title: CYP1A-mediated metabolism of the Janus kinase-3 inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline: structural basis for inactivation by regioselective O-demethylation.
abstract: Here we report the phase I metabolism of the rationally designed Janus kinase-3 (JAK) inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131; JANEX-1). JANEX-1 was metabolized by the cytochrome P450 enzymes CYP1A1 and CYP1A2 in a regioselective fashion to form the biologically inactive 7-O-demethylation product 4-(4'-hydroxyphenyl)-amino-6-methoxy-7-hydroxyquinazoline (JANEX-1-M). Our molecular modeling studies indicated that the CYP1A family enzymes bind and demethylate JANEX-1 at the C-7 position of the quinazoline ring since the alternative binding conformation with demethylation at the C-6 position would result in a severe steric clash with the binding site residues. The metabolism of JANEX-1 to JANEX-1-M in pooled human liver microsomes followed Michaelis-Menten kinetics with V(max) and K(m) values (mean +/- S.D.) of 34.6 +/- 9.8 pmol/min/mg and 107.3 +/- 66.3 microM, respectively. alpha-Naphthoflavone and furafylline, which both inhibit CYP1A2, significantly inhibited the formation of JANEX-1-M in human liver microsomes. There was a direct correlation between CYP1A activities and the magnitude of JANEX-1-M formation in the liver microsomes from different animal species. A significantly increased metabolic rate for JANEX-1 was observed in Aroclor 1254-, beta-naphthoflavone-, and 3-methylcholanthrene-induced microsomes but not in clofibrate-, dexamethasone-, isoniazid-, and phenobarbital-induced microsomes. The formation of JANEX-1-M in the presence of baculovirus-expressed CYP1A1 and 1A2 was consistent with Michaelis-Menten kinetics. The systemic clearance of JANEX-1-M was much faster than that of JANEX-1 (5525.1 +/- 1926.2 ml/h/kg versus 1458.0 +/- 258.6 ml/h/kg). Consequently, the area under the curve value for JANEX-1-M was much smaller than that for JANEX-1 (27.5 +/- 8.0 versus 94.8 +/- 18.4 microM. h; P < 0.001).
authors.affiliation: Department of Pharmaceutical Sciences, Parker Hughes Cancer Center, 2665 Long Lake Road, Suite 330, St. Paul, MN 55113, USA. fatih_uckun@ih.org
authors.forename: Fatih M
authors.initials: FM
authors.lastname: Uckun
authors.forename: Jason
authors.initials: J
authors.lastname: Thoen
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Elise
authors.initials: E
authors.lastname: Sudbeck
authors.forename: Chen
authors.initials: C
authors.lastname: Mao
authors.forename: Ravi
authors.initials: R
authors.lastname: Malaviya
authors.forename: Xing-Ping
authors.initials: XP
authors.lastname: Liu
authors.forename: Chun-Lin
authors.initials: CL
authors.lastname: Chen
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D004285:Dogs; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D066298:In Vitro Techniques; D053616:Janus Kinase 3; D008252:Macaca fascicularis; D008407:Mast Cells / Q000378:metabolism; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology; D008958:Models, Molecular; D010323:Passive Cutaneous Anaphylaxis / Q000187:drug effects; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011799:Quinazolines / Q000378:metabolism* / Q000493:pharmacokinetics / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D013237:Stereoisomerism
chemical_list: D004791:Enzyme Inhibitors; C507936:Jak3 protein, rat; D011799:Quinazolines; D011994:Recombinant Proteins; C116180:WHI P131; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D011505:Protein-Tyrosine Kinases; C507933:JAK3 protein, human; C507935:Jak3 protein, mouse; D053616:Janus Kinase 3