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Design and in vivo Evaluation of Controlled Release Gliclazide Trilayer Matrix Tablets in the Management of Diabetes Mellitus

The purpose of the present study was to develop and optimize multi-layered matrix tablets of Gliclazide trilayer tablets to achieve zero-order drug release for sustained plasma concentration. Gliclazide tablets were prepared by direct compression and consist of middle active layer with different grades of hydroxypropyl methylcellulose (HPMC K 4M, K 15M and K100M), Guar gum and Eudragit L 100. The tablets were also evaluated for physicochemical characteristics and release kinetics. The physicochemical characteristics of the prepared tablets were satisfactory. The developed drug delivery systems showed prolonged drug release rates over a period of 24 h. In vivo bioavailability studies were carried out on the optimized formulation (EF14), mean time to attain peak drug concentration (Tmax) was 6.00 ± 0.14 and 4.02 ± 0.12 h for the optimized and marketed product respectively, while mean maximum drug concentration (Cmax) was 124.80 ± 1.02 ng/ml and 95.12 ± 1.05 ng/ml respectively. AUC0-α and AUC0-t for optimized formulation was significantly higher (p<0.05) as compared to marketed product. A fair correlation between the dissolution profile and bioavailability for the optimized formulation was observed. The results indicate that the approach used could lead to a successful development of a controlled release formulation of the drug. The EF14 was shown significant plasma concentration with controlled release and maintained for 24 hours with patient compliance by reducing the dosage frequency, when compared with marketed product in the efficient management of Diabetes mellitus.


INTRODUCTION
Controlled release pharmaceutical systems have been developed and studied to improve the performance of drugs and to increase their pharmacological effect and reduce any side effect. [1] Several design options are available to control or modulate drug release from a drug delivery system. Most oral controlled release dosage forms fall in the category of matrix, reservoir or multi-layer systems. Lately, multi-layer matrix systems are gaining importance in the design of oral sustained drug delivery systems. A multi-layer system consists, usually, of a hydrophilic matrix core containing the active ingredient and one or two impermeable or semipermeable polymeric coatings (barrier-layer) applied on one or both faces of the core during tableting. [2] The barrier layers delay the interaction of active solute with dissolution medium, by limiting the surface available for the solute release and at the same time controlling solvent penetration rate. [3] In the device, the coat layers prevent the water penetration through the protected core for some duration. After this phase during the subsequent dissolution process, the swollen barriers erode and the surface available for drug release slowly increases. In this way the decrease of delivery rate due to the increase in diffusion path length is counter balanced by the simultaneous increase of the area available for drug release. [4] Hydrophilic polymers have been given considerable attention in the formulation of controlled release drug delivery systems for various drugs. HPC, HPMC and Sodium CMC & Carbopol are a few representative examples of the hydrophilic polymers that have been extensively used in the formulation of controlled release systems. [5] Guar gum is soluble in water; it swells in gastric fluid to produce a highly viscous layer around the tablet through which the drug can slowly diffuse and is used for the fabrication of matrices with uniform drug release characteristics. [6] There have been different approaches to achieve zeroorder drug release from dosage forms for sustained plasma concentration. Among different approaches to achieve zero-order release from hydrophilic matrix technologies, multilayer matrices have been widely evaluated and developed for commercial products under the trade name of Geomatrix. The technology makes use of bilayer or trilayer tablets to modulate the release and to achieve constant release. [7] Gliclazide is an oral hypo glycaemic second-generation sulfonyl urea drug which is useful for a long-term treatment of non-insulin dependent diabetes mellitus (NIDDM). Previous studies showed that gliclazide possesses good general tolerability, low incidence of hypo glycaemia and low rate of secondary failure. Rapid absorption from the GIT is required for the oral hypoglycaemic drug for effective therapy. However, the absorption rate of Gliclazide from the GIT is slow and varied among the subjects. Slow absorption has been suggested to be due to either poor dissolution of Gliclazide due to hydrophobic nature of drug and poor permeability of drug across the GI membrane. [8][9] By incorporation of Gliclazide in the trilayer tablet may control its absorption from GI tract and overcome the variability problems. Thus, this study was undertaken to develop controlled release trilayer matrix tablets of Gliclazide using release retardant and HPMC polymers up to 24 h.

MATERIALS AND METHODS Materials
Gliclazide (80 mg) film coated tablets were purchased from Orion Pharma Ltd, Mumbai. Gliclazide pure drug was generous gift from Aurobindo Pharma Limited, Hyderabad, India. Sodium carboxyl methyl cellulose, HPMC K 4 M, HPMC K 15 M & HPMC K 100 M were obtained from Rubicon labs, Mumbai. Kondagogu gum, Eudragit L 100 and Carnauba wax were gifted from MSN Labs Ltd. Hyderabad. All other chemicals used were of analytical grade.

Methods
The following Pre-compression parameters were conducted: Angle of Repose [10] , Carr's compressibility Index [11] , Bulk Dentistry, Tapped Density [12] , Hausner's Ratio. [13] Formulation of controlled release Gliclazide trilayer matrix tablets The trilayered matrix tablets of Gliclazide were prepared by direct compression method. The first step in the formulation was to develop the middle active layer so as to give at least 90% drug release during 12 hours. The release profile of this layer might not be of constant rate type but would be preferably of constantly falling rate type. This layer would then be sandwiched between barrier layers (Upper & Lower layers) so as to continue the drug release for 24 h.

Preparation of middle active layer
Sixteen formulations (F1-F16) for active layer were prepared by direct compression method using polymers like different HPMC grades, Kondagogu gum, Sodium CMC, DCP. All the formulations were varied in concentration of polymers, talc (1.5 mg) & magnesium stearate (1.5 mg) constituted in all the formulations. These materials were screened through ≠60 and mixed together in motor by using pestle. Final mixtures were compressed by using 12 mm diameter flat punches on a sixteen-station rotary tablet press. Formulation of active layer was depicted in table 1, 2. The prepared tablets were subjected to dissolution studies.

Preparation of upper and lower layers
The barrier layers were formulated employing hydrophobic swellable polymer natural wax i.e. carnauba wax the swelling erosion modelling fillers which include water soluble DCP, EC and Kondagogu gum. The procedure tried to make the compacts was via direct compressions. For the first procedure the wax, Kondagogu gum and the filler was mixed in motor and lubricated with magnesium stearate. Formulation of upper and lower layers was depicted.

Formulation of Gliclazide trilayer tablets
The powder mixtures required for active and barrier layers were weighed accurately and thoroughly mixed using mortar and pestle for about 20 minutes. Initially, the volume of die cavity (12 mm, round) was adjusted equivalence to the weight of trilayered matrix tablets (500 mg). Then the pre-weighed amount of powder equivalent to bottom layer (125 mg) was taken and placed in the die cavity and slightly compressed for uniform spreading. The upper punch was lifted up and 250 mg of the drug containing middle active layer optimized formulation (F14) was placed over the bottom layer in the die cavity and again slightly compressed. The remaining volume of the die cavity was filled with pre-weighed (125 mg) amount of powder equivalent to top layer and compressed with the full force of compression on rotary tablets press to obtain tri-layered tablets. Tri-layered matrix tablets of each composition were compressed and tested for their friability, Hardness, drug content and drug release characteristics with a suitable number of tablets for each test. Evaluation tests of trilayer matrix tablets performed by Hardness, Friability [14] , Weight variation, Drug content /Assay [15] , Swelling & Erosion studies. [16] In-vitro drug release profile In vitro drug release studies for developed trilayer matrix tablets were carried out by using dissolution apparatus II paddle type (Electrolab TDL-08L). The drug release profile was studied in 900ml Phosphate buffer pH 7.2 at 37± 0.5°C temperature. The amount of drug release was determined by UV visible spectrophotometer (Shimadzu UV 1800) at 229 nm.

Drug-excipient compatibility studies Fourier Transform Infrared Spectroscopy (FTIR) and
Differential Scanning Calorimetry (DSC) [17] Stability studies The stability study of the formulated trilayer tablets were carried out under different conditions according to ICH guidelines using stability chamber (REMI make). Accelerated Stability studies were carried out at 40°C/75% RH for the best formulations for 6 months. The tablets were characterized for the hardness, friability, drug content and cumulative % drug release during the stability study period.

Pharmacokinetic study Animal Preparation
Male rabbits were (weighing 2-3 Kg) selected for this study, all the animals were healthy during the period of the experiment. Animals were maintained at room temperature 25°C, Relative Humidity 45% and 12 h alternate light and dark cycle with 100% fresh air exchange in animal rooms, uninterrupted power and water supply and rabbits were fed with standard diet and water ad libitum. The protocol of animal study was approved by the institutional animal ethics committee (IAEC NO: IAEC NO: P44/VCP/IAEC/2015/10/DBP/ AE12/Rabbits).

In vivo study design
The Rabbits were randomly divided into two groups each group contains six animals. The group A was received prepared Gliclazide matrix Tablets (80 mg), marketed product (80 mg) was administered group B with equivalent dose of animal body weight. Blood samples (approximately 0.5 ml) were obtained with syringes by marginal ear vein at 0, 1, 2, 4, 6, 8, 12, 16, 20 and 24 hours post dose. During collection, blood sample has been mixed thoroughly with heparin in order to prevent blood clotting. Plasma was separated by centrifugation of the blood at 5000 rpm in cooling centrifuge for 5 min to 10 minutes and stored frozen at −20°C until analysis.

Preparation of Plasma Samples for HPLC Analysis
Rabbit plasma (0.5 ml) samples were prepared for chromatography by precipitating proteins with 2.5 ml of ice-cold absolute ethanol for each 0.5 ml of plasma. After centrifugation the ethanol was transferred into a clean tube. The precipitate was re suspended with 1 ml of Acetonitrile by vortexing for 1 min. After centrifugation (5000-6000 rpm for 10 min), the Acetonitrile was added to the ethanol and the organic mixture was taken to near dryness by a steam of nitrogen at room temperature.

Pharmacokinetic analysis
The pharmacokinetic parameters employed to evaluate were maximum plasma concentration (Cmax), time to attain Cmax i.e., Tmax and t ½ values, area under plasma concentration-time curve from zero to the last sampling time (AUC0-t), area under plasma concentration-time curve from zero to infinity (AUC0-∞). AUC0-t was calculated by the linear trapezoidal rule and AUC0-∞ from the following formula.

RESULTS AND DISCUSSION Pre-compression parameters
All the powder mixture belonging to different formulations was tested for micrometrics studies in order to determine the flow properties. All the formulations AF14 to HF14 showed good flow properties, the results are summarized in table 3.

TRILAYER TABLETS Preparation of middle active layer
The matrix tablets of Gliclazide were prepared without the barrier layers. All the formulation trails were subjected to in vitro dissolution to determine the release profiles. From the above results, among all the formulations the formulation F14 was decided as optimized formulation for active layer based on the highest drug release i.e. 99.60 ± 5.25 within 12 hours when compared with other preparations (table 4, 5 & figure 4, 5). Formulation F14 was chosen as active layer for further studies.

Evaluation of trilayer matrix tablets of Gliclazide
The Gliclazide trilayer matrix tablets are shown in figure 3. Sustained release tablets generally have hardness in the range of 7-10 kg/cm 2 . In case of trilayer tablets the hardness of the tablets was found to be 7.2 to 7.8 kg/cm 2 . The friability of the formulations was found to be less than 1% and hence the tablets with lower friability may not break during handling on machines and or shipping. All the batches of the tablets complied with the weight variation limits as per the IP. The drug content in different formulation was highly uniform and the results are depicted in table 6. In phosphate buffer pH 6.8, HPMC showed good swelling property. In trilayer tablets of Gliclazide, EF14 showed highest degree of swelling index 219.43%, where as in EF14 showed leased swelling with a swelling index of 134.68%.

Stability studies
Optimized formulation was selected for stability studies on the basis of high cumulative % drug release. Stability studies were conducted for 6 months according to ICH guidelines. From these results it was concluded that, optimized formulation is stable and retained their original properties with minor differences.

Bioavailability Parameters
Mean plasma concentration profiles of prepared gliclazide optimized formulation and Marketed product are presented in figure 11. Gliclazide optimized formulation exhibited as sustained release in vivo when compared with Marketed product. All the pharmacokinetics parameters displayed in dissolution profile and release drug kinetics EF14 was found to be optimized formulation. In vitro studies were conducted for optimized Gliclazide trilayer tablets and marketed product, the results were indicating that the optimized Gliclazide formulation was shown sustained release patterns where marketed product was shown immediate release. In vivo bioavailability studies were carried out on the optimized formulation (EF14), mean time to attain peak drug concentration (Tmax) was 6.00 ± 0.14 and 4.02 ± 0.12 h for the optimized and marketed product respectively, while mean maximum drug concentration (Cmax) was 124.80 ± 1.02 ng/ml and 95.12 ± 1.05 ng/ml respectively. AUC0-α and AUC0-t for optimized formulation was significantly higher (p<0.05) as compared to marketed product. A fair correlation between the dissolution profile and bioavailability for the optimized formulation was observed. The results indicate that the approach used could lead to a successful development of a controlled release formulation of the drug. The HF14 was shown significant plasma concentration with controlled release and maintained for 24 hours with patient compliance by reducing the dosage frequency, when compared with marketed product in the efficient management of Diabetes mellitus.