Major Cytochrome P450 Isoforms Research Articles

A comprehensive evaluation in clinic and physiologically-based pharmacokinetic modeling and simulation to confirm lack of cytochrome P450-mediated drug-drug interaction potential for pomotrelvir.

Pomotrelvir is a new chemical entity and potent direct-acting antiviral inhibitor of the main protease of coronaviruses. Here the cytochrome P450 (CYP)-mediated drug-drug interaction (DDI) potential of pomotrelvir was evaluated for major CYP isoforms, starting with in vitro assays followed by the basic static model assessment. The identified CYP3A4-mediated potential DDIs were evaluated clinically at a supratherapeutic dose of 1050 mg twice daily (b.i.d.) of pomotrelvir, including pomotrelvir coadministration with ritonavir (strong inhibitor of CYP3A4) or midazolam (sensitive substrate of CYP3A4). Furthermore, a physiologically-based pharmacokinetic (PBPK) model was developed within the Simcyp Population-based Simulator using in vitro and in vivo information and validated with available human pharmacokinetic (PK) data. The PBPK model was simulated to assess the DDI potential for CYP isoforms that pomotrelvir has shown a weak to moderate DDI in vitro and for CYP3A4 at the therapeutic dose of 700 mg b.i.d. To support the use of pomotrelvir in women of childbearing potential, the impact of pomotrelvir on the exposure of the representative oral hormonal contraceptive drugs ethinyl estradiol and levonorgestrel was assessed using the PBPK model. The overall assessment suggested weak inhibition of pomotrelvir on CYP3A4 and minimal impact of a strong CYP3A4 inducer or inhibitor on pomotrelvir PK. Therefore, pomotrelvir is not anticipated to have clinically meaningful DDIs at the clinical dose. These comprehensive in vitro, in clinic, and in silico efforts indicate that the DDI potential of pomotrelvir is minimal, so excluding patients on concomitant medicines in clinical studies would not be required.

Evaluation of the Cytochrome P450 3A and P-glycoprotein Drug-Drug Interaction Potential of Futibatinib.

Futibatinib, a selective, irreversible fibroblast growth factor receptor 1-4 inhibitor, is being investigated for tumors harboring FGFR aberrations and was recently approved for the treatment of FGFR2 fusion/rearrangement-positive intrahepatic cholangiocarcinoma. In vitro studies identified cytochrome P450 (CYP) 3A as the major CYP isoform in futibatinib metabolism and indicated that futibatinib is likely a P-glycoprotein (P-gp) substrate and inhibitor. Futibatinib also showed time-dependent inhibition of CYP3A in vitro. Phase I studies investigated the drug-drug interactions of futibatinib with itraconazole (a dual P-gp and strong CYP3A inhibitor), rifampin (a dual P-gp and strong CYP3A inducer), or midazolam (a sensitive CYP3A substrate) in healthy adult participants. Compared with futibatinib alone, coadministration of futibatinib with itraconazole increased futibatinib mean peak plasma concentration and area under the plasma concentration-time curve by 51% and 41%, respectively, and coadministration of futibatinib with rifampin lowered futibatinib mean peak plasma concentration and area under the plasma concentration-time curve by 53% and 64%, respectively. Coadministration of midazolam with futibatinib had no effect on midazolam pharmacokinetics compared with midazolam administered alone. These findings suggest that concomitant use of dual P-gp and strong CYP3A inhibitors/inducers with futibatinib should be avoided, but futibatinib can be concomitantly administered with other drugs metabolized by CYP3A. Drug-drug interaction studies with P-gp-specific substrates and inhibitors are planned.

Evaluation of metabolism and cytochrome P450 mediated interaction liabilities of naringenin

Naringenin is one of the major components of grapefruit juice. It has a broad spectrum of pharmacological activities, and many studies report that grapefruit juice inhibits cytochrome P450 (CYP) 3A4 leading to drug interactions. Naringenin was profiled through various in vitro studies like metabolic stability and glucuronidation in rat and human liver microsomes while, CYP inhibition using human liver microsomes. In addition, pharmacokinetic profiling was conducted upon intravenous (i.v.) and oral administration in rats. Naringenin undergoes both phase I and phase II metabolism in rat liver microsomes, and in human liver microsomes, it is predominantly metabolized by phase II. Glucuronidation which is addition (conjugation) of glucuronic acid to various functional groups is one of the major metabolic pathways of Naringenin. Naringenin, at 1.0 μM and 10.0 μM, did not elicit any appreciable inhibition of the 5 major CYP isoforms (CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6). Oral pharmacokinetic studies at 100, 300,and 1000 mg/kg dose and intravenous pharmacokinetic studies at 1 mg/kg dose were performed in male SD rats. Naringenin exhibited very short half-life (0.27 h) and rapid elimination (Clearance=110.65 mL/min/kg) after i.v. administration. There was saturation in Cmax and exposure beyond 100 mg/kg, and the absolute bioavailability was found to be ≤ 5% at the tested oral doses. This present experiment suggests that naringenin does not substantially inhibit CYP3A4 (or any of the tested five isoforms) isoforms per se. Given the minimal involvement of CYP enzymes in the metabolism of naringenin and minimal inhibition of CYP enzymes (IC50> 10 μM), the potential for drug-drug interactions involving CYP substrates and inhibitors is very minimal in humans.

A A Review on the Potential of Application of CRISPR/Cas9 Technology in CYP3A4 and CYP2D6 Genes: A Way to Move Forward in Identifying New Drug Targets

The cytochrome P450 (CYP) enzymes are of a ubiquitous heme–thiolate proteins family that plays an important role within the biotransformation of endogenous molecules and xenobiotics as today’s market is bio-transformed through CYP-mediated metabolism. Major CYP isoforms involved in most reactions are mainly CYP3A4 and CYP2D6. The CYP3A4 is found most extensively within the human liver and gut in which this CYP-isoform takes part in the phase I transformation of toxins, carcinogens, bile acids, and steroid hormones while CYP2D6 plays a central role in the oxidative metabolism of up to 25% of drugs in common clinical use, although it only accounts for 1-5% of the CYP liver content. With the help of vast gene editing technologies such as the CRISPR/Cas9 system, researchers have found its application to be useful and efficient in gene knockout studies involving genetic variants in association with drug metabolism due to its simplicity and affordability. Hence, this review aims to explore the potential use of CRISPR/Cas9 technology in gene editing in vitro studies particularly in knocking out the gene of interest, mainly drug metabolism enzymes. This article will explore how CRISPR/cas9 facilitates ex vivo assays of drug metabolism studies. The long-term goal for bio-editing is an in vivo treatment without the risk of causing harm to humans and providing a ready-to-be-used drug metabolism assay for ex vivo studies of drug development and pharmacokinetics study at a lower cost.

Simultaneous absolute protein quantification of seven cytochrome P450 isoforms in rat liver microsomes by LC-MS/MS-based isotope internal standard method.

The cytochrome P450 (CYP) enzymes play a pivotal role in drug metabolism. LC-MS/MS-based targeting technology has been applied to the analysis of CYP enzymes, promoting drug development and drug-drug interaction studies. Rat is one of the most commonly used models for drug metabolism assessment, but LC-MS/MS assay quantifying the abundance of CYP enzymes in rats is rarely reported. Herein, an accurate and stable LC-MS/MS based method was developed and validated for the simultaneous quantification of seven major rat CYP isoforms (CYP1A2, 2B1, 2C6, 2C11, 2D1, 2E1, and 3A1) in liver microsomes. The careful optimization of trypsin digestion and chromatography combined with isotope-labeled peptide as internal standard improved the efficiency and accuracy of the analysis. Highly specific surrogate peptides were obtained by a procedure including trypsin digestion for six hours and separated on a Hypersil Gold C18 column (100 × 2.1 mm, 3 μm) using gradient elution for 15 min with a mobile phase of water containing 0.1% formic acid and acetonitrile. In the method validation, linearity, matrix effect, recovery, stability, accuracy, and precision all meet the requirements. Subsequently, this method was applied to detect seven enzymes in rat liver microsomes from four different sources, and the correlation between the abundance and activity of CYP enzymes was further analyzed. The high-throughput detection method provided in this study will provide support for pertinent pharmaceutical research based on rat models.

Liquorice Extract and 18β-Glycyrrhetinic Acid Protect Against Experimental Pyrrolizidine Alkaloid-Induced Hepatotoxicity in Rats Through Inhibiting Cytochrome P450-Mediated Metabolic Activation.

Misuse of pyrrolizidine alkaloid (PA)-containing plants or consumption of PA-contaminated foodstuffs causes numerous poisoning cases in humans yearly, while effective therapeutic strategies are still limited. PA-induced liver injury was initiated by cytochrome P450 (CYP)-mediated metabolic activation and subsequent formation of adducts with cellular proteins. Liquorice, a hepato-protective herbal medicine, is commonly used concurrently with PA-containing herbs in many compound traditional Chinese medicine formulas, and no PA-poisoning cases have been reported with this combination. The present study aimed to investigate hepato-protective effects of liquorice aqueous extract (EX) and 18β-glycyrrhetinic acid (GA, the primary bioactive constituent of liquorice) against PA-induced hepatotoxicity and the underlying mechanism. Histopathological and biochemical analysis demonstrated that both single- and multiple-treatment of EX (500 mg/kg) or GA (50 mg/kg) significantly attenuated liver damage caused by retrorsine (RTS, a representative hepatotoxic PA). The formation of pyrrole-protein adducts was significantly reduced by single- (30.3% reduction in liver; 50.8% reduction in plasma) and multiple- (32.5% reduction in liver; 56.5% reduction in plasma) treatment of GA in rats. Single- and multiple-treatment of EX also decreased the formation of pyrrole-protein adducts, with 30.2 and 31.1% reduction in rat liver and 51.8 and 53.1% reduction in rat plasma, respectively. In addition, in vitro metabolism assay with rat liver microsomes demonstrated that GA reduced the formation of metabolic activation-derived pyrrole-glutathione conjugate in a dose-dependent manner with the estimated IC50 value of 5.07 µM. Further mechanism study showed that GA inhibited activities of CYPs, especially CYP3A1, the major CYP isoform responsible for the metabolic activation of RTS in rats. Enzymatic kinetic study revealed a competitive inhibition of rat CYP3A1 by GA. In conclusion, our findings demonstrated that both EX and GA exhibited significant hepato-protective effects against RTS-induced hepatotoxicity, mainly through the competitive inhibition of CYP-mediated metabolic activation of RTS.

Assessment of Herb-Drug Interaction Potential of Five Common Species of Licorice and Their Phytochemical Constituents

The dried roots and rhizomes of Glycyrrhiza species (G. glabra, G. uralensis and G. inflata), commonly known as licorice, have long been used in traditional medicine. In addition, two other species, G. echinata and G. lepidota are also considered “licorice” in select markets. Currently, licorice is an integral part of several botanical drugs and dietary supplements. To probe the botanicals’ safety, herb-drug interaction potential of the hydroethanolic extracts of five Glycyrrhiza species and their key constituents was investigated by determining their effects on pregnane X receptor, aryl hydrocarbon receptor, two major cytochrome P450 isoforms (CYP3A4 and CYP1A2), and the metabolic clearance of antiviral drugs. All extracts enhanced transcriptional activity of PXR and AhR (>2-fold) and increased the enzyme activity of CYP3A4 and CYP1A2. The highest increase in CYP3A4 was seen with G. echinata (4-fold), and the highest increase in CYP1A2 was seen with G. uralensis (18-fold) and G. inflata (16-fold). Among the constituents, glabridin, licoisoflavone A, glyasperin C, and glycycoumarin activated PXR and AhR, glabridin being the most effective (6- and 27-fold increase, respectively). Licoisoflavone A, glyasperin C, and glycycoumarin increased CYP3A4 activity while glabridin, glyasperin C, glycycoumarin, and formononetin increased CYP1A2 activity (>2-fold). The metabolism of antiretroviral drugs (rilpivirine and dolutegravir) was increased by G. uralensis (2.0 and 2.5-fold) and its marker compound glycycoumarin (2.3 and 1.6-fold). The metabolism of dolutegravir was also increased by G. glabra (2.8-fold) but not by its marker compound, glabridin. These results suggest that licorice and its phytochemicals could affect the metabolism and clearance of certain drugs that are substrates of CYP3A4 and CYP1A2. Supplemental data for this article is available online at https://doi.org/10.1080/19390211.2022.2050875 .

In vitro investigations into the roles of CYP450 enzymes and drug transporters in the drug interactions of zanubrutinib, a covalent Bruton's tyrosine kinase inhibitor.

Zanubrutinib is a highly selective, potent, orally available, targeted covalent inhibitor (TCI) of Bruton's tyrosine kinase (BTK). This work investigated the in vitro drug metabolism and transport of zanubrutinib, and its potential for clinical drug–drug interactions (DDIs). Phenotyping studies indicated cytochrome P450 (CYP) 3A are the major CYP isoform responsible for zanubrutinib metabolism, which was confirmed by a clinical DDI study with itraconazole and rifampin. Zanubrutinib showed mild reversible inhibition with half maximal inhibitory concentration (IC50) of 4.03, 5.69, and 7.80 μM for CYP2C8, CYP2C9, and CYP2C19, respectively. Data in human hepatocytes disclosed induction potential for CYP3A4, CYP2B6, and CYP2C enzymes. Transport assays demonstrated that zanubrutinib is not a substrate of human breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP)1B1/1B3, organic cation transporter (OCT)2, or organic anion transporter (OAT)1/3 but is a potential substrate of the efflux transporter P‐glycoprotein (P‐gp). Additionally, zanubrutinib is neither an inhibitor of P‐gp at concentrations up to 10.0 μM nor an inhibitor of BCRP, OATP1B1, OATP1B3, OAT1, and OAT3 at concentrations up to 5.0 μM. The in vitro results with CYPs and transporters were correlated with the available clinical DDIs using basic models and mechanistic static models. Zanubrutinib is not likely to be involved in transporter‐mediated DDIs. CYP3A inhibitors and inducers may impact systemic exposure of zanubrutinib. Dose adjustments may be warranted depending on the potency of CYP3A modulators.

OpenCYP: An open source database exploring human variability in activities and frequencies of polymophisms for major cytochrome P-450 isoforms across world populations

The open source database “OpenCYP database” has been developed based on the results of extensive literature searches from the peer-reviewed literature. OpenCYP provides data on human variability on baseline of activities and polymophism frequencies for selected cytochrome P-450 isoforms (CYP1A2, CYP2A6, CYP2D6, CYP3A4/3A5 and CYP3A7) in healthy adult populations from world populations. CYP enzymatic activities were generally expressed as the metabolic ratio (MR) between an unchanged probe drug and its metabolite(s) in urine or plasma measured in healthy adults. Data on other age groups were very limited and fragmented, constituting an important data gap. Quantitative comparisons were often hampered by the different experimental conditions used. However, variability was quite limited for CYP1A2, using caffeine as a probe substrate, with a symmetrical distribution of metabolic activity values. For CYP3A4, human variability was dependent on the probe substrate itself with low variability when data considering the dextromethorphan/demethilathed metabolite MR were used and large variability when the urinary 6β-hydroxycortisol/cortisol ratio was used. The largest variability in CYP activity was shown for CYP2D6 activity, after oral dosing of dextromethorphan, for which genetic polymorphisms are well characterised and constitute a significant source of variability.It is foreseen that the OpenCYP database can contribute to promising tools to support the further development of QIVIVE and PBK models for human risk assessment of chemicals particularly when combined with information on isoform-specific content in cells using proteomic approaches.

Evaluation of Erythromycin as a Tool to Assess CYP3A Contribution of Low Clearance Compounds in a Long-Term Hepatocyte Culture

Long-term hepatocyte culture systems such as HepatoPac are well suited to evaluate the metabolic turnover of low clearance (CL) drugs because of their sustained metabolic capacity and longer-term viability. Erythromycin (ERY), a moderate, mechanism-based inhibitor of CYP3A, was evaluated as a tool in the HepatoPac model to assess contribution of CYP3A to the clearance of drug candidates. ERY inhibited CYP3A activity by 58% and 80% at 3 and 10 μM, respectively, for up to 72 hours. At 30 µM, ERY inhibited midazolam hydroxylation by >85% for the entire 144-hour duration of the incubation. Alprazolam CLint was inhibited 58% by 3 μM of ERY, 75% by 15 μM of ERY, 89% by 30 μM of ERY, and 94% by 60 μM of ERY. ERY (30 μM) did not markedly affect CLint of substrates for several other major cytochrome P450 isoforms evaluated and did not markedly inhibit uridine diphosphoglucuronosyl transferase (UGT) isoforms 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, or 2B15 as assessed using recombinant UGTs. ERY only mildly increased CYP3A4 gene expression by 2.1-fold (14% of rifampicin induction) at 120 µM, indicating that at effective concentrations for inhibition of CYP3A activity (30-60 µM), arylhydrocarbon receptor, constitutive androstane receptor, and pregnane-X-receptor activation are not likely to markedly increase levels of other drug-metabolizing enzymes or transporters. ERY at concentrations up to 60 µM was not toxic for up to 6 days of incubation. Use of ERY to selectively inhibit CYP3A in high-functioning, long-term hepatocyte models such as HepatoPac can be a valuable strategy to evaluate the contribution of CYP3A metabolism to the overall clearance of slowly metabolized drug candidates. SIGNIFICANCE STATEMENT: This work describes the use of erythromycin as a selective inhibitor of CYP3A to assess the contribution of CYP3A in the metabolism of compounds using long-term hepatocyte cultures.

Results from Drug-Drug Interaction Studies In Vitro and In Vivo Investigating the Effect of Finerenone on the Pharmacokinetics of Comedications.

In vivo studies were performed with the novel, selective, non-steroidal mineralocorticoid receptor antagonist finerenone to assess the relevance of inductive and/or inhibitory effects on cytochrome P450 (CYP) enzymes observed in vitro. CYP isoenzyme-specific substrates were incubated in vitro with finerenone or its metabolites to investigate reversible and irreversible inhibitory as well as inductive potential. Three crossover studies in healthy male volunteers investigated the effects of finerenone (20mg orally) on the pharmacokinetics of the index substrates midazolam (CYP3A4, n = 30), repaglinide (CYP2C8, n = 28) and warfarin (CYP2C9, n = 24). Finerenone caused direct inhibitory effects on CYP activities in vitro in the rank order CYP2C8, CYP1A1 > CYP3A4 > CYP2C9 and CYP2C19, but not on other major CYP isoforms. Moreover, irreversible inhibition of CYP3A4 was observed. The major metabolites of finerenone demonstrated minor reversible inhibition of CYP1A1, CYP2C9 and CYP3A4 with no hint of time-dependent inhibition of any CYP isoform. Calculations from in vitro data according to regulatory guidelines suggested likely inhibition of CYP2C8 and CYP3A4 in vivo, whereas this was not the case for CYP1A1, CYP2C9 and CYP2C19. Furthermore, finerenone and three of its metabolites were inducers of CYP3A4 in vitro with predicted weak-to-moderate in vivo relevance. Studies in healthy volunteers, prompted by these results, demonstrated no effect of finerenone on CYP isoenzymes for which in vitro data had indicated potential inhibition or induction. Administration of finerenone 20mg once daily confers no risk of clinically relevant drug-drug interactions with substrates of cytochrome P450 enzymes.

In vitro inhibitory effects of ganoderic acid A on human liver cytochrome P450 enzymes

Context Ganoderic acid A (GAA) is usually used to prevent cancers or other diseases, which make it likely to be used with other drugs metabolized by cytochromes P450. Objective This study investigates the effect of GAA on eight major cytochrome P450 isoforms in human liver microsomes. Material and method The effects of GAA (100 μM) on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs) with specific substrates for the CYPs, and the enzyme kinetic parameters were calculated. Results The results showed that GAA inhibited the activity of CYP3A4, 2D6, and 2E1, but did not affect other isoforms. The inhibition of CYP3A4, 2D6, and 2E1 was concentration-dependent with IC50 values of 15.05, 21.83, and 28.35 μM, respectively. Additionally, GAA was not only a non-competitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2D6 and 2E1, with Ki values of 7.16, 10.07, and 13.45 μM. Meanwhile, the inhibition of CYP3A4 was time-dependent, with the KI/Kinact value of 7.91/0.048 μM/min. Discussion and conclusion The in vitro study indicated that GAA has the potential to result in drug-drug interactions with other drugs metabolized by CYP3A4, 2D6, and 2E1. Further clinical studies are needed for the identification of this interaction.

ADMET Evaluation in Drug Discovery. 19. Reliable Prediction of Human Cytochrome P450 Inhibition Using Artificial Intelligence Approaches.

Adverse effects induced by drug-drug interactions may result in early termination of drug development or even withdrawal of drugs from the market, and many drug-drug interactions are caused by the inhibition of cytochrome P450 (CYP450) enzymes. Therefore, the accurate prediction of the inhibition capability of a given compound against a specific CYP450 isoform is highly desirable. In this study, three ensemble learning methods, including random forest, gradient boosting decision tree, and eXtreme gradient boosting (XGBoost), and two deep learning methods, including deep neural networks and convolutional neural networks, were used to develop classification models to discriminate inhibitors and noninhibitors for five major CYP450 isoforms (1A2, 2C9, 2C19, 2D6, and 3A4). The results demonstrate that the ensemble learning models generally give better predictions than the deep learning models for the external test sets. Among all of the models, the XGBoost models achieve the best classification capability (average prediction accuracy of 90.4%) for the test sets, which even outperform the previously reported model developed by the multitask deep autoencoder neural network (88.5%). The Shapley additive explanation method was then used to interpret the models and analyze the misclassified molecules. The important molecular descriptors given by our models are consistent with the structural preferences for inhibitors of different CYP450 isoforms, which may provide valuable clues to detect potential drug-drug interactions in the early stage of drug discovery.

In vitro Inhibitory Effects of Cynaroside on Human Liver Cytochrome P450 Enzymes

Introduction: Cynaroside is a biological component isolated from Lonicera japonica Thunb, and it possesses numerous pharmacological activities. However, whether cynaroside affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. The purpose of this study was to investigate the effects of cynaroside on 8 major CYP isoforms in human liver microsomes (HLMs). Methods: In this study, the inhibitory effects of cynaroside on the 8 human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using HLMs. Results: The results showed that cynaroside inhibited the activity of CYP1A2, 3A4, and 2C9, with IC50 values of 21.74, 15.88, and 16.58 μmol/L, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that cynaroside was not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP1A2 and 2C9, with Ki values of 7.33, 11.60, and 8.09 μmol/L, respectively. In addition, cynaroside is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.049/11.62 μmol/L–1min–1. Conclusion: The in vitro studies of cynaroside with CYP isoforms indicate that cynaroside has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, 3A4, and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.

In Vitro Metabolism of Auriculasin and Its Inhibitory Effects on Human Cytochrome P450 and UDP-Glucuronosyltransferase Enzymes.

Auriculasin has a wide range of pharmacological effects, including anticancer and anti-inflammatory effects. In this work, we explored the metabolic characteristics and inhibitory effect of auriculasin against cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes in vitro. Auriculasin inhibited UGT1A6, UGT1A8, UGT1A10, UGT2B7, CYP2C9, and CYP3A4 strongly at a concentration of 100 μM. Different species showed significant differences in auriculasin metabolism, and metabolic characteristics were similar between pig and human. We identified seven metabolites, and hydroxylated auriculasin was the main metabolite. In addition, CYP2D6, CYP2C9, CYP2C19, and CYP2C8 were the major CYP isoforms involved in the metabolism of auriculasin. Molecular docking studies showed that noncovalent interactions between auriculasin and the CYPs are dominated by hydrogen bonding, π-π stacking, and hydrophobic interactions. Our in vitro study provides insights into the pharmacological and toxicological mechanisms of auriculasin.

In vitro inhibitory effects of kaempferitrin on human liver cytochrome P450 enzymes

Context: Kaempferitrinis (KF) is a bioactive flavonoid and possesses numerous pharmacological activities. However, whether KF affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.Objective: This study investigates the effects of KF on eight major CYP isoforms in human liver microsomes (HLMs).Materials and methods: In vitro, HLMs were used to investigate the inhibitory effects of KF (100 μM) on the eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8), and corresponding probe substrates were used. Enzyme kinetic studies (0–50 μM of KF) were conducted to determine the inhibition mode of KF on CYP enzymes.Results: The results showed that KF inhibited the activity of CYP1A2, 3A4, and 2C9, with IC50 values of 20.56, 13.87, and 14.62 μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that KF was not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP1A2 and 2C9, with Ki values of 7.11, 10.24, and 7.58 μM, respectively. In addition, KF is a time-dependent inhibitor for CYP3A4 with KI/Kinact value of 10.85/0.036 min/μM.Discussion: The in vitro studies of KF with CYP isoforms indicate that KF has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, 3A4, and 2C9.Conclusion: It is recommended that KF should not be used with other drugs metabolized by CYP1A2, 3A4, and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.

Genetic diversity of cytochrome P450 3A with different metabolic activity in domestic cats.

Knowledge on genetic polymorphisms of metabolising enzymes including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP3A131, one of the major CYP isoforms in the feline liver and small intestine. Eight non-synonymous variants and one synonymous variant of feline CYP3A131 were identified in 29 cats. A major non-synonymous type was not observed. Metabolic parameters (Km and Vmax) of dibenzylfluorescein hydroxylation were ranged within about 2 times for the identified non-synonymous variants by using a heterologous coexpression system of CYP3A131 and feline cytochrome P450 reductase in Escherichia coli. The results confirmed the polymorphic nature of CYP3A131 as a basis for effective application of medicines and prevention of adverse reactions in the treatment of domestic cats.

Genetic diversity of cytochrome P450 1A2 with different metabolic activities in domestic cats.

Knowledge of genetic polymorphisms of metabolizing enzymes of medical drugs and xenobiotics including cytochrome P450 (CYP) is very limited in cats. We investigated polymorphisms in CYP1A2, one of the major CYP isoforms in the feline liver. Wild-type and three non-synonymous polymorphic variants, but no synonymous variant, were identified in feline CYP1A2 in 50 alleles of domestic cats in Japan. Metabolic parameters, Km and Vmax, of ethoxyresorufin hydroxylation by CYP1A2 were shown to range within two times for identified non-synonymous variants by using a heterologous coexpression system. The results confirmed the polymorphic nature of CYP1A2 as a basis for effective application of medicines and prevention of adverse reactions in the treatment of domestic cats as well as for hereditary disorders.

Genetic diversity of cytochrome P450 2A with different metabolic activities in domestic cats.

Knowledge of genetic polymorphisms of cytochrome P450 (CYP), the most important xenobiotic metabolizing enzyme, is very limited in cats. Preliminarily, we investigated genetic polymorphisms in CYP2A13, one of the major CYP isoforms in the liver and lung. Four synonymous and three non-synonymous polymorphic variants were identified in feline CYP2A13 in domestic cats in Japan, without an obvious major type. Metabolic parameters, Km and Vmax, of coumarin hydroxylation of CYP2A13 were shown to range within two times for the identified non-synonymous polymorphic variants by using heterologous coexpression system in Escherichia coli. The results confirmed the polymorphic nature of CYP2A13 as a basis for effective application of medicines and prevention of adverse reactions in treatment of domestic cats.

Minor contribution of CYP3A5 to the metabolism of hepatitis C protease inhibitor paritaprevir in vitro

Paritaprevir (PTV) is a non-structural protein 3/4A protease inhibitor developed for the treatment of hepatitis C disease as a fixed dose combination of ombitasvir (OBV) and ritonavir (RTV) with or without dasabuvir.The aim of this study was to evaluate the effects of cytochrome P450 (CYP) 3A5 on in vitro PTV metabolism using human recombinant CYP3A4, CYP3A5 (rCYP3A4, rCYP3A5) and human liver microsomes (HLMs) genotyped as either CYP3A5*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3.The intrinsic clearance (CLint, Vmax/Km) for the production of a metabolite from PTV in rCYP3A4 was 1.5 times higher than that in rCYP3A5. The PTV metabolism in CYP3A5*1/*1 and CYP3A5*1/*3 HLMs expressing CYP3A5 was comparable to that in CYP3A5*3/*3 HLMs, which lack CYP3A5.CYP3A4 expression level was significantly correlated with PTV disappearance rate and metabolite formation. In contrast, there was no such correlation found for CYP3A5 expression level.This study represents that the major CYP isoform involved in PTV metabolism is CYP3A4, with CYP3A5 having a minor role in PTV metabolism. The findings of the present study may provide foundational information on PTV metabolism, and may further support dosing practices in HCV-infected patients prescribed PTV-based therapy.