporation of acetone- ethanol solvent mixture. The microspheres were then separated from liquid paraffin by filtration through Whatmann filter paper No. 44, washed with petroleum ether, and air dried for 12 hours.

 

Formulation of Microspheres

Microspheres were prepared by using the Emulsion-solvent evaporation technique.^4-6 Accurately weighed quantity of the polymers (ethyl Cellulose, eudragit RSPO, and eudragit L 100) was dissolved in acetone – ethanol mixture (8:2) (Table 1).

 

Physicochemical Characterization of Patches

The microspheres were evaluated for the following physico-chemical properties:

Percentage yield of microsphere⁷

After thorough drying, the microspheres were taken and weighed accurately. The percentage yield was then calculated using the formula given below.

 

 

Shape and surface characterization^{7, 8}

The shape and surface characterization of microspheres were observed under Scanning Electron Microscope (Joel model JSM 6400, Tokyo). The microspheres were mounted directly on to the SEM sample stub, using double-sided sticking tape, and coated with gold film (thickness 200 nm) under reduced pressure (0.001 torr) and photographed.

 

Size distribution ^{7, 9}

Microspheres size determination was done by optical microscopy method.  Size distribution plays a very imperative function in influencing the release characteristics of the microspheres.

 

Angle of repose ^{7, 10}

Angle of repose was calculated by static method using funnel. Funnel was kept on triangular stand, which was kept on the horizontal plane. The sample was introduced into the funnel, as the pile forms; it reaches the tip of funnel. The diameter of the pile was noted. The angle of repose () is calculated by the following formula,

 

θ = tan^-1 (h/r)

 

Where,

h = pile height of microspheres; r = radius of the circular are formed by the microspheres on the ground.

Determination of bulk density ¹¹

 

The bulk density was determined by 3-tap method. The bulk density was calculated as per the following formula:  

                W_O

ρ      _-----------

V_O

Where,

ρ = bulk density,

Wo = weight of sample in gm,

Vo= final volume after tapping

 

Drug content analysis ⁷

Accurately weighed microspheres equivalent to 25mg of abacavir sulphate, crushed in glass mortar and pestle and the powdered microspheres were suspended in 100 ml of 0.1N Hcl. After 12 hours the solution was filtered and the filtrate was analyzed for the drug content using UV –Visible spectrophotometer at 268nm.

 

Encapsulation efficiency ¹²

Encapsulation efficiency was calculated using the following formula:

 

 

In- vitro dissolution studies ^{13, 14}

The drug release study was performed using USP XXIII dissolution test apparatus, accurately weighed samples of microspheres (containing approx 100 mg of the drug) were placed in a basket.   The instrument was set at 100 rpm rotation and at 37⁰C 900 ml of 0.1N Hcl was filled as dissolution medium for first 2 hours and phosphate buffer pH 7.4 from the third hour.  Samples were withdrawn at predetermined intervals, from 0.5 – 24 hours filtered and analyzed spectrophotometrically at 268nm using corresponding medium as blank. After each withdrawal the same quantity of fresh medium was replaced immediately. (n=3)

 

Release kinetics and mechanism ¹⁵

To know the mechanism of the drug release from the microspheres, the results obtained from the in-vitro dissolution process were fitted into different kinetic equations as follows:

Qt = K₀ t (Zero Order Kinetics)

Q_t = K_KP tⁿ (Korsmeyer and Peppas equation)

Q_t = K_H t^1/2 (Higuchi’s equation) 

Where, Qt is the percent of drug released at time‘t’, K₀, K_HC, K_KP and K_H are the coefficients of Zero order, Korsmeyer-Peppas and Higuchi’s equations. 

 

Stability studies

The stability of microspheres was assessed by keeping the optimized formulation at different temperature condition, i.e., 4⁰ C (refrigerator condition), room temperature, and 45⁰C for a period of 4 weeks. Throughout the study the microspheres were stored in aluminum foil sealed glass vials. Samples were withdrawn every week and analyzed for the drug content ¹⁶ spectrophotometrically at 268nm.

 

Statistical analysis

To confirm substantial differences in drug release from various formulations One-way analysis of variance (ANOVA) and paired t-test were applied and the differences were measured at p < 0.05 to be statistically significant. 

 

RESULTS AND DISCUSSIONS

Investigation of physicochemical compatibility of drug and polymer

The compatibility between drug and polymers was confirmed by the FTIR spectral analysis which showed that there were no changes observed in any characteristic peaks in the physical mixture of drug and polymer.

 

Physicochemical characterization of patches

Percentage Yield

The percentage yield of microspheres of all formulations was found in the range of 79.55%w/w to 81.55% w/w.

 

Shape and surface characterization

The external morphology of the microspheres was studied by SEM analysis and the photographs of the formulation F2 were shown in Fig 1 and 2. The surface of the microspheres prepared in this combination by the solvent evaporation technique were found to be spherical, rough, and remains individual particles (Fig 1&2) showed little pores on its surface, when it is scanned at higher magnifications (Fig 2).

 

Figure 1:  SEM photographs shows the rough surface of the optimized formulation F – 2 microspheres.

 

Figure 2: SEM image of F-2 microspheres scanned at higher magnifications showing pores.

 

Size distribution

The size of the microspheres was determined by the optical microscopy method. The mean particle size of the microspheres ranged from 25.69µm to 37.93 µm (Table 2).

 

This difference in the mean particle size was influenced by the content and type of the polymer used and its proportion in the formulation. The mean size of the formulations F4, F5, F8 were found to be 37.90, 37.93, and 34.57m respectively. 

 

Table 2: Physicochemical characterization of all the batches.

#	Particle size analysis (µm) a,c	Percentage yield (%)a,b	Bulk density (gms/cc) a,b	Angle of repose SD()a,b	Drug content in % w/w in 25 mg equivalent weights of drug % a,b	Percentage drug entrapped % a,b	Drug loading capacity (%w/w) a,b
F1	33.08 ±21.64	80.21± 01.38	0.707±  00.37	24.740 ± 04.01	20.39± 00.41	81.52± 01.70	41.67± 00.69
F2	26.25± 10.42	81.55± 01.38	0.766±  00.40	26.270± 04.29	22.39± 01.30	89.79± 05.47	40.87± 00.69
F3	25.69± 10.55	79.55± 03.67	0.877±  00.44	24.520± 04.39	20.44± 02.34	81.76± 09.37	41.95± 01.98
F4	37.90± 30.84	80.43± 01.03	0.782±  00.41	28.290± 05.57	20.98± 01.25	83.94± 05.05	41.43± 00.52
F5	37.93± 26.39	81.55± 01.38	0.763± 00.40	24.780± 03.24	21.11± 02.91	84.48± 11.66	40.88± 00.69
F6	26.19± 9.57	80.88± 01.01	0.825± 00.44	24.350± 06.76	19.86± 01.92	79.45± 07.71	41.21± 00.50
F7	27.02± 8.70	80.66± 01.15	0.876± 00.46	24.100± 05.31	20.69± 02.39	82.77± 09.56	41.33± 00.56
F8	34.57± 16.07	81.10± 00.38	0.781± 00.39	23.270± 02.66	20.44± 02.40	81.81± 09.61	41.06± 00.23

^a Mean± SD, ^b n=3, ^c n=50.

 

Figure 3: Comparative release profile of the various formulations from F1-F8.

 

 

This increased mean size was due to the addition of eudragit polymers in different proportions. Actually, addition of one of these eudragit polymers caused a significant increase in the viscosity, thus leading to an increase the emulsion droplet size and produced the bigger sized microspheres than the other formulations. The prepared microspheres were considered to be more suitable for the delivery of abacavir sulphate suggesting that the coating was well completed under the present conditions.

 

Micromeritics studies

The angle of repose was determined by fixed funnel method. The angle of repose was found in the range of 23.27⁰ to 28.29⁰ which revealed that the microspheres of all the batches (F1 to F8) had good flow characteristics and flow rates. The bulk density was in the range of 0.703gm/cm³ to 0.877gm/cm^3. The flow characteristics of all the formulations was determined by the angle of repose, revealed the good flow characteristics and flow rates.

 

Drug content analysis and entrapment efficiency

The drug content values of microspheres were found in the range of 19.860mg to 22.393mg in 25mg equivalent weight of the AS in the prepared microspheres, the determination of drug content showed that even if the polymer composition was changed the process was highly efficient to give microspheres having maximum drug loading. The drug entrapment efficiency was found in the range of 79.45% w/w to 89.79% w/w. The encapsulation efficiency of the drug in all formulations was found to be same, except the formulation coded as F2, showed increased encapsulation efficiency (89.8%) was resulted owing to the addition of HPMC K4M polymer in a higher concentration.

 

In vitro drug release kinetics

For all the formulation F1 to F8 the kinetic drug release data shows that the formulations followed Zero-order drug release as the R² values were found in the range of 0.9269 to 0.9988. The release patterns of various formulations are as follows F2>F5>F7>F1>F8>F6>F3>F4. (Fig 3)

 

Mechanism of drug release

In order to comprehend the complex mechanism of drug release from the microspheres, different mathematical models were used to describe the kinetics of AS release from the microspheres. The decisive factor for selecting the most fitting model was preferred on the source of a goodness-of-fit test.

 

Table 3:  In vitro release kinetic parameters.

 

The release kinetics equation was applied to the entire release period for the reason that very few of the microspheres released more than 90% of the incorporated drug. The result of the different parameters derived from the release models is presented in Table 3.

 

The values show that with respect to Higuchi’s model, F-1, and F-2 coded formulations obeyed the model and thus exhibited diffusion-controlled release. The in-vitro Abacavir sulphate release data were fitted to korsmeyer-peppa’s release model. All the formulations exhibited anomalous (non-Fickian transport) diffusion mechanism. The n values ranged between 0.5 and 0.8. 

 

From the results of in-vitro release studies conducted up to 24 hours,  the inclusion of  eudragit polymers (F3, F4, F6) showed decreased in vitro release of AS (p<0.05). This is due to because of the increase in density of the eudragit polymer matrix even at smaller concentrations results in an increased diffusional path length, which obviously resulted in decreased release rate. But the formulations prepared with HPMC K4M (F2, F5, F7) showed an increased percentage of drug release. This was due to increase in hydrophilicity of the microsphere matrix. Microspheres prepared with HPMC K4M produced a less viscous and erodible layer, ease the progress of the drug diffusion and resulting in a higher drug release rate of 94% extended up to 24 hours. From Fig 3, it was observed that formulation coded as F2 have a propensity to give prolonged sustained release of AS over a period of 24 hrs in contrast to other batches.

 

Stability studies

The stability studies did not reveal any notable changes in the drug content, hence it can be indicated that the optimized formulation was stable in average storage conditions.

 

CONCLUSION:

In the present work efforts have been made to design and evaluate microspheres of abacavir sulphate. All the formulations exhibited anomalous (non-Fickian transport) diffusion mechanism and followed zero order kinetics. The formulation F2 with HPMC K4M was selected as best formulation; with 94 % of controlled drug release at the end of 24 hours with preeminent physico- chemical properties. Hence such a design can be used for the delivery of abacavir sulphate as microspheres in the treatment of HIV. Success of the in vitro drug release studies recommends the product for further in vivo studies in detail for its viability in clinical practice.

 

ACKNOWLEDGEMENT:

We are very thankful to Apotex Laboratories, Bangalore, India for providing the gift sample Abacavir Sulfate. We are very much thankful to the staff and management of The Erode College of Pharmacy for providing necessary facilities for the successful completion of this work.

 

REFERENCES:

1.        Ziagen (abacavir sulfate) tablets and oral solution prescribing information. http://www.gsk.com.au/

2.        Skoog DADouglas A. Skoog (Author)

› Visit Amazon's Douglas A. Skoog Page

Find all the books, read about the author, and more.

See search results for this author

Are you an author? Learn about Author Central

, Holler FJF. James Holler (Author)

› Visit Amazon's F. James Holler Page

Find all the books, read about the author, and more.

See search results for this author

Are you an author? Learn about Author Central

, Nieman TA, Timothy A. Nieman (Author)

› Visit Amazon's Timothy A. Nieman Page

Find all the books, read about the author, and more.

See search results for this author

Are you an author? Learn about Author Central

Authors. In: Principles of Instrumental Analysis. 5th ed.Thomson Brooks/Cole, 1997; pp. 380-426.

3.        Dyer JR. In: Applications of absorption spectroscopy of organic compounds. 1st ed. New Delhi: Prentice-Hall of India, 1969; pp. 33-38.

4.        Kiyoshi N, Tomohiro T, Tsuyoshi M, Kanji T. Preparation and evaluation of a time-controlled release capsule made of Ethyl cellulose for colon delivery of drugs. Journal of Drug Targeting. 3; 1995:83-89.doi: 10.3109/10611869509059209

5.        Amol Paharia, Awesh K Yadav, Gopal Rai, Sunil K Jain, Shyam S Pancholi, Govind P Agrawal. Eudragit coated pectin microspheres of 5-fluorouracil for colon targeting. AAPS Pharm ScTech. 8; 2006:1-7. doi:  10.1208/pt0801012

6.        Jain SK, Gopal R, Saraf DK, Agrawal GP. The preparation and evaluation of Albendazole microspheres for colonic   delivery. Pharm Tech. 2004:66-71.

7.        Gilbert SB, Christopher TR. In: Modern Pharmaceutics. 3rd ed. New York: Marcel Dekker.1996; 470, 598, 603, 638, 864.

8.        Barkai A, Pathak V, Benita S. Polyacrylate (Eudragit retard) microspheres for oral controlled release of nifedipine. I. Formulation design and process optimization. Drug Development and Industrial Pharmacy. 16; 1990: 2057-2075.doi: 10.3109/03639049009023640

9.        Milling Eugene L. Lachman L, Liberman HA, editors. In: The Theory and Practice of Industrial Pharmacy. 2nd ed.Mumbai, India: Varghese Publishing House, 1991:26-27.

10.     Sinha VR, Agarwal MK, Kumria R. Influence of formulation and excipients variables on the pellet properties prepared by extrusion spheronization. Current Drug Delivery. 2; 2005: 1-8.doi: 10.2174/1567201052772898.

11.     Hausberger AG, DeLuca PP. Characterization of biodegradable poly (D, L-lactide-co-glycolide) polymers and microspheres. Journal of Pharmaceutical and Biomedical Analysis.13; 1995:747-760.doi:10.1016/0731-7085(95)01276-Q.

12.     Nappinnai M, Ahamed Ibrahim TS, Natarajan V, Dinesh Kumar S. Formulation and evaluation of ethyl cellulose as retardant for prolonging release of chlorpromazine hydrochloride. The Indian Pharmacist. 5; 2006: 77-79.

13.     Joseph R. Nixon, George A. Agyilirah. Effect of microcapsule core-wall ratio and aggregate size on the properties of tableted microcapsules. Journal of Pharmaceutical Sciences. 73; 1984: 52–54.doi: 10.1002/jps.2600730113

14.     Hiromitsu Y, Yoshio K, Shohei So, Hirofumi T Yoshiaki K. Surface-modified PLGA nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions. Journal of Controlled Release.  102; 2005:373-381. doi:10.1016/j.jconrel.2004.10.010.

15.     Gohel MC, Amin AF.Formulation and optimization of controlled release diclofenac sodium microspheres using factorial design. . Journal of Controlled Release. 51; 1998: 115-122. doi:10.1016/S0168-3659(97)00102-8.

16.     Shabaraya AR, Narayanacharyulia R. Design and evaluation of chitosan microspheres of metoprolol tartrate for sustained release. Indian Journal of  Pharmaceutical  Sciences. 65; 2003: 250-252.

 

 

 

Received on 19.03.2013          Modified on 25.05.2013

Accepted on 06.06.2013         © RJPT All right reserved