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Pharmacogenomics in and its Influence on Pharmacokinetics

  • Guy MontayEmail author
  • Jochen Maas
  • Roland Wesch
Living reference work entry

Abstract

CYP1A2 is involved to a major extent in the metabolism of several drugs (imipramine, clozapine, fluvoxamine, olanzapine, theophylline, acetaminophen, propranolol, and tacrine) as well as of diet components (methylxanthines), endogenous substrates (estrogens), numerous aryl, aromatic and heterocyclic amines, and polycyclic aromatic hydrocarbons. It is inducible, notably by cigarette smoking, diet habits such as consumption of cruciferous vegetables (e.g., broccoli, watercress, collard greens, Brussels sprouts, and mustard) and of charbroiled meats, some drugs (omeprazole, phenytoin, and rifampicin) and is a target enzyme for the development of some cancers. Up to now, more than 25 CYP1A2 alleles have been detected. Probe drugs for CYP1A2 phenotyping are caffeine and theophylline. For safety concerns and drug availability, the preferred probe is caffeine. Caffeine 3-demethylation is mediated by CYP1A2, and accounts for 80% of caffeine clearance. Caffeine is also a probe drug for N-acetyltransferase and xanthine oxidase (Clin Pharmacol Ther 53:203–514, 1993).

II.T.1

Phase I enzymes

 II.T.1.1

CYP1A2

 II.T.1.2

CYP2C9

 II.T.1.3

CYP2C19

 II.T.1.4

CYP2D6

 II.T.1.5

CYP3A

 II.T.1.6

Other CYPS

 II.T.1.6.1

CYP2A6

 II.T.1.6.2

CYP2B6

 II.T.1.6.3

CYP2C8

 II.T.1.6.4

CYP2E1

II.T.2

Phase II enzymes

 II.T.2.1

N-acetyltransferases

 II.T.2.2

Uridine diphosphate glucuronosyltransferases

 II.T.2.3

Methyltransferases

 II.T.2.4

Glutathione S-transferases and sulfotransferases

The understanding of the role of pharmacogenetics in drug metabolism expanded greatly in the 1990s. This is mainly due to technological improvements in gene scanning and gene variant identification. The number of variant alleles identified for genes coding for drug metabolizing enzymes (DME) considerably increased in the early 2000s, and continues to increase. The clinical consequences – or at least genotyping–phenotyping relationships – of DME polymorphisms have not been demonstrated for all variants. In the text below, only those DME allele variants will be mentioned for which significant changes in enzyme activity have been found using probe drugs. Comprehensive information on the nomenclature of cytochrome P450 (CYP) alleles can be found at www.imm.ki.se/CYPalleles and Phase I and Phase II DMEs at www.pharmgkb.org/index.jsp.

Phase I Enzymes

CYP1A2

Purpose and Rationale

CYP1A2 is involved to a major extent in the metabolism of several drugs (imipramine, clozapine, fluvoxamine, olanzapine, theophylline, acetaminophen, propranolol, and tacrine) as well as of diet components (methylxanthines), endogenous substrates (estrogens), numerous aryl, aromatic and heterocyclic amines, and polycyclic aromatic hydrocarbons. It is inducible, notably by cigarette smoking, diet habits such as consumption of cruciferous vegetables (e.g., broccoli, watercress, collard greens, Brussels sprouts, and mustard) and of charbroiled meats, some drugs (omeprazole, phenytoin, and rifampicin) and is a target enzyme for the development of some cancers. Up to now, more than 25 CYP1A2 alleles have been detected. Probe drugs for CYP1A2 phenotyping are caffeine and theophylline. For safety concerns and drug availability, the preferred probe is caffeine. Caffeine 3-demethylation is mediated by CYP1A2, and accounts for 80% of caffeine clearance. Caffeine is also a probe drug for N-acetyltransferase and xanthine oxidase (Kalow and Tang 1993).

Procedure

Phenotyping: A fixed or weight-adjusted dose of caffeine (solution, tablet, and coffee) ranging from 1 to 3 mg/kg is administered. Diet requirements have to be respected (stable xanthine-free diet avoiding beverages such as coffee, tea, cola, chocolate, no food component with CYP1A2-inducing properties) during the test period. As smoking is known to induce CYP1A2, control of stable smoking status is mandatory.

There are two commonly used and robust methods for phenotyping. The first one measures caffeine (1,3,7-methylxanthine) and its N-demethylated metabolite 1,7-dimethylxanthine (paraxanthine) in a plasma or saliva sample collected within 5–7 h post-caffeine dosing (Fuhr and Rost 1994). The second one uses the assay of the metabolites 1-methylurate (1 U), 1-methylxanthine (1X), 5-acetylamino-6-formylamino-3-methyluracil (AFMU), and 1,7-dimethylurate (17 U) levels in urine collected at least for 8 h post-dosing (Campbell et al. 1987; Rostami-Hodjegan et al. 1996).

Commonly used methods for caffeine and metabolite(s) assay in plasma or urine involve an extraction step followed by HPLC with UV detection (Krul and Hageman 1998a; Rasmussen and Bosen 1996; Schreiber-Deturmeny and Bruguerolle 1996). Urine needs to be acidified (pH 3.0–3.5) before sample freezing.

Genotyping: Reduced activity has been reported for CYP1A2*1C and CYP1A2*1F alleles in smoking subjects. Induction of CYP1A2 activity has been associated with these alleles, but the effect of CYP1A2*1F mutation on CYP1A2 activity has not been confirmed (Nordmark et al. 2002). In Caucasians, frequency of the CYP1A2*1C and CYP1A2*1F variants is about 1% and 33%, respectively (Sachse et al. 2003).

Evaluation

Metabolic ratios (MR) used are plasma 17X/137X and urinary (1 U + 1X + AFMU)/17 U.

In controlled conditions, in nonsmoking young and elderly subjects, intraindividual and interindividual variability in 17X/137X MR was about 17% and 47%, respectively, with no effect of age (Simon et al. 2003). A 70-fold range in MR has been observed in smoking and nonsmoking female Caucasian subjects using the urinary MR (Nordmark et al. 1999). Up to 200-fold differences were found using the urinary test. Lower variability is expected using the plasma caffeine test.

Higher CYP1A2 activity in men versus women has been reported, though inconsistently, and in children. Higher MR is usually observed in smokers versus nonsmokers, when population sample size is large. Pregnancy and oral contraceptives intake were found to decrease CYP1A2 activity (Abernathy and Todd 1985; Caubet et al. 2004; Kalow and Tang 1993). CYP1A2 activity was found lower in colorectal patients versus controls (Sachse et al. 2003).

Large variability in CYP1A2 activity explains that its distribution has been described unimodal, bimodal, or trimodal. Poor metabolizers (PM, characterized with a MR <0.12) have been identified in Chinese population and represented about 5% of the population tested, whereas PM could represent 5–10% of Caucasian populations and 14% in Japanese population (Ou-Yang et al. 2000).

Critical Assessment of the Method

Numerous studies have shown good correlation between the 17X/137X plasma MR and caffeine systemic clearance, and plasma MR is considered more robust than the urinary one, since this last one can be affected by the effect of urinary flow on metabolite renal clearances.

Currently, no relationship between CYP1A2 genotype characteristics and CYP1A2 activity, as assessed by the caffeine test, has been usually found. Some associations have been found in specific genetic and environmental conditions (Han et al. 2001). Non-well-controlled conditions for urine sample collection, the effects (induction) linked to environmental factors may overcome the role of CYP1A2 polymorphism, which can explain the paucity of clear associations between CYP1A2 genotyping and phenotyping.

Further investigations are needed to characterize the effect of variants (SNPs, haplotypes) on CYP1A2 activity.

Modifications of the Method

Recent drug assay development involved LC-MS methods (Caubet et al. 2004; Kanazawa et al. 2000). A less practical breath test, using 13C or 14C labeled caffeine, can also be used (Kalow and Tang 1991).

References and Further Reading

  • Abernathy DR, Todd EL (1985) Impairment of caffeine clearance by chronic use of low-dose oestrogen-containing oral contraceptives. Eur J Clin Pharmacol 28:425–428

  • Brentano C, Jaillon P (2001) Variability of cytochrome P450 1A2 activity over time in young and elderly healthy volunteers. Br J Clin Pharmacol 52:601–604

  • Campbell ME, Spielberg SP, Kalow W (1987) A urinary metabolic ratio that reflects systemic caffeine clearance. Clin Pharmacol Ther 42:157–165

  • Caubet MS, Comte B, Brazier JL (2004) Determination of urinary 13C-caffeine metabolites by liquid chromatography–mass spectrometry: the use of metabolic ratios to assess CYP1A2 activity. J Pharm Sci Biomed Anal 34:379–389

  • Fuhr U, Rost LK (1994) Simple and reliable CYP1A2 phenotyping by the paraxanthine/caffeine ratio in plasma and saliva. Pharmacogenetics 4:109–116

  • Han XM, Ou-Yang DS, Lu PX et al (2001) Plasma caffeine metabolite ratio (17X/137X) in vivo associated with G-2964A and C734A polymorphisms of human CYP1A2. Pharmacogenetics 11:429–435

  • Kalow W, Tang BK (1991) Use of caffeine metabolite ratios to explore CYP1A2 and xanthine oxidase activities. Clin Pharmacol Ther 50:508–519

  • Kalow W, Tang BK (1993) The use of caffeine for enzyme assays: a critical appraisal. Clin Pharmacol Ther 53:203–514

  • Kanazawa H, Atsumi R, Matsushima Y et al (2000) Determination of theophylline and its metabolites in biological samples by liquid chromatography–mass spectrometry. J Chromatogr A 870:87–96

  • Krul C, Hageman G (1998a) Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reverse-phase high-performance liquid chromatography. J Chromatogr B 709:24–27

  • Nordmark A, Lundgren S, Cnattingius S et al (1999) Dietary caffeine as a probe agent for assessment of cytochrome P450 1A2 activity in random urine samples. Br J Clin Pharmacol 47:397–402

  • Nordmark A, Lundgren S, Ask B et al (2002) The effect of the CYP1A2*F mutation on CYP1A2 inducibility in pregnant women. Br J Clin Pharmacol 54:504–510

  • Ou-Yang DS, Huang SL, Wang W et al (2000) Phenotypic polymorphism and gender-related differences of CYP1A2 activity in a Chinese population. Br J Clin Pharmacol 49:145–151

  • Rasmussen BB, Bosen K (1996) Determination of urinary metabolites of caffeine for the assessment of cytochrome P450 1A2, xanthine oxidase and N-acetyltransferase activity in humans. Ther Drug Monitor 18:254–262

  • Rostami-Hodjegan A, Nurminen S, Jackson PR, Tucker GT (1996a) Caffeine urinary metabolic ratios as markers of enzyme activity: a theoretical assessment. Pharmacogenetics 6:121–149

  • Sachse S, Bhambra U, Smith G et al (2003) Polymorphisms in the cytochrome P4501A2 gene (CYP1A2) in colorectal cancer patients and control: allele frequencies, linkage disequilibrium and influence on caffeine metabolism. Br J Clin Pharmacol 55:68–76

  • Schreiber-Deturmeny E, Bruguerolle B (1996) Simultaneous high-performance liquid chromatographic determination of caffeine and theophylline for routine drug monitoring in human plasma. J Chromatogr B 677:305–312

  • Shirley KL, Hon YY, Penzak S et al (2003) Correlation of cytochrome P450 1A2 activity using caffeine phenotyping and olanzapine disposition in healthy volunteers. Neuropsychopharmacology 25:961–966

  • Shou M, Korzekwa KR, Brooks EN et al (1997) Role of human hepatic cytochrome P450 1A2 and 3A4 in the metabolic activation of estrone. Carcinogenesis 18:207–214

  • Simon T, Becquemont L, Hamon B et al (2003) Determination of –3858G-A and 164C-A genetic polymorphisms of CYP1A2 in blood and saliva by rapid alleleic discrimination: large difference in the prevalence of the –3858G-A mutation between Caucasians and Asians. Eur J Clin Pharmacol 59:343–346

CYP2C9

Purpose and Rationale

CYP2C9 is involved in the hydroxylation of about 16% of drugs (Schwarz 2003), including drugs with narrow therapeutic index such as anticoagulants (warfarin, acenocoumarol, and phenprocoumon active S-enantiomers), and anticonvulsivants (phenytoin and hexobarbital), as well as numerous antidiabetic agents (i.e., tolbutamide, glibenclamide, and glipizide), antihypertensive drugs (losartan, irbesartan), nonsteroidal anti-inflammatory agents (i.e., diclofenac, ibuprofen, and celecoxib), diuretic (torsemide), and anti-rheumatoid agents (leflunomide).

A couple of CYP2C9 variants – mainly CYP2C9*2 and CYP2C9*3 – code for in vivo decreased activity, and two – CYP2C9*6 and CYP2C9*15 – have been reported to be associated with no activity. In Caucasian populations, CYP2C9*2 and CYP2C9*3 are encountered in 20–25% of subjects, while these genotypes have been found in less than 5% of East Asian subjects (Rosemary and Adithan 2007).

Probe drugs regularly used for CYP2C9 phenotyping are tolbutamide, warfarin, phenytoin, and losartan. Diclofenac, flurbiprofen, phenprocoumon, and torsemide have also been used. For safety concerns, the current preferred probe is tolbutamide, despite some risk of hypoglycemia.

Procedure

Phenotyping: The method measures tolbutamide, its CYP2C9-formed 4′-hydroxylated metabolite hydroxytolbutamide and the subsequent carboxytolbutamide metabolite, the latter formed by dehydrogenase enzymes. The urinary excretion of these two metabolites represented more than 85% dose of administered tolbutamide (Veronese et al. 1990, 1993).

Subjects receive a single oral 500 mg tolbutamide tablet in usual Phase I standard controlled conditions, with care to be paid to blood glucose. Urine is collected from drug intake to 8 or 24 h post-dosing.

The assay of tolbutamide and its metabolites is usually performed using HPLC and UV or fluorescence detection (Csillag et al. 1989; Veronese et al. 1990; Kirchheiner et al. 2002a, b; Hansen and Brosen 1999).

Genotyping: About two-third of Caucasian subjects express the wild genotype C9*1/*1. C9*1/*2 and C9*1/*3 heterozygote variants are expressed in 15–25% and 7–16% of Caucasian subjects, whereas the frequency of other variants is lower: 0.5–2.5%, 1–3%, and <1–1.5% for C9*2/*2, C9*2/*3, and C9*3/*3 variants, respectively (Scordo et al. 2001; Lee et al. 2002a, b; Schwarz 2003). More than 95% of Afro-American subjects express the wild genotype C9*1/*1 (Lee et al. 2002a, b). In Asian populations, CYP2C9*1/*3 is expressed in 2–8% subjects, but CYP2C9*2 is absent or extremely rare (Rosemary and Adithan 2007; Schwarz 2003; Xie et al. 2002). Overall, it has been estimated that 0.2–1% and 2–3% of Caucasian and Asian population could be qualified as PM, respectively (Meyer 2000).

Evaluation

The urinary MR (MR, hydroxytolbutamide + carboxytolbutamide)/tolbutamide is generally used. There is a large interindividual variability in MRs in subjects with the same genotype. Different studies performed with different probe drugs (Yasar et al. 2002a, b; Kirchheiner et al. 2002a, b, 2003a; Lee et al. 2002a, b; Miners and Birkett 1998; Morin et al. 2004), highlighted that a PM status could be given to subjects who are homozygous for CYP2C9*3, or expressing CYP2C9*2/*3 variant, but intermediate situations – from extensive to slow metabolizer status – may vary not only among different allele combinations but also with the probe drug used.

Oral contraceptives were found to inhibit CYP2C9 activity using losartan for phenotyping (Sandberg et al. 2004).

Critical Assessment of the Method

The tolbutamide test has the most convincing ability to discriminate between genotype variants and pharmacokinetics. There could be an analytical issue linked to the urine assay precision, as the urinary concentrations of the parent drug are very low in comparison with those of its metabolites.

To date, the CYP2C9*3 variant has been the only one found influencing significantly drug pharmacodynamics for warfarin, acenocoumarol (Sandberg 2003; Morin et al. 2004; Versuyft et al. 2003), glipizide, and glyburide (Kirchheiner et al. 2002a, b) or drug side effects (Sevilla-Mantilla et al. 2004). Inconstant results were found regarding tolbutamide effects (Kirchheiner et al. 2002a, b; Shong et al. 2002). For anticoagulants, the possession of CYP2C9*2 and CYP2C9*3 variants was associated with decreased warfarin dose requirement in patients, and an increased risk of adverse events such as bleeding (Daly and King 2003). An Afro-American subject with only the CYP2C9*6 variant exhibited serious phenytoin side effects associated with a marked impaired elimination of the drug (Kidd et al. 2001).

The variability of CYP2C9 activity observed among ethnic groups cannot be explained with our current knowledge on CYP2C9 variant alleles distribution (Xie et al. 2002).

Modifications of the Method

Losartan (25 mg dose) has been proposed as a safer alternative to tolbutamide. The determination of losartan/E3174 (oxidized metabolite) ratio in 0–8 h urine or in plasma at 6 h post-dosing have been proposed (Yasar et al. 2002a, b; Sekino et al. 2003). However, in a comparative study in 16 subjects, a better correlation between genotyping and phenotyping was found with tolbutamide, as compared to losartan or flurbiprofen, though there was no subject with the C9*2/*3 or C9*3/*3 variants (Lee et al. 2003).

Recently, a 125 mg tolbutamide dose has been validated, with proposal of the use of just one blood sample collected 24 h post-dosing. Its safer use needs the drug to be assayed using LC-MS/MS methodology (Jetter et al. 2004).

References and Further Reading

  • Csillag K, Vereczkey L, Gachalyi B (1989) Simple high-performance liquid chromatographic method for the determination of tolbutamide and its metabolites in human plasma and urine using photo-diode array detection. J Chromatogr 490:355–363

  • Daly AK, King BP (2003) Pharmacogenetics of anticoagulants. Pharmacogenetics 13:247–252

  • Hansen LL, Brosen K (1999) Quantitative determination of tolbutamide and its metabolites in human plasma and urine by high-performance liquid chromatography and UV detection. Ther Drug Monitor 21:664–671

  • Jetter A, Kinzig-Schippers M, Skott A et al (2004) Cytochrome P450 2C9 phenotyping using low-dose tolbutamide. Eur J Clin Pharmacol 60:165–175

  • Kidd RS, Curry TB, Gallagher S et al (2001) Identification of a null allele of CYP2C9 in an African-American exhibiting toxicity to phenytoin. Pharmacogenetics 11:803–808

  • Kirchheiner J, Bauer S, Meineke I et al (2002a) Impact of CYP2C9 and CYP2C19 polymorphisms on tolbutamide kinetics and the insulin and glucose response in healthy volunteers. Pharmacogenetics 12:101–109

  • Kirchheiner J, Brockmöller J, Meineke I et al (2002b) Impact of CYP2C9 amino acid polymorphisms on glyburide kinetics and on the insulin and glucose response in healthy volunteers. Clin Pharmacol Ther 71:286–296

  • Kirchheiner J, Störmer E, Meisel C et al (2003a) Influence of CYP2C9 genetic polymorphisms on pharmacokinetics of celecoxib and its metabolites. Pharmacogenetics 13:473–480

  • Lee CR, Pieper JA, Hinderliter AL et al (2002a) Evaluation of cytochrome P450 2C9 metabolic activity with tolbutamide in CYP2C9*1 heterozygotes. Clin Pharmacol Ther 72:562–571

  • Lee CR, Goldstein JA, Pieper JA (2002b) Cytochrome P450 2C9 polymorphisms: a comprehensive review of the in vitro and human data. Pharmacogenetics 12:251–263

  • Lee CR, Pieper JA, Reginald FF et al (2003) Tolbutamide, flurbiprofen and losartan as probes of CYP2C9 activity in humans. J Clin Pharmacol 43:84–91

  • Meyer UA (2000) Pharmacogenetics and adverse drug reactions. Lancet 356:1667–1671

  • Miners JO, Birkett DJ (1998) Cytochrome P450 2C9: an enzyme of major importance in human drug metabolism. Br J Clin Pharmacol 45: 525–538

  • Morin S, Bodin L, Loriot MA et al (2004) Pharmacogenetics of acenocoumarol. Clin Pharmacol Ther 75:403–414

  • Rosemary J, Adithan C (2007a) The pharmacogenetics of CYP2C9 and CYP2C19: ethnic variation and clinical significance. Curr Clin Pharmacol 2:93–109

  • Sandberg M, Johansson I, Christensen M et al (2004) The impact of CYP2C9 genetics and oral contraceptives on cytochrome P450 2C9 phenotype. Drug Metab Dispos 32:484–489

  • Schwarz UI (2003) Clinical relevance of genetic polymorphisms in the human CYP2C9 gene. Eur J Clin Invest 33:23–30

  • Scordo MG, Aklillu E, Dahl ML et al (2001) Genetic polymorphism of cytochrome P450 2C9 in a Caucasian and a black African population. Br J Clin Pharmacol 52:447–450

  • Sekino K, Kubota T, Okada Y et al (2003) Effect of the single CYP2C9*3 allele on pharmacokinetics and pharmacodynamics in healthy Japanese subjects. Eur J Clin Pharmacol 59:589–592

  • Sevilla-Mantilla C, Ortega L, Agundez JA et al (2004) Leflunomide acute hepatitis. Dig Liver Dis 36:82–84

  • Shong JH, Yoon YR, Kim KA et al (2002) Effects of CYP2C9 and CYP2C19 genetic polymorphisms on the disposition and blood glucose lowering response to tolbutamide in humans. Pharmacogenetics 12: 111–119

  • Veronese ME, Miners JO, Randles D et al (1990) Validation of the tolbutamide metabolic ratio for population screening with use of sulfaphenazole to produce model phenotypic poor metabolizers. Clin Pharmacol Ther 47:403–411

  • Veronese ME, Miners JO, Ress DLP, Birkett DJ (1993) Tolbutamide hydroxylation in humans: lack of bimodality in 106 healthy subjects. Pharmacogenetics 3:86–93

  • Versuyft C, Robert A, Morin S et al (2003) Genetic and environmental risk factors for oral anticoagulant overdose. Eur J Clin Pharmacol 58: 739–745

  • Xie HG, Prasad HC, Kim RB, Stein CM (2002) CYP2C9 allelic variants: ethnic distribution and functional significance. Adv Drug Deliv Rev 54:1257–1270

  • Yasar U, Dahl ML, Christensen M, Eliasson E (2002a) Intra-individual variability in urinary losartan oxidation ratio: an in vivo marker of CYP2C9 activity. Br J Clin Pharmacol 54:183–185

  • Yasar U, Forslund-Bergengren C, Tybring G et al (2002b) Pharmacokinetics of losartan and its metabolite E-3174 in relation to the CYP2C9 genotype. Clin Pharmacol Ther 71:89–98

CYP2C19

Purpose and Rationale

CYP2C19 contributes to the metabolism of about 8% of drugs (Rogers et al. 2002), including S-mephenytoin, proton pump inhibitors (omeprazole, lansoprazole, and pantoprazole), tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and citalopram), benzodiazepines (diazepam and flunitrazepam), torsemide, fluvastatin, and proguanil. Two main variants – CYP2C19*2 and CYP2C19*3 – are coding for in vivo nil activity, as well as CYP2C19*4, *5, *6, *7, and *8 variants. About 15–20% Asians, 4–7% Black Africans, and 3% Caucasians are PM (Scordo et al. 2004).

Probe drugs used for CYP2C19 phenotyping are mephenytoin, omeprazole, and proguanil. The most currently used probe drug is omeprazole.

Procedure

Phenotyping: The method measures omeprazole, and its CYP2C19-formed 5-hydroxylated metabolite in plasma.

Subjects receive a single oral 20 or 40 mg omeprazole capsule in usual Phase I standard controlled conditions. Plasma can be collected from drug intake up to 24 h post-dosing, or only one plasma sample is collected at 2 or 3 h post-dosing.

The assay of omeprazole and its metabolite is usually performed using HPLC and UV detection (Lagerstrom and Persson 1984; Ieri 1996; Yim et al. 2001; Tybring et al. 1997) or LC-MS/MS assay (Kanazawa et al. 2002).

Genotyping: The two alleles CYP2C19*2 and CYP2C19*3 account for quite all PM in Asians (>99%) and Black Africans, but defective alleles have not been fully characterized in 10–15% Caucasians. The CYP2C19*2 allele is the most frequent in Asian populations (30% in Chinese), as well as in Black Africans (about 17%) and in Caucasians (about 15%) (Xie et al. 2001). The CYP2C19*3 accounts for about 25% of inactive forms in Orientals, and is extremely rare in Caucasians (Scordo et al. 2004; Rosemary and Adithan 2007).

Evaluation

The AUC or plasma ratio of omeprazole to 5-hydroxyomeprazole is used.

As expected, homozygous PM subjects have lower metabolic activity as compared to heterozygous PM subjects, and potential interethnic difference has been noticed within a genotype (Yin et al. 2004).

Decreased CYP2C19 activity has been observed with oral contraceptives containing ethinylestradiol (Tamminga et al. 1999; Laine et al. 2000).

Critical Assessment of the Method

Omeprazole hydroxylation rate correlates with S-mephenytoin hydroxylation rate, which was initially the CYP2C19 probe drug (Andersson et al. 1990; Chang et al. 1995; Balian et al. 1995). The alternate pathway – conversion of omeprazole to its sulfone derivative – that is mediated via CYP3A4, does not influence the CYP2C19 pathway of omeprazole (Balian et al. 1995).

Time-dependent kinetics of omeprazole limits its use for phenotyping during chronic therapy (Gafni et al. 2001). CYP2C19 phenotyping with omeprazole may be affected by age, liver disease, and omeprazole therapy (Kimura et al. 1999).

Interethnic differences observed with different CYP2C19 substrates for subjects with same genotype have been attributed to differences in substrate specificity or enzyme isoforms (Bertilsson et al. 1992). The clearance of omeprazole is higher in Caucasian extensive metabolizers (EM) than in Oriental EM, due to a higher proportion of heterozygous EM in this latter population (Ishizaki et al. 1994).

Modifications of the Method

It has been proposed to use omeprazole for both CYP2C19 and CYP3A4 phenotyping (Gonzalez et al. 2003).

References and Further Reading

  • Andersson T, Regardh CG, Dahl-Puustinen ML, Bertilsson L (1990) Slow omeprazole metabolizers are also poor S-mephenytoin hydroxylators. Ther Drug Monit 12:415–416

  • Balian JD, Sukhova N, Harris JW et al (1995) The hydroxylation of omeprazole correlates with S-mephenytoin metabolism: a population study. Clin Pharmacol Ther 57:662–669

  • Bertilsson L, Lou YQ, Du YL et al (1992) Pronounced differences between native Chinese and Swedish populations in the polymorphic hydroxylations of debrisoquin and S-mephenytoin. Clin Pharmacol Ther 51:388–397

  • Chang M, Dahl ML, Tybring G et al (1995) Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype. Pharmacogenetics 5:358–363

  • Gafni I, Nolte H, Tyndale R et al (2001) Resolving the roles of CYP2C19 and CYP3A4 in the metabolism of omeprazole in vivo using chronic omeprazole and ketoconazole. FASEB J 15:A918

  • Gonzalez HM, Romero EM, Peregrina AA et al (2003) CYP2C19- and CYP3A4-dependent omeprazole metabolism in west Mexicans. J Clin Pharmacol 43:1211–1215

  • Ishizaki T, Sohn DR, Kobayashi K et al (1994) Interethnic differences in omeprazole metabolism in the two S-mephenytoin hydroxylation phenotypes studied in Caucasians and Orientals. Ther Drug Monit 16:214–215

  • Kanazawa H, Okada A, Matsushima Y et al (2002) Determination of omeprazole and its metabolites in human plasma by liquid chromatography–mass spectrometry. J Chromatogr A 949:1–9

  • Kimura M, Ieri I, Wada Y, Mamyia K (1999) Reliability of the omeprazole hydroxylation index and length of therapy. Br J Clin Pharmacol 47:115–119

  • Lagerstrom PO, Persson BA (1984) Determination of omeprazole and its metabolites in plasma and urine by liquid chromatography. J Chromatogr A 309:347–356

  • Laine K, Tybring G, Bertilsson L (2000) No sex-related difference but significant inhibition by oral contraceptives of CYP2C19 activity as measured by the probe drugs mephenytoin and omeprazole in healthy Swedish white subjects. Clin Pharmacol Ther 68:151–159

  • Rogers JF, Nafziger AN, Bertino JS (2002) Pharmacogenetics affects dosing, efficacy, and toxicity of cytochrome P450-metabolized drugs. Am J Med 113:746–750

  • Rosemary J, Adithan C (2007b) The pharmacogenetics of CYP2C9 and CYP2C19: ethnic variation and clinical significance. Curr Clin Pharmacol 2:93–109

  • Scordo MG, Caputi AP, D’Arrigo C et al (2004) Allele and genotype frequencies of CYP2C9, CYP2C19 and CYP2D6 in an Italian population. Pharmacol Res 50:195–200

  • Tamminga WJ, Werner J, Oosterhuis B et al (1999) CYP2D6 and CYP2C19 activity in a large population of Dutch healthy volunteers: indications for oral contraceptive-related gender differences. Eur J Clin Pharmacol 55:177–184

  • Tybring G, Bottiger Y, Wyden J, Bertilsson L (1997) Enantioselective hydroxylation of omeprazole catalyzed by CYP2C19. Clin Pharmacol Ther 62:129–137

  • Xie HG, Kim RB, Wood AJ, Stein CM (2001a) Molecular basis of ethnic differences in drug disposition and response. Annu Rev. Pharmacol Toxicol 41:815–850

  • Yim DS, Jeong JR, Park JY (2001) Assay of omeprazole and omeprazole sulfone by semi-microcolumn liquid chromatography with mixed-function precolumn. J Chromatogr B 754:487–493

  • Yin OQP, Tomlinson B, Chow ALH et al (2004) Omeprazole as a CYP2C19 marker in Chinese subjects: assessment of its gene-dose effect and intrasubject variability. J Clin Pharmacol 44:582–589

CYP2D6

Purpose and Rationale

CYP2D6 is involved significantly in the metabolism of drugs mainly used in CNS (antidepressants, i.e., imipramine, paroxetine, citalopram; neuroleptics, i.e., haloperidol, risperidone), or cardiovascular (β-adrenoceptor blockers, i.e., metoprolol; antiarrhythmics, i.e., propafenone and flecainide) disorders. Significant interethnic and interindividual intraethnic differences in CYP2D6 activity have been found. It is found that 5–10% Caucasians, 6–8% Afro-Americans, and only 1% Asians have reduced CYP2D6 activity, and exhibit the PM phenotype. Expression of CYP2D6 has been shown to be polymorphic with up to now more than 80 genetic variants detected for the encoding gene, with more than 15 encoding for inactive enzyme. Probe drugs for CYP2D6 phenotyping are dextromethorphan, debrisoquin, sparteine, and metoprolol. For safety concerns and drug availability, the preferred probe is dextromethorphan (DM) (Schmid et al. 1985).

Procedure

Phenotyping: The method measures DM and its O-demethylated metabolite, dextrorphan (DX), which is formed by CYP2D6. DM and DX, and other metabolites, are excreted in urine, mainly as glucuronide conjugates.

Subjects receive a single oral 10–30 mg DM (generally hydrobromide salt syrup) dose. Urine is collected from drug intake to 8 h post-dosing. Other collection times (0–6, 0–10, 0–12, or 0–24 h) can be used, but short collection intervals might lead to increased intra-subject variability.

Urine is first hydrolyzed with β-glucuronidase. Then, different methods can be used involving DM and DX extraction, followed either by HPLC and fluorescence detection (Chladek et al. 1999; Hoskins et al. 1997) or capillary gas chromatography (Wu et al. 2003).

Genotyping: The incidence of alleles coding for inactive enzymes varies between populations: three “population specific” alleles are CYP2D6*4 in Caucasians, *10 in Asians, and *17 in Africans (Bertilsson et al. 2002). CYP2D6*3, *4, *5, *6 are the main inactive alleles producing the PM phenotype in Caucasians, with CYP2D6*4 most commonly associated with the PM phenotype. By far, the most frequent null allele – not encoding a functional protein product – is CYP2D6*4 with a frequency of 20–25% in Caucasians (Zanger et al. 2004). The frequency of the *17 allele – associated with decreased enzyme activity – is high in Black Africans and in Black Americans, but practically absent in Caucasian populations (Bapiro et al. 2002; Gaedigk et al. 2002; Zanger et al. 2003). Four potential subgroups – ultrarapid metabolizers (UM), extensive metabolizers (EM), intermediate metabolizers (IM), and poor metabolizers (PM) – have been defined based on the genotype–phenotype relationships.

In Caucasian subjects, it has been recommended for “routine test” to genotype for alleles *1, *3, *4, *5, *6 that allow to detect 86–100% of PM (Sachse et al. 1997). To assign correct phenotype in nearly 100% subjects, *9 and *10 variants should also be determined.

Evaluation

Subjects with a DM/DX MR >0.3 are PM. Subjects with DM/DX <0.03 are EM. Those with 0.03 < MR < 0.3 are IM.

No difference or slightly higher CYP2D6 activity in females has been found when comparing to male subjects (Hägg et al. 2001; McCune et al. 2001).

Relationship between phenotyping and genotyping is investigated by plotting log MR versus CYP2D6 allele combinations (Chou et al. 2003).

Critical Assessment of the Method

The method is widely used due to easy and safe administration. High intrasubject variability limits the test for discriminating between EM and UMs (Zanger et al. 2004).

The method is not appropriate in patients with renal impairment, due to reduced renal excretion of DM glucuronide metabolites. Sparteine has been recommended as a probe for this population and to discriminate between the four phenotypes UM, EM, IM, and PM. The DM/DX MR does not allow for consistent differentiation between CYP2D6 EM with one or two active alleles.

Modifications of the Method

Assays have been developed to determine DM and DX in plasma or saliva (Bolden et al. 2002; Hu et al. 1998; Chladek et al. 2000; Härtter et al. 1996). The use of saliva or plasma for CYP2D6 phenotyping has been developed for subject convenience, or for the development of single point methods to be easily incorporated in the “cocktail methods.” Good correlation between MRs calculated from plasma, saliva samples and those obtained from urine has been observed.

References and Further Reading

  • Bapiro TE, Hasler JA, Ridderström M, Masimirembwa CM (2002) The molecular and enzyme kinetic basis for the diminished activity of the cytochrome P450 2D6*17 variant. Biochem Pharmacol 64: 1387–1398

  • Bertilsson L, Dahl ML, Dalen P, Al-Shurbaji A (2002) Molecular genetics of CYP2D6: clinical relevance with focus on psychotropic drugs. Br J Clin Pharmacol 53:111–122

  • Bolden RD, Hoke SH II, Eichhold TH et al (2002) Semi-automated liquid–liquid back extraction in a 96-well format to decrease sample preparation time for the determination of dextromethorphan and dextrorphan in human plasma. J Chromatogr B 772:1–10

  • Chladek J, Zimova G, Martinkova J, Tuma I (1999) Intra-individual variability and influence of urine collection period on dextromethorphan metabolic ratios in healthy subjects. Fundam Clin Pharmacol 13:508–515

  • Chladek J, Zimova G, Beranek M, Martinkova J (2000) In-vivo indices of CYP2D6 activity: comparison of dextromethorphan metabolic ratios in 4-h urine and 3-h plasma. Eur J Clin Pharmacol 56:651–657

  • Chou WH, Yan FX, Robbins-Weilert DK et al (2003) Comparison of two CYP2D6 genotyping methods and assessment of genotype–phenotype relationships. Clin Chem 49:542–551

  • Gaedigk A, Bradford LD, Marcucci KA, Leeder JS (2002) Unique CYP2D6 activity distribution and genotype–phenotype discordance in black Americans. Clin Pharmacol Ther 72:76–89

  • Hägg S, Spigset O, Dahlqvist R (2001) Influence of gender and oral contraceptives on CYP2D6 and CYP2C19 activity in healthy volunteers. Br J Clin Pharmacol 51:169–173

  • Härtter S, Baier D, Dingemanse J et al (1996) Automated determination of dextromethorphan and its main metabolites in human plasma by high-performance liquid chromatography and column switching. Ther Drug Monit 18:297–303

  • Hoskins JM, Shenfield GM, Gross AS (1997) Modified high-performance liquid chromatographic method to measure both dextromethorphan and proguanil for oxidative phenotyping. J Chromatogr B 696:81–87

  • Hu OY, Tang HS, Chang WH, Hu TM (1998) Novel single-point plasma or saliva dextromethorphan method for determining CYP2D6 activity. J Pharmacol Exp Ther 285:955–960

  • Johansson I, Oscarson M, Yue QY et al (1994) Genetic analysis of the Chinese cytochrome P450D locus: characterization of variant CYP2D6 genes present in subjects with diminished capacity for debrisoquin hydroxylation. Mol Pharmacol 46:452–459

  • McCune J, Lindley C, Decker JL et al (2001) Lack of gender differences and large intrasubject variability in cytochrome P450 activity measured by phenotyping with dextromethorphan. J Clin Pharmacol 41: 723–731

  • Sachse C, Brockmöller J, Bauer S, Roots I (1997) Cytochromes P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences. Am J Hum Genet 60:284–295

  • Schmid B, Bircher J, Preisig R, Küpfer A (1985) Polymorphic dextromethorphan metabolism: co-segregation of oxidative O-demethylation with debrisoquin hydroxylation. Clin Pharmacol Ther 38:618–624

  • Wu YJ, Cheng YY, Zeng S, Ma MM (2003) Determination of dextromethorphan and its metabolite dextrorphan in human urine by capillary gas chromatography without derivatization. J Chromatogr B 784:219–224

  • Zanger UM, Raimindo S, Eichelbaum M (2004) Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry. Naunyn-Schmiedeberg’s Arch Pharmacol 369:23–37

CYP3A

Purpose and Rationale

CYP3A is the predominant P450 subfamily (CYP3A4, CYP3A5, CYP3A7, and CYP3A43) in the human liver, and contributes significantly to the metabolism of many (at least 50%) drugs in numerous therapeutic classes. CYP3A4 is the major CYP present notably and predominantly in the liver and the small intestine, and interindividual variability in the level of its expression is very high – 20-fold or more (Shimada et al. 1994). CYP3A5 shares rather similar tissue distribution with CYP3A4, but is preferentially expressed in the lung. It represents generally a few percentage of total CYP3A as compared to CYP3A4 (exceptions are esophagus and prostate, specific for CYP3A5, and kidney in which CYP3A5 is predominantly expressed). CYP3A4 and CYP3A5 exhibit overlapping substrate specificity, and there is currently no specific CYP3A5 probe drug. CYP3A7 is primarily the major fetal CYP3A enzyme.

Most of drugs biotransformed with CYP3A are also P-glycoprotein substrates (noticeable exceptions are midazolam and nifedipine). CYP3A and P-glycoprotein contribute substantially to the first-pass elimination of highly cleared CYP3A substrates when orally administered. However, CYP3A4 and P-glycoprotein activities are not coordinately regulated in the liver and in the intestine (von Richter et al. 2004).

Currently, 40 and 24 alleles have been identified for CYP3A4 and CYP3A5, respectively. Expression of CYP3A5 varies greatly among individuals (Lamba et al. 2002).

Due to multiple confounding factors, such as those involved in endogenous expression of CYP3A regulatory factors, numerous exogenous factors (environment, diet), the interplay between CYP3A and transporters in regulating drug disposition, the establishment of consistent relationships between CYP3A genotype and phenotype is actually a challenge (Wilkinson 2004). Currently, the value of CYP3A genotyping in drug development is far from being clinically useful.

The most used and validated probe drugs for CYP3A phenotyping are midazolam and 14C–erythromycin (Watkins 1994). Alfentanyl, alprazolam, dapsone, DM, lidocaine, nifedipine, omeprazole, quinine, and verapamil have also been used but less frequently, and CYP3A specificity for some of them has been questioned. The “endogenous” 6β-hydroxycortisol test (measurement of 6β-hydroxycortisol: cortisol ratio in urine) is only useful for detecting CYP3A induction, and may be influenced by renal CYP3A activity.

Due to intraindividual differences in the liver and the intestinal CYP3A activity, phenotyping test results are related to the probe drug route of administration.

Procedure

Phenotyping

Midazolam test: Midazolam is primarily metabolized to 1′-hydroxymidazolam by CYP3A. It is rapidly and completely absorbed after oral administration (Gorski et al. 1998). It is the probe of choice to assess intestinal and hepatic or hepatic CYP3A activities only, after oral (Thummel et al. 1996) or intravenous administration, respectively.

Oral test doses are 2, 5, or 7.5 mg (as a solution). IV doses are 0.015, 0.025, or 0.05 mg/kg, or 1 or 2 mg per subject, as a 2–30-min infusion.

Blood samples are collected over a 6-h period. Numerous GC, GC/MS, HPLC/UV, or LC/MS methods have been developed for plasma midazolam assay (Lepper et al. 2004; Frison et al. 2001).

14 C-erythromycin breath test or ERMBT: CYP3A4 catalyzes the N-desmethylation of [14C N-methyl] erythromycin. The test consists of the measurement of a single breath expired 14CO 2 collection obtained at 20 min following the IV administration of a 0.03 mg dose of 14C–erythromycin (2–4 μCi administered) (Watkins 1994). This test is used for assessing hepatic CYP3A activity.

Genotyping: Allelic CYP3A4 gene variants are rare. No impact of the presence of the most common CYP3A4*1B mutation (with a frequency ranging from 0% in Chinese and Japanese to 45% in Afro-Americans) on midazolam, erythromycin, or nifedipine clearance has been evidenced. Most significant mutations are observed for CYP3A5 and CYP3A7. Further information on polymorphic expression of CYP3A5 and CYP3A7 can be found in the review by Lamba et al. (2002).

Evaluation

A complete pharmacokinetic profile is required to assess midazolam clearance, and is therefore more invasive than the ERMBT; however, the latter requires specific logistics for radiolabeled material use. The midazolam or ERMBT phenotype tests are used for dose individualizing of narrow therapeutic index CYP3A-metabolized drugs such as anticancer agents. The ratio 1′-hydroxymidazolam/midazolam has generally been found not useful for phenotyping.

Within a population of similar demographic and health characteristics, a four- to sixfold range in the metabolic clearance of a CYP3A-drug substrate is usual, with common individual outliers exhibiting high or low activity (Lamba et al. 2002).

Critical Assessment of the Method

Midazolam clearance has been found to correlate with hepatic CYP3A levels (Thummel et al. 1994) as well as ERMBT results (Lown et al. 1992). However weak, inconstant, or lack of correlations between midazolam and ERMBT test results have been observed, which could be explained by binding to different CYP3A active sites. In addition, contrary to the midazolam test, the ERMBT does not capture CYP3A5 activity.

An ethnic difference – that could be drug-specific – in CYP3A4 activity has been observed for few CYP3A4 substrates (alprazolam and nifedipine), with a lower clearance in Asians than in Caucasians (Xie et al. 2001).

CYP3A4 and CYP3A5 genotyping tests could not explain sufficiently the interindividual variability observed in midazolam pharmacokinetics (Eap et al. 2004a).

Modifications of the Method

The combined use of IV midazolam and oral 15N-midazolam or of the ERMBT and oral midazolam tests have been proposed to assess simultaneously the contributions of liver and intestine in CYP3A activity (Gorski et al. 1998; McCrea et al. 1999). The administration of orally given midazolam followed by an intravenous administration has also been validated (Lee et al. 2002c). A low oral 75 μg oral dose has recently been proposed, but needs large-scale validation (Eap et al. 2004b).

Modifications of the ERMBT have been described to improve its predictability in drug clearance estimations in cancer patients (Rivory et al. 2000).

A single blood sample for midazolam assay at 4 h post-dose has been reported as good estimator for IV or oral midazolam clearance determination (Lin et al. 2001).

References and Further Reading

  • Eap CB, Buclin T, Hustert E et al (2004a) Pharmacokinetics of midazolam in CYP3A4- and CYP3A5-genotyped subjects. Eur J Clin Pharmacol 60:231–236

  • Eap CB, Buclin T, Cucchia G et al (2004b) Oral administration of midazolam (75 μg) as an in vivo probe for CYP3A activity. Eur J Clin Pharmacol 60:237–246

  • Frison G, Tedeschi L, Maietti S, Ferrara SD (2001) Determination of midazolam in plasma by solid-phase microextraction and gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 15:2497–2501

  • Gorski JC, Jones DR, Haehner-Daniels BD et al (1998) The contribution of intestinal and hepatic CYP3A to the interaction between midazolam and clarithromycin. Clin Pharmacol Ther 64:133–143

  • Lamba JK, Lin YS, Schuety EG, Thummel KE (2002) Genetic contribution to variable human CYP3A-mediated metabolism. Adv Drug Deliv Rev. 54:1271–1294

  • Lee JI, Chaves-Gnecco D, Amico JA et al (2002c) Application of simultaneous midazolam administration for hepatic and intestinal cytochrome P450 3A phenotyping. Clin Pharmacol Ther 72:718–728

  • Lepper ER, Hicks JK, Verweij J et al (2004) Determination of midazolam in human plasma by liquid chromatography with mass-spectrometric detection. J Chromatogr B 806:305–310

  • Lin YS, Lockwood GF, Graham MA et al (2001) In-vivo phenotyping for CYP3A by single-point determination of midazolam plasma concentration. Pharmacogenetics 11:781–791

  • Lown K, Kolars J, Turgeon K et al (1992) The erythromycin breath test selectively measures P450IIIA in patients with severe liver disease. Clin Pharmacol Ther 52:229–238

  • McCrea J, Prueksaritanont T, Gertz BJ et al (1999) Concurrent administration of the erythromycin breath test (EBT) and oral midazolam as in vivo probes for CYP3A activity. J Clin Pharmacol 39:1212–1220

  • von Richter O, Burk O, Fromm MF et al (2004) Cytochrome P450 3A4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: a comparative analysis in paired tissue specimens. Clin Pharmacol Ther 75:172–183

  • Rivory LP, Slaviero KA, Seale JP et al (2000) Optimizing the erythromycin breath test for use in cancer patients. Clin Cancer Res 6:3480–3485

  • Shimada Y, Yamazki H, Mimura M et al (1994) Inter-individual variations in human liver cytochrome P-450 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 270:413–423

  • Thummel KE, Shen DD, Podoll TD et al (1994) Use of midazolam as a human cyotochrome P450 3A probe: in vitro–in vivo correlations in liver transplant patients. J Pharmacol Exp Ther 271:549–556

  • Thummel KE, O’Shea D, Paine MF et al (1996) Oral first-pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A-mediated metabolism. Clin Pharmacol Ther 59:491–502

  • Watkins PB (1994) Noninvasive tests of CYP3A enzymes. Pharmacogenetics 4:171–184

  • Wilkinson GR (2004) Genetic variability in cytochrome P450 3A5 and in vivo cytochrome P450 3A activity: some answers but still questions. Clin Pharmacol Ther 76:99–103

  • Xie HG, Kim RB, Wood AJ, Stein CM (2001b) Molecular basis of ethnic differences in drug disposition and response. Annu Rev. Pharmacol Toxicol 41:815–850

Other CYPS

This section summarizes succinctly the current knowledge on some other CYPs, its role in drug metabolism and its genetic impact have been more recently investigated as compared to other CYPs.

CYP2A6

CYP2A6, primarily expressed in the liver, is the major CYP (the sole at usual low concentrations) involved in nicotine oxidation, and is also involved in the metabolism of carcinogen or procarcinogen compounds (such as nitrosamines and aflatoxins). A couple of drugs is metabolized by CYP2A6: chlormethiazole, coumarin, disulfiram, halothane, valproic acid, and others (Oscarson 2001). CYP2A6 PM is less than 1% in Caucasians but up to 20% in Orientals (Oscarson 2001; Raunio et al. 2001; Xu et al. 2002). The most “in vivo deficient” alleles for PM status are CYP2A6*2 and CYP2A6*4, rather common in Orientals (15% in Chinese, 20% in Japanese). The important role of CYP2A6 in nicotine metabolism was shown in an epidemiological study, revealing that the CYP2A6 genotype was a major determinant for smoking behavior and susceptibility to tobacco-related lung cancer (Fujieda et al. 2004).

Phenotyping has been performed in some countries with coumarin (not available in all countries), despite some limitations with data accuracy obtained with the analytical methods used (Pelkonen et al. 2000; Cok et al. 2001). The test assesses the amount of 7-hydroxycoumarine (free and conjugated) in urine after ingestion of 2–5 mg coumarine by the subjects. Nicotine has also been used as the probe drug for CYP2A6 in vivo activity testing. Recent investigations using pilocarpine as probe demonstrated that PM status was associated with two inactive CYP2A6 alleles, CYP2A6*4A, CYP2A6*7, CYP2A6*9, or CYP2A6*10 (Endo et al. 2008).

References and Further Reading

  • Cok I, Kocabas NA, Cholerton S et al (2001) Determination of coumarin metabolism in Turkish population. Human Exp Pharmacol 20: 179–184

  • Endo T, Nakajima M, Fukami T et al (2008) Genetic polymorphisms of CYP2A6 affect the in-vivo pharmacokinetics of pilocarpine. Pharmacogenet Genomics 18:761–772

  • Fujieda M, Yamazaki H, Saito T et al (2004) Evaluation of CYP2A6 genetic polymorphisms as determinants of smoking behaviour and tobacco-related lung cancer risk in male Japanese smokers. Carcinogenesis 25:2451–2458

  • Oscarson M (2001) Genetic polymorphisms in the cytochrome P450 2A6 (CYP2A6) gene: implications for interindividual differences in nicotine metabolism. Drug Metab Dispos 29:91–95

  • Pelkonen O, Rautio A, Raunio H, Pasanene M (2000) CYP2A6: a human coumarin 7-hydroxylase. Toxicology 144:139–147

  • Raunio H, Rautio A, Gullstén H, Pelkonen O (2001) Polymorphisms of CYP2A6 and its practical consequences. Br J Clin Pharmacol 52: 357–363

  • Xu C, Goodz S, Sellers E, Tyndale RF (2002) CYP2A6 genetic variation and potential consequences. Adv Drug Deliv Rev. 54:1245–1256

CYP2B6

CYP2B6 has been estimated to represent 1–10% of the total hepatic CYP content. It catalyzes bupropion hydroxylation, S-mephenytoin N-demethylation, and is involved in the metabolism of cyclophosphamide, ifosfamide, mianserin, efavirenz, artemisinin, and propofol (Turpeinen et al. 2006). CYP2B6*6 has been associated with reduced bupropion clearance in vitro (Hesse et al. 2004), but not in vivo whereas a moderate clearance increase was observed with CYP2B6*4 (Kirchheiner et al. 2003b). Multiple gene polymorphisms have resulted in phenotypic null alleles (Lang et al. 2004). Pharmacokinetics of the anti-HIV drug efavirenz has been associated with CYP2B6–G516 T polymorphism (Saitoh et al. 2007).

Bupropion (150 mg dose) has been proposed for phenotyping, but it is recommended to administer body weight-adjusted doses (Faucette et al. 2000). Efavirenz may also be a valuable probe for CYP2B6 (Ward et al. 2003).

References and Further Reading

  • Faucette SR, Hawke L, Lecluyse RL et al (2000) Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab Dispos 28:1222–1230

  • Hesse LM, He P, Krishnaswamy S et al (2004) Pharmacogenetic determinants of interindividual variability in bupropion hydroxylation by cytochrome P450 2B6 in human liver microsomes. Pharmacogenetics 14:225–238

  • Kirchheiner J, Klein C, Meineke I et al (2003b) Bupropion and 4-OH-bupropion pharmacokinetics in relation to genetic polymorphisms in CYP2B6. Pharmacogenetics 13:619–626

  • Lang T, Klein K, Richter T et al (2004) Multiple novel nonsynonymous CYP2B6 gene polymorphisms in Caucasians: demonstration of phenotypic null alleles. JPET 311:34–43

  • Saitoh A, Fletcher C, Brundage R et al (2007) Efavirenz pharmacokinetics in HIV-1-infected children are associated with CYP2B6–G516 T polymorphism. J Acquir Immune Defic Syndr 45:280–285

  • Turpeinen M, Raunio H, Pelkonen O (2006) The functional role of CYP2B6 in human drug metabolism: substrates and inhibitors in vitro, in vivo and in silico. Curr Drug Metabol 7:705–714

  • Ward B, Gorski C, Jones D et al (2003) The cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary and secondary metabolism: implication for HIV/AIDS therapy and utility of efavirenz as a substrate marker of CYP2B6 catalytic activity. JPET 306:287–300

CYP2C8

CYP2C8 is involved in the metabolism of arachidonic acid, all-trans retinoic acid, paclitaxel, amiodarone, amodiaquine, repaglinide, rosiglitazone, torsemide, troglitazone, and zopiclone. Most of these drugs are also metabolized by CYP3A4. Recently, the potential contribution of CYP2C8 to the metabolism of NSAIDs in addition to the well-known CYP2C9 role has been highlighted for ibuprofen (Garcia-Martin et al. 2004). The CYP2C8*3 allele (present in 13% and 2% of Caucasians and Afro-American subjects, respectively) has been shown in vitro deficient for paclitaxel and arachidonic acid metabolism (Dai et al. 2001; Bahadur et al. 2002). For the antidiabetic repaglinide, unexpected in vivo lower exposure was observed in subjects with CYP2C8*1/*3 genotype, without any pharmacological consequences (Niemi et al. 2003). For ibuprofen, reduced clearance of the R(−) enantiomer was related to CYP2C8*3 allele, and reduced clearance of the S(+) enantiomer was influenced by CYP2C8*3 and CYP2C9*3 alleles. In subjects homozygous or double heterozygous for these variants (8% of 130 subjects evaluated), the clearances of ibuprofen were only 7–27% of the clearances observed in subjects with no CYP mutations. A strong association between CYP2C8*3 and CYP2C9*2 occurrence has been characterized in a large Swedish population, highlighting linkage between CYP2C8 and CYP2C9 polymorphisms (Yasar et al. 2002c).

Further in vitro/in vivo investigations are needed to assess the relationship between CYP2C8 (and CYP2C9) polymorphisms and drug metabolic clearance, in order to address the clinical relevance of CYP2C8 genotyping.

References and Further Reading

  • Bahadur N, Leathart JBS, Mutch E et al (2002) CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6α-hydroxylase activity in human liver microsomes. Biochem Pharmacol 64: 1579–1589

  • Dai D, Zeldin DC, Blaisdell JA et al (2001) Polymorphisms in CYP2C8 decrease metabolism of the anticancer drug paclitaxel and arachidonic acid. Pharmacogenetics 11:597–607

  • Garcia-Martin E, Martinez C, Tabarés B et al (2004) Interindividual variability in ibuprofen pharmacokinetics is related to interaction of cytochrome P450 2C8 and 2C9 amino acid polymorphisms. Clin Pharmacol Ther 76:119–127

  • Niemi M, Leathart JB, Neuvonen M et al (2003) Polymorphisms in CYP2C8 is associated with reduced plasma concentrations of repaglinide. Clin Pharmacol Ther 74:380–387

  • Yasar U, Lundgren S, Eliasson E et al (2002c) Linkage between CYP2C8 and CYP2C9 genetic polymorphisms. Biochem Biophys Res Commun 299:25–28

CYP2E1

CYP2E1, an ethanol-inducible CYP, activates some procarcinogens such as nitrosamines, is involved in the metabolism of endogenous substrates (steroids and bile acids), alcohols, xanthines, volatile chemicals (toluene, benzene, and halocarbons), but of few drugs (chlorzoxazone, etoposide, dapsone, and high-dose acetaminophen) (Lieber 1997). Seven alleles, 13 genetic mutations have been described, but no genotyping–phenotyping relationships have been well established to date. Based on safe use and CYP selectivity (though CYP1A1, CYP1A2 have been found involved in its biotransformation in vitro), chlorzoxazone is the only in vivo probe drug to phenotype CYP2E1 activity, toward assessment of its 6-hydroxylation (Ono et al. 1995; Lucas et al. 1999; Ernstgard et al. 2004). Due to dose-dependent metabolism, the dose should be preferably administered on a mg/kg basis (10 mg/kg rather than the common 250 or 500 mg doses). Relatively low intraindividual variability in chlorzoxazone metabolism has been observed. Measurement can be done in urine or in plasma, after enzymatic hydrolysis of 6-chlorzoxazone glucuronide, using HPLC and UV detection or LC/MS/MS methods (Frye and Stiff 1996; Frye et al. 1998; Scoot et al. 1999). The use of plasma metabolite ratio determined with only one plasma sample – at 2 h post-dosing – has been recently validated.

References and Further Reading

  • Ernstgard L, Warholm M, Johanson G (2004) Robustness of chlorzoxazone as an in vivo measure of cytochrome P450 2E1 activity. Br J Clin Pharmacol 58:190–200

  • Frye RF, Stiff DD (1996) Determination of chlorzoxazone and 6-hydroxy chlorzoxazone in human plasma and urine by high performance liquid chromatography. J Chromatogr B 686:291–296

  • Frye RF, Adedoyin A, Mauro K et al (1998) Use of chlorzoxazone as an in vivo probe of cytochrome P450 2E1: choice of dose and phenotypic trait measure. J Clin Pharmacol 38:82–89

  • Kim RB, O’Shea D, Wilkinson GR (1995) Interindividual variability of chlorzoxazone 6-hydroxylation in men and women and its relationship to CYP2E1 genetic polymorphisms. Clin Pharmacol Ther 57:645–655

  • Lieber CS (1997) Cytochrome P-450 2E1: its physiological and pathological role. Physiol Rev. 77:517–544

  • Lucas D, Ferrara R, Gonzalez E et al (1999) Chlorzoxazone: a selective probe for phenotyping CYP2E1 in humans. Pharmacogenetics 9:377–388

  • Ono S, Hatanaka T, Hotta H et al (1995) Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1. Pharmacogenetics 5:143–150

  • Scoot RJ, Palmer J, Lewis IA, Pleasance S (1999) Determination of a “GW cocktail” of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom 13:2305–2319

Phase II Enzymes

With the exception of N-acetyltransferases (detailed below), there are few deficiencies in Phase II drug metabolism enzymes that have resulted in clinically significant effects. Each Phase II enzyme class is most often a superfamily of enzymes, and usually there is large interindividual and interethnic variability in drug conjugations, and overlapping substrate specificity exists for numerous isoenzymes. Despite the crucial role of conjugation enzymes in xenobiotic metabolism, the functional significance of enzyme polymorphism is only known for few substrates. Therefore, with the exception of the caffeine and thiopurine methyltransferase (TPMT) tests (see below), no probe test drug has been yet investigated for in vivo phenotyping and validated to assess phenotyping–genotyping relationships. Nevertheless, some important aspects of enzyme polymorphism on the pharmacokinetics of drugs with narrow therapeutic index are summarized below.

N-Acetyltransferases

Purpose and Rationale

N-acetyltransferases type I (NAT1) and type II (NAT2) catalyze N- and O-acetylation reactions involved in the metabolism of drugs containing arylamino, hydroxyl, sulfhydryl groups and hydrazine structure, and also in environmental carcinogens (such as those present in tobacco smoke, or in diet such as charcoal-broiled food) (Weber and Hein 1985). Pending on the drug, and on the interplay between CYPs and N-acetylases (and other Phase II conjugation enzymes) in xenobiotic metabolism, the impact of subject status “poor acetylator” or “rapid acetylator” on drug activity and/or toxicity may vary, and then is drug specific. NAT1 and NAT2 exhibit a high degree (81%) of amino-acid sequence homology, and share common substrates (Meisel 2002) but coding genes loci are regulated independently. Main NAT2 drug substrates are isoniazid, sulfonamides, procainamide, hydralazine, acebutolol, aminoglutethimide, and dapsone.

Para-aminosalicylic and para-aminobenzoic acids are considered specific substrates for human NAT1, and sulfamethazine, isoniazid, procainamide, and dapsone are considered specific substrates for human NAT2 (Butcher et al. 2002). NAT1 is considered as ubiquitously distributed in the body, whereas NAT2 is expressed in liver and intestinal mucosa.

Polymorphic N-acetylation was first described for isoniazid in the 1950s and is the first example of interindividual pharmacogenetic variability. Until 2007, about 30 and more than 50 variant alleles have been described for NAT1 and NAT2, respectively. At http://N-acetyltransferasenomenclature.louisville.edu overviews on the NAT alleles can be found. The presence of some NAT1 variants, as well as NAT2 variants, has been linked to increased susceptibility to some cancers (notably bladder and colon cancers), and NAT2 polymorphism associated with some drug-induced diseases such as lupus erythematosus (hydralazine and procainamide), Stevens–Johnson or Lyell syndromes (sulfonamides).

Significant interethnic and geographic differences in NAT2 activity have been found. Slow acetylators represent 40–70% Caucasians and 10–20% Asians. High acetylation capacity has been reported in 5% Caucasians (Meyer and Zanger 1997).

Probe drugs for NAT1 phenotyping is PAS, and for NAT2 phenotyping are caffeine, sulfamethazine, procainamide, isoniazid, and dapsone. In vivo testing for NAT2 has been proved useful for drug monitoring to avoid potential side effects generally observed in slow metabolizers (the exception was the anticancer agent amonafide, with myelotoxicity observed in rapid acetylators). The most used test to identify rapid and slow acetylators is the caffeine test, which is described thereafter, though the N-acetylation step takes place after the N-desmethylation of caffeine by CYP1A2 followed by the biotransformation into an unstable intermediate.

Procedure

Phenotyping: Caffeine is metabolized by CYP1A2, NAT2, and xanthine oxidases. The methods could involve the measurement of 5-acetyl-formylamino-3-methyluracil (AFMU), 5-acetyl-amino-3-methyluracil (AAMU, degradation product of AFMU), 1-methyl-xanthine (1MX), and 1-methyluric acid (1MU) in 0–8, 0–12, 0–24 h urine of subjects orally given 200 mg or 2–3 mg/kg caffeine after a xanthine-free regimen. The common MR used is AFMU/1MX, but the AFMU/(AFMU +1MX + 1MU) is more discriminating (Relling et al. 1992; Rostami 1995) and should be used when xanthine-oxidase inhibitors may be present (Fuchs et al. 1999). Other ratios such as AFMU/(1MX + 1MU), or AAMU/1MX, AAMU/(AAMU +1MX + 1MU) have been validated (Tang et al. 1991; Nyeki et al. 2002).

The most common methods to assay caffeine and its metabolite in urine used HPLC with UV detection (Grant et al. 1984; Krul and Hageman 1998b) or mass spectrometry (Baud-Camus et al. 2001).

Genotyping: Mutations of NAT2*5, NAT2*6, NAT2*7, NAT2*14, and NAT2*17 alleles are associated with a slow acetylation phenotype for homozygous subjects (Butcher et al. 2002).

There are large differences among ethnic groups regarding alleles’ frequency. High frequency (>28%) of NAT2*5 alleles has been observed in Caucasians and Africans, and of NAT2*7 in Asians (>10%) and of NAT2*14 in Africans (>8%), this last one being <1% in Caucasians and Asians (Meyer and Zanger 1997).

Evaluation

Caffeine test: Subjects with a AFMU/1MX ratio < 0.55 or a AFMU/(AFMU +1MX + 1MU) ratio < 0.26 are slow acetylators (Fuchs et al. 1999). Higher activity has been observed in black as compared to white subjects (Relling et al. 1992), and a gender effect has generally not been observed (Kashuba et al. 1998).

Critical Assessment of the Method

Depending on the probe drug used and on the experimental method, 2 or 3 acetylator types can be described: slow, intermediate, and rapid; the intermediate one being not always distinguished from the rapid one. Phenotype distribution has been considered as a continuous variable (Meisel 2002). Due to slow postnatal maturation of the acetylation enzymatic systems, the acetylation status is evolving in newborns and infants, and depends on the probe drug used (Rane 1999).

Good relationships between genotyping and phenotyping tests have been reported (Meisel et al. 1997; Kita et al. 2001).

The urinary caffeine test is not based on assays of specific substrates and products of NAT2 (“including” other metabolism pathways involving at least xanthine-oxidases), and is affected by diet habits, xanthine-oxidase inhibitors such as allopurinol (Fuchs et al. 1999), or other drugs (Klebovitch et al. 1995). NAT activities are affected by anti-inflammatory drugs. Of note, acetaminophen is an inhibitor of NAT2 in vivo (Rothen et al. 1998).

Discordances between caffeine and dapsone phenotyping data, and between NAT2 phenotyping status and genotyping have been observed in acutely ill patients infected with HIV (O’Neil et al. 2000), which may be due partly to non-detection of rare NAT2 alleles (Alfirevic et al. 2003).

Modifications of the Method

Some recent references for other used NAT2 phenotyping tests can be found for dapsone in Alfirevic et al. (2003), O’Neil et al. (2000), Queiroz et al. (1997), for sulfamethazine in Hadasova et al. (1996) and Meisel et al. (1997), and for procainamide in Okumura et al. (1997) and Mongey et al. (1999).

References and Further Reading

  • Alfirevic A, Stalford AC, Vilar FJ et al (2003) Slow acetylator phenotype and genotype in HIV-positive patients with sulphamethoxazole hypersensitivity. Br J Clin Pharmacol 55:158–165

  • Baud-Camus F, Marquet P, Soursac M et al (2001) Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry. J Chromatogr B 760:55–63

  • Butcher NJ, Boukouvala S, Sim E, Minchin RF (2002) Pharmacogenetics of the arylamine N-acetyltransferases. Pharmacogenomics J 2:30–42

  • Fuchs P, Haefeli WE, Ledermann HR, Wenk M (1999) Xanthine oxidase inhibition by allopurinol affects the reliability of urinary caffeine metabolic ratios as markers for N-acetyltransferase2 and CYP1A2 activities. Eur J Clin Pharmacol 54:869–876

  • Grant DM, Tang BK, Kalow W (1984) A simple test for acetylator phenotype using caffeine test. Br J Clin Pharmacol 17:459–464

  • Hadasova E, Franke G, Zschiesche M et al (1996) Debrisoquin 4-hydroxylation and sulphamethazine N-acetylation in patients with schizophrenia and major depression. Br J Clin Pharmacol 41: 428–431

  • Kashuba ADM, Bertino JS, Kearns GL et al (1998) Quantitation of three-month intraindividual variability and influence of sex and menstrual cycle phase on CYP1A2, N-acetyltransferase-2, and xanthine oxidase activity determined with caffeine phenotyping. Clin Pharmacol Ther 63:540–551

  • Kita T, Tanigawara Y, Chikizawa S et al (2001) N-acetyltransferase2 genotype correlated with isoniazid acetylation in Japanese tuberculous patients. Biol Pharm Bull 24:544–549

  • Klebovitch I, Rautio A, Salonpää et al (1995) Antipyrine, coumarin and glipizide affect N-acetylation measured by caffeine test. Biomed Pharmacother 49:225–227

  • Krul C, Hageman G (1998b) Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reverse-phase high-performance liquid chromatography. J Chromatogr B 709:27–34

  • Meisel P (2002) Arylamine N-acetyltransferases and drug response. Pharmacogenomics 3:349–366

  • Meisel P, Schroeder C, Wulff K, Siegmund W (1997) Relationship between human genotype and phenotype of N-acetyltransferase (NAT2) as estimated by discriminant analysis and multiple linear regression: 1. Genotype and N-acetylation in vivo. Pharmacogenetics 7:241–246

  • Meyer UA, Zanger UM (1997) Molecular mechanisms of genetic polymorphisms of drug metabolism. Annu Rev. Pharmacol Toxicol 37:269–296

  • Mongey AB, Sim E, Risch A, Hess E (1999) Acetylation status is associated with serological change but not clinically significant disease in patients receiving procainamide. J Rheumatol 26:1721–1726

  • Nyeki A, Buclin T, Biollaz J, Decosterd LA (2002) NAT2 and CYP1A2 phenotyping with caffeine: head-to-head comparison of AFMU vs AAMU in the urine metabolite ratios. Br J Clin Pharmacol 55:62–67

  • Okumura K, Kita T, Chikazawa S et al (1997) Genotyping of N-acetylation polymorphism and correlation with procainamide metabolism. Clin Pharm Ther 61:509–517

  • O’Neil WM, Drobitch RK, MacArthur RD et al (2000) Acetylator phenotype and genotype in patients infected with HIV: discordance between methods for phenotype determination and genotype. Pharmacogenetics 10:171–182

  • Queiroz RH, Dreossi SA, Carvalho D (1997) A rapid, specific, and sensitive method for the determination of acetylation phenotype using dapsone. J Anal Toxicol 21:203–207

  • Rane A (1999) Phenotyping of drug metabolism in infants and children: potentials and problems. Pediatrics 104:640–643

  • Relling MV, Lin JS, Ayers GD, Evans EE (1992) Racial and gender differences in N-acetyltransferase, xanthine oxidase and CYP1A2* activities. Clin Pharmacol Ther 52:643–658

  • Rostami-Hodjegan A, Nurminen S, Jackson PR, Tucker GT (1996b) Caffeine urinary metabolic ratios as markers of enzyme activity: a theoretical assessment. Pharmacogenetics 6:121–149

  • Rothen JP, Haefeli WE, Meyer UA et al (1998) Acetominophen is an inhibitor of hepatic N-acetyltransferase 2 in vitro and in vivo. Pharmacogenetics 8:553–559

  • Tang BK, Kadar D, Qian L et al (1991) Caffeine as a metabolic probe: validation of its use for acetylator phenotyping. Clin Pharmacol Ther 49:648–657

  • Weber WW, Hein DW (1985) N-acetylation pharmacogenetics. Pharmacol Rev. 37:25–79

Uridine Diphosphate Glucuronosyltransferases

Glucuronidation is a potent detoxification pathway. The uridine diphosphate glucuronosyltransferases (UGTs) are involved in the biotransformation of endogenous substances (bilirubin, biliary acids, and steroid hormones) and numerous drugs and carcinogens. Currently, 20 functional UGTs have been characterized with activity mainly expressed in the liver and the GI tract. There are three subfamilies: UGT1A, UGT2A, and UGT2B, with distinct but broad overlapping substrate specificity existing for the different isoforms of each family. UGT1A1 is the most abundant UGT in the liver. Human diseases related to deficient UGT1A1 alleles are the well-characterized inherited unconjugated hyperbilirubinemias, including the Gilbert’s syndrome that affects 6–12% of Caucasian subjects. Exhaustive reviews on roles, tissue patterns of expression, and pharmacogenomics of UGTs can be found in papers from Tukey and Strassburg (2000), Fischer et al. (2001), Guillemette (2003), and Wells et al. (2004).

A decreased clearance has been observed for some drugs metabolized by glucuronidation in patients with Gilbert’s syndrome. A clinically significant impact of UGT polymorphism has to date is only demonstrated for some anticancer agents: clearly for irinotecan, and with contradictory results for flavopiridol (Zhai et al. 2003). UGT1A1 and UGT1A9 are involved in the glucuronidation of the active metabolite SN-38 of irinotecan. The presence of the deficient UGT1A1*28 variant (most frequent variant as compared to UGT1A9 variants) has been clinically linked to a decrease in SN-38 glucuronidation rate and to an increased occurrence of serious side effects, mainly severe diarrhea and neutropenia (Ando et al. 1998; Innocenti et al. 2004; Iyer et al. 2002; Paoluzzi et al. 2004). Variants of UGT1A7 were reported to affect SN-38 glucuronidation but only in vitro (Villeneuve et al. 2003). Other factors, such as polymorphism in drug transporter P-glycoprotein and renal excretion, may play a role in the complex disposition pattern of irinotecan.

References and Further Reading

  • Ando Y, Saka H, Asai G et al (1998) OGT1A1 genotypes and glucuronidation of SN-38, the active metabolite of irinotecan. Ann Oncol 9:845–847

  • Fischer MB, Paine MF, Strelevitz TJ, Wrighton SA (2001) The role of hepatic and extrahepatic UDP–glucuronyltransferases in human drug metabolism. Drug Metab Rev. 33:273–297

  • Guillemette C (2003) Pharmacogenomics of human UDP–glucuronosyltransferase enzymes. Pharmacogenomics J 3:136–158

  • Innocenti F, Undvia SD, Iyer L et al (2004) Genetic variants in the UDP–glucuronosyltransferase 1A1 gene predict the risk of severe neutropenia of irinotecan. J Clin Oncol 8:1382–1388

  • Iyer L, Das S, Janish L et al (2002) UGT1A1*28 polymorphism as a determinant of irinotecan disposition and toxicity. Pharmacogenomics J 2:43–47

  • Paoluzzi L, Singh AS, Price DK, Danesi R (2004) Influence of genetic variants in UGT1A1 and UGT1A9 in the in vivo glucuronidation of SN-38. J Clin Pharmacol 44:854–860

  • Tukey RH, Strassburg CP (2000) Human UDP–glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581–616

  • Villeneuve L, Girard H, Fortier LC et al (2003) Novel functional polymorphisms in the UGT1A7 and UGT1A9 glucuronidating enzymes in Caucasian and African-American subjects and their impact on the metabolism of 7-ethyl-10-hydroxycamptothecin and flavopiridol anticancer drugs. J Pharmacol Exp Ther 307:117–128

  • Wells PG et al (2004) Symposium article: glucuronidation and the UDP–glucuronosyltransferases in health and disease. Drug Metab Dispos 32:281–290

  • Zhai S, Sausville EA, Senderowicz AM et al (2003) Clinical pharmacology and pharmacogenetics of flavopiridol 1-h IV infusion in patients with refractory neoplasms. Anticancer Drugs 14:125–135

Methyltransferases

There are at least four enzymes catalyzing S-, N- and O-methylation using S-adenosylmethionine, but only TPMT polymorphism has been found to have important clinical consequences. To date, no endogenous substrate of TPMT is known. TPMT is involved in the metabolism of mercaptopurine, azathioprine and thioguanine, narrow therapeutic index drugs in use for the treatment of patients with neoplasia or autoimmune disease, or of transplant recipients. About 0.3% of Caucasian subjects have no detectable enzyme activity and 10% intermediate activity (McLeod and Evans 2001). Four alleles TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3C account for 80–95% of Caucasians with intermediate or low enzyme activities. Patients with low inherent TPMT activity are at great risk for severe potentially life-threatening myelosuppressive toxicity with treatment by the above-mentioned drugs, whereas subjects with very high activity might be underdosed (Zhou 2006). Patients with two nonfunctional variant TPMT alleles should receive 5–10% of drug standard doses. TPMT genotyping has proved its usefulness in individualizing mercaptopurine dose in patients, and can replace the phenotyping test: measurement of the erythrocyte enzyme activity, based on the in vitro conversion of 6-mercaptopurine to 6-methylmercaptopurine or 6-thioguanine to 6-methylthioguanine (Innocenti et al. 2000; Evans 2004). A cut-off concentration of 45.5 nmol thioguanine/gHb h−1 for this TPMT phenotyping test has been proposed for assessing the need of the genotyping test (Wusk et al. 2004).

References and Further Reading

  • Evans WE (2004) Pharmacogenetics of thiopurine S-methyltransferase and thiopurine therapy. Ther Drug Monit 26:186–191

  • Innocenti F, Iyer L, Ratain MJ (2000) Pharmacogenetics: a tool for individualizing antineoplastic therapy. Clin Pharmacokinet 39: 315–325

  • McLeod HL, Evans WE (2001) Pharmacogenomics: unlocking the human genome for better drug therapy. Annu Rev. Pharmacol Toxicol 41:101–121

  • Wusk B, Kullak-Ublick GA, Rammert C et al (2004) Thiopurine S-methyltransferase polymorphisms: efficient screening method for patients considering taking thiopurine drugs. Eur J Clin Pharmacol 60:5–10

  • Zhou S (2006) Clinical pharmacogenomics of thiopurine S-methyltransferase. Curr Clin Pharmacol 1:119–128

Glutathione S-transferases and Sulfotransferases

Glutathione and sulfatation conjugations are important pathways for generally detoxifying endogenous substrates and xenobiotics (Commandeur et al. 1995). However, some produced metabolites (i.e., mercapturic acids, O-sulfo conjugates) are toxic by different mechanisms, often by reaction with DNA and other cellular nucleophils.

Eight classes of glutathione- S-transferases (GSTs) have been described. The role of the glutathione pathway and the impact of enzyme polymorphism have been highlighted for detoxification and some disease susceptibility, and routine phenotyping of some GSTs exists for clinical safety measurement, but currently there is no evidence of genotyping or phenotyping usefulness for drug dosage adjustment (Hayes and Strange 2000; Tetlow et al. 2004). GSTs are involved in the detoxification of chemotherapeutics, including platinum derivatives. Polymorphisms in the GSTP1 genotype might become a powerful tool to predict oxaliplatin-induced cumulative neuropathy (Lecomte et al. 2006).

Soluble sulfotransferases are involved in the sulfonation of endogenous substrates (notably steroids, neurotransmitters, and eicosanoids) and numerous xenobiotics (i.e., acetaminophen, and organic-platin anticancer agents). The presence of some sulfotransferases variants could be associated with some cancer risk. Phenotyping tests have been developed for some forms (SULT1A and SULT1A3) by measuring platelet sulfotransferase activity (Glatt and Meinl 2004).

References and Further Reading

  • Commandeur JNM, Stintjes GJ, Vermeulen NPE (1995) Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates. Pharmacol Rev. 47:271–330

  • Hayes JD, Strange RC (2000) Glutathione S-transferase polymorphisms and their biological consequences. Pharmacology 61:154–166

  • Glatt H, Meinl W (2004) Pharmacogenetics of soluble sulfotransferases (SULTs). Naunyn-Schmiedeberg’s Arch Pharmacol 369:55–68

  • Lecomte T, Landi B, Beaune P et al (2006) Glutathione S-transferase P1 polymorphism (Ile 105Val) predicts cumulative neuropathy in patients receiving oxaliplatin-based chemotherapy. Clin Cancer Res 12:3050–3056

  • Tetlow N, Robinson A, Mantle T, Board P (2004) Polymorphism of human mu class glutathione transferases. Pharmacogenetics 14:359–368

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.GMPK/PK Sanofi-AventisVitry-Sur-SeineFrance
  2. 2.R&D Metabolism and PK GermanySanofi Aventis Deutschland GmbHFrankfurt am MainGermany

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