Midostaurin drug interaction profile: a comprehensive assessment of CYP3A, CYP2B6, and CYP2C8 drug substrates, and oral contraceptives in healthy participants.

PURPOSE: Midostaurin, approved for treating FLT-3-mutated acute myeloid leukemia and advanced systemic mastocytosis, is metabolized by cytochrome P450 (CYP) 3A4 to two major metabolites, and may inhibit and/or induce CYP3A, CYP2B6, and CYP2C8. Two studies investigated the impact of midostaurin on CYP substrate drugs and oral contraceptives in healthy participants. METHODS: Using sentinel dosing for participants' safety, the effects of midostaurin at steady state following 25-day (Study 1) or 24-day (Study 2) dosing with 50 mg twice daily were evaluated on CYP substrates, midazolam (CYP3A4), bupropion (CYP2B6), and pioglitazone (CYP2C8) in Study 1; and monophasic oral contraceptives (containing ethinylestradiol [EES] and levonorgestrel [LVG]) in Study 2. RESULTS: In Study 1, midostaurin resulted in a 10% increase in midazolam peak plasma concentrations (Cmax), and 3-4% decrease in total exposures (AUC). Bupropion showed a 55% decrease in Cmax and 48-49% decrease in AUCs. Pioglitazone showed a 10% decrease in Cmax and 6% decrease in AUC. In Study 2, midostaurin resulted in a 26% increase in Cmax and 7-10% increase in AUC of EES; and a 19% increase in Cmax and 29-42% increase in AUC of LVG. Midostaurin 50 mg twice daily for 28 days ensured that steady-state concentrations of midostaurin and the active metabolites were achieved by the time of CYP substrate drugs or oral contraceptive dosing. No safety concerns were reported. CONCLUSION: Midostaurin neither inhibits nor induces CYP3A4 and CYP2C8, and weakly induces CYP2B6. Midostaurin at steady state has no clinically relevant PK interaction on hormonal contraceptives. All treatments were well tolerated.

Interplay of UDP-Glucuronosyltransferase and CYP2C8 for CYP2C8 Mediated Drug Oxidation and Its Impact on Drug-Drug Interaction Produced by Standardized CYP2C8 Inhibitors, Clopidogrel and Gemfibrozil.

BACKGROUND AND OBJECTIVE: Early investigations into drug-drug interactions (DDIs) involving cytochrome P450 2C8 (CYP2C8) have highlighted the complexity of interactions between CYP2C8 substrate drugs, including montelukast, desloratadine, pioglitazone, repaglinide, and cerivastatin (the latter two being OATP1B1 substrates), and standardized CYP2C8 inhibitors such as clopidogrel (Clop) and gemfibrozil (Gem). These interactions have proven challenging to predict based solely on simple CYP inhibition. A hypothesis has emerged suggesting that these substrate drugs first distribute to UDP-glucuronosyltransferase (UGT) before undergoing oxidation by CYP2C8, resulting in bidirectional elimination. The process of drug distribution to UGT is believed to significantly impact these DDIs. This study aims to explore the intricate interplay between UGT and CYP2C8 in the context of DDIs involving CYP2C8 substrates affected by Clop and Gem. METHODS: Plasma-level data for the unchanged drug and its metabolite, drawn from the respective literature, formed the basis of our analysis. We evaluated the enzymatic inhibitory activities of DDIs and utilized simulations to estimate plasma levels of the unchanged victim drug and its metabolite in each DDI. This was accomplished by employing a functional relationship that considered the fractional contributions of CYP2C8 and UGT to clearance, perpetrator-specific inhibitory activities against CYP2C8, and drug distribution to UGT. RESULTS: Our findings emphasize the pivotal role of UGT-mediated distribution in the context of CYP2C8 substrate metabolism, particularly in the complex DDIs induced by Clop and Gem. In these DDIs, Gem exerts inhibitory effects on both UGT and CYP2C8, whereas Clop (specifically its metabolite, Clop-COOH) solely targets CYP2C8. Importantly, the inhibition of CYP2C8 by both Clop and Gem is achieved through a non-competitive mechanism, driven by the actions of their acyl-glucuronides. Clop and Gem exhibit inhibition activities accounting for 85% (pAi,CYP2C8 = 7) and 93% (pAi,CYP2C8 = 15), respectively. In contrast, Gem's inhibition of UGT is relatively modest (50%, pAi,UGT(d) = 2), and it operates through a non-specific, competitive process in drug distribution to UGT. Within this context, our UGT-CYP2C8 interplay model offers an accurate means of predicting the alterations resulting from DDIs, encompassing changes in plasma levels of the unchanged drug and its metabolites, as well as shifts in metabolite formation rates. Our analysis highlights the critical importance of considering the fractional contributions of CYP2C8 and UGT to the victim drug's clearance (fm,CYP2C8; fm,UGT) in DDI prediction. Furthermore, our examination of DDIs involving OATP1B1 substrate drugs underscores that accounting for the hepatic uptake transporters' role in the liver is superfluous in DDI prediction. CONCLUSION: These findings substantially enhance our comprehension of CYP2C8-mediated oxidation and DDIs, holding crucial implications for drug development and the planning of clinical trials involving these inhibitors.

Effect of SHR0302 on the pharmacokinetics of CYP3A4, CYP2C8, CYP2C9 and CYP2C19 probe substrates in healthy volunteers: A cocktail analysis.

AIMS: This study evaluated the effects of SHR0302 on the pharmacokinetics of cytochrome P450 (CYP) probe substrates. METHODS: We performed a single-centre, open-label, three-period drug-drug interaction (DDI) study in 24 healthy subjects (NCT05392127). Subjects received a single oral dose of 5 mg warfarin (CYP2C9), 20 mg omeprazole (CYP2C19) and 15 mg midazolam (CYP3A4) on Days 1, 8 and 22, and received 0.5 mg repaglinide (CYP2C8) on Days 7, 14 and 28. Multiple oral doses of 8 mg SHR0302 were administered once daily from Day 8 to Day 28. RESULTS: The exposure of S-warfarin and repaglinide were comparable before and after SHR0302 administration. AUC of midazolam was not affected by SHR0302, whereas the administration of SHR0302 slightly decreased the Cmax of midazolam by 7.6% (single dose) and 15.7% (once daily for 14 days). The AUC0-t , AUC0-inf , and Cmax of omeprazole were slightly decreased after a single dose of SHR0302 by 19.2%, 21.8% and 23.5%, respectively. In the presence of SHR0302 for 14 days, the AUC0-t , AUC0-inf , and Cmax of omeprazole were marginally reduced by 3.0%, 16.4% and 8.3%, respectively. According to the induction mechanism of the CYP enzyme, for the investigation of the induction effect, the results of multiple administrations of the perpetrator were more reliable than those of the single dose. CONCLUSIONS: The results demonstrated that co-administration of SHR0302 8 mg once daily is unlikely to have a clinically meaningful effect on the exposure of drugs metabolized by CYP3A4, CYP2C8, CYP2C9 and CYP2C19 in healthy subjects.

Comprehensive Analysis of the Sorafenib-Associated Druggable Targets on Differential Gene Expression and ceRNA Network in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the predominant pathological type of liver cancer. Several therapeutic treatments, including sorafenib and regorafenib, have only modestly improved survival in patients with HCC. The aim of this study was to investigate the expression profiles and the regulation of competitive endogenous RNAs (ceRNAs) of the sorafenib-related target genes in HCC. Based on clinical information and expression profiles of HCC clinical samples from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, shared differentially expressed genes (DEGs) were analyzed and identified. Sorafenib-associated DEGs (SADs) were obtained by intersecting the DEGs with the sorafenib target genes from SuperTarget database. The expression patterns of SADs were verified in the Oncomine database. The biological functions of the SADs were annotated by gene set enrichment analysis (GSEA). In addition, a ceRNA network associated with SADs was constructed. Long non-coding RNAs (lncRNAs) in network that were significantly associated with overall survival were identified as prognosis of patients by Cox regression analysis. Finally, the expression levels of prognostic genes in HCC tissues and cell lines were verified using qRT-PCR. Gene expression differential analysis yielded a total of 146 common DEGs were obtained, including 21 upregulated and 125 downregulated DEGs. Among them, ten SADs were detected to be differentially expressed between tumor and normal tissues, including AXL, CYP2C19, CYP2C8, CYP2C9, CYP3A4, FGFR2, GMNN, PDGFRA, and TTK. GSEA analysis grouped them into three categories by function. The first category (CYP2C19, CYP2C8, CYP2C9 and CYP3A4) and second category (GMNN, TTK and EGER2) had the opposite roles in the enriched terms and pathways, while the third class (AXL and PDGFRA) has enrichment terms and pathways that intersect with those of the first and second categories. A ceRNA network associated with SADs was also constructed including 49 lncRNAs, 14 miRNAs, and 8 mRNAs. Three of these lncRNAs, SNHG7, GAS5 and HCP5, were found upregulated in HCC tissues and to be independent predictors in HCC patients. Significant correlations were found in expression between the prognostic lncRNAs and SADs. Ten SADs were systematically identified using expression data from HCC and normal tissues from TCGA and GEO datasets. GSEA analysis provided us with insight into the function of SADs. In the future, we will continue to explore the mechanisms of coordinated regulation of SADs-related prognostic lncRNAs and SADs at the ceRNA axis level and their potential functions in the development of HCC.

Rottlerin renders a selective and highly potent CYP2C8 inhibition to impede EET formation for implication in cancer therapy.

CYP2C8 is a crucial CYP isoform responsible for the metabolism of xenobiotics and endogenous molecules. CYP2C8 converts arachidonic acid to epoxyeicosatrienoic acids (EETs) that cause cancer progression. Rottlerin possess significant anticancer actions. However, information on its CYP inhibitory action is lacking in the literature and therefore, we aimed to explore the same using in silico, in vitro, and in vivo approaches. Rottlerin showed highly potent and selective CYP2C8 inhibition (IC50 < 0.1 µM) compared to negligible inhibition (IC50 > 10 µM) for seven other experimental CYPs in human liver microsomes (HLM) (in vitro) using USFDA recommended index reactions. Mechanistic studies reveal that rottlerin could reversibly (mixed-type) block CYP2C8. Molecular docking (in silico) results indicate a strong interaction could occur between rottlerin and the active site of human CYP2C8. Rottlerin boosted the plasma exposure of repaglinide and paclitaxel (CYP2C8 substrates) by delaying their metabolism using the rat model (in vivo). Multiple-dose treatment of rottlerin with CYP2C8 substrates lowered the CYP2C8 protein expression and up-regulated & down-regulated the mRNA for CYP2C12 & CYP2C11 (rat homologs), respectively, in rat liver tissue. Rottlerin substantially hindered the EET formation in HLM. Overall results of rottlerin on CYP2C8 inhibition and EET formation insinuate further exploration for cancer therapy.

3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction.

Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. The 3D spheroid PHHs from three different donors were treated for 4 days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole, or ß-naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and transporters P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1, and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of five- to sixfold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, whereas the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, whereas the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2, and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug-metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.

Characterisation of cytochrome P450 isoenzyme activity in sheep liver and placental microsomes.

INTRODUCTION: Drug metabolism during pregnancy is a complex process that involves maternal, placental and fetal sites of metabolism. Indeed, there is a lack of clarity provided from drug metabolism in human pregnancy due to ethical limitations. Large animal models of human pregnancy provide an opportunity to quantify activity of phase 1 drug metabolism mediated by cytochrome P450 (CYP) enzymes in the maternal, placental, and fetal compartments. Herein, we have validated a comprehensive assay to quantify maternal, placental, and fetal CYP activity. METHODS: Isolated microsomes from sheep maternal liver, placenta, and fetal liver (140d gestation, term = 150d) were incubated with CYP-specific probe drugs to quantify the activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A. Inhibition studies were performed to validate specificity of probe drugs. The validated assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1 and CYP3A were active in maternal liver. In contrast, only CYP1A2, CYP2C8 and CYP2D6 were active in the placenta, whereas CYP2B6, CYP2C8 and CYP2D6 were active in the fetal liver. Of the placental-specific CYPs validated, CYP1A2 increased in type A compared with type D placentomes, whereas CYP2C8 activity increased in type B compared with type A and C. DISCUSSION: This study has established conditions for compartment-specific CYP activity in the sheep maternal-placental-fetal unit using a validated and standardised experimental workflow. Compartment- and placentome type-specific CYP activity are important considerations when examining drug metabolism in the maternal-placental-fetal unit and in determining the impact of pregnancy complications.

Hydroxychloroquine is Metabolized by Cytochrome P450 2D6, 3A4, and 2C8, and Inhibits Cytochrome P450 2D6, while its Metabolites also Inhibit Cytochrome P450 3A in vitro.

This study aimed to explore the cytochrome P450 (CYP) metabolic and inhibitory profile of hydroxychloroquine (HCQ). Hydroxychloroquine metabolism was studied using human liver microsomes (HLMs) and recombinant CYP enzymes. The inhibitory effects of HCQ and its metabolites on nine CYPs were also determined in HLMs, using an automated substrate cocktail method. Our metabolism data indicated that CYP3A4, CYP2D6, and CYP2C8 are the key enzymes involved in HCQ metabolism. All three CYPs formed the primary metabolites desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) to various degrees. Although the intrinsic clearance (CLint) value of HCQ depletion by recombinant CYP2D6 was > 10-fold higher than that by CYP3A4 (0.87 versus 0.075 µl/min/pmol), scaling of recombinant CYP CLint to HLM level resulted in almost equal HLM CLint values for CYP2D6 and CYP3A4 (11 and 14 µl/min/mg, respectively). The scaled HLM CLint of CYP2C8 was 5.7 µl/min/mg. Data from HLM experiments with CYP-selective inhibitors also suggested relatively equal roles for CYP2D6 and CYP3A4 in HCQ metabolism, with a smaller contribution by CYP2C8. In CYP inhibition experiments, HCQ, DCQ, DHCQ, and the secondary metabolite didesethylchloroquine were direct CYP2D6 inhibitors, with 50% inhibitory concentration (IC50) values between 18 and 135 µM. HCQ did not inhibit other CYPs. Furthermore, all metabolites were time-dependent CYP3A inhibitors (IC50 shift 2.2-3.4). To conclude, HCQ is metabolized by CYP3A4, CYP2D6, and CYP2C8 in vitro. HCQ and its metabolites are reversible CYP2D6 inhibitors, and HCQ metabolites are time-dependent CYP3A inhibitors. These data can be used to improve physiologically-based pharmacokinetic models and update drug-drug interaction risk estimations for HCQ. SIGNIFICANCE STATEMENT: While CYP2D6, CYP3A4, and CYP2C8 have been shown to mediate chloroquine biotransformation, it appears that the role of CYP enzymes in hydroxychloroquine (HCQ) metabolism has not been studied. In addition, little is known about the CYP inhibitory effects of HCQ. Here, we demonstrate that CYP2D6, CYP3A4, and CYP2C8 are the key enzymes involved in HCQ metabolism. Furthermore, our findings show that HCQ and its metabolites are inhibitors of CYP2D6, which likely explains the previously observed interaction between HCQ and metoprolol.

Physiologically Based Pharmacokinetic Modeling for Quantitative Prediction of Exposure to a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Substrate of Cytochrome P450 and Aldehyde Oxidase, Using Chimeric Mice With Humanized Liver.

In a previous study on the human mass balance of DS-1971a, a selective NaV1.7 inhibitor, its CYP2C8-dependent metabolite M1 was identified as a human disproportionate metabolite. The present study assessed the usefulness of pharmacokinetic evaluation in chimeric mice grafted with human hepatocytes (PXB-mice) and physiologically based pharmacokinetic (PBPK) simulation of M1. After oral administration of radiolabeled DS-1971a, the most abundant metabolite in the plasma, urine, and feces of PXB-mice was M1, while those of control SCID mice were aldehyde oxidase-related metabolites including M4, suggesting a drastic difference in the metabolism between these mouse strains. From a qualitative perspective, the metabolite profile observed in PXB-mice was remarkably similar to that in humans, but the quantitative evaluation indicated that the area under the plasma concentration-time curve (AUC) ratio of M1 to DS-1971a (M1/P ratio) was approximately only half of that in humans. A PXB-mouse-derived PBPK model was then constructed to achieve a more accurate prediction, giving an M1/P ratio (1.3) closer to that in humans (1.6) than the observed value in PXB-mice (0.69). In addition, simulated maximum plasma concentration and AUC values of M1 (3429 ng/ml and 17,116 ng·h/ml, respectively) were similar to those in humans (3180 ng/ml and 18,400 ng·h/ml, respectively). These results suggest that PBPK modeling incorporating pharmacokinetic parameters obtained with PXB-mice is useful for quantitatively predicting exposure to human disproportionate metabolites. SIGNIFICANCE STATEMENT: The quantitative prediction of human disproportionate metabolites remains challenging. This paper reports on a successful case study on the practical estimation of exposure (C max and AUC) to DS-1971a and its CYP2C8-dependent, human disproportionate metabolite M1, by PBPK simulation utilizing pharmacokinetic parameters obtained from PXB-mice and in vitro kinetics in human liver fractions. This work adds to the growing knowledge regarding metabolite exposure estimation by static and dynamic models.

A comprehensive analysis of six forms of cytochrome P450 2C (CYP2C) in pigs.

Pigs are an important species used in drug metabolism studies; however, the cytochromes P450 (P450s or CYPs) have not been fully investigated in pigs.In this study, pig CYP2C32, CYP2C33, CYP2C34, CYP2C36, CYP2C42, and CYP2C49 cDNAs were isolated and found to contain open reading frames of 490 or 494 amino acids that shared 64-82% sequence identity with human CYP2C8/9/18/19.Pig CYP2C genes formed a gene cluster in a genomic region that corresponded to that of the human CYP2C cluster; an additional gene cluster was formed by pig CYP2C33a and CYP2C33b distant from the first cluster but located in the same chromosome.Among the tissues analysed, these pig CYP2C mRNAs were preferentially expressed in liver, small intestine, and/or kidney; pig CYP2C49, CYP2C32, CYP2C34, and CYP2C33 mRNAs were the most abundant CYP2C mRNAs in liver, jejunum, ileum, and kidney, respectively.Metabolic assays showed that pig CYP2C proteins (heterologously expressed in Escherichia coli) metabolised typical human CYP2C substrates diclofenac, warfarin, and/or omeprazole.The results suggest that these pig CYP2Cs are functional enzymes able to metabolise human CYP2C substrates in liver and small intestine, just as human CYP2Cs do.

Inhibition of CYP2C8 by Acyl Glucuronides of Gemfibrozil and Clopidogrel: Pharmacological Significance, Progress and Challenges.

The lipid-regulating drug gemfibrozil is a useful medication for reducing high cholesterol and triglycerides in the blood. In addition to oxidation, it undergoes extensive glucuronidation to produce gemfibrozil acyl glucuronide, which is a known mechanism-based inactivator of cytochrome P450 (CYP) 2C8. Such selective and time-dependent inhibition results in clinically important drug-drug interactions (DDI) with the drugs metabolized by CYP2C8. Similarly, the acyl glucuronide of clopidogrel, a widely used antiplatelet agent, is a potent time-dependent inhibitor of CYP2C8 that demonstrated significant DDI with the substrates of CYP2C8. Current progress in atomic-level understanding mostly involves studying how different drugs bind and undergo oxidation in the active site of CYPs. It is not clear how an acyl glucuronide metabolite of the drug gemfibrozil or clopidogrel interacts in the active site of CYP2C8 and selectively inhibit the enzyme. This mini-review summarizes the current knowledge on some of the important clinical DDI caused by gemfibrozil and clopidogrel due to the inhibition of CYP2C8 by acyl glucuronide metabolites of these drugs. Importantly, it examines recent developments and potential applications of structural biology tools to elucidate the binding and orientation of gemfibrozil acyl glucuronide and clopidogrel acyl glucuronide in the active site near heme that contributes to the inhibition and inactivation of CYP2C8.

Drug metabolism and drug transport of the 100 most prescribed oral drugs.

Safe and effective use of drugs requires an understanding of metabolism and transport. We identified the 100 most prescribed drugs in six countries and conducted a literature search on in vitro data to assess contribution of Phase I and II enzymes and drug transporters to metabolism and transport. Eighty-nine of the 100 drugs undergo drug metabolism or are known substrates for drug transporters. Phase I enzymes are involved in metabolism of 67 drugs, while Phase II enzymes mediate metabolism of 18 drugs. CYP3A4/5 is the most important Phase I enzyme involved in metabolism of 43 drugs followed by CYP2D6 (23 drugs), CYP2C9 (23 drugs), CYP2C19 (22 drugs), CYP1A2 (14 drugs) and CYP2C8 (11 drugs). More than half of the drugs (54 drugs) are known substrates for drug transporters. P-glycoprotein (P-gp) is known to be involved in transport of 30 drugs, while breast cancer resistance protein (BCRP) facilitates transport of 11 drugs. A considerable proportion of drugs are subject to a combination of Phase I metabolism, Phase II metabolism and/or drug transport. We conclude that the majority of the most frequently prescribed drugs depend on drug metabolism or drug transport. Thus, understanding variability of drug metabolism and transport remains a priority.

Evaluation of Safety and Clinically Relevant Drug-Drug Interactions with Tucatinib in Healthy Volunteers.

BACKGROUND AND OBJECTIVE: Tucatinib is approved for treatment of human epidermal growth factor receptor 2-positive metastatic breast cancer. Understanding potential drug-drug interactions (DDIs) informs proper dosing when co-administering tucatinib with other therapies. The aim of this study was to evaluate DDIs between tucatinib and metabolizing enzymes and transporters in healthy volunteers. METHODS: Parts A-C assessed the impact of itraconazole (cytochrome P450 [CYP] 3A4 inhibitor), rifampin (CYP3A4/CYP2C8 inducer), or gemfibrozil (CYP2C8 inhibitor) on the pharmacokinetics of a single 300 mg dose of tucatinib administered orally and its primary metabolite, ONT-993. Parts D and E assessed the effect of steady-state tucatinib on the pharmacokinetics of repaglinide (CYP2C8 substrate), tolbutamide (CYP2C9 substrate), midazolam (CYP3A4 substrate), and digoxin (P-glycoprotein substrate). RESULTS: Tucatinib area under the concentration-time curve from time 0 extrapolated to infinity (AUC0-inf) increased by ~ 1.3- and 3.0-fold with itraconazole and gemfibrozil, respectively, and decreased by 48% with rifampin, indicating that tucatinib is metabolized primarily by CYP2C8, and to a lesser extent via CYP3A. Tucatinib was a strong inhibitor of CYP3A (midazolam AUC0-inf increased 5.7-fold), a weak inhibitor of CYP2C8 and P-glycoprotein, and had no impact on CYP2C9-mediated metabolism in humans. Tucatinib was well tolerated, alone and with co-administered drugs. CONCLUSION: The potential DDIs identified here may be mitigated by avoiding concomitant use of tucatinib with strong CYP3A inducers, moderate CYP2C8 inducers, CYP3A substrates with a narrow therapeutic window (modifying substrate dose where concomitant use is unavoidable), and strong CYP2C8 inhibitors (decreasing tucatinib dose where concomitant use is unavoidable), or by reducing the dose of P-glycoprotein substrates with a narrow therapeutic window. TRIAL REGISTRATION: This trial (NCT03723395) was registered on October 29, 2018.

Cytochrome P450 oxidase 2J inhibition suppresses choroidal neovascularization in mice.

INTRODUCTION: Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) leads to blindness. It has been widely reported that increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) diets reduce CNV. Of the three major pathways metabolizing ω-3 (and ω-6 LCPUFA), the cyclooxygenase and lipoxygenase pathways generally produce pro-angiogenic metabolites from ω-6 LCPUFA and anti-angiogenic ones from ω-3 LCPUFA. Howevehr, cytochrome P450 oxidase (CPY) 2C produces pro-angiogenic metabolites from both ω-6 and ω-3 LCPUFA. The effects of CYP2J2 products on ocular neovascularization are still unknown. Understanding how each metabolic pathway affects the protective effect of ω-3 LCPUFA on retinal neovascularization may lead to therapeutic interventions. OBJECTIVES: To investigate the effects of LCPUFA metabolites through CYP2J2 pathway and CYP2J2 regulation on CNV both in vivo and ex vivo. METHODS: The impact of CYP2J2 overexpression and inhibition on neovascularization in the laser-induced CNV mouse model was assessed. The plasma levels of CYP2J2 metabolites were measured by liquid chromatography and tandem mass spectroscopy. The choroidal explant sprouting assay was used to investigate the effects of CYP2J2 inhibition and specific LCPUFA CYP2J2 metabolites on angiogenesis ex vivo. RESULTS: CNV was exacerbated in Tie2-Cre CYP2J2-overexpressing mice and was associated with increased levels of plasma docosahexaenoic acids. Inhibiting CYP2J2 activity with flunarizine decreased CNV in both ω-6 and ω-3 LCPUFA-fed wild-type mice. In Tie2-Cre CYP2J2-overexpressing mice, flunarizine suppressed CNV by 33 % and 36 % in ω-6, ω-3 LCPUFA diets, respectively, and reduced plasma levels of CYP2J2 metabolites. The pro-angiogenic role of CYP2J2 was corroborated in the choroidal explant sprouting assay. Flunarizine attenuated ex vivo choroidal sprouting, and 19,20-EDP, a ω-3 LCPUFA CYP2J2 metabolite, increased sprouting. The combined inhibition of CYP2J2 with flunarizine and CYP2C8 with montelukast further enhanced CNV suppression via tumor necrosis factor-α suppression. CONCLUSIONS: CYP2J2 inhibition augmented the inhibitory effect of ω-3 LCPUFA on CNV. Flunarizine suppressed pathological choroidal angiogenesis, and co-treatment with montelukast inhibiting CYP2C8 further enhanced the effect. CYP2 inhibition might be a viable approach to suppress CNV in AMD.

Metabolism of the active carfentanil metabolite, 4-Piperidinecarboxylic acid, 1-(2-hydroxy-2-phenylethyl)-4-[(1-oxopropyl)phenylamino]-, methyl ester in vitro.

Carfentanil, a µ-opioid receptor (MOR) agonist with an analgesic potency 10,000 times that of morphine, is extensively metabolized to norcarfentanil (M1), 4-Piperidinecarboxylic acid, 1-(2-hydroxy-2-phenylethyl)-4-[(1-oxopropyl)phenylamino]-, methyl ester (M0 in this article), and other low abundant metabolites in human hepatocytes and liver/lung microsomes. M0 possessed comparable MOR activity to carfentanil, and accounted for approximately 12 % of the total carfentanil metabolite formation in human liver microsomes (HLMs). Little is known about the subsequent elimination of M0. This study investigated its metabolic pathway in HLMs, separation and preliminary identification of metabolites by liquid chromatography-tandem mass spectrometry, and possible involvement of cytochrome P450 enzymes in M0 metabolism with kinetic analysis. M0 produced 9 metabolites via N-dealkylation (M1), oxidation (M3, M6-9), N-dealkylation followed by oxidation (M2 and M4), and glucuronidation (M5). Formation of the major metabolite M1 fitted typical Michaelis-Menten kinetics. Recombinant human CYP3A5 showed the highest activity toward M1 formation followed by CYP3A4 and CYP2C8, while M8 was primarily formed by CYP3A4 followed by CYP2C19 and CYP2C8. These findings reveal the main involvement of CYP3A5 and 3A4 in human hepatic elimination of M0 with a kinetic profile similar to carfentanil which may inform development of treatment protocols for carfentanil exposure.

Effects of ginkgo leaf tablet on the pharmacokinetics of rosiglitazone in rats and its potential mechanism.

CONTEXT: Ginkgo leaf tablet (GLT), a traditional Chinese herbal formula, is often combined with rosiglitazone (ROS) for type 2 diabetes mellitus treatment. However, the drug-drug interaction between GLT and ROS remains unknown. OBJECTIVE: To investigate the effects of GLT on the pharmacokinetics of ROS and its potential mechanism. MATERIALS AND METHODS: The pharmacokinetics of 10 mg/kg ROS with 100/200 mg/kg GLT as single-dose and 10-day multiple-dose administration were investigated in Sprague-Dawley rats. In vitro, the effects of GLT on the activity of CYP2C8 and CYP2C9 were determined in recombinant human yeast microsomes and rat liver microsomes with probe substrates. RESULTS: The t1/2 of ROS increased from 2.14 ± 0.38 (control) to 2.79 ± 0.37 (100 mg/kg) and 3.26 ± 1.08 h (200 mg/kg) in the single-dose GLT administration. The AUC0-t (139.69 ± 45.46 vs. 84.58 ± 39.87 vs. 66.60 ± 15.90 h·µg/mL) and t1/2 (2.75 ± 0.70 vs. 1.99 ± 0.44 vs. 1.68 ± 0.35 h) decreased significantly after multiple-dose GLT treatment. The IC50 values of quercetin, kaempferol, and isorhamnetin, GLT main constituents, were 9.32, 7.67, and 11.90 µmol/L for CYP2C8, and 27.31, 7.57, and 4.59 µmol/L for CYP2C9. The multiple-dose GLT increased rat CYP2C8 activity by 44% and 88%, respectively. DISCUSSION AND CONCLUSIONS: The metabolism of ROS is attenuated in the single dose of GLT by inhibiting CYP2C8 and CYP2C9 activity, and accelerated after the multiple-dose GLT treatment via inducing CYP2C8 activity in rats, indicating that the clinical dose of ROS should be adjusted when co-administrated with GLT.

High-resolution accurate mass approach to characterization of SCO-267 metabolites using liquid chromatography hybrid quadrupole Orbitrap mass spectrometry.

RATIONALE: SCO-267 is a potent full agonist of G-protein-coupled receptor 40. As a promising therapeutic agent for type 2 diabetes mellitus, it is necessary to elucidate its metabolite profiles during the stage of drug development for safety considerations. METHODS: The in vitro metabolism was investigated by incubating SCO-267 (5 µM) with liver microsomes and hepatocytes (rat and human). For in vivo metabolism, SCO-267 (10 mg/kg) was orally administered to rats and plasma samples were collected. The metabolites were identified via measurements of accurate mass, elemental composition and product ions using liquid chromatography coupled to hybrid quadrupole Orbitrap high-resolution mass spectrometry (LC-Orbitrap-MS). RESULTS: A total of 19 metabolites were structurally identified. M2 (hydroxyl-SCO-267), M15 (SCO-267-acyl-glucuronide), M16 (desmethyl-SCO-267) and M17 (desneopentyl-SCO-267) were verified with reference standards. M2, M11, M16 and M17 were the major metabolites originating from hydroxylation, O-demethylation and N-dealkylation, respectively. Phenotyping study with recombinant human P450 enzymes demonstrated that hydroxylation (M2 and M11) was mainly catalyzed by CYP2C8 and 3A4; demethylation (M16) was mainly catalyzed by CYP2D6, and less catalyzed by CYP2C8 and 3A4; and N-dealkylation (M17) was exclusively triggered by CYP3A4. CONCLUSIONS: Hydroxylation, O-demethylation, N-dealkylation and acyl glucuronidation were the major metabolic pathways of SCO-267. This study is the first to discover the metabolic fates of SCO-267, which provides a basis for safety assessment of this drug candidate.

Elimination of tucatinib, a small molecule kinase inhibitor of HER2, is primarily governed by CYP2C8 enantioselective oxidation of gem-dimethyl.

PURPOSE: Tucatinib, a small molecule for the treatment of metastatic HER2-positive breast cancer, was extensively metabolized in humans to multiple oxidative metabolites. To fully understand the elimination and biotransformation pathways of tucatinib, we investigated the in vitro and in vivo metabolism of tucatinib, and also conducted a Phase I trial using [14C]tucatinib. METHODS: To identify the responsible enzymes for tucatinib clearance, we investigated the in vitro metabolism of tucatinib including enzyme phenotyping, which facilitated the discovery of several metabolites in human and monkey plasma and excreta, in particular M1 (ONT-993, an aliphatic hydroxylated metabolite). Stereoselective formation of M1 was further investigated in vitro, in vivo, and in silico. RESULTS: In humans, approximately 86% of the total radiolabeled dose was recovered in feces and 4% in urine; in plasma, approximately 76% of radioactivity circulated as parent drug, with 19% attributed to multiple metabolites. The primary isoforms responsible for the elimination of tucatinib were CYP2C8 and CYP3A4/5. CYP2C8 was shown to possess sole catalytic activity for the formation of M1, whereas CYP3A4/5 and aldehyde oxidase catalyzed the formation of the remaining metabolites. Subsequent investigation revealed that M1 was formed in a stereoselective manner. Examination of the enantiomeric ratio of M1 stereoisomers observed in humans relative to cynomolgus monkeys revealed comparable results, suggesting that the enantiomers that comprise M1 were not considered to be unique or disproportionately high in human. CONCLUSION: CYP2C8 and CYP3A4/5 are the primary drug-metabolizing enzymes involved in the in vitro metabolism of tucatinib, which provided the basis to describe human disposition of tucatinib and formation of the observed metabolites.

CYP2C8-Mediated Formation of a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Cytochrome P450 and Aldehyde Oxidase Substrate.

Predicting human disproportionate metabolites is difficult, especially when drugs undergo species-specific metabolism mediated by cytochrome P450s (P450s) and/or non-P450 enzymes. This study assessed human metabolites of DS-1971a, a potent Nav1.7-selective blocker, by performing human mass balance studies and characterizing DS-1971a metabolites, in accordance with the Metabolites in Safety Testing guidance. In addition, we investigated the mechanism by which the major human disproportionate metabolite (M1) was formed. After oral administration of radiolabeled DS-1971a, the major metabolites in human plasma were P450-mediated monoxidized metabolites M1 and M2 with area under the curve ratios of 27% and 10% of total drug-related exposure, respectively; the minor metabolites were dioxidized metabolites produced by aldehyde oxidase and P450s. By comparing exposure levels of M1 and M2 between humans and safety assessment animals, M1 but not M2 was found to be a human disproportionate metabolite, requiring further characterization under the Metabolites in Safety Testing guidance. Incubation studies with human liver microsomes indicated that CYP2C8 was responsible for the formation of M1. Docking simulation indicated that, in the formation of M1 and M2, there would be hydrogen bonding and/or electrostatic interactions between the pyrimidine and sulfonamide moieties of DS-1971a and amino acid residues Ser100, Ile102, Ile106, Thr107, and Asn217 in CYP2C8, and that the cyclohexane ring of DS-1971a would be located near the heme iron of CYP2C8. These results clearly indicate that M1 is the predominant metabolite in humans and a human disproportionate metabolite due to species-specific differences in metabolism. SIGNIFICANCE STATEMENT: This report is the first to show a human disproportionate metabolite generated by CYP2C8-mediated primary metabolism. We clearly demonstrate that DS-1971a, a mixed aldehyde oxidase and cytochrome P450 substrate, was predominantly metabolized by CYP2C8 to form M1, a human disproportionate metabolite. Species differences in the formation of M1 highlight the regio- and stereoselective metabolism by CYP2C8, and the proposed interaction between DS-1971a and CYP2C8 provides new knowledge of CYP2C8-mediated metabolism of cyclohexane-containing substrates.

Lovastatin enhances chemosensitivity of paclitaxel-resistant prostate cancer cells through inhibition of CYP2C8.

Chemotherapy is the mainstay of treatment for prostate cancer, with paclitaxel being commonly used for hormone-resistant prostate cancer. However, drug resistance often develops and leads to treatment failure in a variety of prostate cancer patients. Therefore, it is necessary to enhance the sensitivity of prostate cancer to chemotherapy. Lovastatin (LV) is a natural compound extracted from Monascus-fermented foods and is an inhibitor of HMG-CoA reductase (HMGCR), which has been approved by the FDA for hyperlipidemia treatment. We have previously found that LV could inhibit the proliferation of refractory cancer cells. Up to now, the effect of LV on chemosensitization and the mechanisms involved have not been evaluated in drug-resistant prostate cancer. In this study, we used prostate cancer cell line PC3 and its paclitaxel-resistant counterpart PC3-TxR as the cell model. Alamar Blue cell viability assay showed that LV and paclitaxel each conferred concentration-dependent inhibition of PC3-TxR cells. When paclitaxel was combined with LV, the proliferation of PC3-TxR cells was synergistically inhibited, as demonstrated by combination index <1. Moreover, colony formation decreased while apoptosis increased in paclitaxel plus LV group compared with paclitaxel alone group. Quantitative RT-PCR showed that the combination of paclitaxel and LV could significantly reduce the expression of CYP2C8, an important drug-metabolizing enzyme. Bioinformatics analysis from the TCGA database showed that CYP2C8 expression was negatively correlated with progression-free survival (PFS) in prostate cancer patients. Our results suggest that LV might increase the sensitivity of resistant prostate cancer cells to paclitaxel through inhibition of CYP2C8 and could be utilized as a chemosensitizer for paclitaxel-resistant prostate cancer cells.