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0090-9556/05/3306-749–753$20.00DRUG METABOLISM AND DISPOSITION Copyright 2005 by The American Society for Pharmacology and Experimental Therapeutics ROLE OF CYP2C9 AND ITS VARIANTS (CYP2C9*3 AND CYP2C9*13) IN THE
METABOLISM OF LORNOXICAM IN HUMANS
Yingjie Guo, Yifan Zhang, Ying Wang, Xiaoyan Chen, Dayong Si, Dafang Zhong, College of Life Science, Jilin University, Changchun, China (Y.G., Y.W., D.S., H.Z.); Laboratory of Drug Metabolism and Pharmacokinetics, Shenyang Pharmaceutical University, Shenyang, China (Y.Z., X.C., D.Z.); and School of Pharmacy, University of Otago, Dunedin, New Zealand (J.P.F.) Received January 8, 2005; accepted March 9, 2005 ABSTRACT:
CYP2C9 is an important member of the cytochrome P450 enzyme
postmitochondrial supernatant (S9) from transfected cells were
superfamily with some 12 CYP2C9 alleles (*1-*12) being previously
lower than those from wild-type CYP2C9*1. Mean values of K
reported. Recently, we identified a new CYP2C9 allele with a
V
for CYP2C9*1, *3, and *13 were 1.24, 1.61, and 2.79 ␮M and
Leu90Pro mutation in a Chinese poor metabolizer of lornoxicam [Si
0.83, 0.28, and 0.22 pmol/min/pmol, respectively. Intrinsic clear-
D, Guo Y, Zhang Y, Yang L, Zhou H, and Zhong D (2004) Pharma-
ance values (V
/K ) for variant CYP2C9*3 and CYP2C9*13 on the
cogenetics 14:465–469]. The new allele, designated CYP2C9*13,
basis of CYP2C9 protein levels were separately decreased to 28%
was found to occur in approximately 2% of the Chinese population.
and 12% compared with wild type. In a subsequent clinical study,
To examine enzymatic activity of the CYP2C9*13 allele, kinetic
the AUC of lornoxicam was increased by 1.9-fold and its oral
parameters for lornoxicam 5؅-hydroxylation were determined in
clearance (CL/F) decreased by 44% in three CYP2C9*1/*13 sub-
COS-7 cells transiently transfected with pcDNA3.1 plasmids car-
jects, compared with CYP2C9*1/*1 individuals. This suggests that
rying wild-type CYP2C9*1, variant CYP2C9*3, and CYP2C9*13
the CYP2C9*13 allele is associated with decreased enzymatic ac-
cDNA. The protein levels of cDNA-expressed CYP2C9*3 and *13 in
tivity both in vitro and in vivo.
CYP2C9 constitutes approximately 20% of the cytochrome P450 show decreased enzymatic activity in vitro and in vivo (Crespi et al., protein content of human liver microsomes and is responsible for the 1997; Aithal et al., 1999; Imai et al., 2000; Dickmann et al., 2001; metabolism of many clinically important drugs. These include drugs Kidd et al., 2001; Kirchheiner et al., 2002; Allabi et al., 2004; with a narrow therapeutic index such as warfarin and phenytoin and other routinely prescribed drugs such as acenocoumarol, tolbutamide, Recently, a new CYP2C9 allele designated CYP2C9*13 has been losartan, glipizide, and some nonsteroidal anti-inflammatory drugs identified in a Chinese poor metabolizer of lornoxicam. The allele possesses a T269C transversion in exon 2 of CYP2C9 that leads to a The CYP2C9 gene is highly polymorphic. At least 13 CYP2C9 Leu90Pro substitution. Frequency analysis shows that approximately alleles have been identified to date and most of them are associated 2% of the Chinese populations carry the allele (Si et al., 2004).
with reduced CYP2C9 activity. Among them, CYP2C9*3, with an Genotyping of this poor lornoxicam metabolizer revealed a Ile359Leu mutation, has been most widely studied. In vitro studies CYP2C9*3/*13 genotype with the two mutations located on separate show it has significantly impaired catalytic activity to various alleles. His lornoxicam half-life of about 105 h was markedly longer CYP2C9 substrates relative to the wild type (Takanashi et al., 2000).
than that of other CYP2C9*1/*3 and CYP2C9*1/*1 carriers (half-lives In vivo investigations show that individuals heterozygous and ho- of 5.8 – 8.1 and 3.2– 6.3 h, respectively; Zhang et al., 2005), suggest- mozygous for CYP2C9*3 have reduced intrinsic clearance of warfa- ing that the CYP2C9*13 allele has a larger effect on CYP2C9 meta- rin, phenytoin, and glipizide and are more at risk of clinical toxicity bolic capability than other alleles.
from these drugs (Aithal et al., 1999; Kidd et al., 1999; Ninomiya et CYP2C9 has been shown to be the primary enzyme responsible for al., 2000). Other CYP2C9 alleles such as CYP2C9*2 (Arg144Cys), the biotransformation of the nonsteroidal anti-inflammatory drug lor- CYP2C9*4 (Ile359Thr), CYP2C9*5 (Asp360Glu), CYP2C9*6 (null noxicam to its major metabolite, 5Ј-hydroxylornoxicam, in human allele), CYP2C9*11 (Arg335Trp), and CYP2C9*12 (Pro489Ser) also liver microsomes (Bonnabry et al., 1996; Kohl et al., 2000). Recently,it was reported that lornoxicam 5Ј-hydroxylation by the CYP2C9*3allele was markedly reduced compared with wild type, both in vitro Project supported by the National Natural Science Foundation of China, No.
and in vivo (Iida et al., 2004; Zhang et al., 2005). Thus, lornoxicam is an ideal substrate for the study of CYP2C9 enzyme activity. The purpose of this study was to compare the enzymatic activity of CYP2C9*1, CYP2C9*3, and CYP2C9*13 toward lornoxicam both in ABBREVIATIONS: DMEM, Dulbecco’s modified Eagle’s medium; P450, cytochrome P450; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/
nitro blue tetrazolium; CPR, NADPH-cytochrome P450 reductase; AUC, area under the plasma concentration-time curve; CL/F, oral clearance.
vitro in appropriately transfected COS-7 cells and in vivo in subjects (Invitrogen) as a standard with ImageJ software (National Institutes of Health, with CYP2C9*1/*3, CYP2C9*1/*13, and CYP2C9*1/*1 genotypes.
In Vitro Lornoxicam 5؅-Hydroxylation of Recombinant CYP2C9 Pro-
tein. Lornoxicam 5Ј-hydroxylation activity of the recombinant CYP2C9 pro-
Materials and Methods
tein was determined as described previously (Kohl et al., 2000) with minor Materials. Lornoxicam was purchased from Shangdi Xinshiji Medical
modifications. S9 fraction containing CYP2C9s was incubated with 100 mM Academy (Beijing, China.). 5Ј-Hydroxylornoxicam was provided by the Lab- Tris buffer (pH 7.5), 200 ␮M NADPH, and lornoxicam at 37°C for 1 h in the oratory of Microorganisms, Shenyang Pharmaceutical University (Shenyang, presence or absence of CPR. The reaction was stopped by addition of 500 ␮l China). Dulbecco’s modified Eagle’s medium (DMEM), pcDNA3.1(ϩ), and of methanol and stored overnight at Ϫ20°C to allow complete protein precip- LipofectAMINE 2000 were purchased from Invitrogen (Carlsbad, CA). Fetal itation. After centrifugation for 30 min at 12,000 rpm, the supernatants were bovine serum was purchased from Tianjin, H&Y Bio Co. Ltd. (Tianjing, reduced to 100 ␮l by warming at 65°C and subjected to high performance China). KpnI, XhoI, and DpnI enzymes were purchased from New England liquid chromatography assay. High performance liquid chromatography was Biolabs (Beverly, MA). Pfu DNA polymerase was purchased from Bio Basic Inc. (Toronto, ON, Canada). A pREP9 plasmid containing human CYP2C9*1 Technologies, Palo Alto, CA) using a mobile phase of 0.1 M NaH PO , pH cDNA and E. coli Top 10 were provided by the Department of Pathophysiol- 6.0/acetonitrile (7:3), at a flow rate of 1 ml/min. Detection was by UV ogy and the Laboratory of Medical Molecular Biology, School of Medicine, absorption at 371 nm. Under these conditions, retention times of 5Ј-hydroxy- Zhejiang University (Zhejiang, China). Rabbit anti-human cytochrome P450 lornoxicam and lornoxicam were 6.9 and 11.8 min, respectively. A six-point 2C9 antibody was purchased from Serotec (Oxford, UK). Alkaline phos- standard curve was used to quantify 5Ј-hydroxylornoxicam.
phatase-labeled anti-rabbit IgG, 5-bromo-4-chloro-3-indolyl phosphate/nitro In Vivo Lornoxicam Metabolism. The study was approved by the Inde-
blue tetrazolium (BCIP/NBT) and bovine serum albumin were purchased from pendent Ethics Committee of the People’s Hospital of Liaoning Province Beijing Dingguo Biotechnology Development Center (Beijing, China).
(Shenyang, China). Genotyping of CYP2C9*3 and CYP2C9*13 was carried NADPH was purchased from Roche Molecular Biochemicals (Basel, Switzer- out as described previously (Si et al., 2004; Zhang et al., 2005). Thirteen land). COS-7 cells were kindly donated by the Vaccination Center, Jilin CYP2C9*1 homozygotes, 7 CYP2C9*3 heterozygotes, and 3 CYP2C9*13 University (Changchun, China). Human recombinant NADPH-P450 reductase heterozygotes participated in the phenotyping study. All subjects were in good (CPR) was purchased from MBL International Corporation (Woburn, MA).
health and were required to refrain from all medication and alcohol prior to the All other reagents were of analytical grade.
pharmacokinetic study. In vivo lornoxicam metabolism was performed accord- Construction of Expression Plasmids. CYP2C9*1 cDNA in pREP9 plas-
ing to the method described previously (Zhang et al., 2005). In brief, after a mid was subcloned into pcDNA3.1(ϩ) by digestion with KpnI and XhoI single oral dose of 8 mg of lornoxicam, blood samples were collected before enzymes. Site-directed mutagenesis to introduce the A3 C transition at posi- dosing, and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 13, and 24 h postdose. Plasma tion 1075 (CYP2C9*3) and the T3 C transition at position 269 (CYP2C9*13) concentrations of lornoxicam were determined using a validated liquid chro- was performed using pcDNA3.1(ϩ) plasmids carrying CYP2C9*1 cDNA as matography-tandem mass spectrometry method reported elsewhere (Zeng et the template for polymerase chain reaction amplification by Pfu DNA poly- merase. The specific base transition was introduced into the amplificationproducts by a pair of completely complementary primers containing substi-tuted base. The mutagenic primers for CYP2C9*3 and *13 were 5Ј-CGAGG-TCCAGAGATACCTTGACCTTCTCCCCAC-3Ј and 5Ј-GGAAGCCCTGA-TTGATCCTGGAGAGGAGTTTTCTG-3Ј, respectively (mutations und-erlined). After incubation with DpnI enzyme, the origin templates were dig-ested, but the new amplified polymerase chain reaction products containingsubstituted base remained and were transformed to E. coli Top 10. Clonescontaining the desired nucleotide change were identified by sequencing carriedout by Shanghai Sangon Biological Engineering Technology and Service Co.
Ltd. (Shanghai, China).
Transfection of COS-7 Cells and Preparation of Postmitochondrial
Supernatant (S9). COS-7 cells were seeded into 10-cm culture flasks in
DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, and 100
␮g/ml streptomycin. When cells were 90 to 95% confluent, the culture medium
was replaced with DMEM without penicillin and streptomycin, and the
CYP2C9 expression plasmids (24 ␮g/flask), purified with a QIAGEN plasmid
mini kit (QIAGEN, Valencia, CA), were transfected into COS-7 cells using
LipofectAMINE 2000 at 60 ␮l/flask, as per the manufacturer’s instructions.
Forty-eight hours after transfection, cells were scraped from the culture flask
and washed twice with Ca2ϩ- and Mg2ϩ-free Hanks’ solution. The pellets were
resuspended in 20 mM potassium phosphate buffer, pH 7.4, containing 0.2 mM
EDTA, 1 mM dithiothreitol, and 20% glycerol, and sonicated with twelve 5-s
pulses at 23% power of a Sonics Vibra-Cell sonicator (Sonics & Materials,
Inc., Newtown, CT). The homogenate was centrifuged at 9000g, 4°C for 20
min and the postmitochondrial supernatant (S9 fraction) collected for assay or
storage at Ϫ70°C. Protein concentrations in S9 were determined by the
Bradford method (Bradford, 1976) using bovine serum albumin as standard.
Quantification of CYP2C9 Protein by Western Blotting. S9 fraction (50
FIG. 1. CYP2C9 protein levels in postmitochondrial supernatant (S9) from COS-7 ␮g) and human liver microsomes (10 ␮g) were separated on 10% sodium cells expressing wild-type and variant CYP2C9s. A, immunoblot analysis of re- dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene diflu- combinant human CYP2C9 protein. S9 fraction (50 ␮g) isolated from COS-7 cells oride membrane (Millipore Corporation, Billerica, MA). The membrane was transfected with the wild-type and variant CYP2C9 cDNA was utilized for immu-noblotting using anti-human CYP2C9 antibody. Human liver microsome (10 ␮g) incubated with rabbit anti-human cytochrome P450 2C9 antibody as the was used as positive control. B, protein levels of CYP2C9 were quantified by primary antibody and then with alkaline phosphatase-labeled anti-rabbit IgG as densitometric analysis. The results are expressed as a percentage of the level of the secondary antibody. Bands were visualized by incubation with BCIP/NBT CYP2C9*1. Each bar represents the mean Ϯ S.E.M. of three independent experi- and quantified by microsomes from insect cells expressing human CYP2C9 CYP2C9 VARIANTS AND LORNOXICAM METABOLISM FIG. 2. Michaelis-Menten kinetics of lornoxicam by postmitochondrial supernatant(S9) from COS-7 cells expressing wild-type and variant CYP2C9s. S9 fractioncorresponding to 500 ␮g of protein was incubated with different concentrations oflornoxicam in the absence of CPR. Experimental conditions are described underMaterials and Methods. Each point represents the mean of three independentexperiments. f, CYP2C9*1; Œ, CYP2C9*3; , CYP2C9*13.
FIG. 3. Plasma concentration-time curves of lornoxicam in healthy subjects withgenotype CYP2C9*1/*1 (ࡗ) (n ϭ 13), CYP2C9*1/*3 (Ⅺ) (n ϭ 7), and CYP2C9*1/ Data Analysis. Michaelis-Menten analysis was performed by nonlinear
*13 (‚) (n ϭ 3) after a single oral dose of 8 mg of lornoxicam.
regression curve fitting using the computer program Prism v4.0 (GraphPadSoftware Inc., San Diego, CA). Pharmacokinetic parameters were calculated matic activities were normalized to CYP2C9 protein levels, intrinsic using standard noncompartmental methods. Student’s t test was used for /K ) for variant CYP2C9*3 and CYP2C9*13 intergroup comparison. A value of P Ͻ 0.05 was considered to be statistically were also separately decreased to 28% and 12% compared with wild type. Clearly, the activity of CYP2C9*13 is lower than that of In the in vivo study, the CYP2C9 genotype significantly affected A representative immunoblot of S9 fraction prepared from COS-7 the pharmacokinetics of lornoxicam (Fig. 3; Table 2). The AUC of cells expressing CYP2C9*1, CYP2C9*3, and CYP2C9*13 proteins is lornoxicam increased 1.9-fold and CL/F was decreased by 44% in presented in Fig. 1. All constructs yielded immunodetectable CYP2C9 CYP2C9*1/*13 individuals compared with CYP2C9*1/*1 individu- protein, as did human liver microsomes. The expressed protein level als. Similar changes were found in CYP2C9*1/*3 individuals.
of CYP2C9*1 was 7.51 pmol/mg S9 protein, and the expression levelsof variant CYP2C9*3 and *13 were 69.9% and 35.5%, respectively,of that of CYP2C9*1.
Discussion
The effect of exogenous CPR on lornoxicam 5Ј-hydroxylation was To investigate the catalytic activity of the CYP2C9*13 allele in studied by incubation with recombinant CYP2C9*1. S9 fraction con- vitro, we established a COS cell expression system. This has been taining 3.75 pmol of CYP2C9*1 was mixed with varying amounts of widely applied for functional characterization of P450 alleles contain- CPR and 10 ␮M lornoxicam. When CPR was 0, 5, 12.5, or 25 ␮M, the ing CYP2C9, CYP2D6, CYP2B6, and CYP2E1 (Veronese et al., for CYP2C9*1 was 0.66, 0.74, 0.68, or 0.68 1993; Hu et al., 1997; Marcucci et al., 2002; Jinno et al., 2003).
pmol/min/pmol, respectively. There was no significant alteration in However, due to the low levels of expression in COS-7 cells, we catalytic efficiency of CYP2C9*1 with the increasing CPR concen- failed to quantify CYP2C9 holoenzyme contents by CO-difference tration, indicating that endogenous reductase is enough for lornoxicam spectroscopy. Thus, CYP2C9 proteins were quantified by immuno- metabolism in the COS-7 expression system. Thus, exogenous CPR blotting with microsomes from insect cells expressing human was not utilized in the following kinetic study.
CYP2C9 as standard. This kind of method quantifying P450 was also Michaelis-Menten kinetics of lornoxicam for wild-type and mutant used for the functional characterization of CYP2D6, CYP2B6, and CYP2C9 is shown in Fig. 2. Corresponding kinetic parameters are CYP2E1 allelic variants in the COS expression system (Marcucci et summarized in Table 1. On the basis of protein levels of S9 fraction, al., 2002; Hanioka et al., 2003; Jinno et al., 2003). Our results show both CYP2C9*3 and CYP2C9*13 exhibited lower intrinsic clearance that COS-7 cells can efficiently express active CYP2C9 protein. The of lornoxicam (P Ͻ 0.01) than did wild-type CYP2C9*1 resulting protein levels of cDNA-expressed CYP2C9*3 and *13 in S9 fraction (1.3-fold and 2.3-fold, respectively) and from COS-7 cells were lower than those in wild-type CYP2C9*1. The (76% and 90%, respectively). When the enzy- reduced protein levels in the CYP2C9 variants may contribute to Kinetic parameters for lornoxicam 5Ј-hydroxylation from COS-7 cells expressing wild-type and variant CYP2C9s Each value represents the mean Ϯ S.E.M. of three independent experiments.
** P Ͻ 0.01 versus CYP2C9*1.
Pharmacokinetic parameters of lornoxicam in healthy subjects with CYP2C9*1/*1, CYP2C9*1/*3, and CYP2C9*1/*13 genotype Data are given as mean and 95% confidence interval (in parentheses).
* P Ͻ 0.05, **P Ͻ 0.01 versus CYP2C9*1/*1.
lower transcription, translation efficiency, and protein stability. Oth- Our results show that the activities of both CYP2C9*3 and erwise, although polyclonal antibody was used, the immunoreactivity CYP2C9*13 toward lornoxicam in vitro are compatible with their of mutant CYP2C9s may be altered by mutagenization. Therefore, the activities in vivo, and there is a reasonable correlation between in vitro enzymatic activities were assayed in two ways, on the basis of S9 activity and in vivo metabolic clearance of lornoxicam. Recently, it protein level and CYP2C9 protein level. These data obtained using the was reported that individuals carrying the CYP2C9*3 allele are at risk COS expression system need to be confirmed by a baculovirus sys- of experiencing drug toxicity, especially of drugs with a narrow tem, which is better suited to obtain the quantity of P450 by spectral therapeutic index such as warfarin and phenytoin (Aithal et al., 1999; analysis. These studies are currently under investigation in our labo- Kidd et al., 1999; Ninomiya et al., 2000). By extrapolation, one may speculate that carriers of the CYP2C9*13 allele would experience In this study, the presence of the CYP2C9*3 allele impairs both greater risk from these drugs. Therefore, genotyping for CYP2C9*13 intrinsic clearance and systemic clearance of lornoxicam. A recent may be important to allow individualization of dosing for CYP2C9 report shows that CYP2C9*1/*3 individuals have a 55% decrease in CL/F and a 1.9-fold increase in AUC of lornoxicam compared with In conclusion, the Leu90Pro substitution of CYP2C9*13 markedly CYP2C9*1/*1 individuals (Zhang et al., 2005). The magnitude of decreases the intrinsic clearance of lornoxicam in vitro and in vivo.
these changes in pharmacokinetic parameters is consistent with our in The reduction in activity due to CYP2C9*13 is greater than that due vivo results. Iida et al. (2004) reported that CYP2C9*3 expressed in to CYP2C9*3 in vitro. Whether carriers of the CYP2C9*13 allele may baculovirus-infected insect cells significantly decreased lornoxicam be at greater risk of toxicity from CYP2C9 substrate drugs with a 5Ј-hydroxylation relative to wild type with a 2.3-fold increase in K narrow therapeutic index remains to be confirmed by further in vivo (P Ͻ 0.05) and 76% decrease in V showed a 1.3-fold increase (P Ͼ 0.05) and the V on the basis of CYP2C9 protein level relative to wild type. The reason References
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