Cytochrome P450-mediated Drug Metabolism Research Articles

Inflammatory signaling on cytochrome P450-mediated drug metabolism in hepatocytes.

Cytochrome P450 (CYP450) enzymes are membrane-bound blood proteins that are vital to drug detoxification, cell metabolism, and homeostasis. CYP450s belonging to CYP families 1-3 are responsible for nearly 80% of oxidative metabolism and complete elimination of approximately 50% of all common clinical drugs in humans liver hepatocytes. CYP450s can affect the body's response to drugs by altering the reaction, safety, bioavailability, and toxicity. They can also regulate metabolic organs and the body's local action sites to produce drug resistance through altered drug metabolism. Genetic polymorphisms in the CYP gene alone do not explain ethnic and individual differences in drug efficacy in the context of complex diseases. The purpose of this review is to summarize the impact of new inflammatory-response signaling pathways on the activity and expression of CYP drug-metabolizing enzymes. Included is a summary of recent studies that have identified drugs with the potential to regulate drug-metabolizing enzyme activity. Our goal is to inspire the development of clinical drug treatment processes that consider the impact of the inflammatory environment on drug treatment, as well as provide research targets for those studying drug metabolism.

Alterations of Cytochrome P450-Mediated Drug Metabolism during Liver Repair and Regeneration after Acetaminophen-Induced Liver Injury in Mice.

Acetaminophen (APAP)-induced liver injury (AILI) is the leading cause of acute liver failure in the United States, but its impact on metabolism, therapeutic efficacy, and adverse drug reactions (ADRs) of co- and/or subsequent administered drugs are not fully investigated. The current work explored this field with a focus on the AILI-mediated alterations of cytochrome P450-mediated drug metabolism. Various levels of liver injury were induced in mice by treatment with APAP at 0, 200, 400, and 600 mg/kg. Severity of liver damage was determined at 24, 48, 72, and 96 hours by plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), microRNA miR122, and tissue staining. The expression and activities of CYP3A11, 1A2, 2B10, 2C29, and 2E1 were measured. Sedation efficacy and ADRs of midazolam, a CYP3A substrate, were monitored after APAP treatment. ALT, AST, and miR122 increased at 24 hours after APAP treatment with all APAP doses, whereas only groups treated with 200 and 400 mg/kg recovered back to normal levels at 72 and 96 hours. The expression and activity of the cytochromes P450 significantly decreased at 24 hours with all APAP doses but only recovered back to normal at 72 and 96 hours with 200 and 400, but not 600, mg/kg of APAP. The alterations of cytochrome P450 activities resulted in altered sedation efficacy and ADRs of midazolam, which were corrected by dose justification of midazolam. Overall, this work illustrated a low cytochrome P450 expression window after AILI, which can decrease drug metabolism and negatively impact drug efficacy and ADRs. SIGNIFICANCE STATEMENT: The data generated in the mouse model demonstrated that expression and activities of cytochrome P450 enzymes and correlated drug efficacy and ADRs are altered during the time course of liver repair and regeneration after liver is injured by treatment with APAP. Dose justifications based on predicted changes of cytochrome P450 activities can achieve desired therapeutic efficacy and avoid ADRs. The generated data provide fundamental knowledge for translational research to drug treatment for patients during liver recovery and regeneration who have experienced AILI.

Molecular Modeling Studies on Cytochrome P450-mediated Drug Metabolism.

Drug metabolism studies play a critical role in the optimization of the therapeutic efficacy of newer drug candidates. Many drug candidates and drugs were withdrawn from the pre-clinical/clinical stage or market due to the poor metabolic profiles. The poor metabolic profiles may make the clinical candidates/drugs inactive or toxic. Therefore, it is necessary to optimize the metabolic profiles at the initial phase of drug discovery and development processes. Recently, molecular modeling approaches were found to be useful in the optimization of metabolic profiles of clinical candidates. The molecular modeling approaches were employed in the identification of various metabolic profiles. In the present study, the current status of the research work on drug metabolism based on molecular modeling methodologies has been reviewed. The basics of drug metabolism and its importance in the physiological process of the human body have been described. Moreover, the involvement of molecular modeling approaches like pharmacophore- based modeling, QSAR, molecular docking, virtual screening, quantum chemical analysis, molecular dynamics, etc., in predicting metabolic profiles of therapeutic agents is analyzed. The present review provides computational insights in the prediction of substrate specificity, metabolic activity, SOM, metabolites, toxicity, etc., on cytochrome P450-mediated drug metabolism. The study may help the researchers to design novel drug candidates for the various classes of therapeutic targets with efficient metabolic profiles.

Potential Effects of COVID-19 on Cytochrome P450-Mediated Drug Metabolism and Disposition in Infected Patients.

Coronavirus Disease 2019 (COVID-19) has been a global health crisis since it was first identified in December 2019. In addition to fever, cough, headache, and shortness of breath, an intense increase in immune response-based inflammation has been the hallmark of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) virus infection. This narrative review summarizes and critiques pathophysiology of COVID-19 and its plausible effects on drug metabolism and disposition. The release of inflammatory cytokines (e.g., interleukins, tumor necrosis factor α), also known as ‘cytokine storm’, leads to altered molecular pathophysiology and eventually organ damage in the lung, heart, and liver. The laboratory values for various liver function tests (e.g., alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin) have indicated potential hepatocellular injury in COVID-19 patients. Since the liver is the powerhouse of protein synthesis and the primary site of cytochrome P450 (CYP)-mediated drug metabolism, even a minor change in the liver function status has the potential to affect the hepatic clearance of xenobiotics. It has now been well established that extreme increases in cytokine levels are common in COVID-19 patients, and previous studies with patients infected with non-SARS-CoV-2 virus have shown that CYP enzymes can be suppressed by an infection-related cytokine increase and inflammation. Alongside the investigational COVID-19 drugs, the patients may also be on therapeutics for comorbidities; especially epidemiological studies have indicated that individuals with hypertension, hyperglycemia, and obesity are more vulnerable to COVID-19 than the average population. This complicates the drug-disease interaction profile of the patients as both the investigational drugs (e.g., remdesivir, dexamethasone) and the agents for comorbidities can be affected by compromised CYP-mediated hepatic metabolism. Overall, it is imperative that healthcare professionals pay attention to the COVID-19 and CYP-driven drug metabolism interactions with the goal to adjust the dose or discontinue the affected drugs as appropriate.

Knockdown of Long Noncoding RNAs Hepatocyte Nuclear Factor 1α Antisense RNA 1 and Hepatocyte Nuclear Factor 4α Antisense RNA 1 Alters Susceptibility of Acetaminophen-Induced Cytotoxicity in HepaRG Cells

Acetaminophen (APAP) is a commonly used over-the-counter drug for its analgesic and antipyretic effects. However, APAP overdose leads to severe APAP-induced liver injury (AILI) and even death as a result of the accumulation of N-acetyl-p-benzoquinone imine, the toxic metabolite of APAP generated by cytochrome P450s (P450s). Long noncoding RNAs HNF1α antisense RNA 1 (HNF1α-AS1) and HNF4α antisense RNA 1 (HNF4α-AS1) are regulatory RNAs involved in the regulation of P450 expression in both mRNA and protein levels. This study aims to determine the impact of HNF1α-AS1 and HNF4α-AS1 on AILI. Small hairpin RNAs were used to knock down HNF1α-AS1 and HNF4α-AS1 in HepaRG cells. Knockdown of these lncRNAs altered APAP-induced cytotoxicity, indicated by MTT and LDH assays. Specifically, HNF1α-AS1 knockdown decreased APAP toxicity with increased cell viability and decreased LDH release, whereas HNF4α-AS1 knockdown exacerbated APAP toxicity, with opposite effects in the MTT and LDH assays. Alterations on gene expression by knockdown of HNF1α-AS1 and HNF4α-AS1 were examined in several APAP metabolic pathways, including CYP1A2, CYP2E1, CYP3A4, UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Knockdown of HNF1α-AS1 decreased mRNA expression of CYP1A2, 2E1, and 3A4 by 0.71-fold, 0.35-fold, and 0.31-fold, respectively, whereas knockdown of HNF4α-AS1 induced mRNAs of CYP1A2, 2E1, and 3A4 by 1.3-fold, 1.95-fold, and 1.9-fold, respectively. These changes were also observed in protein levels. Knockdown of HNF1α-AS1 and HNF4α-AS1 had limited effects on the mRNA expression of UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Altogether, our study suggests that HNF1α-AS1 and HNF4α-AS1 affected AILI mainly through alterations of P450-mediated APAP biotransformation in HepaRG cells, indicating an important role of the lncRNAs in AILI. SIGNIFICANCE STATEMENT: The current research identified two lncRNAs, hepatocyte nuclear factor 1α antisense RNA 1 and hepatocyte nuclear factor 4α antisense RNA 1, which were able to affect susceptibility of acetaminophen (APAP)-induced liver injury in HepaRG cells, possibly through regulating the expression of APAP-metabolizing cytochrome P450 enzymes. This discovery added new factors, lncRNAs, which can be used to predict cytochrome P450-mediated drug metabolism and drug-induced toxicity.

Identification of a lncRNA‑associated competing endogenous RNA‑regulated network in clear cell renal cell carcinoma.

Long noncoding RNAs (lncRNAs) act as competing endogenous RNAs (ceRNAs) in the regulation of gene expression in various physiological and pathological processes. The present study aimed to explore the lncRNA-miRNA-mRNA interactions in clear cell renal cell carcinoma (ccRCC) using comprehensive bioinformatics analysis. RNA-seq data were downloaded from the TCGA Data Portal, and the differentially expressed lncRNAs (DElncRs), miRNAs (DEmiRs), and mRNAs (DEGs) between tumoral and control samples were identified using the edgeR package. The correlations between the DemiR/DElncR expression levels and clinical features were evaluated using nonparametric regression analysis. The Kaplan-Meier method was used to identify DElncRs associated with overall survival time. Then, the DElncR-DEmiR interaction pairs were predicted using miRcode and the starBase v2.0 database, and DEmiR-DEG pairs were predicted using the miRTarBase database. Then, a ceRNA-regulated network of ccRCC was constructed based on these interactions. Genes in the network were also assigned to functional categories in the KEGG pathway database. A total of 1,573 DEGs, 37 DelncRs, and 62 DEmiRs were identified. Moreover, several DElncRs were significantly associated with patient clinical variables; for example, TCL6 was significantly associated with tumor grade and AJCC pathological stage. Next, 38 pairs of DElncR-DEmiR interactions (13 DElncRs and 8 DEmiRs) were identified. Among the 8 DEmiRs that target DElncRs, six were found to target DEGs. Based on the identified DElncR-DEmiR interactions and DEmiR-DEG interactions, a ceRNA-regulated network comprising 203 nodes and 221 edges was constructed (with MIC >0.15 and MIC-p2 >0.15). The novel lncRNAs, DGCR5, MYCNOS, and PART1 may participate in the progression of ccRCC through cytochrome P450-mediated drug metabolism.

Enhanced Metabolic Activity of Cytochrome P450 via Carbon Nanocage-Based Photochemical Bionanoreactor.

Recently, the early screening of the genotoxicity of new chemicals and drugs calls for the envelope of micro-/nanoreactors for metabolic study. Herein, a novel light-driven enzymatic bionanoreactor is designed with the gold nanoparticle (NP)-modified carbon nanocage (Au@CNC) as a nanoreactor and meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) as a photosensitizer for cytochrome P450-mediated drug metabolism. By confining the cytochrome P450 3A4 (CYP3A4) enzyme and TCPP inside the pores of Au@CNC, a high metabolic activity is achieved by using 7-ethoxytrifluoromethyl coumarin as the substrate because of the three-dimensional hierarchical porous structure, large surface area, and fast electron transfer capacity of Au@CNC. It is noted that owing to the presence of AuNPs inside CNC, the surface hydrophilicity of CNC is much improved, which further promotes the catalytic activity of the CYP3A4 enzyme. To our knowledge, this is the first attempt to apply CNC as a bionanoreactor for NADPH-free and light-driven in vitro drug metabolism. In addition, the presented bionanoreactor exhibits a variety of advantages in terms of fast response, short assay time (10 min), high sensitivity, and good selectivity, which are expected to expedite drug screening and render potential advances in drug discovery and development.

Isoform-Dependent Changes in Cytochrome P450-Mediated Drug Metabolism after Portal Vein Ligation in the Rat

Background: Surgical removal of complicated liver tumors may be realized in two stages via selective portal vein ligation, inducing the atrophy of portally ligated lobes and the compensatory hypertrophy of nonligated liver lobes. Unlike morphological changes, functional aspects such as hepatic cytochrome P450 (CYP)-mediated drug metabolism remain vaguely understood, despite its critical role in both drug biotransformation and hepatic functional analysis. Our goal was the multilevel characterization of hepatic CYP-mediated drug metabolism after portal vein ligation in the rat. Methods: Male Wistar rats (n = 24, 210–230 g) were analyzed either untreated (controls; n = 4) or 24/48/72/168/336 h (n = 4 each) following portal vein ligation affecting approximately 80% of the liver parenchyma. Besides the weights of ligated and nonligated lobes, pentobarbital (30 mg/kg)-induced sleeping time, CYP1A(2), CYP 2B(1/2), CYP2C(6/11/13), CYP3A(1) enzyme activities, and corresponding isoform mRNA expressions, as well as CYP3A1 protein expression were determined by in vivo sleeping test, CYP isoform-selective assays, polymerase chain reaction, and immunohistochemistry, respectively. Results: Portal vein ligation triggered atrophy in ligated lobes and hypertrophy nonligated lobes. Sleeping time was transiently elevated (p = 0.0451). After an initial rise, CYP1A, CYP2B, and CYP3A enzyme activities dropped until 72 h, followed by a potent increase only in the nonligated lobes, paralleled by an early (24–48 h) transcriptional activation only in nonligated lobes. CYP2C enzyme activities and mRNA levels were bilaterally rapidly decreased, showing a late reconvergence only in nonligated lobes. CYP3A1 immunohistochemistry indicated substantial differences in positivity in the early period. Conclusions: Beyond the atrophy-hypertrophy complex, portal vein ligation generated a transient suppression of global and regional drug metabolism, re-established by an adaptive, CYP isoform-dependent transcriptional response of the nonligated lobes.

Student-directed interactive animation for learning cytochrome P450-mediated drug metabolism

IntroductionIn this study, we introduced a student self-directed interactive animation tool created to enhance student understanding of cytochrome P450 (CYP450) mediated drug metabolism. MethodsThe online learning tool was constructed in HTML5 computer code. It was implemented over four years in a second year pharmacy degree course where CYP450 metabolism was taught. Assessment was by comparing test scores of students using the learning tool with a previous class that did not and through survey data from the student users. ResultsUse of the Cyber-CYP learning tool enhanced test performance on CYP450 metabolism-related questions in all years tested. Survey responses indicated that the learning tool was easy to use and facilitated student learning of CYP450-mediated drug metabolism. ConclusionsThis study has shown that complex and dynamic processes, such as CYP450 metabolism, can be more effectively communicated using student-centered, self-paced and interactive animations.

CYP2D6 Allelic Variants *34, *17-2, *17-3, and *53 and a Thr309Ala Mutant Display Altered Kinetics and NADPH Coupling in Metabolism of Bufuralol and Dextromethorphan and Altered Susceptibility to Inactivation by SCH 66712.

Metabolic phenotype can be affected by multiple factors, including allelic variation and interactions with inhibitors. Human CYP2D6 is responsible for approximately 20% of cytochrome P450-mediated drug metabolism but consists of more than 100 known variants; several variants are commonly found in the population, whereas others are quite rare. Four CYP2D6 allelic variants-three with a series of mutations distal to the active site (*34, *17-2, *17-3) and one ultra-metabolizer with mutations near the active site (*53), along with reference *1 and an active site mutant of *1 (Thr309Ala)-were expressed, purified, and studied for interactions with the typical substrates dextromethorphan and bufuralol and the inactivator SCH 66712. We found that *34, *17-2, and *17-3 displayed reduced enzyme activity and NADPH coupling while producing the same metabolites as *1, suggesting a possible role for Arg296 in NADPH coupling. A higher-activity variant, *53, displayed similar NADPH coupling to *1 but was less susceptible to inactivation by SCH 66712. The Thr309Ala mutant showed similar activity to that of *1 but with greatly reduced NADPH coupling. Overall, these results suggest that kinetic and metabolic analysis of individual CYP2D6 variants is required to understand their possible contributions to variable drug response and the complexity of personalized medicine.

Short-Term Fasting Alters Pharmacokinetics of Cytochrome P450 Probe Drugs: Does Protein Binding Play a Role?

Background and ObjectivesShort-term fasting differentially alters cytochrome P450 (CYP) mediated drug metabolism. This has been established by using CYP-enzyme selective probe drugs. However, the observed effects of fasting on the pharmacokinetics of these probe drugs may also include the effects of altered plasma protein binding of these drugs. Therefore, we studied the effect of short-term fasting on protein binding of five commonly used probe drugs [caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A4), omeprazole (CYP2C19) and S-warfarin (CYP2C9)].MethodsThe free and total plasma concentrations of the five probe drugs were analyzed by LC–MS/MS in samples retrieved in a cross-over study in which nine healthy subjects received an intravenous administration of the cocktail after an overnight fast (control) and after 36 h of fasting.ResultsShort-term fasting increased plasma free fatty acid concentrations from 0.48 mmol/L (control) to 1.29 mmol/L (36 h fasting) (p = 0.012). Short-term fasting did not alter the free fractions of caffeine, metoprolol and omeprazole compared to the control intervention (p > 0.05). Power to detect a difference for midazolam and S-warfarin was low since the majority of free concentrations were below the limit of quantification.ConclusionsThis study demonstrates that short-term fasting does not alter protein binding of the probe drugs caffeine, metoprolol and omeprazole.Electronic supplementary materialThe online version of this article (doi:10.1007/s13318-017-0437-7) contains supplementary material, which is available to authorized users.

Long-term culture of human liver tissue with advanced hepatic functions.

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.

Effect of health foods on cytochrome P450-mediated drug metabolism

BackgroundHealth foods have been widely sold and consumed in Japan. There has been an increase in reports of adverse effects in association with the expanding health food market. While health food-drug interactions are a particular concern from the viewpoint of safe and effective use of health foods, information regarding such interactions is limited owing to the lack of established methods to assess the effects of health food products on drug metabolism. We therefore developed cells that mimicked the activities of cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4, which strongly contribute to drug metabolism in human hepatocytes, and established a system to assess the inhibitory activity of health foods toward P450-mediated metabolism.MethodsWe simultaneously infected HepG2 cells with five P450-expressing adenoviruses (Ad-CYP1A2, Ad-CYP2C9, Ad-CYP2C19, Ad-CYP2D6, and Ad-CYP3A4) to mimic the activity levels of these P450s in human hepatocytes, and named them Ad-P450 cells. The activity levels of P450s in Ad-P450 cells and human hepatocytes were calculated via simultaneous liquid chromatography/tandem mass spectrometry analysis utilizing a P450 substrate cocktail.ResultsWe established Ad-P450 cells mimicking the activity levels of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human hepatocytes. We determined the Km values of P450 substrates and IC50 values of P450 inhibitors in Ad-P450 cells. These values were approximately equivalent to those obtained in previous studies. We investigated the inhibitory effects of 172 health foods that were recently in circulation in Japan on P450-mediated metabolism using Ad-P450 cells. Of the 172 health foods, five products (two products having dietary effects, one turmeric-based product, one collagen-based product, and one propolis-containing product) simultaneously inhibited the five P450s by more than 50%. Another 29 products were also confirmed to inhibit one or more P450s.ConclusionsWe established a comprehensive assessment system to elucidate the effects of health foods on P450-mediated metabolism and identified the inhibitory activity of 34 of 172 health foods toward the drug-metabolizing P450s. Our results may provide useful information to predict health food-drug interactions.

Effect of Short-Term Fasting on Systemic Cytochrome P450-Mediated Drug Metabolism in Healthy Subjects: A Randomized, Controlled, Crossover Study Using a Cocktail Approach.

Background and ObjectiveShort-term fasting can alter drug exposure but it is unknown whether this is an effect of altered oral bioavailability and/or systemic clearance. Therefore, the aim of our study was to assess the effect of short-term fasting on oral bioavailability and systemic clearance of different drugs.MethodsIn a randomized, controlled, crossover trial, 12 healthy subjects received a single administration of a cytochrome P450 (CYP) probe cocktail, consisting of caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A4), omeprazole (CYP2C19) and warfarin (CYP2C9), on four occasions: an oral (1) and intravenous (2) administration after an overnight fast (control) and an oral (3) and intravenous (4) administration after 36 h of fasting. Pharmacokinetic parameters of the probe drugs were analyzed using the nonlinear mixed-effects modeling software NONMEM.ResultsShort-term fasting increased systemic caffeine clearance by 17% (p = 0.04) and metoprolol clearance by 13% (p < 0.01), whereas S-warfarin clearance decreased by 19% (p < 0.01). Fasting did not affect bioavailability.ConclusionThe study demonstrates that short-term fasting alters CYP-mediated drug metabolism in a non-uniform pattern without affecting oral bioavailability.

Effects of 6-paradol, an unsaturated ketone from gingers, on cytochrome P450-mediated drug metabolism

Paradols are unsaturated ketones produced by biotransformation of shogaols in gingers. Among them, 6-paradol has been investigated as a new drug candidate due to its anti-inflammatory, apoptotic, and neuroprotective activities. In this study, the inhibitory effects of 6-paradol on the activities of cytochrome P450 (CYP) enzymes were investigated with human liver microsomes and recombinant CYP isozymes. 6-Paradol showed concentration-dependent inhibitory effects on CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 isozymes, with IC50 values ranging from 3.8 to 21.4µM in recombinant CYP isozymes. However, the inhibition was not potentiated following pre-incubation, indicating that 6-paradol is not a mechanism-based inhibitor. These results suggest that pharmacokinetic drug-drug interactions might occur with 6-paradol, which must be considered in the process of new drug development.

African Genetic Diversity: Implications for Cytochrome P450-mediated Drug Metabolism and Drug Development

Genetic diversity is greater in Africa than in other continental populations. Genetic variability in genes encoding drug metabolizing enzymes may contribute to the high numbers of adverse drug reactions reported in Africa. We reviewed publications (1995–April 2016) reporting frequencies of known cytochrome P450 (CYP) variants in African populations. Using principal components analysis (PCA) we identified CYP alleles of potential clinical relevance with a marked difference in distribution in Africa, compared with Asian and Caucasian populations. These were CYP2B6*6, CYP2C8*2, CYP2D6*3, CYP2D6*17, CYP2D6*29, CYP3A5*6, and CYP3A5*7. We show clearly that there is greater diversity in CYP distribution in Africa than in other continental populations and identify a need for optimization of drug therapy and drug development there. Further pharmacogenetic studies are required to confirm the CYP distributions we identified using PCA, to discover uniquely African alleles and to identify populations at a potentially increased risk of drug-induced adverse events or drug inefficacy.

Enantioselective inhibition of Cytochrome P450-mediated drug metabolism by a novel antithrombotic agent, S002-333: Major effect on CYP2B6

A significant number of new chemical entities (NCEs) fail in drug discovery due to inhibition of Cytochrome P450 (CYP) enzymes. Therefore, to avert costly drug failure at the clinical phase it becomes indispensable to evaluate the CYP inhibition profile of NCEs early in drug discovery. In light of these concerns, we envisioned to investigate the inhibitory effects of S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide], a novel and potent antithrombotic agent, on nine major CYP enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4) of human liver microsomes (HLM). S002-333 exists as racemic mixture of S004-1032 (R-isomer) and S007-1558 (S-isomer), consequently, we further examined the enantioselective differences of S002-333 in the inhibition of human CYP enzymes. Of the CYP enzymes tested, CYP2B6-catalyzed bupropion 6-hydroxylation was inhibited by S002-333 (IC50 ∼ 9.25 ± 2.46 μM) in a stereoselective manner with (S)-isomer showing potent inhibition (IC50 ∼ 5.28 ± 1.25 μM) in contrast to (R)-isomer which showed negligible inhibition on CYP2B6 activity (IC50 > 50 μM). S002-333 and its (S)-isomer inhibited CYP2B6 activity in a non-competitive fashion with estimated Ki values of 10.1 ± 3.4 μM and 5.09 ± 1.05 μM, respectively. No shift in the IC50 value was observed for S002-333 and its isomers when preincubated for 30 min in the presence of NADPH suggesting that neither S002-333 nor its enantiomers are time-dependent inhibitors. Thus, the present findings signified that S002-333 is a potent stereoselective inhibitor of CYP2B6, whereas, inhibition for other CYPs was substantially negligible. These in vitro findings would be useful in deciding the development of S002-333 as a single-enantiomer or as a racemic mixture.

Mechanism of Drug-Drug Interactions Between Warfarin and Statins

The anticoagulant drug warfarin and the lipid-lowering statin drugs are commonly co-administered to patients with cardiovascular diseases. Clinically significant drug-drug interactions (DDIs) between these drugs have been recognized through case studies for many years, but the biochemical mechanisms causing these interactions have not been explained fully. Previous theories include kinetic alterations in cytochrome P-450–mediated drug metabolism or disturbances of drug-protein binding, leading to anticoagulant activity of warfarin; however, neither the enantioselective effects on warfarin metabolism nor the potential disruption of drug transporter function have been well investigated. This study investigated the etiology of the DDIs between warfarin and statins. Liquid chromatography–mass spectrometry methods were developed and validated to quantify racemic warfarin, 6 of its hydroxylated metabolites, and pure enantiomers of warfarin; these methods were applied to study the role of different absorption, distribution, metabolism, and excretion properties, leading to DDIs. Plasma protein binding displacement of warfarin was performed in the presence of statins using equilibrium dialysis method. Substrate kinetics of warfarin and pure enantiomers were performed with human liver microsomes to determine the kinetic parameters (Km and Vmax) for the formation of all 6 hydroxywarfarin metabolites, inhibition of warfarin metabolism in the presence of statins, was determined. Uptake transport studies of warfarin were performed using overexpressing HEK cell lines and efflux transport using human adenocarcinoma colonic cell line cells. Fluvastatin significantly displaced plasma protein binding of warfarin and pure enantiomers; no other statin resulted in significant displacement of warfarin. All the statins that inhibited the formation of 10-hydroxywarfarin, atorvastatin, pitavastatin, and simvastatin were highly potent compared to other statins; in contrast, only fluvastatin was found to be a potent inhibitor of formation of 7-hydroxy warfarin. Uptake and efflux drug transporters do not play any role in these DDIs. The results showed that DDIs between warfarin and statins are primarily caused by cytochrome P-450 inhibition.

Interindividual Variability in Cytochrome P450-Mediated Drug Metabolism.

The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals.

Effect of Dioscorea bulbifera and its Major Bioactive Compound, Diosgenin on CYP450 Mediated Drug Metabolism

Cytochrome P450 (CYP450) inhibition by the bioactive molecules of herbs leading to greater potential for toxicity of co-administered drugs. Diosgenin is an important bioactive steroidal sapogenin from Dioscorea bulbifera belonging to the triterpene group and is of great interest to the pharmaceutical industry. The purpose of this study was, to find whether D. bulbifera and Diosgenin influences the effect on rat CYP450 enzymes and its important isoforms (CYP3A4, CYP2D6) by using high through put screening. CYP450-CO Complex assay was performed in rat pooled liver microsomes to calculate percentage inhibition of test samples on CYP450. IC values were determined by fluorometric assays of CYP3A4 and CYP2D6. Mode of inhibition of Diosgenin on these CYPs were further assessed by molecular docking studies with the Glide (Schrodinger Inc. U.S.A.). In CYP450 inhibition assay, the inhibitory potential of herb extract (40.44±1.28 %) was found to be less than positive control (72.08±1.96 %). In fluorometric assay, herb extract exhibited higher IC values (96.21 ± 1.32 to 180.42±0.12 µg/ml) when compared to positive inhibitors and lower than Diosgenin (172.54±0.52 to 201.86±1.49 µg/ml) on CYP3A4 and CYP2D6. Based on the inhibitory potential, test substances exhibited very less interaction capacity, thereby leading to less significant herb-drug interaction with co-administered drugs.Further clinical studies are required to fully assess the safety of Diosgenin in terms of CYP450.