title: Comparative Metabolism of Batracylin (NSC 320846) and N-acetylbatracylin (NSC 611001) Using Human, Dog, and Rat PreparationsIn Vitro.
abstract: BACKGROUND
  Batracylin is a heterocyclic arylamine topoisomerase inhibitor with preclinical anticancer activity. Marked species differences in sensitivity to the toxicity of batracylin were observed and attributed to differential formation of N-acetylbatracylin by N-acetyltransferase. A Phase I trial of batracylin in cancer patients with slow acetylator genotypes identified a dose-limiting toxicity of hemorrhagic cystitis. To further explore the metabolism of batracylin and N-acetylbatracylin across species, detailed studies using human, rat, and dog liver microsomal and hepatocyte preparations were conducted.
  
  
  METHODS
  Batracylin or N-acetylbatracylin was incubated with microsomes and hepatocytes from human, rat, and dog liver and with CYP-expressing human and rat microsomes. Substrates and metabolites were analyzed by HPLC with diode array, fluorescence, radiochemical, or mass spectrometric detection. Covalent binding of radiolabeled batracylin and N-acetylbatracylin to protein and DNA was measured in 3-methylcholanthrene-induced rat, human, and dog liver microsomes, and with recombinant human cytochromes P450.
  
  
  RESULTS
  In microsomal preparations, loss of batracylin was accompanied by formation of one hydroxylated metabolite in human liver microsomes and five hydroxylated metabolites in rat liver microsomes. Six mono- or di-hydroxy-N-acetylbatracylin metabolites were found in incubations of this compound with 3MC rat liver microsomes. Hydroxylation sites were identified for some of the metabolites using deuterated substrates. Incubation with recombinant cytochromes P450 identified rCYP1A1, rCYP1A2, hCYP1A1 and hCYP1B1 as the major CYP isoforms that metabolize batracylin and N-acetylbatracylin. Glucuronide conjugates of batracylin were also identified in hepatocyte incubations. NADPH-dependent covalent binding to protein and DNA was detected in all batracylin and most N-acetylbatracylin preparations evaluated.
  
  
  CONCLUSIONS
  Microsomal metabolism of batracylin and N-acetylbatracylin results in multiple hydroxylated products (including possible hydroxylamines) and glutathione conjugates. Incubation of batracylin with hepatocytes resulted in production primarily of glucuronides and other conjugates. There was no clear distinction in the metabolism of batracylin and N-acetylbatracylin across species that would explain the differential toxicity.
authors.affiliation: Toxicology and Pharmacology Branch, Developmental Therapeutics Program, DCTD, NCI, Rockville, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Covey
authors.affiliation: Department of Oncology, Division of Oncology Research, Mayo Clinic and Foundation Rochester, MN, USA.
authors.forename: Joel M
authors.initials: JM
authors.lastname: Reid
authors.affiliation: Department of Oncology, Division of Oncology Research, Mayo Clinic and Foundation Rochester, MN, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Buhrow
authors.affiliation: Department of Oncology, Division of Oncology Research, Mayo Clinic and Foundation Rochester, MN, USA.
authors.forename: Mary
authors.initials: M
authors.lastname: Kuffel
authors.affiliation: Department of Oncology, Division of Oncology Research, Mayo Clinic and Foundation Rochester, MN, USA.
authors.forename: Chad
authors.initials: C
authors.lastname: Walden
authors.affiliation: Laboratory for Investigative Toxicology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, USA; Institute for In Vitro Sciences, 30 West Watkins Mill Road, Gaithersburg, USA.
authors.forename: Holger
authors.initials: H
authors.lastname: Behrsing
authors.affiliation: Department of Oncology, Division of Oncology Research, Mayo Clinic and Foundation Rochester, MN, USA.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Ames
keywords: Batracylin; Hepatocytes; Metabolism; Microsomes; N-Acetylbatracylin