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.     

Molecular genetics and epigenetics of the cytochrome P450 gene family and its relevance for cancer risk and treatment

The cytochromes P450 (CYPs) are very efficient catalysts of foreign compound metabolism and are responsible for the major part of metabolism of clinically important drugs. The enzymes are important in cancer since they (a) activate dietary and environmental components to ultimate carcinogens, (b) activate or inactivate drugs used for cancer treatment, and (c) are potential targets for anticancer therapy. The genes encoding the CYP enzymes active in drug metabolism are highly polymorphic, whereas those encoding metabolism of precarcinogens are relatively conserved. A vast amount of literature is present where investigators have tried to link genetic polymorphism in CYPs to cancer susceptibility, although not much conclusive data have hitherto been obtained, with exception of CYP2A6 polymorphism and tobacco induced cancer, to a great extent because of lack of important functional polymorphisms in the genes studied. With respect to anticancer treatment, the genetic CYP polymorphism is of greater importance, where treatment with tamoxifen, but also with cyclophosphamide and maybe thalidomide is influenced by CYP genetic variants. In the present review we present updates on CYP genetics, cancer risk and treatment and also epigenetic aspects of interindividual variability in CYP expression and the use of these enzymes as targets for cancer therapy. We conclude that the CYP polymorphism does not predict cancer susceptibility to any large extent but that this polymorphism might be an important factor for optimal cancer therapy using selected anticancer agents.     

Drugs impact on CYP-450 enzyme family: A pharmacogenetical study of response variation

Pharmacogenetics is the study of genetic basis in the individual response to drugs. A thorough knowledge of this will lead to a future where tailor-made drugs, suiting an individual, can be used. Scandinavian countries have been known for wide usage of pharmacogenetics and the most widely used application is for genotyping CYP2D6 in treating psychiatric illness. The CYP-450 enzyme, a super family of microsomal drug-metabolizing enzymes, is the most important of enzymes that catalyzes phase-I drug metabolism reaction. CYP2D6 is a member of this family and it has been most intensively studied and the best example of pharmacogenetics variation in drug metabolism. Neuro-transmitter and drug acting CNS viz. codeine, dextromethorphan, metoprolol and tryptyline etc. are well metabolized by this enzyme. Thus, CYP2D6 is one of the most important and responsible enzymes which regulates bioavailability and metabolism of drug. Presently 75 alleles of CYP2D6 have been described which are responsible for variance of metabolism and toxicity of drugs. Thus, by determining variance of CYP2D6 using molecular approaches viz., PCR, real-time PCR, DNA micro-array and molecular docking can determine the adverse effects, drug toxicity, bioavailability and therapeutic potential of new drug.     

Prediction of CYP2D6-mediated polymorphic drug metabolism (sparteine type) based on in vitro investigations

Discovery of genetic polymorphism in drug metabolism has contributed a great deal to understanding the variability in dose-concentration relationships introduced by genetic factors, thereby elucidating the mechanisms responsible for unexpected drug reactions. This knowledge should find its way into clinical practice in order to make therapy more efficient and safe. Moreover, genetic factors in drug metabolism should be taken into account during drug development. Therefore, in vitro methods for identifying the metabolic pattern of new compounds during early stages of drug development should be improved. This review summarizes in vitro methods available to identify genetic polymorphism in drug oxidation, in particular the CYP2D6-related polymorphism.     

Characterization of Phase I and Phase II hepatic drug metabolism activities in a panel of human liver preparations.

The role of drug metabolism in drug discovery (lead compound selection) and the traditional role of identifying the enzymes involved in biotransformation pathways (reaction phenotyping) have both relied heavily on the availability and use of a human liver bank. The assessment of drug metabolizing enzyme activity and variability in a series of individual human livers is essential when characterizing the enzymes involved in metabolic pathways (i.e. correlation analysis). In this regard, a human liver bank of 21 samples (14 males, six females, and one unknown) was characterized with respect to the activity of several important drug metabolizing enzymes. The total CYP450 content of the livers ranged from 0.06 to 0.46 nmol/mg microsomal protein. The fold variations found in specific enzyme contents were as follows: CYP1A2 (3x), CYP2A6 (21x), CYP2C9 (8x), CYP2C19 (175x), CYP2D6 (18x), CYP2E1 (5x), CYP3A4 (18x), FMO (2.5x), UDPGT (4x), NAT (7x), COMT (5x), ST (5x), TPMT (3x), and GST (2.5x). In general, the fold variation of the Phase II enzymes was lower compared with the Phase I enzymes, with the exceptions of CYP1A2, CYP2E1, and FMO. Similar data were reviewed from other established liver banks and compared with regard to the relative variability observed in drug metabolizing capacities found in this study.     

人细胞色素P450在大肠杆菌中的功能表达

研究人细胞色素P450（P450,CYP）在大肠杆菌中的功能表达对新药研发,临床药物治疗和药物早期ADME/T性质研究均有重要意义。异源表达人P450使用最多的宿主是大肠杆菌E.coli,然而要获得足量的有催化活性的P450仍是一个难题。结合作者近年研究,对异源表达的研究意义,P450在E.coli中功能表达的策略,高效表达的影响因素和共表达等方面做一评述,指出今后的研究应用方向。     

Human hepatic cell cultures: in vitro and in vivo drug metabolism

Drug metabolism is the major determinant of drug clearance, and the factor most frequently responsible for inter-individual differences in drug pharmacokinetics. The expression of drug metabolising enzymes shows significant interspecies differences, and variability among human individuals (polymorphic or inducible enzymes) makes the accurate prediction of the metabolism of a new compound in humans difficult. Several key issues need to be addressed at the early stages of drug development to improve drug candidate selection: a) how fast the compound will be metabolised; b) what metabolites will be formed (metabolic profile); c) which enzymes are involved and to what extent; and d) whether drug metabolism will be affected directly (drug-drug interactions) or indirectly (enzyme induction) by the administered compound. Drug metabolism studies are routinely performed in laboratory animals, but they are not sufficiently accurate to predict the metabolic profiles of drugs in humans. Many of these issues can now be addressed by the use of relevant human in vitro models, which speed up the selection of new candidate drugs. Human hepatocytes are the closest in vitro model to the human liver, and they are the only model which can produce a metabolic profile of a drug which is very similar to that found in vivo. However, the use of human hepatocytes is restricted, because limited access to suitable tissue samples prevents their use in high throughput screening systems. The pharmaceutical industry has made great efforts to develop fast and reliable in vitro models to overcome these drawbacks. Comparative studies on liver microsomes and cells from animal species, including humans, are very useful for demonstrating species differences in the metabolic profile of given drug candidates, and are of great value in the judicious and justifiable selection of animal species for later pharmacokinetic and toxicological studies. Cytochrome P450 (CYP)-engineered cells (or microsomes from CYP-engineered cells, for example, Supersomes) have made the identification of the CYPs involved in the metabolism of a drug candidate more straightforward and much easier. However, the screening of compounds acting as potential CYP inducers can only be conducted in cellular systems fully capable of transcribing and translating CYP genes.     

Vitamin D: a hormone for all seasons--how much is enough?

Further understanding of its endocrine mechanisms and increased evidence for autocrine/paracrine actions has recently enhanced our knowledge of the biological activities of the vitamin D metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D). The recognition of the contribution of vitamin D depletion to increased risk of osteoporosis, and most importantly the risk of hip fracture in the elderly, has increased the clinical significance of clinical laboratory testing for vitamin D status. Research has revealed that at least three genes contribute to vitamin D activity within tissues. These are the vitamin D receptor as well as two major vitamin D metabolising enzymes, CYP27B1, responsible for synthesis of 1,25(OH)2D and CYP24, responsible for catabolism of vitamin D metabolites. Current research focuses on the contribution of vitamin D metabolism to increasing vitamin D activity. This is of particular interest in bone forming cells where increased 1,25(OH)2D activity has been proposed to contribute to strengthening the skeleton. As well, solid tumours such as prostate, breast and colon cancers are another increasing area of vitamin D research. The major issues for the clinical laboratory in vitamin D testing include defining clinical decision limits for the interpretation of serum 25-hydroxyvitamin D (25OHD) levels and improving the precision and accuracy of this assay.     

Polymorphic Cytochrome P450 Enzymes (CYPs) and Their Role in Personalized Therapy

The cytochrome P450 (CYP) enzymes are major players in drug metabolism. More than 2,000 mutations have been described, and certain single nucleotide polymorphisms (SNPs) have been shown to have a large impact on CYP activity. Therefore, CYPs play an important role in inter-individual drug response and their genetic variability should be factored into personalized medicine. To identify the most relevant polymorphisms in human CYPs, a text mining approach was used. We investigated their frequencies in different ethnic groups, the number of drugs that are metabolized by each CYP, the impact of CYP SNPs, as well as CYP expression patterns in different tissues. The most important polymorphic CYPs were found to be 1A2, 2D6, 2C9 and 2C19. Thirty-four common allele variants in Caucasians led to altered enzyme activity. To compare the relevant Caucasian SNPs with those of other ethnicities a search in 1,000 individual genomes was undertaken. We found 199 non-synonymous SNPs with frequencies over one percent in the 1,000 genomes, many of them not described so far. With knowledge of frequent mutations and their impact on CYP activities, it may be possible to predict patient response to certain drugs, as well as adverse side effects. With improved availability of genotyping, our data may provide a resource for an understanding of the effects of specific SNPs in CYPs, enabling the selection of a more personalized treatment regimen.     

Reduction of toxic metabolite formation of acetaminophen

Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation.     

Targeting cytochrome P450 enzymes: a new approach in anti-cancer drug development

Cytochrome P450s (CYPs) represent a large class of heme-containing enzymes that catalyze the metabolism of multitudes of substrates both endogenous and exogenous. Until recently, however, CYPs have been largely overlooked in cancer drug development, acknowledged only for their role in phase I metabolism of chemotherapeutics. The first successful strategy targeting CYP enzymes in cancer therapy was the development of potent inhibitors of CYP19 (aromatase) for the treatment of breast cancer. Aromatase inhibitors ushered in a new era in hormone ablation therapy for estrogen dependent cancers, and have paved the way for similar strategies (i.e., inhibition of CYP17) that combat androgen dependent prostate cancer. Identification of CYPs involved in the inactivation of anti-cancer metabolites of vitamin D(3) and vitamin A has triggered development of agents that target these enzymes as well. The discovery of the over-expression of exogenous metabolizing CYPs, such as CYP1B1, in cancer cells has roused interest in the development of inhibitors for chemoprevention and of prodrugs designed to be activated by CYPs only in cancer cells. Finally, the expression of CYPs within tumors has been utilized in the development of bioreductive molecules that are activated by CYPs only under hypoxic conditions. This review offers the first comprehensive analysis of strategies in drug development that either inhibit or exploit CYP enzymes for the treatment of cancer.     

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome P450 induction studies

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.     

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.     

Efflux transporters- and cytochrome P-450-mediated interactions between drugs of abuse and antiretrovirals

Multidrug regimens and corresponding drug interactions cause many adverse reactions and treatment failures. Drug efflux transporters: P-gp, MRP, BCRP in conjunction with metabolizing enzymes (CYPs) are major factors in such interactions. Most effective combination antiretrovirals (ARV) therapy includes a PI or a NNRTI or two NRTI. Coadministration of such ARV may induce efflux transporters and/or CYP3A4 resulting in sub-therapeutic blood levels and therapeutic failure due to reduced absorption and/or increased metabolism. A similar prognosis is true for ARV-compounds and drugs of abuse combinations. Morphine and nicotine enhance CYP3A4 and MDR1 expression in vitro. A 2.5 fold rise of cortisol metabolite was evident in smokers relative to nonsmokers. Altered functions of efflux transporters and CYPs in response to ARV and drugs of abuse may result in altered drug absorption and metabolism. Appropriate in vitro models can be employed to predict such interactions. Influence of genetic polymorphism, SNP and inter-individual variation in drug response has been discussed. Complexity underlying the relationship between efflux transporters and CYP makes it difficult to predict the outcome of HAART as such, particularly when HIV patients taking drugs of abuse do not adhere to HAART regimens. HIV(+) pregnant women on HAART medications, indulging in drugs of abuse, may develop higher viral load due to such interactions and lead to increase in mother to child transmission of HIV. A multidisciplinary approach with clear understanding of mechanism of interactions may allow proper selection of regimens so that desired therapeutic outcome of HAART can be reached without any side effects.     

Drug Cocktail Optimization in Chemotherapy of Cancer

Background

In general, drug metabolism has to be considered to avoid adverse effects and ineffective therapy. In particular, chemotherapeutic drug cocktails strain drug metabolizing enzymes especially the cytochrome P450 family (CYP). Furthermore, a number of important chemotherapeutic drugs such as cyclophosphamide, ifosfamide, tamoxifen or procarbazine are administered as prodrugs and have to be activated by CYP. Therefore, the genetic variability of these enzymes should be taken into account to design appropriate therapeutic regimens to avoid inadequate drug administration, toxicity and inefficiency.

Objective

The aim of this work was to find drug interactions and to avoid side effects or ineffective therapy in chemotherapy.

Data sources and methods

Information on drug administration in the therapy of leukemia and their drug metabolism was collected from scientific literature and various web resources. We carried out an automated textmining approach. Abstracts of PubMed were filtered for relevant articles using specific keywords. Abstracts were automatically screened for antineoplastic drugs and their synonyms in combination with a set of human CYPs in title or abstract.

Results

We present a comprehensive analysis of over 100 common cancer treatment regimens regarding drug-drug interactions and present alternatives avoiding CYP overload. Typical concomitant medication, e.g. antiemetics or antibiotics is a preferred subject to improvement. A webtool, which allows drug cocktail optimization was developed and is publicly available on http://bioinformatics.charite.de/chemotherapy.     

Oxidation of human cytochrome P450 1A2 substrates by Bacillus megaterium cytochrome P450 BM3

Cytochrome P450 enzymes (P450s or CYPs) are good candidates for biocatalysis in the production of fine chemicals, including pharmaceuticals. Despite the potential use of mammalian P450s in various fields of biotechnology, these enzymes are not suitable as biocatalysts due to their low stability, low catalytic activity, and limited availability. Recently, wild-type and mutant forms of bacterial P450 BM3 (CYP102A1) from Bacillus megaterium have been found to metabolize various. It has therefore been suggested that CYP102A1 may be used to generate the metabolites of drugs and drug candidates. In this report, we show that the oxidation reactions of typical human CYP1A2 substrates (phenacetin, ethoxyresorufin, and methoxyresorufin) are catalyzed by both wild-type and mutant forms of CYP102A1. In the case of phenacetin, CYP102A1 enzymes show only O-deethylation product, even though two major products are produced as a result of O-deethylation and 3-hydroxylation reactions by human CYP1A2. Formation of the metabolites was confirmed by HPLC analysis and LC–MS to compare the metabolites with the actual biological metabolites produced by human CYP1A2. The results demonstrate that CYP102A1 mutants can be used for cost-effective and scalable production of human CYP1A2 drug metabolites. Our computational findings suggest that a conformational change in the cavity size of the active sites of the mutants is dependent on activity change. The modeling results further suggest that the activity change results from the movement of several specific residues in the active sites of the mutants.     

Cytochrome P4502C9 genotype in Southeast Anatolia and possible relation with some serum tumour markers and cytokines

Substrates for CYP2C9 include fluoxetine, phenytoin, warfarin, losartam and numerous nonsteroidal anti-inflammatory drugs. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino-acid residues 144 Arg/Cys and 359 Ile/Leu of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, the frequency of this allele is, however, rather low. Consistently with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. The polymorphic enzyme CYP2C9 takes part in the metabolism of alkylating agents and polycyclic aromatic hydrocarbons like benzo(a)pyrene, a carcinogen present in tobacco smoke. Although the impact of impaired enzyme activity in metabolism of carcinogens and procarcinogens has not been fully defined, an association of CYP2C9 variant alleles to DNA adduct levels in lung tissues as well as to lung cancer risk have been reported. In this study 64 healthy subjects (44M/22F) were analysed for CYP2C9 genotype with PCR-RFLP and for serum carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA 19-9, CA 15-3, ferritin, IL-6, IL-8 concentrations by chemiluminescence or electrochemiluminescence methods. CYP2C9*1 was found to be the most prevalent allele and CYP2C9*1/CYP2C9*1 was the most frequent genotype represented in 64% of the population in southeastern Anatolia (Gaziantep). Although slight differences in serum tumour marker and cytokine concentrations were observed for CYP2C9 genotypes the differences were statistically insignificant (P > 0.05). This could be due to the complexity of the role of CYP2C9 in benzo(a)pyrene metabolism as well as from other contributing factors like interindividual variability of diverse enzymes participating in the same metabolic pathway, unequal expression of the variant alleles and differences in exposure to carcinogens. However, determination of CYP2C9 phenotypes in a larger group of subjects might clarify these slight differences.     

Advances in Molecular Modeling of Human Cytochrome P450 Polymorphism

Cytochrome P450 (CYP) is a supergene family of metabolizing enzymes involved in the phase I metabolism of drugs and endogenous compounds. CYP oxidation often leads to inactive drug metabolites or to highly toxic or carcinogenic metabolites involved in adverse drug reactions (ADR). During the last decade, the impact of CYP polymorphism in various drug responses and ADR has been demonstrated. Of the drugs involved in ADR, 56% are metabolized by polymorphic phase I metabolizing enzymes, 86% among them being CYP. Here, we review the major CYP polymorphic forms, their impact for drug response and current advances in molecular modeling of CYP polymorphism. We focus on recent studies exploring CYP polymorphism performed by the use of sequence-based and/or protein-structure-based computational approaches. The importance of understanding the molecular mechanisms related to CYP polymorphism and drug response at the atomic level is outlined.     

Contributions of CYP2C9/CYP2C19 genotypes and drug interaction to the phenytoin treatment in the Korean epileptic patients in the clinical setting

We examined the contribution of CYP2C9 and CYP2C19 genotypes and drug interactions to the phenytoin metabolism among 97 Korean epileptic patients to determine if pharmacogenetic testing could be utilized in routine clinical practice. The CYP2C9 polymorphism is a wellknown major genetic factor responsible for phenytoin metabolism. The CYP219 polymorphism, with a high incidence of variant alleles, has a minor influence on phenytoin treated Koran patients. Using a multiple regression model for evaluation of the CYP2C9 and CYP2C19 genotypes, together with other non-genetic variables, we explained 39.6% of the variance in serum phenytoin levels. Incorporation of genotyping for CYP2C9 and CYP2C19 into a clinical practice may be of some help in the determination of phenytoin dosage. However, because concurrent drug treatment is common in patients taking phenytoin and many environmental factors are likely to play a role in drug metabolism, these factors may overwhelm the relevance of CYP polymorphisms in the clinical setting. Further investigations with an approach to dose assessment that includes comprehensive interpretation of both pharmacogenetic and pharmacokinetic data along with understanding of the mechanism of drug interactions in dosage adjustment is warranted.