scholarly journals Interspecies Variation in NCMN-O-Demethylation in Liver Microsomes from Various Species

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2765
Author(s):  
Ziru Dai ◽  
Guibo Sun ◽  
Jiada Yang ◽  
Jie Hou ◽  
Ping Zhou ◽  
...  
Keyword(s):  
Cynomolgus Monkey ◽  
Liver Microsomes ◽  
Specific Inhibitor ◽  
Intrinsic Clearance ◽  
Beagle Dog ◽  
Interspecies Variation ◽  
Human Cytochrome ◽  
Practical Tool ◽  
Cytochrome P450 1A

NCMN (N-(3-carboxy propyl)-4-methoxy-1,8-naphthalimide), a newly developed ratiometric two-photon fluorescent probe for human Cytochrome P450 1A (CYP1A), shows the best combination of specificity and reactivity for real-time detection of the enzymatic activities of CYP1A in complex biological systems. This study aimed to investigate the interspecies variation in NCMN-O-demethylation in commercially available liver microsomes from human, mouse, rat, beagle dog, minipig and cynomolgus monkey. Metabolite profiling demonstrated that NCMN could be O-demethylated in liver microsomes from all species but the reaction rate varied considerably. CYP1A was the major isoform involved in NCMN-O-demethylation in all examined liver microsomes based on the chemical inhibition assays. Furafylline, a specific inhibitor of mammalian CYP1A, displayed differential inhibitory effects on NCMN-O-demethylation in all tested species. Kinetic analyses demonstrated that NCMN-O-demethylation in liver microsomes form rat, minipig and cynomolgus monkey followed biphasic kinetics, while in liver microsomes form human, mouse and beagle dog obeyed Michaelis-Menten kinetics, the kinetic parameters from various species are much varied, while NCMN-O-demethylation in MLM exhibited the highest similarity of specificity, kinetic behavior and intrinsic clearance as that in HLM. These findings will be very helpful for the rational use of NCMN as a practical tool to decipher the functions of mammalian CYP1A or to study CYP1A associated drug-drug interactions in vivo.

In Vitro Metabolism of E2, G2: Novel Bile Acid-Coupling Camptothecin Analogues, in Rat Liver Microsomes

2021 ◽  
Vol 16 ◽  
Author(s):  
Xiangli Zhang ◽  
Qin Shen ◽  
Yi Wang ◽  
Leilei Zhou ◽  
Qi Weng ◽  
...  
Keyword(s):  
Bile Acid ◽  
Phase I ◽  
Rat Liver ◽  
Deoxycholic Acid ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
Rat Liver Microsomes ◽  
Camptothecin Analogues

Background: E2 (Camptothecin - 20 (S) - O- glycine - deoxycholic acid), and G2 (Camptothecin - 20 (S) - O - acetate - deoxycholic acid) are two novel bile acid-derived camptothecin analogues by introducing deoxycholic acid in 20-position of CPT(camptothecin) with greater anticancer activity and lower systematic toxicity in vivo. Objective: We aimed to investigate the metabolism of E2 and G2 by Rat Liver Microsomes (RLM). Methods: Phase Ⅰ and Phase Ⅱ metabolism of E2 and G2 in rat liver microsomes were performed respectively, and the mixed incubation of phase I and phase Ⅱ metabolism of E2 and G2 was also processed. Metabolites were identified by liquid chromatographic/mass spectrometry. Results: The results showed that phase I metabolism was the major biotransformation route for both E2 and G2. The isoenzyme involved in their metabolism had some difference. The intrinsic clearance of G2 was 174.7mL/min. mg protein, more than three times of that of E2 (51.3 mL/min . mg protein), indicating a greater metabolism stability of E2. 10 metabolites of E2 and 14 metabolites of G2 were detected, including phase I metabolites (mainly via hydroxylations and hydrolysis) and their further glucuronidation products. Conclusion: These findings suggested that E2 and G2 have similar biotransformation pathways except some difference in the hydrolysis ability of the ester bond and amino bond from the parent compounds, which may result in the diversity of their metabolism stability and responsible CYPs(Cytochrome P450 proteins).

scholarly journals In vitro and in vivo evaluation of hypothermia on pharmacokinetics and pharmacodynamics of nimodipine in rabbits

2017 ◽  
Vol 46 (1) ◽  
pp. 335-347 ◽  
Author(s):  
Yu-xing Fei ◽  
Tian-hong Zhang ◽  
Jing Zhao ◽  
He Ren ◽  
Ya-nan Du ◽  
...  
Keyword(s):  
Blood Flow ◽  
Cerebral Blood Flow ◽  
Protein Binding ◽  
Plasma Concentrations ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
Pharmacokinetics And Pharmacodynamics ◽  
In Vivo Evaluation

Objective To investigate the effect of hypothermia on the pharmacokinetics and pharmacodynamics of nimodipine in rabbits using in vivo and in vitro methods. Methods Five healthy New Zealand rabbits received a single dose of nimodipine (0.5 mg/kg) intravenously under normothermic and hypothermic conditions. Doppler ultrasound was used to monitor cerebral blood flow, vascular resistance, and heart rate. In vitro evaluations of protein binding, hepatocyte uptake and intrinsic clearance of liver microsomes at different temperatures were also conducted. Results Plasma concentrations of nimodipine were significantly higher in hypothermia than in normothermia. Nimodipine improved cerebral blood flow under both conditions, but had a longer effective duration during the hypothermic period. Low temperature decreased the intrinsic clearance of liver microsomes, with no change in protein binding or hepatocyte uptake of nimodipine. Conclusion Nimodipine is eliminated at a slower rate during hypothermia than during normothermia, mainly due to the decreased activity of cytochrome P450 enzymes. This results in elevated system exposure with little enhancement in pharmacological effect.

scholarly journals In Vitro Interaction of AB-FUBINACA with Human Cytochrome P450, UDP-Glucuronosyltransferase Enzymes and Drug Transporters

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4589 ◽  
Author(s):  
Sunjoo Kim ◽  
Dong Kyun Kim ◽  
Yongho Shin ◽  
Ji-Hyeon Jeon ◽  
Im-Sook Song ◽  
...  
Keyword(s):  
Drug Interactions ◽  
Organic Anion ◽  
Liver Microsomes ◽  
Drug Abusers ◽  
Cancer Resistance ◽  
Mixed Inhibition ◽  
P Glycoprotein ◽  
Human Cytochrome

AB-FUBINACA, a synthetic indazole carboxamide cannabinoid, has been used worldwide as a new psychoactive substance. Because drug abusers take various drugs concomitantly, it is necessary to explore potential AB-FUBINACA-induced drug–drug interactions caused by modulation of drug-metabolizing enzymes and transporters. In this study, the inhibitory effects of AB-FUBINACA on eight major human cytochrome P450s (CYPs) and six uridine 5′-diphospho-glucuronosyltransferases (UGTs) of human liver microsomes, and on eight clinically important transport activities including organic cation transporters (OCT)1 and OCT2, organic anion transporters (OAT)1 and OAT3, organic anion transporting polypeptide transporters (OATP)1B1 and OATP1B3, P-glycoprotein, and breast cancer resistance protein (BCRP) in transporter-overexpressing cells were investigated. AB-FUBINACA inhibited CYP2B6-mediated bupropion hydroxylation via mixed inhibition with Ki value of 15.0 µM and competitively inhibited CYP2C8-catalyzed amodiaquine N-de-ethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation, and CYP2D6-catalyzed bufuralol 1′-hydroxylation with Ki values of 19.9, 13.1, 6.3, and 20.8 µM, respectively. AB-FUBINACA inhibited OCT2-mediated MPP+ uptake via mixed inhibition (Ki, 54.2 µM) and competitively inhibited OATP1B1-mediated estrone-3-sulfate uptake (Ki, 94.4 µM). However, AB-FUBINACA did not significantly inhibit CYP1A2, CYP2A6, CYP3A4, UGT1A1, UGT1A3, UGT1A4, UGT1A6, or UGT2B7 enzyme activities at concentrations up to 100 µM. AB-FUBINACA did not significantly inhibit the transport activities of OCT1, OAT1/3, OATP1B3, P-glycoprotein, or BCRP at concentrations up to 250 μM. As the pharmacokinetics of AB-FUBINACA in humans and animals remain unknown, it is necessary to clinically evaluate potential in vivo pharmacokinetic drug–drug interactions induced by AB-FUBINACA-mediated inhibition of CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, OCT2, and OATP1B1 activities.

scholarly journals Investigation on the metabolic characteristics of isobavachin in Psoralea corylifolia L. (Bu-gu-zhi) and its potential inhibition against human cytochrome P450s and UDP-glucuronosyltransferases

2020 ◽  
Author(s):  
Han Xing ◽  
Jing Yang ◽  
Kaidi Ren ◽  
Zifei Qin ◽  
Peile Wang ◽  
...  
Keyword(s):  
Correlation Analysis ◽  
Kinetic Modelling ◽  
Liver Microsomes ◽  
Hepatic Metabolism ◽  
Relative Activity ◽  
Metabolic Characteristics ◽  
Activity Factor ◽  
Human Cytochrome

Abstract Objectives Isobavachin is a phenolic with anti-osteoporosis activity. This study aimed to explore its metabolic fates in vivo and in vitro, and to investigate the potential drug–drug interactions involving CYPs and UGTs. Methods Metabolites of isobavachin in mice were first identified and characterized. Oxidation and glucuronidation study were performed using liver and intestine microsomes. Reaction phenotyping, activity correlation analysis and relative activity factor approaches were employed to identify the main CYPs and UGTs involved in isobavachin metabolism. Through kinetic modelling, inhibition mechanisms towards CYPs and UGTs were also explored. Key findings Two glucuronides (G1 - G2) and three oxidated metabolites (M1 - M3) were identified in mice. Additionally, isobavachin underwent efficient oxidation and glucuronidation by human liver microsomes and HIM with CLint values from 5.53 to 148.79 μl/min per mg. CYP1A2, 2C19 contributed 11.3% and 17.1% to hepatic metabolism of isobavachin, respectively, with CLint values from 8.75 to 77.33 μl/min per mg. UGT1As displayed CLint values from 10.73 to 202.62 μl/min per mg for glucuronidation. Besides, significant correlation analysis also proved that CYP1A2, 2C19 and UGT1A1, 1A9 were main contributors for the metabolism of isobavachin. Furthermore, mice may be the appropriate animal model for predicting its metabolism in human. Moreover, isobavachin exhibited broad inhibition against CYP2B6, 2C9, 2C19, UGT1A1, 1A9, 2B7 with Ki values from 0.05 to 3.05 μm. Conclusions CYP1A2, 2C19 and UGT1As play an important role in isobavachin metabolism. Isobavachin demonstrated broad-spectrum inhibition of CYPs and UGTs.

scholarly journals CYP-Dependent Metabolism of PF9601N, A New Monoamine Oxidase-B Inhibitor, by C57BL / 6 Mouse and Human Liver Microsomes

2007 ◽  
Vol 10 (4) ◽  
pp. 473 ◽  
Author(s):  
Stefania Dragoni ◽  
Giada Materozzi ◽  
Federica Pessina ◽  
Maria Frosini ◽  
José Luis Marco ◽  
...  
Keyword(s):  
Monoamine Oxidase ◽  
Human Liver ◽  
Formation Rate ◽  
Antioxidant Properties ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
Human Liver Microsomes ◽  
Monoamine Oxidase B ◽  
Mao B

Purpose. The selective monoamine oxidase-B (MAO-B) inhibitor, l-deprenyl, is still used for treating Parkinson's patients, however, a disadvantage of its use lies in the formation of l-amphetamine and l-methamphetamine. Subsequently, this has promoted the design of a novel, more potent, MAO-B inhibitor PF9601N, which also has neuroprotective and antioxidant properties. The aim of this work was to investigate the effect of treatment with PF9601N on its own phase I hepatic metabolism. Kinetic parameters of PF9601N CYP-dependent N-dealkylation reaction was also studied and compared with those of l-deprenyl. Methods. C57BL/6 mice were treated with PF9601N for 4 days. After CYP content and related monooxygenase activities were assayed in liver microsomes of control and treated animals. Results. CYP activities, cytochrome b5 content, NADPH-cytochrome P450 reductase and various monooxygenase activities were unaffected by in vivo PF9601N treatment. With microsomes from both control and treated mice, the PF9601N-dealkylation product, FA72, was the only detected metabolite with its formation rate following an hyperbolic, Michaelis-Menten curve. Among various inhibitors, only ketoconazole inhibited the FA72 formation rate, indicating a major involvement for CYP3A. Apparent Km and Vmax values generated by human liver microsomes were similar to those found with mouse microsomes. Ketoconazole inhibition indicates that CYP3A is one of the major enzymes involved in PF9601N metabolism also by human liver microsomes. In mouse liver microsomes, the intrinsic clearance of PF9601N was significantly lower than that of l-deprenyl suggestive of an improved bioavailability for the former. Conclusion. The observed favourable metabolic profile may suggest suitability of PF9601N for clinical use.

scholarly journals Assessment of inhibitory effects on major human cytochrome P450 enzymes by spasmolytics used in the treatment of overactive bladder syndrome

2017 ◽  
Vol 9 (7) ◽  
pp. 163-177
Author(s):  
Dominik Dahlinger ◽  
Sevinc Aslan ◽  
Markus Pietsch ◽  
Sebastian Frechen ◽  
Uwe Fuhr
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Fold Increase ◽  
Pharmacokinetic Data ◽  
Cytochrome P450 Enzymes ◽  
Trospium Chloride ◽  
Screening Experiments ◽  
Human Cytochrome

Background: The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride on the seven major human cytochrome P450 enzymes (CYP) by using a standardized and validated seven-in-one cytochrome P450 cocktail inhibition assay. Methods: An in vitro cocktail of seven highly selective probe substrates was incubated with human liver microsomes and varying concentrations of the seven test compounds. The major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Enzyme kinetics were estimated by determining IC50 and Ki values via nonlinear regression. Obtained Ki values were used for predictions of potential clinical impact of the inhibition using a static mechanistic prediction model. Results: In this study, 49 IC50 experiments were conducted. In six cases, IC50 values lower than the calculated threshold for drug–drug interactions (DDIs) in the gut wall were observed. In these cases, no increase in inhibition was determined after a 30 min preincubation. Considering a typical dosing regimen and applying the obtained Ki values of 0.72 µM (darifenacin, 15 mg daily) and 7.2 µM [propiverine, 30 mg daily, immediate release (IR)] for the inhibition of CYP2D6 yielded a predicted 1.9-fold and 1.4-fold increase in the area under the curve (AUC) of debrisoquine (CYP2D6 substrate), respectively. Due to the inhibition of the particular intestinal CYP3A4, the obtained Ki values of 14 µM of propiverine (30 mg daily, IR) resulted in a predicted doubling of the AUC for midazolam (CYP3A4 substrate). Conclusions: In vitro/ in vivo extrapolation based on pharmacokinetic data and the conducted screening experiments yielded similar effects of darifenacin on CYP2D6 and propiverine on CYP3A4 as obtained in separately conducted in vivo DDI studies. As a novel finding, propiverine was identified to potentially inhibit CYP2D6 at clinically occurring concentrations.

scholarly journals Comprehensive In Vitro Analysis of Voriconazole Inhibition of Eight Cytochrome P450 (CYP) Enzymes: Major Effect on CYPs 2B6, 2C9, 2C19, and 3A

2008 ◽  
Vol 53 (2) ◽  
pp. 541-551 ◽  
Author(s):  
Seongwook Jeong ◽  
Phuong D. Nguyen ◽  
Zeruesenay Desta
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Competitive Inhibitor ◽  
Major Effect ◽  
Cyp Enzymes ◽  
Human Cytochrome ◽  
Vitro Analysis ◽  
In Vitro Analysis

ABSTRACT Voriconazole is an effective antifungal drug, but adverse drug-drug interactions associated with its use are of major clinical concern. To identify the mechanisms of these interactions, we tested the inhibitory potency of voriconazole with eight human cytochrome P450 (CYP) enzymes. Isoform-specific probes were incubated with human liver microsomes (HLMs) (or expressed CYPs) and cofactors in the absence and the presence of voriconazole. Preincubation experiments were performed to test mechanism-based inactivation. In pilot experiments, voriconazole showed inhibition of CYP2B6, CYP2C9, CYP2C19, and CYP3A (half-maximal [50%] inhibitory concentrations, <6 μM); its effect on CYP1A2, CYP2A6, CYP2C8, and CYP2D6 was marginal (<25% inhibition at 100 μM voriconazole). Further detailed experiments with HLMs showed that voriconazole is a potent competitive inhibitor of CYP2B6 (Ki < 0.5), CYP2C9 (Ki = 2.79 μM), and CYP2C19 (Ki = 5.1 μM). The inhibition of CYP3A by voriconazole was explained by noncompetitive (Ki = 2.97 μM) and competitive (Ki = 0.66 μM) modes of inhibition. Prediction of the in vivo interaction of voriconazole from these in vitro data suggests that voriconazole would substantially increase the exposure of drugs metabolized by CYP2B6, CYP2C9, CYP2C19, and CYP3A. Clinicians should be aware of these interactions and monitor patients for adverse effects or failure of therapy.

scholarly journals Development of SGC-GAK-1 as an orally active in vivo probe for cyclin G associated kinase through cytochrome P450 inhibition

2019 ◽  
Author(s):  
Christopher R. M. Asquith ◽  
James M. Bennett ◽  
Lianyong Su ◽  
Tuomo Laitinen ◽  
Jonathan M. Elkins ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
In Vivo Studies ◽  
Chemical Probe ◽  
Active Chemical ◽  
Rapid Clearance ◽  
Cyclin G ◽  
Orally Active

AbstractSGC-GAK-1 (1), a potent, selective, cell-active chemical probe for cyclin G associated kinase (GAK), was rapidly metabolized in mouse liver microsomes by P450 mediated oxidation. 1 displayed rapid clearance in mice, limiting its utility for in vivo studies. All chemical modifications of 1 that improved metabolic stability led to a loss in GAK activity. However, pretreatment of liver microsomes with the irreversible cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) decreased intrinsic clearance of 1. Coadministration of ABT also greatly improved plasma exposure of 1 in mice, supporting its use as a chemical probe to study the in vivo biology of GAK inhibition.

Carbon-Carbon Bond Cleavage Catalyzed by Human Cytochrome P450 Enzymes: a-Ketol as the Key Intermediate Metabolite in Sequential Metabolism of Olanexidine

2019 ◽  
Vol 13 ◽  
Author(s):  
Yiding Hu ◽  
Yi Xiao ◽  
Zhesui Rao ◽  
Vasant Kumar ◽  
Hanlan Liu ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Bond Cleavage ◽  
Liver Microsomes ◽  
Side Chain ◽  
Cytochrome P450 Enzymes ◽  
Acid Metabolite ◽  
Carbon Bond ◽  
Carbon Carbon Bond ◽  
Human Cytochrome

Background: Carbon-carbon bond cleavage of a saturated aliphatic moiety is rarely seen in xenobiotic metabolism. Olanexidine (Olanedine®), containing an n-octyl (C8) side chain, was mainly metabolized to various shortened side chain (C4 to C6) acid-containing metabolites in vivo in preclinical species. In liver microsomes and S9, the major metabolites of olanexidine were from multi-oxidation on its n-octyl (C8) side chain. However, the carbon-carbon bond cleavage mechanism of n-octyl (C8) side chain, and enzyme(s) responsible for its metabolism in human remained unknown. Methods: A pair of regioisomers of -ketol-containing C8 side chain olanexidine analogs (3,2-ketol olanexidine and 2,3-ketol olanexidine) were synthesized, followed by incubation in human liver microsomes, recombinant human cytochrome P450 enzymes or human hepatocytes, and subsequent metabolite identification using LC/UV/MS. Results: Multiple shortened side chain (C4 to C6) metabolites were identified, including C4, C5 and C6-acid and C6-hydroxyl metabolites. Among 19 cytochrome P450 enzymes tested, CYP2D6, CYP3A4 and CYP3A5 were identified to catalyze carbon-carbon bond cleavage. Conclusions: 3,2-ketol olanexidine and 2,3-ketol olanexidine were confirmed as the key intermediates in carbon-carbon bond cleavage. Its mechanism is proposed that a nucleophilic addition of iron-peroxo species, generated by CYP2D6 and CYP3A4/5, to the carbonyl group caused the carbon-carbon bond cleavage between the adjacent hydroxyl and ketone groups. As results, 2,3-ketol olanexidine formed a C6 side chain acid metabolite. While, 3,2-ketol olanexidine formed a C6 side chain aldehyde intermediate, which was either oxidized to a C6 side chain acid metabolite or reduced to a C6 side chain hydroxyl metabolite.

scholarly journals Towards the Development of an In vivo Chemical Probe for Cyclin G Associated Kinase (GAK)

Molecules ◽  
2019 ◽  
Vol 24 (22) ◽  
pp. 4016 ◽  
Author(s):  
Christopher R. M. Asquith ◽  
James M. Bennett ◽  
Lianyong Su ◽  
Tuomo Laitinen ◽  
Jonathan M. Elkins ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Intrinsic Clearance ◽  
In Vivo Studies ◽  
Chemical Probe ◽  
Improved Stability ◽  
Spectrum Activity ◽  
Rapid Clearance ◽  
Cyclin G

SGC-GAK-1 (1) is a potent, selective, cell-active chemical probe for cyclin G-associated kinase (GAK). However, 1 was rapidly metabolized in mouse liver microsomes by cytochrome P450-mediated oxidation, displaying rapid clearance in liver microsomes and in mice, which limited its utility in in vivo studies. Chemical modifications of 1 that improved metabolic stability, generally resulted in decreased GAK potency. The best analog in terms of GAK activity in cells was 6-bromo-N-(1H-indazol-6-yl)quinolin-4-amine (35) (IC50 = 1.4 μM), showing improved stability in liver microsomes while still maintaining a narrow spectrum activity across the kinome. As an alternative to scaffold modifications we also explored the use of the broad-spectrum cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) to decrease intrinsic clearance of aminoquinoline GAK inhibitors. Taken together, these approaches point towards the development of an in vivo chemical probe for the dark kinase GAK.

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