Isolation and in vitro characterization of a novel immunosuppressive cyclosporine metabolite.
A novel metabolite (M-E) was identified by high-performance liquid chromatography in the serum of cyclosporine-treated renal transplant recipients during a second wave of immunosuppressive activity after disappearance of the initial wave due to the direct effect of CsA. M-E was identified in human serum and porcine bile both by HPLC and by a preparative thin-layer chromatography (TLC). It demonstrated homogeneity with characteristic retention times on C8 and C18 column HPLC systems using a variety of elution systems, and distinctive TLC mobility (Rf 0.35). Metabolite E (M-E) was documented to be a CsA metabolite by radioactive tracer studies, by crossreactivity with a polyclonal sheep antibody in radioimmunoassay, and by the presence of a characteristic mass spectrum. Further, in vitro immunosuppressive assays documented effects of M-E similar to those of CsA. The relative activity of M-E versus CsA was quantitated by potency ratios: for inhibition of normal human mixed lymphocyte culture reactions, the ratio was 0.79 +/- 0.23. Interindividual differences were observed in patient susceptibility to MLR inhibition not only by CsA, as previously reported by others, but also by M-E. There was a lesser effect of M-E compared with CsA in inhibiting proliferation of, and IL-2 generation by, C3H murine splenocytes stimulated with concanavalin A: the potency ratios for both systems were about 0.5, possibly reflecting an interspecies variability in generation of or susceptibility to M-E. These studies suggest that heretofore unidentified metabolites--including, but not limited to, M-E--may play an important role in the immunosuppressive effect of CsA in man.
Roesel
T
T
Department of Surgery, University of Texas Medical School, Houston 77030.
LeGrue
S J
SJ
Liehr
J
J
Kahan
B D
BD
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003429:Cross Reactions; D003524:Cyclosporins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000302:isolation & purification*; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation / Q000187:drug effects; D007959:Lymphocyte Culture Test, Mixed; D013058:Mass Spectrometry; D051379:Mice; D011863:Radioimmunoassay; D013552:Swine
D003524:Cyclosporins; D007166:Immunosuppressive Agents; D007376:Interleukin-2