多梳蛋白抑制复合物2抑制剂的药物专利分析

多梳蛋白抑制复合物2(polycomb repressive complex 2,PRC2)具有组蛋白甲基转移酶活性,其亚基包括zeste基因增强子同源物2(enhancer of zeste homologue 2,EZH2)和胚胎外胚层发育蛋白(embryonic ectoderm development,EED)...     

EZH2基因对卵巢癌的研究现状

EZH2(enhancer of zeste homo log 2)是果蝇zeste基因增强子的人类同源物,属于PcG( Polycomb Group)基因家族的重要成员之一,是一个非常重要的核转录调控因子[1].EZH2基因是最近几年新发现的一种转录阻遏抑制因子,它具有促进多种肿瘤转移的活性.EZH2作为后选癌基因,在许多肿瘤组织和细胞中呈现高表达,对肿瘤的发生和进展起着重要作用[2].     

148份胃癌组织中SOX4和EZH2的表达及临床意义

<正>胃癌是全球常见的恶性肿瘤之一,中国胃癌的发病例数达67.9万,死亡病例仅次于肺癌,位于肿瘤病死率的第二位。胃癌的发病是由多种因素共同作用的结果。上皮-间质转化(EMT)是胃癌侵袭及转移的关键过程,SOX4 (Sry-like highmobility groupbox 4)可以通过调节EMT促进胃癌的进展~([1])。果蝇Zeste同源物增强子2(EZH2)作为表观遗传调控的核心成分,调控基因表达来促进肿瘤的发生发展。甘艺等~([2])研究241例乳腺癌组织及126例癌旁组织中SOX4、EZH2表达情况,结果提示SOX4和EZH2的表达呈正相关。本研究通过免     

EZH2选择性抑制剂GSK126对胶质瘤细胞增殖的影响

目的研究Zeste基因增强子人类同源2（EZH2）抑制剂GSK126对胶质瘤细胞系U87和LN229细胞增殖的影响。方法 GSK126处理U87和LN229细胞后,采用CCK-8和平板克隆实验检测该抑制剂对胶质瘤细胞增殖的影响,用流式细胞仪测定其对细胞周期的影响,Western blot检测H3K27me3、p21、cyclin E和CDK2的表达。结果经GSK126处理后,U87和LN229细胞增殖能力下降（P<0.05）;GSK126处理能够诱导细胞停滞于G₀/G₁期（P<0.05）,p21蛋白表达量上升,而H3K27me3、cyclin E和CDK2表达量下调（P<0.05）。结论 GSK126能显著抑制胶质瘤细胞增殖,并通过上调p21表达来阻滞细胞周期。     

替莫唑胺联合EZH2抑制药GSK343对垂体瘤GH3细胞体外侵袭性的影响

目的 研究替莫唑胺(TMZ)联合zeste基因增强子类同源物2(EZH2)抑制药GSK343对GH3细胞凋亡和侵袭的作用及机制。方法 使用不同浓度的TMZ处理细胞筛选TMZ处理剂量,并将GH3细胞随机分为空白组、TMZ低剂量组(200μmol·L^-1TMZ)、TMZ高剂量组(400μmol·L^-1TMZ)、GSK343组(20μmol·L^-1 GSK343)、TMZ+GSK343组(400μmol·L^-1TMZ和20μmol·L^-1GSK343)。药物处理48 h后用细胞计数试剂盒-8(CCK-8)实验检测细胞存活率,以原位末端标记(TUNEL)法和流式细胞术检测细胞凋亡情况,以Transwell实验检测细胞侵袭能力,以蛋白质印迹法检测细胞相关蛋白表达。结果 空白组、TMZ低剂量组、TMZ高剂量组、GSK343组、TMZ+GSK343组TUNEL阳性细胞率分别为(4.31±0.71)%、(15.36±0.91)%、(22.26±2.13)%、(13.05±0.71)%和(34....     

EZH2在大肠癌中的表达及其临床意义

目的：探讨果蝇zeste基因增强子同源物2（EZH2）蛋白在大肠癌中的表达状况及其临床意义。方法：应用免疫组化S-P法检测大肠癌60例、正常大肠组织10例中EZH2蛋白的表达。结果：EZH2蛋白主要表达于细胞核,大肠癌、正常大肠组织的EZH2蛋白阳性表达率分别是71.7%（44/50）、0.0%（0/10）,EZH2蛋白在大肠癌组织中的表达明显高于在正常大肠组织中的表达（χ2=15.68,P〈0.01）。且EZH2蛋白在大肠癌中的表达与性别、年龄、分化程度、临床分期、大体分型、癌肿大小有关。结论：EZH2异常表达与原大肠癌的发展密切相关,在大肠癌的发展过程中起重要的作用。     

lncRNA和EZH2的相互作用在头颈部恶性肿瘤中的研究进展

头颈部恶性肿瘤已成为第六大常见的人类恶性肿瘤。长链非编码RNA(lncRNA)是非蛋白质编码转录本,长度大于200个核苷酸。lncRNA已被证实可以在头颈部恶性肿瘤的转录、转录后及表观遗传水平上调节基因的表达。研究表明,lncRNA参与了zeste同源增强子2(EZH2)的致癌调控网络,EZH2在癌症的发生发展中起着重要作用。一方面,lncRNA可以直接与EZH2结合,将EZH2招募到基因的启动子区域,并抑制其表达;另一方面,lncRNA也可以作为EZH2效应因子或调控因子而发挥作用。本文对lncRNAEZH2相互作用做一综述,并讨论它们对头颈部恶性肿瘤的影响,更好地了解其在头颈部肿瘤中的作用机制,将为基于lncRNA或EZH2的头颈部恶性肿瘤的诊断和治疗提供新的见解。     

地西他滨联合GSK126通过诱导“病毒模拟”对人膀胱癌T24细胞的抑制作用研究

目的研究DNA去甲基化药物地西他滨与Zeste基因增强子同源物2(enhancer of Zeste Homolog 2,EZH2)抑制剂GSK126联合应用对膀胱癌诱导"病毒模拟"免疫应答的抗肿瘤作用。方法将膀胱癌细胞株T24分为4组,对照组、地西他滨单药组、GSK126单药组、地西他滨与GSK126联合用药组。用细胞倍增时间测定T24细胞的增殖能力,用药物联合分析计算联合指数(CI),用实时荧光定量PCR(qRT-PCR)检测ERV-Fc1、ERV-W、IFIT2、IRF7、MDA5、RGH2和RIG1内源性逆转录病毒基因(endogenous retrovirus,ERV)的表达。结果地西他滨药物联合应用对T24细胞有显著的抑制作用(P<0.05),两者之间具有协同作用(CI<1),7个ERVs的表达上调。结论地西他滨联合GSK126对膀胱癌T24细胞有协同增殖抑制作用,其机制可能与诱导"病毒模拟"有关。     

EZH2对HSC-T6细胞增殖活化的影响及其部分机制研究

目的探讨Zeste基因增强子同源物2(enhancer of zeste homolog 2,EZH2)表达沉默对大鼠肝星状细胞HSC-T6细胞增殖活化的影响,及其部分调控机制。方法应用EZH2的抑制剂DZNep(3-Deazaneplanocin A)作用于TGF-β1诱导活化的HSC-T6细胞,采用Western blot法检测蛋白EZH2、p-ERK、p-AKT和α-SMA的表达;根据EZH2的碱基序列设计并合成小干扰RNA(small interfering RNA,si RNA),通过脂质体LipofectamineTM2000转染到HSC-T6细胞内,应用四甲基偶氮唑盐(MTT)法检测HSC-T6细胞的增殖变化,Western blot法检测蛋白EZH2、p-ERK、p-AKT和α-SMA的表达。结果将DZNep加入TGF-β1诱导活化的HSC-T6细胞后,EZH2蛋白水平明显降低,同时p-ERK、pAKT和α-SMA蛋白水平亦明显降低;将EZH2-si RNA转染活化的HSC-T6细胞内,HSC-T6细胞的增殖可明显被抑制,同时EZH2、p-ERK、p-AKT和α-SMA蛋白水平亦明显降低。结论抑制EZH2的表达可明显抑制HSC-T6细胞的增殖活化,EZH2可能是潜在的治疗肝纤维化的靶点。     

乳腺浸润性导管癌中EZH2、VEGF和MVD的表达及与临床病理学特征的关系

目的探讨果蝇zeste基因增强子同源物2(EZH2)表达及与乳腺浸润性导管癌血管内皮生长因子(VEGF)、血管生成及临床病理特征的关系。方法采用免疫组织化学SP两步法检测90例手术切除的原发性乳腺浸润性导管癌组织中EZH2、VEGF和CD34表达情况,用图像分析软件对EZH2、VEGF进行定量测试,计数400倍镜下CD34标记血管内皮细胞数即微血管密度(MVD),并分析EZH2、VEGF和MVD与乳腺浸润性导管癌临床病理特征的关系。用Spearman相关系数分析EZH2、VEGF及MVD表达的相关性。结果 EZH2在正常乳腺组织、乳腺低级别导管内癌、高级别导管内癌和浸润性导管癌中表达依次升高。90例乳腺浸润性导管癌组织中EZH2和VEGF的表达率分别为86.67%(78/90)和80%(72/90)。EZH2和VEGF蛋白与乳腺浸润性导管癌肿瘤直径、组织学分级、淋巴结转移状况、pTNM分期均有关(P<0.05)。MVD与乳腺浸润性导管癌组织学分级、淋巴结转移状况、pTNM分期均有关(P<0.05)。Spearman相关分析显示EZH2蛋白的表达和VEGF和MVD均呈显著正相关(rp=0.984,P=0.000;rp=0.346,P=0.000),VEGF和CD34的表达呈显著正相关(rp=0.321,P=0.000)。结论 EZH2可能直接参与了乳腺浸润性导管癌的演变过程,是肿瘤发生早期阶段的分子事件,EZH2的高表达促进了VEGF的表达和肿瘤血管密度的增加,进一步促进了肿瘤的发生发展。     

Expression of a skin cholesterol sulfotransferase,St2b2, is a trigger of epidermal cell differentiation

1.?St2b2, a mouse cytosolic sulfotransferase, predominantly catalyses epidermal cholesterol sulfation. St2b2 was found in the basement layer by immunohistochemical analysis of normal mouse skin. The highest expression level was detected in epidermis from 3-day-old mice and then decreased before maturation. There was a good correlation between expression levels of skin St2b2 and a differentiation marker, involucrin.

2.?To understand the role of St2b2 in epidermal cell differentiation, recombinant St2b2 was expressed in primary epidermal cells. The expression of St2b2 enhanced the involucrin expression with an increase of cholesterol sulfate. Furthermore, by down-regulation of the St2b2 gene expression, involucrin was decreased in dorsal skin of 1–3-day-old mice by 67% of the control.

3.?These results strongly suggest a possibility that St2b2 expression plays a trigger of epidermal cell differentiation by controlling cholesterol sulfate level in the cells.     

2-Furoyl-LIGRL-NH2, a potent agonist for proteinase-activated receptor-2, as a gastric mucosal cytoprotective agent in mice

1. Proteinase-activated receptor-2 (PAR(2)), expressed in capsaicin-sensitive sensory neurons, plays a protective role in gastric mucosa. The present study evaluated gastric mucosal cytoprotective effect of 2-furoyl-LIGRL-NH(2), a novel highly potent PAR(2) agonist, in ddY mice and in wild-type and PAR(2)-knockout mice of C57BL/6 background. 2. Gastric mucosal injury was created by oral administration of HCl/ethanol solution in the mice. The native PAR(2)-activating peptide SLIGRL-NH(2), administered intraperitoneally (i.p.) at 0.3-1 micromol kg(-1) in combination with amastatin, an aminopeptidase inhibitor, but not alone, revealed gastric mucosal protection in ddY mice, which was abolished by ablation of capsaicin-sensitive sensory neurons. 3. I.p. administration of 2-furoyl-LIGRL-NH(2) at 0.1 micromol kg(-1), without combined treatment with amastatin, exhibited gastric mucosal cytoprotective activity in ddY mice, the potency being much greater than SLIGRL-NH(2) in combination with amastatin. This effect was also inhibited by capsaicin pretreatment. 4. Oral administration of 2-furoyl-LIGRL-NH(2) at 0.003-0.03 micromol kg(-1) also protected against gastric mucosal lesion in a capsaicin-reversible manner in ddY mice. 5. I.p. 2-furoyl-LIGRL-NH(2) at 0.1-0.3 micromol kg(-1) caused prompt salivation in anesthetized mice, whereas its oral administration at 0.003-1 micromol kg(-1) was incapable of eliciting salivation. 6. In wild-type, but not PAR(2)-knockout, mice of C57BL/6 background, i.p. administration of 2-furoyl-LIGRL-NH(2) caused gastric mucosal protection. 7. Thus, 2-furoyl-LIGRL-NH(2) is considered a potent and orally available gastric mucosal protective agent. Our data also substantiate a role for PAR(2) in gastric mucosal protection and the selective nature of 2-furoyl-LIGRL-NH(2).     

Expression of a skin cholesterol sulfotransferase, St2b2, is a trigger of epidermal cell differentiation

1. St2b2, a mouse cytosolic sulfotransferase, predominantly catalyses epidermal cholesterol sulfation. St2b2 was found in the basement layer by immunohistochemical analysis of normal mouse skin. The highest expression level was detected in epidermis from 3-day-old mice and then decreased before maturation. There was a good correlation between expression levels of skin St2b2 and a differentiation marker, involucrin. 2. To understand the role of St2b2 in epidermal cell differentiation, recombinant St2b2 was expressed in primary epidermal cells. The expression of St2b2 enhanced the involucrin expression with an increase of cholesterol sulfate. Furthermore, by down-regulation of the St2b2 gene expression, involucrin was decreased in dorsal skin of 1-3-day-old mice by 67% of the control. 3. These results strongly suggest a possibility that St2b2 expression plays a trigger of epidermal cell differentiation by controlling cholesterol sulfate level in the cells.     

COX-2抑制剂戊地昔布的合成

目的:合成COX-2抑制剂戊地昔布.方法:以苯乙酸为起始原料,经酰氯化、傅克酰化生成二苯乙酮后,与盐酸羟胺反应生成肟,再环合,磺化,氨化得戊地昔布.结果:得到的产品经IR,1H-NMR,MS和元素分析,证明是戊地昔布.结论:本合成路线简化了操作步骤,缩短反应时间,提高了收率.     

Design, synthesis, and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methylcytidine, a potent inhibitor of hepatitis C virus replication

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.     

Characterization of etoricoxib,a novel,selective COX-2 inhibitor

Etoricoxib is a potent selective COX-2 inhibitor in man. Ex vivo whole-blood assays assessed COX-2 inhibition after oral administration of etoricoxib in single (5-500 mg) and multiple (25-150 mg) once-daily doses to healthy human subjects. A separate study examined ex vivo gastric mucosal PGE2 synthesis after etoricoxib (120 mg qd), naproxen (500 mg bid), or placebo for 5 days. The effect of etoricoxib 120 mg qd on the COX-1-mediated antiplatelet effects of low-dose aspirin (ASA) was also assessed. The mean (time)-weighted average inhibition (WAI) of lipopolysaccharide (LPS)-stimulated PGE2 (COX-2 assay) vcrsus placebo was dose related after single (range: 3.1%-99.1%) and multiple doses (range: 52.5%-96.7%). PGE2 remained significantly inhibited 24 hours postdose at steady state. Inhibition of LPS-stimulated PGE2 showed a strong relationship with etoricoxib plasma concentrations; ex vivo, IC50 was almost identical to in vitro. Multiple dosing of etoricoxib (up to 150 mg qd) showed no important effects on serum TXB2, bleeding time, or platelet aggregation (COX-1-mediated effects). The nonselective nonsteroidal anti-inflammatory (NSAID) naproxen significantly inhibited (approximately 78%) ex vivo prostaglandin synthesis in gastric mucosa; etoricoxib had no effect. Etoricoxib did not interfere with the antiplatelet effects of low-dose ASA, as assessed by serum TXB2 and platelet aggregation. Etoricoxib was generally well tolerated, even at doses above the clinical dose range. Based on these results, etoricoxib is a potent selective inhibitor of COX-2 after single and multiple dosing regimens and does not inhibit prostaglandin synthesis in the gastric mucosa, even at doses above the clinical dose range of 60 to 120 mg.     

2-Methoxyestradiol,a promising anticancer agent

Estrogens occurring naturally in the body are metabolized to catecholestrogens (2- and 4-hydroxyestradiol) by the cytochrome P450 enzymes. 2-Hydroxy catecholestrogens are further metabolized by catechol-O-methyltransferase to 2-methoxyestradiol, which is known to be protective against tumor formation. 2-Methoxyestradiol exhibits potent apoptotic activity against rapidly growing tumor cells. It also possesses antiangiogenic activity through a direct apoptotic effect on endothelial cells. Other molecular mechanisms, including microtubule stabilization by inhibition of the colchicine-binding site, have been reported. The exact mechanism of action of 2-methoxyestradiol is still unclear, but it has been shown to be effective in preventing tumor growth in a variety of cell lines. 2-Methoxyestradiol also possesses cardioprotective activity by inhibiting vascular smooth muscle cell growth in arteries. It has a lower binding affinity for estrogen receptor alpha compared with that of estradiol, and its affinity for estrogen receptor beta is even lower than that of estrogen receptor alpha, thus it has minimal estrogenic activity. 2-Methoxyestradiol is distinct because of its inability to engage estrogen receptors as an agonist, and its unique antiproliferative and apoptotic activities are mediated independently of estrogen receptors alpha and beta. A phase I clinical trial of 2-methoxyestradiol 200, 400, 600, 800, and 1,000 mg/day in 15 patients with breast cancer showed significant reduction in bone pain and analgesic intake in some patients, with no significant adverse effects. Another phase I study of 2-methoxyestradiol 200-1,000 mg/day in combination with docetaxel 35 mg/m2/week for 4-6 weeks performed in 15 patients with advanced refractory metastatic breast cancer showed no serious drug-related adverse effects. A phase II randomized, double-blind trial of 2-methoxyestradiol 400 and 1,200 mg/day in 33 patients with hormone-refractory prostate cancer showed that it was well tolerated and showed prostate specific antigen stabilizations and declines. We have started a phase I clinical trial to explore dosages greater than 1,000 mg/day.     

Pharmacokinetic assessment of a five-probe cocktail for CYPs 1A2, 2C9, 2C19, 2D6 and 3A

AIMS

To assess the pharmacokinetics (PK) of selective substrates of CYP1A2 (caffeine), CYP2C9 (S-warfarin), CYP2C19 (omeprazole), CYP2D6 (metoprolol) and CYP3A (midazolam) when administered orally and concurrently as a cocktail relative to the drugs administered alone.

METHODS

This was an open-label, single-dose, randomized, six-treatment six-period six-sequence William''s design study with a wash-out of 7 or 14 days. Thirty healthy male subjects received 100 mg caffeine, 100 mg metoprolol, 0.03 mg kg⁻¹ midazolam, 20 mg omeprazole and 10 mg warfarin individually and in combination (cocktail). Poor metabolizers of CYP2C9, 2C19 and 2D6 were excluded. Plasma samples were obtained up to 48 h for caffeine, metoprolol and omeprazole, 12 h for midazolam, 312 h for warfarin and the cocktail. Three different validated liquid chromatography tandem mass spectrometry methods were used. Noncompartmental PK parameters were calculated. Log-transformed C_max, AUC_last and AUC for each analyte were analysed with a linear mixed effects model with fixed term for treatment, sequence and period, and random term for subject within sequence. Point estimates (90% CI) for treatment ratios (individual/cocktail) were computed for each analyte C_max, AUC_last and AUC.

RESULTS

There was no PK interaction between the probe drugs when administered in combination as a cocktail, relative to the probes administered alone, as the 90% CI of the PK parameters was within the prespecified bioequivalence limits of 0.80, 1.25.

CONCLUSION

The lack of interaction between probes indicates that this cocktail could be used to evaluate the potential for multiple drug–drug interactions in vivo.