Protein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats.
(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single intravenous doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with organic solvents. The unextractable radioactivity remained associated with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110 degrees C from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-methyl BMS-204352 by liquid chromatography/mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-methyl BMS-204352 with the epsilon-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P450-reductase cofactor NADPH and was attenuated by glutathione. P4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated: 1) P4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and 2) nucleophilic addition of the epsilon-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-methyl BMS-204352 lysine adduct.
Zhang
Donglu
D
Department of Preclinical Candidate Optimization, P.O. BOX 4000, Bristol-Myers Squibb, Princeton, NJ 08543, USA. Donglu.Zhang@BMS.com
Ogan
Marc
M
Gedamke
Richard
R
Roongta
Vikram
V
Dai
Renke
R
Zhu
Mingshe
M
Rinehart
J Kent
JK
Klunk
Lewis
L
Mitroka
James
J
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001798:Blood Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000008:administration & dosage / Q000097:blood; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D045563:Indolequinones / Q000097:blood*; D007211:Indoles / Q000008:administration & dosage / Q000097:blood* / Q000378:metabolism / Q000493:pharmacokinetics*; D007275:Injections, Intravenous; D008297:Male; D011485:Protein Binding / Q000187:drug effects* / Q000502:physiology; D011869:Radioligand Assay; D051381:Rats
C482224:BMS-349821; C424388:BMS204352; D001798:Blood Proteins; D002250:Carbon Radioisotopes; D045563:Indolequinones; D007211:Indoles; C068040:quinone methide; D003577:Cytochrome P-450 Enzyme System