The identity of rabbit-liver methotrexate oxidase

Johns, David G.; Iannotti, A.T.; Sartorelli, Alan C.; Booth, Barbara A.; Bertino, Joseph R.

doi:10.1016/s0926-6593(65)80164-3

Cited by 52 publications

(12 citation statements)

References 6 publications

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“…likely since previous studies of biliary excretion of MTX administered at conventional dose levels in the monkey have not shown the presence of the 7-hydroxy metabolite (12). In studies of the 7-hydroxylation of MTX by the rabbit, in which species the reaction is quantitatively significant even at lower dose levels, the hydroxylation has been shown to be catalyzed by the hepatic metalloflavoprotein aldehyde oxidase (EC 1.2.-3.1); the Michaelis constant for the reaction with the rabbit enzyme is of the order of 0.1 mM (13). The latter concentration would be considerably exceeded in vivo after the administration of high-dose MTX (assuming equal distribution of the drug throughout body water), but would not be achieved after the administration of MTX in the conventional dose range.…”

Section: Wmentioning

confidence: 99%

7-Hydroxymethotrexate as a urinary metabolite in human subjects and rhesus monkeys receiving high dose methotrexate.

Jacobs¹,

Stoller²,

Chabner³

et al. 1976

J. Clin. Invest.

235

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A B S T R A C T Human subjects and rhesus monkeys receiving the antitumor agent methotrexate at the high dose levels recently introduced into clinical use (> 50 mg/kg) excrete significant amounts of the metabolite 7-hydroxymethotrexate. The metabolite is not detected in these species after methotrexate therapy at conventional dose levels. The evidence indicates that in primates, the in vivo conversion of methotrexate to 7-hydroxymethotrexate is a dose-dependent phenomenon, with the enzyme system (s) catalyzing the reaction having a low affinity for the drug. INTRODUCTIONThe folate antagonist methotrexate (MTX; 4-amino-4-deoxy-N10-methylpteroylglutamate) 1 is in general clinical use as a single agent or in combination with other antineoplastic agents in the treatment of choriocarcinoma, Burkitt's lymphoma, acute leukemia of childhood, epidermoid carcinoma of the head and neck, and breast cancer (1, 2). Recently, "high-dose" MTX therapy (> 50 mg MTX/kg) with leucovorin rescue has been shown to be of value in the treatment of osteogenic sarcoma (3, 4). The toxicity of this regimen is significant, with renal failure having been observed, in addition to the gastrointestinal ulceration and bone marrow depression seen with conventional MTX therapy. Earlier studies of the clinical pharmacology of MTX administered at conventional dose levels indicate that in man and other primates, the compound is not metabo-

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Section: Wmentioning

confidence: 99%

7-Hydroxymethotrexate as a urinary metabolite in human subjects and rhesus monkeys receiving high dose methotrexate.

Jacobs¹,

Stoller²,

Chabner³

et al. 1976

J. Clin. Invest.

235

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“…Indeed, human AOX1 has a very low affinity for methotrexate [7]. Rabbit AOX on the other hand has been reported to have extremely high AOX activity with methotrexate [17, 18]. In order to understand the structural determinants responsible for high methotrexate activity, a rabbit homology model was generated and methotrexate was docked in to its active site.…”

Section: Introductionmentioning

confidence: 99%

Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase

Choughule

Joswig-Jones

Jones

2015

Biochemical Pharmacology

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Several drug compounds have failed in clinical trials due to extensive biotransformation by aldehyde oxidase (AOX) (EC 1.2.3.1). One of the main reasons is the difficulty in scaling clearance for drugs metabolised by AOX, from preclinical species to human. Using methotrexate as a probe substrate, we evaluated AOX metabolism in liver cytosol from human and commonly used laboratory species namely guinea pig, monkey, rat and rabbit. We found that the metabolism of methotrexate in rabbit liver cytosol was several orders of magnitude higher than any of the other species tested. The results of protein quantitation revealed that the amount of AOX1 in human liver was similar to rabbit liver. To understand if the observed differences in activity were due to structural differences, we modelled rabbit AOX1 using the previously generated human AOX1 homology model. Molecular docking of methotrexate into the active site of the enzyme led to the identification of important residues that could potentially be involved in substrate binding and account for the observed differences. In order to study the impact of these residue changes on enzyme activity, we used site directed mutagenesis to construct mutant AOX1 cDNAs by substituting nucleotides of human AOX1 with relevant ones of rabbit AOX1. AOX1 mutant proteins were expressed in E. coli. Differences in the kinetic properties of these mutants have been presented in this study.

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“…Johns et al have reported that in rabbits, the hydroxylation of MTX is catalyzed by aldehyde oxidase (EC 1.2.3.1), as determined in studies using the partially puri ed enzyme (14). In this species, several tissues have a strong capacity for 7-OH-MTX formation (15,16). Rhee and Galivan have shown that MTX is converted to 7-OH-MTX in cultured rat hepatic cells (17).…”

Section: Introductionmentioning

confidence: 99%

Variation of Hepatic Methotrexate 7-Hydroxylase Activity in Animals and Humans

Kawa¹,

Sugihara²,

Nakatani³

et al. 1999

IUBMB: Life

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This study deals with individual and species variations in the converting activity of methotrexate (MTX) to 7-hydroxymethotrexate in animals and humans. When MTX 7-hydroxylase was assayed in six human liver cytosols, a 48-fold range of intersubject variation of the activity was observed. The variations were correlated to the concentrations of aldehyde oxidase activity in human subjects assayed with benzaldehyde as a substrate. Species differences of liver MTX 7-hydroxylase activity were also observed. The activity was highest in rabbits, followed by rats, hamsters, and monkeys but was undetectable in dogs. Strain differences of MTX 7-hydroxylase activity based on aldehyde oxidase activity were also observed in rats and mice. The results suggest that aldehyde oxidase functions as MTX 7-hydroxylase in livers of animals and humans, and the observed differences of MTX 7-hydroxylase activity are due to variations in the amount of aldehyde oxidase present.

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The identity of rabbit-liver methotrexate oxidase

Cited by 52 publications

References 6 publications

7-Hydroxymethotrexate as a urinary metabolite in human subjects and rhesus monkeys receiving high dose methotrexate.

7-Hydroxymethotrexate as a urinary metabolite in human subjects and rhesus monkeys receiving high dose methotrexate.

Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase

Variation of Hepatic Methotrexate 7-Hydroxylase Activity in Animals and Humans

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