ABSTRACT
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.
Subject(s)
Cyclosporins/isolation & purification , Immunosuppressive Agents/isolation & purification , Animals , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Cross Reactions , Cyclosporins/metabolism , Humans , In Vitro Techniques , Interleukin-2/biosynthesis , Lymphocyte Activation/drug effects , Lymphocyte Culture Test, Mixed , Mass Spectrometry , Mice , Radioimmunoassay , SwineABSTRACT
High-performance thin-layer chromatography (HPTLC) has advantages for the analysis of cyclosporine (CsA) and its metabolites in peripheral blood not shared by the radioimmunoassay (RIA) or high-performance liquid chromatography (HPLC) methods. While separation by HPTLC and HPLC is based on relative hydrophobicity, HPTLC (unlike HPLC) is capable of concurrent multisample analysis without expensive instrumentation. As distinguished from RIA, HPTLC detects CsA and metabolites as separate components, and does not use radioactive reagents. A novel rhodamine B/alpha-cyclodextrin stain was developed and the characteristic retention factors (Rf values), as determined by the ratio of the migration distance of a component in relation to the solvent front, were determined for the mobile phase heptane: pyridine: ethyl acetate (100: 75: 1, v/v) on aminopropyl-bonded silica gel HPTLC plates: 0.65, cyclosporin D (CsD); 0.60, CsA; 0.50, dihydrocyclosporin C (dhCsC); 0.42, metabolites M-21 and M-17; 0.40, M-1; 0.35, M-E; 0.25, M-D; and 0.22, M-A. Metabolite M-18 showed migration similar to that of M-17 using a mobile phase of heptane: pyridine: acetonitrile (5:2:1, v/v) in the 0.60-0.50 range. The metabolite profiles were obtained in 8 patients receiving the drug for the first time. The HPTLC analytical technique identifies CsA and its metabolites in peripheral blood and offers advantages for pharmacologic monitoring of transplant patients.
Subject(s)
Cyclosporins/blood , Kidney Transplantation , Bile/metabolism , Chromatography, Thin Layer/methods , Cyclodextrins , Cyclosporins/isolation & purification , Cyclosporins/metabolism , Humans , Rhodamines , Structure-Activity RelationshipSubject(s)
Cyclosporins/pharmacology , Lymphocyte Activation/drug effects , Animals , Calmodulin/physiology , Humans , Immunosuppression Therapy , Interferon-gamma/biosynthesis , Interleukin-2/biosynthesis , Lymphocyte Culture Test, Mixed , Lymphokines/biosynthesis , Mice , T-Lymphocytes/drug effectsABSTRACT
Intramolecular general base catalysis by the imidazolyl group was found in the hydrolyses of endo-5-[4;(5')-imidazolyl]-bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate and endo-5-[4'(5')-imidazolyl]bicyclo[2.2.2]oct-endo-2-yl trans-cinnamate in which the imidazolyl and trans-cinnamoyl groups are bound in close proximity to each other by rigid bicyclic rings. The rate constants for the intramolecular general base-catalyzed hydrolyses at 60 degrees are 6.4 X 10(-7) sec-1 for the former and 1.8 X 10(-7) sec-1 for the latter and the deuterium oxide solvent isotope effects are 3.0 for both. On the other hand, no intramolecular catalytic participation of the imidazolyl group was observed in the hydrolyses of the endo-exo isomers, exo- 5-[4'(5')-imidazolyl]bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate and endo-5[4'(5')-imidazolyl]bicyclo[2.2.2]oct-exo-2-yl trans-cinnamate, in which the imidazolyl groups are located far from the trans-cinnamoyl groups. Intramolecular general base-catalyzed hydrolyses by the imidazolyl groups in endo-5[4'(5')-imidazolyl]bicyclo[2.2.1]hept-endo-2-yl trans-cinnamate and endo-5-[4'(5')-imidazolyl]bicyclo[2.2.2]oct-endo-2-yl trans-cinnamate can serve as models of serine esterase-catalyzed hydrolyses.
