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     

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.     

Validation of a total IC50 method which enables in vitro assessment of transporter inhibition under semi-physiological conditions

1.?Accurate predictions of clinical transporter-mediated drug–drug interactions (DDI) from in vitro data can be challenging when compounds have poor solubility and/or high nonspecific binding. Additionally, current DDI predictions for compounds with high plasma–protein binding assume that the unbound fraction in plasma is 0.01, if the experimental value is less than 0.01 or cannot be determined. This approach may result in an overestimation of DDI risk. To overcome these challenges, it may be beneficial to conduct inhibition studies under physiologically relevant conditions.

2.?Here, IC₅₀ values, determined in the presence of 4% bovine serum albumin approximating human plasma albumin concentrations, were successfully used to predict DDI for uptake transporters, OATP1B1/1B3, OCT1/2, OAT1/3 and MATE1/2K.

3.?The IC₅₀ values of reference inhibitors with 4% bovine serum albumin, considered total IC₅₀, were comparable to the predicted values based on nominal IC₅₀ values determined under protein-free conditions and unbound fraction in plasma. Calculation of R-total and C_max/IC_50,total values using total plasma exposure and total IC₅₀ values explained the clinical DDI or absence of it for these inhibitors.

4.?These results suggest that IC₅₀ determinations in the presence of 4% albumin can be used, in the context of clinical total exposure, to predict DDI involving uptake transporters.     

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.     

Assessment of drug–drug interaction potential and PBPK modeling of CC-223, a potent inhibitor of the mammalian target of rapamycin kinase

1.?CC-223 was studied in vitro for metabolism and drug–drug interactions (DDI), and in clinic for interaction with ketoconazole.

2.?In vitro, human metabolites of CC-223 included O-desmethyl CC-223 (M1), keto (M2), N-oxide (M3) and imine (M13), with M1 being the most prominent metabolite.

3.?CC-223 was metabolized by CYP2C9 and CYP3A, while metabolism of M1 was mediated by CYP2C8 and CYP3A. Ketoconazole increased CC-223 and M1 exposure by 60–70% in healthy volunteers.

4.?CC-223 (IC₅₀?≥?27?µM) and M1 (IC₅₀?≥?46?µM) were inhibitors of CYP2C9 and CYP2C19 in human liver microsomes. CC-223 and M1 were moderate inducers of CYP3A in human hepatocytes.

5.?CC-223 was a substrate of BCRP, and M1 was a substrate of P-gp and BCRP. CC-223 was an inhibitor of P-gp (IC₅₀?=?3.67?µM) and BCRP (IC₅₀?=?11.7?µM), but at a clinically relevant concentration showed no inhibition of other transporters examined. M1 is a weak inhibitor of P-gp and BCRP.

6.?PBPK model of CC-223 and M1 was developed and verified using clinical results. Model based predictions of DDI with ketoconazole were in agreement with observed results enabling prospective predictions of DDIs between CC-223 and CYP3A4 inhibitors.     

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.     

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.     

Interactions of crizotinib and gefitinib with organic anion-transporting polypeptides (OATP)1B1, OATP1B3 and OATP2B1: gefitinib shows contradictory interaction with OATP1B3

1.?The drug–drug interaction (DDI) mediated by organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and OATP2B1 has a major impact on the hepatic clearance of drugs. The effects of tyrosine kinase inhibitors (TKIs) on OATPs have not been well studied. In the present study, we evaluated the contribution of OATPs to the hepatic uptake of crizotinib and gefitinib and the interaction of those TKIs with OATPs to estimate DDIs.

2.?To clarify whether crizotinib and gefitinib were substrates for OATPs, we performed uptake studies. We examined the effects of the TKIs on uptake of typical substrates and fluvastatin via OATPs. IC₅₀ and EC₅₀ values of the TKIs were calculated.

3.?OATP1B3- and OATP2B1-mediated crizotinib uptake and OATP2B1-mediated gefitinib uptake were observed. Gefitinib accelerated OATP1B3-mediated [³H]TCA uptake and inhibited OATP2B1-mediated [³H]E₃S uptake. On the other hand, gefitinib inhibited OATP1B1- and OATP2B1-mediated fluvastatin uptake.

4.?We provided basic information to estimate the DDI on OATPs caused by TKIs. The DDI on OATPs caused by gefitinib could occur in a normal clinical situation. And the uptake of crizotinib into the intrahepatocellular environment via OATPs may induce DDI and liver damage. We therefore emphasize the necessity of careful use of TKIs.     

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.

     

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.     

Direct and quantitative evaluation of the human CYP3A4 contribution (fm) to drug clearance using the in vitro SILENSOMES model

1.?Among the different in vitro studies recommended by the regulatory agencies, no gold-standard model can easily and directly measure the quantitative CYP450 contributions to drug biotransformation. In this article, we propose an original strategy, called Silensomes^TM, to produce human liver microsomes silenced for one specific CYP450, thanks to specific mechanism-based inhibitors (MBI).

2.?Using azamulin as a specific CYP3A4 MBI, we demonstrated the proof of concept that CYP3A4 can be totally, specifically (even against 3A5) and permanently (at least for six years) inhibited by our process. Thus, comparing clearance in control and CYP3A4-Silensomes^TM, CYP3A4 contributions were determined for 11 CYP3A4 substrates which correlated with known in vivo contributions and revealed accuracy with less than 10% error. In comparison, contributions determined using recombinant human CYP450 (rhCYP450s) were less accurate (more than 10% error for 30% of the tested CYP3A4 substrates).

3.?This easy and ready-to-use in vitro method combines the advantages of existing models (specificity of rhCYP450s and representativeness of HLM) without their drawbacks. The same strategy could be used to silence other major CYP450s one-by-one to provide a complete direct CYP450 quantitative phenotyping kit.     

In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes

1.?The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes.

2.?Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway.

3.?Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH.

4.?Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite.

5.?This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.     

Quantitative in vitro phenotyping and prediction of drug interaction potential of CYP2B6 substrates as victims

1.?Determination of f_{m, CYP} for a compound is critical to assess the potential risk of a drug candidate as a victim of DDI. Several compounds are identified as CYP2B6 substrates, but the f_{m, CYP2B6} values are not determined quantitatively.

2.?Two methods of reaction phenotyping, the chemical inhibition method and metabolism in rCYP enzymes, were used to determine the relative contributions of the enzymes. Chemical inhibition method was also conducted in the presence of BSA (0.5% w/v).

3.?The results confirm with the earlier studies concerning the identity of the CYP2B6 enzyme. The f_{m, CYP2B6} values for artemisinin, bupropion, clopidogrel, ketamine, selegiline, sertraline and ticlopidine were 0.24, 0.28, 0.15, 0.45, 0.46, 0.42 and 0.54, respectively, in HLM determined by chemical inhibition method. The f_{m, CYP2B6} values for artemisinin, bupropion, clopidogrel, ketamine, selegiline, sertraline and ticlopidine were 0.46, 0.17, 0.15, 0.60, 0.51, 0.66 and 0.77, respectively, in HLM determined by chemical inhibition method in the presence of BSA (0.5% w/v).

4.?Bupropion metabolism is majorly mediated by CYP2C19 (0.41) with a minor contribution from CYP2B6 (0.16) in the presence of BSA. Ticlopidine is a time-dependent inhibitor of both CYP2B6 and CYP2C19 that can inhibit the bupropion metabolism by 50–60%.     

Inhibition of cytochrome P450 by ethambutol in human liver microsomes

Although cytochrome P450 inhibition is the major drug–drug interaction (DDI) mechanism in clinical pharmacotherapy, DDI of a number of well-established drugs have not been investigated. Rifampicin, isoniazid, pyrazinamide and ethambutol combination therapy inhibits clearance of theophylline in patients with tuberculosis. We determined the inhibitory effects of ethambutol on the activities of nine CYP isoforms including CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in pooled human liver microsomes (HLM). As measured by liquid chromatography–electrospray ionization tandem mass spectrometry, ethambutol exhibited strong inhibitory potential against CYP1A2 and CYP2E1, moderate against CYP2C19 and CYP2D6 and weak against CYP2A6, CYP2C9 and CYP3A4, based on the IC₅₀ values. The K_i value of ethambutol for CYP1A2 was 1.4 μM and for CYP2E1 was 2.9 μM. Inhibition of CYP1A2 and CYP2E1 was not increased by preincubation with ethambutol and β-nicotinamideadenine dinucleotide phosphate (NADPH), suggesting that the ethambutol-induced CYP inhibition may not be metabolism-dependent. Kinetic analysis showed that the inhibition of CYP1A2 and CYP2E1 by ethambutol was best fit to a competitive inhibition model. Formation of 1-methylxanthene and 1,3-dimethyluric acid from theophylline in HLM was decreased to 47% and 36%, respectively, by 3.0 μM ethambutol, which is comparable to its IC₅₀ value against CYP1A2. Considering its maximal plasma concentrations of ∼10 μM and long half-life of ∼22 h, our findings raise the possibility that ethambutol causes significant DDIs in clinical situations with drugs with narrow therapeutic index, such as theophylline, in clinical situations.     

In vitro inhibition of human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes by rose bengal: system-dependent effects on inhibitory potential

Abstract

1. Rose bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) is being developed for the treatment of cutaneous melanoma and hepatocellular carcinoma. Interestingly, rose bengal can generate singlet oxygen species upon exposure to light.

2. We evaluated rose bengal as an in vitro inhibitor of cytochrome P450 (CYP) or UDP-glucuronosyltransferase (UGT) enzymes in both human liver microsomes (HLM) and cryopreserved human hepatocytes (CHHs) under both yellow light and dark conditions.

3. Rose bengal directly inhibited CYP3A4/5 and UGT1A6 in HLM under yellow light with inhibitor concentration that causes 50% inhibition (IC₅₀) values of 0.072 and 0.035?μM, respectively; whereas much less inhibition was observed in the dark with the IC₅₀ values increasing 43- and 120-fold, respectively. To determine if a more physiologically-relevant test system could be protected from such an effect, rose bengal was evaluated as an inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5 and UGT enzymes in CHH. All IC₅₀ values were similar (64?±?8?μM) and little to no effect of light on inhibitory potential was observed.

4. Given the IC₅₀ values in CHH increased an order of magnitude compared to HLM and the atypical pharmacokinetics of the drug, the risk of rose bengal to cause clinically relevant drug–drug interactions is likely low, particularly when administered to cancer patients on an intermittent schedule.     

Co-treatment with indole-3-carbinol and resveratrol modify porcine CYP1A and CYP3A activities and expression

1.?Humans and animals are commonly exposed to indole-3-carbinol (I3C) and resveratrol (RES) via food or beverages. Moreover, these compounds have been demonstrated to potentially cause food–drug interactions. However, information about their combined effects is limited. Therefore, we investigated the effects of I3C and RES, both as single compounds and in combination, on cytochrome P450 1A and 3A activity and gene expression.

2.?Using porcine microsomes, we demonstrated that RES caused non-competitive inhibition of CYP1A activity and un-competitive inhibition of CYP3A activity. Compared to the effect of single compounds, co-treatment with I3C and RES increased a degree of inhibition of CYP1A activity.

3.?In porcine primary hepatocytes, treatment with I3C and RES resulted in induction of CYP1A1, CYP1A2 and CYP3A29 mRNA expression.

4.?In conclusion, we demonstrated that both RES and I3C could cause food–drug interactions and that the combined effect could be more potent in doing so.     

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.     

In vivo approach for the evaluation of mechanism-based inhibition of cytochrome P450 3A in rats

1.?There have been no reports showing that the area under the concentration-time curve (AUC) of a probe drug is elevated due to mechanism-based inhibition (MBI) of drug-metabolizing enzymes in animals. This study ascertained that mechanism-based inhibitors reported to induce drug–drug interactions (DDIs) in humans also caused MBI in rats.

2.?Midazolam (MDZ), mainly metabolized by cytochrome P450 3A in rats, and mibefradil, which showed the most intense time-dependent inhibition among the inhibitors tested, were selected as the probe and the inhibitor, respectively. Following pretreatment of mibefradil at 24 h before MDZ administration in rats, the C_max and AUC values of MDZ were significantly elevated in comparison with the control. The free plasma concentration of mibefradil was substantially lower than the IC₅₀ value observed in the in vitro inhibition study, suggesting that the DDI was due to MBI.

3.?It is concluded that the evaluation of MBI in rats in vivo in combination with in vitro data using human enzymes could be useful to evaluate risk in clinical studies.     

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.     

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.