Modulation of cyclosporin A/cyclophilin interactions by drug vehicles

Cyclosporin A (CsA) is a tight-binding inhibitor of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of human cytosolic cyclophilin (Cyp18), the putative receptor for immunosuppressive effects of the drug. We examined the influence of cremophor EL (CEL), a surfactant that has found wide use for CsA formulation, on the kinetics of inhibition of the enzyme by CsA. Stock solutions of CsA in CEL administered into aqueous PPIase assays led to inhibition kinetics reminiscent to those of CsA dissolved in tetrahydrofurane, but caused an increase in the final Ki value of about sevenfold at 0.33% (v/v) CEL. The diminished drug affinity to Cyp18 obtained in experiments using CEL could also be established for analogues of cyclosporin A such as [Ala2]-Cs,[Thr2]-Cs, and [MeAla6]-Cs, exhibiting Ki values 13-16-fold higher than in the absence of CEL. In addition, the time-dependent pattern of inhibition indicate only a minor population of bioactive conformation of CsA in bulky CEL. Conformational reshuffling of the bioinactive [cis-MeLeu9-MeLeu10]-Cs to create an inhibitory fraction of the drug was delayed in the presence of CEL micelles, despite potential ability of micelles exists to catalyze cis/trans isomerizations of N-alkyl peptide bonds. The pattern of inhibition when using cyclophilins distinct in their amino acid sequences to the human enzyme can be rationalized in terms of exceptional high structural requirements of human Cyp18 for the drug conformation.