Formulation and evaluation of inlay tablet of metformin hydrochloride as sustained release and pioglitazone with glibenclamide as immediate release

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1 Research Article ISSN: Available online through Formulation and evaluation of inlay tablet of metformin hydrochloride as sustained release and pioglitazone with glibenclamide as immediate release Palanisamy. P* 1, R. Margret Chandira 1, B. Jaykar 2, A.Pasupathi 1, B. S. Venkateshwarlu 1, M. Kumar 2, M. V. Kumudhavalli 3 1 Department of Pharmaceutics, 2 Department of Pharmaceutical Chemistry, 3 Department of Pharmacutical Analysis, Vinayaka Mission s College of Pharmacy,Vinayaka Missions University, Yercaud Main Road, Kondappanaickenpatty, Salem (D.T) ,Tamilnadu,India Received on: ; Revised on: ; Accepted on: ABSTRACT The present research endeavor is directed towards the development of once daily sustained release matrix tablet of metformin hydrochloride with inlay tablet of pioglitazone glibenclamide as immediate release. Combination therapy concluded that Pioglitazone is effective in improving the glycemic control when added to a combination of glibenclamide and metformin in type 2 DM. Matrix system was based on swellable polymer were selected for sustained the drug release. Different polymers Viz., HPMC, Xanthan Gum, Guar gum, to get the desired release profile over a period for 12 hours. Different batches of both Immediate release(ir) and Sustained release (SR) were prepared by Direct compression and Wet granulation method respectively. In the immediate release layer formulation, disintegration such as sodium starch glycolate at three different concentration (5%, 7.5% and 10% w/w) with 5% concentration crosspovidone of tablet weight were tried to get a desired release profile within 30 minutes. The in-vitro dissolution studies were performed for all the IR formulations. Formulation IR1 to IR3 released of pioglitazone and glibenclamide respectively at the end of 30 minutes with SSG concentration of 10%. So IR3 were selected for final formulation. SR formulations F3 showed release profile were complies with USP at 40% concentration of HPMC-K100M with respect to drug compared with another 11 formulations. Inlay tablet formulation were prepared using Optimum formulation of sustained granules and immediate release granules.the tablets assay, weight variation, hardness, thickness friability, disintegration time and in-vitro dissolution were found to be within the official limits. The dissolution data of the optimized batch was subjected to study the in-vitro release kinetics. The result showed that the IR layer of inlay tablet formulation followed the first order release kinetics and the drug release kinetics of SR layer of Inlay tablet formulation correspond best to Higuchi s model and drug release mechanism as per n value of Korsmeyer&Peppas Model appeared to be a complex mechanism of swelling, diffusion and erosion with zero order release kinetics. KEY WORDS: Metformin hydrochloride, pioglitazone, glibenclamide and triple combination tablet. INTRODUCTION: The convenient oral drug delivery has been known for decades is the most widely utilized route of administration among all the routes. It remains the preferred route of administration in the discovery and development of new drug candidates. The popularity of oral route is attributed to patient acceptance, ease of administration, accurate dosing, cost effective manufacturing methods and generally improve the shelf life of the product [1]. *Corresponding author. Mr. Palanisamy.p*.,M.Pharm, Department of Pharmaceutics, Vinayaka Mission s College of Pharmacy, Yercaud Main Road, Kondappanaickenpatty, Salem (D.T),Tamil Nadu (State), Pin. Code: India Immediate release tablets are designed to disintegrate and release the drug in absence of any controlling features such as coating or other formulation techniques. Despite a rising interest in controlled-release drug delivery systems, the most common tablets are those intended to be swallowed whole, disintegrating and releasing their medicaments rapidly in the gastrointestinal tract.the potential benefits that a sustained release system may bring to us can be appreciated by a consideration of prolonged and efficient delivery of therapeutically effective dosages, patient compliance and localization of the therapy.the bioavailability of drug molecules to the ailing tissue cells is governed by a sequence of pharmacokinetics processes-release, absorption, distribution, metabolism and elimination. In some cases, these processes result in the inefficient bioavailability of the drug to the target tissue cells. The bioavailability to a target tissue can be maximized and the adverse side effects in non-target tissue can be minimized by applying the principles of sustained release system [2-4]. Diabetes is a chronic disease marked by high levels of sugar in the blood. Diabetes mellitus often referred to simple as diabetes, which roughly translates to excessive sweet urine. It is a noninfectious disease. The body systems affected by diabetes mellitus are the nervous, digestive, circulatory, endocrine and urinary systems, but all body system are in some way affect. The formulation once daily

2 sustained release (SR) Matrix tablet of Metformin Hydrochloride with Inlay tablet of Pioglitazone Hydrochloride with Glibenclamide as Immediate release (IR). To obtain the beneficial anti-diabetic effect of triple drug combination on a single tablet with dual release mechanism.to improve the patient compliance by giving the triple combination as a single dosage form. To reduce the dosing frequency, the patient already receiving the Glibenclamide 5mg, Metformin 500mg that two times a day to once daily dosage form.to provide effective, Safe and stable pharmaceutical oral formulation containing both immediate release and sustained release of three anti-diabetic drugs with different mechanism of action to improve glycemic control (5). PREFORMULATION STUDIES OF PURE DRUG AND EXCIPIENTS [6-7] : Preformulation study relates to pharmaceutical and analytical investigation carried out proceeding and supporting formulation development efforts of the dosage form of the drug substance. Preformulation yields basic knowledge necessary to develop suitable formulation for the toxicological use. It gives information needed to define the nature of the drug substance and provide frame work for the drug combination with pharmaceutical recipients in the dosage form. Hence, the following Preformulation studies were performed on the obtained sample of drug. The results are shown in Table. No: 4 & 7. COMPATIBILITY STUDIES [8] : The Active ingredients and excipients were mixed and taken in 2 ml glass vials and sealed. These glass vials are kept at Room Temperature and 40 C / 75% RH for about 1 month. At the interval of 10 days, the samples were withdrawn and analyzed for color change. MATERIALS AND METHODS: Metformin hydrochloride was procured by Alekya Chemicals (Mumbai, India); Glibenclamide and Pioglitazone Hydrochloride was procured by Sri Krishnan Drug Ltd (Bangalore, India); HPMC K 100M, Xantham Gum and Guar Gum was gifted by Laffans Petrochem (Bangalore, India); Povidone K30, Sodium Starch Glycolate, Sodium Lauryl Sulphate, Magnesium stearate, Crosspovidone, Colloidal silicon dioxide, Talc and MCCP ph-102 was gifted by Amishi Drugs (Bangalore, India). SPECTRAL IDENTIFICATION [9] : Excipients are integral components of almost all pharmaceutical dosage forms. The successful formulation of a stable and effective solid dosage form depends on the careful selection of the excipients, which are added to facilitate administration, to promote the consistent release and bioavailability of the drug and protect it from degradation. Infra red spectroscopy is one of the most powerful analytical techniques to identify functional groups of a drug. In the present study, the potassium bromide disc (pellet) method was employed. Chemical stability was confirmed by IR spectrometry. The results are shown in Figure. No: 4-14 FORMULATION DEVELOPMENT Table. No: 1. Formulation of SR Matrix metformin Hydrochloride Tablet Batch. No: F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 Ingredients (mg / tablet) Metformin HcL HPMC K100M Xanthan Gum Guar Gum PVP k MCCP Ph Crosspovidone Magnesium stearate Talc Aerosil Total Weight Table. No:2 Formulation of IR Pioglitazone with Glibenclamide Batch. No: IR1 IR2 IR3 Ingredients (mg / tablet) Glibenclamide Pioglitazone HcL Sodium Starch Glycolate Crosspovidone Sodium Lauryl Sulphate Crosspovidone MCCP ph Talc Magnesium Stearate Total Weight Formulation of the Inlay Tablet: Final Inlay tablets were prepared using optimized sustained formulations of Metformin HCL and immediate release Pioglitazone with Glibenclamide. Initially the granules of metformin was filled in the tablet die optimized tablets of the IR are being placed on the metformin granules and it is compressed as an inlay tablet. POST COMPRESSION PARAMETERS [10-13] : a) Weight Variation Test: Twenty tablets were selected randomly from each batch and weighed

3 individually to check for weight variation. A little variation was allowed in the weight of a tablet according to U.S. Pharmacopoeia. The following percentage deviation in weight variation was allowed.the results are shown in Table. No: 5& 8. Average weight of a tablet 130 mg or less ± 10 >130 mg and <324 mg ± mg or more ± 5 Percentage deviation b) Tablet Dimensions: Thickness and diameter were measured using calibrated Vernier calipers. Five tablets of each formulation were picked randomly and thickness and diameter was measured individually. The results are shown in Table. No: 5 & 8. c) Thickness: The thickness of the tablets was determined by Vernier calipers. Five tablets from each batch were used and the average values were calculated. The results are shown in Table. No: 5 & 8. d) Hardness: Hardness indicates the ability of a tablet to withstand mechanical shocks while handling. The hardness of the tablets was determined using Monsanto hardness tester. It is expressed in kg/cm2. Five tablets were randomly picked and hardness of the tablets was determined. The results are shown in Table. No: 5 & 8. e) Friability test: The friability of tablets was determined by using Roche friabilator. It is expressed in percentage (%). Twenty tablets were initially weighed (Wt) and transferred into friabilator. The friabilator was operated at 25 rpm for 4 minutes or run up to 100revolutions. The tablets were weighed again (WF). The % friability was then calculated by- W (initial)-w (final) %F = W (initial) The results are shown in Table. No: 5 & 8. f) Disintegration test: The disintegration time for immediate release layer was determined using the disintegration apparatus. One tablet was placed in each of six tubes placed in a beaker containing 1000 ml of purified water maintained at 37 ± 20 C and the apparatus was operated. The time taken for the tablets to disintegrate and pass through the mesh was noted. The results are shown in Table. No: 5 & 8. ASSAY: For SR Metformin hydrochloride Tablets: Standard preparation: Prepare a solution of metformin hydrochloride in water having a known concentration of about 10 µg per ml. Sample preparation: Weigh and finally powder 20 tablets. Transfer an accurately weighed of the powder, equivalent to about 100mg of metformin hydrochloride to a 100ml volumetric flask. Add 70 ml of water shake by mechanical means for 15 minutes, Dilute with water to volume and filter, discarding the first 20 ml of the filtrate. Dilute 10ml of the filtrate with water to 100ml and dilute 10ml of the resulting solution with water to 100ml. For IR Pioglitazone & Glibenclamide tablets: Standard preparation: Dissolve 165 mg of Pioglitazone and 50 mg of Glibenclamide in 10 ml of methanol with the aid of ultrasound for 20 minutes, add sufficient methanol to produce 100ml and dilute 5ml of this solution to 50ml standard flask and make up to the mark with methanol. Sample preparation: Weigh and fine powder of 20 tablets transfer an accurately weighed portion of the powder, equivalent to about Pioglitazone 15mg with a mixture of 2ml of water and 50ml of methanol in 100ml of standard flask shake until fully dissolved, and make up to volume with methanol. Chromatographic Conditions: Apparatus : HPLC system Column : C18, 4.6mm 250 cm. 5µ Wavelength : 254nm Injection volume : 20µl. Flow rate : 1.0ml/min Colum Temperature : Ambient Uniformity of content: Content uniformity is based on the assay of the individual content of drug substance in a number of individual dosage units to determine whether the individual content is within the limits. Uniformity of content needed for the dosage unit containing less than 25mg of API or less than 25% of total tablet weight. Content uniformity of the dosage unit was analyzed by as directed in the assay of tablet instead of equivalent weight taken as a whole tablet.

4 IN-VITRO DISSOLUTION STUDIES: Dissolution study of sustained release and immediate release of different tablet formulations were carried out separately. Time Time intervals Temperature : 30 Minutes : 5, 10, 15, 20& 30 Minutes : 37 ± 0.5 C. Dissolution SR Tablets: Apparatus : USP Type II (Paddle) Medium : 6.8 Phosphate buffer Medium Volume : 900ml Speed : 100 RPM Time intervals : 1, 3, 6, 9 & 12 hour. Temperature : 37 ± 0.5 C. Apparatus : UV-VIS Spectrophotometer Wavelength : 232nm The results are shown in Table. No: 10 Dissolution of IR Pioglitazone & Glibenclamide tablets: Dissolution of IR tablets done in two parts. Dissolution medium is separately chosen for two drugs. API based on their individual solubility and their absorption site in the G.I.T. For Pioglitazone Apparatus : Dissolution Apparatus USP Type I (Basket Type) Medium : 0.1 HCL Medium Volume : 900ml Speed : 100 RPM Time : 30 Minutes Time intervals : 5, 10, 15, 20& 30 Minutes Temperature : 37 ± 0.5 C. Chromatographic Conditions: Apparatus : HPLC system Column : C18, 4.6mm 250 cm. 5µ Wavelength : 254nm Detector : UV/PDA Injection volume : 20µl. Flow rate : 1.0ml/min Sample cooler temp. : 30 C For Glibenclamide Apparatus : Dissolution Apparatus USP Type I (Basket Type) Medium : 0.05 M boric acid and 0.05 M potassium chloride solution, prepared by dissolving 3.09 g of boric acid and 3.73g of potassium chloride in approximately 250 ml of water, adjusting with 1 N sodium hydroxide to a ph of 9.5 and diluting with water to 1 L. Medium Volume : 900ml Speed : 100 RPM Chromatographic Conditions: Apparatus : HPLC system Column : C18, 4.6mm 250 cm. 5µ Wavelength : 254nm Detector : UV/PDA Injection volume : 20µl. Flow rate : 1.0ml/min Sample cooler temp. : 30 C The results are shown in Table. No: 10 For Inlay Tablet Swelling and Erosion Studies: The rate of test medium uptake by the polymer was determined by equilibrium weigh gain method. The inlay tablets were accurately weighed (W0), Placed in the basket of dissolution apparatus, rotating at 100 rpm, 37 ± 0.5 C temperature, using ph 6.8 phosphate buffer. After 1, 2, 3, 5, 7, 9 and 12 hours, each basket was removed from the dissolution apparatus, the tablet with the pre-weighted mesh was withdramn from the medium and lightly blotted with tissue paper to remove excess test liquid and then reweighed (W1). After the swelling studies, the wet samples were then dried in an oven at 80 C for 12 Hours time period, allowed cooling in desiccators and finally weighed until constant weight was achieved (final dry weight, W2). The experiment was performed in 6 times for each time point and fresh samples were used for each individual time point. The percentage increase in weight due to absorbed liquid or water uptake was estimated at each time point from the following quation. Swelling % = W1 W0/W0 100, Erosion % = W0 W2/W0 100 The results are shown in Table. No: 12 & Fig. no: 18 & 19 Assay of Inlay tablet: Assay of Inlay tablet containing Metformin HCL and Pioglitazone with Glibenclamide done by two steps. The assay of inlay tablet SR layer containing metformin HCL was analyzed by UV method as directed in the Assay as of SR tablet. The assay of inlay tablet IR layer containing Pioglitazone with Glibenclamide was analyzed by HPLC method as directed in the assay as of IR tablet. Uniformity of content: Content uniformity of the dosage unit was analyzed by as directed in the assay of tablet instead of equivalent weight taken as a whole tablet. In-Vitro Dissolution profile of Inlay tablet: Dissolution medium is separately chosen for all three drugs. API

5 based on their individual solubility and their absorption site in the G.I.T. Separate dissolution condition were decide, one for each API. 1. The dissolution of immediate release layer was studies with dissolution medium of Pioglitazone in 0.1HCL for 30 minutes and Glibenclamide in 9.5 Buffer for 30 minutes. As directed in the assay of IR tablet. 2. The dissolution of sustained release layer was studied with dissolution medium of 6.8 ph phosphate buffer for 12 hours. As directed in the assay of SR tablet. The results are shown in Table. No: 13 & 14 Evaluation of In-Vitro release kinetics: To study kinetics, data obtained from in-vitro release were plotted in various kinetic models. Fig. no: 1 Linearity of Metformin Hydrochloride In-vitro Drug Release Kinetic Studies [18] : The dissolution data were subjected to release kinetic study. Drug dissolution from solid dosage form has been described by kinetic models in which the dissolved amount of drug (Q) is compared to the Drug content (%) function of the test time (t). some analytical and kinetic models of the Q versus t commonly used are Zero order, First order, Hixson Crowell, Higuchi and Korsmeyer Peppas model to study the in-vitro kinetic release mechanism. The results are shown in Table.No: 15 & 16 Figure.No: 20 to 28 Stabilty Study: Stability study of optimized inlay tablet was carried out at room temperature and an accelerated condition of 40 C ± 2 C/75%RH± 5%RH for a period of 6 months. Samples were withdrawn at an interval of 1, 2, 3 & 6 month for evaluation with respect to physical parameters,assay and dissolution studies. The results are shown intable.no: 17 & 18 Fig. no: 2 Linearity of Pioglitazone Hydrochloride RESULT AND DISCUSSION: Calibration curve Table. no: 3 calibration curve of API materials Metformin Pioglitazone Glibenclamide Hydrochloride Hydrochloride Conc. Absorbance Conc. Area Conc. Area (µg/ml) at 232nm (µg/ml) (MAU) (µg/ml) (MAU) Fig. no: 3 Linearity of Glibenclamide

6 Preformulation studies The overall objective of preformulation studies is to generate useful information to the formulator in developing stable and bioavailable dosage forms that can be mass produced. Physical Drug Excipients compatibility studies: The successful formulation of a stable and effective solid dosage form depends on the careful selection of the excipients that are added in the formulation. The drug and excipients must be compatible with one another to produce a product that is stable, Efficacious and easy to administer and safe. The physical compatibility evaluation was performed in visual basis. Fig. no :4 Metformin Hydrochloride The study implies that the drug, polymer and other excipients were physically compatible with each other as there was no change of physical description. Chemical compatibility studies by FT-IR: The FT-IR spectral analysis of the Metformin hydrochloride, Pioglitazone, Glibenclamide, polymers and other excipients was shown from Fig. no: 4 to Fig. no:14. All the characteristic peaks appear for the pure Metformin, Pioglitazone, Glibenclamide and its physical mixture indicating no interaction between the three drugs. All three drugs with polymer also show characteristic peaks so, No interaction between the drug and excipients. Fig. no: 5 Pioglitazone Fig. no: 6 Glibenclamide

7 Fig. no:7 Metformin Hydrochloride + Pioglitazone Fig. no:8metformin Hydrochloride + Glibenclamide Fig. no: 9 Pioglitazone + Glibenclamide

8 Fig. no:10 Metformin Hydrochloride + Pioglitazone + Glibenclamide Fig. no:11 Metformin Hydrochloride + Pioglitazone + Glibenclamide + HPMC K100M Fig. no:12 Metformin Hydrochloride + Pioglitazone + Glibenclamide + Guar Gum

9 Fig. no:13 Metformin Hydrochloride + Pioglitazone + Glibenclamide + Xanthan Gum Fig. no: 14 Metformin Hydrochloride + Pioglitazone + Glibenclamide + All excipients (Inlay Tablet) Pre compression study SR formulation Table. No: 4 pre compression study SR formulation Batch Bulk density* Tapped density* Compressibility Harner s Angle of Moisture code g/cm 3 g/cm 3 index g/cm 3 Ratio* Repose* Content* %w/w F ± ± ± ± ± ±0.568 F ± ± ± ± ± ±0.324 F ± ± ± ± ± ±0.212 F ± ± ± ± ± ±0.234 F ± ± ± ± ± ±0.434 F ± ± ± ± ± ±0.768 F ± ± ± ± ± ±0.868 F ± ± ± ± ± ±0.676 F ± ± ± ± ± ±0.988 F ± ± ± ± ± ±0.121 F ± ± ± ± ± ±0.333 F ± ± ± ± ± ±0.889 *Mean ± SD (n=6)

10 Post compression study of a SR tablets Table. No: 5 post compression study of SR tablets Batch Weight Hardness* Thickness* Friability* Diameter* Drug content* code variation** F1 821± ± ± ± ± ±0.04 F2 815± ± ± ± ± ±0.07 F3 820± ± ± ± ± ±0.03 F4 823± ± ± ± ± ±0.04 F5 831± ± ± ± ± ±0.01 F6 835± ± ± ± ± ±0.03 F7 818± ± ± ± ± ±0.02 F8 820± ± ± ± ± ±0.03 F9 823± ± ± ± ± ±0.05 F10 817± ± ± ± ± ±0.03 F11 824± ± ± ± ± ±0.03 F12 828± ± ± ± ± ±0.02 *Mean ± SD (n=6),**mean ± SD (n=20) Iv-Vitro Dissolution study: Table. No: 6. In-Vitro Dissolution profile SR tablets* Time in hrs F ± ± ± ± ±1.12 F ± ± ± ± ±2.14 F ± ± ± ± ±1.21 F ± ± ± ± ±0.98 F ± ± ± ± ±1.12 F ± ± ± ± ±0.67 F ± ± ± ± ±1.23 F ± ± ± ± ±1.67 F ± ± ± ± ±0.78 F ± ± ± ± ±1.34 F ± ± ± ± ±0.87 F ± ± ± ± ±1.75 *Mean ± SD (n=6) Fig. No: 15 In-Vitro Dissolution profile SR tablets

11 Table.no: 7 pre compression study for IR formulation Batch Bulk density* Tapped density* Compressibility Harner s Ratio* Angle of Repose* Moisture code g/cm 3 g/cm 3 index g/cm 3 Content* %w/w IR ± ± ± ± ± ±0.168 IR ± ± ± ± ± ±0.124 IR ± ± ± ± ± ±0.112 *Mean ± SD (n=6) Post compression study of IR tablets Table. No:8 post compression study of IR tablets Batch Weight Hardness* Thickness* Friability* Diameter* Disintegration code variation** Time in Sec IR ± ± ± ± ± ±0.04 IR ± ± ± ± ± ±0.07 IR ± ± ± ± ± ±0.03 *Mean ± SD (n=6), **Mean ± SD (n=20) Table.no: 9 Assay and Content uniformity of the IR formulation S. No Batch.No Pioglitazone Glibenclamide Assay* Uniformity of content** Assay* Uniformity of content** 1 IR ± ± ± ± IR ± ± ± ± IR ± ± ± ±1.765 *Mean ± SD (n=6),**mean ± SD (n=10) In Vitro Dissolution release Profile and Optimization IR tablets Table. No: 10. In-Vitro dissolution profile of IR tablets Time in Minutes IR1 IR2 IR3 Pioglitazone Glibenclamide Pioglitazone Glibenclamide Pioglitazone Glibenclamide ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.65 *Mean ± SD (n=6) Fig. no: 16In-Vitro Dissolution profile of Pioglitazone IR1 - IR3 Fig. no: 17In-Vitro Dissolution profile of Glibenclamide IR1 - IR3

12 FORMULATION AND EVALUATION OF INLAY TABLETS Table. No: 11Formulation of Inlay tablets Sustained Release mg/ tablet Immediate Release mg/ tablet Layer (F3) layer (IR3) Metformin HCL 500 Glibenclamide 5.05 HPMC K100M 200 Pioglitazone HCL 16.7 PVP k30 40 Sodium Starch Glycolate 7 MCCP Ph Crosspovidone 3.5 Crosspovidone 5 Sodium Lauryl Sulphate 2 Aerosil 2.5 Crosspovidone 1 Magnesium stearate 10 MCCP ph Talc 10 Talc 1 Aerosil 2.5 Magnesium Stearate 0.5 Total Weight 820 Total Weight 70 Evaluation of Inlay tablets Parameters Results Uniformity of Weight** ±7.989 Tablet Thickness* 6.987± Diameter* ±0.012 Hardness* 6.98±0.567 Friability* ±0.065 Dinintegration Time* 2.16±0.065 ** Mean ± SD (n=20),*mean ± SD (n=6) Swelling and Erosion behavior of Inlay Tablets Table. No: 12 Swelling and Erosion behavior of Inlay Tablets Time in Hours % of swelling % of erosion ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.169 Fig. no:19 Erosion Behavior of Inlay Tablet The result showed in the table says that the SR tablet maintained their integrity and gave increased swelling through the course of study. Thus the extent of swelling increases with amount of swellable polymer. Assay and content uniformity of the Inlay tablet Table. No: 13Assay and Drug content uniformity of the Inlay tablet Inlay tablet % of Drug (Assay)* Content uniformity** Metformin Hydrochloride 99.98± Pioglitazone ± ±0.245 Glibenclamide ± ±0.675 *Mean ± SD (n=20),**mean ± SD (n=10) In-Vitro Dissolution Release profile of Inlay tablet Table. No: 14 In-Vitro Dissolution profile of SR and IR layer* Fig. no:18 Swelling Behavior of Inlay Tablet Time in Immediate Release Layer Sustained Release Layer Minutes Cumulative % Time Cumulative % of Drug release in hours of Drug release Pioglitazone Glibenclamide Metformin Hydrochloride ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.809 *Mean ± SD (n=6) In-Vitro Kinetic Study of Inlay Tablet For Metformin hydrochloride Table. No: 15 Data for various kinetic models Time % cum. Drug % cum drug Log % cum. Square root Log time Log % cum. Cube root of (hours) release remaining Drug of time Drug % drug remaining release remaining

13 Fig. no: 20 Zero order kinetics Fig. no: 24Korsmeyer Pappas equation The results showed that the formulation followed the Zero order release kinetics. Drug release kinetics of this formulation correspond best Higuchi s model drug release mechanism as per n value of Korsmeyer& Pappas model appeared to be a complex mechanism of swelling, diffusion and erosion. FOR PIOGLITAZONE & GLIBENCLAMIDE Table. No: 16 data for various kinetic models For Pioglitazone For Glibenclamide Fig. no: 21First order kinetics Time % % Log % % Log (Min) Cumulative to be Cumulative Cumulative to be Cumulative Drug released % Drug Drug released % Drug Release Remaining Release Remaining Fig. no: 22 Higuchi diffusion kinetics Fig. no: 25. First order kinetics for Pioglitazone Fig. no: 23 Hixson Crowell Fig. no: 26. First order kinetics for Glibenclamide

14 Fig. no: 27 Zero order kinetics for Pioglitazone Fig. no: 28 Zero order kinetics for Glibenclamide From the above results obtained for Pioglitazone and Glibenclamide has been observed that the order of release of the drug was followed the First order kinetics. Stability Study: The optimized Inlay tablets were subjected to stability studies and the results were showed in the tables. Table. No: 17 Stability Study at 40 C ± 2 C/75%RH± 5%RH Parameters 1 st Month 2 nd Month 3 rd Month 6 th Month RT 40 C RT 40 C RT 40 C RT 40 C Uniformity of Weight** ± ± ± ± ± ± ± ±1.23 Thickness* 7.1± ± ± ± ± ± ± ±0.183 Diameter* 13.04± ± ± ± ± ± ± ±0.021 Hardness* 6.25± ± ± ± ± ± ± ±0.541 D. Time* 2.34± ± ± ± ± ± ± ±0.352 Friability* 0.12± ± ± ± ± ± ± ±0.989 **Mean ± SD (n=20), *Mean ± SD (n=6) Table. No: 18 Assay and Dissolution profile of Inlay tablet at stability study 40 C ± 2 C/75%RH± 5%RH Intervals in Months Drug % of Drug Content* % of Cum. Release* RT 40 C RT 40 C 1 st Month Metformin HCL ± ± ± ±0.78 Pioglitazone ± ± ± ±0.67 Glibenclamide 99.7± ± ± ± nd Month Metformin HCL ± ± ± ±0.28 Pioglitazone ± ± ± ±0.37 Glibenclamide 99.8± ± ± ± rd Month Metformin HCL ± ± ± ±0.21 Pioglitazone ± ± ± ±0.25 Glibenclamide 99.6± ± ± ± th Month Metformin HCL ± ± ± ±0.01 Pioglitazone ± ± ± ±0.15 Glibenclamide 99.2± ± ± ±0.43 * Mean ± SD (n=6)

15 No significant changes were observed in the physical appearance, colour, hardness, drug content and dissolution studies of Inlay tablet of the optimized batch for both at room temperature (RT) and40 C ± 2 C/75%RH± 5%RH. SUMMARY AND CONCLUSION The present research endeavor is directed towards the development of once daily sustained release matrix tablet of metformin hydrochloride (500mg) with inlay tablet of pioglitazone (15mg) glibenclamide (5mg) as immediate release. Combination therapy concluded that Pioglitazone is effective in improving the glycemic control when added to a combination of glibenclamide and metformin in type 2 DM. Matrix system was based on swellable polymer were selected for sustained the drug release. Different polymers Viz., HPMC, Xanthan Gum, Guar gum, to get the desired release profile over a period for 12 hours. Different batches of both Immediate release(ir) and Sustained release (SR) were prepared by Direct compression and Wet granulation method respectively. In the immediate release layer formulation, disintegration such as sodium starch glycolate at three different concentration (5%, 7.5% and 10% w/w) with 5% concentration crosspovidone of tablet weight were tried to get a desired release profile within 30 minutes. All the formulations were evaluated for physical characteristics, disintegration, In-Vitro dissolution study and stability. Following conclusions have been made from the present study. The possibility of Drug Excipients interaction was investigated by FT-IR. The physical characteristics of all blended formulations were satisfactory. The prepared tablets evaluated for weight variation, hardness, thickness, friability and dinintegration time were found to be within the official limits. The dinintrrgration studies showed that the immediate release formulation IR3 prepared by direct compression technique using SSG of 10% w/w concentration was 14 seconds. The in-vitro dissolution studies were performed for all the IR formulations. Formulation IR1 to IR3 released 95.53%, 96.45% and 96.65% of pioglitazone and %, 96.72% and % of glibenclamide respectively at the end of 30 minutes with SSG concentration of 10%. So IR3 were selected for final formulation. SR formulations F3 showed release profile were complies with USP at 40% concentration of HPMC-K100M with respect to drug compared with another 11 formulations. Inlay tablet formulation were prepared using Optimum formulation of sustained granules and immediate release granules. Initially metformin granules were filled in the die cavity over that immediate release tablet was placed on it and then compressed finally to get a inlay tablet. The tablets assay, weight variation, hardness, thickness friability, disintegration time and in-vitro dissolution were found to be within the official limits. The dissolution data of the optimized batch was subjected to study the in-vitro release kinetics. The result showed that the IR layer of inlay tablet formulation followed the first order release kinetics and the drug release kinetics of SR layer of Inlay tablet formulation correspond best to Higuchi s model and drug release mechanism as per n value of Korsmeyer &Peppas Model appeared to be a complex mechanism of swelling, diffusion and erosion with zero order release kinetics. The triple combination of inlay tablet containing pioglitazone with glibenclamide in immediate disintegrating layer and metformin hydrochloride in sustained release layer were successfully formulated and evaluated. ACKNOWLEDGEMENTS: Authors are thankful to Prof (Dr.).B.Jaykar, Principal Vinayaka Mission s College of Pharmacy, Salem, Tamil nadu and providing all the facilities for this research project. REFERENCES: 1. Remington, The Science and pharmacy practice of pharmacy, 21 st edition volume I & II, Page. no: AnisulQuardir, Karl Kolter, A comparative study of current Super disintegrents, pharmaceutical technology, October Aulton M.E. The science of dosage form design, international edition, second, Churchill Livingston ( Brahmankar DM and Jaiswal SB. Biopharmaceutics and Pharmacokinetics, VallabhPrakashan 1 st Edition; 1995: Colombo PR, BettiniManima. Journal of Parma Science; : Clarke s Isolation and Identification of drugs, 2 nd edition, The pharmaceutical press, London, 1986; Page.No: Regmington : The Science and practice of Pharmacy. 20 th Edition; 2000; Page. No: Amidon, G. E.; Augsburger, L. L.; Physical test methods for powder flow characterizationof pharmaceutical materials :areviewofmethods PharmacopeialForum 25,1999; Page. No: Swarbrick J, Boylan J.C., Encyclopedia of Pharmaceutical Technology, Second Volume-1992; Page. No:

16 10. Banker G.S. Anderson N.R., Tablets chapter 11 in The theory and practice of industrial pharmacy edited BY Lachman Edition, Varghese Publishing House, 1991; Page. No: Fonner Characterization of Granulation in Pharmaceutical dosage forms: Tablets, Volume. No:2, edited by Lieberman H.A., Lachman L., Marcel Dekker; Page.No: Carver,L.D.:ParticleSizeAnalysis,Industrial Research, August1971; Page.No: Agbada, C.O., and P.York.1994, Dehydration oftheophylline monohydrate powder effects ofparticlesize and sample weight,int. J.Pharm.106; Page. No: Source of support: Nil, Conflict of interest: None Declared