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.     

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 metabolism of alectinib,a novel potent ALK inhibitor,in human: contribution of CYP3A enzymes

1.?The in vitro metabolism of alectinib, a potent and highly selective oral anaplastic lymphoma kinase inhibitor, was investigated.

2.?The main metabolite (M4) in primary human hepatocytes was identified, which is produced by deethylation at the morpholine ring. Three minor metabolites (M6, M1a, and M1b) were also identified, and a minor peak of hydroxylated alectinib (M5) was detected as a possible precursor of M4, M1a, and M1b.

3.?M4, an important active major metabolite, was produced and further metabolized to M6 by CYP3A, indicating that CYP3A enzymes were the principal contributors to this route. M5 is possibly produced by CYP3A and other isoforms as the primary step in metabolism, followed by oxidation to M4 mainly by CYP3A. Alternatively, M5 could be oxidized to M1a and M1b via an NAD-dependent process. None of the non-CYP3A-mediated metabolism appeared to be major.

4.?In conclusion, this study suggests that involvement of multiple enzymes in the metabolism of alectinib reduces its potential for drug–drug interactions.     

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.     

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.     

In vitro evaluation of potential drug interactions mediated by cytochrome P450 and transporters for luseogliflozin,an SGLT2 inhibitor

1.?We evaluated potential in vitro drug interactions of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, mediated by CYP inhibition, CYP induction and drug transporters using human liver microsomes, primary hepatocytes and recombinant cells-expressing efflux or uptake transporters, respectively.

2.?Human CYP inhibition studies indicated that luseogliflozin was a weak inhibitor for CYP2C19 with an IC₅₀ value of 58.3?μM, whereas it was not an inhibitor of the other eight major isoforms that were tested. The exposure of primary hepatocytes to luseogliflozin for 72?hrs weakly induced CYP3A4 at a concentration of 10?μM, whereas it did not induce CYP1A2 or CYP2B6 at concentrations of 0.1–10?μM.

3.?An in vitro transport study suggested that luseogliflozin is a substrate for human P-glycoprotein (P-gp), but not for breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, organic anion transporter (OAT) 1 and OAT3, or organic cation transporter (OCT) 2. Luseogliflozin weakly inhibited OATP1B3 with an IC₅₀ value of 93.1?μM, but those for other transporters are greater than 100?μM.

4.?Based on the therapeutic plasma concentration of the drug, clinically relevant drug interactions are unlikely to occur between luseogliflozin and coadministered drugs mediated by CYPs and/or transporters.     

Absorption,distribution, metabolism and excretion (ADME) of the ALK inhibitor alectinib: results from an absolute bioavailability and mass balance study in healthy subjects

1.?Alectinib is a highly selective, central nervous system-active small molecule anaplastic lymphoma kinase inhibitor.

2.?The absolute bioavailability, metabolism, excretion and pharmacokinetics of alectinib were studied in a two-period single-sequence crossover study. A 50?μg radiolabelled intravenous microdose of alectinib was co-administered with a single 600?mg oral dose of alectinib in the first period, and a single 600?mg/67?μCi oral dose of radiolabelled alectinib was administered in the second period to six healthy male subjects.

3.?The absolute bioavailability of alectinib was moderate at 36.9%. Geometric mean clearance was 34.5?L/h, volume of distribution was 475?L and the hepatic extraction ratio was low (0.14).

4.?Near-complete recovery of administered radioactivity was achieved within 168?h post-dose (98.2%) with excretion predominantly in faeces (97.8%) and negligible excretion in urine (0.456%). Alectinib and its major active metabolite, M4, were the main components in plasma, accounting for 76% of total plasma radioactivity. In faeces, 84% of dose was excreted as unchanged alectinib with metabolites M4, M1a/b and M6 contributing to 5.8%, 7.2% and 0.2% of dose, respectively.

5.?This novel study design characterised the full absorption, distribution, metabolism and excretion properties in each subject, providing insight into alectinib absorption and disposition in humans.     

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 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.     

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

Context: Friedelin is a triterpenoid with several biological activities. However, the affects of Friedelin on the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.

Objective: This study investigates the inhibitory effects of Friedelin on the major human liver CYP isoforms (CYP3A4, 1A2, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8).

Materials and methods: First, the inhibitory effects of Friedelin (100?μM) on the eight human liver CYP isoforms were investigated in vitro using human liver microsomes (HLMs), and then enzyme inhibition, kinetic studies, and time-dependent inhibition studies were conducted to investigate the IC₅₀, K_i and K_inact/K_I values of Friedelin.

Results: The results indicate that Friedelin inhibited the activity of CYP3A4 and 2E1, with the IC₅₀ values of 10.79 and 22.54?μM, respectively, but other CYP isoforms were not affected. Enzyme kinetic studies showed that Friedelin is not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1, with K_i values of 6.16 and 18.02?μM, respectively. In addition, Friedelin is a time-dependent inhibitor of CYP3A4 with K_inact/K_i value of 4.84?nM/min.

Discussion and conclusion: The in vitro studies of Friedelin with CYP isoforms suggested that Friedelin has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2E1. Further clinical studies are needed to evaluate the significance of this interaction.     

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.     

Comprehensive evaluation of liver microsomal cytochrome P450 3A (CYP3A) inhibition: comparison of cynomolgus monkey and human

1.?Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug–drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment.

2.?Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC₅₀ values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH.

3.?In the absence of preincubation, the IC₅₀ values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC₅₀ were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem).

4.?Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.     

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.     

Dydrogesterone metabolism in human liver by aldo-keto reductases and cytochrome P450 enzymes

1.?The metabolism of dydrogesterone was investigated in human liver cytosol (HLC) and human liver microsomes (HLM). Enzymes involved in dydrogesterone metabolism were identified and their relative contributions were estimated.

2.?Dydrogesterone clearance was clearly higher in HLC compared to HLM. The major active metabolite 20α-dihydrodydrogesterone (20α-DHD) was only produced in HLC.

3.?The formation of 20α-DHD by cytosolic aldo-keto reductase 1C (AKR1C) was confirmed with isoenzyme-specific AKR inhibitors.

4.?Using recombinantly expressed human cytochrome P450 (CYP) isoenzymes, dydrogesterone was shown to be metabolically transformed by CYP3A4 and CYP2C19.

5.?A clear contribution of CYP3A4 to microsomal metabolism of dydrogesterone was demonstrated with HLM and isoenzyme-specific CYP inhibitors, and confirmed by a significant correlation between dydrogesterone clearance and CYP3A4 activity.

6.?Contribution of CYP2C19 was shown to be clearly less than CYP3A4 and restricted to a small group of human individuals with very high CYP2C19 activity. Therefore, it is expected that CYP2C19 genetic variations will not affect dydrogesterone pharmacokinetics in man.

7.?In conclusion, dydrogesterone metabolism in the liver is dominated primarily by cytosolic enzymes (particularly AKR1C) and secondarily by CYP3A4, with the former exclusively responsible for 20α-DHD formation.     

Oxidative metabolism of koumine is mainly catalyzed by microsomal CYP3A4/3A5

1.?Gelsemium elegans Benth (Loganiaceae) is a toxic plant that can be used for committing suicide besides alleviating pains. Its anti-inflammatory and analgesic effect mainly come from its active ingredient, namely koumine. Koumine, an indole alkaloid, possesses widely pharmacological effects especially inhibition of neuropathic pain.

2.?This study aimed to investigate the metabolic profile of koumine using human liver microsomes (HLMs), selective chemical inhibitors and recombinant human CYP isoforms. Ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) was used to detect and identify metabolites.

3.?Four major metabolites of koumine were found after incubation with HLMs or individual CYP isoforms. The metabolic pathways of koumine included demethylation, dehydrogenation, oxidation and demethyl-dehydrogenation. Chemical inhibition study showed that the inhibitor of CYP3A4/3A5 significantly decreased (93%) the formation of koumine metabolites. Further, CYP3A4/3A5 was shown as the most efficient isoform in biotransformation of koumine, among a series of CYP isoforms tested.

4.?In conclusion, koumine was metabolized into four oxidative metabolites in HLMs. And CYP3A4/3A5 was probably the main contributor to the hepatic oxidative metabolism of koumine.     

Investigation of selective inhibitory effects of glycyrol on human CYP 1A1 and 2C9

1.?Glycyrol is a coumarin derivative isolated from the roots of Glycyrrhiza uralensis called Gamcho in Korea and commonly used as a sweetener in oriental medicine. Glycyrol shows several biological activities, including anti-oxidative, anti-inflammatory, antibacterial, anti-angiogenic, and anti-allergenic properties. Although there have been studies on the biological effects of glycyrol, the inhibitory effects of glycyrol on cytochrome P450 (CYP) activities have not been investigated.

2.?We investigated the inhibitory effects of glycyrol on the activities of CYP isoforms using a cocktail of probe substrates in pooled human liver microsome (HLM) and human recombinant cDNA-expressed CYPs. Glycyrol strongly inhibited CYP1A-mediated phenacetin O-deethylation and CYP2C9-mediated diclofenac 4′-hydroxylation in HLMs, which were the result of competitive inhibition as revealed by a Dixon plot. In addition, glycyrol showed selective inhibition of CYP1A1- and CYP1A2-catalyzed phenacetin O-deethylase activity with a half-maximal inhibitory concentration of (IC₅₀) 1.3 and 16.1?μM in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively.

3.?Glycyrol decreased CYP2C9-catalyzed diclofenac 4′-hydroxylation activity with IC₅₀ values of 0.67?μM in human recombinant cDNA-expressed CYP2C9. This is the first investigation of competitive inhibitory effects on CYP1A1 and CYP2C9 in HLMs.     

The in vitro metabolism of bupropion revisited: concentration dependent involvement of cytochrome P450 2C19

1. The involvement of cytochrome P450 2B6 (CYP2B6) to the in vitro and in vivo metabolism of bupropion has been well studied. In these investigations we performed a detailed in vitro phenotyping study to characterize isoforms other than CYP2B6.

2. A total of nine metabolites were identified (M1–M9) in the incubations with cDNA-expressed P450s (rhCYP) and human liver microsomes (HLM).

3. Incubations in rhCYP identified CYP2B6 as the isoform responsible for the formation of hydroxybupropion (M3). CYP2C19 was involved in bupropion metabolism primarily through alternate hydroxylation pathways (M4–M6) with higher activity at lower substrate concentrations, near 1 µM.

4. The results from HLM inhibition studies using CYP2B6 and CYP2C19 inhibitory antibodies indicated that CYP2B6 contributed to approximately 90% of M3 formation, and CYP2C19 contributed to approximately 70–90% of M4, M5, and M6 formation.

5. Studies using single donor HLM with varying degrees of CYP2B6 and CYP2C19 activities showed a good relationship between M3 formation and CYP2B6 activity and M4/M5 formation and CYP2C19 activity.

6. These results confirmed the principle role of CYP2B6 in hydroxybupropion formation, as a selective CYP2B6 probe. In addition, the new findings revealed that CYP2C19 also contributes to bupropion metabolism through alternate hydroxylation pathways.

     

Assessment of the pulmonary CYP1A1 metabolism of mavoglurant (AFQ056) in rat

1.?AFQ056 phenotyping results indicate that CYP1A1 is responsible for the formation of the oxidative metabolite, M3. In line with the predominant assumption that CYP1A1 is mainly expressed in extrahepatic tissues, only traces of M3 were detected in hepatic systems. The aim of this study was to investigate the pulmonary CYP1A1 mediated metabolism of AFQ056 in rat.

2.?Western blot analysis confirmed that CYP1A1 is expressed in rat lung albeit at low levels. M3 formation was clearly observed in recombinant rat CYP1A1, lung microsomes and lung tissue slices and was strongly inhibited by ketoconazole in the incubations. As CYP3A4 and CYP2C9 metabolites were only observed at trace levels, we concluded that the reduced M3 formation was due to CYP1A1 inhibition.

3.?AFQ056 lung clearance (CL_lung) as estimated from in vitro data was predicted to be negligible (<1% pulmonary blood flow). This was confirmed by in vivo experiments where intravenous and intra-arterial dosing to rats failed to show significant pulmonary extraction.

4.?While rat lung may make a contribution to the formation of M3, it is unlikely to be the only organ involved in this process and further experiments are required to investigate the potential metabolic elimination routes for AFQ056.     

In vitro and in vivo characterisation of the metabolism and disposition of suvorexant in humans

1.?Suvorexant (MK-4305, Belsomra®) is a first-in-class dual orexin receptor antagonist approved in the USA and Japan for the treatment of insomnia. The current studies describe suvorexant’s absorption, disposition and potential for CYP-mediated drug interactions in humans.

2.?Following single oral administration of [¹⁴C]suvorexant to healthy human subjects, 90% of the radioactivity was recovered (66% in faeces, 23% in urine), primarily as oxidative metabolites.

3.?In plasma, suvorexant and M9 were predominant, accounting for 30 and 37% of the total radioactivity, respectively. Metabolite M17 became more prominent (approaching 10%) following multiple daily doses of unlabelled suvorexant. M9 and M17 are not expected to contribute to the pharmacological activity of suvorexant due to reduced orexin receptor binding affinity and limited brain penetration.

4.?CYP3A was determined to be the predominant enzyme mediating suvorexant oxidation. In vitro, suvorexant demonstrated reversible inhibition of CYP3A4 and 2C19 (IC₅₀ ～ 4–5 μM), and weak time-dependent inhibition of CYP3A4 (K_I?=?12 μM, k_inact?=?0.14 min^?1). Suvorexant was also a weak inducer of CYP3A4, 1A2 and 2B6. Given the low plasma concentrations at clinical doses, suvorexant was not anticipated to cause significant drug interactions via inhibition and/or induction of major CYPs in vivo.