Recombinant CYP Enzymes Research Articles

Solanidine is a sensitive and specific dietary biomarker for CYP2D6 activity

BackgroundIndividual assessment of CYP enzyme activities can be challenging. Recently, the potato alkaloid solanidine was suggested as a biomarker for CYP2D6 activity. Here, we aimed to characterize the sensitivity and specificity of solanidine as a CYP2D6 biomarker among Finnish volunteers with known CYP2D6 genotypes.ResultsUsing non-targeted metabolomics analysis, we identified 9152 metabolite features in the fasting plasma samples of 356 healthy volunteers. Machine learning models suggested strong association between CYP2D6 genotype-based phenotype classes with a metabolite feature identified as solanidine. Plasma solanidine concentration was 1887% higher in genetically poor CYP2D6 metabolizers (gPM) (n = 9; 95% confidence interval 755%, 4515%; P = 1.88 × 10–11), 74% higher in intermediate CYP2D6 metabolizers (gIM) (n = 89; 27%, 138%; P = 6.40 × 10–4), and 35% lower in ultrarapid CYP2D6 metabolizers (gUM) (n = 20; 64%, − 17%; P = 0.151) than in genetically normal CYP2D6 metabolizers (gNM; n = 196). The solanidine metabolites m/z 444 and 430 to solanidine concentration ratios showed even stronger associations with CYP2D6 phenotypes. Furthermore, the areas under the receiver operating characteristic and precision–recall curves for these metabolic ratios showed equal or better performances for identifying the gPM, gIM, and gUM phenotype groups than the other metabolites, their ratios to solanidine, or solanidine alone. In vitro studies with human recombinant CYP enzymes showed that solanidine was metabolized mainly by CYP2D6, with a minor contribution from CYP3A4/5. In human liver microsomes, the CYP2D6 inhibitor paroxetine nearly completely (95%) inhibited the metabolism of solanidine. In a genome-wide association study, several variants near the CYP2D6 gene associated with plasma solanidine metabolite ratios.ConclusionsThese results are in line with earlier studies and further indicate that solanidine and its metabolites are sensitive and specific biomarkers for measuring CYP2D6 activity. Since potato consumption is common worldwide, this biomarker could be useful for evaluating CYP2D6-mediated drug–drug interactions and to improve prediction of CYP2D6 activity in addition to genotyping.

In Vitro Metabolism and In Vivo Pharmacokinetics Profiles of Hydroxy-α-Sanshool.

Hydroxy-α-sanshool (HAS) is the predominant active compound in Zanthoxylum bungeanum Maxim (ZBM). Our present work was aimed to explore the in vitro metabolism characteristics, and in vivo pharmacokinetic (PK) profile of HAS. Plasma (human), liver microsomes, and hepatocytes (human, monkey, dog, mouse, and rat) were collected for HAS metabolism studies in vitro and HAS elimination rates in liver microsomes and hepatocytes of different species were investigated. In addition, five recombinant human CYP enzymes were used to identify CYP isoforms of HAS. Finally, the PK properties of HAS in rats in vivo were studied by oral administration (p.o.). The results showed that HAS stably metabolized in human and rat liver microsomes and human hepatocytes, and the binding of HAS to human plasma proteins was nonspecific; HAS has strong inhibitory effects on CYP2C9 and CYP2D6 of human liver microsomes. In addition, in vivo PK study, HAS is rapidly absorbed in rats after oral administration. In conclusion, the in vivo and in vitro metabolic studies of HAS in this study provide data support for its further development and application, and the metabolic profiles of different species can be used as a reference for its safety evaluation.

N, N-dimethyltryptamine forms oxygenated metabolites via CYP2D6 - an in vitro investigation

N, N-dimethyltryptamine (DMT) is a psychedelic compound that has shown potential in the treatment of depression. Aside from the primary role of monoamine oxidase A (MAO-A) in DMT metabolism, the metabolic pathways are poorly understood. Increasing this understanding is an essential aspect of ensuring safe and efficacious use of DMT. This work aimed to investigate the cytochrome 450 (CYP) mediated metabolism of DMT by incubating DMT with recombinant human CYP enzymes and human liver microsomes (HLM) followed by analysis using high-resolution mass spectrometry for metabolite identification. DMT was rapidly metabolised by CYP2D6, while stable with all other investigated CYP enzymes. The metabolism of DMT in HLM was reduced after inclusion of harmine and SKF-525A whereas quinidine did not affect the metabolic rate, likely due to MAO-A residues present in HLM. Analysis of the CYP2D6 incubates showed formation of mono-, di- and tri-oxygenated metabolites, likely as a result of hydroxylation on the indole core. More research is needed to investigate the role of this metabolic pathway in vivo and any pharmacological activity of the proposed metabolites. Our findings may impact on safety issues following intake of ayahuasca in slow CYP2D6 metabolizers or with concomitant use of CYP2D6 inhibitors.

The in vitro/in vivo metabolic pathways analysis of lobetyol, lobetyolin, and lobetyolinin, three polyacetylenes from Codonopsis Radix, by UHPLC-Q/TOF-MS and UHPLC-MS/MS

Polyacetylenes, lobetyol, lobetyolin and lobetyolinin, are responsible for antitumor, antioxidant, anti-inflammatory, immunomodulatory activities of Codonopsis Radix. However, their metabolic pathways are still unknown. The study was purposed to investigate the metabolism of three polyacetylenes in vitro and in vivo by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Moreover, a rapid, sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous quantitative and semi-quantitative determination of lobetyol and its 12 metabolites to investigate the metabolic stability and metabolic phenotypes. A total of 47, 30 and 34 metabolites of lobetyol, lobetyolin and lobetyolinin were found in all samples. These metabolites are produced through extensive pathways, mainly involving oxidation, glucuronidation and glutathione conjugation. Lobetyol showed good metabolic stability in liver microsomes. The results of both recombinant human CYP enzymes and chemical inhibition experiments confirmed that CYP2C19, 1A1, 2C9, and 1A2 are the major isozymes mediating lobetyol metabolism.

RF34 | PMON202 The Human Metabolic Profile of Estetrol

Abstract Objectives Estetrol (E4), a native estrogen produced by the fetal human liver, has four hydroxyl-groups, making it structurally distinct from other estrogens. We determined the human metabolic profile of E4, which is recently marketed combined with drospirenone as a novel contraceptive and is currently in development for the relief of menopausal symptoms. Method: We studied the in vitro metabolism of E4 using human hepatocytes, human liver microsomes and recombinant CYP enzymes in reaction phenotyping studies to evaluate the role of human cytochrome P450 enzymes, UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT). Furthermore, we evaluated the in vivo metabolism of E4 in an open-label, non-randomised phase 1 human absorption, distribution, metabolism, and excretion study, in which six healthy postmenopausal participants received a single oral solution containing 15 mg [14C]-E4. We analysed blood (up to 240h), urine and faecal samples (up to 312h). Metabolite profiling and identification was performed using liquid chromatography with tandem mass spectrometry. Results The in vitro studies showed that E4 undergoes extensive phase 2 metabolism to form glucuronide and sulphate conjugates. UGT2B7 is the key enzyme catalysing the direct glucuronidation of E4 and SULT1E1 is the dominant sulfotransferase for sulphate conjugation in vitro. Unlike for other estrogens, oxidative phase 1 metabolism by P450 enzymes does not play a major role in the metabolism of E4. Following oral administration of E4 to humans, the major metabolites observed in human plasma were E4-16-glucuronide, E4-3-glucuronide and E4-glucuronide-sulfate (respectively 62%, 17% and 9% of radioactivity at the time of maximum plasma concentration [Tmax]). At Tmax, unchanged E4 accounts for only 7.5% of plasma radioactivity. Main metabolites quantified in urine during the first 6 hours after administration were E4-16-glucuronide and E4-3-glucuronide (respectively 77.4% and 16.5% of urine radioactivity). Approximately 69% of the total administered radioactivity was recovered in urine and 22% in faeces. Mass spectrometry showed that E4 was not present in urine but was detected in faeces and that E4 is not converted back into one of the other natural estrogens (estriol, estradiol, estrone). Conclusions Altogether, these data suggest that E4 has a unique metabolic profile. E4 is a terminal end-product of estrogen metabolism, which is not converted back, in vivo, into active metabolites like estriol, estradiol or estrone. Unlike for other estrogens, CYP450 enzymes do not play a major role in the metabolic pathway of E4. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m., Monday, June 13, 2022 12:48 p.m. - 12:53 p.m.

Reevaluate In Vitro CYP3A Index Reactions of Benzodiazepines and Steroids between Humans and Dogs.

Cytochrome P450 3A (CYP3A), the most important class of drug-metabolizing enzymes, participates in the metabolism of half of clinically used drugs. The CYP3A index reactions of dogs, one of the most widely used preclinical nonrodent species, are still poorly understood. This work evaluated the activity and selectivity of 10 CYP3A index reactions, including midazolam (MDZ) 1'- and 4-hydroxylation, alprazolam (APZ) and triazolam (TRZ) α- and 4-hydroxylation, testosterone (T) 6β-hydroxylation, lithocholate (LCA) 6α-hydroxylation, deoxycholate (DCA) 1β- and 5β-hydroxylation, with quantitative reaction phenotyping and kinetic analysis in human and canine recombinant CYP enzymes (rCYPs). In human studies, all reactions are reconfirmed as mixed index reactions of CYP3A with minor contributions from non-CYP3A isoforms. In canine studies, all reactions are also primarily catalyzed by CYP3A12 with lower contributions from CYP3A26. However, the canine CYP2B11 appreciably contributes to the hydroxylation of benzodiazepines except for APZ 4-hydroxylation. The canine CYP3A isoforms have lower activity than human isoforms toward T 6β-hydroxylation and LCA 6α-hydroxylation and both substrates undergo non-CYP3A catalyzed side reactions. DCA 1β- and 5β-hydroxylation are validated as the CYP3A index reactions in both humans and dogs with limited non-CYP3A contributions and side reactions. In conclusion, this work provides a comprehensive overview for the selectivity and activity of in vitro CYP3A index reactions in humans and dogs. The validated CYP3A index reactions between humans and dogs may benefit future practices in drug metabolism and drug interaction studies. SIGNIFICANCE STATEMENT: Dogs are one of the most important nonrodent animals with limited studies of cytochrome P450 enzymes than humans. This work provides the most comprehensive quantitative data to date for the selectivity and activity of CYP3A index reactions in humans and dogs. The canine CYP2B11 was found to appreciably contribute to hydroxylation of midazolam, alprazolam and triazolam, the well-known probes for human CYP3A. Deoxycholate 1β- and 5β-hydroxylation are validated as the CYP3A index reactions in both humans and dogs.

Towards the Elucidation of the Pharmacokinetics of Voriconazole: A Quantitative Characterization of Its Metabolism.

The small-molecule drug voriconazole (VRC) shows a complex and not yet fully understood metabolism. Consequently, its in vivo pharmacokinetics are challenging to predict, leading to therapy failures or adverse events. Thus, a quantitative in vitro characterization of the metabolism and inhibition properties of VRC for human CYP enzymes was aimed for. The Michaelis–Menten kinetics of voriconazole N-oxide (NO) formation, the major circulating metabolite, by CYP2C19, CYP2C9 and CYP3A4, was determined in incubations of human recombinant CYP enzymes and liver and intestine microsomes. The contribution of the individual enzymes to NO formation was 63.1% CYP2C19, 13.4% CYP2C9 and 29.5% CYP3A4 as determined by specific CYP inhibition in microsomes and intersystem extrapolation factors. The type of inhibition and inhibitory potential of VRC, NO and hydroxyvoriconazole (OH–VRC), emerging to be formed independently of CYP enzymes, were evaluated by their effects on CYP marker reactions. Time-independent inhibition by VRC, NO and OH–VRC was observed on all three enzymes with NO being the weakest and VRC and OH–VRC being comparably strong inhibitors of CYP2C9 and CYP3A4. CYP2C19 was significantly inhibited by VRC only. Overall, the quantitative in vitro evaluations of the metabolism contributed to the elucidation of the pharmacokinetics of VRC and provided a basis for physiologically-based pharmacokinetic modeling and thus VRC treatment optimization.

In vitro assessments predict that CYP3A4 contributes to a greater extent than CYP3A5 to prednisolone clearance.

Because several steroid hormones are metabolized to their respective 6β-hydroxy forms by CYP3A4 and CYP3A5, these isoenzymes have been assumed to metabolize the immunosuppressive drug prednisolone, with conflicting results in the literature with respect to their relative importance. A direct study of the metabolism of prednisolone by microsomal CYP3A4 and CYP3A5 is missing. The aim of this in vitro study was to investigate the relative importance of recombinant CYP3A4 and recombinant CYP3A5 in the metabolism of prednisolone and to compare the extent of formation of 6β-OH-prednisolone by the two enzymes. Through in vitro incubations using rCYP3A4 and rCYP3A5 enzymes, intrinsic clearance (CLint ) of prednisolone was determined by the substrate depletion approach. Formation of the metabolite 6β-OH-prednisolone by rCYP3A4 and rCYP3A5, respectively, was compared. Prednisolone concentrations were measured, and its metabolite 6β-OH-prednisolone was identified using a HPLC-MS/MS in-house method. CLint for prednisolone by rCYP3A5 was less than 26% relative to rCYP3A4. Formation of 6β-OH-prednisolone by rCYP3A5 was less than 11% relative to rCYP3A4. The study indicates that 6β-hydroxylation of prednisolone assessed in vitro in recombinant CYP enzymes depends on rCYP3A4 rather than rCYP3A5 and that CYP3A5 may be responsible for the formation of other prednisolone metabolite(s) in addition to 6β-OH-prednisolone.

Physiologically Based Pharmacokinetic (PBPK) Modeling for Betrixaban and the Impact of P-Glycoprotein Inhibition on Betrixaban Pharmacokinetics

Introduction: Betrixaban is a factor Xa inhibitor indicated for the prophylaxis of venous thromboembolism (VTE) in adult patients hospitalized for an acute medical illness. A physiologically based pharmacokinetic (PBPK) model for betrixaban provided the mechanistic understanding of the contribution of P-glycoprotein (P-gp), as well as CYP3A4, to the disposition of betrixaban, and predictions of the effects of drug-drug interactions mediated by P-gp and/or CYP3A4. The PBPK model specifically evaluated contribution of the interaction with the P-gp transporter to the disposition of betrixaban, as well as the effects of co-administration of P-gp inhibitors (ketoconazole, verapamil, clarithromycin) on betrixaban pharmacokinetics (PK). Further, in vitro and clinical studies confirmed the lack of a significant interaction of betrixaban with CYP3A4.Methods: The base PBPK model was developed using physiochemical data, in vitro metabolism andtransport data generated using recombinant CYP enzymes and human hepatocytes, and relevant clinical data (eg, observed renal clearance). The model was verified by comparing model predictions with PK data derived from single oral doses of betrixaban (from 80 to 550 mg) in healthy volunteers. The combined contribution of intestinal and hepatic P-gp transport to the disposition of betrixaban was verified by comparing model predictions to the observed ketoconazole and verapamil clinical drug-drug interaction data from healthy volunteers. The verified PBPK model was prospectively employed to predict the impact of a strong P-gp inhibitor (clarithromycin) and a moderate P-gp inhibitor (diltiazem) on betrixaban PK, and to predict the impact of food intake on betrixaban PK and compare it against the observed data in phase 3 studies. The Simcyp Population-Based Simulator (v15 release 1) was used for all simulations.Results: The major clearance pathway for betrixaban was biliary clearance (assigned to hepatic P-gp) as scaled from uptake data generated in human hepatocytes, accounting for ~80% of clearance after IV administration (CLIV). CYP3A4 played virtually no role in the disposition of betrixaban. The base PBPK model incorporating an intestinal P-gp component within the ADAM model resulted in simulations that predicted concentrations very close to the clinically observed pharmacokinetic non-linearity arising from multiple oral dosing of betrixaban in the dose range of 80 to 550 mg in healthy volunteers. The model predicted that the inhibition of intestinal and hepatic P-gp transport of betrixaban would result in the geometric mean area under the curve (AUC) and maximal concentration (Cmax) ratios due to concurrent ketoconazole treatment of 2.4 and 2.5, respectively, which were consistent with the observed ratios of 2.1 and 2.3. The predicted betrixaban geometric mean AUC and Cmax ratios due to concurrent verapamil treatment were 2.3 and 3.1, respectively, compared with the observed ratios of 3.0 and 4.7. The model qualitatively predicted the negative impact of food intake on betrixaban exposure (Figure). The predicted AUC and Cmax for patients treated with betrixaban in the fed state in the phase 3 study were within 1.5-fold of the observed values. In the final application of the PBPK model to predict the impact of the strong and moderate P-gp inhibitors, the predicted AUC and Cmax ratios due to clarithromycin treatment were 3.76 and 3.11, respectively, and due to diltiazem treatment were 1.1 and 1.1, respectively.Conclusions: The PBPK model for betrixaban adequately predicted the clinically observed PK non-linearity and accounted for the observed drug-drug interaction effects due to co-administration of betrixaban with ketoconazole and verapamil in healthy volunteers. The PBPK model did not predict a role for CYP3A4 in the clearance of betrixaban and accurately predicted a negative food effect. Intestinal and hepatic P-gp were identified as major contributors to the observed non-linearity in the kinetics of betrixaban and observed drug-drug interaction effects with P-gp inhibitors. [Display omitted] DisclosuresLeeds:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Ke:Portola Pharmaceuticals, Inc.: Consultancy. Rowland Yeo:Portola Pharmaceuticals, Inc.: Consultancy. Curnutte:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

Metabolic profiling of norepinephrine reuptake inhibitor atomoxetine

Atomoxetine (ATX), a selective and potent inhibitor of the presynaptic norepinephrine transporter, is used mainly to treat attention-deficit hyperactivity disorder. Although multiple adverse effects associated with ATX have been reported including severe liver injuries, the mechanisms of ATX-related toxicity remain largely unknown. Metabolism frequently contributes to adverse effects of a drug through reactive metabolites, and the bioactivation status of ATX is still not investigated yet. Here, we systematically investigated ATX metabolism, bioactivation, species difference in human, mouse, and rat liver microsomes (HLM, MLM, and RLM) and in mice using metabolomic approaches as mice and rats are commonly used animal models for the studies of drug toxicity. We identified thirty one ATX metabolites and adducts in LMs and mice, 16 of which are novel. In LMs, we uncovered two methoxyamine-trapped aldehydes, two cyclization metabolites, detoluene-ATX, and ATX-N-hydroxylation for the first time. Detoluene-ATX and one cyclization metabolite were also observed in mice. Using chemical inhibitors and recombinant CYP enzymes, we demonstrated that CYP2C8 and CYP2B6 mainly contribute to the formation of aldehyde; CYP2D6 is the dominant enzyme for the formation of ATX cyclization and detoluene-ATX; CYP3A4 is major enzyme responsible for the hydroxylamine formation. The findings concerning aldehydes should be very useful to further elucidate the mechanistic aspects of adverse effects associated with ATX from metabolic angles. Additionally, the species differences for each metabolite should be helpful to investigate the contribution of specific metabolites to ATX toxicity and possible drug-drug interactions in suitable models.

Metabolite identification of the antimalarial naphthoquine using liquid chromatography-tandem high-resolution mass spectrometry in combination with multiple data-mining tools.

Naphthoquine (NQ) is one of important partner drugs of artemisinin-based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2-4 h) and has a long elimination half-life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMSn ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid-phase extraction. For data processing, multiple data-mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1-M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N-oxidation. This study demonstrates that LC-HRMSn in combination with multiple data-mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs.

Charactering the metabolism of cryptotanshinone by human P450 enzymes and uridine diphosphate glucuronosyltransferases in vitro.

Cryptotanshinone (CT) is the main active component in the root of Salvia miltiorrhiza Bunge (SMB) that displays antibacterial, anti-inflammatory and anticancer activities. In this study, we characterized phase I and phase II metabolism of CT in human liver microsomes in vitro and identified the metabolic enzymes (CYPs and UGTs) involved. The metabolites of CT generated by CYPs were detected using LC-MS/MS and the CYP subtypes involved in the metabolic reactions were identified using chemical inhibitors of CYP enzymes and recombinant human CYP enzymes (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Glucuronidation of CT was also examined, and the UGT subtypes involved in the metabolic reactions were identified using recombinant human UGT enzymes (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). After adding NADPH to the human liver microsomes incubation system, CT was transformed into 6 main dehydrogenation and hydroxylation metabolites. CYP2A6, CYP3A4 and CYP2C19 were the major contributors to the transformation of its hydroxylation metabolites. CYP2C19, CYP1A2 and CYP3A4 were the major contributors to the transformation of its hydrogenation metabolites in human liver microsomes. This study showed that the metabolites at m/z of 473 were mediated by UGT1A9 and that the metabolites at m/z of 489 were mediated by UGT2B7 and UGT2B4. CT was extensively metabolized by UGTs following metabolism by CYPs in the liver.

The potent mechanism-based inactivation of CYP2D6 and CYP3A4 with fusidic acid in in vivo bioaccumulation

1. The accumulation of fusidic acid (FA) after multiple doses of FA has been reported on in previous studies but the related mechanisms have not been clarified fully. In the present study, we explain the mechanisms related to the mechanism-based inactivation of CYP2D6 and CYP3A4.2. The irreversible inhibitory effects of FA on CYP2D6 and CYP3A4 were examined via a series of experiments, including: (a) time-, concentration- and NADPH-dependent inactivation, (b) substrate protection in enzyme inactivation and (c) partition ratio with recombinant human CYP enzymes. Metoprolol α-hydroxylation and midazolam 1′-hydroxylation were used as marker reactions for CYP2D6 and CYP3A4 activities, and HPLC-MS/MS measurement was also utilised.3. FA caused to the time- and concentration-dependent inactivation of CYP2D6 and CYP3A4. About 55.8% of the activity of CYP2D6 and 75.8% of the activity of CYP3A4 were suppressed after incubation with 10 μM FA for 15 min. KI and kinact were found to be 2.87 μM and 0.033 min−1, respectively, for CYP2D6, while they were 1.95 μM and 0.029 min−1, respectively, for CYP3A4. Inhibition of CYP2D6 and CYP3A4 activity was found to require the presence of NADPH. Substrates of CYP2D6 and CYP3A4 showed that the enzymes were protected against the inactivation induced by FA. The estimated partition ratio for the inactivation was 7 for CYP2D6 and 12 for CYP3A4.4. FA is a potent mechanism-based inhibitor of CYP2D6 and CYP3A4, which may explain the accumulation of FA in vivo.

The Inhibitory Effect of Telmisartan on the Metabolism of Arachidonic Acid by CYP2C9 and CYP2C8: An in Vitro Study.

Epoxyeicosatorienoic acids (EETs) are generated from arachidonic acid (AA) by CYPs. EETs comprise four regioisomers (14,15-, 11,12-, 8,9-, and 5,6-EET). EETs show potent physiological effects, including vasodilation, anti-inflammation, myocardial preconditioning, and anti-platelet aggregation effects. We recently demonstrated that telmisartan, one of angiotensin II receptor blockers, inhibits AA metabolism by CYP enzymes, including CYP2C8, CYP2C9, and CYP2J2. We conducted studies of AA metabolism using recombinant CYP enzymes to estimate the inhibition constant and the type of inhibition by telmisartan of CYP2C9 and CYP2C8. The contribution ratio (CR) of each CYP enzyme was investigated using human liver microsomes. Dixon and Lineweaver-Burk plots indicated that telmisartan is a mixed inhibitor of both CYP2C9 and CYP2C8; telmisartan did not show a time-dependent inhibition toward these CYP enzymes. Based on the CRs, both CYP2C9 and CYP2C8 are the key enzymes in the metabolism of AA in the human liver. Uptake of telmisartan in the liver by organic anion transporting polypeptide (OATP) 1B3 and the non-linear metabolism in gastrointestinal tract augment the potential of the drug to inhibit the CYP enzymes in the liver.

Pharmacokinetic study of isocorynoxeine metabolites mediated by cytochrome P450 enzymes in rat and human liver microsomes

Isocorynoxeine (ICN) is one of the major bioactive tetracyclic oxindole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks. that is widely used for the treatment of hypertension, vascular dementia, and stroke. The present study was undertaken to assess the plasma pharmacokinetic characteristics of major ICN metabolites, and the role of simulated gastric and intestinal fluid (SGF and SIF), human and rat liver microsomes (HLMs and RLMs), and seven recombinant human CYP enzymes in the major metabolic pathway of ICN. A rapid, sensitive and accurate UHPLC/Q-TOF MS method was validated for the simultaneous determination of ICN and its seven metabolites in rat plasma after oral administration of ICN at 40mg/kg. It was found that 18.19-dehydrocorynoxinic acid (DCA) and 5-oxoisocorynoxeinic acid (5-O-ICA) were both key and predominant metabolites, rather than ICN itself, due to the rapid and extensive metabolism of ICN in vivo. The further study indicated that ICN was mainly metabolized in human or rat liver, and CYPs 2C19, 3A4 and 2D6 were the major enzymes responsible for the biotransformation of ICN to DCA and 5-O-ICA in human. These findings are of significance in understanding of the pharmacokinetic nature of tetracyclic oxindole alkaloids, and provide helpful information for the clinical co-administration of the herbal preparations containing U. rhynchophylla with antihypertensive drugs that are mainly metabolized by CYP3A4 and CYP2C19.

Differences in metabolism of the marine biotoxin okadaic acid by human and rat cytochrome P450 monooxygenases.

The ingestion of seafood contaminated with the marine biotoxin okadaic acid (OA) can lead to diarrhetic shellfish poisoning with symptoms like nausea, vomiting and abdominal cramps. Both rat and the human hepatic cytochrome P450 monooxygenases (CYP) metabolize OA. However, liver cell toxicity of metabolized OA is mainly unclear. The aim of our study was to detect the cellular effects in HepG2 cells exposed to OA in the presence of recombinant CYP enzymes of both rat and human for the investigation of species differences. The results should be set in correlation with a CYP-specific metabolite pattern. Comparative metabolite profiles of OA after incubation in rat and human recombinant CYP enzymes were established by using LC-MS/MS technique. Results demonstrated that metabolism of OA to oxygenated metabolites correlates with detoxification which was mainly catalyzed by human CYP3A4 and CYP3A5. Detoxification by rat Cyp3a1 was lower compared to human CYP3A enzymes and activation of OA by Cyp3a2 was observed, coincident with minor overall conversion capacity of OA. By contrast human and rat CYP1A2 seem to activate OA into cytotoxic intermediates. In conclusion, different mechanisms of OA metabolism may occur in the liver. At low OA doses, the human liver is likely well protected against cytotoxic OA, but for high shellfish consumers a potential risk cannot be excluded.

Comparative metabolism of tussilagone in rat and human liver microsomes using ultra‐high‐performance liquid chromatography coupled with high‐resolution LTQ‐Orbitrap mass spectrometry

Tussilagone is a major component in Tussilago farfara that has been widely used as an anti-tussive herbal medicine for the treatment of bronchitis, cough and asthmatic disorders in the clinic. However, its metabolism has been poorly investigated. In order to clarify its in vitro metabolism, a comparative analysis of its metabolic profile in rat liver microsomes (RLMs) and human liver microsomes (HLMs) was carried out. Further, the cytochrome P450 isoforms (CYPs) involved in the metabolism were investigated. In this work, the biotransformation of tussilagone in RLMs and HLMs was compared using ultra-high-performance liquid chromatography coupled with high-resolution LTQ-Orbitrap mass spectrometry (UHPLC/HRMS) and the CYPs involved in the metabolism were further investigated by recombinant human CYP enzymes. Totally, nine metabolites of tussilagone were identified in RLMs and HLMs based on the proposed MS/MS fragmentation pathways of tussilagone and the accurate MS/MS spectra. Among them, one metabolite (M9) was detected in both RLMs and HLMs while the other eight metabolites were only detected in HLMs. Three hydroxylation metabolites (M6, M7 and M8) were detected in the assay with individual recombinant P450s incubation. M6 was detected in all CYPs except CYP2A6 while M7 and M8 were only observed in CYP3A4. The HR-ESI-MS/MS fragmentation behavior of tussilagone and its metabolic profile in RLMs and HLMs were investigated for the first time. The results demonstrated that the biotransformation of tussilagone involved hydrolysis of ester bonds at C-14 and hydroxylation in the side chains at C-12, C-5' or C-6'. Among the CYPs, CYP3A4 played an important role in the hydroxylation reaction of tussilagone in vitro. Furthermore, the results indicated a species-related difference in the metabolism of tussilagone between RLMs and HLMs. This work provided basic information for the metabolism of tussilagone in RLMs and HLMs, which would help to better understand the pharmacological activities of tussilagone.

In vitro inhibition of CYP2C9-mediated warfarin 7-hydroxylation by iguratimod: possible mechanism of iguratimod-warfarin interaction.

Iguratimod is a novel disease-modifying antirheumatic drug. A blue letter (safety advisory) for drug interaction between iguratimod and warfarin was issued by the Ministry of Health, Labour and Welfare of Japan in May 2013. Iguratimod may affect warfarin metabolism catalyzed by CYP. However, it is not clear whether iguratimod inhibits warfarin oxidation. This study was performed to investigate the effects of iguratimod on warfarin 7-hydroxylation with human liver microsomes (HLMs) and recombinant CYP enzymes. Iguratimod concentration-dependently inhibited R,S-warfarin 7-hydroxylase activity of HLMs with an IC50 value of 15.2 µM. The inhibitory effect was examined with S-warfarin and R-warfarin to determine which enantiomer was more potently inhibited by iguratimod. Iguratimod potently inhibited the S-warfarin 7-hydroxylase activity of HLMs with an IC50 value of 14.1 µM, but showed only slight inhibition of R-warfarin 7-hydroxylation. Furthermore, iguratimod inhibited the S-warfarin 7-hydroxylase activity of recombinant CYP2C9.1 (rCYP2C9.1) and rCYP2C9.3 in a concentration-dependent manner with IC50 values of 10.8 and 20.1 µM, respectively. Kinetic analysis of the inhibition of S-warfarin 7-hydroxylation by iguratimod indicated competitive-type inhibition for HLMs and rCYP2C9.1 but mixed-type inhibition for rCYP2C9.3. The Ki values for HLMs, rCYP2C9.1, and rCYP2C9.3 were 6.74, 4.23, and 14.2 µM, respectively. Iguratimod did not exert metabolism-dependent inhibition of S-warfarin 7-hydroxylation. These results indicated that iguratimod is a potent direct inhibitor of CYP2C9-mediated warfarin 7-hydroxylation and that its inhibitory effect on CYP2C9.1 was more sensitive than that on CYP2C9.3.

Effects of adrenolytic mitotane on drug elimination pathways assessed in vitro.

Mitotane (1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane, o,p'-DDD) represents one of the most active drugs for the treatment of adrenocortical carcinoma. Its metabolites 1,1-(o,p'-dichlorodiphenyl) acetic acid (=o,p'-DDA) and 1,1-(o,p'-dichlorodiphenyl)-2,2 dichloroethene (=o,p'-DDE) partly contribute to its pharmacological effects. Because mitotane has a narrow therapeutic index and causes pharmacokinetic drug-drug interactions, knowledge about these compounds' effects on drug metabolizing and transporting proteins is crucial. Using quantitative real-time polymerase chain reaction, our study confirmed the strong inducing effects of o,p'-DDD on mRNA expression of cytochrome P450 3A4 (CYP3A4, 30-fold) and demonstrated that other enzymes and transporters are also induced (e.g., CYP1A2, 8.4-fold; ABCG2 (encoding breast resistance cancer protein, BCRP), 4.2-fold; ABCB1 (encoding P-glycoprotein, P-gp) 3.4-fold). P-gp induction was confirmed at the protein level. o,p'-DDE revealed a similar induction profile, however, with less potency and o,p'-DDA had only minor effects. Reporter gene assays clearly confirmed o,p'-DDD to be a PXR activator and for the first time demonstrated that o,p'-DDE and o,p'-DDA also activate PXR albeit with lower potency. Using isolated, recombinant CYP enzymes, o,p'-DDD and o,p'-DDE were shown to strongly inhibit CYP2C19 (IC50 = 0.05 and 0.09 µM). o,p'-DDA exhibited only minor inhibitory effects. In addition, o,p'-DDD, o,p'-DDE, and o,p'-DDA are demonstrated to be neither substrates nor inhibitors of BCRP or P-gp function. In summary, o,p'-DDD and o,p'-DDE might be potential perpetrators in pharmacokinetic drug-drug interactions through induction of drug-metabolizing enzymes or drug transporters and by potent inhibition of CYP2C19. In tumors over-expressing BCRP or P-gp, o,p'-DDD and its metabolites should retain their efficacy due to a lack of substrate characteristics.

In vitro inhibition of human CYP1A2, CYP2D6, and CYP3A4 by six herbs commonly used in pregnancy.

Black elderberry, cranberry, fennel, ginger, horsetail, and raspberry leaf, herbs frequently used in pregnancy, were investigated for their in vitro CYP1A2, 2D6, and 3A4 inhibitory potential. Aqueous or ethanolic extracts were made from commercially available herbal products, and incubations were performed with recombinant cDNA-expressed human CYP enzymes in the presence of positive inhibitory controls. Metabolite formation was determined by validated LCMS/MS or HPLC methodologies. IC50 inhibition constants were estimated from CYP activity inhibition plots using non-linear regression. The most potent inhibition was shown for fennel towards CYP2D6 and 3A4 with respective IC50 constants of 23 ± 2 and 40 ± 4 µg/ml, horsetail towards CYP1A2 with an IC50 constant of 27 ± 1 µg/ml, and raspberry leaf towards CYP1A2, 2D6, and 3A4 with IC50 constants of 44 ± 2, 47 ± 8, and 81 ± 11 µg/ml, respectively. Based on the recommended dosing of the different commercial herbal products, clinically relevant systemic CYP inhibitions could be possible for fennel, horsetail, and raspberry leaf. In addition, fennel and raspberry leaf might cause a clinically relevant inhibition of intestinal CYP3A4. The in vivo inhibitory potential of these herbs towards specific CYP enzymes should be further investigated.