Substrate specificity, regulation, and polymorphism of human cytochrome P450 2B6

CYP2B6 is mainly expressed in the liver that has been thought historically to play an insignificant role in human drug metabolism. However, increased interest in this enzyme has been stimulated by the discovery of polymorphic and ethnic differences in CYP2B6 expression, identification of additional substrates for CYP2B6, and evidence for co-regulation with CYP3A4. This paper updates our knowledge about the structure, function, regulation and polymorphism of CYP2B6. CYP2B6 can metabolise approximately 8% of clinically used drugs (n > 60), including cyclophosphamide, ifosfamide, tamoxifen, ketamine, artemisinin, nevirapine, efavirenz, bupropion, sibutramine, and propofol. CYP2B6 is one of the CYP enzymes that bioactivate several procarcinogens and toxicants. This enzyme also metabolizes arachidonic acid, lauric acid, 17beta-estradiol, estrone, ethinylestradiol, and testosterone. Typical substrates of CYP2B6 are non-planar molecules, neutral or weakly basic, highly lipophilic with one or two hydrogen-bond acceptors. The crystal structure of CYP2B6 has not been resolved, while several pharmacophore and homology models of human CYP2B6 have been reported. Human CYP2B6 is closely regulated by constitutive androstane receptor (CAR/NR1I3) which can activate CYP2B6 expression upon ligand binding. Pregnane X receptor and glucocorticoid receptor also play a role in the regulation of CYP2B6. Induction of CYP2B6 may partially explain some clinical drug interactions observed. For example, coadministered carbamazepine decreases the systemic exposure of bupropion. There is a wide interindividual variability in the expression and activity of CYP2B6. Such a large variability is probably due to effects of genetic polymorphisms and exposure to drugs that are inducers or inhibitors of CYP2B6. To date, at least 28 allelic variants and some subvariants of CYP2B6 (*1B through *29) have been described and some of them have been shown to have important functional impact on drug clearance and drug response. For example, the efavirenz plasma levels in African-American subjects with the CYP2B6 homozygous 516T/T genotype are approximately 3-fold higher than individuals carrying the homozygous G/G genotype. The CYP2B6 516T/T genotype is associated with 1.7-fold greater plasma levels of nevirapine in HIV-infected patients. Smokers with the 1459C>T (R487C) variant of CYP2B6 may be more vulnerable to abstinence symptoms and relapse following treatment with bupropion as a smoking cessation agent. Further studies in the structure, function, regulation and polymorphism of CYP2B6 are warranted.

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study

1. The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR.

2. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells.

3. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401.

4. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb–drug interactions.

Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs

Consistent with its highest abundance in humans, cytochrome P450 (CYP) 3A is responsible for the metabolism of about 60% of currently known drugs. However, this unusual low substrate specificity also makes CYP3A4 susceptible to reversible or irreversible inhibition by a variety of drugs. Mechanism-based inhibition of CYP3A4 is characterised by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-, time- and concentration-dependent enzyme inactivation, occurring when some drugs are converted by CYP isoenzymes to reactive metabolites capable of irreversibly binding covalently to CYP3A4. Approaches using in vitro, in silico and in vivo models can be used to study CYP3A4 inactivation by drugs. Human liver microsomes are always used to estimate inactivation kinetic parameters including the concentration required for half-maximal inactivation (K(I)) and the maximal rate of inactivation at saturation (k(inact)).Clinically important mechanism-based CYP3A4 inhibitors include antibacterials (e.g. clarithromycin, erythromycin and isoniazid), anticancer agents (e.g. tamoxifen and irinotecan), anti-HIV agents (e.g. ritonavir and delavirdine), antihypertensives (e.g. dihydralazine, verapamil and diltiazem), sex steroids and their receptor modulators (e.g. gestodene and raloxifene), and several herbal constituents (e.g. bergamottin and glabridin). Drugs inactivating CYP3A4 often possess several common moieties such as a tertiary amine function, furan ring, and acetylene function. It appears that the chemical properties of a drug critical to CYP3A4 inactivation include formation of reactive metabolites by CYP isoenzymes, preponderance of CYP inducers and P-glycoprotein (P-gp) substrate, and occurrence of clinically significant pharmacokinetic interactions with coadministered drugs.Compared with reversible inhibition of CYP3A4, mechanism-based inhibition of CYP3A4 more frequently cause pharmacokinetic-pharmacodynamic drug-drug interactions, as the inactivated CYP3A4 has to be replaced by newly synthesised CYP3A4 protein. The resultant drug interactions may lead to adverse drug effects, including some fatal events. For example, when aforementioned CYP3A4 inhibitors are coadministered with terfenadine, cisapride or astemizole (all CYP3A4 substrates), torsades de pointes (a life-threatening ventricular arrhythmia associated with QT prolongation) may occur.However, predicting drug-drug interactions involving CYP3A4 inactivation is difficult, since the clinical outcomes depend on a number of factors that are associated with drugs and patients. The apparent pharmacokinetic effect of a mechanism-based inhibitor of CYP3A4 would be a function of its K(I), k(inact) and partition ratio and the zero-order synthesis rate of new or replacement enzyme. The inactivators for CYP3A4 can be inducers and P-gp substrates/inhibitors, confounding in vitro-in vivo extrapolation. The clinical significance of CYP3A inhibition for drug safety and efficacy warrants closer understanding of the mechanisms for each inhibitor. Furthermore, such inactivation may be exploited for therapeutic gain in certain circumstances.

Genetic polymorphisms of cytochrome P450 2B6 gene in Han Chinese

Cytochrome P450 (CYP2B6) is an important enzyme that metabolizes more than eight compounds and about 3.0% of therapeutic drugs. The genetic polymorphisms of CYP2B6 have earlier been studied in Caucasian, Japanese and Korean, but the data are lacking for Han Chinese. The aim of this study was to investigate the frequencies of allelic variants of CYP2B6 in healthy Han Chinese and compare with those in other ethnic groups reported in the literature. Polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) method was used to test the five common non-synonymous single nucleotide polymorphisms (SNPs) of CYP2B6 gene, namely, 64C > T, 516G > T, 777C > A, 785A > G and 1459C > T in unrelated healthy Han Chinese (n = 193). The study demonstrated that the frequencies of 64C > T, 516G > T, 777C > A, 785A > G and 1459C > T SNPs in Han Chinese were 0.03, 0.21, 0, 0.28 and 0.003, respectively. The frequencies of all five SNPs tested in female were higher than those in male, but the statistical difference was insignificant (P > 0.05). Compared to the data reported in the literature, the frequencies of common CYP2B6 allelic variants in Chinese are similar to those of other Asian populations including Japanese and Korean, but markedly different from those in Caucasians. These results indicate the presence of marked ethnic difference in CYP2B6 SNP frequencies between Chinese and Caucasian. Further studies are required to explore the impact of these SNPs of CYP2B6 gene on the clinical response (efficacy and toxicity) to drugs that are substrates for CYP2B6 in patients.

Integrated RNA extraction and RT-PCR for semi-quantitative gene expression studies on a microfluidic device

This paper describes the development of a microfluidic methodology, using RNA extraction and reverse transcription PCR, for investigating expression levels of cytochrome P450 genes. Cytochrome P450 enzymes are involved in the metabolism of xenobiotics, including many commonly prescribed drugs, therefore information on their expression is useful in both pharmaceutical and clinical settings. RNA extraction, from rat liver tissue or primary rat hepatocytes, was performed using a silica-based solid-phase extraction technique. Following elution of the purified RNA, amplification of target sequences for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the cytochrome P450 gene CYP1A2, was carried out using a one-step reverse transcription PCR. Once the microfluidic methodology had been optimized, analysis of control and 3-methylcholanthrene-induced primary rat hepatocytes were used to evaluate the system. As expected, GAPDH was consistently expressed, whereas CYP1A2 levels were found to be raised in the drug-treated samples. The proposed system offers an initial platform for development of both rapid throughput analyzers for pharmaceutical drug screening and point-of-care diagnostic tests to aid provision of drug regimens, which can be tailor-made to the individual patient.

Metabolism and transport of oxazphosphorines and the clinical implications

The oxazaphosphorines including cyclophosphamide (CPA), ifosfamide (IFO), and trofosfamide represent an important group of therapeutic agents due to their substantial antitumor and immuno-modulating activity. CPA is widely used as an anticancer drug, an immunosuppressant, and for the mobilization of hematopoetic progenitor cells from the bone marrow into peripheral blood prior to bone marrow transplantation for aplastic anemia, leukemia, and other malignancies. New oxazaphosphorines derivatives have been developed in an attempt to improve selectivity and response with reduced toxicity. These derivatives include mafosfamide (NSC 345842), glufosfamide (D19575, β-D-glucosylisophosphoramide mustard), NSC 612567 (aldophosphamide perhydrothiazine), and NSC 613060 (aldophosphamide thiazolidine). This review highlights the metabolism and transport of these oxazaphosphorines (mainly CPA and IFO, as these two oxazaphosphorine drugs are the most widely used alkylating agents) and the clinical implications. Both CPA and IFO are prodrugs that require activation by hepatic cytochrome P450 (CYP)-catalyzed 4-hydroxylation, yielding cytotoxic nitrogen mustards capable of reacting with DNA molecules to form crosslinks and lead to cell apoptosis and/or necrosis. Such prodrug activation can be enhanced within tumor cells by the CYP-based gene directed-enzyme prodrug therapy (GDEPT) approach. However, those newly synthesized oxazaphosphorine derivatives such as glufosfamide, NSC 612567 and NSC 613060, do not need hepatic activation. They are activated through other enzymatic and/or non-enzymatic pathways. For example, both NSC 612567 and NSC 613060 can be activated by plain phosphodiesterase (PDEs) in plasma and other tissues or by the high-affinity nuclear 3'-5' exonucleases associated with DNA polymerases, such as DNA polymerases and ε. The alternative CYP-catalyzed inactivation pathway by N-dechloroethylation generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde (CAA). Various aldehyde dehydrogenases (ALDHs) and glutathione S-transferases (GSTs) are involved in the detoxification of oxazaphosphorine metabolites. The metabolism of oxazaphosphorines is auto-inducible, with the activation of the orphan nuclear receptor pregnane X receptor (PXR) being the major mechanism. Oxazaphosphorine metabolism is affected by a number of factors associated with the drugs (e.g., dosage, route of administration, chirality, and drug combination) and patients (e.g., age, gender, renal and hepatic function). Several drug transporters, such as breast cancer resistance protein (BCRP), multidrug resistance associated proteins (MRP1, MRP2, and MRP4) are involved in the active uptake and efflux of parental oxazaphosphorines, their cytotoxic mustards and conjugates in hepatocytes and tumor cells. Oxazaphosphorine metabolism and transport have a major impact on pharmacokinetic variability, pharmacokinetic-pharmacodynamic relationship, toxicity, resistance, and drug interactions since the drug-metabolizing enzymes and drug transporters involved are key determinants of the pharmacokinetics and pharmacodynamics of oxazaphosphorines. A better understanding of the factors that affect the metabolism and transport of oxazaphosphorines is important for their optional use in cancer chemotherapy.

Drug bioactivation, covalent binding to target proteins and toxicity relevance

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targetsof reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.

Induction of propranolol metabolism by Ginkgo biloba extract EGb 761 in rats

Ginkgo biloba is one of the most popular herbal medicines in the world, due to its purported pharmacological effects, including memory-enhancing, cognition-improving, and antiplatelet effects. When used in the elderly, Ginkgo has a high potential for interactions with cardiovascular drugs. This study aimed to investigate the effects of the standard Ginkgo biloba extract (EGB 761) treatment on the pharmacokinetics of propranolol and its metabolism to form Ndesisopropylpropranolol (NDP) in rats. We also examined the activity and expression of cytochrome P450 (CYP) 1A and other CYPs in rats treated with EGb 761 at 10 and 100 mg/kg/day for 10 days. A single oral dose of propranolol (10 mg/kg) was administered on day 11 and the concentrations of both propranolol and NDP were determined using validated liquid chromatography-mass spectrometry (LC-MS) methods. The levels of mRNA and protein of various CYPs were determined by RT-PCR and Western blotting analysis, respectively. Pretreatment of EGb 761 at 100 mg/kg, but not 10 mg/kg, for 10 days significantly reduced the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (C max) of propranolol, whereas those values of NDP were significantly increased. CYP1A1, 1A2, 2B1/2, and 3A1 activities and gene expression in the rat liver were significantly increased in a dose-dependent manner by pretreatment with EGb 761. The ex-vivo formation of NDP in liver microsomes from rats pretreated with EGb 761 was markedly enhanced. The formation of NDP from propranolol in liver microsomes was significantly inhibited by α- naphthoflavone (ANF, a selective CYP1A2 inhibitor), but not by quinidine (a CYP2D inhibitor). These results indicated that EGb 761 pretreatment decreased the plasma concentrations of propranolol by accelerated conversion of parental drug to NDP due to induction of CYP1A2. EGb 761 pretreatment also significantly induced CYP2B1/2 and CYP3A1, suggesting potential interactions with substrate drugs for these two enzymes. Further study is needed to explore the potential for gingko-drug interactions and the clinical impact.

Clincial pharmacogenetics and potential application in personalized medicine

The current `fixed-dosage strategy' approach to medicine, means there is much inter-individual variation in drug response. Pharmacogenetics is the study of how inter-individual variations in the DNA sequence of specific genes affect drug responses. This article will highlight current  pharmacogenetic knowledge on important drug metabolizing enzymes, drug transporters and drug targets to understand interindividual variability in drug clearance and responses in clinical practice and potential use in  personalized medicine. Polymorphisms in the cytochrome P450 (CYP) family may have had the most impact on the fate of pharmaceutical drugs. CYP2D6, CYP2C19 and CYP2C9 gene polymorphisms and gene duplications account for the most frequent variations in phase I metabolism of drugs since nearly 80% of drugs in use today are metabolised by these enzymes. Approximately 5% of Europeans and 1% of Asians lack CYP2D6 activity, and these  individuals are known as poor metabolizers. CYP2C9 is another clinically significant drug metabolising enzyme that demonstrates genetic variants. Studies into CYP2C9 polymorphism have highlighted the importance of the CYP2C9*2 and CYP2C9*3 alleles. Extensive polymorphism also occurs in a majority of Phase II drug metabolizing enzymes. One of the most important polymorphisms is thiopurine S-methyl transferases (TPMT) that catalyzes the S-methylation of thiopurine drugs. With respect to drug transport  polymorphism, the most extensively studied drug transporter is  P-glycoprotein (P-gp/MDR1), but the current data on the clinical impact is limited. Polymorphisms in drug transporters may change drug's distribution, excretion and response. Recent advances in molecular research have revealed many of the genes that encode drug targets demonstrate genetic polymorphism. These variations, in many cases, have altered the targets sensitivity to the specific drug molecule and thus have a profound effect on drug efficacy and toxicity. For example, the β2-adrenoreceptor, which is encoded by the ADRB2 gene, illustrates a clinically significant genetic variation in drug targets. The variable number tandem repeat polymorphisms in serotonin transporter (SERT/SLC6A4) gene are associated with response to antidepressants. The distribution of the common variant alleles of genes that encode drug metabolizing enzymes, drug transporters and drug targets has been found to vary among different populations. The promise of pharmacogenetics lies in its potential to identify the right drug at the right dose for the right individual. Drugs with a narrow therapeutic index are thought to benefit more from pharmacogenetic studies. For example, warfarin serves as a good practical example of how pharmacogenetics can be utilized prior to commencement of therapy in order to achieve maximum efficacy and minimum toxicity. As such, pharmacogenetics has the potential to achieve optimal quality use of medicines, and to improve the efficacy and safety of both prospective and licensed drugs.

植物CytP450和抗氧化酶对土壤菲、芘暴露的生态毒理响应

Maize (Zea mays L.) for the tested plants, meadow brown soil as the soil tested in order to microsomal cytochrome P450 content, superoxide dismutase (SOD), catalase (CAT) and peroxidase enzyme (POD) activity of indicators, the soil phenanthrene and pyrene in response to exposure to eco-toxicological studies. The results show that phenanthrene, pyrene exposure can cause detoxification of plant metabolism and antioxidant defense system of the stress response, caused varying degrees of detoxification of plant metabolism and changes in antioxidant capacity. P450 enzyme activity and low concentrations of phenanthrene and pyrene in a single - relevant exposure concentration (r = 0.834, P <0.01), and phenanthrene and pyrene exposure concentration was negatively correlated compound, saying that Ming Fei, pyrene compound exposed to lead detoxification metabolism of a reduced ability to detoxify the metabolism of plants have synergistic toxic effects; SOD activity and phenanthrene and pyrene in a single exposure concentration was negatively correlated, CAT activity and phenanthrene and pyrene in a single - exposure concentration was positively correlated, POD activity and water solubility of the Philippines positively correlated with the total concentration of pyrene in a negative correlation. SOD, CAT and POD activity and phenanthrene and pyrene were positively related to the concentration of compound exposure, saying that Ming Fei, pyrene complex degree of exposure to lead to reduced oxidative damage, oxidative damage of plants with antagonistic effects .

植物CytP450和抗氧化酶对土壤低浓度菲、芘胁迫的响应

Wheat (Triticum aestivum) was chosen to analyze the effect of two polycyclic aromatic hydrocarbons (PAHs), Phenanthrene (PHE) and Pyrene (PY) in brown meadow soil at low concentrations. The effects of PHE and PY were determined by analyzing the changes in activity of Cytochrome P450 (CytP450) and antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Results indicated that both PHE and PY caused changes in activity of CytP450 and the antioxidant enzymes, SOD, POD and CAT. CytP450 activity was significantly stimulated with 1 mg kg-1 of both PHE and PY individually and significantly inhibited with 4 mg kg-1, which showed that pollution stress of PHE or PY can damage the metabolism and detoxification systems of plants. Moreover, as PHE and PY combined at 1 mg kg-1, CytP450 was increased significantly more than when PHE and PY were applied individually, which illustrates obvious synergistic effects. No significant variation were found in activity of SOD in response to individual exposure of PHE or PY in soil, but SOD activity decreased slightly in response to a combined PHE and PY exposure. Great decrease variation was found in CAT and POD activity in response to individual exposure of PHE or PY in soil. No enhanced toxic effects were shown by POD in response to a PHE and PY combined exposure, however CAT showed increased inhibition. From the aspects of metabolism and detoxification as well as antioxidant enzyme activity, our study has provided experimental basis for the pollution diagnosis of PAHs in soils at low concentrations.