Adverse drug events (ADEs) are a common cause of patient morbidity and mortality and are classically thought to result, in part, from variation in expression and activity of hepatic enzymes of drug metabolism. It is now known that alterations in the expression of genes that encode drug- and bile-acid–transporter proteins in both the gut and liver play a previously unrecognized role in determining patient drug response and eventual clinical outcome. Four nuclear receptor (NR) superfamily members, including pregnane X receptor (PXR, NR1I2), constitutive androstane receptor (NR1I3), farnesoid X receptor (NR1H4), and vitamin D receptor (NR1I1), play pivotal roles in drug- and bile-acid– activated programs of gene expression to coordinately regulate drug- and bile-acid transport activity in the intestine and liver. This review focuses on the NR-mediated gene activation of drug and bile-acid transporters in these tissues as well as the possible underlying molecular mechanisms.
Combined structure, function and molecular dynamics studies of human cytosolic sulfotransferases (SULT1A1 and 2A1) have revealed that these enzymes contain a ~30-residue active-site cap whose structure responds to substrates and mediates their interactions. The binding of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) gates access to the active site by a remodeling of the cap that constricts the pore through which acceptors must pass to enter the active site. While the PAPS-bound enzyme spends the majority (~95%) of its time in the constricted state, the pore isomerizes between the open and closed states when the nucleotide (PAPS) is bound. The dimensions of the open and closed pores place widely different steric constraints on substrate selectivity. Nature appears to have crafted these enzymes with two specificity settings – a closed-pore setting that admits a set of closely related structures, and an open setting that allows a far wider spectrum of acceptor geometries. The specificities of these settings seem well matched to the metabolic demands for homeostatic and defensive SULT functions. The departure of nucleotide requires that the cap open. This isomerization dependent release can explain both the product bursts and substrate inhibition seen in many SULTs. Here, the experimental underpinnings of the cap-mechanism are reviewed, and the advantages of such a mechanism are considered in the context of the cellular and metabolic environment in which these enzymes operate.
Xenobiotic compounds undergo a critical range of biotransformations performed by the phase I, II, and III drug-metabolizing enzymes. The oxidation, conjugation, and transportation of potentially harmful xenobiotic and endobiotic compounds achieved by these catalytic systems are significantly regulated, at the gene expression level, by members of the nuclear receptor (NR) family of ligand-modulated transcription factors. Activation of NRs by a variety of endo- and exogenous chemicals are elemental to induction and repression of drug-metabolism pathways. The master xenobiotic sensing NRs, the promiscuous pregnane X receptor and less-promiscuous constitutive androstane receptor are crucial to initial ligand recognition, jump-starting the metabolic process. Other receptors, including farnesoid X receptor, vitamin D receptor, hepatocyte nuclear factor 4 alpha, peroxisome proliferator activated receptor, glucocorticoid receptor, liver X receptor, and RAR-related orphan receptor, are not directly linked to promiscuous xenobiotic binding, but clearly play important roles in the modulation of metabolic gene expression. Crystallographic studies of the ligand-binding domains of nine NRs involved in drug metabolism provide key insights into ligand-based and constitutive activity, coregulator recruitment, and gene regulation. Structures of other, noncanonical transcription factors also shed light on secondary, but important, pathways of control. Pharmacological targeting of some of these nuclear and atypical receptors has been instituted as a means to treat metabolic and developmental disorders and provides a future avenue to be explored for other members of the xenobiotic-sensing NRs.
Cytosolic sulfotransferases (SULTs) are phase II detoxification enzymes that are involved in the biotransformation of a wide variety of structurally diverse endo- and xenobiotics. Single-nucleotide polymorphisms (SNPs) in SULTs can alter the phenotype of the translated proteins. SNPs in some SULTs are fairly uncommon in the population, but some, most notably for SULT isoform 1A1, are commonly found and have been associated with cancer risk for a variety of tumor sites and also with response to therapeutic agents. SNPs in many SULTs vary by ethnicity, another factor that could influence SULT-associated disease risk and pharmacogenetics. This review surveys the current knowledge ofSULTgenetic variability in relation to cancer risk and response to therapy, focusing primarily onSULT1A1.
In 2008, the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) detected unregulated, psychoactive synthetic cannabinoids (SCBs) in purportedly all-natural herbal incense products (often known as K2 or Spice) that were being covertly abused as marijuana substitutes. These drugs, which include JWH-018, JWH-073 and CP-47,497, bind and activate the cannabinoid receptors CB1R and CB2R with remarkable potency and efficacy. Serious adverse effects that often require medical attention, including severe cardiovascular, gastrointestinal and psychiatric sequelae, are highly prevalent with SCB abuse. Consequently, progressively restrictive legislation in the US and Europe has banned the distribution, sale and use of prevalent SCBs, initiating cycles in which herbal incense manufacturers replace banned SCBs with newer unregulated SCBs. The contents of the numerous, diverse herbal incense products was unknown when SCB abuse first emerged. Furthermore, the pharmacology of the active components was largely uncharacterized, and confirmation of SCB use was hindered by a lack of known biomarkers. These knowledge gaps prompted scientists across multiple disciplines to rapidly (1) monitor, identify and quantify with chromatography/mass spectrometry the ever-changing contents of herbal incense products, (2) determine the metabolic pathways and major urinary metabolites of several commonly abused SCBs and (3) identify active metabolites that possibly contribute to the severe adverse effect profile of SCBs. This review comprehensively describes the emergence of SCB abuse and provides a historical account of the major case reports, legal decisions and scientific discoveries of the ″K2/Spice Phenomenon″. Hypotheses concerning potential mechanisms SCB adverse effects are proposed in this review.
Pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR), members of the nuclear receptor superfamily, are two major xeno-sensing transcription factors. They can be activated by a broad range of lipophilic xenobiotics including therapeutics drugs. In addition to xenobiotics, endogenous compounds such as steroid hormones and bile acids can also activate PXR and/or CAR. These nuclear receptors regulate genes that encode enzymes and transporters that metabolize and excrete both xenobiotics and endobiotics. Sulfotransferases (SULTs) are a group of these enzymes and sulfate xenobiotics for detoxification. In general, inactivation by sulfation constitutes the mechanism to maintain homeostasis of endobiotics. Thus, deciphering the molecular mechanism by which PXR and CAR regulateSULTgenes is critical for understanding the roles of SULTs in the alterations of physiological and pathophysiological processes caused by drug treatment or environmental exposures.
Inhibitory Effects of Dibenzocyclooctadiene Lingans from Fructus Schisandrae on CYP2C8-Mediated Paclitaxel Metabolism in Human Liver MicrosomesYan-Yan Zhang, Ling YangLaboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 ChinaDibenzocyclooctadiene lingans are the principle active ingredients of Fructus Schisandrae, which is extensively used as an antitussive, sedative and tonic agent in China, Korea, Japan and Russia. According to Chinese Pharmacopoeia 2005 edition, these lingans are recognized as the major constituents in this Chinese herb medicine. Recently, schisandra extract and some purified lingans have been found to enhance the efficacy of paclitaxel against multidrug resistance tumor cell lines1. In the present study, an in vitro study was undertaken to evaluate the influence of 7 major Schisandra lingans on CYP2C8, which is the primary metabolic enzyme for paclitaxel. Among all these components, gomisin G (IC50=4.3±0.7 µM) exhibited the most potent inhibitory effect on CYP2C8-dependent paclitaxel 6α-hydroxylation in noncompetitive manner, with apparent Ki value of 4.1 µM. Gomisin A (IC50= 8.7±0.8 µM, Ki=8.6 µM) and pregomisin (IC50= 9.3±0.8 µM, Ki=8.0 µM) were also found to potently inhibit CYP2C8 in noncompetitive and competitive manner, respectively. However, schizandrol A (IC50>100 µM), schizandrin A (IC50= 31.8±1.7 µM), B (IC50= 12.6±1.9 µM) and C (IC50>100 µM) were considered less likely to cause drug-drug interactions via inhibition of CYP2C8 activity. Since paclitaxel holds a complex pharmacological and toxicological profile with a narrow therapeutic window2, special attention should be taken on the alteration of paclitaxel pharmacokinetic behavior, when coadministered with the corresponding dibenzocyclooctadiene lingans or the crude extract of Fructus Schisandrae.Reference1. Huang M, Jin J, Sun H, Liu GT. Reversal of P-glycoprotein-mediated multidrug resistance of cancer cells by five schizandrins isolated from the Chinese herb Fructus Schizandrae. Cancer Chemother Pharmacol 2008 62(6):1015-1026.2. Mielke S, Sparreboom A, Steinberg SM, Gelderblom H, Unger C, Behringer D, Mross K. Association of Paclitaxel pharmacokinetics with the development of peripheral neuropathy in patients with advanced cancer. Clin Cancer Res 2005 11(13): 4843-4850.
Abstract: Evodiamine is the main active alkaloid of Evodia rutaecarpa and has been demonstrated to have many pharmacological activities. In the present study, the phase I metabolites of evodiamine and the cytochrome P450 isoforms involved were profiled. Human liver microsomal incubation of evodiamine in the presence of NADPH resulted in the formation of five major metabolites (M-1, M-2, M-3, M-4, M-5). Three major metabolites (M-1, M-3, and M-5) were identified to be monohydroxylated evodiamine and one metabolite (M-4) was identified to be N-demethylated evodiamine by liquid chromatography /mass spectrometry (LC/MS). M-2 was proposed to be 5,6-dehydro evodiamine based on its mass spectrum and retention time. CYP3A4 and CYP1A2 were identified to be the isoform primarily involved in the formation of M-1, M-3, M-4, M-5 in studies with specific P450 inhibitors, recombinant P450s, and correlation analysis. In human liver microsomes, the Km values ranged from 4.7 to 18.4 μM, and the Vm values ranged from 86.3 to 447.5 pmol/min/mg for M-1, M-3, M-4 and M-5. Clarification of the metabolite profile and kinetic properties of evodiamine by human P450s provide important information relevant to the pharmacology and toxicology of evodiamine and E.rutaecarpa.
张延延;张江伟;葛广波;刘勇;杨凌.Metabolism of 7-epi-paclitaxel by Human Cytochrome P450 Enzymes: Effect of Epimer-epimer Interaction on the Metabolic Elimination of Paclitaxel.见：. ISSN:0360-2532 Publication-Date:2008-5-11
-epi-paclitaxel is the principal degradant of paclitaxel under physiological conditions. Human liver microsomal incubation of 7-epi-paclitaxel in the presence of NADPH resulted in the formation of two monohydroxylated metabolites (M-1 and M-2), which were detected by LC/MS. The metabolic sites occurred at the C13 side chain for M-1 and taxane core ring for M-2, respectively. Chemical inhibition studies, correlation studies, and assays with individual cDNA-expressed P450s indicated that formation of M-1 was mediated by CYP3A4 and M-2 was mediated by CYP2C8. The initial rates of formation of paclitaxel metabolites were about 4-fold higher than those of 7-epi-paclitaxel as CYP2C8 being the major metabolic enzyme. Both CYP3A4 and CYP2C8-catalyzed hydroxylation resulted in minor differences of Clint between these epimers; however, the affinity of CYP2C8 was 13-fold higher for 7-epi-paclitaxel than for paclitaxel (Km 1.59 versus 20.32 µM) that indicated stereoselectivity in metabolism. 7-epi-paclitaxel had only a modest effect on the 3''p-hydroxylation of paclitaxel, whereas efficiently suppressed the formation of 6α-hydroxypaclitaxel in a concentration-dependent manner (IC50≈3.03 μM, Ki≈1.8 μM). The present study demonstrated that the possible epimer-epimer interaction might attribute to the saturation of paclitaxel metabolism by CYP2C8. This information could help to understand the disproportional increase of paclitaxel in systemic exposure with dose escalation. To our knowledge, this is the first report of potential uncontrollable risks to patients caused by the fragile stereochemical structural effect of paclitaxel on metabolic stability.
Abstract:Sanguinaria, a benzoc]phenanthridine alkaloid extracted from the root of Sanguinaria canadenis and other poppy-fumaria species, exhibits many biological activities, such as antimicrobial, antiinflammatory, antioxidant, antitumor activities, and singlet oxygen generation potential 1]. As antibiotics or anticancer drug, sanguinaria is likely to be used in combination with various synthetic drug in most case, thus it is necessary to evaluate potential pharmacokinetics drug-drug interactions induced by sanguinaria. An in vitro study revealed that sanguinaria can caused competitive inhibition of human CYP1A2 with Ki of 2 µM 2], but the inhibitory effects on other CYP enzymes remain unreported. In this study, the inhibition of sanguinariato on CYP3A4 and CYP2C8 displayed reversible inhibition. The kinetic parameters of reversible inhibition (Ki) are 2.2 µM for CYP3A4 and 8.82 µM for CYP2C8. Due to the limited pharmacokinetics data of sanguinaria in human, it is nigh impossible to evaluate its potential effects to human from in vitro data, further work will be done in the future.Reference:1] Curr drug metab, 2007; 8:173-1762] Toxicol lett, 2004; 151:375-387
P450 3A4, the most important human CYP enzyme, is responsible for the metabolism of more than 50%-60% of clinically used drugs. Early knowing the site of metabolism will help the medicinal chemist to decide how best to block the metabolism of candidates in drug discovery. Traditionally, the regioselectivity has always been evaluated by estimating the dehydrogenation energy, and/or the binding poses of substrates in the active site of the enzyme. Here, we present a novel strategy for predicting the metabolic regioselectivity mediated by human cytochrome P450 3A4. We have defined a novel type of fragmental-based descriptors that was used together with the E-state molecular descriptors to establish the model for predicting the site of metabolism. With pattern recognition method, 127 substrates of P450 3A4 collected from literatures were employed to develop the model. The cross-validated prediction result is 79% and the accuracy for predicting one right site is higher than 88%, suggesting the efficiency of the newly defined fragmental-based descriptors. The predictability of this model is as good as the recently published QSAR model proposed by Sheridan et al1. 1. Sheridan, R. P.; Korzekwa, K. R.; Torres, R. A.; Walker, M. J., Empirical regioselectivity models for human cytochromes p450 3A4, 2D6, and 2C9. Journal of Medicinal Chemistry 2007, 50, (14), 3173-3184.
Cytochrome P450 2C8 (CYP2C8) is one of the principal drug metabolizing P450 monooxygenases expressed in human liver. It is involved in the metabolism of some therapeutically important drugs such as paclitaxel, repaglinide, rosiglitazone, and cerivastatin. The inhibition of this enzyme in many cases leads to undesired accumulation of the administered therapeutic agent. The purpose of this study is to develop in silico models that can effectively distinguish human CYP2C8 inhibitors from non-inhibitors. We present a data set of more than 200 structurally diverse drug compounds classified for their interaction (inhibitor, non-inhibitor) with CYP2C8. Structures of each drug were characterized by a lot of 2D descriptors. Feature selection was performed using Monte Carlo variable selection (MCVS) method to select key descriptors for modle building. Artificial neural network (ANN) and support vector machaine (SVM) methods were introduced for the classification of inhibitor and non-inhibitor of CYP2C8. The SVM model was better than ANN with the accuracy of 67.2% and sensitivity of 68.6%. This model is further validated by the test set where both accuracy and sensitivity were 65.8% and 66.4%, respectively. The 2D descriptors sufficiently represented the molecular features of CYP2C8 inhibitors. Our model can be used for the prediction of either CYP2C8 inhibitors or non-inhibitors in the early stages of the drug discovery process.
Abstract:Metabolic profiles are pivotal to understand the mechanism of drug biotransformation and elimination, and sequential selection of suitable animal model(s) for pharmacokinetic, toxicological and pharmacological studies. Daphnetin (7,8-dihydroxycoumarin), is an oral medicine for treatment of coagulation disorders and rheumatoid arthritis in China and shows a rapid elimination in rats after intravenous administration with a t1/2 of approximately 15 min and poor bioavailability 1]. Recently, an in vitro study revealed that glucuronidation may play a role in daphnetin metabolism 2], but the in vivo metabolism of daphnetin remains unreported. Herein, we developed a MS-based method for characterization of the metabolites of daphnetin in rat plasma after oral administration at dose of 50 mg/kg bw. Fifteen minutes after daphnetin administration, seven metabolites but no parent were detected from rat plasma, indicating that the metabolism of daphnetin was extensively in rat. These metabolites were identified by comparison of their chromatographic behaviors and ESI-MS/MS spectra to those of daphnetin, an authentic standard 7-O-methyl-8-hydroxycoumarin and three biosynthesized standards, including 7-O glucuronide daphnetin, 8-O glucuronide daphnetin and 7-O-methyl-8-hydroxycoumarin glucuronide. The results showed that glucuronidation played a primary role in metabolic elimination of daphnetin in rat evidenced by the formation of two major monoglucuronides daphnetin (M-1 and M-2) and a diglucuronide daphnetin (M-3). The typical product ions for M-1 and M-2 were m/z 353 and 177, while M-3 showed the fragment ions of m/z 529, 353 and 177. Sulfotransferase(s) was also involved in the metabolism of daphnetin in rat, evidenced by the formation of two trace monosulfates daphnetin (M-4 and M-5) with fragment ions of m/z 257 and 177. Furthermore, glucuronidated (M-6) and sulfated (M-7) derivatives of 7-O-methyl-8-hydroxycoumarin were also detected in rat plasma with the fragment ions m/z 367 and 191 for M-6, and m/z 271 and 176 for M-7, which suggested the overlapping contribution of the conjugation pathways (glucuronidation, methoxylation and sulfation) in daphnetin elimination in rat. These data give us an insight into the mechanism of daphnetin metabolism and elimination, and provide useful information for improving the metabolic stability of this compound. References:1. Qu SY, Wu YJ, Wang YH, and Zuo YX. Metabolism and pharmacokinetics of daphnetin. Yao Xue Xue Bao, 1983; 18: 496–500.2. Liang SC, Ge GB, Liu HX, Zhang YY, et al. Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of daphnetin. Drug Metab Dispos, 2010; 38:973-980.
The subfamily of ABCC transporters consists of 13 members in mammals, including the multidrug resistance-associated proteins (MRPs), sulfonylurea receptors (SURs), and the cystic fibrosis transmembrane conductance regulator (CFTR). These proteins play roles in chemical detoxification, disposition, and normal cell physiology. ABCC transporters are expressed differentially in the liver and are regulated at the transcription and translation level. Their expression and function are also controlled by post-translational modification and membrane-trafficking events. These processes are tightly regulated. Information about alterations in the expression of hepatobiliary ABCC transporters could provide important insights into the pathogenesis of diseases and disposition of xenobiotics. In this review, we describe the regulation of hepatic ABCC transporters in humans and rodents by a variety of xenobiotics, under disease states and in genetically modified animal models deficient in transcription factors, transporters, and cell-signaling molecules.
Electrophilic products of lipid peroxidation are important contributors to the progression of several pathological states. The prototypical α,β–unsaturated aldehyde, 4-hydroxynonenal (HNE), triggers cellular events associated with oxidative stress, which can be curtailed by the glutathione-dependent elimination of HNE. The glutathione transferases (GSTs) are a major determinate of the intracellular concentration of HNE and can influence susceptibility to toxic effects, particularly when HNE and GST levels are altered in disease states. In this article, we provide a brief summary of the cellular effects of HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTs. Some of the key determining characteristics that impart high alkenal activity reside in the unique C-terminal interactions of the GSTA4-4 enzyme. Studies encompassing both kinetic and structural analyses of related isoforms will be highlighted, with additional attention to stereochemical aspects that demonstrate the capacity of GSTA4-4 to detoxify both enantiomers of the biologically relevant racemic mixture while generating a select set of diastereomeric products with subsequent implications. A summary of the literature that examines the interplay between GSTs and HNE in model systems relevant to oxidative stress will also be discussed to demonstrate the magnitude of importance of GSTs in the overall detoxification scheme.
Deoxyschizandrin Could Attenuate Cytochrome P450 3A4-Mediated Bioactivation of Gomisin A in Human Liver MicrosomesYan-Yan Zhang, Ling YangLaboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023 ChinaDibenzocyclooctadiene lignans are discussed as the major absorbed effective ingredients of Schisandra fruit, which is widely used as an antitussive, sedative, tonic agent, and a component of dietary supplement products. Our previous investigations have revealed that biotransformation of the methylenedioxy lignans (e.g. gomisin A) by cytochrome P450 (CYP) 3A can lead to mechanism-based inactivation and reactive ortho-quinone metabolites. In the present study, to gain insight into the clinical value of this herb medicine, a detailed study on the metabolic and inhibitory properties of deoxyschizandrin, another schisandra lignan without methylenedioxy functionality, was conducted. Its influence on the bioactivation of gomisin A was also evaluated. In human liver microsomes (HLMs), deoxyschizandrin underwent C-7 monohydroxylation. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs and enzyme kinetics (Km, 1.6±0.1 μM; Vmax, 623±9.9 pmol/min/mg protein) indicated that its metabolite was generated predominantly by CYP3A4. However, no GSH adduct was observed, which was quite different from gomisin A under the same incubation conditions. Deoxyschizandrin was also found to potently inhibit CYP3A in a competitive manner (Ki, 2.6 μM). IC50 shift studies demonstrated that deoxyschizandrin was not a time and cofactor-dependent inhibitor of CYP3A. Co incubation of deoxyschizandrin (0~100 μM) with gomisin A (50 μM) in HLMs resulted in great reduction of time-dependent inhibitory potential of gomisin A against CYP3A (% activity remaining, 20~80). Moreover, the production of GSH adduct demethylenated gomisin A could be strongly impaired by deoxyschizandrin in vitro. These results collectively demonstrated that deoxyschizandrin might exhibit significant modulatory effects on CYP3A4 activity, thus reducing the bioactivation potential of gomisin A. The presence of deoxyschizandrin in Schisandra fruit could increase the clinical safety of this herb medicine.