Assessment of the inhibition risk of chlorophenol substances on cytochrome P450 via cocktail inhibition assays.

Chlorophenols (CPs) are widespread pollutants in nature. CPs have raised significant concern due to their potential hepatotoxic effects on humans. This research aimed to ascertain the inhibitory potential of eleven CPs (2-CP, 3-CP, 4-CP, 2,4-DCP, 2,3,4-TCP, 2,4,5-TCP, 2,4,6-TCP, 2,3,4,5-TeCP, 2,3,4,6-TeCP, 2,3,5,6-TeCP, and PCP) on nine human CYP isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4). The CPs that inhibit the activity of CYP isoforms were detected with human liver microsomes (HLM) using a cocktail approach in vitro. The results demonstrated that trichlorophenols, tetrachlorophenols, and PCP strongly inhibited CYP2C8 and CYP2C9. The half inhibition concentration (IC50) value of 2,3,4,6-TeCP and PCP for CYP2C8 inhibition was 27.3 µM and 12.3 µM, respectively. The IC50 for the inhibition of 2,4,6-TCP, 2,3,4,6-TeCP and PCP towards CYP2C9 were calculated to be 30.3 µM, 5.8 µM and 2.2 µM, respectively. 2,3,4,6-TeCP, and PCP exhibited non-competitive inhibition towards CYP2C8. 2,4,6-TCP, 2,3,4,6-TeCP, and PCP exhibited competitive inhibition towards CYP2C9. The inhibition kinetics parameters (Ki) were 51.51 µM, 22.28 µM, 37.86 µM, 7.27 µM, 0.68 µM for 2,3,4,6-TeCP-CYP2C8, PCP-CYP2C8, 2,4,6-TCP-CYP2C9, 2,3,4,6-TeCP-CYP2C9, PCP-CYP2C9, respectively. This study also defined clear structure-activity relationships (SAR) of CPs on CYP2C8, supported by molecular docking studies. Overall, CPs were found to cause inhibitory effects on CYP isoforms in vitro, and this finding may provide a basis for CPs focused on CYP isoforms inhibition endpoints.

Cytochrome P450 inhibition to decrease dosage and costs of venetoclax and ibrutinib: A proof-of-concept case study.

The inhibition of cytochrome P450 (CYP) enzymes is the most frequent cause of drug-drug interactions. Many safe, inexpensive and widely available therapeutic drugs can inhibit CYP enzymes (e.g., azoles). Also, the specific potency of inhibition and the targeted CYP enzyme have been well described (e.g., itraconazole strongly inhibits CYP enzyme 3A4 and, in turn, CYP3A4 metabolizes venetoclax and ibrutinib). CYP enzyme inhibitors increase the plasma concentration of other drugs via shared metabolic pathways. We herein present the effects of inhibiting CYP enzymes with itraconazole-venetoclax for the treatment of refractory acute myeloid leukaemia, as well as itraconazole-ibrutinib to treat steroid-refractory acute graft vs. host disease in the same patient. Both of the patient's conditions responded completely. This appears to be a feasible strategy that decreases treatment costs by 75%. Previous Food and Drug Administration recommendations and clinical data support these subsequent dose reductions. Eleven months after the transplant, the patient remains in complete response and with no minimal residual disease. Another patient had been effectively treated before with CYP enzyme inhibition prior to venetoclax-itraconazole administration for orbital myeloid sarcoma. Thus, this case study furthers information on the CYP enzyme inhibition strategy when associated with another costly drug, ibrutinib. The CYP enzyme inhibition strategy could be applied to many more anticancer drugs (e.g., ruxolitinib and ponatinib) and facilitate the availability of expensive oncological treatments in low- and middle-income countries. Also, this strategy could be further generalized by using different CYP enzyme inhibitors with varied pharmacokinetic and pharmacodynamic properties (i.e., grapefruit, azoles and clarithromycin).

Risk assessment of the inhibition of hydroxygenkwanin on human and rat cytochrome P450 by cocktail method.

Hydroxygenkwanin (HGK), a natural flavonoid extracted from the buds of Daphne genkwa Sieb.et Zucc. (Thymelaeaceae), possesses a wide range of pharmacological activities, including anti-inflammatory, antibacterial and anticancer. However, the inhibitory effect of HGK on cytochrome P450 (CYP) remains unclear. This study investigated the potential inhibitory effects of HGK on CYP1A2, 2B1/6, 2C9/11, 2D1/6, 2E1 and 3A2/4 enzymes in human and rat liver microsomes (HLMs and RLMs) by the cocktail approach. HGK exhibited no time-dependent inhibition of CYP activities in HLMs and RLMs. Enzyme inhibition kinetics indicated that HGK was not only a competitive inhibitor of human CYP1A2 and 2C9, but also competitively inhibited rat CYP1A2 and 2C11 activities, with Ki value at 0.84 ± 0.03, 8.09 ± 0.44, 2.68 ± 0.32 and 8.35 ± 0.31 µM, respectively. Further studies showed that the inhibitory effect of HGK on CYP enzymes was weaker than that of diosmetin, which may be related to the substitution of hydroxyl and methoxy in the A and B rings of the flavone skeleton. Therefore, the low Ki values of HGK for CYP1A2 and 2C may lead to potential drug-drug interactions and toxicity.

Structure-based virtual screening of CYP1A1 inhibitors: towards rapid tier-one assessment of potential developmental toxicants.

Cytochrome P450 1A1 (CYP1A1) metabolizes estrogens, melatonin, and other key endogenous signaling molecules critical for embryonic/fetal development. The enzyme has increasing expression during pregnancy, and its inhibition or knockout increases embryonic/fetal lethality and/or developmental problems. Here, we present a virtual screening model for CYP1A1 inhibitors based on the orthosteric and predicted allosteric sites of the enzyme. Using 1001 reference compounds with CYP1A1 activity data, we optimized the decision thresholds of our model and classified the training compounds with 68.3% balanced accuracy (91.0% sensitivity and 45.7% specificity). We applied our final model to 11 known CYP1A1 orthosteric binders and related compounds, and found that our ranking of the known orthosteric binders generally agrees with the relative activity of CYP1A1 in metabolizing these compounds. We also applied the model to 22 new test compounds with unknown/unclear CYP1A1 inhibitory activity, and predicted 16 of them are CYP1A1 inhibitors. The CYP1A1 potency and modes of inhibition of these 22 compounds were experimentally determined. We confirmed that most predicted inhibitors, including drugs contraindicated during pregnancy (amiodarone, bicalutamide, cyproterone acetate, ketoconazole, and tamoxifen) and environmental agents suspected to be endocrine disruptors (bisphenol A, diethyl and dibutyl phthalates, and zearalenone), are indeed potent inhibitors of CYP1A1. Our results suggest that virtual screening may be used as a rapid tier-one method to screen for potential CYP1A1 inhibitors, and flag them out for further experimental evaluations.

Do Inhibitory Metabolites Impact DDI Risk Assessment? Analysis of in vitro and in vivo Data from NDA Reviews Between 2013 and 2018.

Evaluating the potential of new drugs and their metabolites to cause drug-drug interactions (DDIs) is critical for understanding drug safety and efficacy. Although multiple analyses of proprietary metabolite testing data have been published, no systematic analyses of metabolite data collected according to current testing criteria have been conducted. To address this knowledge gap, 120 new molecular entities approved between 2013 and 2018 were reviewed. Comprehensive data on metabolite-to-parent area under the curve ratios (AUCM /AUCP ), inhibitory potency of parent and metabolites, and clinical DDIs were collected. Sixty-four percent of the metabolites quantified in vivo had AUCM /AUCP  ≥ 0.25 and 75% of these metabolites were tested for cytochrome P450 (CYP) inhibition in vitro, resulting in 15 metabolites with potential DDI risk identification. Although 50% of the metabolites with AUCM /AUCP  < 0.25 were also tested in vitro, none of them showed meaningful CYP inhibition potential. The metabolite percentage of plasma total radioactivity cutoff of ≥ 10% did not appear to add value to metabolite testing strategies. No relationship between metabolite versus parent drug polarity and inhibition potency was observed. Comparison of metabolite and parent maximum concentration (Cmax ) divided by inhibition constant (Ki ) values suggested that metabolites can contribute to in vivo DDIs and, hence, quantitative prediction of clinical DDI magnitude may require both parent and metabolite data. This systematic analysis of metabolite data for newly approved drugs supports an AUCM /AUCP cutoff of ≥ 0.25 to warrant metabolite in vitro CYP screening to adequately characterize metabolite inhibitory DDI potential and support quantitative DDI predictions.

An automated cocktail method for in vitro assessment of direct and time-dependent inhibition of nine major cytochrome P450 enzymes - application to establishing CYP2C8 inhibitor selectivity.

We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-ß-glucuronide and clopidogrel acyl-ß-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-ß-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-ß-glucuronide was a strong (>90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 µM, while the selectivity of clopidogrel acyl-ß-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 µM, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting time-dependent inhibition. Moreover, gemfibrozil 1-O-ß-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.

Preliminary in vitro assessment of pharmacokinetic drug-drug interactions of EST64401 and EST64514, two sigma-1 receptor antagonists.

EST64401 and EST64514 are two selective sigma-1 receptor ligands that showed a good profile in a lead optimization process for oral pain treatment. Their potential for pharmacokinetic-based drug-drug interactions was assessed to anticipate clinical interactions.Both compounds showed a low potential for CYP inhibition with percentages of inhibition <50% at 1 µM in recombinant human CYPs (CYP1A2, 2C9, 2C19, 2D6 and 3A4) and IC50 ≥75 µM for CYP3A4 and 2D6 in human liver microsomes.No CYP induction was observed for CYP1A2, 2B6 and 3A4 at concentrations ≤25 µM (EST64401) or ≤50 µM (EST64514) in human hepatocytes using as endpoints CYP activities and mRNA levels.More than one enzyme participated in compound metabolism. The main enzymes involved were CYP3A4 for EST64401 and CYP2D6 besides CYP3A4 for EST64514.Neither EST64401 nor EST64514 seemed to be substrates of P-gp or BCRP in Caco-2 cells (efflux ratio ≤2). Transporter inhibition was observed at concentrations ≥20 µM; EST64401 only inhibiting P-gp at higher concentrations (≥125 µM).Preliminary in vitro interaction studies suggest a similar profile for EST64401 and EST64514. Therefore, other properties will have to be considered for compound differentiation and selection for further development.

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of nafithromycin, a next generation lactone ketolide antibiotic.

Nafithromycin is a next generation lactone ketolide antibiotic slated to enter phase III clinical development in India for the treatment of CABP as a shorter 800 mg-OD X3 day therapeutic regimen. Nafithromycin exhibits potent activity against MDR Streptococcus pneumoniae including erythromycin and telithromycin-resistant resistant strains. Older macrolides/ketolides are reported to be potent inhibitors of CYP3A4/5. To facilitate comparative assessment of drug-drug interaction potential, CYP inhibitory activities of nafithromycin was evaluated in comparison with clarithromycin, telithromycin, cethromycin and solithromycin. CYP inhibitory activities were assessed against key CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and CYP3A4/5) using human liver microsomes. Additionally, time-dependent inhibition (TDI), metabolism-based inhibition (MBI) and k inact /K I activities were also investigated for CYP3A4/5. Nafithromycin did not inhibit key CYP enzymes and was found to be a weak inhibitor of CYP3A4/5. Comparator antibiotics were found to be potent inhibitors with 2- to 50-fold leftward shifts in CYP3A4/5 IC50 values, while such shift was not noted for nafithromycin. k inact /K I ratio of nafithromycin was 3- to 153-fold lower than comparator drugs, further substantiating its lower affinity for CYP3A4/5. In sum, weaker inhibition and lower k inact /K I ratio for CYP3A4/5, points towards nafithromycin's lower propensities towards clinical drug-drug interactions as compared to other macrolides/ketolides antibiotics.

Risk assessment of the chiral pesticide fenamiphos in a human model: Cytochrome P450 phenotyping and inhibition studies.

Fenamiphos (FS) is a chiral organophosphate pesticide that is used to control nematodes in several crops. Enantioselective differences may be observed in FS activity, bioaccumulation, metabolism, and toxicity. Humans may be exposed to FS through occupational and chronic (food, water, and environmental) exposure. FS may cause undesirable CYP450 pesticide-drug interactions, which may impact human health. Here, the CYP450 isoforms involved in enantioselective FS metabolism were identified, and CYP450 inhibition by rac-FS, (+)-FS, and (-)-FS was evaluated to obtain reliable information on enantioselective FS risk assessment in humans. CYP3A4 and CYP2E1 metabolized FS enantiomers, and CYP2B6 may participate in rac-FS metabolism. In addition, rac-FS, (+)-FS, and (-)-FS were reversible competitive CYP1A2, CYP2C19, and CYP3A4/5 inhibitors. High stereoselective inhibition potential was verified; rac-FS and (-)-FS strongly inhibited and (+)-FS moderately inhibited CYP1A2. Stereoselective differences were also detected for CYP2C19 and CYP3A4/5, which were strongly inhibited by rac-FS, (+)-FS, and (-)-FS. Our results indicated a high potential for CYP450 drug-pesticide interactions, which may affect human health. The lack of stereoselective research on the effect of chiral pesticides on the activity of CYP450 isoforms highlights the importance of assessing the risks of such pesticides in humans.

Assessment of effects of repeated oral doses of fedratinib on inhibition of cytochrome P450 activities in patients with solid tumors using a cocktail approach.

PURPOSE: Fedratinib, an oral selective kinase inhibitor with activity against both wild type and mutationally activated Janus kinase 2, has been approved for the treatment of adult patients with intermediate-2 or high-risk myelofibrosis by the US Food and Drug Administration. In vitro studies indicated that fedratinib was an inhibitor of several cytochrome P450 (CYP) enzymes. The primary objective of this study was to evaluate the effects of repeated doses of fedratinib on the activity of CYP2D6, CYP2C19, and CYP3A4 in patients with solid tumors using a CYP probe cocktail. METHODS: An open-label, one-sequence, two-period, two-treatment crossover study was conducted. Patients were administered a single oral dose cocktail of metoprolol (100 mg), omeprazole (20 mg), and midazolam (2 mg) used as probe substrates for CYP2D6, CYP2C19, and CYP3A4 enzyme activities, respectively, without fedratinib on Day -1 or with fedratinib on Day 15. RESULTS: Coadministration of 500 mg once-daily doses of fedratinib for 15 days increased the mean area under the plasma concentration-time curve from time zero to infinity following a single-dose cocktail containing metoprolol (CYP2D6 substrate), omeprazole (CYP2C19 substrate), and midazolam (CYP3A4 substrate) by 1.77-fold (90% confidence interval [CI] 1.27-2.47) for metoprolol, 2.82-fold (90% CI 2.26-3.53) for omeprazole, and 3.84-fold (90% CI 2.62-5.63) for midazolam, respectively. The mean plasma Day 14/Day 1 ratio of 4ß-hydroxycholesterol, an endogenous biomarker of CYP3A4 activity, was 0.59 (90% CI 0.54-0.66), suggesting a net inhibition of CYP3A4 by fedratinib. CONCLUSION: Fedratinib is a weak inhibitor of CYP2D6, and a moderate inhibitor of CYP2C19 and CYP3A4. These results serve as the basis for dose modifications of these CYP substrate drugs when co-administered with fedratinib.

Assessment of in vitro inhibitory effects of novel anti MRSA benzoquinolizine fluoroquinolone WCK 771 (levonadifloxacin) and its metabolite on human liver cytochrome P450 enzymes.

WCK 771 (INN: levonadifloxacin) is a novel antibacterial agent belonging to benzoquinolizine subclass of fluoroquinolones which is under clinical development as a parenteral formulation and its prodrug WCK 2349 (INN: alalevonadifloxacin) as an oral option. Both the drugs have been approved recently in India based on phase III trial completed for ABSSSI.In vitro CYP inhibition potential of levonadifloxacin and its sulfate metabolite (WCK 2146) were assessed in this study. The inhibitory effects of levonadifloxacin and its sulfate metabolite were assessed for seven key human liver CYP isoforms 1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 using human liver microsome (HLM) employing validated LC-MS/MS method.The results showed that levonadifloxacin and its metabolite did not inhibit enzyme activity of any of the key CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) even at supra therapeutic concentrations (12-24X, Clinical Cmax: 25-35µg/mL).These in vitro CYP inhibition studies of levonadifloxacin and its sulfate metabolite indicate lack of potential for pharmacokinetic drug interactions of levonadifloxacin when co-administered with drugs which are substrate of these isoforms. Therefore, further clinical studies evaluating CYP mediated drug-drug interactions are not warranted for levonadifloxacin and alalevonadifloxacin.

Preliminary in Vitro Assessment of the Potential of EST64454, a Sigma-1 Receptor Antagonist, for Pharmacokinetic Drug-Drug Interactions.

EST64454 is a selective sigma-1 receptor ligand intended for orally administered pain treatment that showed a promising profile in the lead optimization process. As part of the preliminary compound profiling, the potential for future drug-drug interactions was explored in vitro. Both direct and time-dependent CYP inhibition for CYP1A2, 2C9, 2C19, 2D6 and 3A4 was studied in human liver microsomes. EST64454 showed a low potential for CYP inhibition (IC50 between 100 and 1000 µM) and as time-dependent inhibitor (IC50 shift mainly around 1). CYP induction studies with HepaRG™ cells revealed no CYP induction at concentrations ≤50 µM, as shown by the CYP1A2, 3A4 and 2B6 activities measured. Reaction phenotyping was assessed after incubation with recombinant human enzymes. Although a very low metabolism was observed, several enzymes catalyzed the formation of metabolites, including CYP3A4, 2C19 and flavin monooxygenases (FMO) 1 and 3. EST64454 was not a P-glycoprotein (P-gp) substrate and was highly permeable in Caco-2 cells. P-gp inhibition was only observed at 200 µM, the highest concentration studied. Preliminary studies suggest that neither CYP nor P-gp interaction of EST64454 would be of any concern for further development. At later stages, the interaction kinetics and the clinical relevance of these findings will be thoroughly evaluated.

Assessment of Inhibition of Bovine Hepatic Cytochrome P450 by 43 Commercial Bovine Medicines Using a Combination of In Vitro Assays and Pharmacokinetic Data from the Literature.

BACKGROUND: There has been a lack of information about the inhibition of bovine medicines on bovine hepatic CYP450 at their commercial doses and dosing routes. OBJECTIVE: The aim of this work was to assess the inhibition of 43 bovine medicines on bovine hepatic CYP450 using a combination of in vitro assay and Cmax values from pharmacokinetic studies with their commercial doses and dosing routes in the literature. METHODS: Those drugs were first evaluated through a single point inhibitory assay at 3 µM in bovine liver microsomes for six specific CYP450 metabolisms, phenacetin o-deethylation, coumarin 7- hydroxylation, tolbutamide 4-hydroxylation, bufuralol 1-hydroxylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation. When the inhibition was greater than 20% in the assay, IC50 values were then determined. The potential in vivo bovine hepatic CYP450 inhibition by those drugs was assessed using a combination of the IC50 values and in vivo Cmax values from pharmacokinetic studies at their commercial doses and administration routes in the literature. RESULTS: Fifteen bovine medicines or metabolites showed in vitro inhibition on one or more bovine hepatic CYP450 metabolisms with different IC50 values. Desfuroylceftiour (active metabolite of ceftiofur), nitroxinil and flunixin have the potential to inhibit one of the bovine hepatic CYP450 isoforms in vivo at their commercial doses and administration routes. The rest of the bovine medicines had low risks of in vivo bovine hepatic CYP450 inhibition. CONCLUSION: This combination of in vitro assay and in vivo Cmax data provides a good approach to assess the inhibition of bovine medicines on bovine hepatic CYP450.

Myclobutanil enantioselective risk assessment in humans through in vitro CYP450 reactions: Metabolism and inhibition studies.

Myclobutanil is a chiral triazole fungicide that is employed worldwide. Although enantiomers have the same physical-chemical properties, they may differ in terms of activity, metabolism, and toxicity. This investigation consisted of in vitro enantioselective metabolism studies that employed a human model to assess the risks of myclobutanil in humans. A LC-MS/MS enantioselective method was developed and validated. The enzymatic kinetic parameters (VMAX, KMapp, and CLINT) determined for in vitro rac-myclobutanil and S-(+)-myclobutanil metabolism revealed enantioselective differences. Furthermore, human CYP450 enzymes did not metabolize R-(-)-myclobutanil. The predicted in vivo toxicokinetic parameters indicated that S-(+)-myclobutanil may be preferentially eliminated by the liver and suffer the first-pass metabolism effect. However, because CYP450 did not metabolize R-(-)-myclobutanil, this enantiomer could reach the systemic circulation and stay longer in the human body, potentially causing toxic effects. The CYP450 isoforms CYP2C19 and CYP3A4 were involved in rac-myclobutanil and S-(+)-myclobutanil metabolism. Although there were differences in the metabolism of the myclobutanil enantiomers, in vitro inhibition studies did not show significant enantioselective differences. Overall, the present investigation suggested that myclobutanil moderately inhibits CYP2D6 and CYP2C9 in vitro and strongly inhibits CYP3A and CYP2C19 in vitro. These results provide useful scientific information for myclobutanil risk assessment in humans.

Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of ZYTP1, a novel poly (ADP-ribose) polymerase inhibitor.

ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications. The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1. ZYTP1 IC50 for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC50 of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam. The mean IC50 values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3-58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC50 of depropylated metabolite was 11.95-24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC50 shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075 min-1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively. Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.

Assessment of CYP-Mediated Drug Interactions for Evocalcet, a New Calcimimetic Agent, Based on In Vitro Investigations and a Cocktail Study in Humans.

Evocalcet is a novel calcimimetic agent for the treatment of secondary hyperparathyroidism (SHPT). This study evaluated the effects of evocalcet on inhibition and induction of cytochrome P450 (CYP) isozymes. Although drug interactions arising from reversible inhibition of CYP isozymes by evocalcet were considered unlikely based on the results of in vitro studies and static model analyses, the potential for evocalcet to cause time-dependent inhibition of CYP3A or induction of several CYP isozymes could not be ruled out. Therefore, a clinical drug-drug interaction (DDI) study to evaluate the effects of evocalcet on the pharmacokinetics (PKs) of probe substrates for CYP isozymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP3A) was conducted in healthy male volunteers using a novel cocktail combination. Evocalcet did not significantly affect the PKs of the probe substrates, confirming that CYP-mediated interactions were unlikely.

In Vitro Assessment of Potential for CYP-Inhibition-Based Drug-Drug Interaction Between Vonoprazan and Clopidogrel.

BACKGROUND AND OBJECTIVES: It was recently proposed that CYP-mediated drug-drug interactions (DDIs) of vonoprazan with clopidogrel and prasugrel can attenuate the antiplatelet actions of the latter two drugs. Clopidogrel is metabolized to the pharmacologically active metabolite H4 and its isomers by multiple CYPs, including CYP2C19 and CYP3A4. Therefore, to investigate the possibility of CYP-based DDIs, in vitro metabolic inhibition studies using CYP probe substrates or radiolabeled clopidogrel and human liver microsomes (HLMs) were conducted in this work. METHODS: Reversible inhibition studies focusing on the effects of vonoprazan on CYP marker activities and the formation of the [14C]clopidogrel metabolite H4 were conducted with and without pre-incubation using HLMs. Time-dependent inhibition (TDI) kinetics were also measured. RESULTS: It was found that vonoprazan is not a significant direct inhibitor of any CYP isoforms (IC50 ≥ 16 µM), but shows the potential for TDI of CYP2B6, CYP2C19, and CYP3A4/5. This TDI was weaker than the inhibition induced by the corresponding reference inhibitors ticlopidine, esomeprazole, and verapamil, based on the measured potencies (kinact/KI ratio and the R2 value). In a more direct in vitro experiment, vonoprazan levels of up to 10 µM (a 100-fold higher concentration than the plasma Cmax of 75.9 nM after taking 20 mg once daily for 7 days) did not suppress the formation of the active metabolite H4 or other oxidative metabolites of [14C]clopidogrel in a reversible or time-dependent manner. Additionally, an assessment of clinical trials and post-marketing data suggested no evidence of a DDI between vonoprazan and clopidogrel. CONCLUSIONS: The body of evidence shows that the pharmacodynamic DDI reported between vonoprazan and clopidogrel is unlikely to be caused by the inhibition of CYP2B6, CYP2C19, or CYP3A4/5 by vonoprazan.

Inhibition of Cytochrome P450 Activities by Extracts of Hyptis verticillata Jacq.: Assessment for Potential HERB-Drug Interactions.

Understanding the potential for adverse drug reactions (ADRs), from herb-drug interactions, is a key aspect of medicinal plant safety, with particular relevance for public health in countries where medicinal plant use is highly prevalent. We undertook an in-depth assessment of extracts of Hyptis verticillata Jacq., via its impact on activities of key cytochrome P450 (CYP) enzymes (CYPs 1A1, 1A2, 1B1, 3A4 and 2D6), its antioxidant properties (determined by DPPH assays) and chemical characterisation (using LC-MS). The dried plant aqueous extract demonstrated potent inhibition of the activities of CYPs 1A1 (7.6 µg/mL), 1A2 (1.9 µg/mL), 1B1 (9.4 µg/mL) and 3A4 (6.8 µg/mL). Further analysis of other crude extracts demonstrated potent inhibition of CYP1A2 activity for a dried plant ethanol extract (1.5 µg/mL), fresh plant ethanol extract (3.9 µg/mL), and moderate activity for a fresh plant aqueous extract (27.8 µg/mL). All four extracts demonstrated strong antioxidant activity, compared to the positive control (ascorbic acid, 1.3 µg/mL), with the dried plant ethanol extract being the most potent (1.6 µg/mL). Analysis of the dried plant aqueous extract confirmed the identity of seven phytochemicals, five lignans and two triterpenes. Individual screening of these phytochemicals against the activity of CYP1A2 identified yatein as a moderate inhibitor (71.9 µM), likely to contribute to the plant extract's potent bioactivity. Further analysis on the impact of this plant on key drug metabolizing enzymes in vivo appears warranted for likely ADRs, as well as furthering development as a potential chemopreventive agent.

Species differences between rat and human in vitro metabolite profile, in vivo predicted clearance, CYP450 inhibition and CYP450 isoforms that metabolize benzanthrone: Implications in risk assessment.

Benzanthrone (BNZ) is a polycyclic aromatic hydrocarbon found in industrial effluent causing skin, respiratory, gastrointestinal, genitourinary, nervous and hemopoietic toxicity. While its toxicity has been well studied, its metabolism in humans has not been investigated. The aim of this study was to characterize species differences in the in vitro metabolism of BNZ in rat and human liver microsomes and to identify the CYP isoforms involved in its metabolism. Upon incubation in liver microsomes, BNZ was found to be a direct substrate of phase I metabolism in both rat and human, undergoing oxidation and reduction. The Km in rat, 11.62 ± 1.49 µM, was two-fold higher than humans (5.97 ± 0.83 µM) suggesting higher affinity for human CYPs. Further, incubation with human rCYPs, BNZ was found to be substrate of multiple CYPs. The predicted in vivo hepatic clearance was 63.55 and 18.91 mL/min/kg in rat and human, respectively, indicating BNZ to be a high clearance compound. BNZ was found to be a moderate inhibitor of human CYP1A2. BNZ metabolism by multiple CYPs indicates that single enzyme genetic polymorphism is unlikely to have profound effect on the toxicokinetics of BNZ and default uncertainty factor of 3.16 might be sufficient to capture the intraspecies kinetic variability.