Gastroretentive Floating Capsules of Risedronate Sodium: Development, Optimization, in vitro and in vivo Evaluation in Healthy Human Volunteers

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1 Bhikshapathi et al: Gastroretentive Floating Capsules of Risedronate Sodium: Evaluation in Healthy Volunteers 2835 International Journal of Pharmaceutical Sciences and Nanotechnology Research Paper Gastroretentive Floating Capsules of Risedronate Sodium: Development, Optimization, in vitro and in vivo Evaluation in Healthy Human Volunteers Darna Bhikshapathi 1 *, Jarathi Arun Kumar 1, Gande Suresh 2, Medipally Viswaja 1 and Bomma Ramesh 2 1 Vijaya college of Pharmacy, Hayathnagar, Hyderabad , Telangana, India. 2 Department of Pharmaceutics, University College of Pharmaceutical Sciences, Kakatiya University, Warangal , Telangana, India. Received January 2, 2015; accepted March 11, 2015 Volume 8 Issue 2 April June 2015 MS ID: IJPSN BHIKSHAPATHI ABSTRACT Background and the purpose of the study: Risedronate sodium inhibits osteoclast bone resorption and modulates bone metabolism. Risedronate has a high affinity for hydroxyapatite crystals in bone and is a potent antiresorptive agent. In the present investigation efforts were made to improve the bioavailability of risedronate sodium by increasing the residence time of the drug through sustained-release matrix capsule formulation via gastroretentive mechanism. Capsules were prepared by wet granulation technique. The influence of gel forming agents, amount of risedronate and total weight of capsules on physical properties, in vitro buoyancy, drug release, FTIR, DSC, X-ray studies were investigated. The release mechanisms were explored and explained by applying zero order, first order, Higuchi and Korsmeyer equations. The selected formulations were subjected to stability study at 40 C/75% RH, 25 C/60% RH for the period of three months. For all formulations, kinetics of drug release from capsules followed Higuchi s square root of time kinetic treatment heralding diffusion as predominant mechanism of drug release. Formulation containing 25 mg HPMC K4M and 75 mg HPMC K100 LV (F-8) showed zero order release profile. There was no significant change in the selected formulation, when subjected to accelerated stability conditions over a period of three months. X-ray imaging in six healthy human volunteers revealed a mean gastric retention period of 5.60 ± 0.77 hrs for the selected formulation. Stable, sustained release effervescent floating capsules of risedronate sodium could be prepared by wet granulation technique. KEYWORDS: Differential Scanning Calorimeter; Risedronate sodium; Gelucire; ; in vivo gastric retention. Introduction Oral route is considered as the most promising route for drug delivery (Santhana Laxmi et al., 2010). Development of oral controlled release systems has been a challenge to formulation scientists because of the difficulty in localizing the system in target areas of the gastrointestinal tract (Harshal et al., 2012). The real challenge in the development of an oral controlledrelease drug delivery system is not just to sustain the drug release but also to prolong the presence of the dosage form within the gastrointestinal tract (GIT) until all the drug is completely released at the desired period of time (Tadros, 2010). One of the novel approaches in the area of oral sustained release drug delivery is gastro retentive drug delivery system (GRDDS) (Sarfaraz et al., 2012). Gastroretentive drug delivery systems (GRDDS) can be retained in the stomach and assist in improving the oral sustained delivery of drugs that have an absorption window in a particular region of the gastrointestinal tract. These systems help in continuously releasing the drug before it reaches the absorption window, thus ensuring optimal bioavailability (Mohan et al., 2011). Gastric retentive dosage forms are designed to be retained in the stomach and prolong the gastric residence time of the drugs. Prolonged gastric retention improves bioavailability, reduces drug waste and improves solubility for drugs that are less soluble in a high ph environment (Sivabalan et al., 2011). These floating delivery systems when reached to stomach, carbon dioxide is liberated by the acidity of gastric contents and is entrapped in the jellified Hydrocolloid (Senthil et al., 2010). Therefore, control of placement of a drug delivery system (DDS) in a specific region of the GI tract offers advantages for a variety of important drugs characterized by a narrow absorption window in the GIT or drugs with a stability problem (Singh and Kim, 2000). The main goal of any drug delivery system is to achieve desired concentration of the drug in blood or tissue, which is therapeutically effective and non toxic for a prolonged period. An appropriately designed sustained or controlled release drug delivery system can be a solution towards solving these problems. Over the last three 2835

2 2836 Int J Pharm Sci Nanotech Vol 8; Issue 2 April June 2015 decades, various attempts have been done to retain the dosage form in the stomach as a way of increasing retention time (Chandrasekhar, 2012). These considerations have led to the development of a unique oral controlled release dosage form with gastroretentive properties. After oral administration, such a drug formulation would be retained in the stomach and release the drug there in a controlled and prolonged manner, so that the drug could be supplied continuously to its absorption sites in the upper gastrointestinal tract (Streubel et al., 2006). Among the various gastro retentive systems, gastric floating drug delivery systems (GFDDS) offer numerous advantages over the gastric retentive systems. (Malay et al., 2012). Bisphophonates, an anti-resorptive agent used in the treatment of Paget s disease and post menopause conditions to decrease calcium degradation in bones. Risedronate sodium, a pyridinyl-bisphosphonate in the form of hemi-pentahydrate with small amounts of monohydrate, inhibits osteoclast bone resorption and modulates bone metabolism. Risedronate has a high affinity for hydroxyapatite crystals in bone and is a potent antiresorptive agent. At the cellular level, risedronate inhibits osteoclasts. The osteoclasts adhere normally to the bone surface, but show evidence of reduced active resorption. The main reason for lack of therapeutic effectiveness is low oral bioavailability (0.63%). The low oral bioavailability of the drug can be attributed to its complexing nature with metal ions and site specific absorption (upper GIT). The plasma half-life of the drug is ~1.5 hr. Absorption after an oral dose is relatively rapid (Tmax ~ 1 hr) and occurs throughout the upper GIT. The site specific absorption (upper GIT) of Risedronate sodium provides a strong rationale for its use as a model drug for gastroretentive dosage form. The principle of buoyancy offers a simple and practical approach to achieve increased residence time in the stomach. The impact of formulation variables on the release rate and release mechanism was also evaluated by the use of mathematical models. Materials and Methods Materials Risedronate sodium (white crystalline powder, freely soluble in water) was a generous gift from Dr. Reddy s Laboratories, Hyderabad, India, Gelucire 50/13, (Waxy solid, M.P.= 50 o C, HLB = 13) was generous gift from Gattefosse, Cedex, France. Hydroxy propyl methyl cellulose (HPMC K4M, 100LV) Polyethylene oxide (POLYOX WSR 303, WSR 301, WSR N60K and WSR N80) were obtained from Colorcon Asia Private Limited (India), Licaps Capsules (Size 3, hard gelatine capsules specially designed for lipid formulations) were obtained as a gift sample from Capsugel, Mumbai, India. All excipients were of USP/NF grades and all other chemicals used were of analytical grades. Methods Drug-excipients interaction study Differential scanning calorimetry The physicochemical compatibilities of the drug and the used excipients were tested by differential scanning calorimetric (DSC) analysis. The DSC thermograms of the pure drug, Gelucire 50/13, drug and gelucire 50/13 mixture, and optimized formulation were derived from a DSC (DSC Q1000 V9.8 Build 296). Accurately weighed (2-4 mg) samples were scanned in the temperature range of o C at constant scanning speed of 5 o C/min in sealed aluminum pans, using nitrogen as purging gas. Fourier Transform Infrared Spectroscopy (FTIR) FTIR spectra for risedronate sodium, gelucire 50/13, drug and gelucire 50/13 mixture and optimized formulation was recorded using a FTIR (Perkin Elmer bx1) samples were prepared using KBr (spectroscopic grade) disks by means of hydraulic pellet press at pressure of seven to ten tons. The samples were scanned from 4000 to 400 cm 1. Preparation of risedronate sodium gastroretentive floating capsules Single unit matrix systems were formulated with help of different low-density polymers, which upon administration would attain a density of less than that of gastric fluids and therefore would float. Initially risedronate sodium and gelucire 50/13 were taken in 10:1 ratio. The gelucire 50/13 was melted in a glass beaker at 60 o C. Risedronate sodium 35 mg was added to the molten mass under stirring to obtain a homogeneous mass. The homogeneous molten mixture carefully triturated with different ratios of low density polymers (Shown in Table 1) in a mortar and pestle. The blend was passed through # 24 mesh to obtain granules. Granules were lubricated with talc and magnesium stearate. The blend equivalent to 35 mg of drug was accurately weighed and manually filled in size 3 empty capsules shells. TABLE 1 Composition (in mg/tablet) of different formulations of risedronate sodium floating capsules. Each formulation includes 0.5% talc and 0.5% magnesium stearate. Formulation Code Drug Gelucire 50/13 WSR303 WSR301 WSR N-60K WSR N80 HPMC K4M HPMC K100 LV F F F F F Lactose Table 1 Contd

3 Bhikshapathi et al: Gastroretentive Floating Capsules of Risedronate Sodium: Evaluation in Healthy Volunteers 2837 Formulation Code Drug Gelucire 50/13 WSR303 WSR301 WSR N-60K WSR N80 HPMC K4M HPMC K100 LV Lactose F F F F F Evaluation of Capsules Average weight of the dosage unit To study weight variation, 10 capsules of each formulation were weighed using an electronic balance (Mettler Toledo, Switzerland). Values are reported in milligrams. Mean and SD value were also calculated. Drug content Capsules were taken and contents were emptied in 100 ml of 0.1 N HCl maintained at 37 o C. The dispersion was agitated for 1 hr on rotary shaker and filtered through whatman filter paper. The filtrate obtained was suitably diluted and analysed for drug content by UV- Visible spectrophotometer (Shimadzu UV-1800) at 260 nm. Determinations were performed in triplicate (n=3). In vitro drug release studies The in vitro drug release study was performed in a 900 ml dissolution medium containing hydrochloric acid (ph of 1.2), maintained at 37 C ± 0.5 C and stirred at 50 rpm using USP dissolution apparatus II. About 5 ml sample was withdrawn through a 0.45 µm filter and replaced with another 5 ml of a suitable fresh dissolution medium at preselected intervals up to 12 h. The amount of the drug was determined by UV- Vis Spectrophotometer at 260 nm. The cumulative percentage drug released was calculated. Kinetic modeling of drug release The dissolution profiles of all the batches were fitted to zero order, first order, Higuchi (Higuchi T, 1961) and Peppas (Siepmann J, Peppas NA, 2001) equation 1-4 respectively. Mt = Mo + Ko t..(i) lnmt = lnmo + K1t..(ii) Mt = Mo KHt 1/2..(iii) Mt/M =Kt n..(iv) In these equations, Mt is the cumulative amount of drug released at any specified time (t), Mo is the dose of the drug incorporated in the delivery system and Mt/Ma is a fraction of drug released at time (t). Ko, K1, KH and K are rate constants for zero order, first order, Higuchi and Korsmeyer model respectively, n is the release exponent. The n value is used to characterize different release mechanisms. If a value of n 0.5, indicates the Fickian release mechanism. The value of n between 0.5 and 1 is an indication of Non-Fickian release mechanism (both diffusion and swelling controlled). When, n 1, it is case-ii transport and this involves polymer dissolution and polymeric chain enlargement or relaxation. Stability studies In this study, one of the optimized formulation F-8 was subjected to 25 C ± 2 C/60 ± 5% RH and 40 C ± 2 C/ 75 ± 5% RH in closed HDPE bottles along with 1 g desiccant and stored in a stability chamber (Newtronic, Mumbai, India) for 3 months. Periodically the capsules were taken out and analyzed for drug content and drug release studies. Abdominal X-ray imaging For in vivo radiographic studies, institutional ethical committee permission was obtained. For the X-ray experiment 35 mg of the drug in formulation F8 was replaced with barium sulfate and its other ingredients were kept constant. This amount was determined experimentally to allow X-ray visibility but not to hinder tablet buoyancy. After overnight fasting, the volunteers were fed with a low calorie food. After 30 min, a barium sulfate-loaded tablet was given to every subject with 200 ml of water. The volunteers were asked to take 200 ml water after every 1 hour. At different time intervals (0.5, 1.5, 4 and 6 hr post-administration of capsule), volunteers were exposed to abdominal X-ray imaging (Genesis 50, Josef Bets chart AG, Brunnen, Switzerland) in standing position. A radiograph was made just before administration of the tablet, at zero time, to ensure the absence of radioopaque material in the stomach. The distance between the source of X-rays and the subject was kept constant for all images. Thus, the observation of the floating tablet movements could be easily observed and the mean gastric retention period could be estimated. Results and Discussion Differential scanning calorimetry The DSC analysis of the Gelucire 50/13 was shown in Fig. 1 and the endothermic peak was obtained at 43 C. The reported Literature value is 48 C. The physical mixture elicited an endothermic peak at 47 C Fig. 2. It was found that the endothermic peaks of physical mixtures as well as Gelucire reflected the characteristic features of risedronate sodium. It indicates that there is no interaction between drug and other excipients used in the formulation. Fourier Transform Infrared Spectroscopy The FT-IR studies revealed that Risedronate sodium is compatible with the excipients used in the formulation. The FT-IR spectrum s of pure drug, Gelucire 50/13, Drug and Gelucire 50/13, HPMC and Optimized Formulation

4 2838 Int J Pharm Sci Nanotech Vol 8; Issue 2 April June 2015 were shown in Fig. 3, 4, 5, 6 and 7 respectively. There were no extra peaks observed in the IR spectrum of the optimized formulation. This established that the drug Risedronate sodium, with all the excipients used in the study showed no interaction and indicated that they were compatible with each other Fig. 1. DSC thermogram of Gelucire 50/13. Fig. 2. DSC thermogram of physical mixture. Fig. 3. FT-IR spectra of Risedronate sodium pure drug.

5 Bhikshapathi et al: Gastroretentive Floating Capsules of Risedronate Sodium: Evaluation in Healthy Volunteers 2839 Fig. 4. FT-IR spectra of Gelucire 50/13. Fig. 5. FT-IR spectra of drug and Gelucire 50/13. Fig. 6. FT-IR spectra of HPMC.

6 2840 Int J Pharm Sci Nanotech Vol 8; Issue 2 April June 2015 Fig. 7. FT-IR spectra of optimized formulation. Physical characterization of Risedronate sodium gastro retentive floating capsules The risedronate sodium granules were prepared and filled in Size 3 capsules and the total weight of the capsule was 140 mg. All the prepared formulations of risedronate sodium were ranged from 137 ± 2.2 to 142 ± 2.4 mg. The drug content of all the formulations ranged in between and (Table 2). TABLE 2 Weight variation and drug content of the prepared hydrophilic capsules, expressed as mean ± SD. Formulation Weight variation (mg) Drug Content (%) F1 137 ± ± 1.58 F2 140 ± ± 2.17 F3 139 ± ± 1.86 F4 140 ± ± 1.45 F5 137 ± ± 2.21 F6 142 ± ± 2.42 F7 140 ± ± 2.22 F8 139 ± ± 1.98 F9 141 ± ± 2.18 F ± ± 1.89 In vitro drug release studies Effect of the polymer grade on the release of risedronate sodium capsules A hydrodynamically balanced system was fabricated using various low density polymers like, HPMC as rate controllers and Gelucire 50/13 as paracellular absorption enhancer. Formulations F1 to F4 prepared with different grades of. The drug release was 77, 92, 98 and 97 % in case of F1, F2, F3 and F4 Fig. 8 and the floating times were 8 hr, 6 hr, 1 hr and 0 h respectively Table 4. Floating time was not desirable hence, release order kinetics were not studied. The floating and release characteristics of the HBS were improved by employing HPMC K4M and HPMC K100LV in various ratios. The formulations F-5, F-6 and F-7 Fig. 9 having HPMC K4M and HPMCK100LV in 3:1 ratio were compared to explore the effect of polymers and amount of the drug release. Percent of the release of drug at the end of 12 hr for F5, 10 hr for F6 and 12 hr for F7 were 47.75%, 56.49% and 77.14% respectively Table 4. These values indicated a decrease of the drug release by increase in HPMC K4M formulation. The optimized formulation (F-8) containing HPMC K4M and HPMCK100LV (17.8:53.5%) polymer showed zero order release and was found to follow predominantly non fickian (anomalous) release coupled diffusion mechanism Table 3. Fig. 8. In-vitro drug release of formulations F1-F4. Fig. 9. In-vitro drug release of formulations F5-F10. TABLE 3 Release order kinetic parameters of risedronate sodium gastro retentive floating capsules. Formula code Zero order First order Higuchi Korsmeyer - Peppas R 2 k R 2 k R 2 k R 2 n F F F F F F

7 Bhikshapathi et al: Gastroretentive Floating Capsules of Risedronate Sodium: Evaluation in Healthy Volunteers 2841 TABLE 4 Floating lag time and release parameters of risedronate sodium gastro retentive floating capsules. Formula code Floating time (Hrs) % Drug Release at the end of 12 Hrs F ± 1.2 F ± 0.64 F ± 1.1 F-4 Not floated ± 0.7 F ± 0.66 F ± 0.96 F ± 1.2 F ± 1.18 F ± 1.32 F ± 2.8 Percent of the drug release at the end of the twelth hour was 98.01% Table 4. R 2 values obtained from zero order equations were (Table 3). The best linearity value found in Higuchi s equation plot were indicating the release of drug from matrix as a square root of time dependent process based on diffusion. The n value was found to be 0.753, indicating non fickian (anomalous) release, coupled diffusion. Formulations containing HPMC EK100LV and HPMC K4M alone were compared to explore the effect of polymers and amount of the drug release. Percent of the release of drug at the end of twelth hour and eighth hour for F-9, and F-10 were 97.8% and 76.1% respectively Table 4. R 2 values obtained from zero order equation for F-9 and F-10 were and respectively Table 3. The best linearity values found in Higuchi s equation plot were and respectively indicating the release of drug from matrix as a square root of time dependent process based on diffusion. The n values for korsmeyer and Peppas equation (F-9 and F-10) were found to be 0.50 and 0.69 respectively, indicating Non-Fickian (anomalous) release, coupled diffusion, and polymer matrix relaxation, 0.5<n<0.89. Thus, it was proposed that these formulations delivered their active compounds by coupled diffusion and erosion. Stability studies of floating capsules of risedronate sodium The stability of optimized formulation (F8) of Risedronate sodium floating capsules was tested for the stability at 25 C/60% RH and 40 C/75% RH for 3 months. The drug release rate of Risedronate sodium Fig. 10 from the capsule showed no significant change during storage for 3 months. Thus, it was concluded that the optimized formulation was stable under these storage conditions for 3 months. Fig. 10. In-vitro drug release of the optimized formulations (F8) after storage for 3 months. Abdominal X-ray imaging The X-ray images was taken at different intervals after administration of the barium sulphate loaded capsules in six healthy human volunteers showed that capsule was more or less at the same position in stomach for the first 3 hrs and moved slightly downwards and remained within the stomach till the end of 6 hrs Fig. 11. The mean gastric retention period was 5.60 ± 0.77 hrs. Fig. 11. Radiographic images showing the presence of a BaSo4 loaded risedronate sodium floating capsules in the stomach at different time intervals.

8 2842 Int J Pharm Sci Nanotech Vol 8; Issue 2 April June 2015 Conclusion Floating capsules of risedronate sodium were prepared by using gelucire 50/13, different grades of WSR, HPMCK4M and HPMC K100LV by wet granulation technique. The prepared capsules were evaluated for their physical characters and found to be within the limits. FTIR and DSC studies revealed that no interaction was observed between drug excipients used in the study. Formulation F8 considered optimized based on physical characters and in vitro drug release and followed the Peppas model with Non-Fickian mechanism. One of the optimized formulations (F8) was subjected for stability studies at 25 C/60% RH and 40 C/75%RH for 3 months and was found to be stable for at least 3 months under these conditions. Further, the optimized formulation F8 subjected for radiological studies and gastric retention time was found to be 5.60 ± 0.77 hr (n = 6). Acknowledgements The authors wish to thank Dr. Reddy s Laboratories, Hyderabad, India, for providing gift sample of risedronate sodium. References Chandrasekhar BB (2012). Floating Systems for Oral Controlled Release Drug Delivery. Int J App Pharm. 4(2):1-13. Harshal PG, Manohar VP, Bharat WT, Vinod MT, Patil VR (2012). Formulation and in-vitro Evaluation of Trifluoperazine Hydrochloride Bilayer Floating Tablet. Int J Pharm Bio Sci. 2(1): Higuchi T (1961). Rate of Release of Medicaments from Ointment Bases Containing Drugs in Suspensions. J Pharm Sci. 50(10): Malay RP, Amit AP, Laxman MP and Natvarlal MP (2012). Intragastric Floating Drug Delivery System of Metformin Hydrochloride as Sustained Release Component and Glimepiride as Immediate Release Component: Formulation and Evaluation. Int J Pharm Sci. 4(3): Mohan G, Rames h B and Kishan V (2011). Development of Sustained Release Floating Drug Delivery System for Norfloxacin: In vitro and In vivo Evaluation. PDA J Pharm Sci Tech. 65(3): Santhana laxmi G, Elango K, Ramesh Kumar K, Farheen F (2012). Formulation and Evaluation of Bilayer Floating Tablets of Trimetazidine Hcl and Metoprolol Succinate. Ind J Pharm Edu Res. 46 (3): Sarfaraz Md, Keerthi CRP, Udupi RH and Doddayya H (2012). Formulation and In-Vitro Evaluation of Bilayer Floating Tablets of Tramadol Hcl. Int J Drug Dev Res. 4(3): Senthil A, Suresh Kumar P, Raju CH and Mohideen S (2010). Formulation and Evaluation of Gastric Oral Floating Tablet of Glipizide. Int J Bio Pharm Res. 1(2): Singh BN and Kim KH (2000). Floating Drug Delivery Systems an Approach to Oral Controlled Drug Delivery via Gastric Retention. J Control Rel. 63(3): Siepmann J, Peppas NA (2001). Modelling of Drug Release from Delivery Systems based on Hydroxypropyl Methylcellulose. Adv Drug Del Res. 48(2001): Sivabalan M, Vani T, Phaneendhar R, Anup J and Nigila G(2011). Formulation and Evaluation of Gastroretentive Glipizide Floating Tablets. Int J Compreh Pharm. 2(1): 1-4. Streubel A, Siepmann J and Bodmeier R(2006). Gastroretentive Drug Delivery System. Expert Opin Drug Deliv. 3(2): Tadros MI(2010). Controlled-release Effervescent Floating Matrix Tablets of Ciprofloxacin Hydrochloride: Development, Optimization and In Vitro In Vivo Evaluation in Healthy Human Volunteers. Eur J Pharm Biopharm. 74: Address correspondence to: Dr. Darna Bhikshapathi, Head, Dept. of Pharmaceutics, Vijaya College of Pharmacy, Hayathnagar, Hyderabad , Telangana, India. Tel: ; dbpathi@yahoo.com