POLYMER MEDIATED DELIVERY OF ANTIDIABETIC DRUG

Abstract

With the aid of engineering and biotechnology, biomedical and pharmaceutical engineering has emerged as a fascinating field with applications in diverse areas such as drug delivery, medical imaging and nanotechnology. In the pharmaceutical industry, novel drug delivery technologies represent a strategic tool for expanding drug markets. These new technologies can address issues associated with current pharmaceuticals, by extending product life or by adding to their performance and acceptability, either by increasing efficacy or improving safety and patient compliance. Oral delivery of drugs, especially proteins, is difficult since bioavailability is limited by the epithelial barriers of the gastrointestinal tract and gastrointestinal degradation by digestive enzymes. Carrier technology with biodegradable polymers offers an intelligent approach for drug delivery by entrapping the drug in a carrier particle such as microspheres or nanoparticles which modulate the release and absorption characteristics. Protein delivery through polymeric microspheres and nanospheres is expected to create innovations and play a critical role in drug delivery. Biodegradable polymer microspheres are preferred because surgical removal of spent device, as in the case of implants, is not required. Further, there is minimal possibility of toxicological problems. Their release rates can be tailored and they degrade in biological fluids to produce biocompatible or non-toxic products in the body, which are removed by normal physiological pathways. Both natural and synthetic biodegradable polymers have been studied for drug delivery purposes. Among the biodegradable polymers, poly-s-caprolactone (PCL) and its derivatives, with a high permeability to many therapeutic drugs and lack of toxicity, are well suited for oral and controlled drug delivery. Considerable research effort has been devoted to developing suitable, painless delivery systems to replace injectable insulin dosage forms. An oral insulin dosage form would be preferred by diabetic patients over the currently available parenteral formulations. Microspheres of PCL were prepared by using by w/o/w double emulsion solvent evaporation technique. The results showed the formation of smooth spherical PCL microspheres. The effect of different solvents (chloroform, dichloromethane and ethyl acetate) in combination with stabilizers (polyvinyl alcohol and hydroxypropyl methylcellulose) on the percent yield and surface morphology of PCL microspheres was studied. It was found that microspheres formed with dichloromethane as the solvent and HPMC as the stabilizer gave the best microspheres in terms of smoothness and were devoid of pits. Other formulational parameters like the stabilizers (PVA or HPMC) and their concentrations in the range of 0.5% to1.5%, polymer concentration in the range of 0.5% to 2.0% and the drug concentration, either 40 IHU/ml or 100 IHU/ml were varied. The responses were evaluated in terms of percent yield, percent entrapment efficiency, particle size and surface morphology. On the basis of these experiments, it was found that PCL microspheres prepared with 1% polymer concentration and 1% solution of HPMC as the stabilizer gave the optimum results of 92.2 ± 0.26% yield and an entrapment efficiency of 65.72 ± 0.14%. Micro BCA Protein Estimation Kit (PIERCE®) was used for the estimation of the amount of drug present in the microspheres. Surface morphology of the microspheres was studied by scanning electron microscopy. The morphology of the microspheres prepared with 1% PCL and 1% HPMC concentration was smooth and spherical, with the average mean particle diameter being 2.33 urn. The changes in morphology of the microspheres, when kept in contact with the release medium, with time were determined by scanning electron microscopy. Characterization of biodegradable polymeric microspheres used for drug delivery is essential to ensure reproducible results oiin-vitro and in-vivo drug release profiles. Different advanced techniques were used to further characterize the microspheres prepared. Confocal laser scanning microscopy was used to determine the drug distribution within the microspheres. This was achieved by fluorescently labeling insulin with FITC and PCL with nile red and viewing the microspheres formed under laser adjusted in the green/red fluorescence mode resulting in two excitation wavelengths at 488 and 514 nm. Drug polymer interaction was studied by differential scanning calorimetry (DSC). The DSC curves of unloaded PCL microspheres and insulin-loaded PCL microspheres were compared. The Tm of PCL polymer was found to be 66°C, and Tm, for the unloaded and insulin-loaded PCL microspheres was found to be 59.7°C and 62°C respectively confirming no major change in the Tm of the polymer, since polymers usually exhibit a melting range instead of a sharp melting point. Residual solvent in the microspheres was determined by gas chromatography. Residual content of dichloromethane was found to be well within the pharmacopoeial limits of 600 ppm and indicated safe use of microspheres for oral administration. The in-vitro insulin release profile from the PCL microspheres was evaluated. An initial burst release, followed by a slower release of insulin from the microspheres was observed. Since PCL is a slow degrading polymer, the predominant mechanism of insulin release from these microspheres is diffusion. The initial burst release may be explained due to the drug desorption from the particle surface. The effect of polymer, stabilizer and drug concentration on the in-vitro release profiles was also studied. It was found that microspheres prepared with increased polymer concentration showed the least drug release. When microspheres were formulated with a low stabilizer concentration in the external water phase, they showed a more rapid insulin release. A correlation was also established between the encapsulation efficiency and the release rate. Results showed that higher the insulin concentration used, higher was the insulin encapsulation within the microsphere which further led to a more rapid release rate. The biological efficacy of the insulin-loaded microspheres was determined after oral administration in diabetic rats and rabbits. In order to determine hypoglycemic effect of the insulin-loaded PCL microspheres, the microspheres were administered orally to overnight fasted diabetic rats and rabbits. Blood glucose concentrations were measured by AccuChek blood glucometer (Roche, Germany) and the changes in blood glucose level versus time profiles were observed. The results showed that the encapsulation of insulin into microspheres prepared with PCL allowed the preservation of its biological activity alongwith its prolongation of action, following its oral administration in fasted and fed diabetic animal models. To further evidence our in-vivo findings, pharmacoscintigraphic evaluation was carried out in the animal models to ascertain the absorption and distribution characteristics of the radiolabeled drug. Insulin was radiolabeled with radionuclide, 99mTc, and encapsulated into the polymeric microspheres. The radiolabeling efficiency of the 99mTc-insulin radiocomplex was evaluated by instant thin layer chromatography (ITLC) and found to be 99.2%. Stability studies of 99mTc-insulin complex revealed that the radiocaomplex retained high stability (97%) both in saline and serum. In order to determine the absorption and distribution characteristics of the radiolabeled insulin, gamma camera imaging of diabetic Male New Zealand rabbits and Sprague-Dawley rats administered with 99mTc-insulin loaded microspheres, free 99mTc-insulin and 99mTc-insulin loaded microspheres administered alongwith ethanol as enhancer was carried out. The gamma camera images showed showed that after 4 hours of oral administration, animals fed with 9 mTc-insulin loaded PCL microspheres showed significantly higher diffusion into the stomach and intestine besides some diffusion in other tissues like paws and visceral organs as compared to those fed with free 99mTc-insulin without loading into PCL microspheres. Biodistribution of free 99mTcinsulin and 99mTc-insulin loaded PCL microspheres in heart, blood, liver, stomach and intestine in Sprague-Dawley rats was studied at various time intervals after its oral administration. It was observed that among all the organs studied, stomach exhibited highest radioactivity per gram organ for both 99mTc-insulin loaded PCL microspheres and free 99mTc- insulin. Onthe contrary, intestine and stomach showed highest radioactivity per whole organ for mTc-insulin loaded microspheres and free 99mTc-insulin respectively. To determine any toxic effects of the insulin-loaded PCL microspheres, toxicity studies were carried out in Sprague-Dawley rats. Histopathological studies of liver, stomach, small intestine and large intestine were carried out. Results showed that two month of repetitive dose of PCL microspheres to experimental animals showed no adverse effect on histopathology of the tissues studied.