Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide

Transcription

1 2756 International Int J Pharm Journal Sci of Nanotech Pharmaceutical Vol 8; Issue Sciences 1 January and Nanotechnology March 2015 Research Paper Volume 8 Issue 1 January March 2015 MS ID: IJPSN NAMDEV Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide Shrishti Namdev*, Kishore Gujar, Satish Mandlik and Preeti Jamkar Department of Pharmaceutics, Sinhgad College of Pharmacy, Vadgaon (Bk), Pune , Maharashtra, India. Received July 23, 2014; accepted December 23, 2014 ABSTRACT The objective of this study is to prepare and characterise repaglinide niosomes using the Factorial Design strategy. Repaglinide is a potent second-generation oral hypoglycemic agent and has short half-life of 1 hour and oral bioavailability of 50%. Preparing Niosomal drug delivery of repaglinide may increase its bioavailability which would lead to better therapeutic effects, reduce the frequency of dosing from twice a day to once a day and decrease side effects. The preliminary study was carried out for selection of surfactant and method of preparation based on least particle size and highest entrapment efficiency. For niosome preparation, organic solvent injection method was selected and span 60, cholesterol were selected as variable. A32 factorial design was used to optimize the effect of amounts of span 60(X1) and cholesterol (X2) which were the independent variables. Particle size (Y1) and entrapment efficiency (Y2) were the dependent variables. Relation between the dependent and independent variables were drawn out from the mathematical equations and response surface plots. Statistical analysis was performed using ANOVA which was found to be significant and quadratic equation was obtained by MLRA. The particle size was found to be in range of nm and entrapment efficiency between 54-88%. Scanning electron microscopy indicated the spherical shape of the niosomes and formation of vesicle. Zeta potential analysis showed negatively charged surface with value of-36.7 mv. In vitro drug release profile showed that drug released fast initially followed by a slow release. In vivo pharmacokinetic study revealed that the niosomal preparation showed significant decrease in blood glucose level when compared to free repaglinide. The developed niosomal system also has potential of maintaining therapeutic level of RPG for longer period of time.thus,the niosomes could be promising carriers for delivery of repaglinide with increased oral bioavailability and reduced dosing frequency. KEYWORDS: Solvent injection method; factorial design; repaglinide; particle size; entrapment efficiency. Introduction Diabetes mellitus is a widespread disease affecting large fraction of population worldwide. As per the current estimate around million (2.8%) people were suffering from diabetes in the year 2000 and it is predicted to be increased approximately upto million (4.4%) by the year 2030 (Wild et al., 2004). Diabetes mellitus is a major risk factor for heart disease, stroke and peripheral vascular diseases and is associated with marked morbidity and mortality (Dunstan et al., 2002). First line of treatment usually comprises dietary control and weight loss, but nearly about 50-70% of patients require an oral antidiabetic drug. Numerous research is going on, in this field either to decrease the cost of the product or increase patient compliance. Vesicular systems offer a novel means of drug delivery that can improve bioavailability of encapsulated drug and provide therapeutic activity in a controlled manner for a prolonged period of time. Niosomes (nonionic surfactant vesicles) are the type of vesicular system that offers several advantages over conventional dosage form and micelles because the particles can act as drug containing reservoirs. Niosomes are non-ionic surfactant vesicles which are formed from self-assembly of hydrated synthetic non-ionic surfactant monomers along with cholesterol and or addition of other lipids. They are vesicular system that offers many similarities to liposomes and can be evaluated as an alternative to liposomes as it avoids some problems associated with liposomes such as chemical instability, variable purity of phospholipids and high cost (Uchegbu and Vyas, 1998, Pratap et al., 2009). They can be used as carriers of hydrophilic and hydrophobic drugs and also they can encapsulate variety of drugs having poor bioavailability (Akhilesh et al., 2012). Niosomes being non-ionic are promising vehicle for drug delivery. Since the drug is encapsulated in the vesicular structure it can be predicted that the existence of the drug in the systemic circulation will be prolonged and which in turn may increase penetration into target tissue thereby reducing the toxicity and enhancing the therapeutic performance 2756

2 Namdev et al: Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide 2757 of the drug (Rangasamy and Ayyasamy, 2008). In recent years, niosomes have been extensively studied for their potential to serve as carriers for delivery of drugs, antigens, hormones and other bioactive agents. In niosomes, the aqueous compartment entraps the soluble drug molecules whereas insoluble drug moiety is entrapped within the bilayer matrix (Ruckmani and Sankar, 2010). A second-generation oral hypoglycemic agent Repaglinide (RPG) is a potent fast and short acting meglitinide analog used widely in the treatment of noninsulin dependent diabetes mellitus (NIDDM). It acts by binding to specific site on beta cells of pancreas and blocks ATP dependent potassium channels which in turn cause opening of calcium channels and resulting in calcium influx and thus induces insulin secretion to stimulate insulin release. As far as the specific properties of the repaglinide are concerned, it has short half-life of about 1 hr and has low bioavailability (50%) and absorption in the upper intestinal tract is poor (DhanaLekshmi et al., 2011). Frequent dosing beforemeal is required due to its short half-life and this may cause various side effects like headache, skeletal muscle pain and gastrointestinal effects (Culy et al., 2001). It is intended to take thrice or quarterly a day. Moreover it produces hypoglycemia after oral administration. Since these drugs are intended to be taken for a long period, patient compliance is also very important and is to be looked at. To overcome these demerits of such a useful drug, Repaglinide our rationale is to prepare repaglinide loaded niosomes which will overcome its low bioavailability and reduce its frequency of dosing by providing sustained release. This will also have an advantage of surpassing the first pass metabolism of repaglinide. These in turn will reduce side effects and thus provide patient compliance. The niosomal system is supposed to improve bioavailability of poorly bioavailable drugs by crossing the anatomical barrier of gastrointestinal tract via transcytosis of M cells of Peyer's patches at the intestinal lymphatic tissues (Rentel et al., 1999). A possible explanation for this sustained release effect (Attia et al., 2007) is that niosomes act as a carrier and a slow release vehicle. The drug is carried by the niosomes through the epithelium into deeper layers of the mucosa, where the encapsulated drug is slowly released. This sustained release effect can improve the bioavailability of drugs with slow and limited absorption and narrow absorption windows. There are various methods of preparation of Niosomes viz thin film hydration technique, organic solvent injection method, reverse phase evaporation method, etc. Considering cost, reproducibility and ease of preparation organic solvent injection method was selected for preparing repaglinide loaded niosomes. Nonionic surfactant Span 60 was used for formation of vesicle and cholesterol was added which provides rigidity and orientational order to the niosomal bilayer (Girigoswami et al., 2006). Particle size and entrapment efficiency were the key parameters involved in the preparation of niosomes. Several preparation and processing variables are involved during niosome preparation which may affect these parameters and hence the performance of the preparation. Thus it becomes extremely difficult to study the effect of interactions between various variables and preparation of niosome by a conventional method. Factorial design and response surface methodology is an important statistical tool to study the effect of several factors influencing responses by varying them simultaneously by carrying out limited number of experiments (Mandlik et al., 2009). Materials and Methods Materials and chemicals: Repaglinide as a core material for the niosome preparation was obtained from Wockhardt Research Centre, Aurangabad (M.S.) India as a gift sample; Span 20, span 40, span 60, span 80 and cholesterol were obtained from LobaChemiePvt. Ltd., Mumbai. All other chemicals were of analytical grade. Distilled water was produced in house using distillation unit. Methodology Drug-excipient compatibility study: Drug Excipient compatibility studies were done in order to evaluate any interaction between drug and polymers used in preparation of niosomes. The physical mixture of repaglinide with formulation excipients in 1:1 ratio was placed in glass vials which were kept at room temperature. After 15 days samples were observed for physical changes like color/appearance and chemical changes were observed by FTIR spectroscopy and DSC studies. Fourier Transform Infrared Spectroscopic Analysis (FTIR): To study the interactions between drug and excipients, FTIR study was carried out. Samples were mixed with IR grade KBr in the ratio of 1:100 and mixtures were compressed to form pellets by applying 10 tons of pressure in hydraulic press. The pellets were scanned over a wave number range of 4000 to 400 cm 1 in FTIR instrument (Perkin Elmer, Spectrum Bx) and spectra were analysed. Thermal Analysis- Differential Scanning Calorimetry (DSC): Thermal analysis was performed using a Mettler Toledo DSC-823 e system with a differential scanning calorimeter equipped with a computerized data station. The differential scanning calorimetry analysis gives an idea about the interaction of various materials at different temperature. It also indicates the possible degradation of the material. DSC detects phase transition such as glass transition (exothermic) crystallization and (endothermic) melting. DSC thermograms were obtained to define the physical state of the drug and excipients. Preparation of Niosomes Preliminary studies were carried out for the selection of method of preparation, surfactant and its concentration range. Repaglinide loaded niosomes were

3 2758 Int J Pharm Sci Nanotech Vol 8; Issue 1 January March 2015 prepared by organic solvent injection technique reported by Pratap et al, 2009 with minor modifications. Briefly, non-ionic surfactant span 60 and cholesterol in different concentration ratios were weighed accurately and this mixture was dissolved in organic phase containing 6 ml chloroform. Repaglinide 10 mg (1:1 ratio of surfactantcholesterol: drug) solubilised in 2 ml methanol was then dissolved in organic phase. The resulting solution was slowly injected through 18 guage needle at a rate of 1 ml/min into 30 ml of the hydrating solution i.e., phosphate buffer (ph 7.4). The solution was stirred continuously on magnetic stirrer and temperature was maintained at o C. As the lipid solution was injected slowly into aqueous phase, the differences in temperature between phases cause rapid vaporization of organic solvent, resulting in spontaneous vesiculation and formation of niosomes. Factorial design of experiment Factorial design is used to evaluate two or more factors simultaneously. It is more efficient and allows the interactions to be detected. To study the effect of input variables on the niosomal preparations 32 factorial design was adopted. In this design, 2 factors were evaluated each at 3 levels and experimental trials were performed using all possible 9 combinations. Amount of surfactant span 60 (X1) and cholesterol (X2) were selected as two independent (input) variables whereas particle size (Y1) and entrapment efficiency (Y2) were chosen as dependent (response) variables. As per the design different preparations of niosomes were prepared by varying the concentration of span 60 and cholesterol in order to select an optimized formula. The experimental design matrices are mentioned in Table 1 and Table 2. TABLE 1 Design matrix for niosome preparations. Independent (Input) Variables Factors Levels Low Medium High X1 (Span 60) X2 (Cholesterol) Dependent (Output) Variables Responses Constraints Y1 Particle Size (nm) Minimum Y2 Entrapment Efficiency (%) Maximum TABLE 2 Preparation of RPG niosomes by 3 2 design. RUN Coded Values Actual Values (mg) X1 X2 X1 X2 R R R R R R R R R Characterisation of niosomal suspension Particle size and polydispersity index (PDI) determination: The mean particle size of niosomes was determined by Photon Cross-correlation Spectroscopy (PCCS) using NanophoxSympatec GmbH, Germany at room temperature by keeping angle of detection at 90 o. The average particle size for all batches was recorded. The polydispersity index reflects the uniformity of particle diameter and it can be used to depict the size distribution of niosomes. The polydispersity index (PDI) value was determined as a measurement of the breadth of the size distribution: a PDI value lower than 0.3 indicates a homogenous and monodisperse population. Surface charge determination (zeta potential): The stability of niosome is directly related to the magnitude of the surface charge. The surface charge of RPG niosomes was determined by Photon Cross-correlation Spectroscopy (PCCS) using Beckman Coulter (DelsaNano) zetasizer. The mean zeta potential of the optimised batch was recorded. Surface morphology study (SEM): The surface morphology of repaglinide loaded niosomes was analysed by taking the micrographs using scanning electron microscopy (SEM, JSM-6330 TF, Joel, Tokyo, Japan). High resolution SEM analysis of the optimized batch was carried out to understand the morphology and the surface characteristics of the niosomes. Entrapment efficiency (EE): Entrapment efficiency was determined by centrifugation method (Ruckmani et al., 2000, Chawda et al., 2011). The formulated niosomes were taken in centrifuge tubes. The temperature of the instrument (REMI-24C BL, Germany) was maintained at 4 o C. This solution was then allowed to centrifuge at rpm for 60 min. Centrifugation lead to formation of pellet. The supernatant was separated and pellet was re-dispersed in 5 ml of phosphate buffer PH 7.4. The amount of drug entrapped in niosome was estimated by UV spectrophotometer (Shimadzu 1800, Japan). Absor-bance of the supernatant was recorded at 278 nm. Encapsulation efficiency (EE) of niosomes was calculated as follows: % EE = Total amount of drug added Amount of drug in supernatant 100 Total amount of drug added In vitro drug release study: On the basis of literature survey phosphate buffer ph 7.4 was used as a dissolution media. Preparation (equivalent to 10 mg of drug) was used. Dissolution studies (Umah Rani and Vignesh, 2012) were conducted in a dissolution apparatus using USP II paddle method. Dialysis method was used to evaluate the in vitro release. Five millilitres of niosome suspension was placed in a dialysis bag (cellophane membrane, molecular weight cut off 10,000-12,000, Hi- Media, India), which was then sealed at both ends. The dialysis bag was dipped into the receptor compartment containing the dissolution medium, 300 ml of phosphate buffer (ph 7.4), was stirred continuously at 100 rpm and

4 Namdev et al: Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide 2759 maintained at 37 C. At selected time intervals, aliquots were withdrawn from the release medium and replaced with the same amount of phosphate buffer. The sample was assayed spectrophotometrically at 279 nm. By determining the amount of drugs released at various time intervals the cumulative % release of drugs versus time (hr) graphs were plotted. Dissolution kinetic studies: To understand the mechanism of drug release, the data obtained from the in-vitro drug release study of niosomes (R2 batch) was fitted into various kinetic model equations like zero order (cumulative % release vs time), first order (log % drug remaining vs time), Higuchi s model (cumulative % drug release vs. square root of time), and the Korsemeyer- Peppas (log cumulative % drug release vs log time) and Hixson- Crowel models (cubic root of drug remaining vs time). (Ruckmani and Sankar, Tamzhaini et al, 2009). The correlation coefficient (r 2 ) and k values were calculated for the linear curve obtained by regression analysis of the above plots. The study was carried out using PCP-Disso-v3 software, Pune India. RP-HPLC method development: The quantitative determination of RPG was performed by reverse phase high performance liquid chromatography (RP-HPLC). A gradient HPLC (Shimadzu LC 2010 CHT, Japan) with VP Pump, variable wavelength programmable UV/VISIBLE Detector SPD-10 A, VP, CTO-10AS Controller and RP-C18 Column Cosmosil, ODS-3V, 250 x 4.6 mm, particle size 5 µ was used. Mobile phase used was a mixture of acetonitrile and ammonium acetate buffer (ph adjusted with ortho phosphoric acid to 4) in a ratio of 80:20 % v/v. The filtered mobile phase was pumped at a flow rate of 1.0 ml/min.; Column temperature was maintained at C. The eluent was detected by UV detector at 230 nm and data were acquired, store and analyzed with software class ODS- 3V. Stability of niosomes: Stability of selected niosome dispersion (R2) was carried out at (4 ± 2 o C) and room temperature for period of 2 months (Adel et al., 2014). Effect of temperature was studied by checking parameters like colour, odour, sedimentation and extent of leakage during the stability period. To determine extent of leakage, % entrapment efficiency after storage of sample was calculated after 2 months and then correlated with the extent of leakage which was calculated as follows: Extent of leakage = (initial % entrapment efficiency) (%retained after storage) Statistical analysis: Statistical optimization was performed using registered Design-Expert 8.0 software (Stat-Ease Inc., MN, USA). In 32 Factorial design, the effects of various independent variables upon measured responses were statistically analysed for multiple linear regression analysis (MLRA) generally depicted in following mathematical model equation which involves effect of independent variables and their interactions for measured responses: Y = 0 + 1X1 + 2X2 + 3X1X2 + 4X X2 2..(1) where Y is the dependent (response) variable, while β0 is the intercept, β1-5 are regression coefficients; X1, X2 are independent variables; X1X2 is interaction between variables and X12, X22 are the quadratic effect. The mathematical polynomial equations were used to draw conclusions after considering the magnitude of coefficient and the mathematical sign it carries (i.e., negative or positive). Positive or negative signs before a coefficient in quadratic models indicate a synergistic effect or an antagonistic effect for the factor. The significance of the factors and their combinations was evaluated by the analysis of variance (ANOVA). The results of the responses were graphically represented using response surface and contour plots: Response surface plots are three dimensional (3-D) graphical representation of a response plotted between two independent variables and one response variable. The use of 3-D response surface plots allows understanding of the behaviour of the system by demonstrating the contribution of the independent variables. Contour plots are the geometric illustration of responses, obtained by plotting one independent variable versus another while holding the magnitude of response level and other variables constant. The slices of response surface are represented by the contour plots. Validation of design by numerical optimization: Numerical optimization was performed in order to predict a Niosome preparation with particle size less than the 200 nm and maximal % yield from the model generated. By numerical optimization method two check point batches (V1 and V2) were prepared to validate the model In vivo Animal study: In vivo pharmacodynamic study was conducted on male wistar rats. All procedures and protocols used in this study were reviewed and approved by the Institutional Animal Ethical Committee (IAEC) with permission number of SCOP/IAEC/ /146. Ethical guidelines were strictly followed during all the experiments. Pharmacodynamic study: To study the anti-diabetic activity of niosomes, streptozotocin induced diabetes model was used. Animals were divided in 4 groups each containing 6 animal. Streptozotocin (40 to 60 mg/kg i.p.) was given to induce diabetes in groups II, III, IV. After 72 hr blood samples were withdrawn from the tail vein of rats to measure glucose levels. Animals with fasting blood glucose levels above 200 mg/dl were considered diabetic and used for further study. On 14th day, blood glucose was estimated to check diabetes is stabilized or not. Rats showing blood glucose level more than 200 mg/dl was used. On 15th day, repaglinide conventional dosage form and repaglinide niosome were given to group III and IV respectively. Blood glucose estimations were determined for following time periods: 2hr, 4hr, 6 hr, 8 hr (Vijayan et al., 2011; Behera et al., 2012). 1. Group I : Normal Control 2. Group II : Diabetic Control (40 mg/kg STZ) 3. Group III : Repaglinide conventional dosage form (2 mg/kg p.o.) 4. Group IV : Repaglinide niosome (2mg/kg p.o.)

5 2760 Int J Pharm Sci Nanotech Vol 8; Issue 1 January March 2015 Results and Discussion Drug-excipient compatibility study: Physical compatability test was carried out with drug and excipients to determine drug-excipient interaction/compatibility. After 15 days samples were evaluated for colour/ appearance for compatibility testing and there is no change observed in appearance. Fourier Transform Infrared Spectroscopic Analysis (FTIR): The FTIR spectrum of Repaglinide is shown in Fig. 1. FTIR spectra of drug-excipient mixtures retained the characteristic functional peaks of the drug, from the report it can be concluded that there is no interaction between drug and excipients Fig. 2. Fig. 1 IR spectrum of pure Repaglinide. Fig. 2 Drug excipients interaction study: IR spectra of A. pure repaglinide, B. drug + cholesterol mixture, C. drug + span 60 mixture. Fig. 3 DSC Thermo-grams of RPG (A), RPG + Cholesterol (B), RPG + Span 60(C).

6 Namdev et al: Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide 2761 Thermal analysis (Differential Scanning Calorimetry): The DSC thermogram of pure Repaglinide indicated a sharp endothermic peak at C corresponding to melting of pure Repaglinide. The peaks broadening as well as change in relative intensities were observed due to dilution of drug in physical mixtures of drug with excipients. It can be concluded that the excipients and drug did not interact with each other. Also the drug did not form a complex with the excipients as the endothermic peaks remained unchanged in position Fig. 3. Particle size determination: The particle size of repaglinide loaded niosomes of the different batches according to the factorial design ranged between 144 to 497 nm. The polydispersity index (PDI) was in the range of 0.21 to for repaglinide loaded niosomes which indicates a narrow vesicle size distribution. The particle size and PDI of all the nine batches of factorial design is shown in Table 3. TABLE 3 Particle size analysis, polydispersity index and entrapment efficiency. Batch code Particle size (nm)* Polydispersity Index* Entrapment efficiency (%) * R ± ± ± 0.21 R ± ± ± 0.33 R ± ± ± 0.87 R ± ± ± 0.14 R ± ± ± 0.42 R ± ± ± 0.23 R ± ± ± 0.56 R ± ± ± 0.76 R ± ± ± 0.47 *values expressed are mean ± S.D (n = 3) Entrapment efficiency: Entrapment efficiency was expressed as fraction of drug incorporated into niosomes relative to total amount of drug used. In the present study the observed percentage entrapment efficiency for all batches were in the range of 54%-88%. It was observed that Entrapment efficiency was significantly affected by the applied processing variables such as concentration of span 60 as well as cholesterol concentration Selection of optimum niosome preparation: Based on the predefined constraints, batch R2 was found to be optimum niosome preparation and it was selected for further characterization. Surface charge Determination (Zeta ( ) Potential): The value of zeta potential of the optimized repaglinide loaded niosomal preparation (R2) was found to be mv which indicates the stability of the prepared niosomes as shown in Fig. 5. The zeta potential value of mv indicates the high negative surface charge on niosomes which leads to high stability because of the anticipated surface repulsion between similar charged particles hence inhibiting aggregation of the particulate niosomes. It has been reported that a physically stable niosome solely stabilized by electrostatic repulsion will have a minimum zetapotential of 30 mv. Surface morphology study (SEM): Scanning electron microscopy of optimized niosomal preparation was performed by Scanning Electron Microscope for determining the surface morphology, size and shape of preparation and also to confirm the formation of vesicles. SEM revealed spherical shape of the particles. The surface of niosomes was smooth, vesicles are nearly uniform and particle size is as desired. The SEM of optimized niosome batch R2 is shown in Fig. 6. In-vitro drug release: The cumulative percentage release of preparations R1-R9 ranges from ± 0.39 to ± 0.26%. The release profile of the RPG from the niosomes in PBS ph 7.4 is shown in Fig. 7. Amount of surfactant and cholesterol affected the drug release profile, drug release decreases with increase in concentrations of cholesterol and increases with increase in concentration of surfactant. All preparations showed burst release initially followed by sustained release; burst release was due to dissolution of surface drug. The drug release at the end of 8 hours was found to be up to 75% for the R2 batch. Fig. 4 Graph of particle size of R2 batch.

7 2762 Int J Pharm Sci Nanotech Vol 8; Issue 1 January March 2015 Fig. 5 Zeta potential deter-mination of R2 batch. Fig. 6 SEM analysis of optimised batch. Fig. 7 Drug release profile.

8 Namdev et al: Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide 2763 Kinetic model fitting of dissolution data: The in vitro release data of optimised preparation R2 was applied to various kinetic models to predict the drug release kinetic mechanism. The first order kinetic model was found to be best fit model. The value of the coefficient of regression suggests the best fit kinetic model. From interpretation given in Table 4 and Fig. 8 indicates the best fitted kinetic model was found to be first order model (Costa and Lobo, 2000). TABLE 4 Various model fittings for optimized formulation. Model r 2 K Zero order st order Matrix Peppas Hixson Crowell HPLC method development: The calibration curve for RPG was found to be linear in the concentration range of μg/ml. Calibration typical chromatogram of RPG is shown in Fig. 9. Under the experimental conditions described, all peaks were well defined and free from tailing. The retention time (Rt) was 6.23 min. Stability studies: The stability study of optimized niosomal suspension was carried out at refrigeration (4 ± 2 o C) and room temperature for period of 2 months. At above conditions, the preparation retained the original colour (white) and odour (Characteristic), no sedimentation or creaming was observed after 2 months. Also, there was no significant change in particle size. These results indicated that prepared niosomes had adequate stability. Fig. 8 In-vitro drug release kinetic study (R2). Fig. 9 Typical chromatogram of RPG (100 µg/ml) at 230 nm using RP HPLC.

9 2764 Int J Pharm Sci Nanotech Vol 8; Issue 1 January March 2015 Extent of leakage: The extent of leakage of drug from the vesicles upon storage was determined after 1 month by HPLC analytical method which is as shown in Table 5. After keeping this preparation for stability studies at refrigeration temperature (4 ± 2 o C) and at room temperature for one month drug retained was determined and found to be 99.78% and 98.04%. This gave an amount of drug leaked from vesicles i.e., 0.22% and 1.96% for refrigeration temperature and room temperature respectively. Hence, it can be claimed that niosomes are more stable at refrigeration temperature. Statistical analysis: Particle Size: To understand the effect of independent variables on particle size of the drug loaded niosomes, statistical analysis was carried out and the quadratic model was found to be adequate model and its polynomial equation is shown below: Y1 = X X X1X X X 2 2..(2) The results of multiple linear regression analysis (MLRA) showed that both the coefficients β1 and β2 bear a positive sign. Therefore, increasing the concentration of either Span 60 or cholesterol is expected to increase the particle size. A good correlation was observed for both variables X1 (Span 60) and X2 (Cholesterol) in vesicle size of drug loaded niosomes (r 2 = ). The statistical evaluation was performed by ANOVA and the model was found to be significant. TABLE 5 Stability study of niosomes Parameters At refrigeration temperature (4 ± 2 o C) At room temperature Time Freshly Prepared After 1 month Freshly Prepared After 1 month Mean area (repaglinide) Amount retained (%) Amount leaked (%) Fig. 10 Chromatogram of freshly prepared niosomal suspension. Fig. 11 Chromatogram of niosomal suspension at refrigeration temperature.

10 Namdev et al: Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide 2765 The response surface plot showing the effect of different proportion of independent variables on the response Y1 (PS) is shown in Fig. 13. It was observed that the relative amount of span 60 and cholesterol was found to play an important role in determining particle size. Particle size of drug loaded niosomal batches were found to increase as concentration of span and cho-lesterol increases. Hydrophobic repaglinide intercalate into the lipid bilayer leading to appreciable cohesion among a polar portion of the membrane, causing reduction in the vesicle size. Therefore, it can be derived that the change of both independent variables had significant effect on response Y1(PS). Entrapment Efficiency: To understand the effect of Span 60 and Cholesterol concentration on EE, the statistical analysis was performed and quadratic model was found to be best fit model. The obtained polynomial equation is shown below. Y2 = X X X1X X X 2 2..(3) The results of multiple linear regression analysis (MLRA) showed that the coefficient β1 bear a positive sign and β2 bears a negative sign. Therefore, increasing the concentration of Span 60 is expected to increase the EE and increasing the concentration of cholesterol decreases the EE. The response surface plot representing the effect of independent variables on EE is shown in Fig. 14. It was clearly indicated that entrapment efficiency decreases with increase in concentrations of cholesterol and surfactant (span 60). The highest entrapment efficiency was found to be at the lowest concentration of span 60 and cholesterol. Therefore, it can be derived that the change of both independent variables had significant effect on response Y2 (%EE). Validation of optimized preparations/check point analysis: By numerical optimization method, two checkpoint batches (V1 and V2) were prepared and evaluated to validate the model's ability to predict response; the experimentally observed values of the responses were compared with that of the predicted values, and the prediction error for the two responses was found to be varying between 3.59 and 1.92%. Table 6 lists the predicted and experimentally observed values of all the response variables, and the percentage error in prognosis. The theoretical (predicted) values were compared with the experimental (observed) values and were found to be in reasonably close agreement. Fig. 12 Chromatogram of niosomal suspension at room temperature. Fig. 13 Response surface plot of Y1 (particle size).

11 2766 Int J Pharm Sci Nanotech Vol 8; Issue 1 January March 2015 Fig. 14 Response surface plot of Y2 (entrapment efficiency). Fig. 15 Hypoglycemic effect of niosomes. TABLE 6 Comparison of experimentally observed values with predicted responses. Check point batches V1 V2 Response variables Predicted value Observed value Residual* % Error** Y1 (nm) Y2 (%) Y1 (%) Y2 (nm) *residual = actual (experimental) value predicted value. **error = [predicted value experimental value/predicted value] 100. TABLE 7 Mean of blood glucose level in rats. Time (hrs) Normal* Diabetic control* Free repaglinide* Niosome Formulation* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.34 *values expressed are mean ± S.D (n = 6)

12 Namdev et al: Preparation and In Vivo Characterization of Niosomal Carriers of the Antidiabetic Drug Repaglinide 2767 In vivo animal studies Pharmacodynamic study: In the present study intraperitoneal injection of streptozotocin (60 mg/kg) produced significant elevation in serum glucose level and produced hyperglycemia. Streptozotocin-induced diabetic rats exhibited hyperglycemia. The repaglinide drug and niosome preparation was studied for antihyperglycemic activity. The blood glucose level was compared with the conventional dosage form, it was found that the blood glucose level first decreased and then increased after 4 hr in conventional dosage form, whereas niosomes showed constant decrease in blood glucose level for 8 hr. Conclusions Repaglinide loaded Niosomes were successfully formulated with average particle size 144 nm and 87% entrapment efficiency for R2 batch. FTIR and DSC studies showed that the drug and polymers were compatible. The application of factorial design revealed that the concentration of span 60 and cholesterol significantly affect the dependent variables, particle size, and entrapment efficiency. Through in vitro release studies it was concluded that the niosomes showed prolonged drug release for 8 hr. It was observed that the increase in the surfactant concentration showed good vesicle formation. SEM studies confirmed that the smooth vesicles were formed properly. The in vivo pharmacodynamic study suggested that the blood glucose level was decreased more in RPG niosomes as compared to free RPG. From the present study, it can be concluded that niosomes can be used efficiently for enhancing bioavailability and reduce the dosing frequency of repaglinide from twice a day to once a day. Acknowledgements We are thankful to Wockhardt Research Centre, Aurangabad (M.S.) India for providing Repaglinide as a gift sample. We acknowledge BCUD Pune University for financing the project. 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Global Prevalence of Diabetes, Estimates for the year 2000 and Projections for Diabetes Care 27: Address correspondence to: Shrishti Namdev, Department of Pharmaceutics, Sinhgad College of Pharmacy, Vadgaon (Bk), Pune , Maharashtra, India. Mob: ; shrishti.namdev@gmail.com