Biliary excretion of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) and metabolites by Fischer 344 rats

Abstract

Purpose: 17-(Allylamino)-17-demethoxygeldanamycin (17AAG), an analogue of the benzoquinone ansamycin geldanamycin, has been extensively studied preclinically and is being evaluated clinically. Studies were performed to define the biliary excretion of 17AAG after i.v. delivery to rats, and to characterize the metabolites of 17AAG observed in rat bile.

Materials and methods: In vivo studies were performed in bile-duct-cannulated Fischer 344 rats given a 10 mg/kg i.v. bolus dose of 17AAG. In vitro studies were performed with cloned human CYPs and microsomal epoxide hydrolase. Biliary excretion of 17AAG and metabolites was quantified by HPLC and followed for 4 h after drug delivery. 17AAG metabolites in bile and in in vitro reaction mixtures were identified with LC/MS/MS.

Results: By 15 min after i.v. delivery of 17AAG, bile contained at least 15 biotransformation products with absorbance spectra similar to that of 17AAG. Of these, metabolites eluting at 2.7, 2.9, and 8.6 min were present in sufficient concentrations to be quantified, although the lack of authentic standards resulted in their being expressed as 17AAG equivalents. Within the first 4 h after drug delivery, biliary excretion accounted for 28.9+/-6.1% of the 10-mg/kg 17AAG dose. 17AAG and 17-(amino)-17-demethoxygeldanamycin (17AG) accounted for 4.1+/-1.0% of the delivered dose, with 17AAG accounting for 2.0+/-0.5% and 17AG accounting for 2.1+/-0.5%. The metabolites eluting at 2.7, 2.9, and 8.6 min accounted for 10.6+/-2.0%, 9.8+/-1.2%, and 1.0+/-0.2%, respectively, of the administered dose. LC/MS/MS analysis of bile demonstrated major metabolites with molecular weights of 545 and 619, corresponding to 17AG and the diol previously described as resulting from metabolism of 17AAG by CYP3A and microsomal epoxide hydrolase. Of the remaining proposed metabolites, ten had a mass and MS/MS spectrum consistent with mono-oxygenated 17AAG metabolites. One of these metabolites has been identified as the epoxide previously described as resulting from CYP3A oxidation of the allyl double bond. Two other proposed metabolites had a mass and MS/MS spectrum consistent with demethylated 17AAG metabolites, and one had a mass and MS/MS spectrum consistent with a di-demethylated 17AAG metabolite. An analogous series of demethylated and oxidized metabolites was also observed for the 17AG metabolite.

Conclusions: Biliary excretion of 17AAG represents a major route of elimination, although most of the material excreted is in the form of metabolites. Bile of rats dosed with 17AAG contained a number of metabolites not previously identified in the plasma or urine of mice treated with 17AAG, but analogous to metabolites described in bile of rats treated with 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG, NSC 707545), another geldanamycin analogue undergoing preclinical evaluation in preparation for subsequent clinical trials.