Decreased Warfarin Clearance Associated With the CYP2C9 R150H (*8) Polymorphism

Abstract

The cytochrome P450 (CYP) 2C9 R150H (*8) allele occurs commonly in African Americans and is associated with lower warfarin dose requirements. We examined whether the CYP2C9*8 allele impacts warfarin clearance through a pharmacokinetic study in warfarin-treated African American patients and an in vitro kinetic study of S-warfarin 7-hydroxylation using cDNA-expressed CYP2C9 enzymes. We observed a 30% reduction in the unbound oral clearance of S-warfarin and 25% lower R- to S-warfarin plasma concentration in patients with the CYP2C9*8 allele (n=12) compared to CYP2C9*1 homozygotes (n=26). Consistent with these findings, the in-vitro intrinsic clearance of S-warfarin was 30% lower with the cDNA-expressed R150H protein compared to the wild-type protein. These data show that the R150H variant of the CYP2C9*8 allele reduces S-warfarin clearance, thus providing clinical and experimental evidence to explain lower warfarin dose requirements with the CYP2C9*8 allele.

Characterization of Inhibition Kinetics of (S)-Warfarin Hydroxylation by Noscapine: Implications in Warfarin Therapy

“…Since the formation of an MI complex is dependent upon catalytic rate, the susceptibility to MBI is expected to be dependent upon the relative activities of cytochrome P450 variants (Polasek and Miners, 2007;Orr et al, 2012). To verify that formation of a reactive metabolite(s) of noscapine by CYP2C9 is responsible for the MBI by noscapine, the kinetics of CYP2C9 inhibition were characterized using CYP2C9.2 and CYP2C9.3 variants that are known to exhibit decreased enzyme activity (Liu et al, 2012). Surprisingly, our results showed that the inactivation of CYP2C9 by noscapine was .2-fold more efficient (i.e., k inact /K I ) for CYP2C9.2 and CYP2C9.3 than for CYP2C9.1.…”

“…At different time points (0-30 minutes), an aliquot (10 ml) was transferred into a secondary incubation system containing (S)-warfarin (25 mM), and 7-hydroxywarfarin concentrations were measured by LC-MS/MS. The concentration of warfarin in the secondary incubation system was determined to be .4 times higher than the apparent K m values obtained from different enzyme sources (Hutzler et al, 2009;Liu et al, 2012). The observed rates of inactivation (k obs ) were determined from the slopes of residual CYP2C9 activity versus time plot.…”

“…Pooled HLMs (0.1 mg/ml) or recombinant enzymes (40 pmol/ml) were incubated with (S)-warfarin (2.5 mM) in the presence of noscapine (0-100 mM) in a NADPH-regenerating system for 20 minutes. The warfarin concentration was selected based on the apparent K m values in pooled HLMs or cDNA-expressed CYP2C9 (2-9 mM) (Liu et al, 2012) and the clinically relevant concentration range (C max , ;5 mM) (Maddison et al, 2013). 7-Hydroxywarfarin formation rates were determined, and the half-maximal inhibitory concentration (IC 50 ) values were estimated.…”

“…7-Hydroxywarfarin concentrations in microsomal samples were determined by an LC-MS/MS method as previously described, with slight modifications (Liu et al, 2012). An Agilent 1200 high-performance liquid chromatography (Agilent Technologies, Santa Clara, CA) interfaced with an Applied Biosystems Qtrap 3200 equipped with an electrospray ion source was used.…”

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59

“…They have been identified as the most common defective alleles and have exhibited reduced metabolic activities, both in vitro and in vivo, for the CYP2C9 substrates, such as warfarin. In addition to these two common alleles, other allelic variants, such as CYP2C9*4 (Lee et al, 2007), CYP2C9*5 (Dickmann et al, 2001), CYP2C9*8 (Liu et al, 2012), CYP2C9*11 (Tai et al, 2005), CYP2C9*12 (O'Brien et al, 2013), CYP2C9*14 (Lee et al, 2014), CYP2C9*35 (Ciccacci et al, 2011), CYP2C9*57 (Nahar et al, 2013), and CYP2C9*58 (Luo et al, 2014), were reported to affect warfarin sensitivity. In this study, we present the case of a Chinese warfarin-sensitive patient who requires only one-half of the normal oral warfarin dose.…”