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 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.     

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.     

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.     

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. II: establishment and evaluation of dexamethasone-pretreated female rats

1.?Cytochrome P450 (CYP) 3A catalysis of testosterone 6β-hydroxylation in female rat liver microsomes was significantly induced, then reached a plateau level after pretreatment with 80?mg?kg^?1?day^?1 dexamethasone (DEX) for 3 days.

2.?Midazolam was mainly metabolized by CYP3A in DEX-treated female rat liver microsomes from an immuno-inhibition study, and the apparent K_m was 1.8?μM, similar to that in human microsomes.

3.?Ketoconazole and erythromycin, typical CYP3A inhibitors, demonstrated extensive inhibition of midazolam metabolism in DEX-treated female rat liver microsomes, and the apparent K_i values were 0.088 and 91.2?μM, respectively. The values were similar to those in humans, suggesting that DEX-treated female rat liver microsomes have properties similar to those of humans.

4.?After oral administration of midazolam, the plasma midazolam concentration in DEX-treated female rats significantly decreased compared with control female rats. The area under the plasma concentration curve (AUC) and elimination half-life were one-11th and one-20th of those of control female rats, respectively.

5.?Using DEX-treated female rats, the effect of CYP3A inhibitors on midazolam pharmacokinetics was evaluated. The AUC and maximum concentration in plasma (C_max) increased when ketoconazole was co-administered with midazolam.

6.?It was shown that the drug–drug interaction that occurs in vitro is also observed in vivo after oral administration of midazolam. In conclusion, the DEX-treated female rat could be a useful model for evaluating drug–drug interactions based on CYP3A enzyme inhibition.     

Role of human cytochrome P450 3A4 in the metabolism of DA–8159, a new erectogenic

1.?The purpose of this paper was to characterize cytochrome P450 (CYP) enzymes involved in N-dealkylation of a new oral erectogenic, DA-8159 to DA-8164, a major circulating active metabolite, in human liver microsomes and to investigate the inhibitory potential of DA-8159 on CYP enzymes.

2.?CYP3A4 was identified as the major enzyme responsible for DA-8159 N-dealkylation to DA-8164 based on correlation analysis and specific CYP inhibitor and antibody-mediated inhibition study in human liver microsomes, and DA-8159 metabolism in cDNA expressed CYP enzymes. There is the possibility of drug-drug interactions when prescribing DA-8159 concomitantly with known inhibitors or inducers of CYP3A4.

3.?DA-8159 was found to be only a very weak inhibitor of eight major CYPs (1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4), the largest inhibition occurring against CYP2D6 (IC₅₀ 67.7?μM) in human liver microsomes. Drug–drug interactions would not be predicted on the basis of DA-8159 inhibiting the metabolism of coadministered drugs.     

Effects of safflower injection on the pharmacodynamics and pharmacokinetics of warfarin in rats

1.?Safflower injection (SI) is extracted from Chinese herbal medicine safflower that comprises many active components. Warfarin is a common anticoagulant and has exhibited drug interactions with several herbal products. This study aimed to investigate the effects of SI on pharmacodynamics and pharmacokinetics of warfarin in rats.

2.?Wistar rats were randomly divided into blank control group, SI group, warfarin control group and SI?+?warfarin group, respectively. In SI and SI?+?warfarin groups, rats were injected with SI (1.6?mL/kg/d, i.p.) for 14?days. Warfarin (0.2?mg/kg) was given orally on the eighth day. Saline was given as control. The blood samples were collected at various time points. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured. UPLC-MS/MS was used to determine the plasma concentrations of S(R)-warfarin, and the pharmacokinetic parameters were calculated.

3.?PT, APTT in SI and SI?+?warfarin rats increased significantly compared with corresponding control rats. The pharmacokinetic parameters including C_max, t_1/2, AUC_0-t and AUC_0-∞ of S-warfarin and R-warfarin in SI?+?warfarin rats were higher than those in warfarin control rats.

4.?These findings suggest that SI significantly increases the anticoagulant effect of warfarin by affecting its pharmacodynamic and pharmacokinetic parameters.     

Influence of grapefruit juice on pharmacokinetics of triptolide in rats grapefruit juice on the effects of triptolide

1.?Triptolide, a major pharmacological component isolated from Tripterygium wilfordii Hook F (TWHF), is a substrate of both CYP3A4 and P-glycoprotein (P-gp).

2.?This study investigates the effects of GFJ on the pharmacokinetics of triptolide in rats.

3.?The pharmacokinetics of orally administered triptolide with or without GFJ pretreatment were investigated. A mechanistic study was also undertaken using the Caco-2 cell transwell model and rat liver microsomes incubation systems to support the in vivo pharmacokinetic data.

4.?The results indicated that coadministration of GFJ could increase the systemic exposure of triptolide significantly, including area under the curve (828.58?±?79.72 versus 541.53?±?45.23?ng·h/mL) and maximum plasma concentration (273.58?±?27.98 versus 193.67?±?10.08?ng/mL). The apparent permeability of triptolide across the Caco-2 cell transwell model increased significantly with the pretreatment of GFJ (from 1.62?±?0.25?×?10^?6 to 2.51?±?0.41?×?10^?6 cm/s), and the metabolic stability of triptolide was also increased from 32.6?±?5.1 to 52.5?±?7.8?min with the pretreatment of GFJ, and the difference was significant (p?5.?In conclusion, GFJ could increase the systemic exposure of triptolide in rats, when GFJ and triptolide was coadministered, and it might work mainly through increasing the absorption of triptolide by inhibiting P-gp, or through slowing down the metabolism of triptolide in rat liver by inhibiting the activity of CYP3A4.     

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. I: evaluation of cynomolgus monkeys as surrogates for humans

1.?Anti-human cytochrome P450 (CYP) 3A4 antiserum completely inhibited midazolam metabolism in monkey liver microsomes, suggesting that midazolam was mainly metabolized by CYP3A enzyme(s) in monkey liver microsomes.

2.?Midazolam metabolism was also inhibited in vitro by typical chemical inhibitors of CYP3A, such as ketoconazole, erythromycin and diltiazem, and the apparent K_i values for ketoconazole, erythromycin and diltiazem were 0.127, 94.2 and 29.6?μM, respectively.

3.?CYP3A inhibitors increased plasma midazolam concentrations when midazolam and CYP3A inhibitors were co-administered orally. However, the pharmacokinetic parameters of midazolam were not changed by treatment with CYP3A inhibitors when midazolam was given intravenously. This suggests that CYP3A inhibitors modified the first-pass metabolism in the liver and/or intestine, but not systemic metabolism.

4.?The drug–drug interaction responsible for CYP3A enzyme(s) inhibition was observed when midazolam and inhibitors were co-administrated orally. Therefore, it was concluded that monkeys given midazolam orally could be useful models for predicting drug–drug interactions in man based on CYP3A enzyme inhibition.     

Investigation of the influence of glycyrrhizin on the pharmacokinetics of celastrol in rats using LC-MS and its potential mechanism

1.?The aim of this study was to investigate the effects of glycyrrhizin on the pharmacokinetics of celastrol in rats.

2.?Twelve male Sprague–Dawley rats were randomly assigned to two groups: control group and test group. Test group was pretreated with glycyrrhizin at a dose of 100?mg/kg/day for 10 days, and then the two groups were orally administered with celastrol at a dose of 1?mg/kg. The concentration of celastrol was determined using a sensitive and reliable LC-MS method.

3.?The results showed that glycyrrhizin could significantly decrease the plasma concentration (from 64.36?ng/mL to 38.42?ng/mL) and AUC_0?t (from 705.39 to 403.43?μg·h/L) of celastrol in rats. To investigate its potential mechanism, the effects of glycyrrhizin on the transport and metabolic stability of celastrol were investigated using Caco-2 cell monolayer transwell model and rat liver microsome incubation systems. The Caco-2 cell monolayer transwell experiments indicated that glycyrrhizin could increase the efflux ratio of celastrol (4.02 versus 6.51). However, the rat liver microsome incubation experiments showed that glycyrrhizin could significantly increase the intrinsic clearance rate of celastrol from 20.3?±?3.37 to 38.8?±?4.18?μL/min/mg protein.

4.?In conclusion, these results indicated that the herb–drug interaction between glycyrrhizin and celastrol might occur when they were coadministered.     

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.     

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.     

Identification of the cytochrome P450 enzymes involved in the metabolism of domperidone

1.?The objective was to identify the major cytochrome P450 enzyme(s) involved in the metabolism of domperidone.

2.?Experiments were performed using human liver microsomes (HLMs), recombinant human cytochrome P450 enzymes, cytochrome P450 chemical inhibitors and monoclonal antibodies directed against cytochrome P450 enzymes.

3.?Four metabolites were identified from incubations performed with HLMs and excellent correlations were observed between the formation of domperidone hydroxylated metabolites (M1, M3 and M4), N-desalkylated domperidone metabolite (M2) and enzymatic markers of CYP3A4/5 (r²?=?0.9427, 0.951, 0.9497 and 0.8304, respectively).

4.?Ketoconazole (1?μM) decreased the formation rate of M1, M2, M3 and M4 by 83, 78, 75 and 88%, respectively, whereas the effect of other inhibitors (quinidine, furafylline and sulfaphenazole) was minimal. Important decreases in the formation rate of M1 (68%), M2 (64%) and M3 (54%) were observed with anti-CYP3A4 antibodies.

5.?Formation of M1, M2 and M3 in HLMs exhibited Michaelis–Menten kinetics (K_m: 166, 248 and 36?μM, respectively). Similar K_m values were observed for M1, M2 and M3 when incubations were performed with recombinant human CYP3A4 (K_m: 107, 273 and 34?μM, respectively).

6.?The data suggest that CYP3As are the major enzymes involved in the metabolism of domperidone.     

Epigallocatechin-3-gallate decreases the transport and metabolism of simvastatin in rats

1.?This study aimed to investigate the potential impact of epigallocatechin-3-gallate (EGCG) on the pharmacokinetic behaviors of simvastatin and its metabolite simvastatin acid and explored the possible role of metabolizing enzymes and transporters of this food–drug interaction.

2.?Female SD rats were intravenously administered with EGCG (5?mg/kg), ketoconazole (10?mg/kg) and rifampin (10?mg/kg), followed by intravenous administration of 2?mg/kg simvastatin. In vitro, the effects of EGCG on Cytochrome P450 enzymes (CYP450) and organic anion transporting polypeptides (OATPs) were studied using human hepatic microsomes and human embryonic kidney 293 (HEK293) cells overexpressing OATP1B1 or OATP1B3. The results showed that areas under concentration–time (AUC) curves of simvastatin and simvastatin acid increased by 2.21- and 1.4-fold while the clearance was reduced by 2.29- and 1.4-fold, respectively, when co-administered with EGCG. In vitro experiments suggested the inhibitory effect of EGCG on CYP enzymes (IC₅₀: 18.37?±?1.36?μM, 26.08?±?1.51?μM for simvastatin and simvastatin acid, respectively). Simvastatin transport by OATP1B1 and OATP1B3 was also inhibited by EGCG (IC₅₀: 8.68?±?1.27?μM and 22.67?±?1.42?μM, respectively).

3.?The presently reported novel food–drug interaction between EGCG and simvastatin involves the inhibition of not only CYP450 enzymes but also OATPs by EGCG.     

Influence of andrographolide on the pharmacokinetics of warfarin in rats

Context: Andrographolide and warfarin are often used together in clinics in China. However, the herb-drug interaction between andrographolide and warfarin is still unknown.

Objective: This study investigates the herb-drug interaction between andrographolide and warfarin in vivo and in vitro.

Materials and methods: A sensitive and reliable LC-MS/MS method was developed for the determination of warfarin in male Sprague-Dawley rats plasma, and then the pharmacokinetics of orally administered warfarin (0.5?mg/kg) with or without andrographolide (30?mg/kg/day for 7?days) pretreatment was investigated. In addition, Sprague-Dawley rat liver microsomes incubation systems were used to support the in vivo pharmacokinetic data and investigate its potential mechanism.

Results: The method validation results showed that a sensitive and reliable LC-MS/MS method was developed for the determination of warfarin in rat plasma samples. The pharmacokinetic results indicated that co-administration of andrographolide could increase the systemic exposure of warfarin significantly, including area under the curve (118.92?±?18.08 vs. 60.58?±?9.46?μg?×?h/mL), maximum plasma concentration (3.32?±?0.41 vs. 2.35?±?0.25?μg/mL) and t_1/2 (22.73?±?3.28 vs. 14.27?±?2.67?h). Additionally, the metabolic stability of warfarin increased from 23.5?±?4.7 to 38.7?±?6.1?min with the pretreatment of andrographolide, and the difference was significant (p?Discussion and conclusion: In conclusion, andrographolide could increase the systemic exposure of warfarin in rats when andrographolide and warfarin were co-administered, and possibly by slowing down the metabolism of warfarin in rat liver by inhibiting the activity of CYP3A4 or CYP2C9.     

The effect of resveratrol on pharmacokinetics of aripiprazole in vivo and in vitro

1.?The objective of this study were to investigate the effect of orally administered resveratrol on the pharmacokinetics of aripiprazole (APZ) in rat, and the inhibitory effects of resveratrol on APZ dehydrogenation activity in liver microsomes and human cytochrome P450 3A4 and 2D6.

2.?Twenty-five healthy male Sprague–Dawley rats were randomly divided into five groups: A (control group), B (multiple dose of 200?mg/kg resveratrol), C (multiple dose of 100?mg/kg resveratrol), D (a single dose of 200?mg/kg resveratrol) and E (a single dose of 100?mg/kg resveratrol). A single dose of 3?mg/kg APZ administered orally 30?min after administration of resveratrol. In addition, CYP2D6*1, CYP3A4*1, human and rat liver microsomes were performed to determine the effect of resveratrol on the metabolism of APZ in vitro.

3.?The multiple dose of 200 or 100?mg/kg resveratrol significantly increased the AUC and C_max of APZ. The resveratrol also obviously decreased the CL, but without any significant difference on t_1/2 in vivo. On the other hand, resveratrol showed inhibitory effect on CYP3A4*1, CYP2D6*1, human and rat microsomes, the IC₅₀ of resveratrol was 6.771, 87.87, 45.11 and 35.59?μmol?l^?1, respectively.

4.?Those results indicated more attention should be paid when APZ was administrated combined with resveratrol.     

Altered hepatic drug-metabolizing activity in rats suffering from hypoxemia with experimentally induced acute lung impairment

1.?Hepatic drug-metabolizing activity was investigated in vitro with liver microsomes prepared from rats suffering from hypoxemia with experimentally induced acute lung impairment (ALI).

2.?Male Wistar rats received an intrabronchial administration of dilute hydrochloride solution for ALI induction. Pooled liver microsomes were prepared for the normal and ALI rats, and the hepatic drug metabolism mediated by cytochrome P450 (CYP) 3?A was examined in an incubation study with the microsomes.

3.?The NADPH-dependent metabolism of midazolam significantly increases in ALI rats as compared with that in normal rats. Testosterone 6β-hydroxylation was also observed to significantly increase in ALI rats.

4.?When the hepatic expression of CYP3A proteins was examined, the protein expression of CYP3A1 was shown to significantly increase and that of CYP3A2 remained unaltered in ALI rats. The hepatic expression of NADPH-cytochrome P450 reductase (POR), a protein mediating electron transfer in CYP-mediated drug metabolism, was also revealed to significantly increases in ALI rats.

5.?With the findings regarding the midazolam elimination, the hepatic drug-metabolizing activity seems to increase in response to acute hypoxemia, partly due to an altered expression of the CYP3A enzymes, and an augmented electron transfer with an increased POR expression is probably involved in the increase.     

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.     

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.     

Effect of CYP2D6 variants on venlafaxine metabolism in vitro

1.?CYP2D6 is an important member of the cytochrome P450 (CYP450) enzyme superfamily, we recently identified 22 CYP2D6 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of venlafaxine in vitro.

2.?The wild-type and 24 CYP2D6 variants were expressed in insect cells, and each variant was characterized using venlafaxine as the substrate. Reactions were performed at 37?°C with 5–500?μM substrate (three variants was adjusted to 1000?μM) for 50?min. By using high-performance liquid chromatography to detect the products, the kinetic parameters K_m, V_max, and intrinsic clearance (V_max/K_m) of O-desmethylvenlafaxine were determined.

3.?Among the 22 CYP2D6 variants, the intrinsic clearance (V_max/K_m) values of all variants were significantly decreased (from 0.2% to 84.5%) compared with wild-type CYP2D6*1. In addition, the kinetic parameters of two CYP2D6 variants could not be detected because they have no detectable enzyme activity.

4.?The comprehensive in vitro assessment of CYP2D6 variants provides significant insights into allele-specific activity towards venlafaxine in vivo.