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 withVmax and Kmvalues (mean ± S.D.) of 34.6 ± 9.8 pmol/min/mg and 107.3 ± 66.3 μM, respectively. α-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-, β-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 μM · h; P < 0.001).
Footnotes
- Abbreviations used are::
- TNF
- tumor necrosis factor
- PTK
- protein tyrosine kinases
- JAK3
- Janus kinase-3
- JANEX-1
- 4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline
- HPLC
- high-performance liquid chromatography
- JANEX-1-M
- 4-(4′-hydroxyphenyl)-amino-6-methoxy-7-hydroxyquinazoline
- P450
- cytochrome P450
- FMO
- flavin-containing monooxygenase
- ARO
- Aroclor 1254
- BNF
- β-naphthoflavone
- CFB
- clofibrate
- DEX
- dexamethasone
- ISO
- isoniazid
- 3-MC
- 3-methylcholanthrene
- PHEN
- phenobarbital
- DMSO
- dimethyl sulfoxide
- MS
- mass spectrometry
- PBS
- phosphate-buffered saline
- LC
- liquid chromatography
- DNP
- dinitrophenyl
- PIPES
- piperazine-N,N′-bis(2-ethanesulfonic acid)
- BSA
- bovine serum albumin
- TEA
- tetraethylammonium
- CL
- clearance
- RT
- retention time
- VDW
- van der Waals
- DDQ
- 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
- Received July 9, 2001.
- Accepted September 27, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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