Metabolism of 5-fluorouracil to an N-cholyl-2-fluoro-beta-alanine conjugate: previously unrecognized role for bile acids in drug conjugation.

Abstract

Recently we demonstrated clinically significant levels of a previously unrecognized metabolite of the anticancer drug 5-fluorouracil (FUra) in bile ofcancer patients. In the present study, reanalysis of bile from these patients demonstrated the presence of not one but two previously unrecognized metabolites. The major unrecognized metabolite was purified by reversed-phase HPLC, after which its molecular weight was determined by fast-atom-bombardment mass spectrometry to be 497. The similarity in HPLC retention times and molecular weights of this FUra derivative and the bile acids N-cholylglycine (Mr 465) and N-cholyltaurine (Mr 515), along with the structural similarity of the FUra catabolite 2-fluoro-,B-alanine and the amino acids glycine and taurine, led to the hypothesis that this metabolite could be a conjugate of 2-fluoro-,B-alanine and cholic acid. This hypothesis was tested and confirmed by hydrolyzing the purified metabolite by cholylglycine hydrolase after which: (i) 2-fluoro-,-alanine was demonstrated by using a sensitive HPLC technique capable of resolving all of the known putative FUra metabolites, and (ii) unconjugated cholic acid was identified by both GC and GC-MS. Additionally, chemically synthesized N-cholyl-2-fluoro-8-alanine was shown to cochromatograph on HPLC and TLC with the purified biiary metabolite. In summary, this study demonstrates a unique, so far as we know, pathway of drug metabolism in man in which an amino acid drug metabolite is conjugated with cholic acid and eliminated into the bile.Furthermore, the finding that 2-fluoro-p-alanine is conjugated to bile acids may provide some insight Into the mechanism of cholestasis that is frequently observed after administration of fluoropyrimidine by hepatic arterial infusion.Although first synthesized 30 years ago, fluoropyrimidines are still widely used in the treatment of a variety of cancers.The anabolism of the fluoropyrimidines to cytotoxic nucleotides has been extensively examined (1, 2) and appears to be responsible for the chemotherapeutic action of these agents. On the other hand, relatively few studies have focused on fluoropyrimidine catabolism. This is despite the fact that >80% of the administered dose is metabolized via the catabolic route (3). Additionally, inadequacies in the analytical techniques previously used for the separation of fluoropyrimidine catabolites has until recently prevented a comprehensive examination of the catabolism of these agents. However, these technical problems have been overcome by the development of a specific HPLC method for the separation of all of the putative fluoropyrimidine metabolites (4).Our laboratory has demonstrated the formation of a 5-fluorouracil (FUra) glucuronide conjugate in isolated rat hepatocytes (5) and the presence of an unidentified FUra metabolite in bile of cancer patients (6). In the present study, further analysis of bile from these patients has shown the presence of two previously unrecognized FUra metabolites. These metabolites did not appear to represent FUra...

Conserved Residues in the Putative Catalytic Triad of Human Bile Acid Coenzyme A:Amino Acid N-Acyltransferase

“…5). This can also be used to explain why ␤-alanine, the carboxylic acid analog of taurine, is a poor substrate of hBAT (7), whereas both 2-fluoro-␤-alanine (a stronger acid) and taurine are excellent substrates (24). The result is also consistent with previous studies on thioesterases, in which the serine acyl-enzyme intermediate was shown to be unstable relative to the cysteine acyl-enzyme intermediate.…”

Assay of urinary α‐fluoro‐β‐alanine by gas chromatography–mass spectrometry for the biological monitoring of occupational exposure to 5‐fluorouracil in oncology nurses and pharmacy technicians

“…Inactive metabolites are derived from initial reduction of the heteroaromatic ring to 5-fluoro-5,6-dihydrouracil and further ring opening to yield the unnatural amino acid α-fluoro-β-alanine (AFBA) as the main product excreted in urine and corresponding to 80-90% of the administered dose. A fraction of AFBA is conjugated in the liver to cholic acid to yield an analog of taurocholic acid, which is possibly associated with the onset of cholestasis in some treated patients (Sweeny et al, 1987).…”

Metabolism of Capecitabine, an Oral Fluorouracil Prodrug:19F NMR Studies in Animal Models and Human Urine

“…The low biliary excretion found in IPRL experiments (2.2% a.d.) and the low recovery of radioactive dose measured in the feces after administration of 14 C-CAP to humans (2.6%) (Judson et al, 1999) suggest that, if glucuronidation occurs in humans, it would be a minor CAP detoxification pathway. Since cholestasis has not been described as a side effect of CAP treatment, it is likely that 5Ј-DFCR-G does not affect the physicochemical properties of bile in contrast to what has been observed with a biliary conjugate of FBAL (Sweeny et al, 1987). The third activation step of CAP leads to the formation of 5-FU that occurs mainly via uridine phosphorylase in rodents and thymidine phosphorylase in humans (Ninomiya et al, 1990).…”