title: Compartmental and enzyme kinetic modeling to elucidate the biotransformation pathway of a centrally acting antitrypanosomal prodrug.
abstract: DB868 [2,5-bis [5-(N-methoxyamidino)-2-pyridyl] furan], a prodrug of the diamidine DB829 [2,5-bis(5-amidino-2-pyridyl) furan], has demonstrated efficacy in murine models of human African trypanosomiasis. A cross-species evaluation of prodrug bioconversion to the active drug is required to predict the disposition of prodrug, metabolites, and active drug in humans. The phase I biotransformation of DB868 was elucidated using liver microsomes and sandwich-cultured hepatocytes from humans and rats. All systems produced four NADPH-dependent metabolites via O-demethylation (M1, M2) and N-dehydroxylation (M3, M4). Compartmental kinetic modeling of the DB868 metabolic pathway suggested an unusual N-demethoxylation reaction that was supported experimentally. A unienzyme Michaelis-Menten model described the kinetics of M1 formation by human liver microsomes (HLMs) (K(m), 11 μM; V(max), 340 pmol/min/mg), whereas a two-enzyme model described the kinetics of M1 formation by rat liver microsomes (RLMs) (K(m1), 0.5 μM; V(max1), 12 pmol/min/mg; K(m2), 27 μM; V(max2), 70 pmol/min/mg). Human recombinant CYP1A2, CYP3A4, and CYP4F2, rat recombinant Cyp1a2 and Cyp2d2, and rat purified Cyp4f1 catalyzed M1 formation. M2 formation by HLMs exhibited allosteric kinetics (S(50), 18 μM; V(max), 180 pmol/mg), whereas M2 formation by RLMs was negligible. Recombinant CYP1A2/Cyp1a2 catalyzed M2 formation. DB829 was detected in trace amounts in HLMs at the end of the 180-min incubation and was detected readily in sandwich-cultured hepatocytes from both species throughout the 24-h incubation. These studies demonstrated that DB868 biotransformation to DB829 is conserved between humans and rats. An improved understanding of species differences in the kinetics of DB829 formation would facilitate preclinical development of a promising antitrypanosomal prodrug.
authors.affiliation: Divisions of Molecular Pharmaceutics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Claudia N
authors.initials: CN
authors.lastname: Generaux
authors.forename: Garrett R
authors.initials: GR
authors.lastname: Ainslie
authors.forename: Arlene S
authors.initials: AS
authors.lastname: Bridges
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Ismail
authors.forename: David W
authors.initials: DW
authors.lastname: Boykin
authors.forename: Richard R
authors.initials: RR
authors.lastname: Tidwell
authors.forename: Dhiren R
authors.initials: DR
authors.lastname: Thakker
authors.forename: Mary F
authors.initials: MF
authors.lastname: Paine
mesh_terms: D000818:Animals; D001711:Biotransformation; D002478:Cells, Cultured; D002490:Central Nervous System / Q000187:drug effects*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003640:Dealkylation; D005260:Female; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology; D008954:Models, Biological; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D014344:Trypanocidal Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D007527:Isoenzymes; D011355:Prodrugs; D011994:Recombinant Proteins; D014344:Trypanocidal Agents; D003577:Cytochrome P-450 Enzyme System