LIQUID-CHROMATOGRAPHY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY AND LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY IDENTIFICATION OF NOVEL METABOLITES OF THE MULTIDRUG-RESISTANCE MODULATOR LY335979 IN RAT BILE AND HUMAN LIVER MICROSOMAL INCUBATIONS

Compound LY335979 is a P-glycoprotein inhibitor currently entering pha se I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from r ats, dogs, monkeys, and humans. Although the parent compound was compl etely metabolized, no prominent metabolite peaks were observed. One pe ak did appear early in the time course, but it did not increase over t ime, In another preliminary experiment, rats were treated iv with [H-3 ]LY335979 (prepared for pharmacology studies), and urine and bile frac tions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the materi al eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent co mpound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites w ere not successful. There was also evidence in both systems of product s derived from cleavage of LY335979 (by both further metabolism and de gradation). LC/NMR was thus used to analyze materials directly in thei r respective matrices. An N-oxide metabolite (LY389551) formed by oxid ation of the quinoline nitrogen was identified in the microsomal incub ations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinolin e ring of LY335979. There have been few reports in the literature of L C/NMR analysis of bile, which is a more complex matrix than either uri ne or microsomal suspensions. However, the HPLC techniques developed i n this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/ NMR. Using a 500-MHz instrument, basic H-1 NMR spectra could be obtain ed in 2-3 hr with approximately 100 ng of material in the LC/NMR micro probe. With approximately 1.5 mu g of material injected onto the colum n, H-1-H-1 correlation spectroscopy spectra could be acquired overnigh t. Along with LC/MS data, the LC/NMR technique facilitated direct iden tification of a number of metabolites of LY335979 at a point at which their identification by traditional methods would not have been pursue d.