Human Drug-metabolizing Enzymes Research Articles

Mixed Ru(II)-Ir(III) Complexes as Photoactive Inhibitors of the Major Human Drug Metabolizing Enzyme CYP3A4.

Cytochrome P450 3A4 (CYP3A4) is a crucial enzyme in human drug metabolism. To garner photochemical control over the inhibition of CYP3A4, a potent Ir(III)-based inhibitor of CYP3A4 was complexed with two Ru(II)-based photocaging groups. Chemical, photochemical, and biological properties of the photocaged inhibitors were characterized. Importantly, mixed Ru(II)-Ir(III) complexes strongly absorb green light, which facilitates the photochemical release of the Ir(III) inhibitor from the Ru(II) caging fragment [Ru(tpy)(Me2bpy)]2+, where tpy = 2,2':6',2″-terpyridine and Me2bpy = 6,6'-dimethyl-2,2'-bipyridine. Emission turn on, type II heme binding, and more potent inhibition under light vs dark conditions were observed. The study also demonstrated that a Ru(II)-Ir(III) conjugate can be photoactivated to exert cytotoxic effects on MCF-7 breast cancer cells upon green light exposure. Additionally, a synthesized analogue with one [Ru(TPA)]2+ fragment (TPA = tris(pyridin-2-ylmethyl)amine) and two Ir(III) centers, although resistant to photochemical release, showed strong inhibition of CYP3A4 both in purified form and in CYP3A4-overexpressing HepG2 cells, with nanomolar potency. These mixed Ru(II)-Ir(III) compounds can permeate cell membranes and inhibit CYP3A4, presenting a new class of bioactive compounds.

Formation of potentially toxic metabolites of drugs in reactions catalyzed by human drug-metabolizing enzymes.

Data are presented on the formation of potentially toxic metabolites of drugs that are substrates of human drug metabolizing enzymes. The tabular data lists the formation of potentially toxic/reactive products. The data were obtained from in vitro experiments and showed that the oxidative reactions predominate (with 96% of the total potential toxication reactions). Reductive reactions (e.g., reduction of nitro to amino group and reductive dehalogenation) participate to the extent of 4%. Of the enzymes, cytochrome P450 (P450, CYP) enzymes catalyzed 72% of the reactions, myeloperoxidase (MPO) 7%, flavin-containing monooxygenase (FMO) 3%, aldehyde oxidase (AOX) 4%, sulfotransferase (SULT) 5%, and a group of minor participating enzymes to the extent of 9%. Within the P450 Superfamily, P450 Subfamily 3A (P450 3A4 and 3A5) participates to the extent of 27% and the Subfamily 2C (P450 2C9 and P450 2C19) to the extent of 16%, together catalyzing 43% of the reactions, followed by P450 Subfamily 1A (P450 1A1 and P450 1A2) with 15%. The P450 2D6 enzyme participated in an extent of 8%, P450 2E1 in 10%, and P450 2B6 in 6% of the reactions. All other enzymes participate to the extent of 14%. The data show that, of the human enzymes analyzed, P450 enzymes were dominant in catalyzing potential toxication reactions of drugs and their metabolites, with the major role assigned to the P450 Subfamily 3A and significant participation of the P450 Subfamilies 2C and 1A, plus the 2D6, 2E1 and 2B6 enzymes contributing. Selected examples of drugs that are activated or proposed to form toxic species are discussed.

Novel Independent Trans- and Cis-Genetic Variants Associated with CYP2D6 Expression and Activity in Human Livers.

Cytochrome P450 2D6 (CYP2D6) is a critical hepatic drug-metabolizing enzyme in humans, responsible for metabolizing approximately 20%-25% of commonly used medications such as codeine, desipramine, fluvoxamine, paroxetine, and tamoxifen. The CYP2D6 gene is highly polymorphic, resulting in substantial interindividual variability in its catalytic function and the pharmacokinetics and therapeutic outcomes of its substrate drugs. Although many functional CYP2D6 variants have been discovered and validated, a significant portion of the variability in the expression and activity of CYP2D6 remains unexplained. In this study, we performed a genome-wide association study (GWAS) to identify novel variants associated with CYP2D6 protein expression in individual human livers, followed by a conditional analysis to control for the effect of functional CYP2D6 star alleles. We also examined their impact on hepatic CYP2D6 activity. Genotyping on a genome-wide scale was achieved using the Illumina Multi-Ethnic Genotyping Array (MEGA). A data-independent acquisition (DIA)-based proteomics method was used to quantify CYP2D6 protein concentrations. CYP2D6 activity was determined by measuring the dextromethorphan O-demethylation in individual human liver s9 fractions. The GWAS identified 44 single nuclear polymorphisms (SNPs) that are significantly associated with CYP2D6 protein expressions with a P value threshold of 5.0 × 10-7 After the conditional analysis, five SNPs, including the cis-variants rs1807493 and rs1062753 and the trans-variants rs4073010, rs729559, and rs80274432, emerged as independent variants significantly correlated with hepatic CYP2D6 protein expressions. Notably, four of these SNPs, except for rs80274432, also exhibited a significant association with CYP2D6 activities in human livers, suggesting their potential as novel and independent cis- and trans-variants regulating CYP2D6. SIGNIFICANT STATEMENT: Using individual human livers, we identified four novel cis- and trans-pQTLs/aQTLs (protein quantitative trait loci/activity quantitative trait loci) of Cytochrome P450 2D6 (CYP2D6) that are independent from known functional CYP2D6 star alleles. This study connects the CYP2D6 gene expression and activity, enhancing our understanding of the genetic variants associated with CYP2D6 protein expression and activity, potentially advancing our insight into the interindividual variability in CYP2D6 substrate medication response.

CYP1B1 Genetic Variants (rs10916 and rs2855658) Are Associated with Increased Risk of Ischemic Stroke in Chinese Han Population

Introduction: Ischemic stroke (IS) is an extremely complex disease caused by the combined action of multiple environmental and genetic factors. CYP1B1 is a member of the cytochrome P450 protein family, and it is an important human drug-metabolizing enzymes. We aimed to explore the association between CYP1B1 genetic variants and IS risk in Chinese Han population. Methods: We recruited 1,150 participants to conduct a “case-control” study. The assessment of association between candidate CYP1B1 genetic variants (rs2855658, rs10916, rs162560, rs2567206) and IS risk was performed by SNPStats online software. In addition, false-positive report probability analysis was used to detect whether the positive findings were just chance or noteworthy observations. Finally, the interaction of candidate SNPs in IS risk was evaluated by multifactor dimensionality reduction. Results: The results showed that CYP1B1-rs2855658 was a risk factor for IS among ≥60-year-old (dominant: p = 0.034; overdominant: p = 0.026), smoking (heterozygote: p = 0.009; dominant: p = 0.004; overdominant: p = 0.012; log-additive: p = 0.003), and drinking participants (homozygous: p = 0.036; dominant: p = 0.019; recessive: p = 0.012; log-additive: p = 0.006). CYP1B1-rs10916 also was a risk factor for IS patients among ≥60-year-old (heterozygote: p = 0.047; overdominant: p = 0.048), smoking (dominant: p = 0.050; overdominant: p = 0.049), and drinking participants (dominant: p = 0.019; overdominant: p = 0.038; log-additive: p = 0.013). Conclusion: CYP1B1-rs10916 and CYP1B1-rs2855658 can increase the IS risk in Chinese Han population who are ≥60 years old, smoking, or drinking alcohol.

Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site.

Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site.

Ir(III)-Based Agents for Monitoring the Cytochrome P450 3A4 Active Site Occupancy.

Cytochromes P450 (CYPs) are a superfamily of enzymes responsible for biosynthesis and drug metabolism. Monitoring the activity of CYP3A4, the major human drug-metabolizing enzyme, is vital for assessing the metabolism of pharmaceuticals and identifying harmful drug-drug interactions. Existing probes for CYP3A4 are irreversible turn-on substrates that monitor activity at specific time points in end-point assays. To provide a more dynamic approach, we designed, synthesized, and characterized emissive Ir(III) and Ru(II) complexes that allow monitoring of the CYP3A4 active-site occupancy in real time. In the bound state, probe emission is quenched by the active-site heme. Upon displacement from the active site by CYP3A4-specific inhibitors or substrates, these probes show high emission turn-on. Direct probe binding to the CYP3A4 active site was confirmed by X-ray crystallography. The lead Ir(III)-based probe has nanomolar Kd and high selectivity for CYP3A4, efficient cellular uptake, and low toxicity in CYP3A4-overexpressing HepG2 cells.

Methylophiopogonanone A is a naturally occurring broad-spectrum inhibitor against human UDP-glucuronosyltransferases: Inhibition behaviours and implication in herb-drug interactions.

Methylophiopogonanone A (MOA) is an abundant homoisoflavonoid in the Chinese herb Ophiopogonis Radix. Recent investigations revealed that MOA inhibited several human cytochrome P450 enzymes (CYPs) and stimulated OATP1B1. However, the inhibitory effects of MOA on phase II drug-metabolizing enzymes, such as human UDP-glucuronosyltransferases (hUGTs), have not been well investigated. Herein, the inhibition potentials of MOA on hUGTs were assessed. The results clearly demonstrated that MOA dose-dependently inhibited all tested hUGTs including UGT1A1 (IC50 = 1.23 μM), one of the most important detoxification enzymes in humans. Further investigations showed that MOA strongly inhibited UGT1A1-catalysed NHPH-O-glucuronidation in a range of biological settings including hUGT1A1, human liver microsomes (HLM) and HeLa cells overexpressing UGT1A1. Inhibition kinetic analyses demonstrated that MOA competitively inhibited UGT1A1-catalysed NHPH-O-glucuronidation in both hUGT1A1 and HLM, with Ki values of 0.52 and 1.22 μM, respectively. Collectively, our findings expanded knowledge of the interactions between MOA and human drug-metabolizing enzymes, which would be very helpful for guiding the use of MOA-related herbal products in clinical settings.

In Silico Prediction of CYP2C8 Inhibition with Machine-Learning Methods

Cytochrome P450 2C8 (CYP2C8) is a major drug-metabolizing enzyme in humans and is responsible for the metabolism of ∼5% drugs in clinical use. Thus, inhibition of CYP2C8, which causes potential adverse drug events, cannot be neglected. The in vitro drug interaction studies guidelines for industry issued by the FDA also point out that it needs to be determined whether investigated drugs are CYP2C8 inhibitors before clinical trials. However, current studies mainly focus on predicting the inhibitors of other major P450 enzymes, and the importance of CYP2C8 inhibition has been overlooked. Therefore, there is a need to develop models for identifying potential CYP2C8 inhibition. In this study, in silico classification models for predicting CYP2C8 inhibition were built by five machine-learning methods combined with nine molecular fingerprints. The performance of the models built was evaluated by test and external validation sets. The best model had AUC values of 0.85 and 0.90 for the test and external validation sets, respectively. The applicability domain was analyzed based on the molecular similarity and exhibited an impact on the improvement of prediction accuracy. Furthermore, several representative privileged substructures such as 1H-benzo[d]imidazole, 1-phenyl-1H-pyrazole, and quinoline were identified by information gain and substructure frequency analysis. Overall, our results would be helpful for the prediction of CYP2C8 inhibition.

Structural Dynamics of Cytochrome P450 3A4 in the Presence of Substrates and Cytochrome P450 Reductase.

Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds and by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects, however, is poorly understood. We have used hydrogen-deuterium exchange mass spectrometry to investigate the impact of binding of CPR and of three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone, and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced forms of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from the suppression of deuterium exchange in several regions of CYP3A4, including regions known to be involved in protein-protein interactions (helix C) and substrate binding and specificity (helices B' and E, and loop K/β1). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted upon binding to CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of ligand binding, allostery, and catalysis in CYP enzymes.

Inhibition of drug-metabolizing enzymes by Jingyin granules: implications of herb-drug interactions in antiviral therapy.

Jingyin granules, a marketed antiviral herbal medicine, have been recommended for treating H1N1 influenza A virus infection and Coronavirus disease 2019 (COVID-19) in China. To fight viral diseases in a more efficient way, Jingyin granules are frequently co-administered in clinical settings with a variety of therapeutic agents, including antiviral drugs, anti-inflammatory drugs, and other Western medicines. However, it is unclear whether Jingyin granules modulate the pharmacokinetics of Western drugs or trigger clinically significant herb–drug interactions. This study aims to assess the inhibitory potency of the herbal extract of Jingyin granules (HEJG) against human drug-metabolizing enzymes and to clarify whether HEJG can modulate the pharmacokinetic profiles of Western drug(s) in vivo. The results clearly demonstrated that HEJG dose-dependently inhibited human CES1A, CES2A, CYPs1A, 2A6, 2C8, 2C9, 2D6, and 2E1; this herbal medicine also time- and NADPH-dependently inhibited human CYP2C19 and CYP3A. In vivo tests showed that HEJG significantly increased the plasma exposure of lopinavir (a CYP3A-substrate drug) by 2.43-fold and strongly prolonged its half-life by 1.91-fold when HEJG (3 g/kg) was co-administered with lopinavir to rats. Further investigation revealed licochalcone A, licochalcone B, licochalcone C and echinatin in Radix Glycyrrhizae, as well as quercetin and kaempferol in Folium Llicis Purpureae, to be time-dependent CYP3A inhibitors. Collectively, our findings reveal that HEJG modulates the pharmacokinetics of CYP substrate-drug(s) by inactivating CYP3A, providing key information for both clinicians and patients to use herb–drug combinations for antiviral therapy in a scientific and reasonable way.

Assessment of CYP2C9 Structural Models for Site of Metabolism Prediction.

Structure-based prediction of a compound's potential sites of metabolism (SOMs) mediated by cytochromes P450 (CYPs) is highly advantageous in the early stage of drug discovery. However, the accuracy of the SOMs prediction can be influenced by several factors. CYP2C9 is one of the major drug-metabolizing enzymes in humans and is responsible for the metabolism of ∼13 % of clinically used drugs. In this study, we systematically evaluated the effects of protein crystal structure models, scoring functions, heme forms, conserved active-site water molecules, and protein flexibility on SOMs prediction of CYP2C9 substrates. Our results demonstrated that, on average, ChemScore and GlideScore outperformed four other scoring functions: Vina, GoldScore, ChemPLP, and ASP. The performance of the crystal structure models with pentacoordinated heme was generally superior to that of the hexacoordinated iron-oxo heme (referred to as Compound I) models. Inclusion of the conserved active-site water molecule improved the prediction accuracy of GlideScore, but reduced the accuracy of ChemScore. In addition, the effect of the conserved water on SOMs prediction was found to be dependent on the receptor model and the substrate. We further found that one of snapshots from molecular dynamics simulations on the apo form can improve the prediction accuracy when compared to the crystal structural model.

Ontogeny of Hepatic Transporters and Drug-Metabolizing Enzymes in Humans and in Nonclinical Species.

The liver represents a major eliminating and detoxifying organ, determining exposure to endogenous compounds, drugs, and other xenobiotics. Drug transporters (DTs) and drug-metabolizing enzymes (DMEs) are key determinants of disposition, efficacy, and toxicity of drugs. Changes in their mRNA and protein expression levels and associated functional activity between the perinatal period until adulthood impact drug disposition. However, high-resolution ontogeny profiles for hepatic DTs and DMEs in nonclinical species and humans are lacking. Meanwhile, increasing use of physiologically based pharmacokinetic (PBPK) models necessitates availability of underlying ontogeny profiles to reliably predict drug exposure in children. In addition, understanding of species similarities and differences in DT/DME ontogeny is crucial for selecting the most appropriate animal species when studying the impact of development on pharmacokinetics. Cross-species ontogeny mapping is also required for adequate translation of drug disposition data in developing nonclinical species to humans. This review presents a quantitative cross-species compilation of the ontogeny of DTs and DMEs relevant to hepatic drug disposition. A comprehensive literature search was conducted on PubMed Central: Tables and graphs (often after digitization) in original manuscripts were used to extract ontogeny data. Data from independent studies were standardized and normalized before being compiled in graphs and tables for further interpretation. New insights gained from these high-resolution ontogeny profiles will be indispensable to understand cross-species differences in maturation of hepatic DTs and DMEs. Integration of these ontogeny data into PBPK models will support improved predictions of pediatric hepatic drug disposition processes. SIGNIFICANCE STATEMENT: Hepatic drug transporters (DTs) and drug-metabolizing enzymes (DMEs) play pivotal roles in hepatic drug disposition. Developmental changes in expression levels and activities of these proteins drive age-dependent pharmacokinetics. This review compiles the currently available ontogeny profiles of DTs and DMEs expressed in livers of humans and nonclinical species, enabling robust interpretation of age-related changes in drug disposition and ultimately optimization of pediatric drug therapy.

Inhibition of drug-metabolizing enzymes by Qingfei Paidu decoction: Implication of herb-drug interactions in COVID-19 pharmacotherapy

Corona Virus Disease 2019 (COVID-19) has spread all over the world and brings significantly negative effects on human health. To fight against COVID-19 in a more efficient way, drug-drug or drug-herb combinations are frequently used in clinical settings. The concomitant use of multiple medications may trigger clinically relevant drug/herb-drug interactions. This study aims to assay the inhibitory potentials of Qingfei Paidu decoction (QPD, a Chinese medicine compound formula recommended for combating COVID-19 in China) against human drug-metabolizing enzymes and to assess the pharmacokinetic interactions in vivo. The results demonstrated that QPD dose-dependently inhibited CYPs1A, 2A6, 2C8, 2C9, 2C19, 2D6 and 2E1 but inhibited CYP3A in a time- and NADPH-dependent manner. In vivo test showed that QPD prolonged the half-life of lopinavir (a CYP3A substrate-drug) by 1.40-fold and increased the AUC of lopinavir by 2.04-fold, when QPD (6 g/kg) was co-administrated with lopinavir (160 mg/kg) to rats. Further investigation revealed that Fructus Aurantii Immaturus (Zhishi) in QPD caused significant loss of CYP3A activity in NADPH-generating system. Collectively, our findings revealed that QPD potently inactivated CYP3A and significantly modulated the pharmacokinetics of CYP3A substrate-drugs, which would be very helpful for the patients and clinicians to avoid potential drug-interaction risks in COVID-19 treatment.

Structure of Cytochrome P450 2C9*2 in Complex with Losartan: Insights into the Effect of Genetic Polymorphism.

The human CYP2C9 plays a crucial role in the metabolic clearance of a wide range of clinical therapeutics. The *2 allele is a prevalent genetic variation in CYP2C9 that is found in various populations. A marked reduction of catalytic activity toward many important drug substrates has been demonstrated by CYP2C9*2, which represents an amino acid variation at position 144 from arginine to cysteine. The crystal structure of CYP2C9*2 in complex with an antihypertensive drug losartan was solved using X-ray crystallography at 3.1-Å resolution. The Arg144Cys variation in the *2 complex disrupts the hydrogen-bonding interactions that were observed between the side chain of arginine and neighboring residues in the losartan complex of CYP2C9 and the wild-type (WT) ligand-free structure. The conformation of several secondary structural elements is affected, thereby altering the binding and orientation of drug and important amino acid side chains in the distal active site cavity. The new structure revealed distinct interactions of losartan in the compact active site of CYP2C9*2 and differed in occupancy at the other binding sites previously identified in the WT-losartan complex. Furthermore, the binding studies in solution using losartan illustrated lower activity of the CYP2C9*2 compared with the WT. Together, the findings yield valuable insights into the decreased hydroxylation activity of losartan in patients carrying CYP2C9*2 allele and provide a useful framework to investigate the effect of a single-nucleotide polymorphism that leads to altered metabolism of diverse drug substrates. SIGNIFICANCE STATEMENT: The *2 allele of the human drug-metabolizing enzyme CYP2C9 is found in different populations and results in significantly reduced activity toward various drug substrates. How the CYP2C9*2 variant induces altered drug metabolism is poorly understood given that the Arg144Cys variation is located far away from the active site. This work yield insight into the effect of distal variation using multitude of techniques that include X-ray crystallography, isothermal titration calorimetry, enzymatic characterization, and computational studies.

Pulmonary Metabolism of Substrates for Key Drug-Metabolizing Enzymes by Human Alveolar Type II Cells, Human and Rat Lung Microsomes, and the Isolated Perfused Rat Lung Model.

Significant pulmonary metabolism of inhaled drugs could have drug safety implications or influence pharmacological effectiveness. To study this in vitro, lung microsomes or S9 are often employed. Here, we have determined if rat and human lung microsomes are fit for purpose or whether it is better to use specific cells where drug-metabolizing enzymes are concentrated, such as alveolar type II (ATII) cells. Activities for major hepatic and pulmonary human drug-metabolizing enzymes are assessed and the data contextualized towards an in vivo setting using an ex vivo isolated perfused rat lung model. Very low rates of metabolism are observed in incubations with human ATII cells when compared to isolated hepatocytes and fewer of the substrates are found to be metabolized when compared to human lung microsomal incubations. Reactions selective for flavin-containing monooxygenases (FMOs), CYP1B1, CYP2C9, CYP2J2, and CYP3A4 all show significant rates in human lung microsomal incubations, but all activities are higher when rat lung microsomes are used. The work also demonstrates that a lung microsomal intrinsic clearance value towards the lower limit of detection for this parameter (3 µL/min/mg protein) results in a very low level of pulmonary metabolic clearance during the absorption period, for a drug dosed into the lung in vivo.

Interactions of drug-metabolizing enzymes with the Chinese herb Psoraleae Fructus.

Psoraleae Fructus (the dried fruits of Psoralea corylifolia), one of the most frequently used Chinese herbs in Asian countries, has a variety of biological activities. In clinical settings, Psoraleae Fructus or Psoraleae Fructus-related herbal medicines frequently have been used in combination with a number of therapeutic drugs for the treatment of various human diseases, such as leukoderma, rheumatism and dysentery. The use of Psoraleae Fructus in combination with drugs has aroused concern of the potential risks of herb-drug interactions (HDI) or herb-endobiotic interactions (HEI). This article reviews the interactions between human drug-metabolizing enzymes and the constituents of Psoraleae Fructus; the major constituents in Psoraleae Fructus, along with their chemical structures and metabolic pathways are summarized, and the inhibitory and inductive effects of the constituents in Psoraleae Fructus on human drug-metabolizing enzymes (DMEs), including target enzyme(s), its modulatory potency, and mechanisms of action are presented. Collectively, this review summarizes current knowledge of the interactions between the Chinese herb Psoraleae Fructus and therapeutic drugs in an effort to facilitate its rational use in clinical settings, and especially to avoid the potential risks of HDI or HEI through human DMEs.

Alteration of drug-metabolizing enzyme activity in palliative care patients: Microdosed assessment of cytochrome P450 3A

Background: Cytochrome P450 3A is the most relevant drug-metabolizing enzyme in humans as it is involved in the elimination of 50% of marketed drugs. Nothing is known about the activity of cytochrome P450 3A in palliative care patients who have complicated symptoms often associated with a terminal illness. Aim: In order to improve drug dosing in end-of-life care and to avoid drug interactions, cytochrome P450 3A activity was determined in patients of a palliative care unit under real-life clinical conditions. Design: As midazolam is an established marker substance for cytochrome P450 3A activity, this single-arm prospective trial was designed to obtain a 4-h pharmacokinetic profile of midazolam after oral administration of a 10-µg dose from each enrolled patient. Plasma concentrations of midazolam and its primary metabolite 1′-hydroxy-midazolam were quantified by mass spectrometry techniques. Cytochrome P450 3A activity was calculated as partial metabolic clearance from a limited sampling area under the curve. All other drugs taken by the participating patients were considered, as well as recent blood test results and patients’ diagnoses. The trial was registered at German Clinical Trials Register (www.drks.de): DRKS00011753. Setting/participants: The trial was carried out at a university palliative care unit under real-life clinical conditions. Every patient admitted to the ward was screened for possible participation, independent of the individual performance status. Results: Partial metabolic clearance of midazolam in palliative care patients was 31.7 ± 32.1 L/h. This was a highly significant 40% reduction (p < 0.0001) in comparison with the cytochrome P450 3A activity of healthy subjects. Conclusion: Dosing of cytochrome P450 3A substrate drugs (e.g. macrolide antibiotics, benzodiazepines, calcium channel blockers) needs to be adjusted in palliative care patients; otherwise, escalation of debilitating symptoms due to drug interactions might occur.

Utility of Chimeric Mice with Humanized Liver for Predicting Human Pharmacokinetics in Drug Discovery: Comparison with in Vitro-in Vivo Extrapolation and Allometric Scaling.

Predicting human pharmacokinetics (PK) such as clearance (CL) and volume of distribution (Vd) is a critical component of drug discovery. These predictions are mainly performed by in vitro-in vivo extrapolation (IVIVE) using human biological samples, such as hepatic microsomes and hepatocytes. However, some issues with this process have arisen, such as inconsistencies between in vitro and in vivo findings; the integration of predicted CYP, non-CYP and transporter-mediated human PK; and the difficulty of evaluating very metabolically stable compounds. Various approaches to solving these issues have been reported. Allometric scaling using experimental animals has also often been used. However, this method has also shown many problems due to interspecies differences, albeit that various correction methods have been proposed. Another approach involves the production of chimeric mice with humanized liver via the transplantation of human hepatocytes into mice. The livers of these mice are repopulated mostly with human hepatocytes and express human drug-metabolizing enzymes and drug transporters, suggesting that these mice are useful for solving the issues of IVIVE and allometric scaling, and more reliably predicting human PK. In this review, we summarize human PK prediction methods using IVIVE, allometric scaling and chimeric mice with humanized liver, and discuss the utility of predicting human PK in drug discovery by comparing these chimeric mice with IVIVE and allometric scaling.

Mathematical Models in the Description of Pregnane X Receptor (PXR)-Regulated Cytochrome P450 Enzyme Induction.

The pregnane X receptor (PXR) is a drug/xenobiotic-activated transcription factor of crucial importance for major cytochrome P450 xenobiotic-metabolizing enzymes (CYP) expression and regulation in the liver and the intestine. One of the major target genes regulated by PXR is the cytochrome P450 enzyme (CYP3A4), which is the most important human drug-metabolizing enzyme. In addition, PXR is supposed to be involved both in basal and/or inducible expression of many other CYPs, such as CYP2B6, CYP2C8, 2C9 and 2C19, CYP3A5, CYP3A7, and CYP2A6. Interestingly, the dynamics of PXR-mediated target genes regulation has not been systematically studied and we have only a few mechanistic mathematical and biologically based models describing gene expression dynamics after PXR activation in cellular models. Furthermore, few indirect mathematical PKPD models for prediction of CYP3A metabolic activity in vivo have been built based on compartmental models with respect to drug–drug interactions or hormonal crosstalk. Importantly, several negative feedback loops have been described in PXR regulation. Although current mathematical models propose these adaptive mechanisms, a comprehensive mathematical model based on sufficient experimental data is still missing. In the current review, we summarize and compare these models and address some issues that should be considered for the improvement of PXR-mediated gene regulation modelling as well as for our better understanding of the quantitative and spatial dynamics of CYPs expression.

Cytochrome P450 in Pharmacogenetics: An Update.

Interindividual variability in drug disposition is a major cause of lack of efficacy and adverse effects of drug therapies. The majority of hepatically cleared drugs are metabolized by cytochrome P450 (CYP) enzymes, mainly in families CYP1, CYP2, and CYP3. Genes encoding these enzymes are highly variable with allele distribution showing considerable differences between populations. Genetic variability of especially CYP2C9, CYP2C19, CYP2D6, and CYP3A5 is known to have clear clinical impact on drugs that are metabolized by these enzymes. CYP1A2, CYP2A6, CYP2B6, CYP2C8, and CYP3A4 all show variability that affects pharmacokinetics of drugs as well, but so far the evidence regarding their clinical implications is not as conclusive. In this review, we provide an up-to-date summary of the pharmacogenetics of the major human drug-metabolizing CYP enzymes, focusing on clinically significant examples.