CHAPTER-3 MATERIALS AND METHODS
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1 75 CHAPTER-3 MATERIALS AND METHODS
2 MATERIALS Drugs used in the present study Lamivudine Zidovudine Stavudine Drug name Source Alchem laboratories, Mumbai, India Excipients and chemicals used in the present study Excipient name Source CAP CAB EC HPMCP HPMC 100K LV Polyox WSR 303 Pharmatose DCL21 Avicel PH 200 Aerosil Talc Silica Gel Pak GM Chemicals, India Eastman chemical company, USA DOW chemical company, USA SHIN-Etsu, Japan DOW chemical company, USA DOW chemical company, USA DMV International, Netherlands FMC Biopolymers, USA Degussa, Germany Luzenac, France Desiccare, Inc., Richland, MS, USA 0.45 µ (Millipore) filter Millipore, USA Eudragit RL100 & RS100 Degussa, Germany HPMC K 100 M Polyox WSR 303 Eudragit (L100) Carbapol 971 Pharmatose DCL 11 Magnesium stearate Avicel PH 200 KOLLIDON CL Starch 1500 Di-sodium hydrogen Phosphate Colorcon Asia Private Ltd, India DOW chemical company, USA Degussa, Germany Lubrizol corporation DMV International, Netherlands Ferro Industrial Chemicals USA. FMC Biopolymers, USA FMC Biopolymers, USA Colorcon Asia Private Ltd, India Rankem, India
3 List of Equipments used in the study Equipment Name 16-station tablet compression machine Electronic balance UV visible spectrophotometer Hardness tester Friability tester FT-IR DSC Sonicator Dissolution apparatus Scanning electron microscope Overhead 3-blade medium duty stirrer Stability chambers HPLC Vortex mixer Manufacturer Cadmach Co, Ahmedabad, India ER182A, Mettler Toledo Schimadzu, UV-1700 E 23 MHT-20, Campbell Electronics, Mumbai, India FTA-20, Campbell Electronics, Mumbai, India Thermo Nicolet 670 spectrometer Mettler Star SW 8.10, USA Power sonic 505, HWASHIN Technology Co, USA LABINDIA, DISSO-2000, Mumbai, India JEOL JSM -5200, USA Model RO 123, Remi, Mumbai Thermolab Scientific Equipments Pvt Ltd, India Shimadzu Scientific Instruments, Japan Remi Equipment, Mumbai, India
4 DOSAGE FORMS SELECTED IN THE PRESENT STUDY Single unit system (matrix tablets) Matrix drug delivery systems consist of a polymer, drug, and other excipients distributed throughout the matrix. This system is dependent on polymer wetting, polymer hydration, and polymer dissolution for the controlled release of drug. At the same time, other soluble excipients or drug substances comprising the tablet will also become wet, dissolve, and diffuse out of the matrix, while insoluble excipients or drug substances will be held in place until the surrounding polymer/excipient/drug complex erodes or dissolves away. Figure 3.1 Schematic representation of drug release from the matrix tablets
5 Microparticles These are particles with size more than 1 m, containing the polymer. At present, there is no universally accepted size range that particles must have in order to be classified as microparticles. However, many workers classify the particles smaller than 1 m, as nanoparticles and those more than 1000 m, as macroparticles. Classification: Microparticles are classified into two groups Mircroparticles Microcapsules (Micrometric Reservoir Systems) Microspheres (Micrometric Matrix Systems) Schematic Representation Microcapsules: Microcapsules are small particles that contain an active agent or core material surrounded by a coating or shell. (Commercial microcapsules typically have a diameter between 3 & 800 micrometer and 10-90% core). Microspheres: Microspheres are solid, spherical particles containing dispersed drug molecules, either in solution or crystalline form, among the polymer molecules.
6 GENERAL METHODS IN THE PREPARATION AND CHARACTERIZATION OF MATRIX TABLETS AND MICROCAPSULES Preparation of Matrix Tablets using direct compression method The drug, polymer(s) and all other excipients sifted through 425 μm sieve (ASTM mesh no 40) and mixed uniformly. The dry mix blend was then pre lubricated with respective excipients and lubricated with magnesium stearate. The lubricated granules were directly compressed on 16-station tablet compression machine using respective punches. (Cadmach Co, Ahmedabad, India) Preparation of Matrix Tablets using Wet granulation method The drug, polymer and other excipients were sifted through 425 μm sieve (ASTM mesh no 40) and mixed uniformly. The dry mix blend was then granulated with respective granulation fluid. The wet granules were dried at 60 C until the complete evaporation of granulation fluid from the granules. The dried granules were again sifted through ASTM mesh no 30. The dried and sifted granules were then pre lubricated with respective excipients and then lubricated with magnesium stearate. The lubricated granules were compressed on 16-station tablet compression machine using respective punches. (Cadmach Co, Ahmedabad, India).
7 Preparation of Microcapsules Microcapsules were prepared by using solvent evaporation method. The drug and polymer were dissolved/dispersed in 25 ml of the organic solvent (Acetone). A 100 ml of heavy liquid paraffin was taken in 250 ml beaker and kept for stirring at rpm. The dispersed /dissolved drug polymer solution was slowly added to the heavy liquid paraffin under stirring condition. Stirring was continued until the complete evaporation of the acetone from the liquid manufacturing vehicle. The prepared microcapsules were filtered and washed with n-hexane to remove the liquid paraffin and to harden the microcapsules. The micro capsules were dried at 40 C for the complete evaporation of the solvent. Fig 3.2 describes the process for the preparation of microcapsules.
8 Solubility determination of drugs Solubility study of the active drug was investigated in four different media as follows: 1) Purified water 2) 0.1 N hydrochloric Acid (HCl), USP 3) Acetate buffer ph 4.5, USP 4) Phosphate buffer ph 6.8 USP Required quantity of above media was transferred in to a volumetric flask and heated up to 37 ±0.5 o C using magnetic stirrer provided with heat. Previously weighed quantity of active drug was added to the above volumetric flask until the saturation point occurs. The total quantity of drug added was recorded. Stirring was continued up to 5 hours at 37 ±0.5 o C. The sample was filtered through 0.45 µm filter. A measured quantity of filtered sample was transferred in to another volumetric flask and further dilutions made. The absorbance was measured using UV visible spectrophotometer (Schimadzu, UV E 23) Construction of standard calibration curves Accurately weighed quantity of active drug was transferred in to the volumetric flask. Required quantity of media was added to the above volumetric flask. Shake the volumetric flask until the complete solubility of the drug and make up the volume with remaining quantity of media. Similarly stock solutions were prepared in all the media. Standard calibration curves in different media were constructed using the above stock solutions. The samples were
9 83 scanned for max at the UV range of nm. After 1 day again the samples were scanned for max. The max at initial and 1 day were compared for the stability of pure drug in the respective media. From the above stock solutions different concentrations of the solutions were prepared and standard calibration curves were prepared by plotting the absorbance values vs concentration Fourier Transform Infrared spectroscopy (FT-IR) The FT-IR spectrums of pure drug, initial formulation and stability samples of both matrix tablets and microcapsules were determined. A FT-IR (Thermo Nicolet 670 spectrometer) was used for the analysis in the frequency range between 4000 and 400 cm -1, and 4 cm -1 resolution. The results were the means of 6 determinations. A quantity equivalent to 2 mg of pure drug was used for the study Differential scanning calorimetry (DSC) Thermal properties of pure drug, initial formulation and stability samples of both matrix tablets and microcapsules were evaluated by Differential scanning calorimetry (DSC) using a Diamond DSC (Mettler Star SW 8.10). The analysis was performed at a rate 5 0 C min -1 from 50 0 C to C temperature range under nitrogen flow of 25 ml min Drug content estimation The drug content of the prepared matrix was determined in triplicate. From each batch, 20 tablets were taken, weighed, crushed and finely powdered. An accurately weighed quantity of this powder was taken and suitably dissolved under sonication (Power sonic 505, HWASHIN Technology Co.) in ph 6.8 phosphate buffer and filtered
10 84 through 0.45µ (Millipore) filters. The sample was analyzed after making appropriate dilutions using the developed analytical method Hardness, weight variation and friability determination The weight variation was determined by taking 20 tablets using an electronic balance (type ER182A, Mettler Toledo). Tablet hardness was determined for 10 tablets using a Monsanto tablet hardness tester (MHT-20, Campbell Electronics, Mumbai, India). Friability was determined by testing 10 tablets in a friability tester (FTA-20, Campbell Electronics) for 300 revolutions at 25 rpm In vitro drug release studies of prepared matrix tablets and microcapsules The in vitro dissolution studies were performed up to 14 hours and more using dissolution apparatus (LABINDIA, DISSO-2000, Mumbai, India). The dissolution medium consisted of phosphate buffer ph 6.8 (900 ml), maintained at 37 ±0.5 C. An aliquot (5 ml) was withdrawn at specific time intervals and filtered through 0.45 µ (Millipore) filter. After appropriate dilution the samples were analyzed and cumulative percentage of the drug released was calculated. 6 tablets from 3 different batches were used in analysis Accelerated stability studies on the prepared formations 102 Selected formulations from prepared formulation were filled in HDPE containers and stored at the following conditions like 40 C/75% RH for about 3 months as per ICH guidelines. The samples were characterized for percent drug content, FTIR and DSC study.
11 Kinetic analysis of dissolution data The release rate and mechanism of drug release from the prepared formulations were analyzed by fitting the dissolution data into the zero-order equation Q = k0t where Q is the amount of drug released at time t, and k0 is the release rate constant, The dissolution data was fitted to the first order equation ln (100 Q) = ln 100 k1t. where k1 is the release rate constant. The dissolution data was fitted to the Higuchi s equation Q = k2 t1/ Statistical Comparison of Dissolution Profiles 95 Dissolution studies of the prepared matrix tablets and microcapsules for all the formulations were determined. A statistical comparison such as similarity factor (f2 factor) among some formulations was used. This statistical model is suitable only when three or more dissolution time points are available. The similarity factor (f2) is a logarithmic reciprocal square root transformation of the sum of squared error and is a measurement of the similarity between two curves in the dissolution. The following equation represents a similarity factor (f2):
12 86 where 1) f2 similarity factor, log is logarithm to base 10, 2) P is number of sampling time points, 3) Σ is the summation of over all time points, 4) μti is the dissolution measurement (in mean percent labeled amount) at time point t of the first batch (test batch) profile, 5) μri is the dissolution measurement (in mean percent labeled amount) at time point t of the second batch (reference batch) profile Encapsulation efficiency (EE) 98 Drug loaded microcapsules (100 mg) were powdered and suspended in water. Then the contents suspended in the water were kept for sonication (Power sonic 505, HWASHIN Technology Co) for about 20 mins and shaked using mechanical shaker (ORBITEX, Scigenics Biotech) for about 20 mins for the complete extraction of drug from the microcapsules. The resultant solution was filtered through 0.45 µm filter. Drug content was determined by UV- visible spectrophotometer (Schimadzu, UV-1700 E 23). The percent entrapment was calculated by using the following formula.
13 Particle size distribution of microcapsules and granules Particle size analysis 99 of the microcapsules was done by sieving method using Indian Standard Sieves (Test Sieves ASTM-E-11) #10, #20, #30, #40, #60 and #80, #100, #120 fitted to a mechanical vibrator-shaker. The fractions were calculated by collecting for each sieve and percent retained and cumulated percent retained were calculated Scanning electron microscopy (SEM) Morphological characterization of the microcapsules was done by using Scanning electron microscope (JEOL JSM -5200). The samples were coated to 200 A thickness with gold-palladium prior to microscopy.
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