Recombinant CYP3A4 Research Articles

Inhibition of Tacrolimus Metabolism by Cannabidiol and Its Metabolites InVitro.

Drug interactions are major causes of interindividual variability in tacrolimus exposure and effect. Tacrolimus, a widely used drug in transplant patients, is metabolized by CYP3A4 and CYP3A5. Cannabidiol (CBD) use after transplant is common. Clinical cases suggest CBD may alter tacrolimus exposure, but the mechanism of this interaction is unknown. We hypothesize that cannabidiol will inhibit tacrolimus metabolism invitro mainly through CYP3A5 inhibition. In pooled human liver microsomes (HLMs) and recombinant (r) CYP3A4 and CYP3A5 enzymes, tacrolimus (1 μM) metabolism was determined using substrate depletion method in the absence (control) and the presence of 10 μM CBD, 7-hydroxyCBD, and 7-carboxyCBD. Ketoconazole (1 μM) served as a positive control for the inhibition of CYP3A. Linear regression analyses were performed to obtain kinetic parameters of the depletion. Tacrolimus depletion half-life was 2.54, 0.922, and 0.351 min with pooled HLMs, rCYP3A4, and rCYP3A5, respectively. In pooled HLMs, CBD and 7-hydroxyCBD increased tacrolimus half-life by 0.8- and 2.3-fold (both p < 0.0001), respectively. In rCYP3A4, CBD, 7-hydroxyCBD, and ketoconazole prolonged tacrolimus half-life by 5.8-, 14-, and 7.7-fold, respectively. In rCYP3A5, CBD, 7-hydroxyCBD, and ketoconazole prolonged half-life by 29.3-, 19.7-, and 0.1-fold, respectively. In all experiments, 7-carboxyCBD had minimal effect on tacrolimus depletion. CBD and 7-hydroxyCBD inhibited tacrolimus metabolism invitro. CBD showed stronger inhibition in rCYP3A5 than rCYP3A4. The demonstrated CYP3A5 selectivity of cannabidiol may contribute to the invitro identification of CYP3A5 substrates in new drug development. Our results support the potential of a clinical drug-drug interaction between CBD and tacrolimus.

Heterotropic Activation of Cytochrome P450 3A4 by Perillyl Alcohol.

Background/Objectives: Perillyl alcohol (POH), a monoterpene natural product derived from the essential oils of plants such as perilla (Perilla frutescens), is currently in phase I and II clinical trials as a chemotherapeutic agent. In this study, we investigated the effect of POH on cytochrome P450 (CYP) activity for evaluating POH-drug interaction potential. Methods: The investigation was conducted using pooled human liver microsomes (HLMs), recombinant CYP3A4 (rCYP3A4) enzymes, and human pluripotent stem cell-derived hepatic organoids (hHOs) employing liquid chromatography-tandem mass spectrometry. Results: POH inhibited the activities of CYP2A6 and CYP2B6 with Ki of 6.35 and 3.78 μM, respectively, whereas it stimulated CYP3A4 activity in pooled HLMs incubated with midazolam (MDZ). In a direct CYP inhibition assay using HLMs, activities of CYP2C9, CYP2C19, and CYP2E1 were also inhibited by POH, with IC50 values greater than 50 μM, but those of CYP1A2, CYP2C8, CYP2D6, and CYP3A4 (testosterone) were not significantly inhibited. In pooled HLMs, the Vmax/Km value of 1'-hydroxy MDZ, but not that of 4-hydroxy MDZ, was increased 2.7-fold by 100 μM POH compared with that in the absence of POH. Moreover, stimulation of MDZ 1'-hydroxylation by CYP3A4 was observed in hHOs and rCYP3A4 with cytochrome b5 but not rCYP3A4 without cytochrome b5. Furthermore, activation of CYP3A4-mediated metabolism by POH was observed in HLMs incubated with fimasartan but not atorvastatin, buspirone, donepezil, nifedipine, or tadalafil, suggesting a substrate-dependent activation of CYP3A4 by POH. Conclusions: POH inhibits CYP2A6 and CYP2B6, but it activates CYP3A4. These findings underscore the need for further evaluation of the interactions of clinical drugs with POH.

Effects of CYP3A4 Variants on Methadone Metabolism InVitro.

In hepatic drug metabolism, cytochrome P450 (CYP450) enzymes, particularly CYP3A4, catalyze the majority of drug biotransformations, accounting for over 50% of the CYP450 family's metabolic capacity. This study aimed to assess the catalytic efficiency of 22 CYP3A4 allelic variants on the invitro oxidative metabolism of methadone. We utilized a baculovirus-insect cell expression system to produce recombinant CYP3A4 variants and subsequently assessed their catalytic activity in the N-demethylation of methadone. Of the 23 tested CYP3A4 allelic variants, CYP3A4*1 represents the wild type. Compared with CYP3A4*1, 12 variants displayed significantly lower intrinsic clearance of methadone, while 3 variants showed increased intrinsic clearance of methadone. Additionally, six variants demonstrated no significant difference in intrinsic clearance of methadone compared to CYP3A4*1, and one variant showed no detectable expression. Our evaluation of the enzymatic activity of CYP3A4 gene polymorphisms on methadone can aid in the personalized clinical use of methadone and facilitate the investigation into the relationship between genetic variations and clinical phenotypes.

Detection and Quantification of Drug-Protein Adducts in Human Liver.

Covalent protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods identifying and quantifying covalent adducts in complex matrices. This study presents a workflow that combines data-dependent and data-independent acquisition (DDA and DIA) based liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect very low abundance adducts resulting from CYP mediated drug metabolism in human liver microsomes (HLMs). HLMs were incubated with raloxifene as a model compound and adducts were detected in 78 proteins, including CYP3A and CYP2C family enzymes. Experiments with recombinant CYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested, including CYPs not subject to time-dependent inhibition by raloxifene. These data suggest adducts can be benign. DIA analysis showed variable adduct abundance in many peptides between livers, but no concomitant decrease of unadducted peptides. This study sets a new standard for adduct detection in complex samples, offering insights into the human adductome resulting from reactive metabolite exposure. The methodology presented will aid mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and those that are benign.

The Effect of Concomitant Administration of Proton Pump Inhibitors on the Pharmacokinetics of CDK4/6 Inhibitors in Rats: Implications for the Evaluation of Hepatic and Transporter-Mediated Drug-Drug Interactions.

Numerous clinical concerns have been expressed regarding the potential worsening of cyclin-dependent kinase 4/6 inhibitor effects in breast cancer patients because of co-administration of proton pump inhibitors. Hence, this study evaluated the effects of proton pump inhibitors on the pharmacokinetics of palbociclib and ribociclib in terms of cytochrome P450 (CYP)3A4 and P-glycoprotein involvement. The effects of omeprazole and rabeprazole on drug metabolism and efflux of these drugs were investigated using molecular docking, metabolic stability assay in rat liver microsomes, human recombinant CYP3A4(rCYP3A4) enzymes, and Caco-2 cell monolayers, and in vivo pharmacokinetics with omeprazole and rabeprazole in (5 and 10 mg/kg) 30 min and 7 days before orally dosing palbociclib and ribociclib (10 mg/kg). Omeprazole and rabeprazole inhibited CYP3A4 enzyme activity in rCYP3A4 baculosomes with a 50-60% inhibition at 30μM. Additionally, both omeprazole and rabeprazole (10µm) significantly reduced the P-glycoprotein-mediated drug efflux of palbociclib and ribociclib. The 7-day pretreatment of omeprazole at a dose of 10 mg/kg resulted in 24% and 26% reductions in palbociclib's mean maximum plasmaconcentration) Cmaxand area under the plasmaconcentration-time curve(AUC0-24 h), respectively. Palbociclib's pharmacokinetics were not significantly altered by the pretreatment with rabeprazole; however, ribociclib pharmacokinetics exhibited an 83.94% increase in AUC0-24 h. The findings indicate that long-term treatment with therapeutic doses of both omeprazole and rabeprazole can alter the pharmacokinetics of palbociclib and ribociclib. The co-administration of rabeprazole may alter the pharmacokinetics of palbociclib and ribociclib via CYP enzyme and P-glycoprotein inhibition.

Drug-induced enzyme activity inhibition and CYP3A4 genetic polymorphism significantly shape the metabolic characteristics of furmonertinib

This study aimed to elucidate the impact of variations in liver enzyme activity, particularly CYP3A4, on the metabolism of furmonertinib. An in vitro enzyme incubation system was established for furmonertinib using liver microsomes and recombinant CYP3A4 baculosomes, with analytes detected by LC-MS/MS. The pharmacokinetic characteristics of furmonertinib were studied in vivo using Sprague-Dawley rats. It was found that telmisartan significantly inhibited the metabolism of furmonertinib, as demonstrated by a significant increase in the AUC of furmonertinib when co-administered with telmisartan, compared to the furmonertinib-alone group. Mechanistically, it was noncompetitive in rat liver microsomes, while it was mixed competitive and noncompetitive in human liver microsomes and CYP3A4. Considering the genetic polymorphism of CYP3A4, the study further investigated its effect on the kinetics of furmonertinib. The results showed that compared to CYP3A4.1, CYP3A4.29 had significantly increased activity in catalyzing furmonertinib, whereas CYP3A4.7, 9, 10, 12, 13, 14, 18, 23, 33, and 34 showed markedly decreased activity. The inhibitory activity of telmisartan varied in CYP3A4.1 and CYP3A4.18, with IC50 values of 8.56 ± 0.90 μM and 27.48 ± 3.52 μM, respectively. The key loci affecting the inhibitory effect were identified as ARG105, ILE301, ALA370, and LEU373. Collectively, these data would provide a reference for the quantitative application of furmonertinib.

Apiole, an important constituent of parsley, is a mixed-type inhibitor of the CYP1A subfamily

Apiole (1-allyl-2,5-dimethoxy-3,4-methylenedioxybenzene) and parsley leaves ethanolic extract containing it inhibit the rat liver microsomal ethoxy- and methoxyresorufin-O-deacetylase activities associated with cytochrome P450 (CYP) 1A1 and 1A2, respectively. Cytochrome P4501A subfamily metabolizes environmental mutagens and several drugs, leading to the formation of mutagenic metabolites. Docking analysis showed that residue Phe123 within the active site of the CYP1A1 enzyme is bound to apiole through a π/π stacking of its benzene ring. In the case of 1A2, its Phe226 interacts with the dioxolane ring of apiole. Furthermore, apiole behaves as a mixed-type inhibitor of bacterial human recombinant CYP1A1. To explore one of the possible biological implications of this inhibitory effect, we tested the capacity of apiole and the parsley ethanolic extract to interfere with the mutagenicity of the promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) metabolized by CYP1A subfamily. As expected, both apiole and the plant extract reduced the number of revertant colonies of Salmonella typhimurium TA98 Ames strain after exposure to MeIQx, reaching a 78 % and 100 % reduction, respectively. Neither apiol nor parsley extract were mutagenic to the TA98 strain. We speculate that consuming apiole, a constituent of edible herbs, in conjunction with the utilization of pharmaceuticals metabolized by the CYP1A subfamily, may result in herb-drug interactions. Furthermore, the consumption of apiole by individuals who regularly ingest fresh vegetables may contribute to the low incidence of cancer observed in those who adhere to such a dietary regimen.

Identification of the human Cytochrome P450 enzymes (P450s) responsible for metabolizing infigratinib to its pharmacologically active Metabolites, BHS697, and CQM157, and assessment of their in vitro inhibition of P450s and UDP-glucuronosyltransferases (UGTs)

Infigratinib, an oral FGFR inhibitor for advanced cholangiocarcinoma, yielded two active metabolites, BHS697 and CQM157, with similar receptor affinity. Our study characterized P450s that are responsible for the metabolism of infigratinib to its two major active metabolites, BHS697 and CQM157. In vitro inhibition of P450s and UGTs by infigratinib, BHS697 or CQM157 was further investigated. The unbound apparent Km values for metabolism of infigratinib to BHS697 by HLM, human recombinant CYP2C8, CYP2C19, CYP2D6 and CYP3A4 enzymes are 4.47, 0.65, 2.50, 30.6 and 2.08 μM, while Vmax values are 90.0 pmol/min/mg protein, 0.13, 0.027, 0.81, and 0.56 pmol/min/pmol protein, respectively. The unbound apparent Km value for metabolism of infigratinib to CQM157 by HLM is 0.049 μM, while the Vmax value is 0.32 pmol/min/mg protein respectively. In HLM, infigratinib displayed moderate inhibition of CYP3A4 and CYP2C19 and weak or negligible inhibition of other P450 isoforms. BHS697 exhibited weak inhibition of CYP2B6, CYP2C9, CYP2C19 and CYP3A4, and no inhibition of CYP2C8 and CYP2D6. CQM157 moderately inhibited CYP2C9 and CYP3A4, and weakly or negligibly inhibited other P450 isoforms. Regarding UGTs, infigratinib moderately inhibited UGT1A4 and weakly inhibited UGT1A1, respectively. BHS697 weakly inhibited UGT1A1. In contrast, CQM157 moderately inhibited both UGT1A1 and UGT1A4. Our findings provide novel insights into the metabolism of and potential DDIs implicating infigratinib.

The influence of drug-induced metabolic enzyme activity inhibition and CYP3A4 gene polymorphism on aumolertinib metabolism

The purpose of this study is to clarify the drug interaction profile of aumolertinib, and the influence of CYP3A4 genetic polymorphism on aumolertinib metabolic characteristics. Through microsomal enzyme reactions, we screened 153 drugs and identified 15 that significantly inhibited the metabolism of aumolertinib. Among them, telmisartan and carvedilol exhibited potent inhibitory activities in rat liver microsomes (RLM) and human liver microsomes (HLM). In vivo, the pharmacokinetic parameters of aumolertinib, including AUC and Cmax, were significantly altered when co-administered with carvedilol, with a notable decrease in the clearance rate CLz/F. Interestingly, the pharmacokinetic parameters of the metabolite HAS-719 exhibited a similar trend as aumolertinib when co-administered. Mechanistically, both telmisartan and carvedilol exhibited a mixed-type inhibition on the metabolism of aumolertinib. Additionally, we used a baculovirus-insect cell expression system to prepare 24 recombinant CYP3A4 microsomes and obtained enzymatic kinetic parameters using aumolertinib as a substrate. Enzyme kinetic studies obtained the kinetic parameters of various CYP3A4 variant-mediated metabolism of aumolertinib. Based on the relative clearance rates, CYP3A4.4, 5, 7, 8, 9, 12, 13, 14, 17, 18, 19, 23, 24, 33, and 34 showed significantly lower clearance rates compared to the wild-type. Among the different CYP3A4 variants, the inhibitory potency of telmisartan and carvedilol on the metabolism of aumolertinib also varied. The IC50 values of telmisartan and carvedilol in CYP3A4.1 were 6.68 ± 1.76 μM and 0.60 ± 0.25 μM, respectively, whereas in CYP3A4.12, the IC50 exceeded 100 μM. Finally, we utilized adeno-associated virus to achieve liver-specific high expression of CYP3A4*1 and CYP3A4*12. In the group with high expression of the less active CYP3A4*12, the magnitude of the drug-drug interaction was significantly attenuated. In conclusion, CYP3A4 genetic polymorphism not only influences the pharmacokinetic characteristics of aumolertinib, but also the inhibitory potency of telmisartan and carvedilol on it.

Comparison of the CYP3A Selective Inhibitors CYP3cide, Clobetasol, and Azamulin for Their Potential to Distinguish CYP3A7 Activity in the Presence of CYP3A4/5.

The CYP3A7 enzyme accounts for ∼50% of the total cytochrome P450 (P450) content in fetal and neonatal livers and is the predominant P450 involved in neonatal xenobiotic metabolism. Additionally, it is a key player in healthy birth outcomes through the oxidation of dehydroepiandrosterone (DHEA) and DHEA-sulfate. The amount of the other hepatic CYP3A isoforms, CYP3A4 and CYP3A5, expressed in neonates is low but highly variable, and therefore the activity of individual CYP3A isoforms is difficult to differentiate due to their functional similarities. Consequently, a better understanding of the contribution of CYP3A7 to drug metabolism is essential to identify the risk that drugs may pose to neonates and developing infants. To distinguish CYP3A7 activity from CYP3A4/5, we sought to further characterize the selectivity of the specific CYP3A inhibitors CYP3cide, clobetasol, and azamulin. We used three substrate probes, dibenzylfluorescein, luciferin-PPXE, and midazolam, to determine the IC50 and metabolism-dependent inhibition (MDI) properties of the CYP3A inhibitors. Probe selection had a significant effect on the IC50 values and P450 inactivation across all inhibitory compounds and enzymes. CYP3cide and azamulin were both identified as MDIs and were most specific for CYP3A4. Contrary to previous reports, we found that clobetasol propionate (CP) was not an MDI of CYP3A5 but was more selective for CYP3A5 over CYP3A4/7. We further investigated CYP3cide and CP's ability to differentiate CYP3A7 activity in an equal mixture of recombinant CYP3A4, CYP3A5, and CYP3A7, and our results provide confidence of CYP3cide's and CP's ability to distinguish CYP3A7 activity in the presence of the other CYP3A isoforms. SIGNIFICANCE STATEMENT: These findings provide valuable insight regarding in vitro testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates. The results presented here also clearly demonstrate the effect that probe selection may have on CYP3A cytochrome P450 inhibition studies.

In vitro and in vivo metabolism of psilocybin's active metabolite psilocin.

In vivo, psilocybin is rapidly dephosphorylated to psilocin which induces psychedelic effects by interacting with the 5-HT2A receptor. Psilocin primarily undergoes glucuronidation or conversion to 4-hydroxyindole-3-acetic acid (4-HIAA). Herein, we investigated psilocybin's metabolic pathways in vitro and in vivo, conducting a thorough analysis of the enzymes involved. Metabolism studies were performed using human liver microsomes (HLM), cytochrome P450 (CYP) enzymes, monoamine oxidase (MAO), and UDP-glucuronosyltransferase (UGT). In vivo, metabolism was examined using male C57BL/6J mice and human plasma samples. Approximately 29% of psilocin was metabolized by HLM, while recombinant CYP2D6 and CYP3A4 enzymes metabolized nearly 100% and 40% of psilocin, respectively. Notably, 4-HIAA and 4-hydroxytryptophol (4-HTP) were detected with HLM but not with recombinant CYPs. MAO-A transformed psilocin into minimal amounts of 4-HIAA and 4-HTP. 4-HTP was only present in vitro. Neither 4-HIAA nor 4-HTP showed relevant interactions at assessed 5-HT receptors. In contrast to in vivo data, UGT1A10 did not extensively metabolize psilocin in vitro. Furthermore, two putative metabolites were observed. N-methyl-4-hydroxytryptamine (norpsilocin) was identified in vitro (CYP2D6) and in mice, while an oxidized metabolite was detected in vitro (CYP2D6) and in humans. However, the CYP2D6 genotype did not influence psilocin plasma concentrations in the investigated study population. In conclusion, MAO-A, CYP2D6, and CYP3A4 are involved in psilocin's metabolism. The discovery of putative norpsilocin in mice and oxidized psilocin in humans further unravels psilocin's metabolism. Despite limitations in replicating phase II metabolism in vitro, these findings hold significance for studying drug-drug interactions and advancing research on psilocybin as a therapeutic agent.

Effect of P. corylifolia on the pharmacokinetic profile of tofacitinib and the underlying mechanism.

This work aimed to explore the mechanisms underlying the interaction of the active furanocoumarins in P. corylifolia on tofacitinib both in vivo and in vitro. The concentration of tofacitinib and its metabolite M8 was determined using UPLC-MS/MS. The peak area ratio of M8 to tofacitinib was calculated to compare the inhibitory ability of furanocoumarin contained in the traditional Chinese medicine P. corylifolia in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4 (rCYP3A4). We found that bergapten and isopsoralen exhibited more significant inhibitory activity in RLMs than other furanocoumarins. Bergapten and isopsoralen were selected to investigate tofacitinib drug interactions in vitro and in vivo. Thirty rats were randomly allocated into 5 groups (n = 6): control (0.5% CMC-Na), low-dose bergapten (20mg/kg), high-dose bergapten (50mg/kg), low-dose isopsoralen (20mg/kg) and ketoconazole. 10mg/kg of tofacitinib was orally intervented to each rat and the concentration level of tofacitinib in the rats were determined by UPLC-MS/MS. More imporrantly, the results showed that bergapten and isopsoralen significantly inhibited the metabolism of tofacitinib metabolism. The AUC(0-t), AUC(0-∞), MRT(0-t), MRT(0-∞) and Cmax of tofacitinib increased in varying degrees compared with the control group (all p < 0.05), but CLz/F decreased in varying degrees (p < 0.05) in the different dose bergapten group and isopsoralen group. Bergapten, isopsoralen and tofacitinib exhibit similar binding capacities with CYP3A4 by AutoDock 4.2 software, confirming that they compete for tofacitinib metabolism. P. corylifolia may considerably impact the metabolism of tofacitinib, which can provide essential information for the accurate therapeutic application of tofacitinib.

Probe Substrate Dependencies in CYP3A4 Allosteric Inhibition: A Novel Molecular Mechanism Involving F-F' Loop Perturbations.

The biochemical basis for substrate dependences in apparent inhibition constant values (Ki) remains unknown. Our study aims to elucidate plausible structural determinants underpinning these observations. In vitro steady-state inhibition assays conducted using human recombinant CYP3A4 enzyme and testosterone substrate revealed that fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and pemigatinib noncompetitively inhibited CYP3A4 with apparent Ki values of 10.2 ± 1.1 and 3.3 ± 0.9 μM, respectively. However, when rivaroxaban was adopted as the probe substrate, there were 2.0- and 3.2-fold decreases in its apparent Ki values. To glean mechanistic insights into this phenomenon, erdafitinib and pemigatinib were docked to allosteric sites in CYP3A4. Subsequently, molecular dynamics (MD) simulations of apo- and holo-CYP3A4 were conducted to investigate the structural changes induced. Comparative structural analyses of representative MD frames extracted by hierarchical clustering revealed that the allosteric inhibition of CYP3A4 by erdafitinib and pemigatinib did not substantially modulate its active site characteristics. In contrast, we discovered that allosteric binding of the FGFR inhibitors reduces the structural flexibility of the F-F' loop region, an important gating mechanism to regulate access of the substrate to the catalytic heme. We surmised that the increased rigidity of the F-F' loop engenders a more constrained entrance to the CYP3A4 active site, which in turn impedes access to the larger rivaroxaban molecule to a greater extent than testosterone and culminates in more potent inhibition of its CYP3A4-mediated metabolism. Our findings suggest a potential mechanism to rationalize probe substrate dependencies in Ki arising from the allosteric noncompetitive inhibition of CYP3A4.

Introducing the Dynamic Well-Stirred Model for Predicting Hepatic Clearance and Extraction Ratio

The well-stirred model (WSM) incorporating the fraction of unbound drug (fu) to account for the effect of plasma binding on intrinsic clearance has been widely used for predicting hepatic clearance under the assumption that drug protein binding reaches equilibrium instantaneously. Our theoretical analysis reveals that the effect of protein binding on intrinsic clearance is better accounted for with the dynamic free fraction (fD), a measure of drug protein binding affinity, which leads to a putative dynamic well-stirred model (dWSM) without the instantaneous equilibrium assumption. Using recombinant CYP3A4 as the in vitro clearance system, we demonstrate that the binding effect of albumin on the intrinsic clearance of both highly bound midazolam and highly free verapamil is fully corrected by their corresponding fD values, respectively. On the other hand, fu only corrects the binding effect of albumin on the intrinsic clearance of verapamil, and yields severe over-correction of the intrinsic clearance of midazolam. The results suggest that the traditional WSM is suitable for highly free drugs like verapamil but not necessarily for highly bound drugs such as midazolam due to the violation of the instantaneous equilibrium assumption or under-estimating the true free drug concentration. In comparison, the dWSM incorporating fD holds true as long as drug elimination follows steady-state kinetics, and hence, it is more broadly applicable to drugs with different protein binding characteristics. Here we demonstrate with 36 diverse drugs, that the dWSM significantly improves the accuracy of predicting human hepatic clearance and liver extraction ratio from in vitro microsomal clearance data, highlighting the importance of drug plasma protein binding kinetics in addressing the under-prediction of hepatic clearance by the WSM.

The effect of shikonin on the metabolism of lapatinib in vitro, and in vivo

PurposeThe purpose of this study was to develop an UPLC-MS/MS assay for simultaneous determination of lapatinib and its metabolites (N-dealkylated lapatinib and O-dealkylated lapatinib) by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and to determine the interaction between shikonin and lapatinib in vitro, in vivo, in silico and its mechanism of action. MethodsA new UPLC-MS/MS method for the determination of the concentrations of lapatinib and its metabolites was developed. In vivo, Sprague-Dawley (SD) rats were given lapatinib with or without shikonin. In vitro, to study the interaction mechanism, rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4.1 were used for determining enzyme kinetics. Lastly, we used in silico molecular docking to investigate the molecular mechanism of inhibition. ResultsThe selectivity, precision, accuracy, stability, matrix effect and recovery of UPLC-MS/MS all met the requirements of quantitative analysis of biological samples. Administration of lapatinib combined with shikonin resulted in significantly increased pharmacokinetic parameters (AUC(0-t) and Cmax) of lapatinib, indicating that shikonin increased the exposure of lapatinib in rats. Moreover, in vitro kinetic measurements indicated that shikonin was a time-independent inhibitor, which inhibited the metabolism of lapatinib through a competitive mechanism in RLMs, while noncompetitive inhibition type in both HLMs and CYP3A4.1. Molecular docking analysis further verified the non-competitive inhibition of shikonin on lapatinib in CYP3A4.1. ConclusionWe developed an UPLC-MS/MS assay for simultaneous determination of lapatinib and its metabolites. It could be successfully applied to the study of pharmacokinetic interaction of shikonin on the inhibition of lapatinib metabolism in vivo and in vitro. In the end, Further studies are needed to determine if such interactions are indeed valid in humans and if the interaction is clinically relevant.

Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 μM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.

The variability in CYP3A4 activity determines the metabolic kinetic characteristics of ketamine

Ketamine is a psychotropic drug that can cause significant neurological symptoms and is closely linked to the activity of the CYP3A4 enzyme. This study aimed to examine the diversity of CYP3A4 activity affects the metabolism of ketamine, focusing on genetic variation and drug-induced inhibition. We used a baculovirus-insect cell expression system to prepare recombinant human CYP3A4 microsomes. Then, in vitro enzyme incubation systems were established and used UPLC-MS/MS to detect ketamine metabolite. In rats, we investigated the metabolism of ketamine and its metabolite in the presence of the CYP3A4 inhibitor voriconazole. Molecular docking was used to explore the molecular mechanism of inhibition. The results showed that the catalytic activity of CYP3A4.5, .17, .23, .28, and .29 significantly decreased compared to CYP3A4.1, with a minimum decrease of 3.13%. Meanwhile, the clearance rate of CYP3A4.2, .32, and .34 enhanced remarkably, ranging from 40.63% to 87.50%. Additionally, hepatic microsome incubation experiments revealed that the half-maximal inhibitory concentration (IC50) of voriconazole for ketamine in rat and human liver microsomes were 18.01 ± 1.20 µM and 14.34 ± 1.70 µM, respectively. When voriconazole and ketamine were co-administered, the blood exposure of ketamine and norketamine significantly increased in rats, as indicated by the area under the concentration-time curve (AUC) and maximum concentration (Cmax). The elimination half-life (t1/2Z) of these substances was also prolonged. Moreover, the clearance (CLz/F) of ketamine decreased, while the apparent volume of distribution (Vz/F) increased significantly. This might be attributed to the competition between voriconazole and ketamine for binding sites on the CYP3A4 enzyme. In conclusion, variations in CYP3A4 activity would result in the stratification of ketamine blood exposure.

The impacts of CYP3A4 genetic polymorphism and drug interactions on the metabolism of lurasidone

The aim of this study was to investigate the impacts of 24 variants of recombinant human CYP3A4 and drug interactions on the metabolism of lurasidone. In vitro, enzymatic reaction incubation system of CYP3A4 was established to determine the kinetic parameters of lurasidone catalyzed by 24 CYP3A4 variants. Then, we constructed rat liver microsomes (RLM) and human liver microsomes (HLM) incubation system to screen potential anti–tumor drugs that could interact with lurasidone and studied its inhibitory mechanism. In vivo, Sprague–Dawley (SD) rats were applied to study the interaction between lurasidone and olmutinib. The concentrations of the analytes were detected by ultra–performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). As the results, we found that compared with the wild-type CYP3A4, the relative intrinsic clearances vary from 355.77 % in CYP3A4.15 to 14.11 % in CYP3A4.12. A series of drugs were screened based on the incubation system, and compared to without olmutinib, the amount of ID–14283 (the metabolite of lurasidone) in RLM and HLM were reduced to 7.22 % and 7.59 %, and its IC50 were 18.83 ± 1.06 μM and 16.15 ± 0.81 μM, respectively. At the same time, it exerted inhibitory effects both through a mixed mechanism. When co-administration of lurasidone with olmutinib in rats, the AUC(0−t) and AUC(0-∞) of lurasidone were significantly increased by 73.52 % and 69.68 %, respectively, while CLz/F was observably decreased by 43.83 %. In conclusion, CYP3A4 genetic polymorphism and olmutinib can remarkably affect the metabolism of lurasidone.

Quantitative Translation of Substrate Intrinsic Clearance from Recombinant CYP1A1 to Humans.

CYP1A1 is a cytochrome P450 family 1 enzyme that is mostly expressed in the extrahepatic tissues. To understand the CYP1A1 contribution to drug clearance in humans, we examined the in vitro-in vivo extrapolation (IVIVE) of intrinsic clearance (CLint) for a set of drugs that are in vitro CYP1A1 substrates. Despite being strong in vitro CYP1A1 substrates, 82% of drugs gave good IVIVE with predicted CLint within 2-3-fold of the observed values using human liver microsomes and hepatocytes, suggesting they were not in vivo CYP1A1 substrates due to the lack of extrahepatic contribution to CLint. Only three drugs (riluzole, melatonin and ramelteon) that are CYP1A2 substrates yielded significant underprediction of in vivo CLint up to 11-fold. The fold of CLint underprediction was linearly proportional to human recombinant CYP1A1 (rCYP1A1) CLint, indicating they were likely to be in vivo CYP1A1 substrates. Using these three substrates, a calibration curve can be developed to enable direct translation from in vitro rCYP1A1 CLint to in vivo extrahepatic contributions in humans. In vivo CYP1A1 substrates are planar and small, which is consistent with the structure of the active site. This is in contrast to the in vitro substrates, which include large and nonplanar molecules, suggesting rCYP1A1 is more accessible than what is in vivo. The impact of CYP1A1 on first-pass intestinal metabolism was also evaluated and shown to be minimal. This is the first study providing new insights on in vivo translation of CYP1A1 contributions to human clearance using in vitro rCYP1A1 data.

Determine the enzymatic kinetic characteristics of CYP3A4 variants utilizing artemether-lumefantrine

Artemether-lumefantrine is an artemisinin-based combination therapy for the treatment of malaria, which are primarily metabolized by cytochrome P450 3A4. Therapeutic difference caused by gene polymorphisms of CYP3A4 may lead to uncertain adverse side effects or treatment failure. The aim of this study was to evaluate the effect of CYP3A4 gene polymorphism on artemether-lumefantrine metabolism in vitro. Enzyme kinetics assay was performed using recombinant human CYP3A4 cell microsomes. The analytes, dihydroartimisinin and desbutyl-lumefantrine, were detected by ultra-performance liquid chromatography tandem mass spectrometry. The results demonstrated that compared to CYP3A4.1, the intrinsic clearance of CYP3A4.4, 5, 9, 16, 18, 23, 24, 28, 31–34 significantly reduced for artemether (58.5%–93.3%), and CYP3A4.17 almost loss catalytic activity. Simultaneously, CYP3A4.5, 14, 17, 24 for lumefantrine were decreased by 56.1%–99.6%, and CYP3A4.11, 15, 18, 19, 23, 28, 29, 31–34 for lumefantrine was increased by 51.7%–296%. The variation in clearance rate indicated by molecular docking could be attributed to the disparity in the binding affinity of artemether and lumefantrine with CYP3A4. The data presented here have enriched our understanding of the effect of CYP3A4 gene polymorphism on artemether-lumefantrine metabolizing. These findings serve as a valuable reference and provide insights for guiding the treatment strategy involving artemether-lumefantrine.