DISCOVERY OF LESS NEPHROTOXIC FK506 ANALOGS AND DETERMINING IMMUNOPHILIN DEPENDENCE OF IMMUNOSUPPRESSANT NEPHROTOXICITY WITH A NOVEL SINGLE-DOSE RAT CISPLATIN POTENTIATION ASSAY

Comparing nephrotoxicity of numerous drug analogs is impractical with chronic in vivo models. We devised a new cisplatin potentiation assay (CISPA) that sensitively detects renal injury as a serum creatinine in crease when only one dose of test compound is followed by cisplatin. R eference nephrotoxins known to act on various sites in kidney tubules, glomeruli or renal papilla were all detected by the CISPA at single d oses that without cisplatin gave little change, which showed that this simple, sensitive assay has broad potential utility for mechanistic s tudies of nephrotoxicity. We used the CISPA both to probe the nephroto xic mode of action of immunosuppressants and to search for safer compo unds. Although several non-nephrotoxic immunosuppressants were inactiv e, cyclosporine, FK506, ascomycin (C21-ethyl-FK506) and rapamycin were nephrotoxic in the CISPA at single doses equal to the daily amounts r equired to reduce creatinine clearance with 14 days of treatment. Simi lar therapeutic indices were derived comparing toxicity by either meth od to prevention of rat ear-heart allograft rejection. C18-OH-ascomyci n, an FK506-binding protein (FKBP) antagonist, reversed in vivo immuno suppression by FK506 and ascomycin in the rat, and pretreatment in the CISPA blocked FK506 and ascomycin nephrotoxicity, which showed a comm on immunophilin dependence. Rapamycin nephrotoxicity was unaffected (a s with cyclosporine), which indicated that binding to FKBP was not req uired, Rapamycin nephrotoxicity thus appears mechanistically unrelated to its immunosuppressive mode of action. Screening with the CISPA ena bled discovery of A-119435, a less nephrotoxic ascomycin analog having a 10-fold higher therapeutic index.