| Rapamycin is an immunosuppressive drug used in organ transplantation. It is metabolised to a number of products. It is not known what the relevance of these metabolites is---if they posses immunosuppressive, or toxic activity; what their concentrations are in blood; if they should be monitored clinically in transplant patients. To answer these questions, rapamycin metabolites were tested extensively. First, metabolites were generated using a microbial system expressing cytochrome P450 3A enzyme. The metabolites were purified and five metabolites were structurally identified by high performance liquid chromatography with mass spectrometry detection (HPLC-MS). They have been named according to the structural change(s) they posses: 27-, 39-O-di-demethylrapamycin, C(1--14)-hydroxyrapamycin, C(15--27)hydroxyrapamycin, 16-O-demethylrapamycin and 39-O-demethylrapamycin. The immunosuppressive activity of these metabolites was evaluated using the mixed lymphocyte reaction. The immunosuppressive activity of the metabolites ranged from 0.1% to 17.3% of rapamycin. The toxic activity of these metabolites was investigated in an in vitro vascular endothelial vasoactive substance release assay; results confirm that it is not an important mode of toxicity for rapamycin or its metabolites. A method was developed for measurement of five metabolites in clinical and research specimens by HPLC-MS. A study investigating differences in canine portal and systemic pharmacokinetics of rapamycin revealed rapamycin oral dosing produces an appropriate pharmacokinetic profile for liver and portal vein embolised pancreatic islet transplantation, as differences between portal and systemic peak levels were negligible. Rapamycin and rapamycin metabolite levels were also measured in liver transplant patients on a compassionate release program. None of the five metabolites evaluated were present in concentrations that exceeded 30% of rapamycin in these liver transplant patients. Overall, given the new information on the concentrations and immunosuppressive activities, the five metabolites tested do not contribute significantly to immunosuppression. While further study of the metabolites of rapamycin should be pursued, at this time rapamycin alone should be measured for therapeutic drug monitoring and dosage adjustment in transplant patients. |
