Repaglinide is an antidiabetic drug in the class of medications known as meglitinides, and was invented in 1983. Repaglinide is an oral medication used in addition to diet and exercise for blood sugar control in type 2 diabetes mellitus. The mechanism of action of repaglinide involves promoting insulin release from β-islet cells of the pancreas; like other antidiabetic drugs.  The present investigation involved the formulation of the alginate microspheres of Repaglinide (model drug) using calcium chloride as a cross linking agent by inotropic gelation method. Microspheres were prepared by using 2%, 2.2% sodium alginate concentrations. Polymers (HPMC, Ethyl cellulose, Carbopol 934P) were used in combination concentration to prepare Microspheres. Microspheres were evaluated for micromeritic properties like angle of repose, bulk density, tapped density, Carr’s index, Hausner’s ratio and for drug content. The in vitro drug release study was done for microspheres All formulations. The mean particle size, In vitro Buoyancy, Encapsulation efficiency%, Percentage yield (%) were within limits. Floating Microspheres of Repaglinide improves patient compliance by decreasing dosing frequency. Gastric retention time is increased because of buoyancy. Enhanced absorption of drugs which solubilise only in stomach. Drug releases in controlled manner for prolonged period, site-specific drug delivery.

Keywords: Repaglinide, Antidiabetic, Floating microspheres, Inotropic gelation method, Sodium alginate, Carbopol 934P, HPMC, Ethyl cellulose

INTRODUCTION

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. To obtain maximum therapeutic efficacy, it becomes necessary to deliver the agent to the target tissue in the optimal amount in the right period of time there by causing little toxicity and minimal side effects [1,2].

Repaglinide lowers blood glucose by stimulating the release of insulin from the beta islet cells of the pancreas. It achieves this by closing ATP-dependent potassium channels in the membrane of the beta cells. This depolarizes the beta cells, opening the cells’ calcium channels, and the resulting calcium influx induces insulin secretion [3]. Repaglinide has a 56% bioavailability when absorbed from the gastrointestinal tract. Bioavailability is reduced when taken with food; the maximum concentration decreases by 20%. The protein binding of repalglinide to albumin is greater than 98%. repaglinide is primarily metabolized by the liver - specifically CYP450 2C8 and 3A4 - and to a lesser extent via glucuronidation. Metabolites of repaglinide are inactive and do not display glucose-lowering effects and it is 90% excreted in the feces and 8% in the urine. 0.1% is cleared unchanged in the urine [4].

The word new or novel in the relation to drug delivery system is a search for something out of necessity [5]. An appropriately designed sustained or controlled release drug delivery system can be major advance toward solving the problem associated with the existing drug delivery system. The aim of any drug delivery system is to afford a therapeutic amount of drug to the proper site in the body to attain promptly, and then maintain the desired drug concentration [6]. Oral drug delivery has been known for decades as the most widely used route of administration among  all  the  routes  that  have  been  explored  for  the systemic delivery [7]. All controlled release systems have limited applications if the systems cannot remain in the vicinity of the absorption site. Floating drug delivery systems were first described by Davis in 1968. It is possible to prolong the gastric residence time of drugs using these systems [1]. Several techniques are used to design gastro retentive dosage forms. These include floating, swelling, inflation, adhesion, high-density systems and low-density systems that increase the gastric residence time [7]. Gastric retention is useful for drugs which (i) act locally; (ii) have a narrow absorption window in the small intestinal region; (iii) unstable in the intestinal environment; (iv) low solubility at high pH environment [8].

MATERIALS AND METHODS

Materials

Repaglinide from  Rakshit Drug PVT LTD., Hyderabad, HPMC, Ethyl cellulose, Carbopol 934P, Sodium Alginate, Sodium bicarbonate, Calcium chloride, Acetic acid, Glutaraldehyde, Merck Specialities Pvt Ltd, Mumbai, India.

Equipments

Weighing Balance, Automatic dissolution test apparatus,Brookfield digital viscometer, Sartorious digital IR balance, Scanning electron microscope, Magnetic stirrer, DissolutionApparatus, UV-Visible Spectrophotometer, pH meter, FT-IR Spectrophotometer.

METHODOLOGY

Preparation of 0.1n Hcl (Ph 1.2)

Take 8.6ml of HCL in a 1000ml volumetric flask and make up the volume with distilled water.

Preparation calibration curve

100 mg of Repaglinide pure drug was dissolved in 15 ml of Methanol and volume make up to 100 ml with 0.1N HCL (stock solution-1). 10 ml of above solution was taken and make up with 100 ml by using 0.1 N HCL(stock solution-2 i.e. 100 μg/ml). From this take 0.2, 0.4, 0.6, 0.8 and 1.0 ml of solution and make up to 10ml with 0.1 N HCL to obtain 2, 4, 6 ,8, and 10 μg/ml of Repaglinide solution. The absorbance of the above dilutions was measured at 241 nm by using UV-Spectrophotometer taking 0.1N HCL as blank. Then a graph was plotted by taking Concentration on X-Axis and Absorbance on Y-Axis which gives a straight-line Linearity of standard curve was assessed from the square of correlation coefficient (R2) which determined by least-square linear regression analysis. The experiment was performed in triplicate and based on average absorbance; the equation for the best line was generated. The results of standard curve preparation are shown in Table 1 and Figure 1.

Preparation of microspheres

The floating microspheres were prepared by Ionic gelation technique using the formulation showed in Table 1. A solution of sodium alginate is prepared. The gelation medium was prepared by dissolving calcium chloride in 2% glacial acetic acid and was added to solution. In this method cross-linking agent & polymer in combination were dispersed in the purified water to form a homogeneous polymer mixture. Resultant solution was extruded drop wise with the help of syringe and needle into aqueous calcium chloride solution and stirred at 100 rpm. The drug was added to the polymer dispersion and mixed thoroughly on a magnetic stirrer to form a homogeneous dispersion. The homogenous alginate solution was extruded using syringe needle into the gelation medium. Then, microsphere was collected and washed with distilled water twice, dried at room temperature for 24 h and  dried at 60°C for 2 h in a hot air oven and stored in dessicator.

Micromeritic properties of microspheres

The microspheres are characterized by their micromeritic properties, such as particle size, tapped density, compressibility index, true density, and flow property [9]. The tapping method was used to calculate tapped densities and percentage compressibility index. Tapped densities and percentage compressibility index can be calculated using following equation:

Tapped density:

  D_t = M/V_t                                                                 (1)

Where, M = mass of the powder

V_t = tapped volume of powder

Carr’s compressibility index

I = (tapped density - bulk density) / (tapped density) × 100  

I = (D_t - D_b) / D_t ×100

Where, D_t = Tapped density of the powder (g)

D_b = Bulk density of the powder (mL)

The angle of repose (f) of the microspheres, which measures the resistance to particle flow, was measured using fixed funnel method and calculated as per following equation:2

θ = tan^-1 h/r                                         (2)

Where, = angle of repose

h = height (cm)

r = radius (cm) 

Results of micromeritic properties like Angle of Repose, Compressibility Index

Determination of mean particle size: The particle size was measured using an optical microscope and the mean particle size was calculated by measuring 200 particles with the help of a calibrated ocular micrometer. A small amount of dry microspheres was suspended in purified water (10 ml). A small drop of suspension thus obtained was placed on a clean glass slide. The slide containing microspheres was mounted on the stage of the microscope and diameter of at least 100 particles was measured using a calibrated optical micrometer.

Incorporation efficiency (IE): To determine the incorporation efficiency, 10 mg microspheres were thoroughly triturated and dissolved in minimum amount of ethanol. The resulting solution was made up to 100 ml with 0.1 N HCL and filtered. Drug content was analyzed spectrophotometrically at 320 nm. Calculation was done as per equation 3:

    % Incorporation efficiency = (Actual drug content) / (Theoretical content) × 100                                                                     (3)

Percentage buoyancy: The floating test was carried out to investigate the floatability of the prepared microspheres.  To assess the floating properties, the microspheres were placed in 0.1 N HCL containing 0.02% v/v Tween 20 surfactant (pH 2.0, 100 ml) to simulate gastric conditions. The use of 0.02% Tween 20 was to account for the wetting effect of the natural surface-active agents, such as phospholipids in the GIT. The mixture was stirred at 100 rpm in a magnetic stirrer. After 12 h, the layer of buoyant microparticles was pipetted and separated by filtration. Particles in the sinking particulate layer were separated by filtration. Particles of both types were dried in an oven at 65°C until constant weight. Both the fractions of microspheres were weighed, and buoyancy was determined by the weight ratio of floating particles to the sum of floating and sinking particles. Despite the solution being stirred for 12 h, the microspheres still floated, indicating that the microspheres exhibit an excellent buoyancy effect. Density values of the microspheres (3) were less than that of the gastric fluid (1.004 g/cm³), further supporting the floating nature. The in vitro floating test was conducted on the drug-loaded microspheres. Results of percentage buoyancy calculated using equation 4:

      % Buoyancy = (Weight of floating microspheres) / (Initial weight of microspheres) × 100                                                       (4)

Yield: Production yield of microspheres containing a drug was determined by the weight ratio of the dried microspheres to the loading amount of the drug and Polymer. Production yield was calculated using equation 5:

    % Production yield = (Total weight of the microsphere) / (Total weight of drug and polymer) × 100                                           (5)

In vitro drug release study: The release rate of Repaglinide from microspheres was determined using USP dissolution testing apparatus I (Basket type). The dissolution test was performed using 900 ml of 0.1 N HCL, at 37 ± 0.5°C and 100 rpm [8]. Microspheres equivalent to 25 mg were used for the test. Aliquots (5 ml) were withdrawn at hourly intervals for 12 h. Samples were replaced by its equivalent volume of dissolution medium. The samples were filtered through Whatman filter paper and solutions were analyzed using UV spectrophotometer (Shimadzu 1700 UV/V is double beam Spectrophotometer Kyoto, Japan).

In vitro drug release studies:

Apparatus                                   --      USP-I, Basket Method

Dissolution Medium           --             p H 0.1N HCL   

RPM                              --      100

Sampling intervals (hrs).    --             1, 2, 3, 4, 5, 6, 8, 10 & 12.

Temperature                -- 37°c + 0.5°C

Where, Mt is the amount of drug released at time t, M ∞ is the amount of drug released after infinite time, Kt is a kinetic constant and n is the diffusional exponent indicative of the drug release mechanism. Calculated from the slope of the plot of log of fraction of drug released.

Drug-Excipient compatibility studies

Fourier Transform Infrared (FTIR) spectroscopy: Drug excipient interaction studies are significant for the successful formulation of every dosage form. Fourier Transform Infrared (FTIR) Spectroscopy studies were used for the assessment of physicochemical compatibility and interactions, which helps in the prediction of interaction between drug and other excipients. In the current study 1:1 ratio was used for preparation of physical mixtures used for analyzing of compatibility studies. FT-IR studies were carried out with a Bruker, ATR FTIR facility using direct sample technique [10].

SEM (Scanning Electron Microscope) studies: The surface morphology of the layered sample was examined by using SEM (JEOL Ltd., Japan). The small amount of powder was manually dispersed onto a carbon tab (double adhesive carbon coated tape) adhered to an aluminum stubs were coated with a thin layer (300A) of gold by employing POLARON - E 3000 sputter coater [11]. The samples were examined by SEM with direct data capture of the images onto a computer.

RESULTS AND DISCUSSION

The present work was designed to developing Floating Microspheres of Repaglinide using various polymers. All the formulations were evaluated for physicochemical properties and in vitro drug release studies.

Standard graph of Repaglinide in 0.1N HCL: The scanning of the 10µg/ml solution of Repaglinide in the ultraviolet range (200-400 nm) against 0.1 N HCL the maximum peak observed at l_max as 241 nm. The standard concentrations of Repaglinide (2-10 µg/ml) was prepared in 0.1N HCL showed good linearity with R² value of 0.999, which suggests that it obeys the Beer-Lamberts law.

Evaluation Parameters (Tables 2 & 3, Figures 2-5)

The floating property of the microspheres was calculated from the fractional amount of drug and polymer density of the microspheres. As shown in Table 2, the Floating efficiency of the microspheres. As the concentration of HPMC increases in formulation the floating lag time decreases and % drug release is retard as the concentration. Ethyl cellulose acts as floating enhancer.

The high levels of sodium alginate lead to increased encapsulation efficiency. The percentage yield (%) is more for F5-F8 Formulations.

The % drug release of formulations (F5 to F8) containing HPMC, Ethyl Cellulose, Carbopol  depends on the concentration of Sodium Alginate (2.2%). In that F7 formulation was maximum drug release (99.87%) was showed at 12 h (Table 6).

Data of in vitro release studies of formulations which were showing better drug release were fit into different equations to explain the release kinetics of Repaglinide release. The data was fitted into various kinetic models such as zero, first order kinetics; higuchi and korsmeyer peppas mechanisms (Figures 8-11).

1.   Jain NK (2002) .Controlled. And Novel. Drug Delivery. CBS. Publishers. New.Delhi, India, pp: 236.

2.   Chein. YW (1992). Novel drug delivery systems. Marcel Dekker Inc, New York 50: 139-177.

3.   Sam MT, Gayathri. SD, Prasanth VV, Vinod B (2008) NSAIDs as microspheres. The Internet. J. Pharmacol 6: 67-73.

4.   Li SP, Kowalski CR, Feld.KM, Grim WM (1988) Recent advances in microencapsulation technology and equipment. Drug Dev Ind Pharm 14: 353-376.

5.   Nasa P, Mahant S, Sharma D (2010) Floating systems: A novel approach towards. gastroretentive drug delivery systems. Int J Pharmacy Sci 2: 27.

6.   Gayathridevi M, Adlin J, Nesalin J, Mani TT (2016) Floating microsphere: A review. IJRPC 6: 501-510.

7.   Audumbar DM, Rithesh S, Manoj KP (2015) Floating microspheres: A novel approach in drug delivery system GCC. J Sci Technol 1: 134-153.

8.   Griffith GH, Owen GM, Kirkman S, Shields R (l966) Measurement of rate of gastric emptying using Chromium-51. Lancet 1: 1244-1245.

9.   Kavitha K, Sudhir K, Yadav, Mani T (2010) The need of floating drug delivery system. Res J Pharma Biol Chem Sci 1: 396. [9]

10. Wilding IR, Coupe AJ, Davis SS (2001) The role of g-scintigraphy in oral drug delivery. Adv Drug Del Rev 46: 103-124.

11. Hafeez A, Maurya A, Singh J, Mittal A, Rana L (2013) An overview on floating microsphere: Gastro retention floating drug delivery system. (FDDS) J Phytopharmacol 2: 1-12.