PREPARATION, CHARACTERIZATION AND EVALUATION OF RANITIDINE MUCOADHESIVE MICROSPHERES FOR PROLONGED GASTRIC RETENTION

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1 Page1269 Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: Journal home page: INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH PREPARATION, CHARACTERIZATION AND EVALUATION OF RANITIDINE MUCOADHESIVE MICROSPHERES FOR PROLONGED GASTRIC RETENTION Rajesh K S*, Srilakshmi N, Das Arun Kumar, Pavani A, Raja Reddy R Department of Pharmaceutics, C M College of Pharmacy, Jawaharlal Nehru Technological University, Hyderabad. ARTICLE INFO Article history Received 08/11/2013 Available online 30/12/2013 Keywords H 2 -receptor antagonist, Sodium Alginate; Carbopol, HPMC, CMC, Gastric retention. Corresponding author Rajesh K.S Department of Pharmaceutics, C M College of Pharmacy, Jawaharlal Nehru Technological University, Hyderabad. rajesh_kadavath@yahoo.com Mobile: ABSTRACT The oral route of drug administration constitutes the most convenient and preferred means of drug delivery to systemic circulation of the body. Various conventional dosage forms have been employed for this purpose but they lack to provide the desired bioavailability of the drug due to poor residence time and faster clearance resulting in decreased bioavailability. Formulating multiparticulate system with mucoadhesive polymers may provide a significant increase in the residence time. Ranitidine hydrochloride is a H 2 -receptor antagonist which inhibits basal and stimulated secretion of gastric acid, reducing both volume and acid and pepsin content of the secretion. It is generally marketed as dosage forms including tablets, capsules, syrup and injections wherein the bioavailability is less. The aim of the present study was to overcome the drawbacks of the conventional dosage forms by formulating mucoadhesive microspheres of Ranitidine hydrochloride with Sodium alginate and copolymers such as Carbopol, CMC and HPMC using ionic gelation method, and to determine its effect on gastric retention. The prepared microspheres were characterized for particle size, surface morphology, degree of swelling, encapsulation efficiency, In-vitro mucoadhesion, drug release and stability studies. Formulations F-VII displayed the best results with Entrapment efficiency 74.6, Mucoadhesion 82 % at the end of first hour and Drug release at the end of 8hr85.7. In-vitro studies concluded that Carbopol and HPMC based microspheres were better than Carbopol and CMC based microspheres for the delivery of Ranitidine. Stability studies indicated that the formulation was stable at ±2 C. Please cite this article in press as Rajesh K.S, et al. Preparation, characterization and evaluation of ranitidine mucoadhesive microspheres for prolonged gastric retention. Indo American Journal Of Pharm Research.2013:3(12). Copy right 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page1270 INTRODUCTION Oral administration of most of the drugs in conventional dosage forms has short-term limitations due to their inability to restrain and localize the system in gastro-intestinal tract. Incomplete release of the drug and a shorter residence time of dosage forms in the upper gastro-intestinal tract, a prominent site for absorption of many drugs will lead to lower bioavailability. A well designed controlled drug delivery system can overcome some of the problems of conventional therapy and enhance the therapeutic efficacy of a given drug [1]. The aim of the present study was to overcome the drawbacks of the conventional dosage forms by formulating mucoadhesive microspheres of Ranitidine hydrochloride using ionic gelation method and to determine the effect on gastric retention when mucoadhesive polymers are used in combination. Ranitidine hydrochloride is indicated for duodenal ulcer associated with Helicobacter pylori infection, post-operative ulcer, oesophageal reflux disease including long-term management of healed oesophagitis, symptomatic relief in gastro-oesophageal reflux disease and Zollinger-Ellison Syndrome. Half life of the drug is 2-3 hours. The principal route of excretion is urine, with approximately 30-70% of the orally administered dose collected in the urine as unchanged drug in 24 hours [2]. Ranitidine, an H 2 antagonist which unlike other drugs has no anti adrenergic action, does not increase prolactin secretion or spare estradiol from hepatic metabolism, thus there is no effect on male sexual function or gynaecomastia. It has lower permeability to brain thus having lesser propensity to cause CNS side effects. It does not significantly inhibit the metabolism of other drugs. Overall incidence of side effects including headache, diarrhoea/constipation, and dizziness is lower [3]. MATERIALS AND METHODS Materials: Ranitidine hydrochloride, obtained as a gift sample from Chandra Labs (Hyderabad), was employed as a model drug. Sodium Alginate Extra pure was obtained from Finar Chem Ltd, Ahmedabad. Mucoadhesive polymers Carbopol, HPMC and CMC were procured from Essel Fine Chem, Mumbai. All the other chemicals used were of analytical grade. Method: Preparation of mucoadhesive microspheres by Ionic Gelation Technique Ranitidine microspheres were formulated by using Ionic Gelation technique using Sodium Alginate as a matrix polymer. Sodium Alginate was dissolved in of water and the mucoadhesive polymer and drug was dispersed with the polymer solution. The dispersed content was placed drop wise in 100 of 10% Calcium chloride solution maintained at ±5 o C while stirring at 100 rpm. Stirring at room temperature was continued for 1-2 hrs to produce spherical microspheres. The microspheres so obtained were retained for at least 1 hour in the calcium chloride solution so as to provide stability and rigidity to the microspheres. The microspheres were then separated from calcium chloride solution by filtration through Whatmann filter paper No-44, collected and washed thrice with distilled water and dried using vacuum filter. The product was then dried in hot air oven at ±2 0 C and finally airdried for 24 hours to obtain microspheres. Table1: Formulation Table for Ranitidine Mucoadhesive Microspheres Ingredient F-I F-II F-III F-IV F-V F-VI F-VII F-VIII F-IX F-X F-XI Ranitidine Hydrochloride Sodium Alginate Carbopol HPMC CMC Distilled Water Eleven batches of microspheres were prepared by taking different drug: polymer ratios with same amount of drug and four different polymers. The formulation batches were designated as F- I, F-II, F-III, F-IV, F-V, F-VI, F-VII, F-VIII, F-IX, F-X and F-XI. Drug and polymer in different proportions were weighed and co dissolved at room temperature into water with vigorous agitation to form uniform drug polymer dispersion. Yield of microspheres: The prepared microspheres were collected and weighed. The measured weight was divided by the total amount of all nonvolatile compounds (solid material) which were used for preparation of microspheres. Weight of microspheres % Yield = X 100 Weight of solid starting material

3 Page1271 Micromeritic properties [4]: Microspheres were characterized for micromeritic properties, such as particle size, true density, tapped density, compressibility index, Hausner s ratio and flow properties. From bulk density and tapped density compressibility index was determined using the following formula. Carr s Index (CI): Carr s index is measured using the values of bulk density and tapped density. The following equation is used to find the Carr s index (TD-BD) CI = x 100 TD Where TD = Tapped density BD = Bulk density Angle of repose: The manner in which stresses are transmitted through a bead and the beads response to applied stress are reflected in the various angles of friction and response. The method used to find the angle of repose is to pour the microspheres through a conical funnel on a level, flat surface and measure diameter of the cone. tanθ = h/r Where, h= height of the heap r= Radius of the heap Particle size analysis: Particle size of different batches of microspheres was determined by optical microscopy. The projected diameter of microspheres from each batch was determined using stage micrometer equipped with optical microscope. The instrument was calibrated and found that 1unit of eyepiece micrometer was equal to 12.5μm. Nearly about 100 Micro particles sizes were calculated under 10x magnifications. Analysis was carried out by observing the slide containing microspheres under the microscope. The average particle size of the microspheres was expressed as diameter. The average particle size was determined by using the Edmondson s equation: nd D mean = n Where,n Number of microspheres observed d Mean size range in µm. Swelling index [5]: This technique was used for characterization of mucoadhesive microspheres. Microspheres (50) were placed in a china dish containing 10mL of distilled water and microspheres were allowed to swell at 37 0 C by placing them in a stability chamber for 3 hrs. Changes in weight variation between initial weight of microspheres and weight due to swelling were measured by taking weight periodically after blotting with filter paper. The swelling index of the microsphere is calculated by using the formula:- Mass of swollen microspheres - Mass of dry microspheres Swelling index = 100 Mass of dry microspheres In-vitro mucoadhesion test (Wash-off test) [6]: The mucoadhesive property of the microspheres prepared using different polymers were evaluated by an in vitro mucoadhesion testing method known as the wash-off method. A goat stomach mucosa (2 2cm) is tied onto the glass slide using a thread. In this method 50 microspheres are spread onto wet rinsed tissue specimen previously soaked in saline solution. The prepared slide is hung onto one of the grooves of a USP II tablet disintegrating test apparatus. The disintegrating test apparatus is switched on and the tissue specimen is given up and down movements for 8 hrs in the beaker of the disintegration test apparatus, which contains the stimulated gastric fluid (ph 1.2). The microspheres remaining at the surface of gastric mucosa are then counted after every 1 hr, and the percentage of the remaining microspheres is calculated. The percentage mucoadhesion is calculated by the following formula: Number of adhered microsphere Percent mucoadhesion = 100 Number of applied microspheres

4 Page1272 Fig: 1 Wash-off test for Mucoadhesive Microspheres Determination of Entrapment Efficiency [7, 8]: To determine the incorporation efficiency accurately, 100 of microspheres were taken, crushed thoroughly, triturated and suspended in a 10 of methanol for 12 hrs. The suspension was finally sonicated for 2 hrs and filtered to separate shell fragments. Drug contents were suitably diluted before analyzed against blank spectrophotometrically at 315 nm. Drug entrapment efficiency for each batch was calculated in terms of percentage drug entrapment (PDE) as per the following formula. Practical yield PDE = X 100 Theoretical Drug Loading In vitro drug release studies [9, 10]: The release rate of ranitidine from mucoadhesive microspheres was determined in a United States Pharmacopoeia (USP) XXIV basket type dissolution apparatus. Drug loaded microspheres (weight equivalent to of drug) were introduced into the 900 of 0.1 M HCl. The dissolution fluid was maintained at 37 ± 2 O C at a rotation speed of 100 rpm. Perfect sink conditions prevailed during the drug release study. Five milliliters samples were withdrawn at regular intervals for 12hrs and analyzed spectrophotometrically at 315nm. The initial volume of dissolution fluid was maintained by adding 5 of fresh dissolution fluid after each withdrawal. Compatibility Studies: In order to check the compatibility of drug in the formulation, FTIR spectra of the formulations along with the drug and other excipients were compared using Shimadzu FTIR 8400 spectrophotometer. In the present study, IR absorption spectra of the pure drug and the final formulation were taken in the range of cm -1 using KBr disc method, 1-2 of the substance to be examined was triturated with -400 (specified quantity) of finely powered and dried potassium bromide. Surface Morphology: Scanning electron microscopy (SEM) was performed to characterize the surface of formed microspheres. Microspheres were mounted directly onto the sample stub and coated with gold film under reduced pressure. This film acts as a conducting medium on which a stream of electron was allowed to flow and then photograph was taken with SEM. RESULTS Spectroscopic Studies A solution of 10µg/ of Ranitidine Hydrochloride was scanned in the range of 200 to 400nm. The drug exhibited λmax at 315nm in simulated gastric fluid (0.1N HCl) ph 1.2 and had good reproducibility. The absorbance of stock solution containing 10, 20, 40, 60, 80 and 100µg/ was determined at 315nm. Correlation between the concentration and absorbance i.e., regression factor was found to be near to 0.996, with a slope of and intercept of

5 Page1273 Average Particle Size in µm Fig 2: Standard graph of Ranitidine in simulated gastric fluid ph 1.2 Percentage recovery yield The percentage practical yields slightly decreased as the carbopol concentration in the formulation increased. The maximum yield was found to be 90.55% for F-III. The percentage yield of each formulation is given in Table-2 Table 2: % Yield of Microspheres S. No Formulation Wt. Obtained % Yield 1 I II III IV V VI VII VIII IX X XI Particle Size Analysis The mean particle size increased with increasing polymer concentration which is due to a significant increase in the viscosity, thus leading to an increased droplet size and finally a higher microsphere size. Microspheres prepared from sodium alginate (F-I, II and III) exhibited a size range of µm Microspheres prepared from sodium alginate along with three different copolymers (F- IV, V and VI) exhibited a size range of µm, microspheres prepared from sodium alginate along with carbopol and HPMC as copolymers (F-VII, VIII and IX) exhibited a size range between µm and microspheres prepared from sodium alginate along with carbopol and CMC as copolymer (F-X and XI) exhibited a size range of µm.The effect of polymer concentration on average particle size is shown in figure F-I F-II F-III F-IV F-V F-VI F-VII F-VIII F-IX F-X F-XI Fig 3: Plot of Average particle size and Formulation

6 Page1274 Micromeritic Properties The values of angle of repose were found in the range of o to 30 o which are within the normal acceptable range of 20 o to 40 o. The microspheres showed reasonably good flow potential. This is further substantiated by the value of Compressibility Index which was in the range 11.8 to 15.7, indicating good flow characteristics of the prepared microspheres. This also implies that the microspheres are non-aggregated. The improved micrometric properties of the prepared microspheres when compared to that pure drug alone, suggest that they can be easily handled and filled into a capsule. Swelling Study The swelling ratio is expressed as the percentage of water in the hydrogel at any instant during swelling. Swellability is an important characteristic as it affects mucoadhesion as well as drug release profiles of polymeric drug delivery systems. Swellability is an indicative parameter for rapid availability of drug solution for diffusion with greater flux. Swellability data revealed that amount of polymer plays an important role in solvent transfer. It can be concluded from the data shown in Table 4 that with an increase in mucoadhesive polymer concentration, the percentage of swelling also increases. Thus we can say that amount of polymer directly affects the swelling ratio. As the polymer to drug ratio increased, the percentage of swelling increased. The percentage swelling of the prepared microspheres of formulation F-I to F-XI is displayed in figure 4. Formulation Number Pure Drug Bulk density (gm/) Table 3: Micromeritic property of prepared formulation Tapped Density (gm/) Carr`s Index Hausner s Ratio Angle of repose ( o ) Poor Type of Flow Ranitidine F-I Excellent F-II Good F-III Good F-IV Good F-V Good F-VI Good F-VII Good F-VIII Good F-IX Good F-X Good F-XI Good Table 4: Percentage swelling of the prepared microspheres Time in Hrs Degree of Swelling F-I F-II F-III F-IV F-V F-VI F-VII F-VIII F-IX F-X F-XI

7 Page1275 Fig 4: Swelling Index profile of Formulations F-I to F-XI In-Vitro Mucoadhesion test The percentage mucoadhesion increased with the increase in concentration of mucoadhesive polymer. It was found that Sodium alginate microspheres (F-I - F-III) showed less mucoadhesion property when compared to microspheres formulated using mucoadhesive polymers. Higher mucoadhesion of carbopol microspheres may be attributed to the higher molecular weight and viscosity of carbopol than the two polymers. Furthermore, HPMC being non-ionic possesses low hydrogen bonding capability with mucus membrane. Fig 5: Effect of polymer on mucoadhesion (F-I to F-XI) Entrapment Efficiency The values for entrapment efficiency are shown in Table 5. From the figures, it is clear that as the concentration of mucoadhesive polymer increases, the entrapment efficiency increases. However, it was noted that carbopol had higher effect on entrapment efficiency than HPMC and CMC.

8 Page1276 Table 5: Mucoadhesion and Entrapment Efficiency of the prepared microspheres Sr No. Formulation Code Entrapment Efficiency 1. F-I F-II F-III F-IV F-V F-VI F-VII F-VIII F-IX F-X F-XI 54.3 This may be attributed to higher molecular weight of carbopol than the two polymers. Increase in the molecular weight of the mucoadhesive polymer increases the encapsulation efficiency of the microspheres due to formation of more intact matrix network. In-Vitro Drug Release Studies Dissolution studies of all the formulations were carried out using dissolution apparatus USP XXIV basket. The dissolution studies were conducted by using 0.1N HCl as dissolution medium, ph1.2. The Cummulative percentage drug release vs time graph is given below. Table 6: In-Vitro release profile of Ranitidine Mucoadhesive microspheres % Cummulative Drug Release Time in Hrs F-I F-II F-III F-IV F-V F-VI F-VII F-VIII F-IX F-X F-XI Fig 6: Comparison of In Vitro release profile of Ranitidine Mucoadhesive microspheres In-Vitro Drug Release Kinetics In order to understand the mechanism and kinetic of drug release and release rate kinetics of the drug from dosage form, the in-vitro drug dissolution data obtained was fitted to various mathematical models. The coefficient of determination (R) was used as an indicator of the best fitting for each of the models considered. The mechanism of release for the optimized formulations was determined by finding the R 2 value for each kinetic model viz Higuchi, Korsmeyer-Peppas and Hixson-Crowell corresponding to the release data of formulations.

9 Page1277 Table 7: Model fitting for release profiles Higuchi Hixson- Formulation Korsmeyer - Peppas Matrix Crowell Code R 2 R 2 n R 2 Best Fit Model F-I Hixson-Crowell F-II Peppas F-III Peppas F-IV Peppas F-V Peppas F-VI Peppas F-VII Peppas F-VIII Peppas F-IX Peppas F-X Peppas F-XI Peppas For most of the formulations the R 2 value of Korsmeyer-Peppas and Hixson-Crowellmodel is very near to 1. The values are compiled in Table 7. The kinetic data analysis of F-I showed that the best suited model was Hixson-Crowellwhereas for the remaining formulations (F-II to F-XI) the best suited model was Korsmeyer-Peppas (R 2 = to 0.999) whereas release exponent value (n) ranged from to From the coefficient of determination and release exponent values, it can be suggested that the mechanism of drug release follows Korsmeyer-Peppas model along with non-fickian diffusion mechanism, leading to the conclusion that release mechanism of drug followed combination of diffusion and erosion. CHARACTERIZATION OF MICROSPHERES Compatibility Studies Drug polymer compatibility studies were carried out using Fourier Transform Infra Red spectroscopy to establish any possible interaction of Model drug with the polymers used in the formulation. The FTIR spectra of Ranitidine showed characteristic absorption band at cm -1 which represents the presence of N-H stretching of secondary amines. The weak absorption band at cm -1 represents the presence of furan ring. Ranitidine showed the characteristic Nitro band at cm -1, the absorption band at cm -1 represents C-H band. The FT-IR spectrum of the optimized formulation was compared with the FT-IR spectra of the pure drug. The results indicated that the Nitro band showed sharp shift in the peak which may be due to reaction with polymers, other characteristic absorption peaks of the pure model drug have appeared in the formulated microspheres, without any significant change in their position after successful encapsulation, indicating no chemical interaction between model drug and polymers used. Fig 7: FTIR Spectra of Pure Drug

10 Page1278 Fig 8: FTIR Optimized Formulation (F-VII) Table 8: Comparison of Wave number of pure drug and Optimized Formulation Wave Number Wave Number (cm -1 ) in S No. (cm -1 ) in Pure Optimized Formulation(F- Frequency Assignment Drug VII) N-H stretching of Secondary amine Presence of Furan ring Nitro band C-H band Surface Morphology: Scanning electron microscopy images of F-II and F-VII are as shown in Figure 4 and 5 which indicates that the microspheres of F-II were discrete, uniform and spherical with regular surface with lesser stability. Scanning Electron microscopy images of the optimized formulation (F-VII) indicates that the microspheres were smooth, stable, uniform and spherical with smooth surface. Fig 9: SEM images of Sodium Alginate Beads (F-II)

11 Page1279 Fig 10: SEM images of Optimized Formulation (F-VII) Stability Studies of Ranitidine Hydrochloride Microspheres: The optimized formulation (F-VII) of Ranitidine Hydrochloride microspheres was then filled in empty capsule away from light and subjected to short-term stability testing by storing them at two different temperatures and relative humidity in stability chamber. ±2 C temperature and 65±5% RH 40±2 C temperature and 65±5% RH The formulations were then studied for the percent drug content at regular interval of time. Table 9: Stability studies of Ranitidine Hydrochloride microspheres at ±2 C temperature and 65±5% RH Time Colour Percent drug content First day Yellow days Yellow days Yellow days Yellow 73.8 Table 10: Stability studies of Ranitidine Hydrochloride microspheres at 37±2 C temperature and 65±5% RH Time Colour Percent drug content First day Yellow days Yellow days Yellow days Yellow 59.9 CONCLUSION The present study has been a satisfactory attempt to formulate Mucoadhesive Microspheres of Ranitidine Hydrochloride with a view of improving its oral bioavailability and giving a sustained release of the drug. From all the parameters studied, it can be concluded that the formulation containing Sodium alginate, Carbopol and HPMC in 1:0.75:0. (Formulation-VII) is the best formulation for prolonged drug release. Swelling and Mucoadhesive property may be considered important to optimize a formulation. Formulation-VII showed better swelling characteristics associated with better mucoadhesive characteristics when compared to remaining formulations. In the FTIR spectra of the optimized formulation the Nitro band showed sharp shift in the peak which may be due to reaction with polymers, other characteristic absorption peaks of the pure model drug have appeared in the formulated microspheres, without any significant change in their position indicating that the excipients used were compatible. The formulation showed sustained release up to 12 hrs. At the end of 12hr the formulation showed cumulative drug release of 85.7%. Fitting the dissolution values into kinetic models indicated that the formulation followed Korsmeyer Peppas model. From the coefficient of determination and release exponent values, it can be suggested that the mechanism of drug release follows Korsmeyer-Peppas model along with non-fickian diffusion mechanism leading to the conclusion that a release mechanism of drug followed combination of diffusion and spheres erosion Thus, the formulated mucoadhesive microspheres seem to be a potential candidate for gastro retentive sustained drug delivery of Ranitidine Hydrochloride. Further detailed stability studies and in vivo bioavailability studies are to be done to establish

12 Page1280 the efficacy of these formulations. In vitro in vivo correlation study is to be done to establish the guarantee of efficacy and bioavailability of the formulation. DISCUSSION The present novel mucoadhesive microsphere approach for Ranitidine HCl can be used for prolonged gastric retention thus reducing the frequency of administration. This approach can be extended to various drugs used in clinical practice so as to improve bioavailability of poorly absorbed drugs in GI. ACKNOWLEDGEMENT With a deep sense of appreciates, we acknowledge C M College of Pharmacy, Hyderabad, for providing unconditional and necessary support for completing this work. We also like to thank Chandra Labs, Hyderabad for providing the drug as gift sample and conducting FTIR and SEM studies. It was an inspirational practice to work under your Institute. The authors also acknowledge the contribution of Prof. Analía Odetto, from IES Tafí del Valle Anexo Amaicha, Argentina, for final editing of the English version. REFERENCES: 1. Jain NK, Controlled and Novel drug delivery,04 Edition,New Delhi, CBS Publisher, Pg: Brogden RN, Carmine AA, Heel RC, Speight TM, Avery GS Ranitidine: a review of its pharmacology and therapeutic use in peptic ulcer disease and other allied diseases. Pg: Tripathi KD, Essentials of Medical Pharmacology, 6th edition, New Delhi, Jaypee Brothers Medical Publishers (P) Ltd, Pg: Martin A., Micrometrics in Physical Pharmacy. 4th edition. Lea Febiger, Philadelphia Moy AC, Mathew ST, Mathapan R, Prasanth VV. Microsphere-An Overview. International Journal of Pharmaceutical and Biomedical Sciences 2011; 2(2): Shiva Kumar Yellanki et al., Design and Characterization of Amoxicillin trihydrate Mucoadhesive Microspheres for Prolonged Gastric retention, International Journal of Pharmaceutical Sciences and Drug Research 2010; 2(2): Patil HS, Patil MP, Tekade BW Patil VR, Formulation and In-vitro evaluation of floating microspheres of acyclovir, Arch Pharm Sci and Res 2009; 1 (1): Deepaa M.K., Karthikeyanb M.,Cefpodoxime Proxetil Floating Microspheres: Formulation and In Vitro Evaluation Iranian J. Pharm. Sci. 5, 69 (2009). 9. The United States Pharmacopoeia XXIV, United States Pharmacopoeial Convention, Rockville, (2000). 10. Singh N., Kim K.H.Floating drug delivery systems: an approach to oral controlled drug delivery via gastric retention J. Control Feb 3; 63(3): Submit your next manuscript to IAJPR and take advantage of: Access Online first Double blind peer review policy No space constraints Rapid publication International recognition Submit your manuscript at: editorinchief@iajpr.com