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Dual antiplatelet therapy with clopidogrel combined to aspirin reduces ischemic events in a wide range of patients with cardiovascular disease. However, a large interindividual variability has been observed in the pharmacodynamics response of clopidogrel with lower platelet inhibition associated with increased risk for major adverse cardiovascular events. Multiple mechanisms have been proposed for the variable response to clopidogrel, among which the impact of pharmacogenetic seems to play an important role. This article reviews the role of genetic polymorphisms in the clinical impact of oral inhibitors of the platelet P2Y12 adenosine diphosphate (ADP) receptor.

Recently, some authors have examined the possibility of an interaction between proton pump inhibitors (especially omeprazole) and clopidogrel. Pharmacodynamically, this interaction is proven and leads to a reduction in anti-aggregation activity. Nevertheless, the clinical implications are not fully clarified. We are all waiting for a randomised prospective study assessing specifically this issue. Meanwhile, caution is recommended when prescribing PPI in patients taking clopidogrel.

Clopidogrel has a known biological variability that has been consistently associated with recurrence of coronary ischemic events in clinical studies. Among the tests that are currently available, quantification of the phosphorylation status of the vasodilator phosphoprotein (VASP assay) is probably the most specific assay to evaluate the inhibition of the P2Y12 receptor by clopidogrel. A genetic polymorphism of the cytochrome 2C19 has been associated with the biological efficacy of clopidogrel and is also associated with recurrent ischemic events. The VASP assay and the 2C19 genotyping are candidates for the identification of patients at risk; this is the focus of the present review.

A 33-year-old man was hospitalized unconscious on suspicion of acute diazepam and hexobarbital intoxication, a barbiturate not marketed in France. Its quantification was developed by gas chromatography coupled with mass spectrometry detection and validated on one-hundred microL of plasma extracted by a liquid/liquid method. Hexobarbital and diazepam concentrations in initial plasma samples were at toxic levels, respectively 15 900 ng/mL and 13 800 ng/mL. Hexobarbital toxicokinetic was studied during 175 h and analysed with a non-compartmental model. Half-life value of 61,8 h was found, being much longer than already published hexobarbital half-lives (between 3.2 h and 6.9 h). The reasons of this long half-life were discussed according to metabolism and pharmacogenetic.

The use of vitamin K antagonists (VKA) is challenging because of their narrow therapeutic index and a high bleeding risk. These drugs are widely prescribed for the prophylaxis and treatment of many thrombo-embolic disorders. The management of patients with VKA represents a public health problem according to the high number of deaths and hospitalizations in relation to hemorrhagic complications. Monitoring is required because of the large inter-individual variability. The identification of factors, in particular genetic factors, influencing the response to VKA will improve the safety of VKA treatment. In addition to demographic, clinical, biological factors and drug interactions, genetic factors can explain a large part of the inter-individual variability. The main enzyme responsible for VKA metabolism is the hepatic cytochrome P450 2C9 (CYP2C9). Vitamin K epoxide reductase complex subunit I (VKORC1) is a key enzyme in the vitamin K cycle and is the pharmacological target of VKA. Genetic variations affecting both CYP2C9 and VKORC1 are associated with a significant decrease in the VKA dose requirement and an increased risk of bleeding. CYP2C9 and VKORC1 genotyping may identify a subgroup of patients with an early response at the induction of VKA therapy, potentially leading to a high bleeding risk. On the opposite, rare mutations in VKORC1 can explain high dose requirement and pharmacodynamic resistance. Genotyping CYP2C9 and VKORC1 variants before treatment initiation could allow the development of dosing protocols and the identification of patients at high risk of bleeding complications.

Single nucleotide polymorphisms (SNPs) in genes encoding cytochrome P450 enzyme 2C9 (CYP2C9) and Vitamin K epoxide reductase subunit I (VKORC1) make a significant contribution to the inter-individual variability in the maintenance dose of vitamin K antagonists (warfarin, acenocoumarol, phenprocoumon). Doses requirements in CYP2C9*2 and CYP2C9*3 heterozygotes are reduced by 8-16% and 20-36%, respectively. SNP g-1639a in VKORC1 is also associated with vitamin K antagonists dosage since heterozygotes ga and homozygotes aa require respectively 21-28% and 27-56% less warfarin or acenocoumarol than homozygotes gg. CYP2C9 and VKORC1 account for up to half the variability in vitamin K antagonists requirements and incorporating genotying data for these two genes into dosing algorithms could lead to a safer anticoagulation therapy.

Oral anti-vitamin K (AVK) anticoagulants constitute the first cause of iatrogenic accidents in France because of narrow therapeutic index and bleeding risk. The wide interindividual variation in AVK response is partly genetically determined. The main enzyme responsible for the metabolism of AVK is the hepatic cytochrome P450 2C9 (CYP2C9). Vitamine K epoxide reductase complex subunit I (VKORC1) is a key enzyme in the vitamin K cycle, cofactor required for the activation of vitamin K-dependent clotting factors, and is the target enzyme of AVK inhibition. Genetic variations affecting both CYP2C9 and VKORC1 are associated with variability in drug response and may explain differences in dose requirements. Genotyping for CYP2C9 and VKORC1 variants before initiation of treatment may allow clinicians to develop dosing protocols and identify the patients at a higher risk for bleeding complications.

Vitamin K antagonists (VKA) are difficult to use because of a narrow therapeutic index and of a marked inter- and intra individual variability among patients in the required dosage. Beside well known demographic or environmental factors (advanced age, co-morbid conditions, acute illnesses, concomitant drugs, vitamin K intake), genetic single nucleotide polymorphisms (SNPs) have been identified as strongly affecting the maintenance dosage and its variability. First, SNPs of vitamin K epoxide reductase complex subunit-1 (VKORC1) gene have been identified, affecting the enzyme shown as one of the target of VKA. Secondly, SNPs of cytochrome P450 2C9 (CYP2C9) gene have been shown to decrease the catabolism of coumarin derivatives (acenocoumarol, warfarin). Several recent studies have shown that being carrier of at least one mutated allele of either VKORC1 or CYP2C9 (CYP2C9*2, CYP2C9*3) allele is associated with a hypersensitivity to VKA therapy, i.e. a lower maintenance dose. Moreover, it has been associated with an increased risk of over-anticoagulation, a longer time to achieve the maintenance dose and an increased risk of bleeding. Finally, the combined analysis of VKORC1 and CYP2C9 SNPs with age may account for more than 50% of the individual variability of the warfarin maintenance dosage. Predicting models of warfarin maintenance dosage taking into account these individual parameters are currently developed.

HERITABILITY INDICATORS: Genetic studies of tobacco use can be useful to understand the physiopathology of nicotine dependence and potentially to prevent it. Twin and adoption studies have clearly shown the role of genetic factors in tobacco use at different stages. Genetic factors account for 55% (range: 11-84%) of the smoking initiation and 61% (range: 52-71%) for persistence. Age at onset and intensity of smoking are also influenced by genetic factors. Estimation of the heritability of initiation/persistence of smoking varies by gender. It is estimated as 66%/61% for women and 49%/61% for men respectively. In adolescent twin studies, heritability estimated the liability of lifetime or current use of tobacco to be more than 80%, while the heritability for initiation being between 11% and 59%. Heavy smoking is also influenced by genetic factors, especially when patients are co-abusing alcohol or coffee. Genetics findings - Advances in molecular genetics identified different candidate genes for tobacco use mainly involving neurotransmission of neuromodulators. Because of the brain reward effects of nicotine on the mesolimbic system, the genes involved in the dopaminergic transmission receive specific attention. Genetic polymorphisms of the dopamine D1, D2, D4 and D5 receptors, dopamine transporter (DAT1) and dopamine B-hydroxylase (DBH) have been associated at least once with clinical aspects of tobacco use (initiation, dependence and intensity) and temperament traits as novelty seeking, the latter being lower in smokers and thus considered as a vulnerable marker in accordance with the reinforcement effect of nicotine. Regarding interaction between nicotine use and anxiety and depression, the gene encoding for the serotonin transporter (5-HTT) may constitute a candidate gene. Because of interindividual bioavailability of nicotine, genetic polymorphisms of metabolism enzymes have also been analysed. Some variants of the cytochrome P450 seem to be more frequent among dependent smokers than controls or ever smokers (CYP2A6) and heavier smokers (CYP2D6). Genetic research might be suitable for a therapeutic approach and identify subjects at high risk for nicotine dependence.

Pharmacogenetics, a discipline still in its infancy, is the study of genetically determined variations in how individuals respond to drugs. Mutations may affect drug metabolism, transmembrane transport into cells, or target receptors. Genetic polymorphisms affecting drug metabolism were the first to be identified. Genetic factors control the activity of phase I reactions involving cytochrome (CYP) P450 isoenzymes. Three CYP families catalyze drug metabolism in humans. Their genes have been identified and polymorphisms have been described in various populations, leading to either high activity ("extensive metabolizer" phenotype) or low activity ("poor metabolizer" phenotype). The CYP2C9 polymorphism illustrates the potential clinical importance of pharmacogenetics. This enzyme catalyzes the metabolism of the coumarinic oral anticoagulants acenocoumarol and warfarin. The homozygous mutant genotype CYP 2C9 *31*3, present in 0, 7% of Caucasians, leads to low enzyme activity and thus to the accumulation of these drugs in the body; this in turn increases the anticoagulant activity and induces a higher risk of bleeding. In three clinical studies of patients and healthy volunteers, we found that this CYP2C9 *3 mutant allele was responsible for 14% of the variability in the response to these drugs. Then, by studying the genetic polymorphism of the receptor site of oral anticoagulants--the vitamin K epoxide reductase multiprotein complex--we showed that a combination of the two genetic variants (CYP2C9 and the receptor site) was responsible for 50% of the variability. These data suggest that patients who have both genetic polymorphisms could be at an increased risk of bleeding during oral anticoagulant therapy.

Allelic variants of the gene coding for cytochrome P450 isoform 2C9 (CYP2C9), 2C9*2 and 2C9*3, were shown to increase sensitivity to warfarin in adults. In the elderly, the maintenance dose is influenced by acquired factors including comorbidities and polymedication. The aim of our purpose was to investigate whether a genetic factor, such as cyp2c9 genotype, does influence the warfarin maintenance dose in very elderly patients.In-patients treated with warfarin were recruited with the following inclusion criteria: i/ 75 years-old or over; ii/ a stable INR within the therapeutic range (INR 2.0-3.0). Genotypes were coded as numbers of alleles for each of the three polymorphisms, namely 2C9*1 (wild-type), 2C9*2, and 2C9*3.CYP2C9 genotype was performed in 126 patients, mean age 87 +/-6 years (75-103), 29 males-97 females. The mean daily dose of warfarin was 3.0 +/-1.4 mg, with 3.1 mg in patients with the wild-type *1/*1 genotype (n =80), 2.7 mg in *1/*2 heterozygotes (n =20), 2.9 mg in *1/*3 heterozygotes (n =18), 1.2 mg in *2/*2 homozygotes (n =2), 2.3 mg in compound heterozygotes *2/*3 (n =6). The relationships between dose and potential factors were assessed using the correlation coefficient test for age and Fischer exact tests for the categorical variables. The only factors significantly linked to the dose were the numbers of 2C9*1 and 2C9*2 alleles.In elderly patients, a genetic influence on response to warfarin does exist as in younger patients.

Single nucleotide polymorphisms (SNPs) can significantly affect human phenotypes. Detection of allelic variant carriers has become a major goal for clinical pharmacologists in order to study phenotype-genotype relationships. However, there is a crucial need for rapid, and validated pharmacogenetic tests. The aim of the study was to validate a new fluorescence PCR strategy for cytochrome P450 2C9 (CYP2C9) and multidrug resistance gene (MDR1) genotyping. Results of CYP2C9 and MDR1 genotypes determined with reference techniques were compared to those obtained by allelic discrimination assays employing fluorescent TaqMan probes. Sixteen subjects carrying CYP2C9*2 and CYP2C9*3 allelic variants (heterozygous and homozygous) previously identified by sequencing and 55 subjects previously genotyped for MDR1 exon 26 (C3435T) SNP by conventional PCR-RFLP were genotyped with fluorescent PCR. Fluorescent PCR gave 100 % accuracy with the results obtained with reference genotyping strategies for each of the 3 SNPs. Genotyping results with fluorescent PCR repeated on three consecutive occasions remained constant over time for each of the 3 SNPs. Allelic discrimination assays based on fluorescent PCR gave entire satisfaction for CYP2C9 and MDR1 genotyping. This reliable genotyping strategy can be easily used in clinical practice and should be further developed for additional SNPs identification.

To determine the viability and differentiation of human hepatocytes immunoprotected by encapsulation and transplanted in rats without immunosuppression.Freshly isolated human hepatocytes were encapsulated in hollow fibers and transplanted in the peritoneal cavity of immunocompetent rats. The fibers were explanted for analysis at D3, D7 and D14 following transplantation. Morphological features under light and electron microscopy and gene expression were compared to those of non-transplanted encapsulated hepatocytes (D0). Human cytochrome P450 3A and albumin mRNAs were quantified by Northern blot. Cytochrome P450 3A proteins were detected by Western blot and cytochrome P450 3A enzyme activity was assessed by measuring the formation of 6beta-hydroxytestosterone by high performance liquid chromatography.Transplanted hepatocytes were more than 60 % viable and exhibited morphological criteria of hepatocytic differentiation up to D7. Albumin and cytochrome P450 3A transcripts were also detected up to D14. At D3 and D7, albumin mRNA levels were of 30 %, compared to control D0 hepatocytes, while cytochrome P450 3A5 and cytochrome P450 3A4 mRNA levels were 65 % and 0 %, respectively. Cytochrome P450 3A immunoreactivity was detected by Western blot up to D14 and 6beta-hydroxylase activity was 17 % at D3 compared to D0, supporting with disappearance of cytochrome P450 3A4 mRNA.Human hepatocytes remain viable for a short period, following encapsulation and intraperitoneal transplantation in rat. Other experimental conditions need to be tested to prevent or delay a decrease in hepatocyte specific gene expression.

Testosterone 16 alpha hydroxylase is a liver microsome-bound, dependent monooxygenase cytochrome P 450. In contrast with the rat and the mouse, 16 alpha hydroxylase activity appeared to be very low in the bovine liver and was very similar to the human values. By contrast, the level of cytochrome P 450 in the liver protein is very similar in all species. In the bovine species, the activity of the enzyme was 15.14 +/- 7.05 pmol/(min x mg of microsomal protein) and the level of cytochrome P 450 was 0.845 +/- 0.196 nmol/mg of microsomal protein.

Aryl hydrocarbon hydroxylase (AHH) activity and epoxyde hydrase (EH) activity have been found in Rat liver nucleoli obtained from untreated (C) and methylcholanthrene (MC) pretreated Rats. Electron microscopic observations of nucleolar preparations did not reveal significant contamination either by intact nuclei or by nuclear membranes. Very low but detectable activity of NADPH cytochrome C reductase was found in the nucleoli. Nucleolar preparations revealed little AHH activity (12-18 pmoles/min/mg). AHH was inducible by MC in nuclei but not in nucleoli. The presence of EH in nucleoli was demonstrated with phenanthrene 9,10-oxide (550-620 pmoles/min/mg) and benzopyrene 4,5-oxide (92-116 pmoles/min/mg). These values were lower than those obtained using intact nuclei. The addition of TCPO (10(-4) M) inhibited EH activity.

The effects of thyroid status upon cyt. P 450 concentration and ethoxycoumarin deethylase, benzopyrene hydroxylase and UDP-glucuronosyltransferase activities in the liver microsome fraction were far more important in partially hepatectomized rats than in control animals. The partial hepatectomy simultaneously lowered the MFO enzymes activities in the hepatic microsome fraction and made them more sensitive to thyroid hormones effects.

In vitro cytochrome P-450-dependent monooxygenase activity was studied in hepatic and duodenal microsomes from the Japanese quail (Coturnix coturnix) with two substrates, 7-ethoxycoumarin and coumarin. The rate of formation of 7-hydroxycoumarin--as the main metabolic product--was followed by fluorometry. 7-Ethoxycoumarin O-de-ethylase activity (7-ECOD) was shown in liver and duodenum, but coumarin 7-hydroxylase (Cou 7-OH) was found only in liver. The requirement for some cofactors (NADPH), the inhibitory action of CO, optimal pH and temperature, kinetic constants (Km, Vm, and the spectral dissociation constant, Ks) were determined in all cases. Specific activities were found in the range 1-4 nmole/mg/min, 0.1-1 nmole/mg/min, and 0.5-3 nmole/mg/min for hepatic and duodenal 7-ECOD and hepatic Cou 7-OH, respectively. The coumarin 7-hydroxylase activity showed some striking features, inhibition by excess of substrate and high inhibition by alcohols.

The trace elements are surrounding factors which are able to act on the yield of benzo(a)pyrene hydroxylation reaction of the hepatic tissue. This action was studied for each element and for various concentrations. The action of these elements may occurred to arylhydrocarbon hydroxylase by activating or inhibiting it, to epoxyde hydrase or to glutathione S epoxydase, favouring or inhibiting the way epoxyde dihydrodiol, and finally to arylhydrocarbon hydroxylase induction. Our results, obtained in vitro on hepatic tissue with many metallic salts, showed that some trace elements may have a cocarcinogenic action.

Nabam (fungicide dithiocarbamate) has been incorporated with the diet of rats during six months (the doses were: 0, 10, 50, 100, 500, 1000 and 2000 ppm). It decreases significantly the hepatic microsomal enzymes activity (aniline hydroxylase and aminopyrine N-demethylase and the liver P 450 content, but the cytochrome b 5 concentration doesn't seem to be modified. The microsomal lipidic and proteic content is only modified with the highest Nabam dose. Several hypotheses may be proposed to explain the Nabam effects: one is the inhibition of the monooxygenases and their biosynthesis repression.