PT  - JOURNAL ARTICLE
AU  - Kirk R. Henne
AU  - Thuy B. Tran
AU  - Brooke M. VandenBrink
AU  - Dan A. Rock
AU  - Divesh K. Aidasani
AU  - Raju Subramanian
AU  - Andrew K. Mason
AU  - David M. Stresser
AU  - Yohannes Teffera
AU  - Simon G. Wong
AU  - Michael G. Johnson
AU  - Xiaoqi Chen
AU  - George R. Tonn
AU  - Bradley K. Wong
TI  - Sequential Metabolism of AMG 487, a Novel CXCR3 Antagonist, Results in Formation of Quinone Reactive Metabolites That Covalently Modify CYP3A4 Cys239 and Cause Time-Dependent Inhibition of the Enzyme
AID  - 10.1124/dmd.112.045708
DP  - 2012 Jul 01
TA  - Drug Metabolism and Disposition
PG  - 1429--1440
VI  - 40
IP  - 7
4099  - http://dmd.aspetjournals.org/content/40/7/1429.short
4100  - http://dmd.aspetjournals.org/content/40/7/1429.full
SO  - Drug Metab Dispos2012 Jul 01; 40
AB  - CYP3A4-mediated biotransformation of (R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-N-(pyridin-3-ylmethyl)-2-(4-(trifluoromethoxy)phenyl)acetamide (AMG 487) was previously shown to generate an inhibitory metabolite linked to dose- and time-dependent pharmacokinetics in humans. Although in vitro activity loss assays failed to demonstrate CYP3A4 time-dependent inhibition (TDI) with AMG 487, its M2 phenol metabolite readily produced TDI when remaining activity was assessed using either midazolam or testosterone (KI = 0.73–0.74 μM, kinact = 0.088–0.099 min−1). TDI investigations using an IC50 shift method successfully produced inhibition attributable to AMG 487, but only when preincubations were extended from 30 to 90 min. The shift magnitude was ∼3× for midazolam activity, but no shift was observed for testosterone activity. Subsequent partition ratio determinations conducted for M2 using recombinant CYP3A4 showed that inactivation was a relatively inefficient process (r = 36). CYP3A4-mediated biotransformation of [3H]M2 in the presence of GSH led to identification of two new metabolites, M4 and M5, which shifted focus away from M2 being directly responsible for TDI. M4 (hydroxylated M2) was further metabolized to form reactive intermediates that, upon reaction with GSH, produced isomeric adducts, collectively designated M5. Incubations conducted in the presence of [18O]H2O confirmed incorporation of oxygen from O2 for the majority of M4 and M5 formed (>75%). Further evidence of a primary role for M4 in CYP3A4 TDI was generated by protein labeling and proteolysis experiments, in which M4 was found to be covalently bound to Cys239 of CYP3A4. These investigations confirmed a primarily role for M4 in CYP3A4 inactivation, suggesting that a more complex metabolic pathway was responsible for generation of inhibitory metabolites affecting AMG 487 human pharmacokinetics.