Metabolites of alanosine, an antitumor antibiotic

Abstract The metabolism of alanosine. dl -2-amino-3-( N -hydroxy, N -nitrosamino) propionic acid (NSC-143647), a new antitumor antibiotic, was studied in mice, rats, monkeys and dogs. Urine is the principal excretory vehicle for the drug in these four species. In rats, unchanged alanosine is the principal excretory product. In the other species, a second, more acidic component accounts for the major fraction of the drug-derived radioactivity in urine; this product retains the characteristic u.v. spectrum and both carbons, 1 and 3, of alanosine: it is chromatographically and spectrally indistinguishable from the compound generated by the action of NADH and malate dehydrogenase on the product resulting from the incubation of l -alanosine with l -glutamate oxaloacetate transaminase (GOT) (EC 2.6.1.1) and α-ketoglutarate. On the basis of this evidence, this metabolite is concluded to be the α-hydroxy counterpart of l -alanosine. The antibiotic was susceptible to transamination in vitro by extract of organs rich in GOT; heart was pre-eminent in this regard, and α-ketoglutaric acid was found to be the preferred α-keto partner in the reaction. Crystalline GOT catalyzed an identical reaction in vitro , and the product, like oxaloacetic acid, was susceptible to enzymatic condensation with acetyl CoA, in the presence of citrate synthase. Inability to detect the -α- keto analogue of alanosine. 2-oxo-3-( N -hydroxy, N -nitrosamino) propionic acid, in tissues and excreta is attributed to the facile decomposition of this metabolite in vivo . In vitro , alanosine was susceptible to decarboxylation by homogenates of mouse brain and by purified l -glutamate decarboxylase (EC 4.1.1.15) from Escherichia coli . No evidence could be adduced for denitrosation of the antibiotic nor for reduction of the nitroso function in a system containing hepatic microsomes and NADPH. However, l -amino acid oxidase (EC 1.4.3.2) and high concentrations of pyridoxal phosphate catalyzed the deamination of alanosine at alkaline pH. In confirmation of the observations of Hurlbert et al , ¦ Proc. Am. Ass. Cancer Res. 18 , 234,(1977)¦. alanosine was found to be used by phosphoribosylaminoimi-dazole-succinocarboxamide synthetase (EC 6.3.2.6) as a fraudulent substrate. Also observed was the condensation of alanosine with IMP. catalyzed by a partially purified preparation of adenylosuccinate synthetase (EC 6.3.4.4) from rabbit muscle; this anabolite exhibited chromatographic properties quite similar to adenylosuccinic acid. Inasmuch as a substantial percentage of the administered dose of alanosine was found to associate with carcass-macromolecules for protracted periods, attempts were made to determine the basis for this fate. Equivalent labeling was produced irrespective of whether dl -[1- 14 C] or dl -[3- 14 C] alanosine was the injectate, so that reutilization of metabolically generated [ 14 C]O 2 is not likely to explain macromolecular retention of the antibiotic. In vitro , no esterification of alanosine to tRNA was observed, but the drug did bind to tRNA in an ATP-independent reaction. In vivo , ten times more dl -[3- 14 C] alanosine was incorporated into the hemoglobin of animals recovering from phenylhydrazine anemia than was observed in their saline-treated counterparts. Isolated reticulocytes incorporated only minor amounts of purified dl -[I- 14 C] alanosine into their molecules; this process was insensitive to inhibition by cycloheximide. In vitro , alanosine (used in lieu of l -aspartic acid) neither supported nor inhibited globin synthesis by rabbit reticulocyte lysates. These results leave unsettled the question of whether macromolecular association of alanosine reflects incorporation or affiliation.