In vitro inhibitory effects of dihydromyricetin on human liver cytochrome P450 enzymes

Context: Dihydromyricetin (DHM) is the most abundant and active flavonoid component isolated from Ampelopsis grossedentata (Hand-Mazz) W.T. Wang (Vitaceae) and it possesses numerous pharmacological activities. However, whether DHM affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

Materials and methods: The inhibitory effects of DHM on eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) were investigated in vitro using human liver microsomes (HLMs).

Results: The results showed that DHM could inhibit the activity of CYP3A4, CYP2E1 and CYP2D6, with IC₅₀ values of 14.75, 25.74 and 22.69?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that DHM was not only a non-competitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2E1 and CYP2D6, with Ki values of 6.06, 9.24 and 10.52?μM, respectively. In addition, DHM is a time-dependent inhibitor for CYP3A4 with K_I/K_inact value of 12.17/0.057?min^?1?μM^?1.

Discussion and conclusion: The in vitro studies of DHM with CYP isoforms indicate that DHM has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2D6. Further clinical studies are needed to evaluate the significance of this interaction.     

In vitro inhibitory effects of sophocarpine on human liver cytochrome P450 enzymes

Abstract

1.?Sophocarpine is a biologically active component isolated from the foxtail-like sophora herb and seed that is often orally administered for the treatment of cancer and chronic bronchial asthma. However, whether sophocarpine affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

2.?In this study, the inhibitory effects of sophocarpine on the eight human liver CYP isoforms (CYP1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs).

3.?The results indicate that sophocarpine could inhibit the activity of CYP3A4 and 2C9, with the IC₅₀ values of 12.22 and 15.96?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that sophocarpine is not only a noncompetitive inhibitor of CYP3A4 but also a competitive inhibitor of CYP2C9, with K_i values of 6.74 and 9.19?μM, respectively. Also, sophocarpine is a time-dependent inhibitor of CYP3A4 with K_inact/K_I value of 0.082/21.54?μM^?1?min^?1.

4.?The in vitro studies of sophocarpine with CYP isoforms suggested that sophocarpine has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.     

Effects of catalpol on the activity of human liver cytochrome P450 enzymes

Abstract

1. Catalpol possesses numerous pharmacological activities, and however, little data available for the effects of catalpol on the activity of human liver cytochrome P450 (CYP) enzymes.

2. This study investigates the inhibitory effects of catalpol on the main human liver CYP isoforms. In this study, the inhibitory effects of catalpol on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8 and 3A4 were investigated in human liver microsomes.

3. The results indicated that catalpol could inhibit the activity of CYP3A4, CYP2E1 and CYP2C9, with IC₅₀ values of 14.27, 22.4 and 14.69?μM, respectively, but those other CYP isoforms were not affected. Enzyme kinetic studies showed that catalpol was not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1 and CYP2C9, with Ki values of 7.40, 10.75 and 7.37?μM, respectively. In addition, catalpol is a time-dependent inhibitor for CYP3A4, with maximum inactivation (kinact) and 50% maximum inactivation (K_I) values of 0.02?min^?1 and 1.86?μM, respectively.

4. The in vitro studies of catalpol with CYP isoforms suggest that catalpol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4, CYP2E1 and CYP2C9. Further in vivo studies are needed in order to evaluate the significance of this interaction.     

Inhibitory effects of curculigoside on human liver cytochrome P450 enzymes

1.?Curculigoside possesses numerous pharmacological activities, and however, little data available for the effects of curculigoside on the activity of human liver cytochrome P450 (CYP) enzymes.

2.?This study investigates the inhibitory effects of curculigoside on the main human liver CYP isoforms. In this study, the inhibitory effects of curculigoside on the eight human liver CYP isoforms 1A2, 2A6, 2E1, 2D6, 2C9, 2C19, 2C8, and 3A4 were investigated in human liver microsomes.

3.?The results indicated that curculigoside could inhibit the activity of CYP1A2, CYP2C8, and CYP3A4, with IC₅₀ values of 15.26, 11.93, and 9.47?μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that curculigoside was not only a noncompetitive inhibitor of CYP1A2, but also a competitive inhibitor of CYP2C8 and CYP3A4, with Ki values of 5.43, 3.54, and 3.35?μM, respectively. In addition, curculigoside is a time-dependent inhibitor for CYP1A2, with kinact/K_I values of 0.056/6.15?μM^?1?min^?1.

4.?The in vitro studies of curculigoside with CYP isoforms suggest that curculigoside has the potential to cause pharmacokinetic drug interactions with other coadministered drugs metabolized by CYP1A2, CYP2C8, and CYP3A4. Further in vivo studies are needed in order to evaluate the significance of this interaction.     

In vitro evaluation of the inhibition and induction potential of olaparib,a potent poly(ADP-ribose) polymerase inhibitor,on cytochrome P450

1.?In vitro studies were conducted to evaluate potential inhibitory and inductive effects of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, on cytochrome P450 (CYP) enzymes. Inhibitory effects were determined in human liver microsomes (HLM); inductive effects were evaluated in cultured human hepatocytes.

2.?Olaparib did not inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6 or CYP2E1 and caused slight inhibition of CYP2C9, CYP2C19 and CYP3A4/5 in HLM up to a concentration of 100?μM. However, olaparib (17–500?μM) inhibited CYP3A4/5 with an IC₅₀ of 119?μM. In time-dependent CYP inhibition assays, olaparib (10?μM) had no effect against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 and a minor effect against CYP3A4/5. In a further study, olaparib (2–200?μM) functioned as a time-dependent inhibitor of CYP3A4/5 (K_I, 72.2?μM and K_inact, 0.0675?min^?1). Assessment of the CYP induction potential of olaparib (0.061–44?μM) showed minor concentration-related increases in CYP1A2 and more marked increases in CYP2B6 and CYP3A4 mRNA, compared with positive control activity; however, no significant change in CYP3A4/5 enzyme activity was observed.

3.?Clinically significant drug–drug interactions due to olaparib inhibition or induction of hepatic or intestinal CYP3A4/5 cannot be excluded. It is recommended that olaparib is given with caution with narrow therapeutic range or sensitive CYP3A substrates, and that prescribers are aware that olaparib may reduce exposure to substrates of CYP2B6.     

Screening of drug metabolizing enzymes for fusidic acid and its interactions with isoform-selective substrates in vitro

1.?Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro.

2.?The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including K_i, K_i′, V_max, and IC₅₀ were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates.

3.?FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with K_is of 13.9 and 38.6?μM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA.

4.?FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug–drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.     

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes

1.?The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes.

2.?100?μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC₅₀) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00?μM, and the inhibition kinetic parameters (K_i) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11?μM, respectively.

3.?Metabolism of morusin exhibited significant species differences. The quantities of M₁ from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The K_m values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83?μM, and V_max for these species were 0.09, 1.23, 1.43, 0.15, and 0.75?nmol/min/mg, respectively. CL_int (intrinsic clearance) values (V_max/K_m) for morusin obeyed the following order: monkey?>?rat?>?minipig?>?dog?>?human. CL_H (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12?mL/min/kg body weight, respectively.

4.?This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.     

Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems

Abstract

1.?Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems.

2.?Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC₅₀ values of 615.2?μM and 669.5?μM in HLM and rCYP3A4 systems, respectively. IC₅₀-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC₅₀ in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables K_I and K_inact were 485.8?μM and 0.05?min^–1, respectively.

3.?Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products.

4.?In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.     

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities

1.?The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically.

2.?The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with K_i of 35.95?±?6.96 and 60.56?±?3.53?μmol/l and CYP3A4 (inhibited by genistein with K_i of 23.25?±?5.85?μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (K_i of 57.69?±?2.36?μmol/l) and equol (K_i of 38.47?±?2.32?μmol/l).

3.?Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.     

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305

1.?5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors.

2.?It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy.

3.?The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4.

4.?Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, K_I?=?10.8?±?2.14?μM) and testosterone 6β-hydroxylation (CYP3A4, K_I = 9.1?±?2.82?μM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively.

5.?In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug–drug interactions in potent multidrug therapy.     

In vitro study on the effect of peucedanol on the activity of cytochrome P450 enzymes

ContextPeucedanol is a major extract of Peucedanum japonicum Thunb. (Apiaceae) roots, which is a commonly used herb in paediatrics. Its interaction with cytochrome P450 enzymes (CYP450s) would lead to adverse effects or even failure of therapy.ObjectiveThe interaction between peucedanol and CYP450s was investigated.Materials and methodsPeucedanol (0, 2.5, 5, 10, 25, 50, and 100 μM) was incubated with eight human liver CYP isoforms (CYP1A2, 2A6, 3A4, 2C8, 2C9, 2C19, 2D6, and 2E1), in pooled human liver microsomes (HLMs) for 30 min with specific inhibitors as positive controls and untreated HLMs as negative controls. The enzyme kinetics and time-dependent study (0, 5, 10, 15, and 30 min) were performed to obtain corresponding parameters in vitro.ResultsPeucedanol significantly inhibited the activity of CYP1A2, 2D6, and 3A4 in a dose-dependent manner with IC₅₀ values of 6.03, 13.57, and 7.58 μM, respectively. Peucedanol served as a non-competitive inhibitor of CYP3A4 with a K_i value of 4.07 μM and a competitive inhibitor of CYP1A2 and 2D6 with a K_i values of 3.39 and 6.77 μM, respectively. Moreover, the inhibition of CYP3A4 was time-dependent with the K_i/K_inact value of 5.44/0.046 min/μM.Discussion and conclusionsIn vitro inhibitory effect of peucedanol on the activity of CYP1A2, 2A6, and 3A4 was reported in this study. As these CYPs are involved in the metabolism of various drugs, these results implied potential drug-drug interactions between peucedanol and drugs metabolized by CYP1A2, 2D6, and 3A4, which needs further in vivo validation.     

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. K_I and k_inact 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.     

Impact of CYP2C8*3 polymorphism on in vitro metabolism of imatinib to N-desmethyl imatinib

Abstract

1.?Imatinib is metabolized to N-desmethyl imatinib by CYPs 3A4 and 2C8. The effect of CYP2C8*3 genotype on N-desmethyl imatinib formation was unknown.

2.?We examined imatinib N-demethylation in human liver microsomes (HLMs) genotyped for CYP2C8*3, in CYP2C8*3/*3 pooled HLMs and in recombinant CYP2C8 and CYP3A4 enzymes. Effects of CYP-selective inhibitors on N-demethylation were also determined.

3.?A single-enzyme Michaelis–Menten model with autoinhibition best fitted CYP2C8*1/*1 HLM (n?=?5) and recombinant CYP2C8 kinetic data (median?±?SD K_i?=?139?±?61?µM and 149?µM, respectively). Recombinant CYP3A4 showed two-site enzyme kinetics with no autoinhibition. Three of four CYP2C8*1/*3 HLMs showed single-enzyme kinetics with no autoinhibition. Binding affinity was higher in CYP2C8*1/*3 than CYP2C8*1/*1 HLM (median?±?SD K_m?=?6?±?2 versus 11?±?2?µM, P=0.04). CYP2C8*3/*3 (pooled HLM) also showed high binding affinity (K_m?=?4?µM) and single-enzyme weak autoinhibition (K_i?=?449?µM) kinetics. CYP2C8 inhibitors reduced HLM N-demethylation by 47–75%, compared to 0–30% for CYP3A4 inhibitors.

4.?In conclusion, CYP2C8*3 is a gain-of-function polymorphism for imatinib N-demethylation, which appears to be mainly mediated by CYP2C8 and not CYP3A4 in vitro in HLM.     

Pogostone inhibits the activity of CYP3A4, 2C9, and 2E1 in vitro

ContextPogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination.ObjectiveTo investigate the effect of pogostone on the activity of human CYP450s.Materials and methodsThe effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0–100 μM pogostone and various concentrations of substrates.ResultsPogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the IC₅₀ values of 11.41, 12.11, and 14.90 μM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the K_i value of 5.69 μM and the KI/K_inact value of 5.86/0.056/(μM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the K_i value of 6.46 and 7.67 μM and was not affected by the incubation time.Discussion and conclusionsThe inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further in vivo investigations to validate the potential interaction.     

IC50-based approaches as an alternative method for assessment of time-dependent inhibition of CYP3A4

1. The predictive utility of two in vitro methods (empirical IC₅₀-based and mechanistic k_inact/K_I) for the assessment of time-dependent cytochrome P450 3A4 (CYP3A4) inhibition has been compared.

2. IC₅₀ values were determined at multiple pre-incubation time points over 30?min for five CYP3A4 time-dependent inhibitors (verapamil, diltiazem, erythromycin, clarithromycin, and azithromycin). The ability of IC₅₀ data obtained following pre-incubation to predict k_inact/K_I parameters was investigated and its utility was assessed relative to the conventional k_inact/K_I model using 50 reported clinical drug–drug interactions (DDIs). Models with either hepatic or hepatic with intestinal components were explored.

3. For low/medium potency time-dependent inhibitors, 81% of the predicted k_inact/K_I(unbound) from IC₅₀ data were within an order of magnitude of the actual values, in contrast to 50% of potent inhibitors. An underprediction trend and >?50% of false-negatives were observed when IC₅₀ data were used in the DDI hepatic prediction model; incorporation of the intestine improved the prediction accuracy. On the contrary, 86% of the DDI studies were predicted within twofold using k_inact/K_I mechanistic approach and the combined hepatic and intestinal model.

4. Use of the empirical IC₅₀ approach as an alternative to the mechanistic k_inact/K_I model for in vivo DDI prediction is limited and is best restricted to preliminary investigations.

     

Identification of the cytochrome P450 isoforms involved in the O-demethylation of 4-nitroanisole in human liver microsomes

1. 4-Nitroanisole is O-demethylated to 4-nitrophenol by human liver microsomes. Kinetic studies indicate that this metabolic route is mediated by two cytochrome P450 isoforms, one with a K_m = 2.1 μM and the other with a K_m = 220 μM. 2. Chemical inhibition and correlation studies in human liver microsomes indicate that the low K_m enzyme is CYP2A6 and the high K_m enzyme is CYP2E1 suggesting that 4-nitroanisole is not a general cytochrome P450 substrate. 3. Studies using expressed recombinant cytochrome P450s indicated that all the cytochrome P450s investigated metabolized 4-nitroanisole but CYP2A6 and CYP2E1 produced the highest rates. Kinetic studies with these two isoforms produced a K_m for CYP2A6 of 9 μM and 54 μM for CYP2E1. 4. The involvement of these two isoforms in the O-demethylation of 4-nitroanisole can be rationalized in terms of a hydrogen bond interaction with the nitro group and the active site of CYP2A6 and a hydrophobic interaction with the active site of CYP2E1.     

Retrospective use of PBPK modelling to understand a clinical drug–drug interaction between dextromethorphan and GSK1034702

1.?In a clinical trial, a strong drug–drug interaction (DDI) was observed between dextromethorphan (DM, the object or victim drug) and GSK1034702 (the precipitant or perpetrator drug), following single and repeat doses. This study determined the inhibition parameters of GSK1034702 in vitro and applied PBPK modelling approaches to simulate the clinical observations and provide mechanistic hypotheses to understand the DDI.

2.?In vitro assays were conducted to determine the inhibition parameters of human CYP2D6 by GSK1034702. PBPK models were populated with the in vitro parameters and DDI simulations conducted and compared to the observed data from a clinical study with DM and GSK1034702.

3.?GSK1034702 was a potent direct and metabolism-dependent inhibitor of human CYP2D6, with inhibition parameters of: IC_50?=_?1.6?μM, K_inact?=?3.7?h^?1 and K_I?=?0.8?μM. Incorporating these data into PBPK models predicted a DDI after repeat, but not single, 5?mg doses of GSK1034702.

4.?The DDI observed with repeat administration of GSK1034702 (5?mg) can be attributed to metabolism-dependent inhibition of CYP2D6. Further, in vitro data were generated and several potential mechanisms proposed to explain the interaction observed following a single dose of GSK1034702.     

A simplified approach to predict CYP3A-mediated drug–drug interactions at early drug discovery: validation with clinical data

Abstract

1. The present study evaluates which factors should be incorporated into a simplified approach to reasonably predict CYP3A-mediated drug–drug interaction (DDI) at an early drug discovery stage.

2. CYP3A IC₅₀ values were obtained using human liver microsomes (HLM) and hepatocytes. Plasma and microsomal protein binding and in vitro hepatocyte partition coefficient (K_p) were also determined for 10 drugs. Therapeutic human maximum plasma concentrations (C_max) were retrieved from the literature. DDI predictions were performed using an equation incorporating the fraction of the substrate metabolized by CYP3A with the total or free plasma C_max, with or without correction for hepatocyte K_p.

3. Based on the K_i data from HLM, the use of total C_max provided a prediction of DDI within 2-fold of the observed clinical values for 9 out of 10 drugs.

4. In comparison, free drug corrections for both C_max and K_i values from HLM led to an underprediction of DDI (>3-fold error for five drugs).

5. Data from hepatocytes showed, in general, lower prediction accuracy than data from HLM.

6. CYP3A-mediated DDIs can be predicted with a high level of accuracy based on K_i estimates from HLM data and the total therapeutic plasma C_max of the inhibitors. This approach should be widely applicable to the assessment of clinically significant DDIs risk in early drug discovery programs     

Preclinical evaluation of the potential for cytochrome P450 inhibition and induction of the selective ALK inhibitor,alectinib

1.?A novel selective anaplastic lymphoma kinase (ALK) inhibitor, alectinib, has shown remarkable efficacy and safety in patients with ALK-positive non-small-cell lung cancer (NSCLC). The purpose of this study was to evaluate in vitro the potential to inhibit and induce cytochrome P450 (CYP) isoforms for alectinib and its major metabolite M4.

2.?Alectinib and M4 did not show the meaningful direct inhibition of six major CYP isoforms (CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4) in human liver microsomes (HLM). Alectinib, but not M4, competitively inhibited CYP2C8, by which few marketed drugs are exclusively metabolized, with an inhibition constant of 1.98?μM.

3.?Out of the seven CYP isoforms in HLM, alectinib and M4 showed time-dependent inhibition (TDI) of only CYP3A4, which suggests low TDI potential due to low inactivation efficiency.

4.?Alectinib exhibited quite smaller induction of mRNA expression of CYP1A2, 2B6 and 3A4 genes in human hepatocytes compared to the respective positive controls, suggesting a low potential of enzyme induction.

5.?In summary, the risk of alectinib causing drug-drug interactions with coadministered drugs is expected to be low due to the weak potential of CYP inhibition and induction estimated in the preclinical studies.     

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1.?Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.

2.?UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.

3.?UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC₅₀ 0.391?±?0.013?μM; K_i 0.185?±?0.015?μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 2.651?±?0.201?μM; K_i 1.334?±?0.146?μM).

4.?OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC₅₀ 0.336?±?0.013?μM; K_i 0.176?±?0.007?μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 5.468?±?0.697?μM; K_i 6.298?±?0.891?μM).

5.?Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.