Characterization of in vitro metabolites of cudratricusxanthone A in human liver microsomes

Cudratricusxanthone A (CTXA), isolated from the roots of Cudrania tricuspidata, exhibits several biological activities; however, metabolic biotransformation was not investigated. Therefore, metabolites of CTXA were investigated and the major metabolic enzymes engaged in human liver microsomes (HLMs) were characterized using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). CTXA was incubated with HLMs or human recombinant CYPs and UGTs, and analysed by an LC‐MS/MS equipped electrospray ionization (ESI) to qualify and quantify its metabolites. In total, eight metabolites were identified: M1–M4 were identified as mono‐hydroxylated metabolites during Phase I, and M5–M8 were identified as O‐glucuronidated metabolites during Phase II in HLMs. Moreover, these metabolite structures and a metabolic pathway were identified by elucidation of MSⁿ fragments and formation by human recombinant enzymes. M1 was formed by CYP2D6, and M2–M4 were generated by CYP1A2 and CYP3A4. M5–M8 were mainly formed by UGT1A1, respectively. While investigating the biotransformation of CTXA, eight metabolites of CTXA were identified by CYPs and UGTs; these data will be valuable for understanding the in vivo metabolism of CTXA. Copyright © 2015 John Wiley & Sons, Ltd.     

Metabolism and transfer of the mycotoxin zearalenone in human intestinal Caco-2 cells

The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10–200 μM ZEA showed efficacious metabolism of the toxin. α-zearalenol and β-zearalenol were the measured preponderant metabolites (respectively 40.7 ± 3.1% and 31.9 ± 4.9% of total metabolites, after a 3 h exposure to 10 μM ZEA), whereas ZEA-glucuronide and α-zearalenol glucuronide were less produced (respectively 8.2 ± 0.9% and 19.1 ± 1.3% of total metabolites, after a 3 h exposure to 10 μM ZEA). Cell production of reduced metabolites was strongly inhibited by α-and β-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited α-hydroxysteroid dehydrogenase type II and β-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, α-zearalenol was preponderantly found at the basal side, whereas β-zearalenol and both glucuronides were preferentially excreted at the apical side. As α-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.     

UGT2B7 C802T和G211T基因多态性对癫痫患者丙戊酸代谢的影响

目的:检测尿苷二磷酸葡糖醛酰转移酶UGT2B7 C802T和G211T等位基因在癫痫患者中的分布和突变频率,探讨UGT2B7 C802T和G211T基因型对癫痫患者丙戊酸代谢的影响。方法:直接化学发光法测定丙戊酸血药浓度,PCR-RFLP技术检测UGT2B7 C802T和G211T基因多态性,PCR扩增产物直接测序验证基因型检测方法的可靠性。结果:102例癫痫患者中UGT2B7 C802T位点野生型CC14例,杂合突变型和纯合突变型CT、TT分别为46例和42例;UGT2B7 G211T位点的野生型GG78例,突变型GT、TT分别为23例和1例;位点802CC野生基因型患者服用单位剂量(mg·kg^-1)后的血药浓度为(3.02±1.32) μg·kg·ml^-1·mg^-1,CT基因型患者为(2.11±1.26) μg·kg·ml^-1·mg^-1,TT基因型患者为(2.31±1.25) μg·kg·ml^-1·mg^-1,CT、TT患者血药浓度较CC患者明显偏低,差异有统计学意义。位点211GG基因型患者服用单位剂量(mg·kg^-1)引起的血药浓度为(2.28±1.32) μg·kg·ml^-1·mg^-1,GT基因型患者为(2.30±1.38) μg·kg·ml^-1·mg^-1,GG型与GT型患者间无统计学差异。结论:UGT2B7 C802T基因多态性与丙戊酸的血药浓度有显著相关性,UGT2B7 G211T位点基因多态性与丙戊酸的血药浓度无显著相关性。临床上个体血药浓度的差异可能与UGT2B7 C802T基因多态性有关。     

Comparison of Inhibition Capability of Scutellarein and Scutellarin Towards Important Liver UDP‐Glucuronosyltransferase (UGT) Isoforms

Scutellarin is an important bioactive flavonoid extracted from Erigeron breviscapus (Vant.) Hand‐Mazz, and scutellarein is the corresponding aglycone of scutellarin. The present study aims to compare the inhibition potential of scutellarin and scutellarein towards several important UDP‐glucuronosyltransferase (UGT) isoforms, including UGT1A1, UGT1A6, UGT1A9 and UGT2B7. It was demonstrated that scutellarein exerted stronger inhibition towards the tested UGT isoforms than scutellarin. Furthermore, the inhibition kinetic type and parameters (K_i) were determined for the scutellarein's inhibition towards these UGT isoforms. Competitive inhibition of scutellarein towards all these UGT isoforms was demonstrated, and the K_i values were calculated to be 0.02, 5.0, 5.8 and 35.9 μM for UGT1A1, 1A6, 1A9 and 2B7, respectively. Using in vivo maximum plasma concentration of scutellarein in rat, the in vitro–in vivo extrapolation was performed to predict in vivo situation, indicating the most possible in vivo adverse effects due to the inhibition of scutellarein towards UGT1A1. All these results remind us to monitor the utilization of scutellarin and scutellarein, and the herbs containing these two components. Copyright © 2013 John Wiley & Sons, Ltd.     

Sodium 2-propenyl thiosulfate derived from garlic induces phase II detoxification enzymes in rat hepatoma H4IIE cells

There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 μmol/L of NPTS or 2PTS for 48 hours at 37°C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase II inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 μmol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone.     

芫花氯仿萃取物对大鼠及人肝微粒体UGTs及UGT1A1活性影响

考察芫花氯仿萃取物对尿苷二磷酸葡萄糖醛酸转移酶(UGTs)及UGT1A1活性的影响,为预测芫花致肝损伤可能发生机制提供实验依据。采用体外肝微粒体孵育模型,分别以4-硝基酚和β-雌二醇为底物检测UGTs及UGT1A1活性;利用UV和HPLC测定底物或代谢物含量。结果表明,HPLC测得氯仿萃取物中3种黄酮类成分芹菜素、羟基芫花素和芫花素质量分数分别为1.00%,6.40%,18.38%;UV法测得总二萜质量分数为37.39%。与空白组相比,在大鼠肝微粒体(RLM)孵育体系,氯仿萃取物能显著抑制UGTs活性,而在人肝微粒体(HLM)孵育体系,抑制作用不明显;对UGT1A1,在RLM和HLM孵育体系中,氯仿萃取物均表现为中等强度的抑制作用(以芫花素计,IC50=8.76,10.36μmol·L-1);抑制类型分别为非竞争性抑制和反竞争性抑制。综上,氯仿萃取物对UGTs及UGT1A1活性均可产生抑制作用且存在种属差异性,推测这种抑制作用可能是芫花致肝损伤的机制之一。     

6种黄连生物碱对鼠肝微粒体UGTs及UGT1A1活性影响的体内外研究

为探讨黄连与其他药物代谢性相互作用的机制,对6种黄连生物碱与尿苷二磷酸葡萄糖醛酸转移酶(UGTs)及UGT1A1的相互作用进行了考察。采用大小鼠肝微粒体,以及生物碱小鼠体内诱导后的肝微粒体,构建微粒体体外孵育模型,以4-硝基酚(4-NP)为底物检测UGTs活性,β-雌二醇为底物检测UGT1A1活性,利用UV和HPLC测定底物或代谢物含量。结果,在大鼠体外实验中,小檗碱、表小檗碱、黄连碱及药根碱均能显著抑制UGTs活性,其中表小檗碱抑制作用最强;对UGT1A1,药根碱表现为弱的抑制作用(IC50≈227μmol·L-1),而黄连碱和巴马汀则呈显著的激活作用。小鼠体外实验中,小檗碱、黄连碱、药根碱和巴马汀对UGTs均呈显著的抑制作用;而6种生物碱对UGT1A1均表现为显著的激活作用。小鼠体内诱导实验中,只有小檗碱对UGTs,药根碱对UGT1A1活性呈现显著升高作用,其他生物碱的作用不明显。综上,黄连生物碱在对UGT的作用上显示出明显的种属和体内外的差异,同时生物碱结构的变化对UGT活性也会产生较大的影响,这种影响可能是黄连与其他药物发生代谢性相互作用的原因之一。     

羟基芫花素对UGTs及UGT1A1活性抑制作用种属差异的体外研究

考察羟基芫花素对尿苷二磷酸葡萄糖醛酸转移酶(UGTs)及UGT1A1活性的影响,为预测其与其他药物的代谢性相互作用提供理论借鉴。该实验采用体外肝微粒体孵育模型,以4-硝基酚(4-NP)为底物检测UGTs活性;β-雌二醇为底物检测UGT1A1活性,利用UV和HPLC测定底物或代谢物含量。结果表明,在大鼠、小鼠和人肝微粒体(HLM)孵育体系,羟基芫花素能显著抑制UGTs活性;对UGT1A1,在小鼠肝微粒体(MLM)孵育体系中,羟基芫花素几乎无抑制作用(IC50=190μmol·L-1);在大鼠肝微粒体(RLM)和重组酶(r UGT1A1)孵育体系中,羟基芫花素表现为中等强度的抑制作用(IC50=10.93,20.07μmol·L-1),抑制类型分别为竞争性抑制和线性混合型抑制;在HLM孵育体系中,羟基芫花素表现为弱抑制作用(IC50=76.31μmol·L-1),抑制类型为竞争性抑制;其抑制强弱顺序为RLMr UGT1A1HLMMLM。综上,羟基芫花素对不同肝微粒体孵育体系中UGTs及UGT1A1活性均可产生抑制作用且存在种属差异性,提示羟基芫花素可能存在基于UGT1A1酶的药物相互作用。该研究可为合理开发利用羟基芫花素提供实验依据,并为研究药物在临床上的联合用药提供理论借鉴。     

Molecular characterization of UDP-glucuronosyltransferases 3A and 8A in cynomolgus macaques

UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. The cynomolgus macaque is a nonhuman primate species widely used in drug metabolism studies. The molecular characteristics of UGTs have been extensively investigated in humans, but they remain to be elucidated in cynomolgus macaques. In this study, cynomolgus macaque UGT3A1, UGT3A2, and UGT8A1 cDNAs were isolated and characterized. Amino acid sequences deduced from cynomolgus UGT3A1, UGT3A2, and UGT8A1 cDNAs were highly identical with their human orthologs (93, 96, and 99%, respectively) and were closely clustered in a phylogenetic tree. In the genome, cynomolgus UGT3A and UGT8A genes were located in the regions corresponding to those of their human orthologs. Among the 10 tissue types analyzed, expression of cynomolgus UGT3A1 and UGT3A2 mRNAs was detected in liver, kidney, and testis; the UGT3A1 and UGT3A2 mRNAs were most abundant in liver and testis, respectively. Cynomolgus UGT8A1 was most abundantly expressed in kidney, followed by brain, jejunum, and testis. These results suggest that cynomolgus UGT3As and UGT8A1 have molecular similarities to their human orthologs.     

Identification of UGTs and BCRP as potential pharmacokinetic determinants of the natural flavonoid alpinetin

1. Alpinetin is a natural flavonoid showing a variety of pharmacological effects such as anti-inflammatory, anti-tumor and hypolipidemic activities. Here, we aim to determine the roles of UDP-glucuronosyltransferases (UGTs) and breast cancer resistance protein (BCRP) in disposition of alpinetin.

2. Glucuronidation potential of alpinetin was evaluated using pooled human liver microsomes (pHLM), pooled human intestine microsomes (pHIM) and expressed UGT enzymes supplemented with the cofactor UDPGA. Activity correlation analyses with a bank of individual HLMs were performed to identify the main contributing UGT isozymes in hepatic glucuronidation of alpinetin. The effect of BCRP on alpinetin disposition was assessed using HeLa cells overexpressing UGT1A1 (HeLa1A1) cells.

3. Alpinetin underwent extensive glucuronidation in pHLM and pHIM, generating one glucuronide metabolite. Of 12 test UGT enzymes, UGT1A3 was the most active one toward alpinetin with an intrinsic clearance (CL_int?=?V_max/K_m) value of 66.5?μl/min/nmol, followed by UGT1A1 (CL_int?=?48.6?μl/min/nmol), UGT1A9 (CL_int?=?21.0?μl/min/nmol), UGT2B15 (CL_int?=?16.7?μl/min/nmol) and UGT1A10 (CL_int?=?1.60?μl/min/nmol). Glucuronidation of alpinetin was significantly correlated with glucuronidation of estradiol (an activity marker of UGT1A1), chenodeoxycholic acid (an activity marker of UGT1A3), propofol (an activity marker of UGT1A9) and 5-hydroxyrofecoxib (an activity marker of UGT2B15), confirming the important roles of UGT1A1, UGT1A3, UGT1A9 and UGT2B15 in alpinetin glucuronidation. Inhibition of BCRP by its specific inhibitor Ko143 significantly reduced excretion of alpinetin glucuronide, leading to a significant decrease in cellular glucuronidation of alpinetin.

4. Our data suggest UGTs and BCRP as two important determinants of alpinetin pharmacokinetics.