In vitro metabolism of midazolam, triazolam, nifedipine, and testosterone by human liver microsomes and recombinant cytochromes p450: role of cyp3a4 and cyp3a5.

Midazolam, triazolam (TRZ), testosterone, and nifedipine have all been widely used as probes for in vitro metabolism of CYP3A. We used these four substrates to assess the contributions of CYP3A4 and CYP3A5 to in vitro biotransformation in human liver microsomes (HLMs) and in recombinant enzymes. Recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) both produced 1-OH and 4-OH metabolites from midazolam and triazolam, 6 beta-hydroxytestosterone from testosterone, and oxidized nifedipine from nifedipine. Overall, the metabolic activity of CYP3A5 was less than that of CYP3A4. Ketoconazole potently inhibited midazolam, triazolam, testosterone, and nifedipine metabolite formation in HLMs and in rCYP3A4. The inhibitory potency of ketoconazole in rCYP3A5 was about 5- to 19-fold less than rCYP3A4 for all four substrates. In testosterone interaction studies, testosterone inhibited 1-OH-TRZ formation, but significantly activated 4-OH-TRZ formation in HLMs and rCYP3A4 but not in rCYP3A5. Oxidized nifedipine formation was inhibited by testosterone in rCYP3A4. However, in rCYP3A5, testosterone slightly activated oxidized nifedipine formation at lower concentrations, followed by inhibition. Thus, CYP3A4 and CYP3A5 both contribute to midazolam, triazolam, testosterone, and nifedipine biotransformation in HLMs, with CYP3A5 being metabolically less active than CYP3A4 in general. Because the inhibitory potency of ketoconazole in rCYP3A5 is substantially less than in rCYP3A4 and HLMs, CYP3A5 is probably less important than CYP3A4 in drug-drug interactions involving ketoconazole and CYP3A substrates.     

Effect of single‐walled carbon nanotubes on cytochrome P450 activity in human liver microsomes in vitro

Single‐walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH‐P‐ and SO‐SWCNTs) on human CYP activity. In addition, other nano‐sized carbon materials, such as carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S‐mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6β‐hydroxylation and midazolam 1′‐hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO‐SWCNT. Dextromethorphan O‐demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO‐SWCNT. Other CYP activities, however, were not attenuated by SO‐SWCNT. FH‐P‐SWCNT, carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ at 0.1 mg/ml had no effect on CYP activities. The K_i values for testosterone 6β‐hydroxylation, midazolam 1′‐hydroxylation, and dextromethorphan O‐demethylation in liver microsomes were 136, 34, and 56 μg/ml, respectively. SO‐SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO‐SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.     

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.     

Midazolam α-Hydroxylation by Human Liver Microsomes in vitro: Inhibition by Calcium Channel Blockers,Itraconazole and Ketoconazole

Abstract: The inhibitory effects of five calcium channel blockers (diltiazem, isradipine, mibefradil, nifedipine and verapamil) and three azole antifungal agents (itraconazole, hydroxyitraconazole and ketoconazole) on the α-hydroxylation of midazolam, a probe drug for CYP3A4-mediated interactions in humans, were studied in vitro using human liver microsomes. IC₅₀ and K_i values were determined for each inhibitor. The kinetics of the formation of α-hydroxymidazolam were best described by simple Michaelis-Menten kinetics. The estimated values of V_max and K_m were 696 pmol min.^?1 mg^?1 and 7.46 μmol l^?1, respectively. All the compounds studied inhibited midazolam α-hydroxylation activity in a concentration-dependent manner, but there were marked differences in their relative inhibitory potency. Ketoconazole was the most potent inhibitor of midazolam α-hydroxylation (IC₅₀ 0.12 μmol l^?1), being 10 times more potent than itraconazole (IC₅₀ 1.2 μmol l^?1). The inhibitory effect of hydroxyitraconazole (IC₅₀ 2.3 μmol l^?1) was almost as large as that of itraconazole. Among the calcium channel blockers, mibefradil was the most potent inhibitor of the α-hydroxylation of midazolam, with an IC₅₀ value (1.6 μmol l^?1) similar to that of itraconazole. The other calcium channel blockers were much weaker inhibitors than mibefradil: verapamil exhibited a modest inhibitory effect with an IC₅₀ of 23 μmol l^?1, while isradipine, nifedipine and diltiazem, with IC₅₀ values ranging from 57 to >100 μmol l^?1, were weak inhibitors. This rank order of potency against the α-hydroxylation of midazolam was verified by the K_i values. With the exception of diltiazem, these in vitro results conform with the observed interaction potential of these agents with midazolam and many other CYP3A4 substrates in vivo in man.     

The xenobiotic inhibitor profile of cytochrome P4502C8

AIMS: To investigate inhibition of recombinant CYP2C8 by: (i) prototypic CYP isoform selective inhibitors (ii) imidazole/triazole antifungal agents (known inhibitors of CYP), and (iii) certain CYP3A substrates (given the apparent overlapping substrate specificity of CYP2C8 and CYP3A). METHODS: CYP2C8 and NADPH-cytochrome P450 oxidoreductase were coexpressed in Spodoptera frugiperda (Sf21) cells using the baculovirus expression system. CYP isoform selective inhibitors, imidazole/triazole antifungal agents and CYP3A substrates were screened for their inhibitory effects on CYP2C8-catalysed torsemide tolylmethylhydroxylation and, where appropriate, the kinetics of inhibition were characterized. The conversion of torsemide to its tolylmethylhydroxy metabolite was measured using an h.p.l.c. procedure. RESULTS: At concentrations of the CYP inhibitor 'probes' employed for isoform selectivity, only diethyldithiocarbamate and ketoconazole inhibited CYP2C8 by > 10%. Ketoconazole, at an added concentration of 10 microM, inhibited CYP2C8 by 89%. Another imidazole, clotrimazole, also potently inhibited CYP2C8. Ketoconazole and clotrimazole were both noncompetitive inhibitors of CYP2C8 with apparent Ki values of 2.5 microM. The CYP3A substrates amitriptyline, quinine, terfenadine and triazolam caused near complete inhibition (82-91% of control activity) of CYP2C8 at concentrations five-fold higher than the known CYP3A Km. Kinetic studies with selected CYP3A substrates demonstrated that most inhibited CYP2C8 noncompetitively. Apparent Ki values for midazolam, quinine, terfenadine and triazolam ranged from 5 to 25 microM. CONCLUSIONS: Inhibition of CYP2C8 occurred at concentrations of ketoconazole and diethyldithiocarbamate normally employed for selective inhibition of CYP3A and CYP2E1, respectively. Some CYP3A substrates have the capacity to inhibit CYP2C8 activity and this may have implications for inhibitory drug interactions in vivo.     

Inactivation kinetics and residual activity of CYP3A4 after treatment with erythromycin

This study aimed to characterize the inactivation kinetics of cytochrome P450 3A4 (CYP3A4) by erythromycin, which involves mechanism‐based inhibition (MBI), in detail. In addition to an MBI assay based on the conventional method in which erythromycin and recombinant CYP3A4 were pre‐incubated for 15 min, the study also evaluated the long‐term MBI kinetics of this reaction by pre‐incubation for 120 min. Mechanism‐based inhibition profiles were obtained using three typical substrates, testosterone, midazolam and nifedipine. In the long‐term assay, erythromycin evoked a time‐dependent biphasic reduction in enzyme activity, but some residual activity (α) was detected in the terminal phase. The inactivation rate constant obtained in the presence of 30 μm erythromycin using nifedipine as a substrate was 1.44‐fold higher than that acquired using testosterone, while there was no difference among the α values obtained with the three substrates. In the short‐term assay, time‐dependent monophasic inactivation was observed. To extrapolate these data to in vivo , the extent of the increase in the area under the curve (AUC ratio) induced by erythromycin was estimated from the results of the conventional short‐term experiment and the long‐term experiment examining residual activity. The AUC ratio estimated from the long‐term kinetics (2.92) was closer to the clinically reported values (3.3–4.42). In conclusion, the relatively long‐term evaluation of the kinetics of CYP3A4 inactivation revealed that the enzyme was not fully inactivated by erythromycin. To improve the estimation of the extent of the drug–drug interactions induced by MBI from in vitro data, longer‐term investigations of the target enzyme's inactivation profile might be necessary.     

Simultaneous evaluation of substrate‐dependent CYP3A inhibition using a CYP3A probe substrates cocktail

Cytochrome P450 (P450) 3A (CYP3A) is an enzyme responsible for the metabolism of therapeutic drugs such as midazolam, nifedipine, testosterone and triazolam. It is involved in 40% of all cases of P450‐mediated metabolism of marketed drugs. Therefore, it is important to evaluate the CYP3A‐mediated drug interaction potential of new chemical entities (NCEs). In the past, one P450 isoform‐specific probe substrate has been used at a time to evaluate the degree of inhibition of P450 isoforms by using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). However, CYP3A enzymes have been shown to have a multi‐substrate binding site. Therefore, multiple CYP3A substrates should be used to evaluate precisely the drug interaction potential of NCEs with the enzyme CYP3A. In this study, a method of screening NCEs for their potential to inhibit by CYP3A enzyme activity was developed. It involves the employment of a CYP3A substrate cocktail (including midazolam, testosterone and nifedipine). The concentration of each CYP3A probe substrate in vitro was optimized (0.1 μm for midazolam, 2 μm for testosterone and 2 μm for nifedipine) to minimize mutual drug interactions among probe substrates. The method was validated by comparing inhibition data obtained from the incubation of CYP3A with each individual substrate with data from incubation with a cocktail of all three substrates. The CYP3A inhibition profiles from the substrate cocktail approach were similar to those from the individual substrates approach. This new method could be an effective tool for the robust and accurate screening of the CYP3A inhibition potential of NCEs in drug discovery. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Selegiline Metabolism and Cytochrome P450 Enzymes:In vitro Study in Human Liver Microsomes*

Although being a drug therapeutically used for a long time, the enzymatic metabolism of selegiline has not been adequately studied. In the current work we have studied the cytochrome P450 (CYP)‐catalyzed oxidative metabolism of selegiline to desmethylselegiline and l‐methamphetamine and the effects of selegiline, desmethylselegiline and l‐methamphetamine on hepatic CYP enzymes in human liver microsomes in vitro. The apparent K_m values for desmethylselegiline and l‐methamphetamine formation were on an average 149 μM and 293 μM, and the apparent V_max values, 243 pmol/min./mg and 1351 pmol/min./mg, respectively. Furafylline and ketoconazole, the known reference inhibitors for CYP1A2 and CYP3A4, respectively, inhibited the formation of desmethylselegiline with K_i value of 1.7 μM and 15 μM. Ketoconazole inhibited also the formation of l‐methamphetamine with K_i of 18 μM. Fluvoxamine, an inhibitor of CYP1A2, CYP2C19 and CYP3A4, inhibited the formation of desmethylselegiline and l‐methamphetamine with K_i values of 9 and 25 μM, respectively. On the basis of these results we suggest that CYP1A2 and CYP3A4 contribute to the formation of desmethylselegiline and that CYP3A4 participates in the formation of l‐methamphetamine. In studies with CYP‐specific model activities, both selegiline and desmethylselegiline inhibited the CYP2C19‐mediated S‐mephenytoin 4′‐hydroxylation with average IC₅₀ values of 21 μM and 26 μM, respectively. The K_i for selegiline was determined to be around 7 μM. Selegiline inhibited CYP1A2‐mediated ethoxyresorufin O‐deethylation with a K_i value of 76 μM. Inhibitory potencies of selegiline, desmethylselegiline and l‐methamphetamine towards other CYP‐model activities were much lower. On this basis, selegiline and desmethylselegiline were shown to have a relatively high affinity for CYP2C19, but no evidence about selegiline metabolism by CYP2C19 was obtained.     

In vitro inhibition and induction of human cytochrome P450 enzymes by SIPI5357, a potential antidepressant

This study investigated the in vitro drug–drug interaction potential of SIPI5357, an arylalkanol‐piperazine derivative used in the treatment of depression. Drug–drug interaction occurs via inhibition or induction of enzymes involved in their metabolism. In human liver microsomes, SIPI5357 showed the strongest inhibition of CYP2D6, followed by CYP3A4 (testosterone) and CYP2C8. Inhibition was observed in a concentration‐dependent manner, with IC₅₀ values of 18.45 µM, 36.63 µM (CYP3A4/testosterone), 89.23 µM, respectively. SIPI5357 was predicted not to cause significant metabolic drug–drug interaction via inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2E1 or CYP3A4 (midazolam) because the IC₅₀ values for these enzymes were both >100 µM (200 times maximum plasma concentration [C_max]). SIPI5357 showed a mixed model inhibition of CYP2D6 (K_i = 11.12 µM). The value of [I]/K_i for CYP2D6 inhibition by SIPI5357 is below the FDA cut‐off value of 0.1; it is therefore reasonable to assume that SIPI5357 will not cause significant CYP2D6 inhibition. However, positive controls (50 µM omeprazole and 25 µM rifampin) caused the anticipated CYP induction, but the highest concentration of SIPI5357 (5 µM; 10 times plasma C_max) had a minimal effect on CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that SIPI5357 is not an inducer of these enzymes. However, significant induction of CYP2B6 was observed at 0.5 µM and 5 µM. In conclusion, SIPI5357 might cause drug–drug interaction via induction of CYP2B6. The in vivo drug–drug interaction potential deserves further investigation. Copyright © 2015 John Wiley & Sons, Ltd.     

Terfenadine t-butyl hydroxylation catalyzed by human and marmoset cytochrome P450 3A and 4F enzymes in livers and small intestines

1.?Roles of human cytochrome P450 (P450) 3A4 in oxidation of an antihistaminic drug terfenadine have been previously investigated in association with terfenadine–ketoconazole interaction. Several antihistamine drugs have been recently identified as substrates for multiple P450 enzymes. In this study, overall roles of P450 3A4, 2J2, and 4F12 enzymes in terfenadine t-butyl hydroxylation were investigated in small intestines and livers from humans, marmosets, and/or cynomolgus monkeys.

2.?Human liver microsomes and liver and small intestine microsomes from marmosets and cynomolgus monkeys effectively mediated terfenadine t-butyl hydroxylation. Ketoconazole and N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine (a P450 4A/F inhibitor) almost completely and moderately inhibited these activities, respectively, in human liver microsomes; however, these chemicals did not show substantially suppression in marmoset liver. Anti-human P450 3A and 4F antibodies showed the roughly supportive inhibitory effects.

3.?Recombinant P450 3A4/90 and 4F12 showed high terfenadine t-butyl hydroxylation activities with substrate inhibition constants of 84–144?μM (under 26–76?μM of K_m values), in similar manners to liver and intestine microsomes.

4.?These results suggest that human and marmoset P450 3A4/90 and 4F12 in livers or small intestines played important roles in terfenadine t-butyl hydroxylation. Marmosets could be a model for humans during first pass extraction of terfenadine and related substrates.     

Comparison of the inhibitory effects of azole antifungals on cytochrome P450 3A4 genetic variants

Cytochrome P450 (CYP) 3A4 is one of the major drug-metabolizing enzymes. Genetic variants of CYP3A4 with altered activity are one of the factors responsible for interindividual differences in drug metabolism. Azole antifungals inhibit CYP3A4 to cause clinically significant drug-drug interactions. In the present quantitative study, we investigated the inhibitory effects of three azole antifungals (ketoconazole, voriconazole, and fluconazole) on testosterone metabolism by recombinant CYP3A4 genetic variants (CYP3A4.1 (WT), CYP3A4.2, CYP3A4.7, CYP3A4.16, and CYP3A4.18) and compared them with those previously reported for itraconazole. The inhibition constants (K_i) of ketoconazole, voriconazole, and fluconazole for rCYP3A4.1 were 3.6 nM, 3.2 μM, and 16.1 μM, respectively. The K_i values of these azoles for rCYP3A4.16 were 13.9-, 13.6-, and 6.2-fold higher than those for rCYP3A4.1, respectively, whereas the K_i value of itraconazole for rCYP3A4.16 was 0.54-fold of that for rCYP3A4.1. The other genetic variants had similar effects on the K_i values of the three azoles, whereas a very different pattern was seen for itraconazole. In conclusion, itraconazole has unique characteristics that are distinct from those shared by the other azole anti-fungal drugs ketoconazole, voriconazole, and fluconazole with regard to the influence of genetic variations on the inhibition of CYP3A4.     

Differential inhibitory effects of phenytoin,diclofenac, phenylbutazone and a series of sulfonam ides on hepatic cytochrom e P4502C activity in vitro,and correlation with som e m olecular descriptors in the dwarf goat (Caprus hircus aegagrus).

1. The aim of the present study was to investigate the potency of various sulfonamides to inhibit tolbutamide hydroxylation (a CYP2C activity) in hepatic microsomal fractions and hepatocytes of the dwarf goat. Also a number of suggested substrates for human CYP2C9 was investigated. 2. From Dixon plots (microsomal fractions) it was observed that all compounds were competitive inhibitors of tolbutamide hydroxylation. Phenytoin (PT) showed the lowest K_i. K_i for the sulfonamides ranged between 205 and 4546 μM, sulfadoxine having the lowest K_i followed by sulfadimethoxine, sulfamoxole, sulfadimidine and sulfaphenazole. 3. In hepatocytes sulfaphenazole and diclofenac were the most potent inhibitors. 4. Out data indicate that PT, diclofenac (DF) and phenylbutazone (PBZ) are relative strong competitive inhibitors of tolbutamide hydroxylation and they are probably also substrates for the same enzyme. Differential inhibition of tolbutamide hydroxylation by sulfonamides was observed. 5. Correlation of structural parameters with the inhibition constant or the inhibition in hepatocytes showed that molecular volume, polarisability and molecular surface area are important parameters in determining the rate of inhibition of tolbutamide hydroxylation by sulfonamides in both microsomes and hepatocytes. In addition, log P_oct are also involved oct in determining inhibition constants in microsomal fractions.     

Inhibitory Effects of Silibinin on Cytochrome P‐450 Enzymes in Human Liver Microsomes

Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P‐450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7–300 μM) at 37° in 0.1 M KH₂PO₄ buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC₅₀ values were determined for each substrate at respective K_M concentrations. Silibinin had little effect (IC₅₀>200 μM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)‐mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC₅₀=173 μM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC₅₀=29 μM and 46 μM) and S(?)‐warfarin 7‐hydroxylation (CYP2C9; IC₅₀=43 μM and 45 μM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (K_i,c=2.3 μM and 2.4 μM). Inhibition was competitive for S(?)‐warfarin 7‐hydroxylation (K_i,c=18 μM and 19 μM) and mainly non‐competitive for denitronifedipine oxidation (K_i,n=9 μM and 12 μM). With therapeutic silibinin peak plasma concentrations of 0.6 μM and biliary concentrations up to 200 μM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.     

Synthesis and Kinetic Testing of Tetrahydropyrimidine-2-thione and Pyrrole Derivatives as Inhibitors of the Metallo-β-lactamase from Klebsiella pneumonia and Pseudomonas aeruginosa

Metallo‐β‐lactamases (MBLs), produced by an increasing number of bacterial pathogens, facilitate the hydrolysis of many commonly used β‐lactam antibiotics. There are no clinically useful antagonists against MBLs. Two sets of tetrahydropyrimidine‐2‐thione and pyrrole derivatives were synthesized and assayed for their inhibitory effects on the catalytic activity of the IMP‐1 MBL from Pseudomonas aeruginosa and Klebsiella pneumoniae. Nine compounds tested ( 1a , 3b , 5c , 6b , 7a , 8a , 11c , 13a , and 16a ) showed micromolar inhibition constants (K_i values range from ～20–80 μm ). Compounds 1c , 2b , and 15a showed only weak inhibition. In silico docking was employed to investigate the binding mode of each enantiomer of the strongest inhibitor, 5c (K_i = 19 ± 9 μm ), as well as 7a (K_i = 21 ± 10 μm ), the strongest inhibitor of the pyrrole series, in the active site of IMP‐1.     

In-vitro metabolism of glycyrrhetinic acid by human and rat liver microsomes and its interactions with six CYP substrates

Objectives Glycyrrhetinic acid is the main metabolite of glycyrrhizin and the main active component of Licorice root. This study was designed to investigate the in‐vitro metabolism of glycyrrhetinic acid by liver microsomes and to examine possible metabolic interactions that glycyrrhetinic acid may have with other cytochrome P450 (CYP) substrates. Methods Glycyrrhetinic acid was incubated with rat liver microsomes (RLM) and human liver microsomes (HLM). Liquid chromatography tandem mass spectrometry was used for glycyrrhetinic acid or substrates identification and quantification. Key findings The K_m and V_max values for HLM are 33.41 µm and 2.23 nmol/mg protein/min, respectively; for RLM the K_m and V_max were 24.24 µm and 6.86 nmol/mg protein/min, respectively. CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. Other human CYP isoforms have minimal or no activity toward glycyrrhetinic acid. The interactions of glycyrrhetinic acid and six CYP substrates, such as phenacetin, diclofenac, (S)‐mephenytoin, dextromethorphan, chlorzoxazone and midazolam were also investigated. The inhibitory action of glycyrrhetinic acid was observed in CYP2C9 for 4‐hydroxylation of diclofenac, CYP2C19 for 4′‐hydroxylation of (S)‐mephenytoin and CYP3A4 for 1′‐hydroxylation of midazolam with half maximal inhibitory concentration (IC50) values of 4.3‐fold, 3.8‐fold and 9.6‐fold higher than specific inhibitors in HLM, respectively. However, glycyrrhetinic acid showed relatively little inhibitory effect (IC50 > 400 µm ) on phenacetin O‐deethylation, dextromethorphan O‐demethylation and chlorzoxazone 6‐hydroxylation. Conclusions The study indicated that CYP3A4 is likely to be the major enzyme responsible for glycyrrhetinic acid metabolism in HLM while CYP2C9 and CYP2C19 are considerably less active. The results suggest that glycyrrhetinic acid has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP2C9, CYP2C19 and CYP3A4 substrates.     

Assessment of CYP3A‐mediated drug–drug interaction potential for victim drugs using an in vivo rat model

The present study aims to determine if an in vivo rat model of drug–drug interaction (DDI) could be useful to discriminate a sensitive (buspirone) from a ‘non‐sensitive’ (verapamil) CYP3A substrate, using ketoconazole and ritonavir as perpetrator drugs. Prior to in vivo studies, ketoconazole and ritonavir were shown to inhibit midazolam hydroxylation with IC₅₀ values of 350 ± 60 nm and 11 ± 3 nm , respectively, in rat liver microsomes (RLM). Buspirone and verapamil were also shown to be substrates of recombinant rat CYP3A1/3A2. In the rat model, the mean plasma AUC_0‐inf of buspirone (10 mg/kg, p.o.) was increased by 7.4‐fold and 12.8‐fold after co‐administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. The mean plasma AUC_0‐inf of verapamil (10 mg/kg, p.o.) was increased by 3.0‐fold and 4.8‐fold after co‐administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. Thus, the rat DDI model correctly identified buspirone as a sensitive CYP3A substrate (>5‐fold AUC change) in contrast to verapamil. In addition, for both victim drugs, the extent of DDI when co‐administered was greater with ritonavir compared with ketoconazole, in line with their in vitro CYP3A inhibition potency in RLM. In conclusion, our study extended the rat DDI model applicability to two additional victim/perpetrator pairs. In addition, we suggest that use of this model would increase our confidence in estimation of the DDI potential for victim drugs in early discovery. Copyright © 2013 John Wiley & Sons, Ltd.     

INDIRECT PARASYMPATHOMIMETIC ACTIVITY OF THE CLASS III ANTIARRHYTHMIC SUBSTANCE / -SOTALOLIN VITRO: REVERSIBLE INHIBITION OF CHOLINESTERASE ISOENZYMES FROM BLOOD AND THE HUMAN CENTRAL NERVOUS SYSTEM

Inhibitory effects of the class III antiarrhythmic compound / -sotalol on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes of both erythrocytes and the human caudate nucleus and on serum cholinesterase (ChE; EC 3.1.1.8) were studiedin vitrousing a spectrophotometric kinetic assay with acetylthiocholine (ASCh) as substrate. Sotalol concentrations in the assays varied from 0.32 to 3.2m . All isoenzymes studied were inhibited by / -sotalol in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ASCh concentrations revealed that / -sotalol reduced substrate affinity (apparent Michaelis constant, KM, increased) of serum ChE, but did not change the enzyme's maximal rate of ASCh hydrolysis (V_max). Thus, / -sotalol inhibition of serum ChE was of the competitive type (rate constant for reversible competitive inhibition: K_i=0.51m ). In contrast, / sotalol reduced the maximal reaction velocity of the AChE isoenzyme from the central nervous system (caudate nucleus), but had no influence on substrate affinity of the enzyme (K_Mwith ASCh unchanged) indicating purely non-competitive inhibition kinetics (rate constant of reversible non-competitive inhibition: K_i′=0.44m ). / -sotalol inhibition of erythrocyte AChE was of mixed competitive/non-competitive type (K_i=0.31m , K_i′=0.49m ). Non-competitive / -sotalol inhibition of caudate nucleus AChE and the non-competitive component of erythrocyte AChE inhibition cannot be overcome by increased concentrations of the cholinergic transmitter acetylcholine (ACh). Peak / -sotalol plasma levels as described in the literature for both humans (15μ ) and experimental animals (dogs: 18μ ; rats: 260μ ) as well as maximal myocardial concentrations of the substance (dogs: 46μ ; rats: 478μ ) are in the range of about 2% to 100% of the sotalol inhibition rate constants determined in the present paper for cholinesterase isoenzymesin vitro. Thus, / -sotalol inhibition of ACh hydrolysisin vivomay contribute to both the well known antiarrhythmic potential and proarrhythmic side effects of the compound.     

Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates

BACKGROUND AND PURPOSE

Imatinib, a cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro.

EXPERIMENTAL APPROACH

Amodiaquine N-deethylation and midazolam 1′-hydroxylation were used as marker reactions for CYP2C8 and CYP3A4/5 activity. Direct, IC₅₀-shift, and time-dependent inhibition were assessed with human liver microsomes.

KEY RESULTS

Inhibition of CYP3A4 activity by imatinib was pre-incubation time-, concentration- and NADPH-dependent, and the time-dependent inactivation variables K_I and k_inact were 14.3 µM and 0.072 min⁻¹ respectively. In direct inhibition experiments, imatinib and N-desmethylimatinib inhibited amodiaquine N-deethylation with a K_i of 8.4 and 12.8 µM, respectively, and midazolam 1′-hydroxylation with a K_i of 23.3 and 18.1 µM respectively. The time-dependent inhibition effect of imatinib was predicted to cause up to 90% inhibition of hepatic CYP3A4 activity with clinically relevant imatinib concentrations, whereas the direct inhibition was predicted to be negligible in vivo.

CONCLUSIONS AND IMPLICATIONS

Imatinib is a potent mechanism-based inhibitor of CYP3A4 in vitro and this finding explains the imatinib–simvastatin interaction and suggests that imatinib could markedly increase plasma concentrations of other CYP3A4 substrates. Our results also suggest a possibility of autoinhibition of CYP3A4-mediated imatinib metabolism leading to a less significant role for CYP3A4 in imatinib biotransformation in vivo than previously proposed.     

Human UDP‐Glucuronosyltransferases 1A1, 1A3, 1A9, 2B4 and 2B7 are Inhibited by Diethylstilbestrol

Inhibition of UDP‐glucuronosyltransferases (UGTs) can result in many undesired side effects. Diethylstilbestrol (DES), a synthetic oestrogen famous for its multiple toxicities, was once widely administered to women in high dosages and now still gains application in clinics. This study investigated in vitro inhibitory effects of DES on catalytic activities of human UGTs, aiming at disclosing new potential toxic mechanisms on the basis of interactions between DES and metabolizing enzymes. DES (10 μM) could decrease activities of UGT1A1, 1A3, 1A9, 2B4 and 2B7 in catalysing 4‐methylumbelliferone (4‐Mu) glucuronidation. Further kinetic analyses showed that inhibition of these UGTs followed competitive (UGT1A1 and 1A9), mixed (UGT1A3 and 2B4) and non‐competitive (UGT2B7) mechanisms, with K_i values ranging from 0.91 to 4.1 μM. The inhibition potentials of UGT1A9 and 2B7 in human liver microsomes (HLM) were further tested by employing propofol and zidovudine as probe substrates, respectively. The inhibition of human liver microsomal UGT1A9 followed mixed mechanism, with the K_i value of 3.5 μM and α of 4.1. On the other hand, DES displayed non‐competitive inhibition against UGT2B7 in HLM, with the K_i value of 9.8 μM. The risks of in vivo inhibition of human UGTs were also predicted by calculation of plasma C/K_i values. Results suggest that DES can trigger in vivo inhibition of UGT1A1, 1A3, 1A9, 2B4 and 2B7 after the intravenous administration in high doses.