Cytochrome P450 Enzymes and Drug Metabolism in Humans

Human cytochrome P450 (CYP) enzymes, as membrane-bound hemoproteins, play important roles in the detoxification of drugs, cellular metabolism, and homeostasis. In humans, almost 80% of oxidative metabolism and approximately 50% of the overall elimination of common clinical drugs can be attributed to one or more of the various CYPs, from the CYP families 1–3. In addition to the basic metabolic effects for elimination, CYPs are also capable of affecting drug responses by influencing drug action, safety, bioavailability, and drug resistance through metabolism, in both metabolic organs and local sites of action. Structures of CYPs have recently provided new insights into both understanding the mechanisms of drug metabolism and exploiting CYPs as drug targets. Genetic polymorphisms and epigenetic changes in CYP genes and environmental factors may be responsible for interethnic and interindividual variations in the therapeutic efficacy of drugs. In this review, we summarize and highlight the structural knowledge about CYPs and the major CYPs in drug metabolism. Additionally, genetic and epigenetic factors, as well as several intrinsic and extrinsic factors that contribute to interindividual variation in drug response are also reviewed, to reveal the multifarious and important roles of CYP-mediated metabolism and elimination in drug therapy.     

Novel Natural Inhibitors of CYP1A2 Identified by in Silico and in Vitro Screening

Inhibition of cytochrome P450 (CYP) is a major cause of herb-drug interactions. The CYP1A2 enzyme plays a major role in the metabolism of drugs in humans. Its broad substrate specificity, as well as its inhibition by a vast array of structurally diverse herbal active ingredients, has indicated the possibility of metabolic herb-drug interactions. Therefore nowadays searching inhibitors for CYP1A2 from herbal medicines are drawing much more attention by biological, chemical and pharmological scientists. In our work, a pharmacophore model as well as the docking technology is proposed to screen inhibitors from herbal ingredients data. Firstly different pharmaphore models were constructed and then validated and modified by 202 herbal ingredients. Secondly the best pharmaphore model was chosen to virtually screen the herbal data (a curated database of 989 herbal compounds). Then the hits (147 herbal compounds) were continued to be filtered by a docking process, and were tested in vitro successively. Finally, five of eighteen candidate compounds (272, 284, 300, 616 and 817) were found to have inhibition of CYP1A2 activity. The model developed in our study is efficient for in silico screening of large herbal databases in the identification of CYP1A2 inhibitors. It will play an important role to prevent the risk of herb-drug interactions at an early stage of the drug development process.     

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis,Characterization and Comparison

Cytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.     

In Silico Prediction of the Metabolic Resistance of Vitamin D Analogs against CYP3A4 Metabolizing Enzyme

The microsomal cytochrome P450 3A4 (CYP3A4) and mitochondrial cytochrome P450 24A1 (CYP24A1) hydroxylating enzymes both metabolize vitamin D and its analogs. The three-dimensional (3D) structure of the full-length native human CYP3A4 has been solved, but the respective structure of the main vitamin D hydroxylating CYP24A1 enzyme is unknown. The structures of recombinant CYP24A1 enzymes have been solved; however, from studies of the vitamin D receptor, the use of a truncated protein for docking studies of ligands led to incorrect results. As the structure of the native CYP3A4 protein is known, we performed rigid docking supported by molecular dynamic simulation using CYP3A4 to predict the metabolic conversion of analogs of 1,25-dihydroxyvitamin D2 (1,25D2). This is highly important to the design of novel vitamin D-based drug candidates of reasonable metabolic stability as CYP3A4 metabolizes ca. 50% of the drug substances. The use of the 3D structure data of human CYP3A4 has allowed us to explain the substantial differences in the metabolic conversion of the side-chain geometric analogs of 1,25D2. The calculated free enthalpy of the binding of an analog of 1,25D2 to CYP3A4 agreed with the experimentally observed conversion of the analog by CYP24A1. The metabolic conversion of an analog of 1,25D2 to the main vitamin D hydroxylating enzyme CYP24A1, of unknown 3D structure, can be explained by the binding strength of the analog to the known 3D structure of the CYP3A4 enzyme.     

Xenobiotic Metabolism: The Effect of Acute Kidney Injury on Non-Renal Drug Clearance and Hepatic Drug Metabolism

Acute kidney injury (AKI) is a common complication of critical illness, and evidence is emerging that suggests AKI disrupts the function of other organs. It is a recognized phenomenon that patients with chronic kidney disease (CKD) have reduced hepatic metabolism of drugs, via the cytochrome P450 (CYP) enzyme group, and drug dosing guidelines in AKI are often extrapolated from data obtained from patients with CKD. This approach, however, is flawed because several confounding factors exist in AKI. The data from animal studies investigating the effects of AKI on CYP activity are conflicting, although the results of the majority do suggest that AKI impairs hepatic CYP activity. More recently, human study data have also demonstrated decreased CYP activity associated with AKI, in particular the CYP3A subtypes. Furthermore, preliminary data suggest that patients expressing the functional allele variant CYP3A5*1 may be protected from the deleterious effects of AKI when compared with patients homozygous for the variant CYP3A5*3, which codes for a non-functional protein. In conclusion, there is a need to individualize drug prescribing, particularly for the more sick and vulnerable patients, but this needs to be explored in greater depth.     

Examination of the Impact of CYP3A4/5 on Drug–Drug Interaction between Schizandrol A/Schizandrol B and Tacrolimus (FK-506): A Physiologically Based Pharmacokinetic Modeling Approach

Schizandrol A (SZA) and schizandrol B (SZB) are two active ingredients of Wuzhi capsule (WZC), a Chinese proprietary medicine commonly prescribed to alleviate tacrolimus (FK-506)-induced hepatoxicity in China. Due to their inhibitory effects on cytochrome P450 (CYP) 3A enzymes, SZA/SZB may display drug–drug interaction (DDI) with tacrolimus. To identify the extent of this DDI, the enzymes’ inhibitory profiles, including a 50% inhibitory concentration (IC₅₀) shift, reversible inhibition (RI) and time-dependent inhibition (TDI) were examined with pooled human-liver microsomes (HLMs) and CYP3A5-genotyped HLMs. Subsequently, the acquired parameters were integrated into a physiologically based pharmacokinetic (PBPK) model to quantify the interactions between the SZA/SZB and the tacrolimus. The metabolic studies indicated that the SZB displayed both RI and TDI on CYP3A4 and CYP3A5, while the SZA only exhibited TDI on CYP3A4 to a limited extent. Moreover, our PBPK model predicted that multiple doses of SZB would increase tacrolimus exposure by 26% and 57% in CYP3A5 expressers and non-expressers, respectively. Clearly, PBPK modeling has emerged as a powerful approach to examine herb-involved DDI, and special attention should be paid to the combined use of WZC and tacrolimus in clinical practice.     

The Combination of the M2 Muscarinic Receptor Agonist and Chemotherapy Affects Drug Resistance in Neuroblastoma Cells

One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.     

Effect of PEGylation on the Drug Release Performance and Hemocompatibility of Photoresponsive Drug-Loading Platform

Coronary stenosis has been one of the most common heart diseases that drastically increases the risk of fatal disorders such as heart attack. Angioplasty using drug coated balloons (DCB) has been one of the most safe and promising treatments. To minimize the risk of thrombosis of such DCBs during intervention, a different approach that can secure high hemocompatibility under blood flow is necessary. Here we report a method of improving the photoresponsive platform’s hemocompatibility by conjugating polyethylene glycol (PEG), onto the functional groups located at the balloon surface. In this study, latex microbeads were used as models for balloons to enable precise observation of its surface under microscopy. These beads were decorated with PEG polymers of a variety of lengths and grafting densities, along with the Cy5-Photoclevable (PC) linker conjugate to mimic drugs to be loaded onto the platform. Results showed that PEG length and grafting density are both critical factors that alter not only its hemocompatibility, but also the drug load and release efficiency of such platform. Thus, although further investigation is necessary to optimize the tradeoff between hemocompatibility, drug load, and release efficiency, it is safe to conclude that PEGylation of DCB surface is an effective method of enhancing and maintaining high hemocompatibility to minimize the risk of thrombosis during angioplasty.     

Severe Adverse Drug Reactions to Quetiapine in Two Patients Carrying CYP2D6*4 Variants: A Case Report

We report two cases of patients who developed severe adverse drug reactions including persistent movement disorders, nausea, and vertigo during treatment with quetiapine at maximum daily doses ranging between 300 and 400 mg. The extensive hepatic metabolism of quetiapine is mainly attributed to cytochrome P450 3A4 (CYP3A4). However, there is recent evidence supporting the idea of CYP2D6 playing a role in the clearance of the quetiapine active metabolite norquetiapine. Interestingly, both patients we are reporting of are carriers of the CYP2D6*4 variant, predicting an intermediate metabolizer phenotype. Additionally, co-medication with a known CYP2D6 inhibitor and renal impairment might have further affected quetiapine pharmacokinetics. The herein reported cases could spark a discussion on the potential impact of a patient’s pharmacogenetic predisposition in the treatment with quetiapine. However, further studies are warranted to promote the adoption of pharmacogenetic testing for the prevention of drug-induced toxicities associated with quetiapine.     

The Effect of Chronic Iloperidone Treatment on Cytochrome P450 Expression and Activity in the Rat Liver: Involvement of Neuroendocrine Mechanisms

In order to achieve a desired therapeutic effect in schizophrenia patients and to maintain their mental wellbeing, pharmacological therapy needs to be continued for a long time, usually from the onset of symptoms and for the rest of the patients’ lives. The aim of our present research is to find out the in vivo effect of chronic treatment with atypical neuroleptic iloperidone on the expression and activity of cytochrome P450 (CYP) in rat liver. Male Wistar rats received a once-daily intraperitoneal injection of iloperidone (1 mg/kg) for a period of two weeks. Twenty-four hours after the last dose, livers were excised to study cytochrome P450 expression (mRNA and protein) and activity, pituitaries were isolated to determine growth hormone-releasing hormone (GHRH), and blood was collected for measuring serum concentrations of hormones and interleukin. The results showed a broad spectrum of changes in the expression and activity of liver CYP enzymes, which are important for drug metabolism (CYP1A, CYP2B, CYP2C, and CYP3A) and xenobiotic toxicity (CYP2E1). Iloperidone decreased the expression and activity of CYP1A2, CP2B1/2, CYP2C11, and CYP3A1/2 enzymes but increased that of CYP2E1. The CYP2C6 enzyme remained unchanged. At the same time, the level of GHRH, GH, and corticosterone decreased while that of T₃ increased, with no changes in IL-2 and IL-6. The presented results indicate neuroendocrine regulation of the investigated CYP enzymes during chronic iloperidone treatment and suggest a possibility of pharmacokinetic/metabolic interactions produced by the neuroleptic during prolonged combined treatment with drugs that are substrates of iloperidone-affected CYP enzymes.     

Effects of Ospemifene on Drug Metabolism Mediated by Cytochrome P450 Enzymes in Humans in Vitro and in Vivo

The objective of these investigations was to determine the possible effects of the novel selective estrogen receptor modulator, ospemifene, on cytochrome P450 (CYP)-mediated drug metabolism. Ospemifene underwent testing for possible effects on CYP enzyme activity in human liver microsomes and in isolated human hepatocytes. Based on the results obtained in vitro, three Phase 1 crossover pharmacokinetic studies were conducted in healthy postmenopausal women to assess the in vivo effects of ospemifene on CYP-mediated drug metabolism. Ospemifene and its main metabolites 4-hydroxyospemifene and 4′-hydroxyospemifene weakly inhibited a number of CYPs (CYP2B6, CYP2C9, CYP2C19, CYP2C8, and CYP2D6) in vitro. However, only CYP2C9 activity was inhibited by 4-hydroxyospemifene at clinically relevant concentrations. Induction of CYPs by ospemifene in cultured human hepatocytes was 2.4-fold or less. The in vivo studies showed that ospemifene did not have significant effects on the areas under the plasma concentration-time curves of the tested CYP substrates warfarin (CYP2C9), bupropion (CYP2B6) and omeprazole (CYP2C19), demonstrating that pretreatment with ospemifene did not alter their metabolism. Therefore, the risk that ospemifene will affect the pharmacokinetics of drugs that are substrates for CYP enzymes is low.     

Species-Dependent Metabolism of Benzo[c]Chrysene Mediated by c-DNA-Expressed Human,Rodent and Fish Cytochrome P450 Enzymes

The metabolism of benzo[c]chrysene (B[c]Ch) with various cytochrome P450 (CYP) enzymes including rat 1A1, 1A2, 2B1 and 2E1, human 1A1, 1A2, 2A6, 1B1, 3A4 and 2E1, mouse 1B1, and scup fish 1A1 expressed in Chinese hamster V79 cells has been investigated to clarify the role of individual enzymes in the regioselective oxidation of B[c]Ch and the species dependency. In six cell lines expressing individual CYP enzymes from four different species B[c]Ch was metabolized to several isomeric phenols and trans?dihydrodiols. However, cell lines expressing human 3A4, 2A6 and 2E1 or rat 1A2, 2B1 and 2E1 were metabolically in-competent towards B[c]Ch. Among the trans?dihydrodiols the 9,10-isomer could be detected in cells expressing human, rat and fish CYP 1A1 and to a minor extent in cells with human 1A2, but not in cells expressing human and mouse CYP 1B1. The latter two cell lines produced high amounts of the bay region 3,4-dihydrodiol, whereas the K-region 7,8-dihydrodiol was a minor metabolite. Oxidation of B[c]Ch to the 1,2-dihydrodiol could not be catalyzed by any of the CYP enzymes investigated except fish 1A1. Our results suggest that metabolic activation of B[c]Ch is initiated predominantly by CYP 1A1 to result selectively in the formation of fjord region 9,10-dihydrodiol 11,12-epoxides regardless of the species involved. The activation of B[c]Ch appears to be limited by a low regioselectivity for the 9,10-oxidation.     

Influence of CYP1A2 and NAT2 Metabolic Phenotypes on Smokers' Urinary Mutagenicity

The influence of the metabolic phenotypes NAT2 and CYP1A2 on urinary mutagenicity of 118 smokers was studied. Mutagenicity of urine samples was determined by Ames test (preincubation plate incorporation assay on YG1024 Salmonella typhimurium strain with S9 mix). Urinary nicotine plus its metabolites were determined to check cigarette smoke intake. The N -acetyltransferase (NAT2) and cytochrome P450 1A2 (CYP1A2) phenotypes were measured by the molar ratio of urinary caffeine metabolites, determined by HPLC analysis. Urinary mutagenicity was significantly higher in smokers CYP1A2 extensive (EM) than in CYP1A2 poor metabolizers (PM) (Mann-Whitney U test, p = 0.020). Linear multiple regression analysis shows that an increase in urinary mutagenicity levels was significantly related to cigarette smoke intake and to CYP1A2 N -hydroxylation activity ( t = 5.06, p < 0.001, and t = 2.33, p = 0.021), but not to NAT2 acetylation phenotype. In conclusion, phenotypic differences in metabolic activation of tobacco smoke mutagens are able to modulate the presence of mutagens in urine of cigarette smokers and, consequently, the potential genotoxic risk.     

A medicinal-chemistry-guided approach to selective and druglike sigma 1 ligands

Based on a medicinal-chemistry-guided approach, three novel series of druglike cycloalkyl-annelated pyrazoles were synthesized and display high affinity (pKi>8) for the sigma1 receptor. Structure-affinity relationships were established, and the different scaffolds were optimized with respect to sigma1 binding and selectivity versus the sigma2 receptor and the hERG channel, resulting in selective compounds that have Ki values (for sigma1) in the subnanomolar range. Selected compounds were screened for cytochrome P450 inhibition (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4), metabolic stability (rat and human liver microsomes), and cell-membrane permeability (Caco-2). They showed favorable in vitro ADME properties as well as favorable calculated druglike and experimental physicochemical properties. Furthermore, compounds 7 f and 17 a, for example, displayed high selectivity (affinity) for the sigma1 receptor against a wide range of other receptors (>60). With these valuable tool compounds in hand, we are further exploring the role of the sigma1 receptor in relevant animal models corresponding to such medicinal indications as drug abuse, pain, depression, anxiety, and psychosis.     

Developmental Toxicity and Biotransformation of Two Anti-Epileptics in Zebrafish Embryos and Early Larvae

The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5¼- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish.     

CYP2E1 in Alcoholic and Non-Alcoholic Liver Injury. Roles of ROS,Reactive Intermediates and Lipid Overload

CYP2E1 is one of the fifty-seven cytochrome P450 genes in the human genome and is highly conserved. CYP2E1 is a unique P450 enzyme because its heme iron is constitutively in the high spin state, allowing direct reduction of, e.g., dioxygen, causing the formation of a variety of reactive oxygen species and reduction of xenobiotics to toxic products. The CYP2E1 enzyme has been the focus of scientific interest due to (i) its important endogenous function in liver homeostasis, (ii) its ability to activate procarcinogens and to convert certain drugs, e.g., paracetamol and anesthetics, to cytotoxic end products, (iii) its unique ability to effectively reduce dioxygen to radical species causing liver injury, (iv) its capability to reduce compounds, often generating radical intermediates of direct toxic or indirect immunotoxic properties and (v) its contribution to the development of alcoholic liver disease, steatosis and NASH. In this overview, we present the discovery of the enzyme and studies in humans, 3D liver systems and genetically modified mice to disclose its function and clinical relevance. Induction of the CYP2E1 enzyme either by alcohol or high-fat diet leads to increased severity of liver pathology and likelihood to develop ALD and NASH, with subsequent influence on the occurrence of hepatocellular cancer. Thus, fat-dependent induction of the enzyme might provide a link between steatosis and fibrosis in the liver. We conclude that CYP2E1 has many important physiological functions and is a key enzyme for hepatic carcinogenesis, drug toxicity and liver disease.