常用中药单体及其制剂对CYP3A活性的体外抑制作用研究

摘 要 目的:研究常用中药单体及其制剂对CYP3A活性的抑制作用。方法: 在体外肝微粒体孵育体系中加入底物睾酮和不同浓度的中药单体或其制剂,以高效液相色谱法检测6β羟基睾酮的生成量,计算CYP3A酶活性。用GraphPad Prism v5.0软件,按照非线性回归计算出药物对CYP3A抑制作用的IC₅₀值。结果:不同中药单体及其制剂对CYP3A抑制作用的IC₅₀值如下：大黄酸IC₅₀值为36.74 μmol·L^-1;大黄素IC₅₀值为23.09 μmol·L^-1、芦荟大黄素IC₅₀值为23.91 μmol·L^-1,蜂胶IC₅₀值为60.3 μg·mL^-1;水飞蓟素胶囊IC₅₀值为24.5μg·mL^-1;丹参酮ⅡA磺酸钠注射液IC₅₀值为0.14%(v/v);五味子酯甲IC₅₀值为0.56 μmol·L^-1。结论: 大黄中的蒽醌、五味子酯甲、蜂胶、水飞蓟胶囊以及丹参酮ⅡA磺酸钠注射液在体外对鼠或人肝CYP3A酶具有抑制作用,临床上使用相关制剂时应密切关注其可能引起的药物相互作用。     

和厚朴酚、厚朴酚、栀子苷、绿原酸和黄芪甲苷对人和大鼠体外CYP1A2、CYP3A和CYP2D的抑制作用

目的 观察和厚朴酚、厚朴酚、栀子苷、绿原酸和黄芪甲苷5种中药成分体外对人和大鼠肝CYP1A2、CYP3A和CYP2D的抑制作用。方法 在人和大鼠肝微粒体孵育体系中,分别以非那西丁、咪达唑仑和右美沙芬为探针,应用HPLC检测受试物对探针代谢产物生成量的影响,评估5种中药成分对CYP1A2、CYP3A和CYP2D在该体系中的活性影响,并计算得到抑制率和IC₅₀。结果 和厚朴酚对人和大鼠CYP1A2、CYP2D的IC₅₀值分别为5.5、3.9、35.3和46.7 μmol·L^-1;厚朴酚对人CYP1A2、大鼠CYP1A2和CYP2D的IC50值分别为23.8,29.1和39.9 μmol·L^-1;栀子苷、绿原酸和黄芪甲苷对3种CYP酶亚型的IC₅₀均>100 μmol·L^-1;和厚朴酚对人和大鼠CYP3A的IC₅₀均>100 μmol·L^-1;厚朴酚对人CYP3A、CYP2D和大鼠CYP3A的IC₅₀均>100 μmol·L^-1。结论 和厚朴酚体外对人和大鼠CYP1A2和CYP2D有抑制作用,厚朴酚体外对人CYP1A2、大鼠CYP1A2和CYP2D有抑制作用,均呈浓度依赖性。     

银杏内酯B对血小板活化因子刺激的大鼠中性粒细胞功能的影响银杏内酯B对血小板活化因子刺激的大鼠中性粒细胞功能的影响

目的 研究银杏内酯B对血小板活化因子(PAF)刺激的大鼠中性粒细胞粘附、趋化及脱颗粒功能的影响。方法 从大鼠外周血分离中性粒细胞,用MTT比色法、Boyden小室法及β-葡萄糖苷酸酶释放法分别检测PAF诱导的粒细胞粘附、趋化及脱颗粒反应。结果10 μmol·L^-1 银杏内酯B可显著抑制中性粒细胞的粘附反应;1～1 000 nmol·L^-1 可剂量依赖性抑制10 nmol·L^-1 PAF诱发的粒细胞趋化反应,其IC₅₀为4.84 nmol·L^-1; 0.01～10 μmol·L^-1可抑制1 μmol·L^-1 PAF诱发的粒细胞释放β-葡糖苷酸酶,其IC₅₀为3.56 μmol·L^-1。结论银杏内酯B能够抑制PAF刺激的大鼠中性粒细胞粘附、趋化及脱颗粒反应。     

牛膝多糖硫酸酯体外和体内抗艾滋病病毒作用

牛膝多糖硫酸酯(Achyranthes bidentata polysaccharide sulfate，ABPS)为牛膝多糖经硫酸酯化修饰后获得的产物，其结构清楚、理化性质稳定及分子质量小，具有抗单纯疱疹病毒、柯萨奇病毒和乙型肝炎病毒等作用。本文研究其在体外和体内的抗艾滋病病毒1型(human immunodeficiency virus type 1，HIV-1)活性。体外采用 ³H掺入法和酶联免疫吸附法测定ABPS对HIV-1逆转录酶和整合酶的活性，其半数抑制浓度(50% inhibiting concentrations,IC₅₀)分别为(2.948±0.556) μmol·L^-1和(0.155±0.030) μmol·L^-1， 但25 μmol·L^-1 ABPS对HIV-1蛋白酶无效。ABPS在MT-4细胞培养内对HIV-1实验病毒株IIIB和在人外周血单核细胞培养内对AZT耐药病毒株的急性感染有较强的抑制作用， 但在HIV-1 IIIB慢性感染的H9细胞培养中则无效。血清药理学研究表明，单次腹腔注射大鼠ABPS 125 mg·kg^-1或连续腹腔注射小鼠ABPS 3 mg·kg^-1(qd×20)后鼠血清在MT-4细胞培养内对HIV-1 P24抗原都有抑制作用，但口服ABPS生物利用度低，灌胃大鼠ABPS 2 g·kg^-1或灌胃小鼠ABPS 300 mg·kg^-1无效。实验结果提示ABPS有抗HIV-1作用，有可能用于防治HIV-1感染，值得进一步研究。     

III类抗心律失常药RP58866对豚鼠及犬内向整流及瞬时外向钾电流的作用

目的:研究III类抗心律失常药RP58866 对I_K1 ,瞬时外向钾电流(Ito) 的作用。方法:用豚鼠和犬离体心肌细胞及全细胞电压钳技术。结果:在- 100 m V 时,RP58866 以浓度依赖方式明显减少了豚鼠心室肌细胞I_K1,其IC₅₀为(3-4±0-8) μmol·L^-1。在犬心室肌细胞,RP58866 可明显抑制Ito( 在100 μmol·L^-1 时减少87% ±2-1% ),其IC₅₀为(2-3±0-5) μmol·L^-1 。结论:RP58866 对心肌细胞的IK1 和Ito 均有抑制作用,而不是一种特殊的IK1抑制剂。     

应用假病毒技术研究HIV-1复制抑制剂

建立以HIV-1复制为靶点的细胞水平假病毒药理筛选模型，并运用该模型筛选化合物库中随机选取的化合物。细胞水平的HIV-1假病毒药理筛选模型是应用水泡性口膜炎病毒的外壳糖蛋白(vesicular stomatitis virus glycoprotein，VSV-G)包装HIV-1核心建立的，简称VSVG/HIV模型。细胞被VSVG/HIV感染后，病毒携带的报告基因的表达水平(荧光素酶活性或GFP阳性细胞比例)反映了HIV-1的复制水平。对HIV-1复制有抑制作用的化合物应用VSVG/MLV模型对其特异性地进一步检测。被感染细胞中报告基因的表达水平与病毒稀释度呈显著的剂量依赖效应。阳性药物齐多夫定(zidovudine，AZT)、拉米夫定(lamivudine，3TC)、去羟基苷(didanosine，DDI)可剂量依赖性地抑制HIV-1的复制，其IC₅₀分别为48.5 nmol·L^-1、 0.13 μmol·L^-1和1.73 μmol·L^-1，与文献报道一致。在随机筛选的500个化合物中有3个化合物可以抑制HIV-1的复制，IC₅₀分别为1.92、 5.38和3.39 μmol·L^-1，而对MLV的复制无影响。VSVG/HIV假病毒系统是一种安全、有效的针对HIV-1复制的药理筛选模型。当将此模型与VSVG/MLV模型联合使用时，可判断化合物作用的特异性。实验表明：3个化合物，2-甲硫基-5-(4-甲基苯并)酰胺基-1,3,4-噻二唑(化合物A)、N-(3-羟基苯基)-2-(4-异丁基苯基)丙酰胺(化合物B)和N-(4-甲基吡啶基)-4-甲基苯磺酰胺(化合物C)可以特异性抑制HIV-1的复制。     

二苯乙烯类化合物对蛋白激酶C的抑制作用

报道15种二苯乙烯类化合物对蛋白激酶C(PKC)活性的影响。其中从中药金雀根中分得的3种二苯乙烯类低聚体α-viniferin,kobophenol A 和 miyabenolC,它们抑制PKC的IC₅₀分别为62.5,52.0和27.5μmol·L^-1。另外6种含酚羟基的二苯乙烯化合物对PKC也显示不同程度的抑制作用,但当酚羟基全甲基化或全乙酰化后其抑制作用大大降低甚至消失。酶动力学研究证明miyabenolC对PKC的抑制作用属于非竞争性抑制。     

狼毒大戟中松香烷型二萜内酯的抗肿瘤活性研究

摘 要 目的： 对狼毒大戟中松香烷型二萜内酯类化学成分进行分离和结构鉴定,将得到的单体化合物与6种不同的人肿瘤细胞株相互作用探讨其抗肿瘤活性。方法: 通过硅胶、ODS等柱色谱与1D、2D-NMR波谱学测试技术结合,从狼毒大戟中分离并确定4个松香烷型二萜内酯类化合物：jolkinolide A (1)、jolkinolide B (2)、17 hydroxyjolkinolide A (3)、17-hydroxyjolkinolide B (4)。采用噻唑蓝比色法（MTT法）分别测定其与6种人肿瘤细胞株：肝癌HepG-2、乳腺癌MCF-7、胃癌SGC 7901、胃癌BGC-823、胃癌MGC-803、宫颈癌Hela相互作用结果,并计算抑制率与半抑制率(IC₅₀)。结果: 化合物2对肝癌HepG-2、乳腺癌MCF-7、宫颈癌Hela细胞的增殖有显著得抑制作用IC₅₀均＜25 μmol·L^-1,化合物4对乳腺癌MCF 7细胞的增殖有显著抑制作用IC₅₀＜30 μmol·L^-1。结论: 狼毒大戟中松香烷型二萜内酯类化合物具有显著的抗肿瘤活性。     

红花黄酮成分抑制血小板激活因子介导的血小板活化作用

目的　观察红花黄酮成分杨梅素(Myr)和山奈酚(Kae)对血小板激活因子(PAF)诱导的兔洗涤血小板聚集、5 HT释放及血小板内游离钙离子浓度升高的影响。方法　以比浊法测定家兔洗涤血小板(WRP)聚集,邻苯二甲醛(OPT)荧光法测定5-HT浓度,Fura-2荧光探针测定血小板内游离钙离子浓度。结果　Myr和Kae体外呈浓度依赖性地抑制PAF诱发的WRP聚集及5-HT释放。Myr抑制WRP聚集的IC₅₀ 为17.5 μmol·L^-1 ;抑制5-HT释放的IC₅₀ 为64.1μmol·L^-1 。Kae抑制聚集、释放作用的IC₅₀ 分别为73.7μmol·L^-1 和128μmol·L^-1 ;同时Myr和Kae均能明显抑制PAF引起的血小板内游离钙增高。结论　Myr和Kae可抑制PAF诱导的血小板活化作用     

1-(3-酞酰亚胺基-2-氧丁基)-4-取代苯基哌嗪的合成及抗HIV-1逆转录酶活性

目的合成新型的非核苷类(双杂环苯基)化合物,并观察其抗HIV1-逆转录酶(HIV1-RT)活性。方法以氮芥盐酸盐为起始原料,与不同取代苯胺反应,得到相应的不同取代的哌嗪盐酸盐,并与1-溴-3-酞酰亚胺基-2-丁酮(4)缩合,得到目标化合物。结果合成11个目标化合物(5～15)。经¹HNMR,红外和元素分析确定结构。结论经HIV逆转录酶P-66蛋白测定,化合物11,14,10和13有一定抑制HIV1-RT活性,其IC₅₀分别为29.80,35.20,43.77和63.76 μmol·L^-1。     

Viral entry as the primary target for the anti-HIV activity of chicoric acid and its tetra-acetyl esters

The antiviral activity of L-chicoric acid against HIV-1 has been attributed previously to the inhibition of HIV-1 integration. This conclusion was based on the inhibition of integrase activity in enzymatic assays and the isolation of a resistant HIV strain with a mutation (G140S) in the integrase gene. Here we show that the primary antiviral target of L-CA and its analogs in cell culture is viral entry. L- and D-chicoric acid (L-CA and D-CA) and their respective tetra-acetyl esters inhibit the replication of HIV-1 (III(B) and NL4.3) and HIV-2 (ROD) in MT-4 cells at a 50% effective concentration (EC(50)) ranging from 1.7 to 70.6 microM. In a time-of-addition experiment, L-CA, D-CA, L-CATA, and D-CATA were found to interfere with an early event in the viral replication cycle. Moreover, L-CA, D-CA, and their analogs did not inhibit the replication of virus strains that were resistant toward polyanionic and polycationic compounds at subtoxic concentrations. Furthermore, HIV-1 strains resistant to L-CA and D-CA were selected in the presence of L-CA and D-CA, respectively. Mutations were found in the V2, V3, and V4 loop region of the envelope glycoprotein gp120 of the L-CA and D-CA-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain. Recombination of the gp120 gene of the L-CA and D-CA resistant strain in a NL4.3 wild-type molecular clone fully rescued the phenotypic resistance toward L-CA and D-CA. No significant mutations were detected in the integrase gene of the drug-resistant virus strains. Although inhibition of HIV integrase activity by L-CA and its derivatives was confirmed in an oligonucleotide-driven assay, integrase carrying the G140S mutation was inhibited to the same extent as the wild-type integrase.     

HIV integrase inhibitors: from diketoacids to heterocyclic templates: a history of HIV integrase medicinal chemistry at Merck West Point and Merck Rome (IRBM)

Replication of the human immunodeficiency virus (HIV) is dependent upon the enzyme HIV integrase (IN), one of three essential enzymes encoded in the viral genome. HIV-1 IN catalyzes the insertion of the proviral DNA into the host genome (strand transfer). HIV-1 IN therefore presents an attractive chemotherapeutic target for the treatment of HIV infection and AIDS that could provide patients and physicians with an additional option for treatment. Assays were developed to identify inhibitors of IN strand transfer. Diketoacid lead compounds were explored and developed into a variety of heterocyclic templates that are potent inhibitors of integrase strand transfer with suitable medicinal chemical properties for treating HIV infection and AIDS. The 1,6-naphthyridine L-870810 (Antiviral activity in cells IC(95) NHS = 102 nM, n=237), was shown to be efficacious in reducing viral RNA by 1.7 log units after doses of 400mg BID to HIV infected patients. Optimization of physical properties led to L-900564, an inhibitor of HIV IN that has excellent cell potency in the presence of protein (Antiviral activity in cells IC(95) NHS = 16 nM, n=15), excellent activity against mutants raised to HIV integrase inhibitors, and a very good pharmacokinetic profile.     

Discovery of a Potent HIV Integrase Inhibitor that Leads to a Prodrug with Significant anti-HIV Activity

Worldwide research efforts in drug discovery involving HIV integrase have produced only one compound, raltegravir, that has been approved for clinical use in HIV/AIDS. As resistance, toxicity and drug-drug interactions are recurring issues with all classes of anti-HIV drugs, the discovery of novel integrase inhibitors remains a significant scientific challenge. We have designed a lead HIV-1 strand transfer (ST) inhibitor (IC(50) 70 nM), strategically assembled on a pyridinone scaffold. A focused structure-activity investigation of this parent compound led to a significantly more potent ST inhibitor, 2 (IC(50) 6 ± 3 nM). Compound 2 exhibits good stability in pooled human liver microsomes. It also displays a notably favorable profile with respect to key human cytochrome P450 (CYP) isozymes and human UDP glucuronosyl transferases (UGTs). The prodrug of inhibitor 2, i.e., compound 10, was found to possess remarkable anti-HIV-1 activity in cell culture (EC(50) 9 ± 4 nM, CC(50) 135 ± 7 μM, therapeutic index = 15,000).     

具有抗HIV-1活性的新核糖体失活蛋白-栝楼蛋白(英文)

目的:研究新核糖体失活蛋白-栝楼蛋白是否具有抗HIV-1活性。方法:合胞体抑制实验以光镜检查,以ELISA测定HIV-1 p24抗原表达水平,用间接免疫荧光技术测定HIV抗原阳性细胞的百分率。结果:栝楼蛋白对HIV-1诱导C8166细胞形成合胞体有显著的抑制作用。栝楼蛋白显著地抑制了HIV-1急性感染T细胞中p24核心抗原的表达水平和减少了HIV抗原阳性细胞的百分率。栝楼蛋白抑制合胞体形成和HIV抗原阳性细胞的IC_(50)分别是5μg·L~(-1)和0.09mg·L~(-1)。栝楼蛋白不影响慢性感染T细胞中HIV-1 p24抗原表达水平。结论:栝楼蛋白是具有抗HIV-1活性的新的核糖体失活蛋白。     

Preferential inhibition of the magnesium-dependent strand transfer reaction of HIV-1 integrase by alpha-hydroxytropolones

Integration is a crucial step in the life cycle of human immunodeficiency virus type 1 (HIV-1); therefore, inhibitors of HIV-1 integrase are candidates for antiretroviral therapy. Two 7-hydroxytropolone derivatives (alpha-hydroxytropolones) were found to inhibit HIV-1 integrase. A structure-activity relationship investigation with several tropolone derivatives from The National Cancer Institute compound repository demonstrated that the 7-hydroxy group is essential for integrase inhibition. alpha-Hydroxytropolones preferentially inhibit strand transfer and are inhibitory both in the presence of magnesium or manganese. Lack of inhibition of disintegration in the presence of magnesium coupled with results from different cross-linking assays suggests alpha-hydroxytropolones as interfacial inhibitors. We propose that alpha-hydroxytropolones chelate the divalent metal (Mg2+ or Mn2+) in the enzyme active site. The most active compound against HIV-1 integrase in biochemical assays [2,4,6-cycloheptatrien-1-one, 2,7-dihydroxy-4-isopropyl (NSC 18806) IC50 = 4.8 +/- 2.5 microM] exhibits weak cytoprotective activity against HIV-1(IIIB) in a cell-based assay. alpha-Hydroxytropolones represent a new family of inhibitors for the development of novel drugs against HIV infection.     

苯并异硒唑酮磺酰胺衍生物对环氧酶的抑制作用

目的　研究苯并异硒唑酮磺酰胺衍生物对环氧酶的抑制作用。方法　放免法;RT-PCR法。结果　化合物A和B对COX-1和COX-2的代谢产物TXB2和PGE2的IC₅₀ 比值分别为1000和560 ,化合物A和B可抑制LPS诱导的大鼠腹腔巨噬细胞COX-2 mRNA生成,而对COX-1的mRNA生成无影响。结论　化合物A和B为COX-2选择性抑制剂,可抑制COX-2 mRNA的生成