Genetic Polymorphisms and in Vitro Functional Characterization of CYP2C8, CYP2C9, and CYP2C19 Allelic Variants

Abstract

Genetic variations in CYP 2C (CYP2C) subfamily, CYP2C8, CYP2C9, and CYP2C19 contribute to interindividual variability in the metabolism of clinically used drugs. Changes in the drug metabolizing activity of CYP2C members may cause unexpected and serious adverse drug reactions and inadequate therapeutic effects. Therefore, CYP2C gene polymorphism is used as a genome biomarker for predicting responsiveness to administered drugs. The most direct method for understanding the extent of the effects of CYP2C gene polymorphism on drug pharmacokinetics is by evaluating the blood and urine concentrations of the drug in subjects. However, in vivo tests are highly invasive, and considering the risk of adverse drug reactions, the burden on the patient may be significant. In addition, examining the functions of rare variant enzymes with an allele frequency of ≤1% requires at least several hundred subjects. Furthermore, it is extremely difficult to evaluate the functions of all variant enzymes in an in vivo test. On the other hand, in vitro enzyme activity can be evaluated using a heterologous expression system to avoid the aforementioned problems. In vitro tests are extremely important as they complement in vivo information. This review focuses on recent findings of in vitro studies on 3 highly polymorphic CYP2C members: CYP2C8, CYP2C9, and CYP2C19.

INTRODUCTION

Individual differences exist in the expression of drug effects and adverse reactions. Therefore, it is often a struggle to design doses of drugs, particularly drugs that have narrow therapeutic ranges and are associated with serious adverse reactions. Various factors can cause differences in the expression of drug effects among individuals. These include age, sex, concomitant drugs, diet, and smoking. Currently, gene polymorphism is considered as one of the factors that significantly influence such differences.^1,2) Therefore, diversity in the genes that code for proteins involved in pharmacokinetic and pharmacodynamic processes causes differences in the expression of drug effects and adverse reactions. Such proteins therefore include those involved in drug absorption, distribution, metabolism, and excretion/elimination, as well as those located at the site of drug action. The study of the causal relationship between drug response and gene polymorphism is pharmacogenomics (PGx). PGx is therefore important in establishing individualized drug therapies based on gene polymorphism. Changes in the functions of the CYP enzyme system due to gene polymorphism can cause changes in the blood concentrations of administered drugs. An example of gene polymorphism is single nucleotide polymorphism (SNP). Changes in the drug metabolizing activity of CYP may be the cause of unexpected and serious adverse drug reactions as well as inadequate therapeutic effects. Therefore, CYP gene polymorphism is used as a genome biomarker for predicting responsiveness to administered drugs.³⁾ As a result, by selecting therapeutic agents based on the gene polymorphism in a particular patient and by adjusting drug doses to suit individual needs, safer and more effective individualized drug therapies can be administered.

The CYP2C is a subfamily of the CYP molecular species in humans and is comprised of four members: CYP2C8, CYP2C9, CYP2C18, and CYP2C19.^4–7) CYP2C8, CYP2C9, and CYP2C19 are particularly important in drug metabolic reactions and are involved in the metabolism of approximately 20% of the drugs on the market.⁵⁾ They have at least 77% amino acid sequence homology; however, they have different substrate specificities.⁶⁾ A large number of gene polymorphisms of these molecular species have been identified (The Human Cytochrome P450 Allele Nomenclature Database; http://www.cypalleles.ki.se/). This is the cause of individual differences in drug pharmacokinetics, therapeutic effects, and expression of adverse drug reactions. Therefore, clarifying the differences in the functions of variant enzymes due to gene polymorphism of CYP2C8, CYP2C9, and CYP2C19 is extremely important for understanding variations in drug responses.

The most direct method for understanding the extent of the effects of CYP2C gene polymorphism on a drug’s pharmacokinetics is by evaluating the blood and urine concentrations of the drug in subjects. However, in vivo tests are highly invasive and considering the risk of adverse drug reactions, the burden on the patient may be significant. Also, the function of rare variant enzymes with an allele frequency of 1% or less, requires at least several hundred subjects. Furthermore, it is extremely difficult to evaluate the functions of all the variant enzymes in an in vivo test. On the other hand, in vitro enzyme activity can be evaluated using a heterologous expression system to avoid the aforementioned problems. In vitro tests are extremely important as they complement in vivo information. Previously, in vitro evaluations of the functions of the CYP2C subfamily variant enzymes were conducted using a variety of expression systems including bacteria, yeast, baculoviruses, and mammalian cells.⁸⁾ It has been reported that values for enzyme kinetic parameters obtained from using various cDNA expression systems differ from each other.⁸⁾ However, using the cDNA expression system of mammalian cells and conducting simultaneous analyses on all CYP2C variants under the same conditions would result in a more accurate evaluation. The protein translation and modification processes in the cDNA expression system of mammalian cells are similar to those in humans. On the other hand, CYP expressed by baculovirus-mediated system can be expressed at levels of 300–1000 pmol/g total cell lysate, and would be useful to evaluate functional changes of CYP2C variants derived from gene polymorphism.^9,10) Several studies made use of recombinant CYP2C proteins by mammalian cell- or baculoviruses-expression system (Tables 1–3). Below is a review on these studies.

Table 1. CYP2C8 Variants and in Vitro Metabolism of CYP2C8 Substrates

CYP2C8 allele	CYP2C8 proteins#	Amino-acid changes	Expression system	Substrates	Effect reduction CLint or activity versus CYP2C8.1	References
CYP2C8*1	CYP2C8.1
CYP2C8*2	CYP2C8.2	I269F	Insect	Paclitaxel	48% of CLint ratio	66)
			Insect	Repaglinide	78% of CLint ratio	66)
			Insect	Ibuprofen	Increased activity	66)
			COS-7	Paclitaxel	143% of CLint ratio	22)
			COS-7	Amodiaquine	110% of CLint ratio	22)
CYP2C8*3	CYP2C8.3	R139K, K399R	Insect	Paclitaxel	64% of CLint ratio	66)
			Insect	Repaglinide	1.31-Fold higher CLint	66)
			Insect	Ibuprofen	Reduced activity	66)
			HepG2	Paclitaxel	Lower activity	67)
			HepG2	Amodiaquine	No effects	67)
			COS-7	Paclitaxel	68% of CLint ratio	22)
			COS-7	Amodiaquine	85% of CLint ratio	22)
CYP2C8*4	CYP2C8.4	I264M	Insect	Paclitaxel	30% of CLint ratio	66)
			Insect	Repaglinide	80% of CLint ratio	66)
			Insect	Ibuprofen	Reduced activity	66)
			COS-7	Paclitaxel	81% of CLint ratio	22)
			COS-7	Amodiaquine	79% of CLint ratio	22)
CYP2C8*5		159Frameshift
CYP2C8*6	CYP2C8.6	G171S	COS-1	Paclitaxel	No effects	68)
			COS-7	Paclitaxel	94% of CLint ratio	22)
			COS-7	Amodiaquine	39% of CLint ratio	22)
CYP2C8*8	CYP2C8.8	R186G	COS-1	Paclitaxel	20% of wild-type activity	68)
			COS-7	Paclitaxel	65% of CLint ratio	22)
			COS-7	Amodiaquine	32% of CLint ratio	22)
CYP2C8*9	CYP2C8.9	K247R	COS-1	Paclitaxel	No effects	68)
			COS-7	Paclitaxel	123% of CLint ratio	22)
			COS-7	Amodiaquine	52% of CLint ratio	22)
CYP2C8*10	CYP2C8.10	K383N	COS-1	Paclitaxel	No effects	68)
			COS-7	Paclitaxel	91% of CLint ratio	22)
			COS-7	Amodiaquine	39% of CLint ratio	22)
CYP2C8*12	CYP2C8.12	461delV	COS-7	Paclitaxel	192% of CLint ratio	22)
			COS-7	Amodiaquine	47% of CLint ratio	22)
CYP2C8*13	CYP2C8.13	I223M	COS-7	Paclitaxel	67% of CLint ratio	22)
			COS-7	Amodiaquine	22% of CLint ratio	22)
CYP2C8*14	CYP2C8.14	A238P	COS-7	Paclitaxel	13% of CLint ratio	22)
			COS-7	Amodiaquine	No activity	22)

Paclitaxel, 6α-hydroxylation; Repaglinide, 3′-hydroxylation; Ibuprofen, 2-hydroxylation; Amodiaquine, N-deethylation. ^# The name for the corresponding proteins have a period between the name of the gene product and the allele number (e.g. CYP2C8.3). If the allele is unable to produce full length protein, no protein name will be assigned (e.g. CYP2C8*5 (475delA, 159Frameshift)).

Table 2. CYP2C9 Variants and in Vitro Metabolism of CYP2C9 Substrates

CYP2C9 allele	CYP2C9 proteins	Amino-acid changes	Expression system	Substrates	Effect reduction CLint or activity versus CYP2C9.1	References
CYP2C9*1	CYP2C9.1
CYP2C9*2	CYP2C9.2	R144C	COS-7	S-Warfarin	32% of CLint ratio	47)
			COS-7	Tolbutamide	42% of wild-type activity	47)
			HepG2	S-Warfarin	Lower Vmax and CLint	69)
			HepG2	Tolbutamide	No effects	69)
			Insect	S-Warfarin	68% of CLint ratio	70)
			Insect	Glimepiride	82% of CLint ratio	49)
			Insect	Tolbutamide	47% of CLint ratio	51)
			Insect	Losartan	46% of CLint ratio	53)
			COS-7	S-Warfarin	25% of CLint ratio	71)
CYP2C9*3	CYP2C9.3	I359L	COS-7	S-Warfarin	21% of wild-type activity	47)
			COS-7	Tolbutamide	28% of wild-type activity	47)
			Insect	S-Warfarin	4% of CLint ratio	70)
			Insect	S-Warfarin	Higher Km, Lower Vmax and CLint	72,73)
			COS-7	Tolbutamide	Higher Km, Lower CLint	74)
			Insect	Glimepiride	16% of CLint ratio	49)
			Insect	Tolbutamide	11% of CLint ratio	51)
			Insect	Losartan	20% of CLint ratio	53)
			COS-7	S-Warfarin	8% of CLint ratio	71)
CYP2C9*4	CYP2C9.4	I359T	COS-7	S-Warfarin	16% of wild-type activity	47)
			COS-7	Tolbutamide	22% of wild-type activity	47)
CYP2C9*5	CYP2C9.5	D360E	COS-7	S-Warfarin	19% of wild-type activity	47)
			COS-7	Tolbutamide	24% of wild-type activity	47)
			Insect	S-Warfarin	Higher Km, Lower CLint	72)
CYP2C9*7	CYP2C9.7	L19I	COS-7	S-Warfarin	47% of CLint ratio	47)
			COS-7	Tolbutamide	69% of wild-type activity	47)
CYP2C9*8	CYP2C9.8	R150H	COS-7	S-Warfarin	41% of CLint ratio	47)
			COS-7	Tolbutamide	74% of wild-type activity	47)
			Insect	Glimepiride	9% of CLint ratio	49)
			Insect	Tolbutamide	20% of CLint ratio	51)
			Insect	Losartan	26% of CLint ratio	53)
			COS-7	S-Warfarin	46% of CLint ratio	71)
CYP2C9*9	CYP2C9.9	H251R	COS-7	S-Warfarin	82% of CLint ratio	47)
			COS-7	Tolbutamide	96% of wild-type activity	47)
CYP2C9*10	CYP2C9.10	E272G	COS-7	S-Warfarin	27% of CLint ratio	47)
			COS-7	Tolbutamide	61% of wild-type activity	47)
CYP2C9*11	CYP2C9.11	R335W	COS-7	S-Warfarin	41% of wild-type activity	47)
			COS-7	Tolbutamide	71% of wild-type activity	47)
			Insect	Glimepiride	61% of CLint ratio	49)
			Insect	Tolbutamide	102% of CLint ratio	51)
			Insect	Losartan	58% of CLint ratio	53)
			COS-7	S-Warfarin	10% of CLint ratio	71)
CYP2C9*12	CYP2C9.12	P489S	COS-7	S-Warfarin	32% of CLint ratio	47)
			COS-7	Tolbutamide	48% of wild-type activity	47)
CYP2C9*13	CYP2C9.13	L90P	COS-7	S-Warfarin	16% of wild-type activity	47)
			COS-7	Tolbutamide	No activity	47)
			COS-7	Tolbutamide	Higher Km, Lower CLint	74)
			Insect	Glimepiride	3% of CLint ratio	49)
			Insect	Tolbutamide	0.3% of CLint ratio	51)
			Insect	Losartan	8% of CLint ratio	53)
CYP2C9*14	CYP2C9.14	R125H	COS-7	S-Warfarin	8% of wild-type activity	47)
			COS-7	Tolbutamide	No activity	47)
			Insect	Glimepiride	6% of CLint ratio	49)
			Insect	Tolbutamide	19% of CLint ratio	51)
			Insect	Losartan	14% of CLint ratio	53)
CYP2C9*16	CYP2C9.16	T299A	COS-7	S-Warfarin	29% of wild-type activity	47)
			COS-7	Tolbutamide	15% of wild-type activity	47)
			Insect	Glimepiride	4% of CLint ratio	49)
			Insect	Tolbutamide	9% of CLint ratio	51)
			Insect	Losartan	17% of CLint ratio	53)
CYP2C9*17	CYP2C9.17	P382S	COS-7	S-Warfarin	32% of CLint ratio	47)
			COS-7	Tolbutamide	60% of wild-type activity	47)
CYP2C9*18	CYP2C9.18	I359L, D397A	COS-7	S-Warfarin	No activity	47)
			COS-7	Tolbutamide	No activity	47)
CYP2C9*19	CYP2C9.19	Q454H	COS-7	S-Warfarin	46% of CLint ratio	47)
			COS-7	Tolbutamide	67% of wild-type activity	47)
			Insect	Glimepiride	0.1% of CLint ratio	49)
			Insect	Tolbutamide	0.1% of CLint ratio	51)
			Insect	Losartan	7% of CLint ratio	53)
CYP2C9*20	CYP2C9.20	G70R	COS-7	S-Warfarin	27% of wild-type activity	47)
			COS-7	Tolbutamide	63% of wild-type activity	47)
CYP2C9*21	CYP2C9.21	P30L	COS-7	S-Warfarin	No activity	47)
			COS-7	Tolbutamide	No activity	47)
CYP2C9*22	CYP2C9.22	N41D	COS-7	S-Warfarin	31% of wild-type activity	47)
			COS-7	Tolbutamide	43% of wild-type activity	47)
CYP2C9*23	CYP2C9.23	V76M	COS-7	S-Warfarin	47% of CLint ratio	47)
			COS-7	Tolbutamide	95% of wild-type activity	47)
			Insect	Glimepiride	7% of CLint ratio	49)
			Insect	Tolbutamide	18% of CLint ratio	51)
			Insect	Losartan	15% of CLint ratio	53)
CYP2C9*24	CYP2C9.24	E354K	COS-7	S-Warfarin	Low expression, No activity	47)
			COS-7	Tolbutamide	Low expression, No activity	47)
			HEK293		No expression	75)
CYP2C9*26	CYP2C9.26	T130R	COS-7	S-Warfarin	No activity	47)
			COS-7	Tolbutamide	23% of wild-type activity	47)
CYP2C9*27	CYP2C9.27	R150L	COS-7	S-Warfarin	58% of CLint ratio	47)
			COS-7	Tolbutamide	106% of wild-type activity	47)
			Insect	Glimepiride	15% of CLint ratio	49)
			Insect	Tolbutamide	50% of CLint ratio	51)
			Insect	Losartan	30% of CLint ratio	53)
CYP2C9*28	CYP2C9.28	Q214L	COS-7	S-Warfarin	9% of wild-type activity	47)
			COS-7	Tolbutamide	22% of wild-type activity	47)
CYP2C9*29	CYP2C9.29	P279T	COS-7	S-Warfarin	54% of CLint ratio	47)
			COS-7	Tolbutamide	72% of wild-type activity	47)
			Insect	Glimepiride	36% of CLint ratio	49)
			Insect	Tolbutamide	53% of CLint ratio	51)
			Insect	Losartan	38% of CLint ratio	53)
CYP2C9*30	CYP2C9.30	A477T	COS-7	S-Warfarin	8% of wild-type activity	47)
			COS-7	Tolbutamide	20% of wild-type activity	47)
CYP2C9*31	CYP2C9.31	I327T	COS-7	S-Warfarin	33% of wild-type activity	47)
			COS-7	Tolbutamide	52% of wild-type activity	47)
			Insect	Glimepiride	14% of CLint ratio	49)
			Insect	Tolbutamide	34% of CLint ratio	51)
			Insect	Losartan	28% of CLint ratio	53)
			COS-7	S-Warfarin	20% of CLint ratio	71)
CYP2C9*32	CYP2C9.32	V490F	COS-7	S-Warfarin	92% of CLint ratio	47)
			COS-7	Tolbutamide	165% of wild-type activity	47)
CYP2C9*33	CYP2C9.33	R132Q	COS-7	S-Warfarin	No activity	47)
			COS-7	Tolbutamide	No activity	47)
			Insect	Glimepiride	8% of CLint ratio	49)
			Insect	Tolbutamide	0.2% of CLint ratio	51)
			Insect	Losartan	7% of CLint ratio	53)
CYP2C9*34	CYP2C9.34	R335Q	COS-7	S-Warfarin	20% of CLint ratio	47)
			COS-7	Tolbutamide	96% of wild-type activity	47)
			Insect	Glimepiride	11% of CLint ratio	49)
			Insect	Tolbutamide	36% of CLint ratio	51)
			Insect	Losartan	69% of CLint ratio	53)
CYP2C9*35	CYP2C9.35	R125L,	COS-7	S-Warfarin	No activity	47)
		R144C	COS-7	Tolbutamide	No activity	47)
CYP2C9*36	CYP2C9.36	START1V	Insect	Glimepiride	146% of CLint ratio	49)
			Insect	Tolbutamide	237% of CLint ratio	51)
			Insect	Losartan	354% of CLint ratio	53)
CYP2C9*37	CYP2C9.37	D49G	Insect	Glimepiride	75% of CLint ratio	49)
			Insect	Tolbutamide	22% of CLint ratio	51)
			Insect	Losartan	27% of CLint ratio	53)
CYP2C9*38	CYP2C9.38	G96A	Insect	Glimepiride	64% of CLint ratio	49)
			Insect	Tolbutamide	50% of CLint ratio	51)
			Insect	Losartan	29% of CLint ratio	53)
CYP2C9*39	CYP2C9.39	G98V	Insect	Glimepiride	10% of CLint ratio	49)
			Insect	Tolbutamide	4% of CLint ratio	51)
			Insect	Losartan	19% of CLint ratio	53)
CYP2C9*40	CYP2C9.40	F110S	Insect	Glimepiride	103% of CLint ratio	49)
			Insect	Tolbutamide	46% of CLint ratio	51)
			Insect	Losartan	20% of CLint ratio	53)
CYP2C9*41	CYP2C9.41	K119R	Insect	Glimepiride	75% of CLint ratio	49)
			Insect	Tolbutamide	61% of CLint ratio	51)
			Insect	Losartan	17% of CLint ratio	53)
CYP2C9*42	CYP2C9.42	R124Q	Insect	Glimepiride	3% of CLint ratio	49)
			Insect	Tolbutamide	4% of CLint ratio	51)
			Insect	Losartan	15% of CLint ratio	53)
CYP2C9*43	CYP2C9.43	R124W	Insect	Glimepiride	0.7% of CLint ratio	49)
			Insect	Tolbutamide	0.3% of CLint ratio	51)
			Insect	Losartan	4% of CLint ratio	53)
CYP2C9*44	CYP2C9.44	T130M	Insect	Glimepiride	15% of CLint ratio	49)
			Insect	Tolbutamide	62% of CLint ratio	51)
			Insect	Losartan	15% of CLint ratio	53)
CYP2C9*45	CYP2C9.45	R132W	Insect	Glimepiride	8% of CLint ratio	49)
			Insect	Tolbutamide	12% of CLint ratio	51)
			Insect	Losartan	8% of CLint ratio	53)
CYP2C9*46	CYP2C9.46	A149T	Insect	Glimepiride	23% of CLint ratio	49)
			Insect	Tolbutamide	69% of CLint ratio	51)
			Insect	Losartan	31% of CLint ratio	53)
CYP2C9*47	CYP2C9.47	P163L	Insect	Glimepiride	115% of CLint ratio	49)
			Insect	Tolbutamide	38% of CLint ratio	51)
			Insect	Losartan	40% of CLint ratio	53)
CYP2C9*48	CYP2C9.48	I207T	Insect	Glimepiride	67% of CLint ratio	49)
			Insect	Tolbutamide	34% of CLint ratio	51)
			Insect	Losartan	28% of CLint ratio	53)
CYP2C9*49	CYP2C9.49	I222V	Insect	Glimepiride	51% of CLint ratio	49)
			Insect	Tolbutamide	29% of CLint ratio	51)
			Insect	Losartan	36% of CLint ratio	53)
CYP2C9*50	CYP2C9.50	P227S	Insect	Glimepiride	29% of CLint ratio	49)
			Insect	Tolbutamide	22% of CLint ratio	51)
			Insect	Losartan	20% of CLint ratio	53)
CYP2C9*51	CYP2C9.51	I284V	Insect	Glimepiride	91% of CLint ratio	49)
			Insect	Tolbutamide	156% of CLint ratio	51)
			Insect	Losartan	80% of CLint ratio	53)
CYP2C9*52	CYP2C9.52	T299R	Insect	Glimepiride	3% of CLint ratio	49)
			Insect	Tolbutamide	3% of CLint ratio	51)
			Insect	Losartan	11% of CLint ratio	53)
CYP2C9*53	CYP2C9.53	P317S	Insect	Glimepiride	87% of CLint ratio	49)
			Insect	Tolbutamide	63% of CLint ratio	51)
			Insect	Losartan	36% of CLint ratio	53)
CYP2C9*54	CYP2C9.54	S343R	Insect	Glimepiride	95% of CLint ratio	49)
			Insect	Tolbutamide	54% of CLint ratio	51)
			Insect	Losartan	28% of CLint ratio	53)
CYP2C9*55	CYP2C9.55	L361I	Insect	Glimepiride	11% of CLint ratio	49)
			Insect	Tolbutamide	42% of CLint ratio	51)
			Insect	Losartan	7% of CLint ratio	53)
CYP2C9*56	CYP2C9.56	L387V	Insect	Glimepiride	81% of CLint ratio	49)
			Insect	Tolbutamide	96% of CLint ratio	51)
			Insect	Losartan	89% of CLint ratio	53)
CYP2C9*57	CYP2C9.57	N204H	No reports of in vitro study
CYP2C9*58	CYP2C9.58	P337T	Insect	Diclofenac	54% of CLint ratio	52)
			Insect	Tolbutamide	16% of CLint ratio	52)
			Insect	Losartan	23% of CLint ratio	52)
CYP2C9*59	CYP2C9.59	I434F	Insect	Diclofenac	19% of CLint ratio	50)
			Insect	Tolbutamide	6% of CLint ratio	50)
			Insect	Losartan	16% of CLint ratio	50)
CYP2C9*60	CYP2C9.60	L467P	Insect	Diclofenac	11% of CLint ratio	48)
			Insect	Tolbutamide	8% of CLint ratio	48)

S-Warfarin, 7′-hydroxylation; Tolbutamide, methylhydroxylation; Diclofenac, 4′-hydroxylation; Losartan, oxidation; Glimepiride, hydroxylation.

Table 3. CYP2C19 Variants and in Vitro Metabolism of CYP2C19 Substrates

CYP2C19 allele	CYP2C19 proteins	Amino-acid changes	Expression system	Substrates	Effect reduction CLint or activity versus CYP2C19.1B	References
CYP2C19*1A#	CYP2C19.1A		COS-7	Clopidogrel	115% of CLint ratio	65)
			COS-7	S-Mephenytoin	89% of CLint ratio	65)
CYP2C19*1B#	CYP2C19.1B	I331V		Clopidogrel
				S-Mephenytoin
CYP2C19*5A	CYP2C19.5A	R433W	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*5B	CYP2C19.5B	I331V, R433W	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*6	CYP2C19.6	R132Q, I331V	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*8	CYP2C19.8	W120R	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*9	CYP2C19.9	R144H, I331V	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*10	CYP2C19.10	P227L, I331V	COS-7	Clopidogrel	43% of CLint ratio	65)
			COS-7	S-Mephenytoin	7% of CLint ratio	65)
CYP2C19*11	CYP2C19.11	R150H, I331V	COS-7	Clopidogrel	109% of CLint ratio	65)
			COS-7	S-Mephenytoin	78% of CLint ratio	65)
CYP2C19*13	CYP2C19.13	I331V, R410C	COS-7	Clopidogrel	107% of CLint ratio	65)
			COS-7	S-Mephenytoin	99% of CLint ratio	65)
CYP2C19*14	CYP2C19.14	L17P, I331V	COS-7	Clopidogrel	56% of CLint ratio	65)
			COS-7	S-Mephenytoin	65% of CLint ratio	65)
CYP2C19*15	CYP2C19.15	I19L, I331V	COS-7	Clopidogrel	74% of CLint ratio	65)
			COS-7	S-Mephenytoin	78% of CLint ratio	65)
CYP2C19*16	CYP2C19.16	R442C	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*18	CYP2C19.18	R329H, I331V	COS-7	Clopidogrel	91% of CLint ratio	65)
			COS-7	S-Mephenytoin	105% of CLint ratio	65)
CYP2C19*19	CYP2C19.19	S51G, I331V	COS-7	Clopidogrel	40% of CLint ratio	65)
			COS-7	S-Mephenytoin	16% of CLint ratio	65)
CYP2C19*22	CYP2C19.22	R186P, I331V	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*23	CYP2C19.23	G91R, I331V	COS-7	Clopidogrel	66% of CLint ratio	65)
			COS-7	S-Mephenytoin	235% of CLint ratio	65)
CYP2C19*24	CYP2C19.24	I331V, R335Q	COS-7	Clopidogrel	No activity	65)
			COS-7	S-Mephenytoin	No activity	65)
CYP2C19*25	CYP2C19.25	I331V, F448L	COS-7	Clopidogrel	27% of CLint ratio	65)
			COS-7	S-Mephenytoin	36% of CLint ratio	65)
CYP2C19*26	CYP2C19.26	D256N, I331V	COS-7	Clopidogrel	65% of CLint ratio	65)
			COS-7	S-Mephenytoin	42% of CLint ratio	65)
CYP2C19*28	CYP2C19.28	I19L, I331V, V374I	COS-7	Clopidogrel	141% of CLint ratio	65)
			COS-7	S-Mephenytoin	99% of CLint ratio	65)
CYP2C19*29	CYP2C19.29	K28I, I331V	No reports of in vitro study
CYP2C19*30	CYP2C19.30	R73C
CYP2C19*31	CYP2C19.31	H78Y, I331V
CYP2C19*32	CYP2C19.32	H99R, I331V
CYP2C19*33	CYP2C19.33	D188N, I331V
CYP2C19*34	CYP2C19.34	P3S, F4L

Clopidogrel, 2-oxidation; S-Mephenytoin, 4′-hydroxylation. ^# Two CYP2C19 wild-type alleles CYP2C19*1A and CYP2C19*1B (I331V) have been reported. Most of CYP2C19 variant alleles harbor I331V substitution. Therefore, CYP2C19.1B was used as wild-type.

1. CYP2C8

CYP2C8 accounts for approximately 7% of the total CYP enzymes in the liver. CYP2C8 is an important enzyme responsible for the metabolism of 60 or more types of drugs used in clinical practice.^7,11) Drugs that are metabolized by CYP2C8 include anticancer (e.g. paclitaxel), antidiabetic (e.g. repaglinide and pioglitazone), lipid-lowering (e.g. fluvastatin), and antimalarial drugs (e.g. amodiaquine). Since the year 2000, CYP2C8 gene polymorphism has been identified in various races^12,13) and to date, CYP2C8*1A to *14 alleles have been reported (http://www.cypalleles.ki.se/cyp2c8.htm). The CYP2C8*3 allele is the most common CYP2C8 allele in Caucasians after the wild type CYP2C8*1 allele.¹⁴⁾ It has been reported that the plasma repaglinide concentration in individuals with the CYP2C8*3 allele is approximately 50 to 60% lower than that in individuals with the CYP2C8*1 allele. Furthermore, in 2004, Ishikawa et al. studied the CYP2C8 gene sequences of a patient who developed serious rhabdomyolysis after she was administered cerivastatin, which is a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor.¹⁵⁾ The data obtained from the study showed that the patient was homozygous for the CYP2C8*5/*5 variant type. The CYP2C8*5 allele contains the nucleotide deletion 475delA that leads to frameshift and premature termination. It is established that cerivastatin is mainly metabolized by CYP2C8.¹⁶⁾ It is therefore considered highly likely that in CYP2C8*5 homozygotes, the metabolizing function of CYP2C8 disappears and plasma cerivastatin concentration increases. This results in the onset of serious adverse reactions to cerivastatin.

Paclitaxel is a typical CYP2C8 substrate and a very effective anticancer drug for various solid cancers, including breast, ovarian, and non-small cell lung cancers. Paclitaxel is used worldwide; however, its use is associated with individual differences in the expression of serious adverse reactions.^17,18) Peripheral neuropathy and neutropenia are some of the reported adverse reactions. There are also individual differences in the therapeutic effect of paclitaxel as well as the general responses to it. Paclitaxel is metabolized in the liver to its 6α-hydroxy and 3′p-hydroxy metabolites through CYP2C8 and CYP3A hydroxylation reactions, respectively.¹⁹⁾ These are followed by oxidative metabolism by the same enzymes and then biliary secretion of the parent compound and the metabolites. The 6α-hydroxy metabolite forms approximately 60% of the secreted metabolites. This indicates that CYP2C8 is the main enzyme that metabolizes paclitaxel; therefore, the activity of paclitaxel may be decreased by CYP2C8. Therefore, CYP2C8 gene polymorphism is thought to be one of the causes of diverse paclitaxel responses. Many clinical studies are underway to investigate the relevance of the polymorphism. In previous research, it was reported that the risks of serious peripheral neuropathy and neutropenia are approximately 3 and 1.7 times higher, respectively, in individuals with the CYP2C8*3 allele than in those with the wild type CYP2C8 allele.^20,21) There have been many cases in which treatment has been stopped due to the onset of various adverse reactions caused by a reduced paclitaxel clearance. These depended on whether patients had the CYP2C8 variant allele or not. However, it has not been clarified which type of CYP2C8 variant allele alters enzyme activity or the extent of the alteration that could be caused. Therefore, individualized paclitaxel therapy based on gene polymorphism is not yet underway.

To assess the functional differences between the enzymatic activities of CYP2C8 variants in vitro, several studies have used a range of heterologous expression systems including insect and mammalian cells (Table 1). Recently, Tsukada et al. created an expression plasmid for the wild type CYP2C8*1 and 11 types of CYP2C8 variants (CYP2C8*2–CYP2C8*4, CYP2C8*6, and CYP2C8*8–CYP2C8*14), with the aim of clarifying the specific functions of enzyme proteins derived from the CYP2C8 variant alleles using in vitro experiments.²²⁾ Each of the variant proteins was expressed on COS-7 cells. Changes in enzyme function were evaluated in the metabolism of paclitaxel and amodiaquine using the respective microsomal fractions. The results indicated that, for both substrates, CYP2C8.11 enzyme activity disappeared. There was also significant reduction in intrinsic clearance of paclitaxel with CYP2C8.8, CYP2C8.13, and CYP2C8.14, than with CYP2C8.1. Therefore, in people with the aforementioned variant alleles, paclitaxel metabolism will be delayed owing to a reduced function of the CYP2C8 enzyme. This may increase the risk of peripheral neuropathy. On the other hand, the metabolism of paclitaxel was found to significantly increase with CYP2C8.2, which may reduce the therapeutic effect of paclitaxel. In addition, changes in the metabolism of paclitaxel and amodiaquine differ significantly with CYP2C8.6, CYP2C8.8, CYP2C8.9, CYP2C8.10, and CYP2C8.12, which demonstrates that the variant enzymes are substrate specific.

2. CYP2C9

Warfarin is a widely prescribed anticoagulant used for the long-term treatment and prevention of thromboembolic events and shows great interindividual variability in the drug dose required to achieve a therapeutic effect.²³⁾ Therefore, after the initial dose of warfarin is administered, it takes time for the optimal maintenance dose to be determined. It is therefore common for bleeding complications to occur after the initial dose has been administered. However, advances in PGx research suggest that it may be possible to predict individual differences in warfarin responsiveness from patients’ gene information. As a result, the clinical usefulness of this practice is being investigated.^24–27) Warfarin is usually administered as a racemic mixture in clinical practice; however, the anticoagulant action of the S form is 3 to 5 times stronger than that of the R form.²⁸⁾ Therefore, the action of the CYP2C9 enzyme, which almost exclusively metabolizes the S form, is attracting attention as an important factor for defining individual differences in patient responses to warfarin.^29,30)

CYP2C9 accounts for approximately 20% of the total CYP enzymes in the liver.^7,31) In addition to warfarin, CYP2C9 catalyzes the metabolism of approximately 15% of the drugs in clinical use. Overall, CYP2C9 metabolizes over 100 types of drugs including antidiabetic (e.g. tolbutamide and glimepiride), antihypertensive (e.g. losartan), and antiepileptic (e.g. phenytoin) drugs, as well as nonsteroidal anti-inflammatory drugs (e.g. diclofenac).^1,32,33) To date over 60 different types of gene polymorphism (CYP2C9*1A to *60) of CYP2C9, mainly SNP, have been reported (http://www.cypalleles.ki.se/cyp2c9.htm). This suggests that individual differences exist in the responses to drugs that are metabolized by CYP2C9.³³⁾ In particular, reductions in the metabolic activities of the CYP2C9*2 (430C>T, R144C) and CYP2C9*3 (1075 A>C, I359L) variant alleles on warfarin have been clarified from a large number of in vitro/in vivo studies.^27,30,34,35) Higashi et al. reported that in patients with the CYP2C9*2 and CYP2C9*3 variants, the metabolism of warfarin is delayed and its half-life is prolonged.³⁶⁾ As a result, it takes longer than normal to reach a steady state and a stable anticoagulant state. In addition, even with an average dose of warfarin, it is acknowledged that there is a high risk of an excessive anticoagulant effect.^37–39) This could result in adverse events such as life-threatening hemorrhage, which would require a dose reduction. Lindh et al. investigated the factors that affect warfarin dosing using a meta-analysis.⁴⁰⁾ The authors reported that in patients with CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3, and CYP2C9*3/*3 variants, it is necessary to reduce the dose of warfarin by 19.6, 33.7, 36.0, 56.7, and 78.1%, respectively. These estimations were made in reference to warfarin metabolism in patients with the wild type CYP2C9*1/*1. The effect of CYP2C9*3 is particularly notable in the Japanese population. Reports indicate that it is necessary to reduce the dose of warfarin by 50 and 90% in Japanese patients with the CYP2C9*1/*3 and CYP2C9*3/*3 variants, respectively.^41,42) Furthermore, it is recognized that CYP2C9*5, CYP2C9*8, CYP2C9*11, and CYP2C9*13 reduce the clearance of warfarin in humans.^42–46) However, there are still many unknown aspects in terms of the effects of CYP2C9 gene polymorphism on the pharmacokinetics of warfarin.

To assess the functional differences between the enzymatic activities of CYP2C9 variants in vitro, several studies have used a range of heterologous expression systems including baculovirus and mammalian cells (Table 1). Recently, Niinuma et al. created an expression plasmid for the wild type CYP2C9*1 and 31 types of CYP2C9 variants (CYP2C9*2–CYP2C9*5, CYP2C9*7–CYP2C9*14, CYP2C9*16–CYP2C9*24, and CYP2C9*26–CYP2C9*35), with the aim of clarifying the functional properties of enzyme proteins derived from the CYP2C9 variant allele using in vitro experiments.⁴⁷⁾ The authors evaluated changes in enzyme activity in the metabolism of S-warfarin and tolbutamide. The results showed that 7-hydroxylation of S-warfarin by CYP2C9.18, CYP2C9.21, CYP2C9.24, CYP2C9.26, CYP2C9.33, and CYP2C9.35 completely disappeared. The authors also determined enzyme kinetic parameters by conducting metabolic reactions with various substrate concentrations. It was also observed that when 40 µM of S-warfarin was reacted, the enzyme activities of 12 types of the variants (CYP2C9.3, CYP2C9.4, CYP2C9.5, CYP2C9.11, CYP2C9.13, CYP2C9.14, CYP2C9.16, CYP2C9.20, CYP2C9.22, CYP2C9.28, CYP2C9.30, and CYP2C9.31) were below the detection limit. It was clarified that the aforementioned 18 types of variants showed significantly reduced enzyme activity. Additionally, the Michaelis–Menten kinetics for S-warfarin hydroxylation were determined for CYP2C9.1–CYP2C9.17, CYP2C9.19–CYP2C9.20, CYP2C9.22–CYP2C9.23, CYP2C9.27–CYP2C9.32, and CYP2C9.34. Three variants (CYP2C9.10, CYP2C9.23, and CYP2C9.34) exhibited significantly lower K_m than the wild type. The V_max values for CYP2C9.23 and CYP2C9.32 were significantly increased, whereas those of 11 variants (CYP2C9.2, CYP2C9.7, CYP2C9.8, CYP2C9.9, CYP2C9.10, CYP2C9.12, CYP2C9.17, CYP2C9.19, CYP2C9.27, CYP2C9.29, and CYP2C9.34) were significantly decreased, relative to the wild-type enzyme.

Recently, Cai and colleagues force expressed the CYP2C9*1 allele to *60 variants in insect cells and comprehensively analyzed the kinetic parameters for each of the variant enzymes in the metabolism of losartan, glimepiride, tolbutamide, and diclofenac.^48–53) They observed that it was difficult to analyze warfarin metabolism using insect cells. It was therefore indicated that mammalian cells have to be used in expressing CYP2C9*1 to *60 variants to analyze the metabolism of warfarin.

3. CYP2C19

The expression level of CYP2C19 accounts for only 1% of the total CYP enzymes in the liver.⁵⁴⁾ However, CYP2C19 is an important enzyme because it is involved in the metabolism of approximately 10% of the drugs used in clinical practice.^55,56) CYP2C19 also exhibits gene polymorphism.⁵⁷⁾ To date, 30 or more different types of CYP2C19 variants (CYP2C19*1A to *35) have been reported and many of them affect drug metabolism (http://www.cypalleles.ki.se/cyp2c19.htm). It has been reported that CYP2C19*2 is present in all human races while CYP2C19*3 occurs mostly in Asians. Base substitution in these variants generates splicing abnormalities and stop codons, which eliminate enzymatic activity. Therefore, CYP2C19*2 and CYP2C19*3 are the main causative allele for slow drug metabolism in poor metabolizers (PMs).^58,59) This demonstrates the low metabolic function of CYP2C19. In Caucasians, there is an abnormality on the CYP2C19 promoter; therefore, there is a high incidence of elevated CYP2C19*17 enzyme activity in Caucasians.^60,61) Patients having the CYP2C19*17 allele are therefore ultrarapid metabolizers and they demonstrate a higher metabolic function than extensive metabolizers (EMs) do. Individuals who are EMs are homozygous for the wild type CYP2C19*1 allele. It is therefore evident that racial differences exist in CYP2C19 gene polymorphism and that the latter significantly affects drug metabolism and therapy.

Recently, it has been suggested that CYP2C19 gene polymorphism affects the therapeutic effect of the antiplatelet drug clopidogrel. Clopidogrel is a prodrug and it requires a two-stage metabolic activation by CYP2C19. Clopidogrel acts by irreversibly binding to the platelet P2Y12 receptor to inhibit adenosine diphosphate-dependent platelet aggregation. CYP2C19 plays a key role in the first stage of clopidogrel metabolism to 2-oxo-clopidogrel.⁶²⁾ It has been shown that the area under the blood concentration–time curve (AUC) of clopidogrel is approximately 3 times higher in PMs than in EMs.⁶³⁾ In addition, the AUCs of the active metabolites of clopidogrel in PM are approximately 30% lower than that in EM.⁶⁴⁾ Based on this information, the inhibition of platelet aggregation by clopidogrel in PM would be 10–30% lower than that in EM. It has been clarified that among patients taking clopidogrel, the incidence of death from cardiovascular disorders, myocardial infarction, and stroke is approximately 1.5 times higher in PMs than in EMs.⁶⁴⁾

Recently, Takahashi et al. created in expression plasmids for the wild types CYP2C19*1A and CYP2C19*1B, and 19 types of CYP2C19 variant alleles (CYP2C19*5A, CYP2C19*5B, CYP2C19*6, CYP2C19*8, CYP2C19*9, CYP2C19*10, CYP2C19*11, CYP2C19*13, CYP2C19*14, CYP2C19*15, CYP2C19*16, CYP2C19*18, CYP2C19*19, CYP2C19*22, CYP2C19*23, CYP2C19*24, CYP2C19*25, CYP2C19*26, and CYP2C19*28), with the aim of clarifying the specific functions of enzyme proteins derived from the alleles using in vitro experiments.⁶⁵⁾ Each of the variant proteins was expressed on COS-7 cells and enzyme activities during the metabolism of clopidogrel and S-mephenytoin were measured using the respective microsomal fractions. Using 40 µM of clopidogrel as substrate, it was observed that the 2-oxidation of clopidogrel disappeared with CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.16, CYP2C19.22, and CYP2C19.24. As a result, patients with the aforementioned variants may show reduced concentrations of the active metabolites of clopidogrel. This suggests that such patients may also experience a reduction in the therapeutic effect of clopidogrel. In addition, the intrinsic clearance of clopidogrel was significantly reduced with CYP2C19.10, CYP2C19.14, CYP2C19.19, CYP2C19.23, CYP2C19.25, and CYP2C19.26, more than with the wild type allele. This suggests that there may also be a reduction in the therapeutic effect of clopidogrel in patients with these variants. It was observed that, in the metabolic experiments on S-mephenytoin and clopidogrel, almost all the variants studied showed similar results in both studies. The results obtained for CYP2C19.23 were different in the two studies. This indicates that CYP2C19.23 shows substrate specificity.

CONCLUSION

The CYP2C subfamily is an important enzyme group responsible for metabolizing many of the drugs used in clinical practice. To date, many CYP2C variants have been described. Available reports suggest that CYP2C gene polymorphism affects drug response, including the pharmacokinetics, therapeutic effects, and onset of adverse reactions of drugs. However, changes in the enzyme functions of some other identified CYP2C variants have not been clarified yet. Therefore, detailed analyses of the functional changes caused by such variants must be conducted to facilitate individualized drug therapy.

In vitro analysis of enzyme protein functions using the cDNA expression system is an effective method that enables easy evaluation of changes in variant enzyme functions. This method is extremely important in terms of complementing in vivo information.⁸⁾ Conversely, it must be said that there are limitations on accurately predicting in vivo CYP2C enzyme functions based on the results of in vitro tests. It is expected that the results from both tests will differ depending on the in vivo protein expression level and stable variants. However, the CYP2C protein expression level is extremely low when using mammalian cells⁸⁾; therefore, it is difficult to evaluate CYP holoprotein expression levels and stabilities using a CO difference spectrum assay. There is therefore a need to develop a system with a high level of CYP expression in mammalian cells. This is because evaluating changes in enzyme function based on actual CYP2C holoprotein levels is important for the application of gene polymorphism in individualized drug therapy.

We expect that this report and others on rare genetic variants of drug-metabolizing enzymes will be used for predicting drug responses based on gene polymorphism. This will enable optimal and individualized drug therapies to be administered to ensure that successful therapeutic outcomes are achieved.

This review of the author’s work was written by the author upon receiving the 2016 Pharmaceutical Society of Japan Award for Divisional Scientific Promotion.

Acknowledgments

This review of our recent findings commemorates my acceptance of the Pharmaceutical Society of Japan Award for Divisional Scientific Promotions. I also wish to express my sincere thanks to all of my colleagues and collaborators for their kind support of my research. This work was supported in part by a Grant from the Ministry of Health, Labour and Welfare (MHLW) of Japan (‘Initiative to facilitate development of innovative drug, medical devices, and cellular & tissue-based products’), the Smoking Research Foundation, and the Japan Research Foundation for Clinical Pharmacology.

Conflict of Interest

The author declares no conflict of interest.

REFERENCES

• 1) Evans WE, Relling MV. Pharmacogenomics: translating functional genomics into rational therapeutics. Science, 286, 487–491 (1999).
• 2) Zhou SF, Liu JP, Chowbay B. Polymorphism of human cytochrome P450 enzymes and its clinical impact. Drug Metab. Rev., 41, 89–295 (2009).
• 3) Zanger UM, Klein K, Thomas M, Rieger JK, Tremmel R, Kandel BA, Klein M, Magdy T. Genetics, epigenetics, and regulation of drug-metabolizing cytochrome P450 enzymes. Clin. Pharmacol. Ther., 95, 258–261 (2014).
• 4) Chen Y, Goldstein JA. The transcriptional regulation of the human CYP2C genes. Curr. Drug Metab., 10, 567–578 (2009).
• 5) Rendic S. Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metab. Rev., 34, 83–448 (2002).
• 6) Ridderström M, Zamora I, Fjellström O, Andersson TB. Analysis of selective regions in the active sites of human cytochromes P450, 2C8, 2C9, 2C18, and 2C19 homology models using GRID/CPCA. J. Med. Chem., 44, 4072–4081 (2001).
• 7) Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther., 270, 414–423 (1994).
• 8) Hiratsuka M. In vitro assessment of the allelic variants of cytochrome P450. Drug Metab. Pharmacokinet., 27, 68–84 (2012).
• 9) Asseffa A, Smith SJ, Nagata K, Gillette J, Gelboin HV, Gonzalez FJ. Novel exogenous heme-dependent expression of mammalian cytochrome P450 using baculovirus. Arch. Biochem. Biophys., 274, 481–490 (1989).
• 10) Gonzalez FJ, Kimura S, Tamura S, Gelboin HV. Expression of mammalian cytochrome P450 using baculovirus. Methods Enzymol., 206, 93–99 (1991).
• 11) Lai XS, Yang LP, Li XT, Liu JP, Zhou ZW, Zhou SF. Human CYP2C8: structure, substrate specificity, inhibitor selectivity, inducers and polymorphisms. Curr. Drug Metab., 10, 1009–1047 (2009).
• 12) Aquilante CL, Niemi M, Gong L, Altman RB, Klein TE. PharmGKB summary: very important pharmacogene information for cytochrome P450, family 2, subfamily C, polypeptide 8. Pharmacogenet. Genomics, 23, 721–728 (2013).
• 13) Daily EB, Aquilante CL. Cytochrome P450 2C8 pharmacogenetics: a review of clinical studies. Pharmacogenomics, 10, 1489–1510 (2009).
• 14) Niemi M, BacKman JT, Neuvonen M, Neuvonen PJ. Effects of gemfibrozil, itraconazole, and their combination on the pharmacokinetics and pharmacodynamics of repaglinide: potentially hazardous interaction between gemfibrozil and repaglinide. Diabetologia, 46, 347–351 (2003).
• 15) Ishikawa C, Ozaki H, Nakajima T, Ishii T, Kanai S, Anjo S, Shirai K, Inoue I. A frameshift variant of CYP2C8 was identified in a patient who suffered from rhabdomyolysis after administration of cerivastatin. J. Hum. Genet., 49, 582–585 (2004).
• 16) Mück W. Clinical pharmacokinetics of cerivastatin. Clin. Pharmacokinet., 39, 99–116 (2000).
• 17) Somlo G, Doroshow JH, Synold T, Longmate J, Reardon D, Chow W, Forman SJ, Leong LA, Margolin KA, Morgan RJ Jr, Raschko JW, Shibata SI, Tetef ML, Yen Y, Kogut N, Schriber J, Alvarnas J. High-dose paclitaxel in combination with doxorubicin, cyclophosphamide and peripheral blood progenitor cell rescue in patients with high-risk primary and responding metastatic breast carcinoma: toxicity profile, relationship to paclitaxel pharmacokinetics and short-term outcome. Br. J. Cancer, 84, 1591–1598 (2001).
• 18) Taniguchi R, Kumai T, Matsumoto N, Watanabe M, Kamio K, Suzuki S, Kobayashi S. Utilization of human liver microsomes to explain individual differences in paclitaxel metabolism by CYP2C8 and CYP3A4. J. Pharmacol. Sci., 97, 83–90 (2005).
• 19) Rahman A, Korzekwa KR, Grogan J, Gonzalez FJ, Harris JW. Selective biotransformation of taxol to 6 alpha-hydroxytaxol by human cytochrome P450 2C8. Cancer Res., 54, 5543–5546 (1994).
• 20) Hertz DL, Motsinger-Reif AA, Drobish A, Winham SJ, McLeod HL, Carey LA, Dees EC. CYP2C8*3 predicts benefit/risk profile in breast cancer patients receiving neoadjuvant paclitaxel. Breast Cancer Res. Treat., 134, 401–410 (2012).
• 21) Leskelä S, Jara C, Leandro-García LJ, Martínez A, García-Donas J, Hernando S, Hurtado A, Vicario JC, Montero-Conde C, Landa I, López-Jiménez E, Cascón A, Milne RL, Robledo M, Rodríguez-Antona C. Polymorphisms in cytochromes P450 2C8 and 3A5 are associated with paclitaxel neurotoxicity. Pharmacogenomics J., 11, 121–129 (2011).
• 22) Tsukada C, Saito T, Maekawa M, Mano N, Oda A, Hirasawa N, Hiratsuka M. Functional characterization of 12 allelic variants of CYP2C8 by assessment of paclitaxel 6alpha-hydroxylation and amodiaquine N-deethylation. Drug Metab. Pharmacokinet., 30, 366–373 (2015).
• 23) Rettie AE, Tai G. The pharmocogenomics of warfarin: closing in on personalized medicine. Mol. Interv., 6, 223–227 (2006).
• 24) Caraco Y, Blotnick S, Muszkat M. CYP2C9 genotype-guided warfarin prescribing enhances the efficacy and safety of anticoagulation: a prospective randomized controlled study. Clin. Pharmacol. Ther., 83, 460–470 (2008).
• 25) Gage BF, Eby C, Johnson JA, Deych E, Rieder MJ, Ridker PM, Milligan PE, Grice G, Lenzini P, Rettie AE, Aquilante CL, Grosso L, Marsh S, Langaee T, Farnett LE, Voora D, Veenstra DL, Glynn RJ, Barrett A, McLeod HL. Use of pharmacogenetic and clinical factors to predict the therapeutic dose of warfarin. Clin. Pharmacol. Ther., 84, 326–331 (2008).
• 26) International Warfarin Pharmacogenetics C, Klein TE, Altman RB, Eriksson N, Gage BF, Kimmel SE, Lee MT, Limdi NA, Page D, Roden DM, Wagner MJ, Caldwell MD, Johnson JA. Estimation of the warfarin dose with clinical and pharmacogenetic data. N. Engl. J. Med., 360, 753–764 (2009).
• 27) Johnson JA, Gong L, Whirl-Carrillo M, Gage BF, Scott SA, Stein CM, Anderson JL, Kimmel SE, Lee MT, Pirmohamed M, Wadelius M, Klein TE, Altman RB. Clinical pharmacogenetics implementation consortium guidelines for CYP2C9 and VKORC1 genotypes and warfarin dosing. Clin. Pharmacol. Ther., 90, 625–629 (2011).
• 28) O’Reilly RA. Studies on the optical enantiomorphs of warfarin in man. Clin. Pharmacol. Ther., 16, 348–354 (1974).
• 29) Rettie AE, Korzekwa KR, Kunze KL, Lawrence RF, Eddy AC, Aoyama T, Gelboin HV, Gonzalez FJ, Trager WF. Hydroxylation of warfarin by human cDNA-expressed cytochrome P450: a role for P-4502C9 in the etiology of (S)-warfarin-drug interactions. Chem. Res. Toxicol., 5, 54–59 (1992).
• 30) Wadelius M, Pirmohamed M. Pharmacogenetics of warfarin: current status and future challenges. Pharmacogenomics J., 7, 99–111 (2007).
• 31) Wrighton SA, Stevens JC. The human hepatic cytochromes P450 involved in drug metabolism. Crit. Rev. Toxicol., 22, 1–21 (1992).
• 32) Rettie AE, Jones JP. Clinical and toxicological relevance of CYP2C9: drug–drug interactions and pharmacogenetics. Annu. Rev. Pharmacol. Toxicol., 45, 477–494 (2005).
• 33) Schwarz UI. Clinical relevance of genetic polymorphisms in the human CYP2C9 gene. Eur. J. Clin. Invest., 33 (Suppl. 2), 23–30 (2003).
• 34) Lee CR, Goldstein JA, Pieper JA. Cytochrome P450 2C9 polymorphisms: a comprehensive review of the in-vitro and human data. Pharmacogenetics, 12, 251–263 (2002).
• 35) Wang B, Wang J, Huang SQ, Su HH, Zhou SF. Genetic polymorphism of the human cytochrome P450 2C9 gene and its clinical significance. Curr. Drug Metab., 10, 781–834 (2009).
• 36) Higashi MK, Veenstra DL, Kondo LM, Wittkowsky AK, Srinouanprachanh SL, Farin FM, Rettie AE. Association between CYP2C9 genetic variants and anticoagulation-related outcomes during warfarin therapy. JAMA, 287, 1690–1698 (2002).
• 37) Aithal GP, Day CP, Kesteven PJ, Daly AK. Association of polymorphisms in the cytochrome P450 CYP2C9 with warfarin dose requirement and risk of bleeding complications. Lancet, 353, 717–719 (1999).
• 38) Ogg MS, Brennan P, Meade T, Humphries SE. CYP2C9*3 allelic variant and bleeding complications. Lancet, 354, 1124 (1999).
• 39) Takahashi H, Echizen H. Pharmacogenetics of CYP2C9 and interindividual variability in anticoagulant response to warfarin. Pharmacogenomics J., 3, 202–214 (2003).
• 40) Lindh JD, Holm L, Andersson ML, Rane A. Influence of CYP2C9 genotype on warfarin dose requirements—a systematic review and meta-analysis. Eur. J. Clin. Pharmacol., 65, 365–375 (2009).
• 41) Takahashi H, Kashima T, Nomoto S, Iwade K, Tainaka H, Shimizu T, Nomizo Y, Muramoto N, Kimura S, Echizen H. Comparisons between in-vitro and in-vivo metabolism of (S)-warfarin: catalytic activities of cDNA-expressed CYP2C9, its Leu359 variant and their mixture versus unbound clearance in patients with the corresponding CYP2C9 genotypes. Pharmacogenetics, 8, 365–373 (1998).
• 42) Takahashi H, Wilkinson GR, Caraco Y, Muszkat M, Kim RB, Kashima T, Kimura S, Echizen H. Population differences in S-warfarin metabolism between CYP2C9 genotype-matched Caucasian and Japanese patients. Clin. Pharmacol. Ther., 73, 253–263 (2003).
• 43) Cavallari LH, Langaee TY, Momary KM, Shapiro NL, Nutescu EA, Coty WA, Viana MA, Patel SR, Johnson JA. Genetic and clinical predictors of warfarin dose requirements in African Americans. Clin. Pharmacol. Ther., 87, 459–464 (2010).
• 44) Kim HS, Lee SS, Oh M, Jang YJ, Kim EY, Han IY, Cho KH, Shin JG. Effect of CYP2C9 and VKORC1 genotypes on early-phase and steady-state warfarin dosing in Korean patients with mechanical heart valve replacement. Pharmacogenet. Genomics, 19, 103–112 (2009).
• 45) Liu Y, Jeong H, Takahashi H, Drozda K, Patel SR, Shapiro NL, Nutescu EA, Cavallari LH. Decreased warfarin clearance associated with the CYP2C9 R150H (*8) polymorphism. Clin. Pharmacol. Ther., 91, 660–665 (2012).
• 46) Scott SA, Jaremko M, Lubitz SA, Kornreich R, Halperin JL, Desnick RJ. CYP2C9*8 is prevalent among African-Americans: implications for pharmacogenetic dosing. Pharmacogenomics, 10, 1243–1255 (2009).
• 47) Niinuma Y, Saito T, Takahashi M, Tsukada C, Ito M, Hirasawa N, Hiratsuka M. Functional characterization of 32 CYP2C9 allelic variants. Pharmacogenomics J., 14, 107–114 (2014).
• 48) Dai DP, Li CB, Wang SH, Cai J, Geng PW, Zhou YF, Hu GX, Cai JP. Identification and characterization of a novel CYP2C9 allelic variant in a warfarin-sensitive patient. Pharmacogenomics, 16, 1475–1486 (2015).
• 49) Dai DP, Wang SH, Geng PW, Hu GX, Cai JP. In vitro assessment of 36 CYP2C9 allelic isoforms found in the Chinese population on the metabolism of glimepiride. Basic Clin. Pharmacol. Toxicol., 114, 305–310 (2014).
• 50) Dai DP, Wang SH, Li CB, Geng PW, Cai J, Wang H, Hu GX, Cai JP. Identification and functional assessment of a new CYP2C9 allelic variant CYP2C9*59. Drug Metab. Dispos., 43, 1246–1249 (2015).
• 51) Dai DP, Wang YH, Wang SH, Geng PW, Hu LM, Hu GX, Cai JP. In vitro functional characterization of 37 CYP2C9 allelic isoforms found in Chinese Han population. Acta Pharmacol. Sin., 34, 1449–1456 (2013).
• 52) Luo SB, Li CB, Dai DP, Wang SH, Wang ZH, Geng PW, Cai J, Jiang ZL, Pu CW, Shang K, Yuan XM, Cao YP, Hu GX, Cai JP. Characterization of a novel CYP2C9 mutation (1009C>A) detected in a warfarin-sensitive patient. J. Pharmacol. Sci., 125, 150–156 (2014).
• 53) Wang YH, Pan PP, Dai DP, Wang SH, Geng PW, Cai JP, Hu GX. Effect of 36 CYP2C9 variants found in the Chinese population on losartan metabolism in vitro. Xenobiotica, 44, 270–275 (2014).
• 54) Kawakami H, Ohtsuki S, Kamiie J, Suzuki T, Abe T, Terasaki T. Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection. J. Pharm. Sci., 100, 341–352 (2011).
• 55) Desta Z, Zhao X, Shin JG, Flockhart DA. Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clin. Pharmacokinet., 41, 913–958 (2002).
• 56) Gardiner SJ, Begg EJ. Pharmacogenetics, drug-metabolizing enzymes, and clinical practice. Pharmacol. Rev., 58, 521–590 (2006).
• 57) Helsby NA, Burns KE. Molecular mechanisms of genetic variation and transcriptional regulation of CYP2C19. Front. Genet., 3, 206 (2012).
• 58) De Morais SM, Wilkinson GR, Blaisdell J, Meyer UA, Nakamura K, Goldstein JA. Identification of a new genetic defect responsible for the polymorphism of (S)-mephenytoin metabolism in Japanese. Mol. Pharmacol., 46, 594–598 (1994).
• 59) de Morais SM, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA. The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans. J. Biol. Chem., 269, 15419–15422 (1994).
• 60) Rudberg I, Mohebi B, Hermann M, Refsum H, Molden E. Impact of the ultrarapid CYP2C19*17 allele on serum concentration of escitalopram in psychiatric patients. Clin. Pharmacol. Ther., 83, 322–327 (2008).
• 61) Sibbing D, Koch W, Gebhard D, Schuster T, Braun S, Stegherr J, Morath T, Schomig A, von Beckerath N, Kastrati A. Cytochrome 2C19*17 allelic variant, platelet aggregation, bleeding events, and stent thrombosis in clopidogrel-treated patients with coronary stent placement. Circulation, 121, 512–518 (2010).
• 62) Kazui M, Nishiya Y, Ishizuka T, Hagihara K, Farid NA, Okazaki O, Ikeda T, Kurihara A. Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metab. Dispos., 38, 92–99 (2010).
• 63) Kim KA, Park PW, Hong SJ, Park JY. The effect of CYP2C19 polymorphism on the pharmacokinetics and pharmacodynamics of clopidogrel: a possible mechanism for clopidogrel resistance. Clin. Pharmacol. Ther., 84, 236–242 (2008).
• 64) Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, Brandt JT, Walker JR, Antman EM, Macias W, Braunwald E, Sabatine MS. Cytochrome P450 polymorphisms and response to clopidogrel. N. Engl. J. Med., 360, 354–362 (2009).
• 65) Takahashi M, Saito T, Ito M, Tsukada C, Katono Y, Hosono H, Maekawa M, Shimada M, Mano N, Oda A, Hirasawa N, Hiratsuka M. Functional characterization of 21 CYP2C19 allelic variants for clopidogrel 2-oxidation. Pharmacogenomics J., 15, 26–32 (2015).
• 66) Yu L, Shi D, Ma L, Zhou Q, Zeng S. Influence of CYP2C8 polymorphisms on the hydroxylation metabolism of paclitaxel, repaglinide and ibuprofen enantiomers in vitro. Biopharm. Drug Dispos., 34, 278–287 (2013).
• 67) Soyama A, Saito Y, Hanioka N, Murayama N, Nakajima O, Katori N, Ishida S, Sai K, Ozawa S, Sawada JI. Non-synonymous single nucleotide alterations found in the CYP2C8 gene result in reduced in vitro paclitaxel metabolism. Biol. Pharm. Bull., 24, 1427–1430 (2001).
• 68) Hichiya H, Tanaka-Kagawa T, Soyama A, Jinno H, Koyano S, Katori N, Matsushima E, Uchiyama S, Tokunaga H, Kimura H, Minami N, Katoh M, Sugai K, Goto Y, Tamura T, Yamamoto N, Ohe Y, Kunitoh H, Nokihara H, Yoshida T, Minami H, Saijo N, Ando M, Ozawa S, Saito Y, Sawada J. Functional characterization of five novel CYP2C8 variants, G171S, R186X, R186G, K247R, and K383N, found in a Japanese population. Drug Metab. Dispos., 33, 630–636 (2005).
• 69) Rettie AE, Wienkers LC, Gonzalez FJ, Trager WF, Korzekwa KR. Impaired (S)-warfarin metabolism catalysed by the R144C allelic variant of CYP2C9. Pharmacogenetics, 4, 39–42 (1994).
• 70) Rettie AE, Haining RL, Bajpai M, Levy RH. A common genetic basis for idiosyncratic toxicity of warfarin and phenytoin. Epilepsy Res., 35, 253–255 (1999).
• 71) Du H, Wei Z, Yan Y, Xiong Y, Zhang X, Shen L, Ruan Y, Wu X, Xu Q, He L, Qin S. Functional characterization of human CYP2C9 allelic variants in COS-7 cells. Front. Pharmacol., 7, 98 (2016).
• 72) DicKmann LJ, Rettie AE, Kneller MB, Kim RB, Wood AJ, Stein CM, Wilkinson GR, Schwarz UI. Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans. Mol. Pharmacol., 60, 382–387 (2001).
• 73) Hanatani T, Fukuda T, Onishi S, Funae Y, Azuma J. No major difference in inhibitory susceptibility between CYP2C9.1 and CYP2C9.3. Eur. J. Clin. Pharmacol., 59, 233–235 (2003).
• 74) Guo Y, Wang Y, Si D, Fawcett PJ, Zhong D, Zhou H. Catalytic activities of human cytochrome P450 2C9*1, 2C9*3 and 2C9*13. Xenobiotica, 35, 853–861 (2005).
• 75) Herman D, Dolzan V, Ingelman-Sundberg M. Characterization of the novel defective CYP2C9*24 allele. Drug Metab. Dispos., 35, 831–834 (2007).