title: Characterization of 1-Aminobenzotriazole and Ketoconazole as Novel Inhibitors of Monoamine Oxidase (MAO): An In Vitro Investigation.
abstract: OBJECTIVE
  1-Aminobenzotriazole, a known time-dependent inhibitor of cytochrome P450 (CYP) enzymes, and ketoconazole, a strong inhibitor of the human CYP3A4 isozyme, are used as standard probe inhibitors to characterize the CYP and/or non-CYP-mediated metabolism of xenobiotics. In the present investigation, 1-Aminobenzotriazole and ketoconazole are characterized as potent monoamine oxidase (MAO) inhibitors in vitro using mouse, rat and human liver microsomes and S9 fractions.
  
  
  METHODS
  Inhibition potential of 1-aminobenzotriazole and ketoconazole was studied in mice, rat and human liver microsomes, S9 fractions, MAO-A and MAO-B expressed enzymes by monitoring the formation of 4-hydroxyquinoline (4-HQ) from kynuramine, a specific substrate of MAO by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mechanism of MAO inhibition was studied by incubating varying concentration of kynuramine with mouse, rat and human S9 fractions at varying concentration of 1-aminobenzatriazole and ketoconazole and monitoring the formation of 4-HQ.
  
  
  RESULTS
  1-aminobenzotriazole and ketoconazole inhibited both MAO isozymes (MAO-A and MAO-B) with more specificity towards MAO-B. Kynuramine substrate kinetics in mouse, rat and human S9 fractions with varying 1-aminobenzotriazole and ketoconazole concentrations showed decreased maximum rate (Vmax) for 4-HQ formation without affecting the Michaelis-Menten constant (Km). A non-competitive inhibition model was constructed and inhibition constants (Ki) for 1-aminobenzotriazole (7.87 ± 0.61, 8.61 ± 0.92, 65.2 ± 1.61 µM for mice, rat and humans, respectively) and ketoconazole (0.12 ± 0.01, 2.04 ± 0.08, 5.52 ± 0.47 µM for mice, rat and humans, respectively) were determined.
  
  
  CONCLUSIONS
  1-Aminobenzotriazole and ketoconazole are characterized as non-competitive inhibitors of mice, rat and human MAO in vitro and the extent of their MAO inhibition potential is species specific. 1-Aminobenzotriazole or ketoconazole can be used as a probe inhibitor in vitro for screening the involvement of MAO-dependent metabolism of new chemical entities (NCE) in early drug discovery.
authors.affiliation: Department of Pharmaceutical Sciences, MCPHS University, Boston, MA, 02115, USA.|Center for Pharmacometrics and Systems Pharmacology, University of Florida, Orlando, FL, 32827, USA.
authors.forename: Abdul Naveed
authors.initials: AN
authors.lastname: Shaik
authors.affiliation: Department of Pharmaceutical Sciences, MCPHS University, Boston, MA, 02115, USA.
authors.forename: Barbara W
authors.initials: BW
authors.lastname: LeDuc
authors.affiliation: ADME Biology, GVK Biosciences Pvt. Ltd., Survey Nos. 125 (Part) and 126, IDA Mallapur, Hyderabad, Telangana, 500 076, India. ansarali.khan@gvkbio.com.
authors.forename: Ansar A
authors.initials: AA
authors.lastname: Khan
mesh_terms: D000818:Animals; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007654:Ketoconazole / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D008995:Monoamine Oxidase / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D014230:Triazoles / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D008996:Monoamine Oxidase Inhibitors; D014230:Triazoles; C033020:1-aminobenzotriazole; D051544:Cytochrome P-450 CYP3A; D008995:Monoamine Oxidase; D007654:Ketoconazole
keywords: Human Liver Microsome; Ketoconazole; Mouse Liver Microsome; Rasagiline; Selegiline