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1. Drug Metab Dispos. 2012 Apr 19. [Epub ahead of print]

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.

Henne KR, Tran TB, Vandenbrink BM, Rock DA, Aidasani DK, Subramanian R, Mason AK,
Stresser DM, Teffera Y, Wong SG, Johnson MG, Chen X, Tonn GR, Wong BK.

1 Amgen, Inc;

CYP3A4-mediated biotransformation of AMG 487 was previously shown to generate an
inhibitory metabolite linked to dose- and time-dependent pharmacokinetics in
humans. Though 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 (K(I)=0.73-0.74 μM, k(inact)=0.088-0.099 min(-1)). TDI
investigations employing an IC(50) shift method successfully produced inhibition
attributable to AMG 487, but only when pre-incubations were extended from 30 to
90 min. The shift magnitude was ≈3x for midazolam activity, but no shift was
observed for testosterone activity. Subsequent partition ratio determinations
conducted for M2 using recombinant CYP3A4 showed inactivation was a relatively
inefficient process (r=36). CYP3A4-mediated biotransformation of [(3)H]-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 [(18)O]-water confirmed incorporation of oxygen from
O(2) 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, where M4 was found covalently bound to Cys239 of CYP3A4. These
investigations confirmed a primarily role for M4 in CYP3A4 inactivation,
suggesting a more complex metabolic pathway was responsible for generation of
inhibitory metabolites affecting AMG 487 human pharmacokinetics.

PMID: 22517972 [PubMed - as supplied by publisher]