Metabolic transformations of antitumor imidazoacridinone, C-1311, with microsomal fractions of rat and human liver

The imidazoacridinone derivative C-1311 is an antitumor agent in Phase II clinical trials. The molecular mechanism of enzymatic oxidation of this compound in a peroxidase model system was reported earlier. The present studies were performed to elucidate the role of rat and human liver enzymes in metabolic transformations of this drug. C-1311 was incubated with different fractions of liver cells and the reaction mixtures were analyzed by RP-HPLC. We showed that the drug was more sensitive to metabolism with microsomes than with cytosol or S9 fraction of rat liver cells. Incubation of C-1311 with microsomes revealed the presence of four metabolites. Their structures were identified as dealkylation product, M0, as well as a dimer-like molecule, M1. Furthermore, we speculate that the hydroxyl group was most likely substituted in metabolite M3. It is of note that a higher rate of transformation was observed for rat than for human microsomes. However, the differences in metabolite amounts were specific for each metabolite. The reactivity of C-1311 with rat microsomes overexpressing P450 isoenzymes, of CYP3A and CYP4A families was higher than that with CYP1A and CYP2B. Moreover, the M1 metabolite was selectively formed with CYP3A, whereas M3 with CYP4A. In conclusion, this study revealed that C-1311 varied in susceptibility to metabolic transformation in rat and human cells and showed selectivity in the metabolism with P450 isoenzymes. The obtained results could be useful for preparing the schedule of individual directed therapy with C-1311 in future patients.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human,Monkey, Dog,Rat, and Mouse Liver Microsomes

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH₃-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH₃-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH₃-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH₃-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH₃-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH₃-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH₃-PPD, especially at the C-20 hydroxyl group.     

Human,rat and crocodile liver microsomal monooxygenase activities measured using diazepam and nifedipine: effects of CYP3A inhibitors and relationship to immunochemically detected CYP3A apoprotein

Nifedipine oxidase and diazepam C₃-hydroxylase were tested as activities for selectively measuring CYP3A enzymes using liver microsomes from male and female human organ donors, male and female Wistar rats and male and female estuarine crocodiles. The association between CYP3A enzymes and these monooxygenations was confirmed for the human samples. Male rat samples had lower specific contents of CYP3A apoprotein than the human samples but had equivalent (nifedipine) or higher (diazepam) monooxygenase specific activities. CYP3A apoprotein was undetectable in female rat samples which had very low activities towards both substrates. Enzyme inhibition studies showed that diazepam C₃-hydroxylase of male rat liver was attributable to CYP3A but corresponding results for female rats suggested a contribution from non-CYP3A enzyme. Western blotting with immunochemical detection using anti-CYP3A4 IgG suggested the presence of putative CYP3A apoprotein in male and female crocodile liver samples and inhibition studies with diazepam as substrate suggested the presence of CYP3A subfamily monooxygenase activity in these enzyme preparations. Results for nifedipine oxidase with male and female rat liver and male crocodile liver suggested major contributions to catalysis from non-CYP3A enzymes. Inhibition studies suggested that a higher proportion of nifedipine oxidase in female crocodile liver may be attributable to the putative CYP3A enzyme(s) than in male crocodile liver. These results show the need for care in the assessment of CYP3A activity of fractionated tissues when using these substrates in cross-species studies and where gender is a variable.     

Metabolism of medroxyprogesterone acetate (MPA) via CYP enzymes in vitro and effect of MPA on bleeding time in female rats in dependence on CYP activity in vivo

Medroxyprogesterone acetate (MPA) is a drug commonly used in endocrine therapy for advanced breast cancer, although it is known to cause thrombosis as a serious side effect. Recently, we found that cytochrome P450 3A4 (CYP3A4) mainly catalyzed the metabolism of MPA via CYP in human liver microsomes. However, the metabolic products of MPA in humans and rats have not been elucidated. In addition, it is not clear whether thrombosis could be induced by MPA itself or by its metabolites. In this study, we determined the overall metabolism of MPA as the disappearance of the parent drug from an incubation mixture, and identified the enzymes catalyzing the metabolism of MPA via CYP in rats. Moreover, the effects of CYP-modulators on MPA-induced hypercoagulation in vivo were examined. Intrinsic clearance of MPA in rat liver microsomes was increased by treatment with CYP3A-inducers. The intrinsic clearance of MPA in liver microsomes of rats treated with various CYP-inducers showed a significant correlation with CYP3A activity, but not CYP1A activity, CYP2B activity or CYP2C contents. Among the eight recombinant rat CYPs studied, CYP3A1, CYP3A2 and CYP2A2 catalyzed the metabolism of MPA. However, since CYP3A2 and CYP2A2 are male-specific isoforms, CYP3A1 appears to be mainly involved in the metabolism of MPA in liver microsomes of female rats. In an in vivo study, pretreatment of female rats with SKF525A, an inhibitor of CYPs including CYP3A1, significantly (p < 0.05) enhanced MPA-induced hypercoagulation, whereas pretreatment with phenobarbital, an inducer of CYPs including CYP3A1, reduced it. These findings suggest that CYP-catalyzed metabolism of MPA is mainly catalyzed by CYP3A1 and that MPA-induced hypercoagulation is predominantly caused by MPA itself in female rats.     

UFLC-MS/MS研究胡椒碱在不同种属肝微粒体中的代谢稳定性和代谢表型

应用体外肝微粒体孵育体系,考察胡椒碱在人、SD大鼠、小鼠、恒河猴和比格犬5个种属肝微粒体中的代谢稳定性,比较代谢的种属差异,确定其在人肝微粒体中的代谢表型。通过UFLC-MS/MS检测方法,测定胡椒碱在各个种属肝微粒体中孵育后的剩余浓度,考察他们的代谢稳定性及体外代谢动力学参数。采用化学抑制法考察胡椒碱在人肝微粒体中的代谢表型。结果表明胡椒碱在人、SD大鼠、小鼠、恒河猴和比格犬的肝微粒体中,半衰期T1/2分别为31. 36、48. 46、138. 60、147. 45、165. 00 min;体外固有清除率CLint分别为0. 0442、0. 0286、0. 0100、0. 0094、0. 0084m L/(m L·mg);在人肝微粒体中,胡椒碱主要被CYP3A4和CYP2C9酶代谢。推测胡椒碱在各种肝微粒体中的代谢均相对较稳定,其中大鼠和人的肝微粒体代谢性质最相近,在后续的实验中可以考虑用大鼠的代谢结果预测人的代谢结果;人肝微粒体中参与胡椒碱代谢的酶主要有CYP3A4和CYP2C9。     

Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation

In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790±690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed K_m and V_max values in the CYP2A6 reconstituted system were 1.48±0.37 μM and 3360±180 pmol product/min/nmol CYP, respectively.     

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

Several P450 enzymes localized in the endoplasmic reticulum and thought to be involved primarily in xenobiotic metabolism, including mouse and rat CYP1A1 and mouse CYP1A2, have also been found to translocate to mitochondria. We report here that the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces enzymatically active CYP1A4/1A5, the avian orthologs of mammalian CYP1A1/1A2, in chick embryo liver mitochondria as well as in microsomes. P450 proteins and activity levels (CYP1A4-dependent 7-ethoxyresorufin-O-deethylase and CYP1A5-dependent arachidonic acid epoxygenation) in mitochondria were 23-40% of those in microsomes. DHET formation by mitochondria was twice that of microsomes and was attributable to a mitochondrial soluble epoxide hydrolase as confirmed by Western blotting with antiEPHX2, conversion by mitochondria of pure 11,12 and 14,15-EET to the corresponding DHETs and inhibition of DHET formation by the soluble epoxide hydrolase inhibitor, 12(-3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). TCDD also suppressed formation of mitochondrial and microsomal 20-HETE. The findings newly identify mitochondria as a site of P450-dependent arachidonic acid metabolism and as a potential target for TCDD effects. They also demonstrate that mitochondria contain soluble epoxide hydrolase and underscore a role for CYP1A in endobiotic metabolism.     

Characterization of metabolites and cytochrome P450 isoforms involved in the microsomal metabolism of aconitine

Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. The current study investigated the metabolism of aconitine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of aconitine in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS(n)) and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Various selective inhibitors of CYP were used to identify the isoforms of CYP, that involved in the metabolism of aconitine. A total of at least six metabolites were found and characterized in rat liver microsomal incubations. Result showed that the inhibitor of CYP 3A had an inhibitory effect on aconitine metabolism in a concentration-dependant manner, the inhibitor of CYP1A1/2 had a modest inhibitory effect, whereas inhibitors of CYP2B1/2, 2D and 2E1 had no obvious inhibitory effects on aconitine metabolism. Aconitine might be metabolized by CYP 3A and CYP1A1/2 isoforms in rat liver microsome.     

An inhibition study of beauvericin on human and rat cytochrome P450 enzymes and its pharmacokinetics in rats

Beauvericin is a secondary metabolite natural product from microorganisms and has been shown to have a new potential antifungal activity. In this study, the metabolism and inhibition of beauvericin in human liver microsomes (HLM) and rat liver microsomes (RLM) were investigated. The apparent K_m and V_max of beauvericin in HLM were determined by substrate depletion approach and its inhibitory effects on cytochromes P450 (CYP) activities were evaluated using probe substrates, with IC₅₀ and the (K_i) values were 1.2 μM (0.5 μM) and 1.3 μM (1.9 μM), respectively for CYP3A4/5 (midazolam) and CYP2C19 (mephenytoin). Similarly, beauvericin was also a potent inhibitor for CYP3A1/2 (IC₅₀: 1.3 μM) in RLM. Furthermore, the pharmacokinetics of beauvericin in the rat were studied after p.o administration alone and co-administration with ketoconazole, which indicated a pharmacodynamic function may play a role in the synergistic effect on antifungal activity.     

Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive

A synthetic androgen 7α-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in ‘androgen replacement therapy’ for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24 h prior to the injection of tritium-labeled MENT (³H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30 min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6 h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of ³H-MENT was investigated after subcutaneous injection of ³H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72 h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30 h.Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of ³H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P₄₅₀ (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7α-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16α-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7α-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, ³H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3-HSD enzymes.     

中药降脂方与阿托伐他汀代谢性相互作用的体外研究

目的：本研究旨在评估降脂中药复方2（Fang-2）及其6个饮片（虎杖、泽泻、甘草、苍术、厚朴和夏枯草）的水提物对肝脏细胞色素CYP3A4的抑制作用,及对阿托伐他汀的代谢性药物相互作用。方法：（1）应用中药标准化制备技术,制备降脂中药复方2及其6个饮片的水提物;采用超速离心法制备肝微粒体。（2）评价降脂中药复方2及其饮片对CYP3A4抑制作用。（3）评价降脂中药复方2及其饮片的水提物对阿托伐他汀的代谢的影响。结果：（1）体外实验的结果提示复方2的水提物显著地抑制了微粒体CYP3A4的活性,其IC50值为9．884mg／mL。虎杖水提物对微粒体CYP3A4的活性具有显著的抑制作用,其IC50值为0．5491mg／mL。（2）阿托伐他汀是首过代谢CYP3A4的底物,在肝脏微粒体中大于70％的母体被代谢,其体外清除率Clint和半数清除时间分别为41．10mL／mg／min和60．43分钟。降脂中药复方2水提物与肝微粒体孵育后,阿托伐他汀的首过代谢显著减慢。降脂中药复方2及其6个饮片水提取物对CYP3A4的抑制强度与避免阿托伐他汀首过代谢的潜力成正比。结论：本文首次应用体外方法研究了在临床上共同使用与PCI术后的阿托伐他汀和降脂中药复方2之间的代谢性相互作用,揭示了后者及其饮片通过抑制CYP3A4改善了前者的强烈的首过代谢,从而可能优化临床治疗方案。     

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A.Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine.In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preclinical metabolism of LB42908, a novel farnesyl transferase inhibitor, and its effects on the cytochrome P450 isozyme activities

Metabolism of LB42908, a novel farnesyl transferase inhibitor, was investigated for preclinical development. In vitro hepatic metabolism of LB42908 gave rise to at least 9 metabolites via phase I biotransformation pathways, which were characterized by HPLC-UV, LC-MS, and LC-MS/MS analyses. N-Dealkylation was shown to be a major phase I metabolic pathway. Species-specific in vitro metabolism of LB42908 was studied in liver fractions of rat, dog, monkey, and human. Order of metabolic stability is human≈dog>rat≈monkey in both S9 and microsomal fractions. Tissue-specific metabolism of LB42908 in various tissue homogenates of rats demonstrated that the liver was the major organ responsible for phase I metabolism of LB42908. The results from both qualitative and quantitative metabolism studies such as metabolic profiling and metabolic clearance indicated that dog would be the animal model of choice for preclinical toxicology studies. In addition, LB42908 was a potent CYP3A4 inhibitor in human liver microsomes and induced the activities of several CYP isozymes, implying that it has the potential for drug-drug interactions. Repeated dosing of LB42908 in rats did not significantly affect its own metabolism, indicating that long-term administration of LB42908 would not alter its pharmacokinetic profiles.     

Investigation of cytochrome P450 inhibitory properties of maslinic acid,a bioactive compound from Olea europaea L., and its structure–activity relationship

Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with IC₅₀ value at 46.1 and 62.3 µM, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with IC₅₀ values more than 100 µM. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with K_i of 18.4, 98.7 and 66.3 µM, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high K_i values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs.     

Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5β-cholestane-3α,7α,12α-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The V_max for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmol×min), respectively. The K_m was 6 μM for CYP3A4 and 32 μM for CYP3A5. Cytochrome b₅ increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5β-cholestane-3α,7α,12α-triol. A strong correlation was observed between formation of 25-hydroxylated 5β-cholestane-3α,7α,12α-triol and CYP3A levels (r²=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 μM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes.     

3,3′,4,4′-Tetrachlorobiphenyl oxidation in fish,bird and reptile species: relationship to cytochrome P450 1A inactivation and reactive oxygen production

Previously we showed that the polychlorinated biphenyl 3,3′,4,4′-tetrachlorobiphenyl (TCB) caused a release of reactive oxygen species (ROS) from cytochrome P450 1A (CYP1A) of the fish scup (Stenotomus chrysops), and from rat and human CYP1A1. This was linked to a TCB- and NADPH-dependent oxidative inactivation of the enzyme, which in scup and rat was inversely related to the rates of TCB oxidation. We examined the relationship between rates of TCB oxidation, CYP1A inactivation and ROS production in liver microsomes from additional vertebrate species, including skate (Raja erinacea), eel (Anguilla rostrata), killifish (Fundulus heteroclitus), winter flounder (Pleuronectes americanus), chicken (Gallus domesticus), cormorant (Phalacrocorax auritus), gull (Larus argentatus), and turtle (Chrysemys picta picta). TCB oxidation rates were induced in all fish and birds treated with aryl hydrocarbon receptor agonists. Induced rates of TCB oxidation were <1 pmol/min/mg microsomal protein in all fish, and 6–14 pmol/min/mg in the birds. In all species but one, TCB oxidation rates correlated positively with EROD rates, indicating likely involvement of CYP1A in TCB oxidation. Incubation of liver microsomes of most species with TCB+NADPH resulted in an immediate (TCB-dependent) inhibition of EROD, and a progressive loss of EROD capacity, indicating an oxidative inactivation of CYP1A like that in scup. NADPH stimulated production of ROS (H₂O₂ and/or O₂⁻) by liver microsomes, slightly in some species (eel) and greatly in others (chicken, turtle). Among the birds and the fish, NADPH-stimulated ROS production correlated positively with EROD activity. TCB caused a significant stimulation of ROS production by liver microsomes of flounder, killifish, cormorant and gull, as well as scup. The stimulation of CYP1A inactivation and ROS generation indicates an uncoupling of CYP1A by TCB in many species, and when compared between species, the rates of CYP1A inactivation correlated inversely with rates of TCB oxidation. Some feature(s) of binding/active site topology may hinder TCB oxidation, enhancing the likelihood for attack of an oxidizing species in the active site.     

Herb-drug interaction between Styrax and warfarin: Molecular basis and mechanism

BackgroundStyrax, one of the most famous folk medicines, has been frequently used for the treatment of cardiovascular diseases and skin problems in Asia and Africa. It is unclear whether Styrax or Styrax-related herbal medicines may trigger clinically relevant herb-drug interactions.PurposeThis study was carried out to investigate the inhibitory effects of Styrax on human cytochrome P450 enzymes (CYPs) and to clarify whether this herb may modulate the pharmacokinetic behavior of the CYP-substrate drug warfarin when co-administered.Study DesignThe inhibitory effects of Styrax on CYPs were assayed in human liver microsomes (HLM), while the pharmacokinetic interactions between Styrax and warfarin were investigated in rats. The bioactive constituents in Styrax with strong CYP3A inhibitory activity were identified and their inhibitory mechanisms were carefully investigated.MethodsThe inhibitory effects of Styrax on human CYPs were assayed in vitro, while the pharmacokinetic interactions between Styrax and warfarin were studied in rats. Fingerprinting analysis of Styrax coupled with LC-TOF-MS/MS profiling and CYP inhibition assays were used to identify the constituents with strong CYP3A inhibitory activity. The inhibitory mechanism of oleanonic acid (the most potent CYP3A inhibitor occurring in Styrax) against CYP3A4 was investigated by a panel of inhibition kinetics analyses and in silico analysis.ResultsIn vitro assays demonstrated that Styrax extract strongly inhibited human CYP3A and moderately inhibited six other tested human CYPs, as well as potently inhibited warfarin 10-hydroxylation in liver microsomes from both humans and rats. In vivo assays demonstrated that compared with warfarin given individually in rats, Styrax (100 mg/kg) significantly prolonged the plasma half-life of warfarin by 2.3-fold and increased the AUC_(0-inf) of warfarin by 2.7-fold when this herb was co-administrated with warfarin (2 mg/kg) in rats. Two LC fractions were found with strong CYP3A inhibitory activity and the major constituents in these fractions were characterized by LC-TOF-MS/MS. Five pentacyclic triterpenoid acids (including epibetulinic acid, betulinic acid, betulonic acid, oleanonic acid and maslinic acid) present in Styrax were potent CYP3A inhibitors, and oleanonic acid was a competitive inhibitor against CYP3A-mediated testosterone 6β-hydroxylation.ConclusionStyrax and the pentacyclic triterpenoid acids occurring in this herb strongly modulate the pharmacokinetic behavior of warfarin via inhibition of CYP3A.