Preparation and In Vitro Evaluation of Lamivudine Entrapped MOI Microspheres for Oral Administration

 

Nayak Bhabani Shankar*1, Nayak Udaya Kumar2, Patro K Balakrishna2 and Rout Prasant Kumar1

 

1Jeypore College of Pharmacy, Rondapalli, Jeypore-764002, Koraput, Orissa, India

2Glenmark R and D Unit, Navi Mumbai, India

*Corresponding Author E-mail: bhabani143@yahoo.co.in


 



ABSTRACT:

The current  study concern with  the evaluation of natural gum moi as noble sustain release rate controlling materials in microsphere formulation prepared by solvent evaporation technique using lamivudine as the model drug. Microspheres were evaluated for various parameters such as yield percentage, particle size analysis, sphericity measurement, drug entrapment efficiency, loose surface crystal study, in vitro drug release profile and release kinetics study. Effect of drug: gum ratio (1:6, 1:10 w/w with respect to drug weight) on in vitro drug release profile was investigated The rate limiting capacity of moi gum was compared with guar gum as control by keeping all the parameters constant. The gum produced microspheres have satisfactory size (24- 32µm) and acceptable morphological properties. Microspheres of moi gum exhibit sustained action beyond 10 hr in comparison to guar gum but the combination of both the gum in 1:1 ratio demonstrate an additional sustained action. Drug release from the F1, F2, F4 and F6 seems to best fit to Higuchi square root kinetics indicating the diffusion controlled release where as the F3 follows zero order kinetic and f5 shows korsemeyer-peppas model. The release mechanism was found fickian for all the formulations as polymer matrix was used as rate retarding material. It was concluded that the gum possesses substantial rate controlling properties with the combination of guar gum and could be used for sustained drug delivery.

 

KEY WORDS:   circularity factor, microsphere, fickian diffusion

 


 

INTRODUCTION:

One of the common methods of controlling the rate of drug release is micro encapsulation where a drug material is coated with a polymer substance. As a result the process become invincible to safety hazards, toxicity and decreases the cost of production making the techniques reproducible, economically and ecologically at an industrial scale. On the topic of polymer, researchers investigated various natural, semi synthetic and synthetic polymer materials but the synthetic polymers showed specific limitations and the combination of synthetic and natural polymer is being complicated and increases the cost of formulation1. In view of the cost effectiveness, ecofriendly, potentially degradable with easy availability of the plant and high demand of gum through out the world, the gum obtained from Lannea coromandelica (Houtt.) Merrill (Anacardiaceae)2 was investigated for its suitability for used as a rate controlling material for lamivudine microsphere. Past research therefore acknowledged various natural gum like agar,

 

konjac, guar gum, chitosan, xanthan, sodium alginate and lotus bean gum etc for potential pharmaceutical and biomedical application3. These particular explicates the rationale why proposed article concerns the evaluation of natural gums for sustained drug delivery. Moi gum is yellowish white color in fresh and on drying become dark. It is used in paper industry, cloth sizing, preparation of varnish and as preservative in costal area of orissa, Tamilnadu and kerala4. Lamivudine is an active antiretroviral drug belongs to non-nucleosides reverse transcriptase inhibitor. Lamivudine treatment has gained immense popularity in the AIDS treatment in the present era5. Thus, the present study proceeds with an objective of preparation and evaluation of moi encapsulated lamivudine  microspheres of varying solubility within water soluble gum in an absolute aqueous environment and compared with single guar gum and both moi + guar gum (1:1 ratio) microspheres which corroborate the rate retarding  properties of moi for lamivudine delivery.

 

MATERIAL AND METHODS:

Lamivudine was received as a gift sample from GlaxoSmithKline, Mumbai. Moi gum was obtained from Ganjam Orissa and authenticated by RRL Bhubaneswar, Orissa. Ethanol and guar gum were procured from SD.


Table 1: Comparative study of various physical parameters for microspheres containing lamivudine.

Parameters

F1

F2

F3

F4

F5

F6

Particle size (µm)(X±SEM)

25.08±0.006

31.24±0.012

28.29±0.007

30.12±0.009

23.76±0.006

29.48±0.014

Circularity factor(X±SEM)

1.00±0.008

1.00±0.006

1.00±0.004

1.00±0.009

1.00±0.008

1.00±0.005

Yield (%)  (X±SEM)

69±0.012

79.23±0.014

87.25±0.023

82.58±0.024

88.52±0.051

86.65±0.041

Drug entrapment efficiency (%) (X±SEM)

70.12±0.014

87.34±0.016

74.37±0.019

89.36±0.012

91.09±0.019

85.11±0.011

Loose surface crystal study (% of total drug)(X±SEM)

18.21±0.012

22.25±0.011

18.45±0.015

16.66±0.022

15.88±0.014

21±0.012

 

 

 

 

 

 

 

 

 

 All the results are mean ± standard error mean (n=3). Values were significant at 95 % confidence interval (P<0.05)

F1: Lamivudine: moi (1:6); F2: Lamivudine: moi (1:10); F3: Lamivudine : guar gum (1:6); F4: Lamivudine: guar gum (1:10);                             F5: Lamivudine: guar gum + Moi (1:6); F6: Lamivudine: guar gum + moi (1:10)


 


Fine chemicals Kolkata. All other chemicals were purchased from local supplier in A.R. and L.R. Grade as required.

 

Purification of natural gum:

The collected gum was soaked with distilled water and shaken for 4-5 hr and passed through muslin. The mucilage obtained was precipitated out with 95% ethanol (1:1 v/ v) by continuous stirring. The coagulated mucilage as a white mass floating on ethanol was transferred to an evaporating disc and treated successively with ethanol6. The coagulated mass was dried in oven at 40-50 ° C, powdered by passing through sieve and stored in air tight containers (yield =29.87 % w/w).

 

Preparation of microspheres:

The gum moi was dissolved uniformly in 15 ml of distilled water using mechanical stirrer maintaining the speed at 500-600 rpm. To this solution the desired lamivudine was mixed in suitable proportions and the entire mixture was stirred for 1.5 hr at 900 rpm with diminutive warming. Thus obtained microspheres were dried for 3 hr in hot air oven at 60° C and stored for further study7. The same method was espoused for preparation of guar gum and guar gum+ moi mixture.

 

Process variables and Process optimization:

The following process variables were investigated (concentration of moi; concentration of guar gum; concentration of moi +guar gum; variation of drug loading; stirring speed and stirring time) and the different batches thus produced were analyzed for size, shape, ease of preparation, drug content, sphericity, loose crystal study and drug release. On the basis of the result obtained the process parameters were optimized as follows:-

 

Moi concentration – 84 % w/v and 90 % w/v

Guar gum concentration – 84 % w/v and 90 % w/v

Drug load – 16 % w/w and 10 % w/w

Stirring time and speed – 1.5 hr & 900 rpm

Drying condition – oven drying for 3hr at 60°C

 

Different batches of microspheres were then prepared by using the optimized process variables and the only variation followed was use of different polymers. Six set of formulations were prepared using moi (F1, F2); guar

 

gum (F3, F4) and moi +guar gum (F5, F6) at concentration (6%, 10%). The final formulations were subjected to several characterization studies.

 

Table 2: In vitro drug release profile of different microsphere formulations.

Time (hr)

F1

F2

F3

F4

F5

F6

1

51.06

76.46

57.42

70.01

36.02

55.20

2

45.36

76.98

60.36

70.18

36.77

56.14

3

42.34

77.27

59.06

68.70

30.61

54.84

4

52.75

77.39

59.58

71.49

30.28

55.08

5

50.45

77.62

60.36

71.76

31.77

55.20

6

47.19

77.92

60.96

70.99

31.18

55.49

7

47.03

77.39

60.79

71.16

30.94

55.08

8

50.07

77.04

61.65

70.39

30.61

55.73

9

56.63

76.98

62.69

71.49

30.77

55.37

All datas are expressed in percent cumulative drug release.

Verified with one way ANOVA the results were found to be significant at 5 % level of significance.

F1: Lamivudine: moi (1:6)   F4: Lamivudine : guar gum (1:10)

F2: Lamivudine: moi (1:10)    F5: Lamivudine : guar gum + Moi (1:6)

F3: Lamivudine : guar gum (1:6)F6: Lamivudine : guar gum + moi (1:10)

 

Figure 1: Particle size analysis of Lamivudine loaded gum microspheres.

F1: Lamivudine: moi (1:6) F4: Lamivudine : guar gum (1:10)

F2: Lamivudine: moi (1:10)F5: Lamivudine : guar gum + Moi (1:6)

F3:Lamivudine:guar gum (1:6)F6:Lamivudine:guar gum + moi (1:10)

Characterization of Microspheres:

Percentage Yield and Drug entrapment efficiency (DEE)8 :

The microspheres were evaluated for percentage yield and percent drug entrapment. The yield was calculate as per equation-1,

Percentage yield

= Weight of microsphere recovered X 100……. (1)

                Weight (drug + polymer)                                      

 

Drug loaded microspheres (100 mg) were powdered and suspended in 100 ml water solvent system. The resultant dispersion was kept for 20 min for complete mixing with continuous agitation and filtered through a 0.45 µm membrane filter. The drug content was determined spectrophotometrically (UV-Visible-1700, Shimadzu, Japan) at 270 nm using a regression equation derived from the standard graph (r2 = 0.9978). The drug entrapment efficiency (DEE) was calculated by the equation-2,

 

DEE = (Pc / Tc) X 100          …………….. (2)

Pc is practical content, Tc is the theoretical content. All the formulations were analyzed in triplicate (n=3).

 

Particle size analysis :

The size of the prepared microspheres was measured by the optical microscopy method using a calibrated stage micrometer8. Particle size was calculated by using equation-3,

 

Xg = 10 x [(ni x log Xi) / N]   ……….. (3)

Xg is geometric mean diameter, ni is number of particle in range, xi is the mid point of range and N is the total number of particles. All the experimental units were analyzed in triplicate (n=3).

 

Sphericity determination9:

The particle shape was measure by computing circulatory factor (S). The tracing obtained from optical microscopy were used to calculate Area (A) and perimeter (P).This will indicate the approximate shape of the prepared microsphere calculated by this equation-4,

 

S =           P2

          12.56 * A              ……… [4]

 

Loose surface crystals study:

The prepared microspheres were evaluated by loose surface crystal study to observe the excess drug present on the surface of microspheres. From each batch, 100 mg of microspheres was shaken in 20 ml of double distilled water for 5 minute and then filtered through whatman filter paper 41. The amount of drug present in filtrate was determined spectroscopically and calculated as a percentage of total drug content10.  

 

In-vitro drug release study11 :

The USP rotating – paddle Dissolution Rate apparatus (Veego, Mumbai) was used to study drug release from the microspheres. The dissolution parameters [ 100mg microsphere ; 37± 2°C ; 50 rpm ; 900ml of 0.01 n Hcl; n=3; coefficient of variation< 0.05] were maintained for all the six formulations. 3 ml of aliquot were withdrawn at specified intervals and after suitable dilution assayed by Shimadzu UV-VIS PharmSpec 1700 spectrophotometer at 270 nm. The data for percent drug release was fitted for zero order12, first order13; Higuchi matrix equation14 and korsemeyer-peppas model15. Kinetic model had described drug dissolution from solid dosage form where the dissolved amount of drug is a function of test time. The criterion for selecting the most appropriate model was chosen on the basis of goodness of fit test.

 

Statistical Analysis:

Statistical data analyses were performed using the one way ANOVA at 5 % level of significance (p < 0.05) and standard error mean (SEM) at 95 % confidence interval6.

 

RESULTS:

The microspheres thus obtained during experiment were found to be spherical and without aggregation. The mean geometric particle size was found in a range of 23.76 to 31.34 µm having circularity factor as 1.00, which confirm their sphericity represented in table 1. The particle size distribution of all the formulation was presented in figure -1. The percentage yield of all the formulations was found to be satisfactory and each formulation demonstrated high drug entrapment efficiency (DEE), as summarized in table 1. The F5 showed higher DEE among all the formulations. These loose surface crystal studies lend a hand to estimate the excess amount of drug attached on the surface of microspheres after a successful drug entrapment. The study was executed with various prepared formulations and the results were tabularized in table- 1. The in vitro drug release profiles for all the batches were condensed in Table-2. All the identify formulations showed constant release profile. The in vitro drug release profile was presented in Figure-2.To recognize the kinetics of drug release from microspheres, release data was analyzed according to different kinetic models.Table-3 explains the  drug release from F1, F2, F4 and F6 formulations seems to fit best in Higuchi square roots model. The formulation F3 obeys zero order kinetics, while the release data of F5 was followed Korsmeyer-Pappas model. In the present study, the values of release rate exponent (n) were found to range 0.22 to 0.38 for all six formulations. For a sphere a value of n  0.43 is known to indicate predominantly fickian drug release (Peppas NA. et al, 1989). So according to current study the mechanism of drug release from spherical microspheres was distinctly fickian diffusion. Statistical verification with one way ANOVA method attested the fact that the drug release data were found significant for F (502.0197) at 5% level of significance (p< 0.05).

 

DISCUSSIONS:

Gum obtained from Lannea coromandelica after purification was an amorphous free flowing powder with a light black color. It exhibited good solubility in water and gave viscous solution on standing. With comparison between the corresponding ratios of gums, the drug release profile of moi gum was found to be more sustained than guar gum but the mixture of both moi and guar gum(1:1 w/w) exhibits better sustained effect than single gum result. The kinetic analysis predict that,



Table 3: Release Kinetic Study of Selected Formulations.

Correlation coefficient ( r)

Kinetic Models

F1 (X±SD)

F2 (X±SD)

F3 (X±SD)

F4 (X±SD)

F5 (X±SD)

F6 (X±SD)

Zero order

0.6352 ±0.014

0.7252 ±0.019

0.9791 ±0.021

0.6941 ±0.023

0.8978 ±0.025

0.7871 ±0.027

First order

0.7397 ±0.028

0.7982 ±0.032

0.8021 ±0.011

0.7724 ±0.021

0.8641 ±0.089

0.8559 ±0.054

Higuchi square root

0.9564 ±0.025

0.9881 ±0.035

0.8914 ±0.054

0.9832 ±0.024

0.9205 ±0.035

0.9577 ±0.014

Pappas equation

0.8771 ±0.024

0.6187 ±0.021

0.7807 ±0.019

0.7456 ±0.017

0.9832 ±0.017

0.626 ±0.015

Diffusion release exponent (n)

0.33

0.34

0.27

0.31

0.22

0.38

Values were expressed as mean ± standard deviation (n=3) F1: Lamivudine: moi (1:6);  F2: Lamivudine: moi (1:10); F3: Lamivudine : guar gum (1:6);  F4: Lamivudine : guar gum (1:10); F5: Lamivudine : guar gum + Moi (1:6); F6: Lamivudine : guar gum + moi (1:10)


 

formulation F3 obeys zero order kinetics while release data of F1, F2, F4 and F6 fit the best to higuchi square root indicating that diffusion was main factor controlling drug release rate and not influenced by process  variable. On the other hand, F5 showed drug release by Korsmeyer and Pappas model. The mechanism of drug release from spherical microspheres was distinctly fickian diffusion for all the formulations. Gum moi was found more effective than guar gum in sustaining the drug release rate but the drug: gum mixture (moi+ guar gum) in 1:6 ratios (F5) retarded significantly lamivudine release beyond 10 hours.

 

Figure 2: Drug release profile of prepared lamivudine loaded gum microspheres.

F1: Lamivudine: moi (1:6) F4: Lamivudine : guar gum (1:10)

F2: Lamivudine: moi (1:10)F5: Lamivudine : guar gum + Moi (1:6)

F3:Lamivudine:guar gum (1:6)F6:Lamivudine:guar gum + moi (1:10)

 

CONCLUSION:

Sustained release microspheres containing water soluble drug were successfully prepared employing modified solvent evaporation technique, with natural water soluble polymer, namely, moi, the use of copolymer further prolongs the release of the drug. The drug: gum mixture (1:6 w/w) i.e. F5 formulation showed good encapsulation efficiencies, smaller particle size and maximum prolongation of drug release. Hence, further studies can be extended taking moi as the release controlling copolymer. Considering the end product, the microspheres could be administered as prepared or could be compressed into tablet or filled in capsule shell. The entire process is feasible in an industrial scale and demands pilot study.

ACKNOWLEDGEMENTS:

Authors wish to thank GlaxoSmithKline (Mumbai, India) for providing gift sample of lamivudine. We are also thankful to the staff of regional research laboratory (RRL), Bhubaneswar, orissa for their relentless cooperation.

 

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Received on 22.08.2008       Modified on 12.09.2008

Accepted on 15.11.2008      © RJPT All right reserved

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008; Page 437-440