IJCRR - 4(18), September, 2012
Pages: 138-147
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FORMULATION AND EVALAUTION OF SUSTAINED RELEASED AZITHROMYCIN MICROSPHERE BY EMULSION SOLVENT EVAPORATION TECHNIQUE
Author: Preeti. R. Meshram, Suvarna P. Gajare, Manoj A. Bhadane
Category: Healthcare
Abstract:Azithromycin is macrolide antibiotic is effective against a wide variety of bacteria such as Hemophilus influenzae, Streptococcus pneumoniae, Mycoplasma pneumoniae, Staphylococcus aureus, mycobacterium avium, and many others. The aim of present work was to formulate sustained released azithromycin microsphere to overcome the problem related to drug like insoluble in water, bitter taste and gastrointestinal side-effects. Microspheres were prepared by varying drug polymer ratio using emulsion solvent evaporation technique. Characterization of microspheres was done by Differential Scanning Calorimetry (DSC), X-Ray diffractometry (XRD), Surface Morphology (SEM), Stability studies.
Keywords: Sustained Released Microsphere, Azithromycin, Solvent evaporation technique, Ethyl Cellulose, Ethyl Acetate
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INTRODUCTION
The objective of any drug delivery system is to provide drug in therapeutic amount to the proper site in the body to achieve immediately and then maintain the desired drug concentration. These idealized objectives are achieved by appropriately developed sustained release drug delivery which also has diverse applicability and merits. There are certain drugs having large dose they may cause GI side effect so to avoid this drug can be formulated in sustained form to release slowly in lower GI tract. The sustained release drug delivery includes the application of physical and polymer chemistry. These polymers slowly release the drug in biosystem and maintain drug blood level within therapeutic range for longer duration. Some of the products characterize the drug permeation through the appropriate biological membrane and any first pass metabolic effects prior to the entry of drug into systemic circulation. The fact that the absorption and release rate of the drug from the dosage form, is one of the interesting and most recent developments in pharmaceutical field1, 2 . Microencapsulation is a process whereby relatively thin coating of polymers are applied to small particles of solid or droplets of liquid and dispersions. The microencapsulation processes produce small particles ranging in size from 1 to 1000 µm. There are different names for these particles: microparticle, microsphere, microcapsule and micromatrix. The microparticle system has become an indispensable part of the sustained drug delivery fields for the past few decades since it can readily be adapted for various administration methods3, 4, 5 .
MATERIALS AND METHODS
Procurement of drug and excipients: The drug Azithromycin and polymer Ethyl cellulose (50 cps) were gifted from Zim Laboratories Ltd, Nagpur, where other AR grade excipients like Polyvinyl alcohol, Dichloromethane, Chloroform, Ethyl acetate, Sodium hydroxide and Dibasic sodium phosphate were supplied by Loba chemical, Mumbai. Microspheres were prepared by using mechanical stirrer of Remi Motors Ltd, Mumbai. Evaluation and characterization of microspheres was carried out by using equipments like USP Tablet dissolution apparatus Type II (TDT 08L), UVVisible double beam Spectrophotometer (UV1800), FTIR (8400SCCE), X Ray diffractometer (PW1700), DSC (61000), Scanning Electron Microscope ( JSM 6380 A), Light Microscope and Stage Micrometer. METHODS Preliminary study 6, 7, 8: The Preformulation study like Preliminary identification test (melting point, solubility) characterization of drug and polymer and their interaction study were performed. Spectroscopic studies 9, 10, 11: UV Spectroscopy studies involved Determination of λ max and preparation of standard curve of Azithromycin at 244 nm. The IR spectrum of the drug sample was recorded by IR spectroscopy. The drug polymer interaction was carried out using FT-IR Apparatus. Formulation of microspheres: The formulations were prepared by using solvents ethyl acetate at different drug: polymer ratio (1:1, 1:1.5, and 1:2). Method of preparation 12, 13, 14: In the O/W emulsion solvent evaporation method, the polymer (ethyl cellulose) was dissolved in internal organic phase of ethyl acetate. Accurately weighed quantity 1g of Azithromycin was dispersed or dissolved in the polymer solution. This resulting mixture was poured slowly with stirring into 100 ml of a 0.015% w/v aqueous solution of polyvinyl alcohol. The emulsion was then stirred continuously at 700 rpm for 1 hr to evaporate the solvent. The microspheres were recovered by vacuum filtration, washed with 200 ml of deionized water and dried at room temperature. The compositions of Azithromycin microspheres using ethyl acetate as solvent were given in Table 1. Evaluation of microspheres15, 16: Angle of repose: Angle of repose is defined as the maximum angle possible between the surface of pile of powder and horizontal plane. The angle of repose for the microspheres of each formulation was determined by the funnel method. The microspheres were allowed to flow out of the funnel orifice on a plane paper kept on the horizontal surface. It forms a pile of microspheres on the paper. The angle of repose was calculated by substituting the values of the base radius ‘R’ and pile height ‘H’ in the following equation Bulk density of all batches of microspheres was determined by pouring gently 2 g of sample through a glass funnel into a 10 ml graduated cylinder. The volume occupied by the sample was recorded. Bulk density was calculated as per given formula:
Tapped Density:
The tapped density was determined by pouring 2 g of microspheres through a glass funnel into a 10 ml graduated cylinder. The cylinder was tapped from height of 2 inches until a constant volume was obtained. Volume occupied by the sample after tapping was recorded .The values for tapped density was calculated as per given formula:
Compressibility index:
The compressibility indices of the formulation blends were determined using Carr’s compressibility index formula.
Hausner Ratio:
It provides an indication of the degree of densification which could result from vibration of feed hopper. Lower the Hausner ratio better is the flowability. It was calculated as per given formula.
Determination of particle size 17:
The particle size was determined using stage micrometer. The diameters of about 300 microspheres were measured and the average particle size was determined. Estimation of drug loading18, 19: For determination of drug content, microspheres equivalent to 100 mg were weighed and dissolved in 100ml of acetone. After suitable dilutions were with phosphate buffer (pH 6.0), the resulting solution was analyzed spectrophotometrically at 244 nm.
In-vitro dissolution study20, 21, 22: The study was carried out using dissolution apparatus USP Type-I (Rotating Basket type). Accurately weighed microspheres equivalent to 200 mg of Azithromycin were taken in muslin cloth and it was kept in baskets. Dissolution study was carried out in phosphate buffer pH 6.0 at 50 RPM at temp 37 ± 0.5° c. During dissolution study 10 ml of aliquot was withdrawn at a time intervals of 1 to 12 hr and same was replaced with equal volume of fresh medium. The withdrawn samples were filtered through Whatmann filter paper and absorbances were measured at 244 nm. Drug concentration in the samples was determined from the calibration curve. Characterization of microspheres: Differential Scanning Calorimetry 9, 10, 11, 27:
The DSC measurements were performed on a differential scanning colorimeter with thermal analyzer. All accurately weighed samples (about 10 mg of Azithromycin, ethyl cellulose, physical mixtures, and formulation) were placed in a sealed aluminium pan, before heating under nitrogen flow (10 ml/min) at a scanning rate of 20 ° c per min from 100 to 300 ° c an empty aluminium pan was used as reference. X-Ray diffractometry 13, 14, 15, 23: The powder X-ray diffraction pattern of Azithromycin and polymer were obtained using Phillips X-ray diffractometer with a Ni-filtered CuKα-radiation at a scanning speed of 10 °/min at 2θ.The graph was plotted in 2 theta angle Vs intensity count. Surface Morphology 24 The microspheres were coated with Platinum by ion sputtering using Autofine coater. The microspheres were kept on the sample holder and the scanning electron micrographs were taken. Stability studies 35 The study was carried out by storing the microspheres in glass bottle 40 ° c and 75% RH for 30 days. These samples were collected on 7th, 14th , 21st, 28th day and analyzed for changes drug content and in-vitro dissolution studies. RESULTS Preformulation studies: The melting point of azithromycin was found in the range of 119 – 121 ° c. The wavelength of maximum absorbance (λ max) found was 244 nm. Evaluation of microspheres: All the formulations show angle of repose value in the range of 19.72 to 20.42. The values for bulk density were found in the range of 0.352 to 0.450. The values for tapped density were found to range from 0.414 to 0.510. The compressibility index values were found in the range of 11.76 to 14.99 respectively. Hausner’s ratio was ranging from 1.13 to 1.17. The average particle size of microcapsules is found to be within 151.263 to 171.342 µm. The % encapsulation efficiency is found to be in the range of 73.50 to 75. 60%. In – vitro dissolution study, % cumulative drug release from formulation in 12 hrs was decreased from 80.13 to 69.98 % represented in Table 2. The dissolution data for formulations C1, C2, and C3 was fitted to various drug release kinetic models like Zero order, First order, Higuchi Matrix and Korsemeyer Peppas, Hixon-Crowel model. Kinetic Treatment to Dissolution Data proved important information about rate constants (K), correlation coefficients (R) obtained for various models were tabulated in Table 3. Characterization of microspheres: The FT-IR spectra of pure drug - Azithromycin, ethyl cellulose, physical mixture and formulation (C1) shows major functional groups of Azithromycin (methyl substituted nitrogen on lactone ring) in spectra of individual drugs as well as in spectra of physical mixture and formulation (C1). FT-IR of Formulation C1 presented in Fig 1. DSC curve presented in fig 2 of Azithromycin shows a single endothermic peak at 121 ° c, due to melting of the drug. Optimized formulation C1 which contains Azithromycin and ethyl cellulose, the thermogram indicates characteristic peaks for melting of Azithromycin at 121 ° c. The XRD pattern presented in fig 3 of formulation exhibited halo pattern with less intense and denser peaks compared to plain Azithromycin, Scanning Electron Microscopy results were presented in fig 4a and 4b. Accelerated stability studies (AST) was carried for optimized batch (C1) by exposing it to 40° c/75% RH for one month and analyzed the samples at the interval of 7,14,21,28 days and the samples was analyzed for drug content and invitro dissolution study. The stability studies show drug content after 28 day was 74.14 where percent cumulative drug release 80.55. DISCUSSION The XRD scan of Azithromycin showed intense peaks of crystallinity. Diffractogram of azithromycin showed high intensity peaks between 2θ of 10-200 values demonstrating the crystalline nature of drug. No intense peaks were observed in diffractogram of ethyl cellulose which indicates amorphous nature. Thus, preformulation studies indicated that there is no interaction between Azithromycin and ethyl cellulose. Physical evaluation of microsphere formulation for angle of repose, Compressibility index and Hausner’s ratio showed that they had good flow properties. The microsphere formulated using ethyl acetate as internal organic phase or solvent in ratio1: 1 (C1) showed better encapsulation efficiency than other formulations. All the formulations showed diffusion exponent (n) varying from 0.524 to 0.668. From the n values of all formulations it can be concluded that as the concentration and viscosity of polymer was increased the value of diffusion exponent also increases. From the dissolution profile of formulations C1, C2 and C3, It can be concluded that formulation C1 shows better drug release (80%in 12 hrs) than C2 and C3. Formulations C1 to C3 showed retardation of Azithromycin as concentration of ethyl cellulose increased. Total release in 12 hrs was decreased from 80% to 70%. The IR data of Azithromycin loaded ethyl cellulose microsphere also showed the peaks same as that the peaks in IR spectrum of pure drug, this suggested that there was no chemical interaction between ethyl cellulose and Azithromycin. The DSC results also support IR spectrometry results indicating absence of drug - polymer interactions. The XRD pattern of formulation C1 exhibited halo pattern with less intense and denser peaks compared to plain Azithromycin. This indicated that Azithromycin is dispersed at the molecular level in the blend of polymeric matrix. The SEM photographs showed that the microspheres were spherical in nature and had a smooth surface. SEM photographs revealed the absence of crystals of drug on the surface of microspheres and uniform distribution of the drug within the microspheres.
The stability study conducted for observing effect of temperature on the formulation showed that there is no significant change in drug content and in-vitro drug release of optimized formulation. CONCUSION From the investigation carried out and results obtained, it was observed that the % of drug released from microspheres was sustained. As the GI side effects of Azithromycin are related to its high dose which can be minimized by formulating as microspheres. It would also helps to improve bioavailability because of absorption window and microencapsulation helps to mask the bitter taste of drug. Hence from this investigation, we can propose that the objective of the study is achieved. ACKNOWLEDGEMENT Authors are grateful to Zim Laboratoris Nagpur and Loba chemical, Mumbai, for providing the gift sample of drug and chemicals.
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ABBREVIATION IR:
Infrared spectroscopy DSC: Differential Scanning Calorimetry XRD: X-Ray diffractometry SEM: Surface Morphology UV: Ultraviolet Visible double beam Spectrophotometer K: Rate constants R: Correlation coefficients AST: Accelerated stability studies
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