Development of Lipid based drug delivery systems (LBDDS) for immunomodulators and their evaluation using experimental animal models

Abstract

Immunosuppressive drugs or immunosuppressive agents are drugs that inhibit or prevent activity of the immune system. They are used in immunosuppressive therapy to prevent the rejection of transplanted organs (like liver or kidney) and tissues (e.g., bone marrow, heart, kidney, liver), and to treat autoimmune diseases or diseases that are most likely of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupuserythematosus, sarcoidosis, focal segmental glomerulosclerosis, Crohn's disease, Behcet's Disease, pemphigus, and ulcerative colitis). Immunology is one of the most rapidly developing areas of medical biotechnology research and has great promises with regard to the prevention and treatment of a wide range of disorders. The success of solid organ transplantation lies in the appropriate utilization of immunosuppressive medications. Objectives of present investigation were to improve solubility, dissolution rate and their by bioavailability or efficacy of immunosuppressant drugs (tacrolimus and sirolimus) by development of lipid based drug delivery systems. Tacrolimus, a lipophilic 23-member macrolide lactone isolated from Streptomyces tsukubaensis (molecular weight of 803.5 Da). The half-life of tacrolimus in human is 8.7– 11.3 h. Tacrolimus has low solubility and is a subtract of P-gp efflux pump and cytochrome P450 3A4 enzyme system (CYP3A4). The mean bioavailability is approximately 21%. Sirolimus is a macrocyclic lactone produced by the Streptomyces hygroscopicus. Sirolimus is practically insoluble in water and contains no ionizable functional groups. Sirolimus is metabolized by the CYP3A4. The oral bioavailability of sirolimus is approximately 14%. From the various available formulation strategies, lipid based drug delivery system was selected. Solid dispersion and self-emulsifying drug delivery system were developed as lipid based drug delivery systems. Solid dispersion refers to a group of solid products consisting of at least two different components, generally a hydrophilic matrix and a hydrophobic drug. Solid dispersions (SD) have attracted considerable interest as an efficient means of improving the dissolution rate and hence the bioavailability of a range of hydrophobic drugs. Major reported mechanisms for improving solubility are reduction in particle size, improved wetting property and conversion of crystalline state to amorphous state. Self-emulsifying or micro emulsifying (SMEDDS) formulation contains oil (synthetic or natural) with hydrophilic or lipophilic surfactants and co-surfactant. SMEDDS improve as well as normalize drug absorption, which is particularly beneficial for low therapeutic index drug like tacrolimus. These formulations enhanced absorption by a number of mechanisms like improving drug solubility and maintaining drug in solution state throughout GI tract, inhibition of P-glycoprotein-mediated drug efflux and pre-absorptive metabolism by gut membrane-bound cytochrome enzymes and also by promotion of lymphatic transport. Solid dispersion of tacrolimus and sirolimus has been prepared by using gelucire 44/14 and gelucire 50/13 along with lactose monohydrate as an adsorbent with a view that adsorbent will prevent or reduce recrystallization of drug or carrier on storage. Carrier was selected based on phase solubility study. Solid dispersions were prepared at three different ratios (1:3, 1:5 and 1:7) of drug to carrier. Prepared dispersions were characterized by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD). Mathematical modeling of in-vitro dissolution data indicated the best fitting with Korsemeyer– Peppas model. Optimised batches were subjected for stability study (40 0C / 75 %RH) and found stable for at least 90 days. Moreover dissolution of optimised batch showed no significant effect of dissolution media on drug release rate and almost similar dissolution profile after storage period. XRD and DSC were also performed after storage period to evaluate any possible physicochemical changes. Wherever possible data were treated using student t-test and f2 test (similarity factor for dissolution). Self-emulsifying or self-micro emulsifying drug delivery systems have been prepared for tacrolimus and sirolimus using various oils, surfactants and co-surfactant. Excipients were selected based on solubility study and there after emulsification study. In tacrolimus SMEDDS composition of oil, surfactant and co-surfactant were selected based on pseudo ternary phase diagram where as for sirolimus SMEDDS they were selected based on dispersibility/emulsification study. Various in-vitro tests like percentage transmittance, emulsification time, cloud point, precipitation and thermodynamic stabilities were used to find out optimized formulations. Optimized liquid SMEDDS were characterized by particle size analysis and converted in solid by using Florite RE as an adsorbent which is further characterized by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and particle size analysis. The optimized liquid and solid SMEDDS showed significantly (P<0.05) higher drug release than the marketed capsule (only for tacrolimus) and pure API powder (for tacrolimus and sirolimus). Optimised liquid SMEDDS and solid SMEDDS showed particle size less than 300 nm. The solid state characterization of solid-SMEDDS by SEM, DSC, FTIR and XRD revealed the absence of crystalline drug in the solid-SMEDDS. XRD study was also repeated after 45 days storage period to evaluate any physical changes in solid SMEDDS. Liquid and solid optimised formulations found stable for at least 90 days (25 0C/60 %RH; 40 0C/75 % RH) and showed almost similar dissolution profile in different dissolution media and after storage period. In comparison among solid dispersion, liquid SMEDD and solid SMEDD of tacrolimus and sirolimus liquid SMEDDS showed significantly (P<0.05) higher dissolution rate than other two formulations. Moreover globule size of liquid SMEDDS was also smaller compared to solid SMEDDS. For ex-vivo and in-vivo evaluation liquid SMEDDS was selected. Optimised tacrolimus liquid SMEDDS was subjected for intestinal permeability study, in-vitro metabolism study and pharmacokinetics study and found superior in all three studies compared to marketed preparation and plain drug powder. Liquid SMEDDS of sirolimus was evaluated for pharmacodynamic study. At last, were developed two delivery systems (solid dispersion and SMEDDS) for tacrolimus and sirolimus. Liquid SMEDDS of tacrolimus showed improve dissolution rate and pharmacokinetics parameters than plain tacrolimus powder and marketed preparation. Liquid SMEDDS of sirolimus exhibited improve dissolution rate and in vivo efficacy than home made suspension of sirolimus.