In-vitro diffusion studies of Tacrolimus from an ointment formulation (Tacroz')

摘要

Tacrolimus is an immunosuppressant discovered in 1987 from the fermentation broth of a Japanese soil sample containing the bacterium Streptomyces tsukubaensis. It is used for Immunosuppression, Ulcerative colitis and for Dermatological use. Dermatologically, Tacrolimus Ointment is used for the treatment of eczema, especially in atopic dermatitis. The important advantage of Tacrolimus Ointment is, it does not cause skin thinning (atrophy).;Tacrolimus for topical use is a relatively new and the mechanism of action for the same is still unknown. Being an immunosuppressant, it acts as a calcineurin channel blocker which inhibits T-Lymphocyte activation by first binding to intracellular protein FKBP-12. Resulting complex inhibits the phosphate activity of calcineurin.;The purpose of this thesis is to determine the diffusion profile of a commercially available Tacrolimus ointment through cellulose membrane. In vitro studies were performed in triplicates with jacketed Franz diffusion cell and cellulose membrane. The samples collected from jacketed Franz diffusion cell were analyzed using a modified and validated HPLC method. The separation was achieved by C18 Column at 60°C. The detection wavelength of 210nm was used. Acetonitrile (100%) was used as a mobile phase with flow rate of 0.5mL/min, sample run time was 15mins and injection volume was 20microL. The validation was carried out by determining parameters like LOD, LLOQ and linearity, etc. LOD and LLOQ was found to be 0.2mg/mL and the linearity was determined from 0.2mg/mL to 100mg/mL respectively.;The receiver solution used to carry out Franz diffusion were Phosphate Buffer Saline (PBS) Solution, acetonitrile and mixtures of PBS and acetonitrile in three different ratios (25:75, 50:50, 75:25). The flux (Jss) and cumulative release for all the experiments was calculated. The flux (J ss) was found to be 0.0074microg/cm2/min with acetonitrile as receiver solution, 0.0013microg/cm'/min with PBS and acetonitrile in ratio 25:75 and 0.001 1microg/cm2/min with 50:50 ratio of PBS Solution and acetonitrile as receiver solution. The percent cumulative release was found to be 21.87% after 24hrs with acetonitrile as receiver solution, 4.52% after 4hrs for PBS and acetonitrile in ratio 25:75 and with PBS and acetonitrile in 50:50 ratio 12.80% after 6hrs.;The diffusion was not observed with PBS solution in receiver and 75:25 ratio of PBS and acetonitrile was not performed due to precipitation of phosphates (monosodium phosphate monohydrate and disodium phosphate heptahydrate) while mixing the two components in the proportion.;The results show that the diffusion of the tacrolimus across the membrane is very slow. Tacrolimus being highly lipophilic, ointment base used for the same consists of white petroleum, mineral oil, polyethylene carbonate, white wax and paraffin.;In conclusion, the release of tacrolimus from an ointment base could possibly be the rate limiting step. This also governs the diffusion of tacrolimus across the membrane. There is a possibility of degradation and deterioration of an ointment as it the formulation was stored on benchtop conditions. The data obtained also inform us that the flux decreased with respect to time.