尿苷二磷酸葡萄糖醛酸转移酶的研究进展

目的:介绍尿苷二磷酸葡萄糖醛酸转移酶(UGT)的最近研究进展;方法:查阅了大量相关文献,总结了UGT的功能、诱导、底物及其基因研究等内容;结果:UGT是一种最重要的Ⅱ相代谢酶,对它的研究已经深入到基因水平,且可从分子水平上去解释它的作用机理;结论:人们对UGT有了更深更全面的了解。     

尿苷二磷酸葡萄糖醛酸转移酶与内源性物质的相互作用

尿苷二磷酸葡萄糖醛酸转移酶(UGT)是人体重要的II相代谢酶,代谢药物的同时也代谢许多重要的内源性物质,如胆红素、甲状腺激素、雌激素、雄激素、胆汁酸和5-羟色胺等。该酶对许多内源性物质的代谢是灭活和清除这些内源性物质的关键步骤,能够防止内源性物质累积引发的毒性反应,或及时终止内源性激素的信号防止肿瘤的发生。然而,内源性物质对UGT酶也会产生影响,特别是在一些生理病理条件下,某些内源性物质能够抑制UGT酶活性,影响其参与的代谢反应。将就内源性物质和UGT酶的相互作用做一综述,以引起人们对UGT酶和内源性物质相互作用的关注。关键词:药物代谢;尿苷二磷酸葡萄糖醛酸转移酶;内源性物质     

尿苷二磷酸葡萄糖醛酸转移酶的转录调节与疾病相关性的研究进展

葡萄糖醛酸结合反应是体内重要的Ⅱ相代谢途径,主要由尿苷二磷酸葡萄糖醛酸转移酶（ UGT ）催化。尿苷二磷酸葡萄糖醛酸转移酶能参与多种内源性物质如胆红素、胆汁酸、甲状腺激素等的代谢,也能参与多种药物如阿片类镇痛药、非甾体抗炎药等药物的代谢,在代谢解毒方面起着重要作用。近年来对尿苷二磷酸葡萄糖醛酸转移酶的研究越来越深入,尿苷二磷酸葡萄糖醛酸转移酶与不同疾病的研究受到普遍关注。本文就转录因子介导的尿苷二磷酸葡萄糖醛酸转移酶的分子调节机制及其与不同疾病的相关性研究进行综述。     

尿苷二磷酸葡萄糖醛酸转移酶基因多态性对药物代谢影响的研究进展

由尿苷二磷酸葡萄糖醛酸转移酶(UDP-glucuronosyltransferase,UGT)催化完成的葡萄糖醛酸结合反应是生物体内重要的Ⅱ相代谢途径,它与毒性或活性物质结合形成葡萄糖醛酸苷,将内源性、外源性化合物通过胆汁或肾脏排出体外。UGT是一个超家族酶,因主要利用UDP-尿苷二磷酸葡糖醛酸为糖基供体而得名。人类UCT广泛分布于体内的各种组织,包括肾、脑、皮肤、肠、脾、胸腺、心脏等,其中以     

人尿苷二磷酸葡萄糖醛酸转移酶基因多态性与相关疾病易感性

尿苷二磷酸葡萄糖醛酸转移酶(UGT)是体内重要的药物代谢Ⅱ相酶,具有明显的基因多态性。评价基因多态性对疾病易感性影响的重要性,建立基因多态性数据库并进行致病基因-疾病易感性的种群研究具有深远意义。本文拟就人UGT基因多态性及相关疾病易感性进行简述。     

孤儿核受体对尿苷二磷酸葡萄糖醛酸转移酶基因转录调节的研究进展

尿苷二磷酸葡萄糖醛酸转移酶（UDP-glucuro-nosyltransferase,UGT）属于Ⅱ相药物代谢酶。它能催化葡醛酸与其相应底物结合,是化学物质在生物体内进行Ⅱ相生物转化时最重要的一类酶。越来越多的研究发现,孕烷X受体（pregnaneXreceptor,PXR）、组成型雄甾烷受体（constitutiveandrostanere-ceptor,CAR）等孤儿核受体（orphannuclearreceptors）及其他转录因子能调节UGT基因的转录,这些受体及因子分布和活性的差异可导致UGT酶表达和分布的差异。此外,研究表明化合物通过核受体（NR）介导的UGT酶表达量的变化可能是影响化合物体内代谢的一条重要途径。由于UGT酶表达量与激素平衡、药物疗效、药物毒副作用及肿瘤等多种疾病发病预后密切相关,所以研究核受体对Ⅱ相代谢酶的调节作用在预测药物相互作用、指导临床合理用药及人类疾病的预防等方面都具有重要意义。     

腺苷蛋氨酸对L02细胞尿苷二磷酸葡萄糖醛酸转移酶表达的影响

目的探讨腺苷蛋氨酸(S-adenosyl-L-methionine,SAMe)对尿苷二磷酸葡萄糖醛酸转移酶(Uridine Diphosphate Glucuronsyltrans-ferase,UGT)表达的调节作用。方法体外培养L02细胞加入药物分别处理0～72 h后应用RT-PCR法观察SAMe对UGT mRNA的作用。结果 0.5 mmol/L的SAMe在24～72 h可上调UGTmRNA的表达,0.1～1 mmol/L的SAMe都能上调UGTmRNA的表达,但0.5mmol/L的作用最强,强于苯巴比妥。结论 SAMe可以上调UGTmRNA的表达,从而促进胆红素代谢。     

尿苷二磷酸葡萄糖醛酸转移酶的调控及其介导的中药-药物相互作用研究进展

尿苷二磷酸葡萄糖醛酸转移酶(UGT)催化的葡萄糖醛酸化反应是的是Ⅱ相代谢中重要的代谢反应之一,对于维持内源性化合物如胆红素、胆汁酸的动态平衡和药物、致癌物等外源性化合物的处置过程起着至关重要的作用。尿苷二磷酸葡萄糖醛酸转移酶的表达和酶活性受多维机制的调控。深入研究其调控网络以及尿苷二磷酸葡萄糖醛酸转移酶介导的相关中药-药物相互作用,对于临床更安全、有效的使用中药提供了指导。因此,本文总结了尿苷二磷酸葡萄糖醛酸转移酶的转录前、转录水平、翻译后修饰等分子调节机制,以及由尿苷二磷酸葡萄糖醛酸转移酶介导的中药-药物相互作用相关的研究进展。     

尿苷二磷酸葡醛酸转移酶的代谢类型及影响因素

尿苷二磷酸葡醛酸转移酶 (UGT)是一类Ⅱ相代谢酶 ,能催化葡醛酸与其相应底物结合。该过程是机体的重要排泄途径之一。目前 ,有 15种人类UGT被确证有活性 ,而且对它们的底物选择性方面有了进一步认识 ,但UGT的酶学研究相对于细胞色素P4 5 0还比较落后 ,有待于进一步提高。     

尿苷二磷酸葡醛酸转移酶介导的酪氨酸激酶抑制剂药物相互作用研究进展

近年来由代谢酶和转运体介导的酪氨酸激酶抑制剂(TKIs)的药物相互作用(DDI)已成为临床治疗的一个重要问题。除CYP450酶,尿苷二磷酸葡醛酸转移酶(UGTs)是参与TKIs代谢的另一类代谢酶,而且在体外多数TKI对UGTs呈抑制作用。TKIs与UGTs底物或抑制剂联合用药可能发生潜在的有临床意义的DDI。本文将重点研究UGTs介导的TKIs的药物-药物相互作用以及UGT1A基因型对TKIs的药物相互作用的影响,并探讨解决策略,以期为临床医师和药师对TKIs的安全合理应用提供参考。     

The effect of valproic acid on drug and steroid glucuronidation by expressed human UDP-glucuronosyltransferases

Valproic acid glucuronidation kinetics were carried our with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid was typified by high K(m) values with microsomes and expressed UGTs (2.3-5.2mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively (K(i)=1.6+/-0.06mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.     

Regulation of UDP-glucuronosyltransferase (UGT) 1A1 by progesterone and its impact on labetalol elimination

The authors recently reported the increased oral clearance of labetalol in pregnant women. To elucidate the mechanism of the elevated oral clearance, it was hypothesized that female hormones, at the high concentrations attainable during pregnancy, enhance hepatic metabolism of labetalol. Labetalol glucuronidation, which is the major elimination pathway of labetalol, was characterized by screening six recombinant human UGTs (UGT1A1, 1A4, 1A6, 1A9, 2B4, and 2B7) for their capacity to catalyse labetalol glucuronidation. The effect of female hormones (progesterone, oestradiol, oestriol, or oestrone) on the promoter activities of relevant UDP glucuronosyltransferases (UGT) was investigated using a luciferase reporter assay in HepG2 cells. The involvement of oestrogen receptor α (ERα) and pregnane X receptor (PXR) was examined by co-transfecting ERα- or PXR-constructs. UGT1A1 and UGT2B7 were identified as the major UGT enzymes producing labetalol glucuronides (trace amount of glucuronide conjugate was formed by UGT1A9). The activities of the UGT1A1 promoter containing PXR response elements were enhanced by progesterone, but not by oestrogens, indicating PXR-mediated induction of UGT1A1 promoter activity by progesterone. Results from semi-quantitative real-time polymerase chain reaction (PCR) assays are consistent with the above findings. This effect of progesterone on UGT1A1 promoter activities was concentration dependent. Promoter activities of UGT2B7 were not affected by either oestrogens or progesterone. The results suggest a potential role for progesterone in regulating labetalol elimination by modulating the expression of UGT1A1, leading to enhanced drug metabolism during pregnancy.     

Molecular cloning and functional analysis of minipig UDP-glucuronosyltransferase 1A6

Abstract

1.?UDP-glucuronosyltransferase 1A6 (UGT1A6) plays important roles in the glucuronidation of numerous drugs, environmental pollutants, and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies because many of their physiological characteristics are similar to those of humans. The aim of the present study was to examine similarities and differences in the enzymatic properties of UGT1A6 between humans and minipigs.

2.?Minipig UGT1A6 (mpUGT1A6) cDNA was cloned by the RACE method, and the corresponding proteins were expressed in insect cells. The enzymatic function of mpUGT1A6 was analyzed by the kinetics of serotonin glucuronidation.

3.?Amino acid homology between human UGT1A6 (hUGT1A6) and mpUGT1A6 was 79.9%. The kinetics of serotonin glucuronidation by recombinant hUGT1A6 and mpUGT1A6 enzymes fit the Michaelis–Menten equation. The K_m, V_max, and CL_int values of hUGT1A6 were 10.5?mM, 4.04?nmol/min/mg protein, and 0.39?µL/min/mg protein, respectively. The K_m value of mpUGT1A6 was similar to that of hUGT1A6, whereas the V_max and CL_int values of mpUGT1A6 were approximately 2-fold higher than those of hUGT1A6.

4.?These results suggest that the enzymatic properties of UGT1A6 enzymes are moderately different between humans and minipigs.     

UGT2B17拷贝数变异的研究进展

尿苷二磷酸葡萄糖醛酸基转移酶（UDP-g1ucuronosyltransferase,UGT）是人体内重要的Ⅱ相代谢酶,UDP-尿苷二磷酸葡萄糖醛酸为其主要的糖基供体。UGT为多基因编码并且含有大量同工酶的超基因家族,包括UGT1和UGT2两个亚家族。UGT2B17是UGT2B家族的一员,越来越多的研究发现UGT2B17基因存在普遍遗传缺失现象,其造成的遗传差异对疾病的影响已引起了众多关注。UGT2B17基因拷贝数变异（Copy Number Variants,CNV）源自4号染色体上一段约120 kb的DNA序列缺失和插入,CNV发生频率高,种族差异明显,与器官移植、骨质疏松、肿瘤发生,乃至兴奋剂检测密切相关。UGT2B17的CNV研究及功能意义具有重大遗传药理学及药物基因组学意义。我们将从UGT2B17的基因拷贝数变异与种族差异、移植排斥反应与肿瘤发生发展等几个方面进行探讨。     

Ritonavir and dexamethasone induce expression of CYP3A and P-glycoprotein in rats

1. The consequences of extended exposure to the human immunodeficiency viral protease inhibitor ritonavir (RIT) on the expression and function of CYP3A isoforms in the liver and in enteric mucosal cells, and on the expression of the efflux transport protein P-glycoprotein (P-gp) in enteric mucosa and in brain microvessel endothelial cells, were evaluated in rat. Dexamethasone (DEX), a known inducer of CYP3A and P-gp in rodents, served as a positive control.

2. Male CD-1 rats received RIT (20?mg?kg^?1), DEX (80?mg?kg^?1) or vehicle by oral/duodenal gavage once daily for 3 days.

3. Compared with vehicle control, CYP3A activity in liver microsomes (intrinsic clearance for triazolam hydroxylation in vitro) was increased by a factor of 2–4 by RIT, and by 10–14-fold by DEX. Similar increases were observed in expression of immunoactive CYP3A protein. Overall, maximum reaction velocity and immunoactive protein were highly intercorrelated (r² = 0.89). Both RIT and DEX also increased function and expression of enteric CYP3A, although to a more modest extent (about 1.7-fold for RIT, about 3.3-fold for DEX).

4. Enteric P-gp expression was equally induced (by 2.8-fold) by both RIT and DEX. P-gp expressed in brain microvessel endothelial cells was increased by a factor of 1.3 by both compounds.

5. Thus, increased expression of CYP3A isoforms and of P-gp occurs with 3 days of exposure to RIT in rats. Qualitatively similar changes occur in human cell culture models and in clinical studies, and might contribute to drug interactions involving RIT (and other antiretroviral agents) in humans.     

In vitro metabolism of eupatilin by multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, is extensively metabolized to eupatilin glucuronide, 4-O-desmethyleupatilin and 4-O-desmethyleupatilin glucuronide in human liver microsomes. This study characterized the human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for the metabolism of eupatilin. The specific CYPs responsible for O-demethylation of eupatilin to the major metabolite, 4-O-desmethyleupatilin were identified using a combination of correlation analysis, immuno-inhibition, chemical inhibition in human liver microsomes and metabolism by human cDNA-expressed CYP enzymes. UGT enzymes involved in the eupatilin glucuronidation were identified using pooled human liver microsomes and human cDNA-expressed UGT enzymes. Eupatilin was predominantly metabolized by CYP1A2 and, to a lesser extent, CYP2C8 mediated O-demethylation of eupatilin to 4-O-desmethyleupatilin. Eupatilin glucuronidation was catalysed by UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT1A10.     

Assessment of UDP-glucuronosyltransferase catalyzed formation of Picroside II glucuronide in microsomes of different species and recombinant UGTs

1. This study compared the hepatic glucuronidation of Picroside II in different species and characterized the glucuronidation activities of human intestinal microsomes (HIMs) and recombinant human UDP-glucuronosyltransferases (UGTs) for Picroside II.

2. The rank order of hepatic microsomal glucuronidation activity of Picroside II was rat > mouse > human > dog. The intrinsic clearance of Picroside II hepatic glucuronidation in rat, mouse and dog was about 10.6-, 6.0- and 2.3-fold of that in human, respectively.

3. Among the 12 recombinant human UGTs, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 catalyzed the glucuronidation. UGT1A10, which are expressed in extrahepatic tissues, showed the highest activity of Picroside II glucuronidation (K_m?=?45.1 μM, V_max?=?831.9 pmol/min/mg protein). UGT1A9 played a primary role in glucuronidation in human liver microsomes (HLM; K_m?=?81.3 μM, V_max?=?242.2 pmol/min/mg protein). In addition, both mycophenolic acid (substrate of UGT1A9) and emodin (substrate of UGT1A8 and UGT1A10) could inhibit the glucuronidation of Picroside II with the half maximal inhibitory concentration (IC₅₀) values of 173.6 and 76.2 μM, respectively.

4. Enzyme kinetics was also performed in HIMs. The K_m value of Picroside II glucuronidation was close to that in recombinant human UGT1A10 (K_m?=?58.6 μM, V_max?=?721.4 pmol/min/mg protein). The intrinsic clearance was 5.4-fold of HLMs. Intestinal UGT enzymes play an important role in Picroside II glucuronidation in human.

     

Optimization of a UDP-glucuronosyltransferase assay for trout liver S9 fractions: activity enhancement by alamethicin,a pore-forming peptide

1.?An existing assay for UDP-glucuronosyltransferase (UGT) activity in trout liver microsomes was optimized using trout liver S9 fractions. Individual experiments were conducted to determine the time dependence of UGT activity as well as optimal levels of S9 protein, uridine 5'-diphosphoglucuronic acid (UDPGA), substrate (p-nitrophenol) and alamethicin, a pore-forming agent added to eliminate latency.

2.?Addition of Mg^2+?(to 1?mM) or bovine serum albumin (BSA; to 2% w/v) had variable effects on activity, but these effects were minor. Eliminating alamethicin from the system resulted in very low levels of activity. A portion of this activity could be recovered by adding Triton X-100 or Brij 58; however, the optimal concentration range for either detergent was very narrow.

3.?When expressed on a pmol/min/g liver basis, UGT activities determined using this updated assay were substantially higher than those reported previously for uninduced trout.

4.?These results clearly demonstrate the advantages of using alamethicin for the removal of latency in UGT activity studies with trout and may have broad implications for the study of UGTs in other fish species.     

Role for protein kinase C delta in the functional activity of human UGT1A6: implications for drug–drug interactions between PKC inhibitors and UGT1A6

1. Many UDP-glucuronosyltransferases (UGTs) require phosphorylation by protein kinase C (PKC) for glucuronidation activity. Inhibition of UGT phosphorylation by PKC inhibitor drugs may represent a novel mechanism for drug–drug interactions.

2. The potential for PKC-mediated inhibition of human UGT1A6, an isoform involved in the glucuronidation of drugs such as acetaminophen (paracetamol) and endogenous substrates including serotonin, was evaluated using various cell model systems.

3. Of ten different PKC inhibitors screened for their effects on acetaminophen glucuronidation by human LS180 colon cells, only rottlerin (PKC δ selective inhibitor; IC₅₀?=?9.0?±?1.2 μM) and the non-selective PKC inhibitors (calphostin-C, curcumin and hypericin) decreased glucuronidation by more than 50%.

4. Using UGT1A6-infected Sf9 insect cells, calphostin-C and hypericin showed three times more potent inhibition of serotonin glucuronidation in treated whole cells versus cell lysates. However, both curcumin and rottlerin showed significant direct inhibition and so (indirect) PKC effects could not be differentiated in this model system.

5. Of nine PKC isoforms co-expressed with UGT1A6 in human embryonic kidney 293T cells only PKC δ increased protein-normalized UGT1A6-mediated serotonin glucuronidation significantly (by 63% ± 4%).

6. These results identify an important role for PKC δ in UGT1A6-mediated glucuronidation and suggest that PKC δ inhibitors could interfere with glucuronidation of UGT1A6 substrates.