Human cytochrome P450 1A1 is a novel target gene of liver X receptor α

Cytochrome P450 (CYP) 1A1 is involved in the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) and is induced by several compounds, including PAHs. The induction of CYP1A1 mediated by the aryl hydrocarbon receptor (AhR) has been well investigated; however, little has been reported on the mechanisms of CYP1A1 induction mediated by factors other than AhR. In this study, we investigated the involvement of liver X receptor alpha (LXRα) in the induction of CYP1A1. TO-901317, an LXRα ligand, induced CYP1A1 mRNA in a dose-dependent fashion. Luciferase reporter assays using HepG2 cells showed that TO-901317 was capable of activating the promoter of the CYP1A1 gene and that a direct repeat 4 (DR4) motif located in a region from -452 to -467 was required for the induction of CYP1A1 through LXRα. Specific binding of LXRα to this DR4 motif was confirmed by gel shift and chromatin immunoprecipitation assays. Co-treatment of HepG2 cells with TO-901317 and 2,3,7,8-tetrachlorodibenzo-p-dioxin, a typical AhR ligand, caused the synergistic induction of CYP1A1 mRNA. Thus, we propose that the expression of CYP1A1 is regulated by LXRα as well as by AhR, suggesting that exposure to both LXRα and AhR ligands can result in the alteration of individual susceptibility to environmental carcinogens metabolically activated by CYP1A1.     

In vitro metabolism of a novel PPARγ agonist,KR-62980, and its stereoisomer,KR-63198, in human liver microsomes and by recombinant cytochrome P450s

1.?KR-62980 and its stereoisomer KR-63198 are novel and selective peroxisome proliferator-activated receptor gamma (PPARγ) modulators with activity profiles different from that of rosiglitazone. This study was performed to identify the major metabolic pathways for KR-62980 and KR-63198 in human liver microsomes.

2.?Human liver microsomal incubation of KR-62980 and KR-63198 in the presence of a β-nicotinamide adenine dinucleotide phosphate (NADPH)-generating system resulted in hydroxy metabolite formation. In addition, the specific cytochrome P450s (CYPs) responsible for KR-62980 and KR-63198 hydroxylation were identified by using a combination of chemical inhibition in human liver microsomes and metabolism by recombinant P450s. It is shown that CYP1A2, CYP2D6, CYP3A4, and CYP3A5 are the predominant enzymes in the hydroxylation of KR-62980 and KR-63198.

3.?The intrinsic clearance through hydroxylation was consistently and significantly higher for KR-62980 than for KR-63198, indicating metabolic stereoselectivity (CL_int of 0.012?±?0.001 versus 0.004?±?0.001 μl min^?1 pmol^?1 P450, respectively).

4.?In a drug–drug interaction study, KR-62980 and KR-63198 had no effect on the activities of the P450s tested (IC₅₀?>?50 μM), suggesting that in clinical interactions between KR-62980 and KR-63198 the P450s tested would not be expected.     

Comparison between recombinant P450s and human liver microsomes in the determination of cytochrome P450 Michaelis–Menten constants

1. Non-linear dose–exposure (supra-proportionality) occurs when plasma drug concentrations increase in a non-linear fashion with increasing dose. To predict the likelihood of this, an understanding is required of the K_M, which reflects a drug ability to saturate a specific enzyme involved in its metabolism.

2. This study assessed the accuracy of K_M and V_max determinations for compounds using a substrate-depletion approach with those determined using the product-formation approach, using both recombinant human cytochrome P450 (CYP) enzymes and human liver microsomes.

3. For the vast majority of the compounds studied, the K_M’s using recombinant CYPs and human liver microsomes in the two approaches predicted within two-fold. Further comparisons between the K_M and V_max-values were made between those measured using the product-formation approach and those estimated following simultaneous fitting of the Michaelis–Menten equation to all substrate depletion plots. In each case values were comparable.

4. In conclusion, the current study showed the substrate-depletion approach can be used to estimate K_M and V_max using both human liver microsomes and recombinant P450s. Estimation of these parameters during early discovery will aid in the understanding of dosages at which non-linearity may occur, but potentially aid predictions of likely clinical drug–drug interactions.

     

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.     

Enzyme kinetics of imperatorin and its cytochrome P450 phenotyping in liver microsomes北大核心CSCD

OBJECTIVE: To investigate enzyme kinetic characteristics of imperatorin in rat liver microsomes (RLM) or human liver microsomes (HLM), and to identify the reaction phenotyping of human recombinant cytochrome P450 enzyme (CYP) mediated phase I metabolism. METHODS: Imperatorin was incubated at 37°C with HLM or RLM in the presence or absence of nicotinamide adenine dinucleotide phosphate (NADPH) or uridine 5'-diphosphoglucuronic acid (UDGPA). The concentrations of imperatorin in the incubation systems were determined with LC-MS/MS to evaluate its metabolic stability and enzymatic kinetics. The CYP phenotyping of imperatorin was identified using a panel of human recombinant CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and also using a group of specific inhibitors in HLM. RESULTS: Imperatorin was metabolically eliminated in the presence of NADPH in HLM or RLM. The elimination rates for HLM and RLM in 30 min were 69.7% and 94.5%, respectively, and elimination half-life (t1/2) values were 18.9±0.6 and (2.8±0.4)min, respectively. The extrapolated hepatic clearance parameters (Clh) were 16.9±0.1 and (51.9±0.4)mL·min-1-kg-1. The Michaelism-Menten parameters (Km) were 13.60±0.16 and (14.00± 0.24)umol·L-1 and maximum velocity (Vmax) were (2928±96) and (8434±27)nmol·min-1·g-1, respectively. The metabolic elimination of imperatorin in RLM was quicker than in HLM. The results of CYP phenotyping indicated that CYP1A2, CYP2B6, CYP2C19 and CYP3A4 were the major CYP isoforms involved in the imperatorin metabolism. Their individual contributions assessed using the method of total normalized rate were 20.4%, 7.3%,10.5% and 61.8%, respectively. CONCLUSION: Imperatorin is mainly eliminated by CYP mediated metabolism in HLM and RLM. CYP1A2 and CYP3A4 are the major responsible enzymes with a contribution rate above 20%.     

Assessment of a novel β2-adrenoceptor agonist, trantinterol, for interference with human liver cytochrome P450 enzymes activities

The effect of a novel β₂-adrenoceptor agonist, trantinterol on the activities of cytochrome P450 (CYP450) was investigated with human liver microsomes and human cryohepatocytes in order to assess the potential for drug-drug interactions. The ability of trantinterol to inhibit CYP450 activities was evaluated in vitro in human liver microsomes. Trantinterol did not inhibit CYP2C19, CYP2D6, and CYP3A4/5 (IC₅₀ > 100 μM). It acted as a weak inhibitor of CYP1A2 and CYP2C9 with IC₅₀ of 70.8 and 81.9 μM, respectively. No time-dependent inhibitions were observed in the present research. To evaluate CYP450 induction, human cryohepatocytes (n = 3) were used and treated once daily for 3 days with trantinterol (0.01, 0.1, and 1 ng/ml), after which CYP450 activities were measured. At concentration of 0.01 ng/ml, which is close to the C_max at maximal recommended doses (50 μg), trantinterol was about 8% as effective as omeprazole (CYP1A2 inducer) only with donor 2. At concentration of 1 ng/ml, trantinterol was about 3.6 ± 3.1% as effective as rifampin (CYP3A4/5 inducer). These in vitro results indicated that, at pharmacological relevant concentrations, trantinterol will not produce clinically significant CYP450 inhibition or induction.     

Inhibition of cytochrome P450 and uridine 5′-diphospho-glucuronosyltransferases by MAM-2201 in human liver microsomes

MAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors and is increasingly used as an illicit recreational drug. The inhibitory effects of MAM-2201 on major drug-metabolizing enzymes such as cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) have not yet been investigated although it is widely abused, sometimes in combination with other drugs. We evaluated the inhibitory effects of MAM-2201 on eight major human CYPs (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six UGTs (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) of pooled human liver microsomes; we thus explored potential MAM-2201-induced drug interactions. MAM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, and UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with K _i values of 5.6, 5.4 and 5.0 µM, respectively. MAM-2201 exhibited mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-de-ethylation with K _i and k _inact values of 1.0 µM and 0.0738 min^?1, respectively. In human liver microsomes, MAM-2201 (50 µM) negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. Based on these in vitro results, we conclude that MAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP3A4, and UGT1A3.     

Roles of human cytochrome P450 3A4/5 in dexamethasone 6β-hydroxylation mediated by liver microsomes and humanized liver in chimeric mice metabolically suppressed with azamulin

The urinary metabolic ratio of 6β-hydroxydexamethasone to dexamethasone reportedly acts as a noninvasive marker for human cytochrome P450 (P450) 3A4/5, which is induced by rifampicin in humanized-liver mice. In the current study, the pharmacokinetics of dexamethasone in humanized-liver mice after intravenous administration (10 mg/kg) were investigated using azamulin (a time-dependent P450 3A4/5 inhibitor). After intravenous dexamethasone administration, significant differences were observed in the time-dependent plasma and 24-h urinary concentrations of 6β-hydroxydexamethasone between untreated humanized-liver mice and humanized-liver mice treated with azamulin (daily oral doses of 15 mg/kg for 3 days). The mean ratios of 6β-hydroxydexamethasone to dexamethasone for the maximum concentrations, the areas under the plasma concentration-versus-time curves, and urinary concentrations were significantly lower in the azamulin-treated group (59%, 58%, and 41% of the untreated values, respectively). 6β-Hydroxydexamethasone formation was suppressed by 93% by replacing control human liver microsomes with P450 3A4/5-inactivated liver microsomes. These results suggest that the oxidation of dexamethasone in humans is mediated mainly by P450 3A4/5 (which is suppressed by azamulin), and that humanized-liver mice orally treated with azamulin may constitute an in vivo model for metabolically inactivated P450 3A4/5 in human hepatocytes transplanted into chimeric mice.     

Drug–drug interactions in the metabolism of imidafenacin: Role of the human cytochrome P450 enzymes and UDP-glucuronic acid transferases,and potential of imidafenacin to inhibit human cytochrome P450 enzymes

Imidafenacin (IM), 4-(2-methyl-1H-imidazol-1-yl)-2,2-diphenylbutanamide, is a newly synthesized antimuscarinic drug developed for the treatment of overactive bladder. To predict clinically relevant drug interactions in the metabolism of IM, the paper investigated: (1) the major enzymes responsible for the metabolism of IM, (2) the effects of concomitant drugs on the inhibition of metabolism of IM, and (3) the effects of IM and its metabolites on the inhibition of human cytochrome P450 (CYP). The elimination of IM and production of oxidative metabolites were mainly catalysed by recombinant CYP3A4, and the elimination of IM by human liver microsomes (HLM) was markedly inhibited by co-incubation with ketoconazole. The production of the N-glucuronide metabolite was only catalysed by recombinant UGT1A4. Clinically established CYP3A4 inhibitors including itraconazole, ketoconazole, erythromycin and clarithromycin inhibited the elimination of IM in HLM. IM and its major metabolites did not affect the activities of CYP enzymes in vitro. The results suggest that the major enzymes responsible for the metabolism of IM are CYP3A4 and UGT1A4, and oxidative metabolism of IM is reduced by concomitant administration of CYP3A4 inhibitors. In contrast, IM and its metabolites have no inhibitory effect on the CYP-mediated metabolism of concomitant drugs.     

Identification of human cytochrome P450 enzymes involved in the metabolism of a novel к-opioid receptor agonist, nalfurafine hydrochloride

Nalfurafine hydrochloride (TRK-820) exhibits strong к-opioid agonistic activity and is a new antipruritic agent for uremic pruritus. This study was performed to identify the human hepatic cytochrome P450 isoforms involved in the metabolic conversion of nalfurafine to the decyclopropylmethylated form, de-CPM, using human liver microsomes and E. coli membrane fractions expressing human P450 isoforms. Samples were analysed by liquid chromatography with a radioactivity detector and liquid chromatography-tandem mass spectrometry. The metabolism of nalfurafine by human liver microsomes exhibited a biphasic kinetic profile. Experiments examining the metabolism by E. coli membrane fractions expressing human P450 isoforms indicated that CYP1A1, 2C8, 2C19 and 3A4 had the ability to produce de-CPM. In experiments with human liver microsomes that examined the inhibition of nalfurafine metabolism by anti-human P450 antibodies, anti-CYP3A4 antibody predominantly, and anti-CYP2C8 and 2C19 antibodies moderately, inhibited de-CPM formation. From these results, CYP3A4 appeared to be the major isoform involved in the metabolic decyclopropylmethylation of nalfurafine, while CYP2C8 and 2C19 most likely play a minor role in the formation of de-CPM.     

Identification of cytochrome P450 enzymes involved in the metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a designer drug,in human liver microsomes

1. The metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a novel designer drug, to its demethylated major metabolite 4′-hydroxy-pyrrolidinopropio-phenone (HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes.

2. CYP2C19 catalysed the demethylation with apparent K_m and V_max values of 373.4 ± 45.1?μM and 6.0 ± 0.3?pmol?min^?1?pmol^?1 CYP, respectively (mean ± SD). Both CYP2D6 and HLM exhibited clear biphasic profiles with apparent K_m,1 values of 1.3 ± 0.4 and 22.0 ± 6.5?μM, respectively, and V_max,1 values of 1.1 ± 0.1 pmol?min^?1?pmol^?1 CYP and 169.1 ± 20.5?pmol?min^?1?mg^?1 protein, respectively.

3. Percentages of intrinsic clearances of MOPPP by particular CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively.

4. MOPPP, HO-PPP and the standard 3′,4′-methylenedioxy-pyrrolidinopropio-phenone (MDPPP) were separated and analysed by liquid chromatography–mass spectrometry in the selected-ion monitoring (SIM) mode.

5. The CYP2D6 specific chemical inhibitor quinidine (3?μM) significantly (?p<0.0001) inhibited HO-PPP formation by 91.8 ± 0.5% (mean ± SEM) in incubation mixtures with HLM and 2?μM MOPPP.

6. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MOPPP demethylation.     

Determination of cytochrome P450 enzymes involved in the metabolism of (−)-terpinen-4-ol by human liver microsomes

1. The in vitro metabolism of (?)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes.

2. The biotransformation of (?)-terpinen-4-ol was investigated by gas chromatography–mass spectrometry. (?)-Terpinen-4-ol was found to be oxidized to (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol, major metabolic product by human liver microsomal P450 enzymes. The formation of metabolites of (?)-terpinen-4-ol was determined by relative abundance of mass fragments and retention times on GC.

3. CYP2A6 in human liver microsomes was a major enzyme involved in the oxidation of (?)-terpinen-4-ol by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 had the highest activity for oxidation of (?)-terpinen-4-ol. Second, oxidation of (?)-terpinen-4-ol was inhibited by (+)-menthofuran. Finally, there was a good correlation between CYP2A6 maker activity and (?)-terpinen-4-ol oxidation activities in liver microsomes of 10 human samples.

4. Kinetic analysis showed that the V_max/K_m values for (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol.

5. Human recombinant CYP2A6 catalysed (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with V_max values of 13.9 nmol/min/nmol P450 and apparent K_m values of 91 μM.

     

Identification of cytochrome P450 enzymes involved in the metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a designer drug,in human liver microsomes

The metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3′,4′-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent K_m and V_max values of 120.0?±?13.4?µM and 3.2?±?0.1?pmol/min/pmol?CYP, respectively (mean?±?standard deviation). CYP2D6 catalysed the demethylenation with apparent K_m and V_max values of 13.5?±?1.5?µM and 1.3?±?0.1 pmol/min/pmol?CYP, respectively. HLM exhibited a clear biphasic profile with an apparent K_m,1 value of 7.6?±?9.0 and a V_max,1 value of 11.1?±?3.6?pmol/min/mg?protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4′-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3?µM) significantly (p?相似文献   

In vitro characterization of 4′-(p-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s

1.?4′-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC.

2.?TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms.

3.?Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K_m and V_max values were 2.46?µM and 85.1?pmol/min/mg protein for M1 and 9.98?µM and 32.1?pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism.     

Tobacco smoke-dependent changes in cytochrome P450 1A1, 1A2, and 2E1 protein expressions in fetuses, newborns, pregnant rats, and human placenta

Tobacco smoke (TS) was described as a mixture of numerous cytochrome P450 (P450) substrates, inducers, and inhibitors. These inducers and inhibitors may modify drug clearance and xenobiotic or endogenous metabolism affecting P450s expression. In the present study, the effect of gestation and TS on: (1) cytochrome P450 CYP1A1, CYP1A2, and CYP2E1 protein expressions, and (2) cytochrome P450-linked microsomal enzyme activities, were studied in fetal rat liver, rat, and human placenta and in newborn and adult rat hepatic and extrahepatic tissues. Non-pregnant and pregnant 4-month-old female Wistar rats were exposed to TS (500, 1,000, or 1,500 mg carbon monoxide per m³ air) in a toxicological chamber for 3 weeks (6 h daily, 5 days weekly). Human placentas were sampled from non-smoking, passive smoking, or active smoking primiparas. The efficacy of exposure was assessed by measuring urine cotinine levels. The TS-dependent inductory effect on the expression of CYP1A1 and 1A2 and related monooxygenase activities, and the inhibitory/inductory effect on CYP2E1 expression in rat tissues were observed. Pregnancy was associated with decreased levels of constitutive CYP1A1 and 2E1 in hepatic and extrahepatic tissues, TS-inducible CYP1A2 expression in the liver, and CYP1A1 expression in lungs and heart, but had no inhibitory effect on TS-inducible CYP1A1 and 2E1 expression, EROD, and P450-cooperated enzyme activities in the liver, kidney, and, in the latter case, in the heart. The presence of TS-induced CYP1A1 protein was confirmed in rat and human placenta and showed in newborn liver and lungs. CYP1A2 and 2E1 proteins were detectable in fetal rat liver. It was concluded that the expression of CYP1A1, 1A2, and 2E1, which metabolize some drugs and activate carcinogens, is controlled by age-, pregnancy-, and tissue-specific regulatory mechanisms in rats. Gestational differences in the regulation of expression of CYP1A subfamily members are not excluded. CYP1A1 and 2E1, but not CYP1A2 inductory mechanisms seem to be functional in fetal liver at day 21 of pregnancy but they appeared to be uninducible under a TS exposure. In TS-exposed pregnant females and fetuses the effects of metabolic activation of CYP1A1 and 1A2 substrates might be reduced because of lower CYP expressions or poor induction, respectively.This paper was presented partially at the following congresses: 7th European ISSX Meeting, Budapest 1999; Eurotox 1999, Oslo; 11th World Conference on Tobacco OR Health, Chicago, 2000; EUROTOX 2001, Istanbul; 6th International ISSX Meeting, Munich, 2001     

Safety, tolerability, and pharmacokinetics of etamicastat, a novel dopamine-β-hydroxylase inhibitor, in a rising multiple-dose study in young healthy subjects

Background: Activation of the sympathetic nervous system is an important feature in hypertension and congestive heart failure. A strategy for directly modulating sympathetic nerve function is to reduce the biosynthesis of norepinephrine (noradrenaline) via inhibition of dopamine-β-hydroxylase (DβH).Objective: To assess the safety, tolerability, and pharmacokinetics of etamicastat (BIA 5–453), a new DβH inhibitor, following repeated dosing.Methods: A double-blind, randomized, placebo-controlled study was conducted in healthy young male volunteers. Participants received once-daily doses of placebo or etamicastat 25, 50, 100, 200, 400, or 600 mg, for 10 days.Results: Etamicastat underwent N-acetylation to its metabolite BIA 5–961. Etamicastat and BIA 5–961 maximum concentrations were achieved at 1–3 and 2–4 hours, respectively, after dosing. Elimination half-lives ranged from 18.1 to 25.7 hours for etamicastat and 6.7 to 22.5 hours for BIA 5–961. Both etamicastat and BIA 5-961 followed linear pharmacokinetics. The extent of systemic exposure to etamicastat and BIA 5–961 increased in an approximately dose-proportional manner, and steady-state plasma concentrations were attained up to 9 days of dosing. Etamicastat accumulated in plasma following repeated administration. The mean observed accumulation ratio was 1.3–1.9 for etamicastat and 1.3–1.6 for BIA 5–961. Approximately 40%of the etamicastat dose was recovered in urine in the form of parent compound and BIA 5–961. There was a high variability in pharmacokinetic parameters, attributable to different N-acetyltransferase-2 (NAT2) phenotype. Urinary excretion of norepinephrine decreased following repeated administration of etamicastat. Etamicastat was generally well tolerated. There was no serious adverse event or clinically significant abnormality in clinical laboratory tests, vital signs, or ECG parameters.Conclusion: Etamicastat was well tolerated. Etamicastat undergoes N-acetylation, which is markedly influenced by NAT2 phenotype. NAT2 genotyping could be a step toward personalized medicine for etamicastat.Trial Registration: EudraCT No. 2007-004142-33     

Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter‐system extrapolation factors

The ‘relative activity factor’ (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless ‘inter‐system extrapolation factor’ (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro–in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL_int). Values of ISEF for S‐warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL_int values derived from the kinetic values V_max and K_m of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes?. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes? were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S‐warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended. Copyright © 2011 John Wiley & Sons, Ltd.     

The effect of interferon-α on the expression of cytochrome P450 3A4 in human hepatoma cells

Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 μM) esculetin for 8 h prevented cell death and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8 h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 μM esculetin for 8 h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.     

In vitro cytochrome P450 monooxygenase and prostaglandin H-synthase mediated aflatoxin B1 biotransformation in guinea pig tissues: Effects of β-naphthoflavone treatment

In the present study, we examined the effects of treating guinea pigs with -naphthoflavone (BNF) on aflatoxin B₁ (AFB₁) metabolism by microsomal cytochrome P450 monooxygenase (P450) and prostaglandin H synthase (PHS) in liver, lung and kidney tissues. After BNF treatment, microsomal 7-ethoxyresorufin 0-deethylase activity was induced 13-, 25- and 11-fold in lung, kidney and liver, respectively, confirming that the BNF treatment protocol was effective at inducing monooxygenase activity. Treatment of guinea pigs with BNF did not change [³H]AFB₁-DNA binding catalyzed by microsomal PHS or P450 in lung, kidney or liver. In contrast, AFM₁ formation by P450 was significantly increased in microsomes from all three organs. The data indicate that BNF-inducible P450 isozymes of the P4501A class are responsible for the biotransformation of AFB₁ to non-toxic metabolites. Guinea pig kidney microsomes could also catalyze NADPH-dependent formation of aflatoxicol (AFL), a metabolite usually produced by a cytosolic steroid dehydrogenase. Renal microsomal AFL formation was not altered by prior BNF treatment. The results in the present study suggest that BNF may alter the bioactivation of AFB₁ in guinea pig tissues.by inducing P450 activity, leading to the formation of less reactive metabolite.     

In vitro inhibition and induction of human cytochrome P450 enzymes by mirabegron,a potent and selective β3-adrenoceptor agonist

1. The potential for mirabegron, a β₃-adrenoceptor agonist for the treatment of overactive bladder, to cause drug–drug interactions via inhibition or induction of cytochrome P450 (CYP) enzymes was investigated in vitro.

2. Mirabegron was shown to be a time-dependent inhibitor of CYP2D6 in the presence of NADPH as the IC₅₀ value in human liver microsomes decreased from 13 to 4.3 μM after 30-min pre-incubation. Further evaluation indicated that mirabegron may act partly as an irreversible or quasi-irreversible metabolism-dependent inhibitor of CYP2D6. Therefore, the potential of mirabegron to inhibit the metabolism of CYP2D6 substrates in vivo cannot be excluded. Mirabegron was predicted not to cause clinically significant metabolic drug–drug interactions via inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 because the IC₅₀ values for these enzymes both with and without pre-incubation were >100 μM (370 times maximum human plasma concentration [C_max]).

3. Whereas positive controls (100 µM omeprazole and 10 µM rifampin) caused the anticipated CYP induction, the highest concentration of mirabegron (10 µM; 37 times plasma C_max) had minimal effect on CYP1A2 and CYP3A4/5 activity, and CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that mirabegron is not an inducer of these enzymes.