INTRODUCTION:

Micro particulate delivery systems are painstaking and acknowledged as reliable means to deliver the drug to the target site with specificity, if modified and to maintain the desired concentration at the site of interest without untoward effect. It has greater advantages over the conventional delivery system includes, increased bioavailability, reduced fluctuation in steady state levels, subject variability and drug induced toxicity and side effect^1-3. Lamivudine is an active antiretroviral drug belongs to non-nucleosides reverse transcriptase inhibitor⁴. Lamivudine treatment has gained immense popularity in the AIDS treatment in the present era⁴. Hence the present study proceeds with an objective of preparation and evaluation of lamivudine microspheres and to assess the in vivo performance of prepared sustained release lamivudine microspheres and to

compare it with conventional lamivudine tablet after administering orally to New Zealand white rabbit species. Further, the work was extended to establish a correlation between in vitro and in vivo data.

MATERIAL AND METHODS:

Materials

Lamivudine was received as a gift sample from GlaxoSmithKline Ltd. Mumbai. Ethyl cellulose and Acrycoat S-100 was obtained from LOBA chemicals, Kolkata and Corel Pharma Ahmadabad, India respectively All other chemicals and solvents used were of analytical grade and procured from an authorized dealer, USP XXI paddle type dissolution apparatus, FT-IR (Shimadzu IR spectrophotometer, Model 840, Japan) and UV-Visible spectrophotometer (UV-1700, Shimadzu, Japan) were the instruments employed in the current study.

Preparation of microspheres⁵.

This is the method widely used in the microencapsulation process. Concisely the polymer ethyl cellulose was dissolved in methanol to get a clear solution. The drug lamivudine was added and dissolved in the polymer solution. The resultant mixture was then stirred at 900 rpm for 1 hr to evaporate the volatile substance. The formed microspheres were collected and air dried for 3 hour and stored in desiccator for further use.

Percentage yield (% yield)⁶.

The yield was calculated as the weight of the microspheres recovered from each batch divided by total weight of drug and polymer used to prepare that batch by 100.

Drug Entrapment Study⁶.

Drug loaded microspheres (100 mg) were powdered and suspended in 100 ml. The drug entrapment efficiency (DEE) was calculated by the equation

DEE = (Pc / Tc) X 100

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

Drug content estimation⁶.

Drug loaded microspheres (100 mg) were powdered and suspended in 100 ml methanolic: water (1:99 v/v) solvent. 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 spectrophotometer) at 270 nm³ using a regression equation derived from the standard graph (r²=0.9978).

Particle size analysis⁷.

Xg = 10 x [(n_i x log X_i) / N]

The micro sphere size distribution was determined by the optical microscopy method using a calibrated stage micrometer (µm) was calculated by using equation

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

Determination of sphericity⁸.

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

S = p²/12.56 * A

Where h= wall thickness, r = arithmetic mean radius of microspheres, d1=density of core material, d2= density of coat material, p= proportion of medicament in microsphere. The experiment was repeated thrice (n=3).

Fourier Transform Infrared Radiation measurement (FTIR)⁹.

The FTIR spectral measurements were taken at ambient temperature using IR spectrophotometer (shimadzu, model 840, Japan). Two mg of pure drug, empty microspheres and drug loaded microspheres were selected separately.

Scanning electron microscopy (SEM)¹⁰.

Scanning electron microscopy (Stereo scan S250 MK III, Cambridge, UK) was carried out to study the morphological characteristics of lamivudine microspheres. The dried microspheres were coated with gold foil (100 A°) under an argon atmosphere in a gold coating unit and Scanning electron micrographs in both higher and lower resolutions were observed.

In vitro drug release profile¹¹.

Invitro drug release study was carried out in USP XXI paddle type dissolution test apparatus using 0.01 M Hcl as dissolution medium. Volume of dissolution medium was 900 ml and bath temperature was maintained at 37±1° throughout study. Paddle speed was adjusted to 50 rpm. An interval of 1 hr, 5 ml of sample was withdrawn with replacement of 5 ml fresh medium and analyzed for lamivudine content by UV-Visible spectrophotometer at 270 nm³. All the experimental units were analyzed in triplicate (n=3).

In vitro drug release kinetics

In order to study the exact mechanism of drug release from microspheres, drug release data was analyzed according to zero order kinetic¹²., first order kinetic¹³., Higuchi square root equation¹⁴., Hixon-crowell equation¹³.. The criterion for selecting the most appropriate model was chosen on the basis of goodness of fit test. All the experimental units were analyzed in triplicate (n=3).

In vivo experimentation

Experimental design

The animals were divided into 4 groups containing 6 animals each and one animal was used as control. The animals were kept fasted for overnight. Water was given ad libitum during fasting and throughout experiment. The rabbits were not anesthetized during or prior to the experiment and were administered the formulations with an oral cannula. They swallowed easily without any difficulties. The weight of individual animal was recorded. The procedures employed in this study were approved by Institutional Ethical committee North Bengal University, Darjeeling by issuing registration No: HPI/ 07/60/ IAEC/ 0008.

Experimental Procedure^{15- 20}

One group was fed with standard lamivudine tablet (Lamivir) at a dose of 3 mg/kg. Other three groups were fed with prepared lamivudine microspheres (A2, B2 and C4) and marked as test A, B and C. One animal was kept as control. Blood samples (3ml) were collected from marginal ear vein of the control animal using xylene into centrifuge tubes containing 0.4 ml of 2.5 % (w/v) sodium citrate solution. The same method was followed for each group (both standard and test) at an interval of 30 min, 1 hr, 2, 4, 6,8,10 and 12th hour during study. The blood samples withdrawn as above were transferred to a series of graduated centrifuge tube containing 0.4 ml of 2.5 w/v sodium citrate solution. The samples were centrifuged immediately at 2500 rpm for 5 min in cooling centrifuge machine. The plasma was separated and transferred into other set of sample tubes and stored in -20 °C until assayed. An un-dose plasma sample was kept as blank sample at same condition. About 1 ml plasma was mixed with 1.5 ml of Trichloro acidic acid (15 % w/v), shaken well for 3 min and centrifuged again at 4000 rpm for 15 min. The analysis of plasma samples were analyzed by UV-Visible spectrophotometer (UV-1700, Shimadzu, Japan.

Invitro - Invivo Correlation (IVIVC)

There are four levels of IVIVC that have been described in the FDA guidance, which include levels A, B, C, and multiple C²¹.. Here the correlation was established according to Drewe and Guitard basing on (degree A)²².The parameters compared were cumulative absorption profile to that of in vitro dissolution profile i.e. Correlation of the amount of drug dissolved to that of respective fraction of dose absorbed, time taken for 50% dissolution to that of 50% absorbed (T₅₀), In vitro dissolution rate constant(K) Vs Area Under Curve (AUC) and Mean dissolution time (MDT) versus mean residence time (MRT)

CONCLUSIONS:

The Lamivudine loaded microspheres prepared by solvent evaporation method with various polymers ethyl cellulose (A), S-100 (B) and Ethylcellulose (C) in different ratios. The percentage yield of all the formulation was found to be satisfactory except B1 and C1 and drug entrapment efficiency (DEE) of all the formulations were found to be more than 80 %, as abridged in Table 1. The drug content data was represented in the same table. The microspheres obtained under these conditions were found to be spherical and without aggregation and mean geometric particle size was found in a range of 24 to 79 µm epitomized in Table 1. The particle size distribution for all the formulations were graphically signified in Figure1. The lamivudine loaded microspheres obtained by various ratios, having circularity factor as 1.00, which confirm their sphericity, as represented in Table 1. The In-vitro lamivudine releases from microspheres prepared by different ratios [A1-A4, B1-B4, and C1-C4] were studied and results were condensed in Table 2. All the formulations were found to release lamivudine in a controlled manner for a prolonged period over 10 hour. Verified with one way ANOVA the invitro drug release results were found significant at 5% level of significance (p<0.05). The comparative drug release profile of different formulations were presented in Figure 2.The shape of lamivudine microsphere as evidenced from the scanning electron microscope photomicrograph was spherical and uniformly distributed shown in Figure 3. To illustrate the kinetic of drug release from microspheres, release data was analyzed according to different kinetic equations depicted in text. Release data of A1, B1, B2 and B3 obeys zero order kinetic, whereas, A2, A4, B4, C2 and C3 seems to best fit in Higuchi square root kinetic model and A2, C1 and C4 releases drug following Hixon-Crowell cube root equation kinetic epitomized in Table 3. Verified with one way ANOVA the results were found to be significant at 5% level of significance P<0.05. The spectrums of micro particles formed with various ratios were compared with the spectrum of original Lamivudine. The interaction study between the lamivudine: ethyl cellulose and/or S-100 in different formulations were performed using FT-IR spectrophotometer and Four bands appeared in Lamivudine spectrum at 3445.9, 2930.92, 1736.5 and 1637.7 cm^-1, due to the formation of N-H, O-H, C=O, C=N linkage respectively, identified in the formulation spectrum, confirming no interaction between the drug and the polymer represented in Figure 4 to 7. The selected formulations i.e. A2, B2 and C4 were examined in invivo rabbit model. In comparison to the Lamivir the T _max of all the microspheres were increased from 1 hr to 4 hr confirms its sustaining property. The figure 8 indicates that the A2 formulation has lowest concentration among all and after 4 hr it was sustained over 12 hr which facilitates its sustained activity in body. On the basis of extended release A2 microsphere formulation was selected for invitro-invivo correlation study. The cumulative percentage of drug dissolved and cumulative fraction of drug absorbed were compared. The graphical analysis confirms, a good degree of correlation (r²=0.9680) fulfilling our objective. Good correlation was observed between the release rate constant and AUC of the formulation also between the mean dissolution time and mean resident time of the designed formulation epitomized in table 4. Degree A level of correlation was established from the results. Thus, the formulation has the potential to liberate lamivudine following Fickian diffusion mechanism having good degree of in-vitro- in-vivo correlation

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Received on 20.06.2008 Modified on 20.07.2008

Research J. Pharm. and Tech. 1(4): Oct.-Dec. 2008;Page 353-356