μM In Human Liver Microsomes Research Articles

Evaluation of the inhibitory effect of azoles on pharmacokinetics of lenvatinib in rats both in vivo and in vitro by UPLC-MS/MS.

Lenvatinib is a multitargeted tyrosine kinase inhibitor used in the treatment of a variety of solid tumors. This study aims to investigate the potential pharmacokinetic interactions between lenvatinib and various azoles (ketoconazole, voriconazole, isavuconazole and posaconazole) when orally administered to rats. A total of 30 Sprague-Dawley rats were randomly allocated into five groups and administered 20 mg/kg of ketoconazole, voriconazole, isavuconazole and 30 mg/kg of posaconazole and 0.5% CMC-Na, through gavage for a duration of 7 days prior to the commencement of the experiment. On the final day, the rats were given 10 mg/kg of lenvatinib. The blood concentration of lenvatinib was determined using UPLC-MS-MS. In vitro lenvatinib were incubated with azoles and rat liver microsomes (RLMs) or human liver microsomes (HLMs). Molecular docking was lastly used to examine the binding strength of the enzymes and ligands with Autodock Vina. AUC and Cmax of lenvatinib significantly increased with each of the azoles (p < 0.05), whereas CLz/F decreased 0.83-flod, 0.41-fold (p < 0.05) and 0.72-fold (p < 0.01) in voriconazole, isavuconazole and ketoconazole in rats. The IC50 of lenvatinib with the azoles were 0.237, 1.300, 0.355 and 2.403 μM in RLMs and 0.160, 1.933, 3.622 and 1.831 μM in HLMs. Molecular docking analysis suggested that azoles exhibited a strong binding ability towards the target enzymes. It is imperative to acknowledge the potential drug-drug interactions mediated by CYP3A4 between azoles and lenvatinib, as these interactions hold significant implications for their clinical utilization.

Effect of apatinib on the pharmacokinetics of tramadol and O-desmethyltramadol in rats.

Since the combination of anticancer drugs and opioids is very common, apatinib and tramadol are likely to be used in combination clinically. This study evaluated the effects of apatinib on the pharmacokinetics of tramadol and its main metabolite O-desmethyltramadol in Sprague-Dawley (SD) rats and the inhibitory effects of apatinib on tramadol in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP2D6.1. The samples were determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The in vivo results showed that compared with the control group, apatinib increased the AUC(0-t), AUC(0-∞) and Cmax values of tramadol and O-desmethyltramadol, and decreased the values of VZ/F and CLz/F. In addition, the MRT(0-t), MRT(0-∞) values of O-desmethyltramadol were increased. In vitro, apatinib inhibited the metabolism of tramadol by a mixed way with IC50 of 1.927 µM in RLMs, 2.039 µM in HLMs and 15.32 µM in CYP2D6.1. In summary, according to our findings, apatinib has a strong in vitro inhibitory effect on tramadol, and apatinib can increase the analgesic effect of tramadol and O-desmethyltramadol in rats.

Impact of Heterotropic Allosteric Modulation on the Time-Dependent Inhibition of Cytochrome P450 3A4.

The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam (MDZ), and on the determination in vitro time-dependent inhibition (TDI) of CYP3A4 using human liver microsomes (HLM). As the concentration of midazolam increased to 250 µM in HLMs, homotropic cooperativity resulted in a decrease in the 1'-hydroxymidazolam to 4-hydroxymidazolam ratio to a maximum of 1.1. The presence of varying concentrations of testosterone, progesterone (PGS), or carbamazepine (CBZ) in HLMs with MDZ could recapitulate the effect of homotropic cooperativity such that the formation rates of the 1'hydroxymidazolam and 4-hydroxymidazolam were equal even at low concentrations of MDZ. The presence of PGS (10 or 100 µM) and CBZ (100 or 1000 µM) in in vitro TDI determination of four known CYP3A4 time-dependent inactivators (clarithromycin, troleandomycin, mibefradil, raloxifene) simultaneously decreased potency and inactivation rate constant, resulting in fold changes in inactivation efficiency on average of 1.6-fold and 13-fold for the low and high concentrations of allosteric modulator tested, respectively. The formation of a metabolic-intermediate complex (MIC) for clarithromycin and troleandomycin decreased in the presence of the allosteric modulators in a concentration-dependent manner, reaching a new steady state formation that could not be overcome with increased incubation time. Maximum reduction of the MIC formed by clarithromycin was up to ∼91%, while troleandomycin MIC decreased up to ∼31%. These findings suggest that the absence of endogenous allosteric modulators may contribute to the poor translation of HLM-based drug-drug interaction predictions. SIGNIFICANCE STATEMENT: The reported overprediction of in vitro human liver microsome time-dependent inhibition of CYP3A4 and observed drug interactions in vivo remains an issue in drug development. We provide characterization of allosteric modulators on the CYP3A4 metabolism of the prototypical substrate midazolam, demonstrating the ability of the modulators to recapitulate the homotropic cooperativity of midazolam. Furthermore, we demonstrate that allosteric heterotropic cooperativity of CYP3A4 can impact the time-dependent inhibition kinetics of known mechanisms-based inhibitors, providing a potential mechanism to explain the overprediction.

Effects of drug-drug interactions and CYP3A4 variants on alectinib metabolism.

In this study, the effects of 17 CYP3A4 variants and drug-drug interactions (DDI) with its mechanism on alectinib metabolism were investigated. In vitro incubation systems of rat liver microsomes (RLM), human liver microsomes (HLM) and recombinant human CYP3A4 variants were established. The formers were used to screen potential drugs that inhibited alectinib metabolism and study the underlying mechanism, and the latter was used to determine the dynamic characteristics of CYP3A4 variants. Alectinib and its main metabolite M4 were quantitatively determined by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The results showed that compared with CYP3A4.1, only CYP3A4.29 showed higher catalytic activity, while the catalytic activity of CYP3A4.4, .7, .8, .12, .14, .16, .17, .18, .19, .20, .23, and .24 decreased significantly. Among them, the catalytic activity of CYP3A4.20 is the lowest, only 2.63% of that of CYP3A4.1. Based on the RLM incubation system in vitro, 81 drugs that may be combined with alectinib were screened, among which 18 drugs had an inhibition rate higher than 80%. In addition, nicardipine had an inhibition rate of 95.09% with a half-maximum inhibitory concentration (IC50) value of 3.54 ± 0.96μM in RLM and 1.52 ± 0.038μM in HLM, respectively. There was a mixture of non-competitive and anti-competitive inhibition of alectinib metabolism in both RLM and HLM. In vivo experiments of Sprague-Dawley (SD) rats, compared with the control group (30mg/kg alectinib alone), the AUC(0-t), AUC(0-∞), Tmax and Cmax of alectinib administered in combination with 6mg/kg nicardipine were significantly increased in the experimental group. In conclusion, the metabolism of alectinib was affected by polymorphisms of the CYP3A4 gene and nicardipine. This study provides reference data for clinical individualized administration of alectinib in the future.

Studies on the inhibitory effect of isavuconazole on flumatinib metabolism in vitro and in vivo.

As the validated agent for the treatment of chronic myelogenous leukemia (CML), flumatinib is a novel oral tyrosine kinase inhibitor (TKI) with higher potency and selectivity for BCR-ABL1 kinase compared to imatinib. Many patients experience aspergillosis infection and they may start using isavuconazole, which is an inhibitor of CYP3A4. However, there is no study on their interaction in vitro and in vivo. In the present study, the concentrations of flumatinib and its major metabolite M1 were rapidly determined using an stable ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. The half-maximal inhibitory concentration (IC50) was 6.66μM in human liver microsomes (HLM), while 0.62μM in rat liver microsomes (RLM) and 2.90μM in recombinant human CYP3A4 (rCYP3A4). Furthermore, the mechanisms of inhibition of flumatinib in human liver microsomes, rat liver microsomes and rCYP3A4 by isavuconazole were mixed. Moreover, ketoconazole, posaconazole, and isavuconazole showed more potent inhibitory effects than itraconazole, fluconazole, and voriconazole on HLM-mediated flumatinib metabolism. In pharmacokinetic experiments of rats, it was observed that isavuconazole could greatly change the pharmacokinetic parameters of flumatinib, including AUC(0-t), AUC(0-∞), Cmax and CLz/F, but had no effect on the metabolism of M1. According to the results of in vitro and in vivo studies, the metabolism of flumatinib was inhibited by isavuconazole, suggesting that isavuconazole may raise the plasma concentration of flumatinib. Thus, it is important to take special care of the interactions between flumatinib and isavuconazole in clinical applications.

Inhibitory Effects of Major Cannabinoids and their Metabolites on Oxazepam Metabolism

Cannabis and cannabis‐derived products are increasingly used for recreational and medical purposes including anxiety and epilepsy and are often concurrently used with other types of conventional medications. The concomitant use of cannabis with other therapeutic drugs has been shown to increase the likelihood of deleterious drug‐drug interactions. Oxazepam is a well‐known benzodiazepine used in clinical practice as an anxiolytic, sedative, and anticonvulsant agent. Oxazepam is a racemic mixture of the S‐and R‐enantiomers and is primarily metabolized via glucuronidation in an enantiomeric‐specific manner, where S‐oxazepam is mainly glucuronidated by UDP‐glucuronosyltransferase (UGT) 2B15 and R‐oxazepam is glucuronidated primarily by UGT1A9 and UGT2B7. Recent comprehensive in vitrostudies using probe substrates have shown that the major cannabinoids Δ9‐ tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) can inhibit several of the primary hepatic and renal UGT enzymes. The objective of the present study was to evaluate the potential inhibitory effect of cannabinoids and their major circulating metabolites, 11‐hydroxy‐Δ9‐tetrahydrocannabinol (11‐OH‐THC), 11‐ nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol (11‐COOH‐THC), and 11‐nor‐Δ9‐tetrahydrocannabinol‐carboxylic acid glucuronide (THC‐COOH‐Gluc) on the UGT enzymes involved in R,S‐oxazepam metabolism. We hypothesize that major cannabinoids and their metabolites will inhibit R,S‐oxazepam metabolism, leading to decreased clearance. To investigate potential drug‐drug interactions, cannabinoids and their major plasma metabolites were screened as inhibitors of R‐ and S‐oxazepam glucuronidation in human liver microsomes (HLM). In vitro inhibition screening assays with 10 μM or 100 μM of CBD and 11‐OH‐THC inhibited both R‐ and S‐oxazepam glucuronidation in HLM by up to 90%. The IC50 values 11‐OH‐THC and CBD inhibition of R‐oxazepam glucuronidation were 10.0 ± 7.8 μM and 7.0 ± 4.2 μM, respectively, in HLM; the respective IC50 values for the inhibition of S‐oxazepam glucuronidation by 11‐OH‐THC and CBD were 15.0 ± 6.4 μM and 52.2 ± 22.6 μM in HLM. These data indicate that co‐administration of oxazepam with cannabis may result in inhibition of oxazepam clearance, suggesting a strong potential for drug‐drug interactions in vivo.

UGT1A1 and UGT1A3 activity and inhibition in human liver and intestinal microsomes and a recombinant UGT system under similar assay conditions using selective substrates and inhibitors

In vitro enzyme kinetics and inhibition data was compared for UGT1A1 and UGT1A3 isoforms under similar assay conditions using human liver microsomes (HLM), human intestinal microsomes (HIM) and recombinant UGT (rUGT) enzyme systems. UGT1A1 catalysed β-estradiol 3-β-D-glucuronide formation showed allosteric sigmoidal kinetics in all enzyme systems; while UGT1A3 catalysed CDCA 24-acyl-β-D-glucuronide formation exhibited Michaelis–Menten kinetics in HLM, substrate inhibition kinetics in HIM and rUGT systems. Corresponding K m or S 50 concentrations of β-estradiol and CDCA were employed in the respective UGT inhibition studies. Atazanavir inhibited the production of β-estradiol 3-β-D-glucuronide with values of 0.54 µM and 0.16 µM in HLM and rUGT1A1, respectively. But its inhibition potential was not observed in HIM, indicating potential cross-talk with other high-affinity intestinal UGT isozymes. On the other hand, zafirlukast, a pan UGT inhibitor, exhibited moderate inhibition in HIM with an value of 16.70 µM. Lithocholic acid, inhibited the production of CDCA 24-acyl-β-D-glucuronide with values of 1.68, 1.84, and 12.42 µM in HLM, rUGT1A3, and HIM, respectively. These results indicated that HLM, HIM, and rUGTs may be used as complementary in vitro systems to evaluate hepatic and intestinal UGT mediated DDIs at the screening stage.

In vitro modulatory effects of ginsenoside compound K, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol on uridine 5′-diphospho-glucuronosyltransferase activity and expression

We explored the inhibitory effect of ginsenoside compound K (CK), 20(S)-protopanaxadiol (PPD), and 20(S)-protopanaxatriol (PPT) on six uridine 5′-diphospho-glucuronosyltransferase (UGT) enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) activities in human liver microsomes (HLMs) and 10 UGT enzyme (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B10, 2B15, and 2B17) activities in recombinant UGT isoforms. PPD was a potent inhibitor of UGT1A3 activity with half-maximal inhibitory concentration values of 5.62 and 3.38 μM in HLMs and recombinant UGT1A3, respectively. UGT1A3 inhibition by CK and PPD was competitive with inhibitory constant (K i) values of 17.4 and 1.21 μM, respectively, and inhibition by PPT was non-competitive with a K i value of 8.07 μM in HLMs. PPD exhibited more than 3.4-fold selectivity for UGT1A3 inhibition compared with other UGT isoforms inhibition, while CK and PPT showed more than 2.16- and 2.21-fold selectivity, respectively. PPD did not significantly increase the mRNA expression of UGT1A1, 1A3, 1A4, 1A9, and 2B7 in hepatocytes. Given the low plasma concentrations of PPD in healthy human subjects and the absence of induction potential on UGT isoforms, we conclude that PPD cause no pharmacokinetic interactions with other co-administered drugs metabolised by UGT1A3.

Inhibitory effect of propafenone on vortioxetine metabolism in vitro and in vivo

Vortioxetine, as a new therapeutic drug of major depressive disorder (MDD), was approved for MDD in the USA. The significance of our study in this paper was to explore the inhibitory effect of propafenone on the metabolism of vortioxetine through the in vivo and in vitro experiments. In the vitro experiments, we added a series of concentrations of propafenone into an incubation system as the inhibitors and calculated the half-maximal inhibitory concentration (IC50) of propafenone on vortioxetine metabolism in human liver microsomes (HLMs) and rat liver microsomes (RLMs). Twelve male Sprague-Dawley (SD) rats were included in the vivo experiments. We randomly divided them into Group A (control group) and Group B (90 mg/kg propafenone). 30 min later, a single oral dose of 4.0 mg/kg vortioxetine was administrated to each rat. Then, we used ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to determine the concentrations of vortioxetine and its metabolite Lu AA34443. From our results, it indicated that propafenone inhibited the metabolic rate of vortioxetine in the vitro studies with the IC50 of 0.48 μM and 16.5 μM for HLMs and RLMs, respectively. And, propafenone could competitively inhibit vortioxetine in both HLMs and RLMs for the inhibitory mechanisms. Moreover, a single oral dose of 90 mg/kg propafenone obviously enchanced the exposure of vortioxetine in rats, but not Lu AA34443. Combined with the vitro and vivo data, propafenone showed the inhibitory effect on vortioxetine metabolism. Thus, more attention to the safety of vortioxetine in clinic should be paid when taking it with propafenone in combination for the therapy.

Glabrone as a specific UGT1A9 probe substrate and its application in discovering the inhibitor glycycoumarin

UDP-glucuronosyltransferase 1A9 (UGT1A9) is one of the most important UGT isoforms, and plays an important role in the metabolic elimination of therapeutic drugs via glucuronidation. Herbal medicines affecting the activity of UGT1A9 may influence the metabolism of related drugs, thus causing herb-drug interactions and even adverse effects. However, few methods are available to evaluate the activity of UGT1A9. In this study, a natural product glabrone was discovered as an isoform-specific probe substrate for UGT1A9. The Vmax and Km values of glabrone were 362.6 nmol/min/mg protein and 17.2 μM for human liver microsomes (HLMs), and 382.3 nmol/min/mg protein and 16.6 μM for recombinant human UGT1A9, respectively. Glabrone 7-O-glucuronide, the UGT1A9 metabolite of glabrone, was prepared by using a plant glucuronosyltransferase UGT88D1, and the structure was identified by NMR spectroscopy. Using glabrone as a probe, we established a rapid HPLC method to screen UGT1A9 inhibitors from 54 natural products isolated from the Chinese herbal medicine licorice. Among them, glycycoumarin was found as a potent UGT1A9 inhibitor with an IC50 value of 6.04 μM. In rats, the pretreatment of glycycoumarin (4 mg/kg, i.p.) for 3 days could remarkably increase the plasma concentrations of dapagliflozin while decrease the concentrations of dapagliflozin-O-glucuronide after administration of dapagliflozin (1 mg/kg, i.v.), which is mainly metabolized by UGT1A9. The results indicated the potential risk of herb-drug interactions between licorice and UGT1A9-metabolizing drugs.

Selective inhibitory effects of HYIpro-3-1 on CYP1A2 in human liver microsomes.

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro-3-1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC-MS/MS using a cocktail assay. Among the four compounds, HYIpro-3-1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC50 values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro-3-1 is not dependent on preincubation time. HYIpro-3-1 showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation, based on the Lineweaver-Burk plot, with a Ki value of 0.05 μM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro-3-1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.

Investigation of the effects of axitinib on the pharmacokinetics of loperamide and its main metabolite N-demethylated loperamide in rats by UPLC-MS/MS

The epidemic of loperamide abuse and misuse in the patients for the alternative to opioids has become an increasing worldwide concern and has led to considerations about the potential for drug-drug interactions between loperamide and other combined drugs, especially inhibitors of cytochrome P450 (CYP450) enzymes, such as axitinib. This study assessed the effects of axitinib on the metabolism of loperamide and its main metabolite N-demethylated loperamide in rats and in rat liver microsomes (RLM), human liver microsomes (HLM) and recombinant human CYP3A4*1. The concentrations of both compounds were determined by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The exposures (AUC(0-t), AUC(0-∞) and Cmax) of loperamide and N-demethylated loperamide showed a conspicuous increase when loperamide was co-administered with axitinib. The Tmax of loperamide increased while CLz/F decreased under the influence of axitinib. In vitro, axitinib inhibited loperamide metabolism with the IC50 of 18.34 μM for RLM, 1.705 μM for HLM and 1.604 μM for CYP3A4*1, and it was confirmed as a non-competitive inhibitor in all enzymes. Taken together, the results indicated that axitinib had an obvious inhibitory impact on loperamide metabolism both in vivo and in vitro. Thus, more attention should be paid to the concurrent use of loperamide and axitinib to reduce the risk of unexpected clinical outcomes.

Inhibitory effect of resveratrol on the pharmacokinetic of ibrutinib by UPLC–MS/MS

Objective: To investigate the impact of resveratrol on the metabolism of ibrutinib in vitro and in vivo.Methods: In vitro, rat liver microsomes (RLM) and human liver microsomes (HLM) were used to study. In vivo, 18 male SD rats were randomly divided into three groups (n = 6): ibrutinib and the multiple dose of 100 mg/kg resveratrol for consecutive 7 days (Group A), ibrutinib and the single dose of 100 mg/kg resveratrol (Group B), ibrutinib (Group C). Processed samples were analyzed by UPLC–MS/MS.Results: Resveratrol showed inhibition on RLM and HLM in vitro. The IC50 of resveratrol was 8.745 µM in RLM and 7.789 µM in HLM. Furthermore, Groups A and B both increased the AUC and reduced the CLz/F. The Cmax of Group A and the MRT(0–t) of Group B were significantly improved.Conclusions: Resveratrol inhibits the pharmacokinetic of ibrutinib in vitro and in vivo. It is necessary to pay more attention to adjust the dose of the drug when resveratrol is used in combination with ibrutinib.

A Specific Probe Substrate for Evaluation of CYP4A11 Activity in Human Tissue Microsomes and a Highly Selective CYP4A11 Inhibitor: Luciferin-4A and Epalrestat.

The specificity of cytochrome P450 4A11 (CYP4A11) against luciferin-4A O-demethylation in human liver microsomes (HLMs) and human renal microsomes (HRMs) and selectivity of CYP4A11 inhibition by epalrestat were investigated. Kinetic analysis of luciferin-4A O-demethylation yielded Vmax and S50 values of 39.7 pmol/min per milligram protein and 43.2 μM for HLMs (Hill coefficient 1.24) and 39.4 pmol/min per milligram protein and 33.8 μM for HRMs (Hill coefficient 1.34), respectively. Among the selective CYP inhibitors tested, HET0016 (CYP4 inhibitor) exclusively inhibited luciferin-4A O-demethylation by HLMs and HRMs. Furthermore, anti-CYP4A11 antibody nearly abolished the activity of both tissue microsomes. Luciferin-4A O-demethylase activity of HLMs was significantly correlated with lauric acid ω-hydroxylase activity, a marker of CYP4A11 activity (r = 0.904, P < 0.0001). Next, effects of epalrestat on CYP-mediated drug oxidations were examined. Epalrestat showed the most potent inhibition against CYP4A11 (IC50 = 1.82 μM) among the 17 recombinant enzymes tested. The inhibitory effect of epalrestat on CYP4A11 was at least 10-fold stronger than those on CYP4F2, CYP4F3B, and CYP4F12. For known CYP4 inhibitors, in contrast, HET0016 inhibited the activities of CYP4A11 and CYP4F2 (IC50 = 0.0137-0.0182 μM); 17-octadecynoic acid reduced activities of CYP4A11, CYP4F2, CYP4F3B, and CYP4F12 to a similar extent (IC50 = 5.70-17.7 μM). Epalrestat selectively and effectively inhibited the CYP4A11 activity of HLMs (IC50 = 0.913 μM) and HRMs (IC50 = 0.659 μM). These results indicated that luciferin-4A O-demethylase activity is a good CYP4A11 marker of HLMs and HRMs, and that epalrestat is a more selective CYP4A11 inhibitor compared with known CYP4 inhibitors.

Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes.

Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP) enzyme activities in human liver microsomes were evaluated using liquid chromatography-tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation with a Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4′-hydroxylation (Ki, 3.7 μM; kinact, 0.102 min−1), CYP2C8-catalyzed amodiaquine N-deethylation (Ki, 10.7 μM; kinact, 0.082 min−1), and CYP3A4-catalyzed midazolam 1′-hydroxylation (Ki, 23.0 μM; kinact, 0.050 min−1) in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1′-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.

AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 µM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 µM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

In Vitro Assessment of CYP-Mediated Drug Interactions for Kinsenoside, an Antihyperlipidemic Candidate.

Kinsenoside, the herb-derived medicine isolated from the plant Anoect chilus, has diverse pharmacological actions, and it is considered to be a promising antihyperlipidemic drug candidate. This study evaluates the effects of kinsenoside on CYP enzyme-mediated drug metabolism in order to predict the potential for kinsenoside-drug interactions. Kinsenoside was tested at different concentrations of 0.1, 0.3, 1, 3, 10, 30, and 100 µM in human liver microsomes. The c Cktail probe assay based on liquid chromatography-tandem mass spectrometry was conducted to measure the CYP inhibitory effect of kinsenoside. Subsequently, the metabolism profiles of amlodipine and lovastatin in human liver microsomes were analyzed following co-incubation with kinsenoside. The concentration levels of the parent drug and the major metabolites were compared with the kinsenoside-cotreated samples. The effect of kinsenoside was negligible on the enzyme activity of all the CYP isozymes tested even though CYP2A6 was slightly inhibited at higher concentrations. The drug-drug interaction assay also showed that the concomitant use of kinsenoside has a non-significant effect on the concentration of lovastatin or amlodipine, and their major metabolites. So, it was concluded that there is almost no risk of drug interaction between kinsenoside and CYP drug substrates via CYP inhibition.

Selective Inhibition of Bakuchicin Isolated from Psoralea corylifolia on CYP1A in Human Liver Microsomes.

Bakuchicin is a furanocoumarin isolated from Psoralea corylifolia and shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs) and human recombinant cDNA-expressed CYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetin O-deethylation with an IC50 value of 0.43 μM in HLMs. It was confirmed by human recombinant cDNA-expressed CYP1A1 and CYP1A2 with a K i value of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs.

In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4.

Glycyrrhetinic acid (GA) has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450) cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs) and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 μM in HLMs and 7.498 μM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver–Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of ursolic acid

This study aims to characterize the glucuronidation kinetics of ursolic acid (UA) in human liver microsomes (HLMs) and intestinal microsomes (HIMs) and identify the main UDP-glucuronosyltransferases (UGTs) involved. In our present study, only one type of UA glucuronide was observed after incubation with HLMs and HIMs respectively and was identified as a UA hydroxyl O-glucuronide. The glucuronidation of UA can be shown in HLMs and HIMs with Km values of 3.29 ± 0.16 and 3.74 ± 0.22 μM and Vmax values of 0.33 ± 0.03 and 0.42 ± 0.03 nmol/min/(mg protein). Among the 12 recombinant UGT enzymes investigated, UGT1A3 and UGT1A4 were identified as the major enzymes catalyzing the glucuronidation of UA [Km values of 2.58 ± 0.12 and 4.66 ± 0.60 μM, Vmax values of 0.72 ± 0.01 and 1.00 ± 0.06 nmol/min/(mg protein)]. The chemical inhibition study showed that the IC50 for hecogenin inhibition of UA glucuronidation was 51.79 ± 4.32 μM in HLMs. And chenodeoxycholic acid inhibited UA glucuronidation in HLMs with an IC50 of 28.26 ± 2.91 μM. In addition, UA glucuronidation in a panel of eight HLM was significantly correlated with telmisartan glucuronidation (r2 = 0.7660, p < 0.01) and trifluoperazine glucuronidation (r2 = 0.5866, p < 0.01) respectively. These findings collectively indicate that UGT1A3 and UGT1A4 were the main enzymes responsible for the glucuronidation of UA in human.