Drug metabolizing enzyme activities versus genetic variances for drug of clinical pharmacogenomic relevance

Enzymes are critically important in the transportation, metabolism, and clearance of most therapeutic drugs used in clinical practice today. Many of these enzymes have significant genetic polymorphisms that affect the enzyme's rate kinetics. Regarding drug metabolism, specific polymorphisms to the cytochrome (CYP) P450 enzyme family are linked to phenotypes that describe reaction rates as "ultra", "intermediate", and "poor," as referenced to "extensive" metabolizers that are assigned to wildtype individuals. Activity scores is an alternate designation that provides more genotype-to-phenotype resolution. Understanding the relative change in enzyme activities or rate of clearance of specific drugs relative to an individual's genotypes is an important component in the interpretation of pharmacogenomic data for personalized medicine. Currently, the most relevant drug metabolizing enzymes are CYP 2D6, CYP 2C9, CYP 2C19, thiopurine methyltransferase (TPMT) and UDP-glucuronosyltransferase (UGT). Each of these enzymes is reactive to a host of different drug substrates. Pharmacogenomic tests that are in routine clinical practice include CYP 2C19 for clopidogrel, TPMT for thiopurine drugs, and UDP-1A1 for irinotecan. Other tests where there is considerable data but have not been widely implemented includes CYP 2C9 for warfarin, CYP 2D6 for tamoxifen and codeine, and CYP 2C19 for the proton pump inhibitors.     

细胞色素P450基因多态性与药物代谢研究进展

细胞色素P450（cytochrome P450,CYP450）在人体药物代谢过程中起着非常重要的作用并参与代谢80%以上的临床药物。由于CYP450在不同种族和不同人群中存在基因多态性,从而造成药物反应的个体差异,一度成为药物基因组学研究的热点。通过查阅国外相关文献,综述了近年来关于CYP1A2、CYP2C9、CYP2C19、CYP2D6和CYP3A4五种主要的药物代谢酶的基因多态性和药物代谢的研究进展,为临床指导个体化用药、避免药物不良反应和新药研发提供科学参考依据。     

An evaluation of the P450 inhibition and induction potential of daptomycin in primary human hepatocytes

Two in vitro studies assessed the potential of daptomycin (Cubicin), a newly marketed antibiotic, to affect the cytochrome P450 (CYP450) isoforms in primary cultured human hepatocytes. Both induction and inhibition of isoforms 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 were evaluated. The highest concentrations of daptomycin used in both the induction and inhibition assays were approximately eight-fold higher than the peak total drug concentration (50-60 microg/mL), or the peak free drug concentration (estimated 5-6 microg/mL), in plasma at the clinical dose regimen of 4 mg/kg qd. Results in primary human hepatocytes indicate that daptomycin, at concentrations up to 400 microg total drug/mL, demonstrated no biologically significant induction of any of the CYP450 isoform activities in comparison with the negative control or known inducers. At daptomycin concentrations up to 40 microg free drug/mL, no biologically significant inhibition of the activities of these CYP450 isoforms was observed as compared with known inhibitors. The human hepatocyte results demonstrate that daptomycin has no effects on hepatic CYP450-mediated drug metabolism and, therefore, suggest that daptomycin is unlikely to show potential for pharmacokinetic interactions with concomitantly administered drugs that are metabolized by CYP450 isoforms.     

Probing the world of cytochrome P450 enzymes

Variability in drug response can be attributed in part to variability in the activity of drug-metabolizing enzymes. One of the most important drugmetabolizing enzyme systems in humans is the cytochrome P450 (CYP) enzyme family, which is responsible for the oxidative metabolism of numerous endogenous compounds and xenobiotics. The clinical relevance of factors that influence CYP-mediated metabolism can be appreciated by estimating in vivo enzyme activity (i.e., the phenotype) through the use of "probe drugs," which are drugs predominately or exclusively metabolized by an individual CYP enzyme. Thus, the use of probe drugs alone or in combination (i.e., the cocktail approach) can provide an invaluable tool to explore the clinical relevance of genetic and nongenetic factors that affect CYP enzyme activity and thereby contribute substantially to variability in response to therapeutic drugs.     

Metabolism, Pharmacogenetics, and Metabolic Drug–Drug Interactions of Antipsychotic Drugs

1. Antipsychotic drugs are extensively metabolised by cytochrome P450 (CYP) enzymes.2. Dispositions of a number of antipsychotic drugs have been shown to cosegregate with polymorphism of CYP2D6.3. Metabolic drug–drug interactions have frequently been observed when antipsychotics are coadministered with other drugs.4. Many antipsychotic drugs are converted to active metabolites which can contribute to the therapeutic or side effects of the parent drug.5. Information concerning the individual CYP isoenzymes involved in the metabolism of antipsychotic drugs is important for the safe clinical use of this group of drugs.     

Evaluation of the activity of CYP2C19 in Gujrati and Marwadi subjects living in Mumbai (Bombay)

Background  

Inherited differences in the metabolism and disposition of drugs, and genetic polymorphisms in the targets of drug therapy (e.g., receptors), can greatly influence efficacy and toxicity of medications. Marked interethnic differences in CYP2C19 (a member of the cytochrome P-450 enzyme superfamily catalyzing phase I drug metabolism) which affects the metabolism of a number of clinically important drugs have been documented. The present study evaluated the activity of CYP2C19 in normal, healthy Gujrati and Marwadi subjects by phenotyping (a western Indian population).     

The influence of cytochrome P450 pharmacogenetics on disposition of common antidepressant and antipsychotic medications

Since the identification of all the major drug-metabolising cytochrome P450 (CYP) enzymes and their major gene variants, pharmacogenetics has had a major impact on psychotherapeutic drug therapy. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is an important reason for drug therapy failure. Variability in CYP activity may be caused by various factors, including endogenous factors such as age, gender and morbidity as well as exogenous factors such as co-medication, food components and smoking habit. However, polymorphisms, present in most CYP genes, are responsible for a substantial part of this variability. Although CYP genotyping has been shown to predict the majority of aberrant phenotypes, it is currently rarely performed in clinical practice.     

Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans

CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.     

The AmpliChip CYP450 genotyping test: Integrating a new clinical tool

The AmpliChip CYP450 Test, which analyzes patient genotypes for cytochrome P450 (CYP) genes CYP2D6 and CYP2C19, is a major step toward introducing personalized prescribing into the clinical environment. Interest in adverse drug reactions (ADRs), the genetic revolution, and pharmacogenetics have converged with the introduction of this tool, which is anticipated to be the first of a new wave of such tools to follow over the next 5-10 years. The AmpliChip CYP450 Test is based on microarray technology, which combines hybridization in precise locations on a glass microarray and a fluorescent labeling system. It classifies individuals into two CYP2C19 phenotypes (extensive metabolizers [EMs] and poor metabolizers [PMs]) by testing three alleles, and into four CYP2D6 phenotypes (ultrarapid metabolizers [UMs], EMs, intermediate metabolizers [IMs], and PMs) by testing 27 alleles, including seven duplications. CYP2D6 is a metabolic enzyme with four activity levels (or phenotypes): UMs with unusually high activity; normal subjects, known as EMs; IMs with low activity; and PMs with no CYP2D6 activity (7% of Caucasians and 1-3% in other ethnic groups). Levels of evidence for the association between CYP2D6 PMs and ADRs are relatively reasonable and include systematic reviews of case-control studies of some typical antipsychotics and tricyclic antidepressants (TCAs). Evidence for other phenotypes is considerably more limited. The CYP2D6 PM phenotype may be associated with risperidone ADRs and discontinuation due to ADRs. Venlafaxine, aripiprazole, duloxetine, and atomoxetine are newer drugs metabolized by CYP2D6 but studies of the clinical relevance of CYP2D6 genotypes are needed. Non-psychiatric drugs metabolized by CYP2D6 include metoprolol, tamoxifen, and codeine-like drugs. CYP2C19 PMs (3-4% of Caucasians and African Americans, and 14-21% of Asians) may require dose adjustment for some TCAs, moclobemide, and citalopram. Other drugs metabolized by CYP2C19 are diazepam and omeprazole. The future of pharmacogenetics depends on the ability to overcome serious obstacles, including the difficulties of conducting and publishing studies in light of resistance from grant agencies, pharmaceutical companies, and some scientific reviewers. Assuming more studies are published, pharmacogenetic clinical applications may be compromised by economic factors and the lack of physician education. The combination of a US FDA-approved test, such as the AmpliChip CYP450 Test, and an FDA definition of CYP2D6 as a 'valid biomarker' makes CYP2D6 genotyping a prime candidate to be the first successful pharmacogenetic test in the clinical environment. One can use microarray technology to test for hundreds of single nucleotide polymorphisms (SNPs) but, taking into account the difficulties for single gene approaches such as CYP2D6, it is unlikely that very complex pharmacogenetic approaches will reach the clinical market in the next 5-10 years.     

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.     

Phyllanthus urinaria extract attenuates acetaminophen induced hepatotoxicity: Involvement of cytochrome P450 CYP2E1

Acetaminophen is a commonly used drug for the treatment of patients with common cold and influenza. However, an overdose of acetaminophen may be fatal. In this study we investigated whether mice, administered intraperitoneally with a lethal dose of acetaminophen, when followed by oral administration of Phyllanthus urinaria extract, may be prevented from death. Histopathological analysis of mouse liver sections showed that Phyllanthus urinaria extract may protect the hepatocytes from acetaminophen-induced necrosis. Therapeutic dose of Phyllanthus urinaria extract did not show any toxicological phenomenon on mice. Immunohistochemical staining with the cytochrome P450 CYP2E1 antibody revealed that Phyllanthus urinaria extract reduced the cytochrome P450 CYP2E1 protein level in mice pre-treated with a lethal dose of acetaminophen. Phyllanthus urinaria extract also inhibited the cytochrome P450 CYP2E1 enzymatic activity in vitro. Heavy metals, including arsenic, cadmium, mercury and lead, as well as herbicide residues were not found above their detection limits. High performance liquid chromatography identified corilagin and gallic acid as the major components of the Phyllanthus urinaria extract. We conclude that Phyllanthus urinaria extract is effective in attenuating the acetaminophen induced hepatotoxicity, and inhibition of cytochrome P450 CYP2E1 enzyme may be an important factor for its therapeutic mechanism.     

Cytochrome allelic variants and clopidogrel metabolism in cardiovascular diseases therapy

Clopidogrel and aspirin are among the most prescribed dual antiplatelet therapies to treat the acute coronary syndrome and heart attacks. However, their potential clinical impacts are a subject of intense debates. The therapeutic efficiency of clopidogrel is controlled by the actions of hepatic cytochrome P450 (CYPs) enzymes and impacted by individual genetic variations. Inter-individual polymorphisms in CYPs enzymes affect the metabolism of clopidogrel into its active metabolites and, therefore, modify its turnover and clinical outcome. So far, clinical trials fail to confirm higher or lower adverse cardiovascular effects in patients treated with combinations of clopidogrel and proton pump inhibitors, compared with clopidogrel alone. Such inconclusive findings may be due to genetic variations in the cytochromes CYP2C19 and CYP3A4/5. To investigate potential interactions/effects of these cytochromes and their allele variants on the treatment of acute coronary syndrome with clopidogrel alone or in combination with proton pump inhibitors, we analyze recent literature and discuss the potential impact of the cytochrome allelic variants on cardiovascular events and stent thrombosis treated with clopidogrel. The diversity of CYP2C19 polymorphisms and prevalence span within various ethnic groups, subpopulations and demographic areas are also debated.     

Polymorphisms and phenotypic analysis of cytochrome P450 2D6 in the Tibetan population

The cytochrome P450 2D6 enzyme (CYP2D6) metabolizes about 25% of prescribed drugs in the endoplasmic reticulum, and genetic polymorphisms in CYP2D6 can greatly affect its activity and lead to differences among individuals in drug efficacy and adverse drug reactions. To investigate genetic polymorphisms in CYP2D6 among Tibetan Chinese, we directly sequenced the whole gene in 96 unrelated, healthy Tibetans from The Tibet Autonomous Region of China and screened for genetic variants in the promoter, exons, introns, and 3′UTR. We detected fifty-one genetic polymorphisms in CYP2D6, and 16 of them are novel. The allele frequencies of CYP2D6*1, *2, *5, *10, *41, and *49 were 0.25, 0.43, 0.02, 0.29, 0.02, and 0.01, respectively. The frequency of CYP2D6*10, a putative poor-metabolizer allele, was lower in our sample population compared with that in the Han Chinese population (p < 0.001). In addition, haplotype analysis allowed 15 CYP2D6 haplotypes to be classified into three groups. In conclusion, our results provide basic information about CPY2D6 alleles in Tibetans and suggest that the enzymatic activities of CYP2D6 may differ among the diverse ethnic populations of China. Our results provide a basis for safer drug administration and better therapeutic treatment among Tibetans.     

人类细胞色素P450与药物氧化代谢遗传多态性分子机制的研究现状

近年,在表型及基因型上均发现存在药物氧化代谢多态性,特别是对于人类细胞色素Ｐ４５０氧化酶与药氧化代谢遗传多态性的关系进行了深入的研究。有关ＣＹＰ２Ｄ６、ＣＹＰ２Ｃ１９等的突变已大多被鉴定;ＣＹＰ１Ａ１、ＣＹＰ１Ａ２等在表型存在多态性而确切的遗传机制尚不清楚。     

Mutation probability of cytochrome P450 based on a genetic algorithm and support vector machine

The support vector machine (SVM), an effective statistical learning method, has been widely used in mutation prediction. Two factors, i.e., feature selection and parameter setting, have shown great influence on the efficiency and accuracy of SVM classification. In this study, according to the principles of a genetic algorithm (GA) and SVM, we developed a GA-SVM program and applied it to human cytochrome P450s (CYP450s), which are important monooxygenases in phase I drug metabolism. The program optimizes features and parameters simultaneously, and hence fewer features are used and the overall prediction accuracy is improved. We focus on the mutation of non-synonymous single nucleotide polymorphisms (nsSNPs) in protein sequences that appear to exhibit significant influences on drug metabolism. The final predictive model has a quite satisfactory performance, with the prediction accuracy of 61% and cross-validation accuracy of 73%. The results indicate that the GA-SVM program is a powerful tool in optimizing mutation predictive models of nsSNPs of human CYP450s.     

CYP2A6 Polymorphisms Associate with Outcome of S-1 Plus Oxaliplatin Chemotherapy in Chinese Gastric Cancer Patients

Gastric carcinoma is a heterogeneous malignant disease involving genetic factors. To identify predictive markers for gastric cancer treatment in Chinese patients, we evaluated the association between polymorphisms of the gene encoding cytochrome P450 2A6(CYP2A6) and outcomes of S-1 plus oxaliplatin(SOX) chemotherapy treatment. Clinical data on 60 consecutive gastric cancer patients receiving SOX regimen were collected prospectively. We sequenced all exons of CYP2A6 and a total of 22 different polymorphisms were detected in the present study. Comprehensive analyses of these genetic polymorphisms were performed to determine their association with both safety and efficacy of SOX regimen. Our results showed that polymorphisms of CYP2A6 were associated with the safety and efficacy of SOX treatment. Among them, missense mutations CYP2A6 rs60823196 and rs138978736 could be possible risk factors(P 0.05) for severe diarrhea induced by SOX, whereas CYP2A6 rs138978736 could be a conceivable predictor for overall survival of patients treated with SOX adjuvant chemotherapy. Further large-scale randomized prospective studies are warranted to confirm these findings.     

The role of polymorphic cytochrome P450 enzymes in drug design,development and drug interactions with a special emphasis on phenotyping

Inhibitors of some cytochrome P450s (CYPs) are used to design target-specific drugs. CYPs belonging to families 1–4 play important roles in drug metabolism and therapeutics. Some isozymes of CYPs also activate pro-carcinogens into their carcinogenic forms. Approximately 40–50% human CYP-dependent drug metabolism is carried out by polymorphic CYPs resulting in therapeutic failure and adverse reactions. Phenotyping of CYPs involved in the metabolism of a drug is important to understand the potential of clinical interactions and to predict the possible individual variations due to genetic polymorphisms of certain CYPs.     

Quantitation of cytochrome P450 mRNA levels in human skin

There is considerable interindividual variation in man's ability to metabolize drugs and foreign compounds. These differences can partly be attributed to genetic polymorphisms that result in the generation of multiple phenotypes with different drug-metabolizing capabilities. Genetically derived differences can easily be assessed by genotyping assays in cases where the polymorphism has been identified. However, many of the polymorphisms that result in these are not known, secondly not all the differences can be attributed to genetic polymorphisms, hence genotyping methods cannot be employed. We have therefore, developed real-time (Taqman) PCR assays to quantitate levels of P450 mRNAs in human tissues. These assays are highly sensitive, reproducible, and specific and will allow quantitation of P450 mRNA levels in various human tissues. We have applied these assays to quantitate cytochrome P450 mRNA levels in human skin samples from 27 healthy volunteers. The expression of 13 P450s was assessed. The major enzymes detected were CYP1B1, CYP2B6, CYP2D6, and CYP3A4 with mean values of 2.5, 2.6, 2.7, and 1.1 fg/18S rRNA in 50ng total RNA, respectively. Lower levels of CYP2C18, CYP2C19, and CYP3A5 were also detected while CYP1A2, 2A6, and 2C8 were below limits of detection. There was interindividual variation in the levels of mRNA among the 27 subjects studied although Poisson analysis showed data to be normally distributed, except for CYP2B6, as some individuals completely lacked CYP2B6 mRNA.     

CYP2D6 polymorphisms in patients with porphyrias

The cytochrome P-450 (CYP) isoenzymes, a superfamily of heme proteins which are the terminal oxidases of the mixed function oxidases system, metabolize more than 70% of all clinically approved drugs. The highly polymorphic CYP2D6 isoform metabolizes more than 25% of most common drugs, and the phenotypes of the 70-plus allelic variants range from compromised to excessive enzymatic activity. Porphyrias are a group of inherited or acquired metabolic disorders of heme biosynthesis, due to a specific decrease in the activity of one of the enzymes of the heme pathway. Clinical signs and symptoms of porphyrias are frequently associated with exposure to precipitating agents, including clinically approved drugs. CYP enzymes, including CYP2D6, participate in the metabolism of some porphyrinogenic drugs, leading to the deregulation of heme biosynthesis. Considering that some of the drugs not recommended for use in porphyric patients are metabolized by CYP2D6, the presence of CYP2D6 polymorphisms in porphyric patients would influence the triggering of the disease when these individuals receive a precipitating agent that is metabolized by CYP2D6. To investigate CYP2D6 polymorphisms in porphyric patients, healthy Argentinean volunteers, porphyric patients, and a group of individuals with high levels of iron were studied. Results indicated that the CYP2D6*3 and CYP2D6*4 alleles, in particular, would be linked to the onset of disease. Predictive genotyping for CYP2D6 in porphyric patients holds promise as a method to improve the clinical efficacy of drug therapy and to personalize drug administration for these patients.