Acenocoumarol pharmacokinetics in relation to cytochrome P450 2C9 genotype

BACKGROUND AND OBJECTIVES: Cytochrome P450 (CYP) 2C9 is one of the major CYP enzymes involved in the biotransformation of drugs, among others, the oral anticoagulant acenocoumarol. The enzyme has several polymorphisms, with the CYP2C9*2 and CYP2C9*3 variants most commonly present in white patients. Patients with the CYP2C9*3 variant are known to require a lower maintenance dose of racemic acenocoumarol. We investigated the impact of the polymorphisms CYP2C9*2 and CYP2C9*3 on the pharmacokinetics of R- and S-acenocoumarol. METHODS AND RESULTS: In the first study 26 healthy volunteers with the genotype *1/*1 (n = 9), *1/*2 (n = 7), *1/*3 (n = 6), *2/*3 (n = 3), and *2/*2 (n = 1) were given 8 mg of racemic acenocoumarol as a single oral dose. Plasma R- and S-acenocoumarol concentrations were assayed at 4, 7, and 24 hours. Mean plasma S-acenocoumarol concentrations at 7 hours were higher in subjects with a variant allele; the differences were significant (P =.01) for the *1/*3 and *2/*3 genotypes. In the second study, the oral pharmacokinetics of acenocoumarol was investigated in 6 subjects (*1/*1 [n = 3] and *1/*3 [n = 3]). The mean oral clearance of S-acenocoumarol was 45% lower in the CYP2C9*1/*3 genotypes (10.9 +/- 3.0 L/h versus 19.8 +/- 3.1 L/h, P =.02). Plasma half-life was prolonged from 1.0 +/- 0.2 hours to 2.0 +/- 0.7 hours (P =.09). R-acenocoumarol pharmacokinetics did not differ between the genotypes. There was no difference in mean international normalized ratio at 24 hours, which was 1.2 in both groups. In vitro enzyme kinetics showed reduced (85%) intrinsic activity of the *3 enzyme to catalyze the hydroxylations of S-acenocoumarol. The lower activity resulted from higher Michaelis-Menten constant (2-fold) and lower maximum rate of metabolism by an enzyme-mediated reaction (by 70%). The activity of the *2 enzyme was 50% of the wild-type one. CONCLUSION: The results show S-acenocoumarol pharmacokinetics to be dependent on CYP2C9 polymorphism. In particular, the presence of the CYP2C9*3 allele impairs oral clearance of the coumarin.     

Frequency of cytochrome P450 2C9 allelic variants in the Chinese and French populations

Cytochrome P450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of different drugs with low therapeutic index such as oral anticoagulants. CYP2C9*2 and CYP2C9*3 are two single nucleotide polymorphic allelic variants. The frequency of these alleles in different ethnic populations is extremely variable. In this study, we compared the frequencies of CYP2C9 allelic variants among 394 Chinese living in Shanghai to 151 French Caucasians living in Paris. The allelic frequencies of CYP2C9 variants of the Chinese and the French subjects were 0.963, 0.001, 0.036 and 0.77, 0.15, 0.08 for CYP2C9*1, CYP2C9*2, CYP2C9*3, respectively. Chinese CYP2C9*3 allelic frequency was twice as lower as the French subjects, but three times higher than Korean (0.036 vs. 0.011). The CYP2C9*2 allele could be detected in only one Chinese subject, whereas it represented the major allelic variant in French Caucasians. The low frequency of the CYP2C9*2 and CYP2C9*3 allelic variants in Chinese subjects does not justify their detection in clinical practice, unlike French Caucasians.     

Clinical consequences of cytochrome P450 2C9 polymorphisms

The gene coding for the cytochrome P450 (CYP) enzyme 2C9 (CYP2C9) carries numerous inherited polymorphisms. Those coding for R144C (*2) and I359L (*3) amino acid substitutions have both significant functional effects and appreciable high population frequencies, and their in vivo consequences have been studied in humans with regard to drug metabolism. This review summarizes present knowledge about the pharmacokinetics, drug responses, and outcomes of clinical studies in individuals with different CYP2C9 genotypes. Tentative estimates of how CYP2C9 genotyping might be applied to dose adjustments in clinical therapy were based on dose-related pharmacokinetic parameters such as clearance or trough drug concentrations. Mean clearances in homozygous carriers of the *3 allele were below 25% of that of the wild type for S -warfarin, tolbutamide, glipizide, celecoxib, and fluvastatin. In the more frequent heterozygous carriers (genotype *1/*3), the clearances were between 40% and 75%. In these cases in which individual dosages are derived from clinical drug effects, such as for the oral anticoagulants, the pharmacogenetics-based dose adjustments showed a good correlation with the genotype-specific empirically derived doses. In addition to its role in pharmacokinetics, CYP2C9 contributes to the metabolism of fatty acids, prostanoids, and steroid hormones, and it may catalyze potentially toxic bioactivation reactions. However, our current understanding of the role of CYP2C9 in biotransformation of endogenous signaling molecules and in drug toxicity is relatively meager.     

Risk factors associated with the development of gastroduodenal ulcers due to the use of NSAIDs

The risk of gastrointestinal mucosal injury with non-steroidal anti-inflammatory drugs (NSAIDs) is dose-dependent. Epidemiological studies have clearly demonstrated a rank order of risk of ulcer complications for commonly used NSAIDs, with ibuprofen consistently associated with the lowest risk and piroxicam with the highest. Antacids, H2 receptor antagonists and misoprostol all have drawbacks as prophylaxis. Of the cyclo-oxygenase (COX)-2 selective NSAIDs, rofecoxib is associated with a lower risk of gastrointestinal toxicity but there is uncertainty about the long-term risk associated with celecoxib. Rofecoxib has been associated with a significantly higher incidence of myocardial infarction than naproxen that may counteract the benefit of greater gastrointestinal safety. At over-the-counter doses, the short duration of use and the low dose reduce the risk of a serious adverse event compared with chronic use at prescribed doses. Intermittent therapy with low-dose NSAIDs has proved extremely safe and it has not been determined whether COX-2 selective agents offer a safety advantage compared with such treatment.     

Allelic variants of cytochrome P450 2C9 modify the interaction between nonsteroidal anti-inflammatory drugs and coumarin anticoagulants

INTRODUCTION: Cytochrome P450 (CYP) plays a key role in the metabolism of coumarin anticoagulants and nonsteroidal anti-inflammatory drugs (NSAIDs). Because CYP2C9 is a genetically polymorphic enzyme, genetic variability could play an important role in the potential interaction between NSAIDs and coumarins. We investigated whether NSAIDs were associated with overanticoagulation during therapy with coumarins and evaluated the effect of the CYP2C9 polymorphisms on this potential interaction. METHODS: We conducted a population-based cohort study among patients of an anticoagulation clinic who were treated with acenocoumarol or phenprocoumon between April 1, 1991, and May 31, 2003, and whose CYP2C9 status was known. Patients were followed up until an international normalized ratio (INR) of 6.0 or greater was reached or until the end of treatment, death, or the end of the study. Proportional hazards regression analysis was used to estimate the risk of an INR of 6.0 or greater in relation to concomitant use of a coumarin anticoagulant and NSAIDs after adjustment for several potentially confounding factors. To study effect modification by CYP2C9 genotype, stratified analyses were performed for wild-type patients and patients with a variant genotype. RESULTS: Of the 973 patients in the cohort, 415 had an INR of 6.0 or greater. Several NSAIDs increased the risk of overanticoagulation. The risk of overanticoagulation was 2.98 (95% confidence interval, 1.09-7.02) in coumarin-treated patients taking NSAIDs with a CYP2C9*2 allele and 10.8 (95% confidence interval, 2.57-34.6) in those with a CYP2C9*3 allele. CONCLUSIONS: Several NSAIDs were associated with overanticoagulation. For NSAIDs that are known CYP2C9 substrates, this risk was modified by allelic variants of CYP2C9. More frequent INR monitoring of patients taking NSAIDs is warranted.     

Common genetic variants of microsomal epoxide hydrolase affect warfarin dose requirements beyond the effect of cytochrome P450 2C9

BACKGROUND: Warfarin dose response is partially explained by the polymorphisms in the cytochrome P450 (CYP) 2C9 gene, affecting S -warfarin clearance, as well as by age and body weight. We examined the influence on warfarin dose requirements of candidate genes encoding microsomal epoxide hydrolase (mEH), as well as glutathione S -transferase A1 (GSTA1) components of vitamin K epoxide reductase and the gamma-glutamylcarboxylase (GGCX) gene. METHODS: We studied the effects of CYP2C9, mEH, GSTA1, and GGCX genotypes on warfarin maintenance doses, accounting for age, weight, vitamin K plasma concentrations and concurrent medications, in 100 patients undergoing therapeutic anticoagulation. RESULTS: Allele frequencies were 76.5%, 12.5%, and 11% for CYP2C9*1 , *2 , and *3 , respectively; 75% and 25% for mEH T 612 C; 75.8% and 24.2% for mEH A 691 G; 73.5% and 26.5% for GSTA1 T 631 G; and 70.5% and 29.5% for GGCX G 8762 A. Warfarin doses differed among the CYP2C9 ( 2C9*1 , 2C9*2 , and 2C9*3 ) genotype groups: 6.3 +/- 1.9 mg/d, 5.3 +/- 1.8 mg/d, and 3.8 +/- 1.7 mg/d, respectively (F = 4.83, P < .01). There were no differences in any of the other genotype groups. Among the 62 wild-type CYP2C9 patients, variant mEH T 612 C homozygotes required higher doses than heterozygotes and wild-type patients (7.5 +/- 2.9 mg/d, 6.5 +/- 4.2 mg/d, and 6.0 +/- 2.6 mg/d, respectively [F = 3.57, P = .03]). The odds ratio for requiring greater than 7 mg/d in variant mEH T 612 C patients versus wild-type patients was 3.14 (95% confidence interval, 1.47-6.67), accounting for CYP2C9. CONCLUSIONS: Variant mEH T 612 C genotypes are associated with warfarin doses of greater than 50 mg/wk beyond the effect of CYP2C9.     

Effects of cytochrome P450 2C9 polymorphisms on phenprocoumon anticoagulation status

OBJECTIVE: Our objective was to assess whether there is an association between the presence of allelic variants of the gene for cytochrome P450 (CYP) 2C9 and anticoagulation problems during the initial phase of phenprocoumon treatment. METHODS: A prospective follow-up study was performed at 2 anticoagulation clinics in The Netherlands. Included subjects started phenprocoumon during the study period, had their first check of the international normalized ratio (INR) on the third or fourth day of therapy, and had an indication for the low therapeutic range (INR, 2.0-3.5). CYP2C9 genotypes ( CYP2C9*1 , CYP2C9*2 , and CYP2C9*3 ) were assessed, and data on indication, INR checks, comedication, and comorbidity were collected. RESULTS: After genotyping, 284 subjects were available for analysis. Of these, 186 (65.5%) were homozygous carriers of the CYP2C9 wild-type allele ( CYP2C9*1/*1 ), 61 (21.5%) were carriers of the CYP2C9*2 allele, and 37 (13.0%) were carriers of the CYP2C9*3 allele. Compared with homozygous CYP2C9*1/*1 subjects, carriers of CYP2C9*2 or *3 had an increased risk of severe overanticoagulation (INR >6.0). The hazard ratio for CYP2C9*2 versus CYP2C9*1/*1 was 3.09 (95% confidence interval [CI], 1.56 to 6.13; P=.001), and the hazard ratio for CYP2C9*3 versus CYP2C9*1/*1 was 2.40 (95% CI, 1.03 to 5.57; P=.042). Carriers of CYP2C9*2 also had a lower chance to achieve stability in the follow-up period. The hazard ratio for CYP2C9*2 versus CYP2C9*1/*1 was 0.61 (95% CI, 0.43 to 0.85; P=.003). Carriers of the CYP2C9*2 or *3 allele needed a significantly lower phenprocoumon dosage compared with homozygous CYP2C9*1/*1 subjects. CONCLUSION: The presence of at least 1 CYP2C9*2 or *3 allele in phenprocoumon users is associated with an increased risk of severe overanticoagulation. Similar to warfarin and acenocoumarol, phenprocoumon had a lower dosage requirement in carriers of CYP2C9*2 or *3 compared with that in CYP2C9 wild-type subjects.     

Enantiospecific effects of cytochrome P450 2C9 amino acid variants on ibuprofen pharmacokinetics and on the inhibition of cyclooxygenases 1 and 2

OBJECTIVE: According to in vitro data, the polymorphic cytochrome P450 enzyme 2C9 (CYP2C9) may be the major S-ibuprofen hydroxylase. In humans, there are 2 variants of CYP2C9 with a high population frequency. We studied their impact on ibuprofen pharmacokinetics and on the inhibition of cyclooxygenases 1 and 2. METHODS: Kinetics of an oral dose of 600 mg racemic ibuprofen were studied in 21 healthy volunteers with all combinations of the CYP2C9 variants *2 (arginine144cysteine) and *3 (isoleucine359leucine). Blood concentrations of racemic ibuprofen and of S-(+)-ibuprofen and R-(-)-ibuprofen were measured by HPLC, and thromboxane B(2) and prostaglandin E(2) were measured with use of an enzyme immunoassay. Data were evaluated with a population pharmacokinetic model that integrated pharmacogenetic information. RESULTS: The pharmacokinetics of racemic and of S-ibuprofen depended on the CYP2C9 isoleucine359leucine amino acid polymorphism: population mean S-ibuprofen clearances were 3.25 L/h (95% confidence interval [CI], 2.84 to 3.73), 2.38 L/h (95% CI, 2.09 to 2.73), and 1.52 L/h (95% CI, 1.33 to 1.74) in carriers of the CYP2C9 genotypes *1/*1, *1/*3, and *3/*3, respectively. The CYP2C9 variant *2 exhibited no significant effect. Ex vivo formation of thromboxane B(2), reflecting cyclooxygenase type 1 inhibition, depended significantly on the CYP2C9 polymorphism. The maximal inhibition of thromboxane B(2) formation and the area under the effect-time curve were larger in carriers of the slow CYP2C9 genotypes *1/*3, *2/*3, and *3/*3 than in *1/*1 carriers; the same trend was observed for prostaglandin E(2), reflecting cyclooxygenase type 2 inhibition. CONCLUSIONS: The reduced S-ibuprofen total clearance accompanied by increased pharmacodynamic activity may have medical impact in patients receiving ibuprofen.     

Different dosage regimens of rabeprazole for nocturnal gastric acid inhibition in relation to cytochrome P450 2C19 genotype status

OBJECTIVE: For the treatment of gastroesophageal reflux disease, intragastric pH should be lower than 4.0 for no more than 4 hours a day (<16.7%). We aimed to develop optimal dosage regimens for rabeprazole to control nocturnal acidity in relation to cytochrome P450 (CYP) 2C19 genotypes. METHODS: Fifteen Helicobacter pylori -negative volunteers, comprising 5 homozygous extensive metabolizers (EMs), 6 heterozygous EMs, and 4 poor metabolizers (PMs) of CYP2C19, took placebo and rabeprazole, at a dose of 20 or 40 mg once daily (at 10 pm ) for 8 days. Plasma rabeprazole concentrations and 24-hour intragastric pH were determined on days 7 and 8, respectively. Because the nocturnal intragastric pH was lower than 4.0 for more than 16.7% of the time with once-daily regimens in homozygous EMs and heterozygous EMs, they were administered 20 mg rabeprazole twice daily (8 am and 10 pm ) or 10 mg rabeprazole 4 times daily (8 am , 12:30 pm , 6 pm , and 10 pm ). RESULTS: With 40 mg rabeprazole once daily, the median percent time with nocturnal pH lower than 4.0 was less than 16.7% in PMs (9.5% [range, 3.0%-31.1%]) but not in homozygous EMs (45.3% [range, 29.0%-52.2%]) ( P = .043) and heterozygous EMs (41.3% [range, 33.0%-59.0%]) ( P = .043). The mean plasma rabeprazole concentrations differed among the different CYP2C19 genotype groups. With 20 mg rabeprazole twice daily and 10 mg rabeprazole 4 times daily, the median percent times with nocturnal pH lower than 4.0 were 5.0% (range, 0.0%-42.0%) and 1.0% (range, 5.0%-7.1%) in heterozygous EMs and 62.0% (range, 10.8%-68.3%) and 14.7% (range, 0.0%-41.7%) in homozygous EMs, respectively, and plasma concentrations were sustained longer than with the once-daily regimens. CONCLUSIONS: We propose that rabeprazole dosage regimens for sufficient acid inhibition are 20 mg once daily for PMs, 20 mg twice daily for heterozygous EMs, and 10 mg 4 times daily for homozygous EMs or heterozygous EMs.     

Interindividual variability in ibuprofen pharmacokinetics is related to interaction of cytochrome P450 2C8 and 2C9 amino acid polymorphisms

OBJECTIVE: Our objective was to identify genetic factors related to interindividual variability in the pharmacokinetics of ibuprofen and its enantiomers. METHODS: The time course for ibuprofen plasma concentration was measured by HPLC in 130 healthy individuals who received a single oral dose of 400 mg racemic ibuprofen. Genomic deoxyribonucleic acid was analyzed for common mutations at CYP2C8 and CYP2C9 genes that cause amino acid substitutions. RESULTS: Ibuprofen clearance values were 4.04 L/h (95% confidence interval [CI], 3.61-4.47 L/h), 2.79 L/h (95% CI, 2.07-3.52 L/h), and 0.40 L/h (95% CI, 0.37-0.43 L/h) for carriers of CYP2C8 genotypes *1/*1, *1/*3, and *3/*3, respectively, and 4.43 L/h (95% CI, 3.94-4.92 L/h), 3.26 L/h (95% CI, 2.53-3.99 L/h), 2.91 L/h (95% CI, 1.52-4.30 L/h), 2.05 L/h (95% CI, 0-6.37 L/h), 1.83 L/h (95% CI, 1.24-2.41 L/h), and 1.13 L/h (95% CI, 0.58-1.66 L/h) for carriers of the CYP2C9 genotypes *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. The P values for comparison across nonmutated, heterozygous, and homozygous genotypes were as follows: P <.001 for CYP2C8*3, P <.005 for CYP2C9*2, and P <.001 for CYP2C9*3. The main genetic factor for reduced clearance of R-(-)-ibuprofen is the CYP2C8*3 allele, whereas the clearance for S-(+)-ibuprofen is influenced by CYP2C8*3 and CYP2C9*3 alleles to a similar extent. The CYP2C9*2 allele was associated with low clearance only when it was present in combination with the CYP2C8*3 allele. As compared with individuals with no mutations, individuals with the common genotype CYP2C8*1/*3 plus CYP2C9*1/*2 (19% of the population) displayed decreased ibuprofen clearance (mean, 65% [95% CI, 42%-89%]; P <.001). Individuals homozygous or double-heterozygous for CYP2C8*3 and CYP2C9*3 variant alleles (8% of the population) had extremely low ibuprofen clearance rates, with values ranging from 7% to 27% of the mean clearance rates among noncarriers of mutations (P <.001). No enantiospecific reduction of ibuprofen clearance was observed. CONCLUSION: Low ibuprofen clearance occurs in a substantial proportion of healthy subjects, is not enantiospecific, and is strongly linked to CYP2C8 and CYP2C9 polymorphisms.     

Baseline blood flow and bradykinin-induced vasodilator responses in the human forearm are insensitive to the cytochrome P450 2C9 (CYP2C9) inhibitor sulphaphenazole

A substantial portion of the vasodilator response elicited by bradykinin in the human forearm is unaffected by the combined inhibition of nitric oxide (NO) synthases and cyclo-oxygenases. The cytochrome P450 (CYP) 2C9 inhibitor sulphaphenazole was recently identified as a potent inhibitor of NO- and prostacyclin (PGI2)-independent relaxation in porcine coronary arteries. The aim of the present study was to determine the effect of sulphaphenazole on basal and bradykinin-induced NO/PGI2-independent changes in the forearm blood flow (FBF) of healthy subjects. Eleven healthy male volunteers participated in this placebo-controlled study. Test agents were infused into the brachial artery and FBF was measured by bilateral venous occlusion plethysmography. Sulphaphenazole (0.02-2 mg/min) alone did not affect basal blood flow. Inhibition of the NO synthases by NG-monomethyl-L-arginine (L-NMMA; 4 micromol/min) and cyclo-oxygenases by ibuprofen (1200 mg, orally) reduced FBF to 48 +/- 7% in the absence and 50 +/- 8% in the presence of sulphaphenazole (2 mg/min; P=not significant). After pretreatment with L-NMMA (16 micromol/min) and ibuprofen (1200 mg, orally), sulphaphenazole (6 mg/min) did not substantially inhibit bradykinin-induced vasodilation. We conclude that CYP2C9-derived metabolites (i) are not involved in the regulation of baseline blood flow, and (ii) do not mediate bradykinin-induced NO/PGI2-independent vasorelaxation in the human forearm. However, determining the contribution of this enzyme to regulation of blood flow in pathological conditions associated with endothelial dysfunction requires further studies.     

Artemisinin autoinduction is caused by involvement of cytochrome P450 2B6 but not 2C9

AIM: Our goal was to investigate whether artemisinin autoinduction is caused by an increase in cytochrome P450 (CYP) 2B6 or CYP2C9 activities, we evaluated the effects of multiple-dose artemisinin administration on S-mephenytoin N-demethylation in healthy subjects. METHODS: Fourteen subjects, 6 poor metabolizers of CYP2C19 and 8 extensive metabolizers, received a single oral dose of 200 mg racemic mephenytoin (CYP2B6 in vivo marker) before (day -28) and during multiple-dose artemisinin administration (250 mg/d orally for 9 days and 500 mg on the tenth day). A 500-mg single dose of artemisinin was administered on day -28. The CYP2C9 in vivo marker tolbutamide was administered on day -28 and on days 7, 12, and 17 to monitor the minor involvement of CYP2C9 in S-mephenytoin N-demethylation. RESULTS: Artemisinin oral clearance increased 5.3-fold (P <.001) by the tenth day of administration. Its pharmacokinetics was not different in the 2 CYP2C19 phenotypes. The oral clearance of R-mephenytoin increased 1.7-fold (P <.05) in both phenotypes during the period of artemisinin administration. The area under the concentration-time curve ratio of S-nirvanol/S-mephenytoin, an index of CYP2B6 activity, increased 1.9-fold (P <.05) in CYP2C19 poor metabolizers during artemisinin multiple-dose administration, whereas the urinary excretion ratio of hydroxytolbutamide plus carboxytolbutamide/tolbutamide remained constant during the study period. CONCLUSIONS: These results indicate that artemisinin induces the N-demethylation of S-mephenytoin probably by an increased capacity of CYP2B6. The autoinduction phenomenon of artemisinin may, therefore, be attributed, at least in part, to induction of CYP2B6, because this is the isozyme primarily involved in its metabolism. In addition, artemisinin alters the disposition of R-mephenytoin by an unidentified isozyme.     

Major role of human liver microsomal cytochrome P450 2C9 (CYP2C9) in the oxidative metabolism of celecoxib, a novel cyclooxygenase-II inhibitor

In vitro studies were conducted to identify the cytochromes P450 (CYP) involved in the oxidative metabolism of celecoxib. The hydroxylation of celecoxib conformed to monophasic Michaelis-Menten kinetics (mean +/- S.D., n = 4 livers, K(m) = 3.8 +/- 0.95 microM, V(max) = 0.70 +/- 0.45 nmol/min/mg protein) in the presence of human liver microsomes, although substrate inhibition was significant at higher celecoxib concentrations. The treatment of a panel of human liver microsomal samples (n = 16 subjects) with antibodies against CYP2C9 and CYP3A4 inhibited the formation of hydroxy celecoxib by 72 to 92% and 0 to 27%, respectively. The presence of both antibodies in the incubation suppressed the activity by 90 to 94%. In addition, the formation of hydroxy celecoxib significantly correlated with CYP2C9-selective tolbutamide methyl hydroxylation (r = 0.92, P <. 001) and CYP3A-selective testosterone 6beta-hydroxylation (r = 0.55, P <.02). In contrast, correlation with activities selective for other forms of CYP was weak (r 相似文献   

Involvement of cytochrome P450 2C9, 2E1 and 3A4 in trimethadione N-demethylation in human microsomes

BACKGROUND AND OBJECTIVES: Trimethadione (TMO), an antiepileptic drug, may be used as a candidate for estimating hepatic drug-oxidizing activity. While TMO metabolism is mainly catalysed by CYP2C9, CYP2E1 and CYP3A4 the contribution of the different isoforms is unclear. In this study, we determined the percentage contribution of the three CYPs (CYP2C9, CYP2E1 and CYP3A4) to TMO N-demethylation. METHOD: We used human liver microsomes and human recombinant CYPs expressed in human B-lymphoblast cells and baculovirus-infected insect cells. RESULTS: The mean Km, Vmax and Vmax/Km values of TMO N-demethylation in human microsomes were 3.66 (mm), 503 (pmol/min/mg) and 2.61 (mL/h/mg), respectively. In the microsomes from human B-lymphoblast cells or baculovirus-infected insect cells, CYP 2C9, CYP 2E1 and CYP3A4 exhibited similar Km and higher Vmax in baculovirus-infected insect cells than B-lymphoblast cells. In baculovirus-infected insect cells, CYP2C9, CYP2E1 and CYP3A4 exhibited activities of 32, 286 and 77 pmol/min/pmol CYP, respectively. No CYP activity catalysed by CYP1A2 and 2D6 were detected in the two human cDNA expressed CYP isoforms. CONCLUSION: TMO is metabolized not only by CYP2E1 but also CYP3A4 and CYP2C9. The order of this metabolism is as follows: CYP2E1 > CYP3A4 > CYP2C9.     

Slow chloroguanide metabolism in Tanzanians compared with white subjects and Asian subjects confirms a decreased CYP2C19 activity in relation to genotype

BACKGROUND: We have previously found decreased CYP2C19 activity in Tanzanians tested with mephenytoin and omeprazole in relation to genotype when compared with white and Asian subjects. OBJECTIVE: We investigated the impact of CYP2C19 genotype and phenotype on chloroguanide (INN, proguanil) metabolism to its metabolites cycloguanil and 4-chlorophenylbiguanide. METHODS: A single oral chloroguanide dose was given to 25 healthy Tanzanian subjects with CYP2C19 genotypes (CYP2C19*1, CYP2C19*2, and CYP2C19*3). Homozygous wild-type and mutated genotype groups were chosen randomly, but the heterozygous genotype group was chosen with a range in phenotype. We used a novel HPLC method for drug determination. RESULTS: Pharmacokinetics of chloroguanide did not differ between groups. Maximum plasma concentration (Cmax) and area under the plasma concentration versus time [AUC(0-infinity)] for cycloguanil was significantly lower (t test P < .05) in the homozygously mutated group compared with the homozygously wild-type group. There were similar significant group differences of median urinary excretion. The chloroguanide/cycloguanil ratio closely correlated (r(s) = .87) with omeprazole metabolic ratio, confirming that Tanzanian subjects are generally slower CYP2C19 metabolizers. It also confirms that CYP2C19 genotype and phenotype predicts cycloguanil formation. In addition, a 3-hour plasma sample metabolic ratio also seems to be a proper time for omeprazole phenotyping in Tanzanian subjects. Because the plasma concentrations of cycloguanil and 4-chlorophenylbiguanide covary (r(s) = .89), it is now suggested that their formation be catalyzed by the same enzyme (ie, CYP2C19) through a common intermediate, the structure of which is also presented. CONCLUSIONS: As shown in an earlier study, also with a third substrate, Tanzanians have a lower capacity to form cycloguanil than white and Asian subjects. Individuals with two mutated alleles have lower metabolic capacity than individuals with two wild-type alleles or individuals in the heterozygous group, which may lead to chloroguanide therapeutic failure. This knowledge should be important when selecting appropriate patients and doses of chloroguanide in different populations.     

A novel intronic mutation, 2988G>A, with high predictivity for impaired function of cytochrome P450 2D6 in white subjects

BACKGROUND: Individuals with the cytochrome P450 (CYP) 2D6 intermediate metabolizer (IM) phenotype have low residual enzyme activity and compose about 10% to 15% of white populations. Their identification is clinically relevant but remains unsatisfactory because of incomplete characterization of the major allele involved, termed CYP2D6*41 (-1584C, R296C, S486T). METHODS: To search for novel mutations, resequencing of the entire CYP2D6 gene was performed in selected individuals. Genotype-phenotype correlation analysis was done in a population sample of 308 white subjects phenotyped with sparteine and previously genotyped for all major alleles. RESULTS: A total of 16 novel polymorphic positions were identified, of which 7 were located within 2.4 kilobases of previously uncharacterized 2D7-2D6 intergenic sequence and 9 were located within intronic regions. The novel mutation 2988G>A in intron 6 appeared to be specifically associated with the IM phenotype. Further analysis in the population sample demonstrated that 2988G>A was strongly linked to allele *41 but not to any other alleles including *1, *2, *2xN, *4, *6, *7, *8, *9, *10, and *35. The overall frequency of the novel polymorphism was 8.4% in the normal white population. Compared with conventionally determined *41, 2988G>A was shown to have improved predictivity for the IM phenotype. With 2988G>A being taken into account, alleles *1, *2, and *35 (-1584G, V11M, R296C, S486T) were found to be phenotypically equivalent. CONCLUSIONS: CYP2D6 genotyping can be considerably simplified by using 2988G>A as a marker for *41 and by omitting genotyping for the functionally equivalent alleles *2 and *35.     

Impact of the ultrarapid metabolizer genotype of cytochrome P450 2D6 on metoprolol pharmacokinetics and pharmacodynamics

INTRODUCTION: In the treatment of heart failure and hypertension with metoprolol, ultrarapid metabolizers (UMs) may not achieve optimal target concentrations with recommended doses. We compared metoprolol pharmacokinetics and effects in UMs with extensive metabolizers (EMs) and with poor metabolizers (PM) as an additional reference group. METHODS: After a single dose of 100 mg metoprolol, pharmacokinetics, resting and exercise heart rate, and blood pressure were analyzed in relation to the CYP2D6 genotypes. We included 12 UMs, 13 EMs, and 4 PMs (healthy volunteers). CYP2D6 genotyping covered alleles *1 to *6 , *9 , *10 , *35 , and *41 and the duplications. beta 1 -Adrenergic receptor polymorphisms Ser49Gly and Arg389Gly were included as factors possibly interfering with the pharmacokinetic-pharmacodynamic relationship of metoprolol. RESULTS: Median total metoprolol clearance values were 31, 168, and 367 L/h and median maximum plasma concentrations were 260, 118, and 67 microg/L in PMs, EMs, and UMs, respectively ( P < .0001). At 6 hours after administration, metoprolol reduced the exercise heart rate by median values of 31, 21, and 18 beats/min in PMs, EMs, and UMs, respectively ( P = .01). Blood pressure did not significantly differ according to CYP2D6 . CONCLUSIONS: A linear relationship between the number of active CYP2D6 genes and metabolic clearance of metoprolol was found and the the median clearances differed by more than 10-fold between the PM and the UM groups. Metoprolol pharmacodynamics, however, differed only by less than 2-fold, and there was only a marginal difference in metoprolol efficacy on heart rate between the EM and UM groups.