镉对大鼠四种器官乳酸脱氢酶同工酶的影响

目的：观察镉对大鼠器官乳酸脱氢酶（LDH）同工酶的影响;方法：采用聚丙稀酰胺凝胶垂直板电泳法结合光密度扫描法,分析在四种镉中毒浓度（0．2mg／kg、0.4mg／kg、0．8mg／kg和1.6mg／kg）下的SD大鼠心、肝、肾和脑中LDH同工酶的变化。结果：随着镉中毒浓度的升高,心脏LDH各同工酶的活性显著升高,而肾脏LDH各同工酶的活性显著下降,脑中的LDH1和LDH2的活性出现先升高后下降现象。镉对肝脏中LDH各同工酶的影响不显著。结论：镉可能对不同器官中LDH同工酶的H、M亚基的基因表达影响不一。     

镉对大鼠四种器官乳酸脱氢酶同工酶的影响

目的：观察镉对大鼠器官乳酸脱氢酶（ＬＤＨ）同工酶的影响；方法：采用聚丙稀酰胺凝胶垂直板电泳法结合光密度扫描法，分析在四咱镉中毒浓度（０．２ｍｇ／ｋｇ、０．４ｍｇ／ｋｇ、０．８ｍｇ／ｋｇ和１．６ｍｇ／ｋｇ）下的ＳＤ大鼠心、肝、肾和脑中ＬＤＨ同工酶的变化。结果：随着镉中毒浓度的升高，心脏ＬＳＨ各同工酶的活性显著升高，而肾脏ＬＤＨ各同工酶的活性显著下降，脑中的ＬＤＨ１和ＬＤＨ２的活性出现选知高后下降现象     

盐酸小檗碱对亚硝酸盐中毒小鼠脑急性缺氧的保护作用

目的观察盐酸小檗碱对小鼠脑急性缺氧的保护作用。方法利用亚硝酸钠（NaNO2）中毒和断头实验复制缺氧模型，用盐酸小檗碱3个剂量组即2．0mg／kg、4．0mg／kg、8．0mg／kg连续灌胃6d，观察小鼠存活时间、断头张口次数和喘息时间；用试剂盒测量脑组织超氧化物岐化酶（SOD）、丙二醛（MDA）、一氧化氮（NO）和乳酸脱氢酶（LDH）含量。HE染色观察脑组织形态学变化。结果盐酸小檗碱能明显延长急性缺氧条件下小鼠的存活时间（P〈0．05，P〈0．01）、断头喘息时间（P〈0．05，P〈0．01），提高缺氧小鼠脑组织SOD和LDH的活性，减少MDA和NO的含量；镜下可见模型组小鼠脑膜出血，皮质细胞坏死，脑水肿；盐酸小檗碱治疗组小鼠除脑膜血管扩张外未见明显异常。结论盐酸小檗碱对亚硝酸盐中毒小鼠脑急性缺氧具有一定的保护作用。     

儿童血清LDH及同工酶分析

目的分析和研究儿童乳酸脱氢酶（LDH）的临床检测意义，并设置出景德镇市妇幼保健院的正常参考范围。方法445名健康儿童（儿童组）和体检健康成人592名（成人组），空腹取血后立即用连续监测法在BaekmanCX5型生化分析仪测定LDH活性；LDH同工酶用醋酸纤维薄膜电泳分离，氯化硝基四氮唑蓝染色，贝克曼光密度计扫描出百分率。结果儿童参考范围：LDH1 96．1～254．5U／L，LDH，26．2％～32．8％，LDH2 34．0％～37．4％，LDH3 17．9％～22．3％，LDH4 6．8％～11．0％，LDH5 4．7％～7．1％；成人：LDH 71．0～204．0U／L，LDH1 20．3％～32．1％，LDH2 28．2％～38．8％，LDH3 13．7％～21．7％，LDH4 8．7％～16．7％，LDH5 7．4％～16．0％。儿童与成人的LDH活力有显著性差异（P〈0.01）。儿童与成人的LDH同工酶的分布顺序一致，即：LDH2〉LDH1〉LDH3〉LDH4〉LDH5，但儿童LDH1和LDH2〉成人；LDH3基本一致；LDH4和LDH5〈成人。结论儿童LDH同工酶盲目参照成人的标准缺乏科学性，各地应建立本地区幼儿、儿童的LDH同工酶的参考值，为临床诊断、治疗提供可靠的指标。     

过氯酸钠抑制法测定LDH1活性

乳酸脱氢酶同工酶1（LDH1）的测定对心肌梗塞等疾病的诊断及预后具有重要价值。目前国内多使用电泳分离后计算相对活性、操作繁琐、费时且需特殊仪器，直接测定LDH1活性的试剂盒均为国外进口，价格昂贵。我们参考文献[1，2]，用过氨酸钠抑制LDH2－5，用LDH试剂盒在生化分析仪上测定LDH1活性。该法简单、快速、特异性好。原理：LDH有五种同工酶，LDH2－5都含有M亚基，当过氨酸钠达到一定浓度时，所含M亚基的同工酶均被抑制，剩余活力即为LDH1活性。反应式为：1材料和方法1．1仪器BT－224半自动生化分析仪（意大利产）。1．2试剂…     

AOPP病人血清ChE、CK—MB和LDH活性的变化

目的 探讨急性有机磷农药中毒（AOPP）病人血清胆碱酯酶（ChE）、肌酸激酶同工酶（CK—MB）、乳酸脱氢酶（LDH）活性变化及其意义。方法 检测38例重度AOPP病人人院第1、3、7天血清ChE、CK—MB和LDH活性,以10例健康成人作为对照。结果 与对照组比较,AOPP组病人血清ChE降低,CK—MB、LDH升高,差异有显著性（F=142．515～513．542,q=4．505～42．764,P〈0．05、0．01）;与第1天比较,人院第3天、第7天AOPP组病人血清ChE升高,CK—MB、LDH降低,差异有显著性（q=17．665～45．655,P〈0．05、0．01）。随治疗进展,血清ChE、CK—MB和LDH活性呈逐渐恢复趋势。结论 血清ChE水平不能作为临床评价有机磷农药中毒程度的有效指标,而CK—MB和LDH可作为有机磷农药中毒程度、病情和预后的判断指标。     

河豚毒素单用及与indoxacarb联合应用的镇痛抗炎作用

目的：观察河豚毒素单用及河豚毒素与indoxacarb联合应用的镇痛抗炎作用。方法：实验于2004-09／2005-01在北京大学医学部神经精神药理实验室完成。选择ICR小鼠240只，按随机数字表法分为24组，每组10只。①醋酸扭体实验：110只小鼠分为阴性对照组、阳性对照组（吗啡，5mg／kg）、单用河豚毒素组（0．79，0．39，0．19μg／kg）、联合给药组[河豚毒素（0．19μg／kg）＋indoxacarb（1．4，2．8，28mg／kg），河豚毒素（0．19，0．39，0．79μg／kg）＋indoxacarb（5．6mg／kg）]。给药40min后，腹腔注射致痛剂6g／L冰醋酸溶液（0．01mL／g）。观察并记录15min内小鼠扭体反应次数。扭体抑制率（％）：（阴性对照组平均扭体次数-给药组平均扭体次数）／阴性对照组平均扭体次数&;#215;100％。②甲醛实验：50只小鼠分为阴性对照组、阳性对照组（吗啡，5mg／kg）、单用河豚毒素组（0．19μg／kg）、联合给药组[河豚毒素（0．19μg／kg）＋indoxacarb（1．4，2．8mg／kg）]。给药40min后，小鼠右后足背皮下注射致痛剂9．2g／L甲醛（生理盐水配制）20μL／只。将小鼠舔、咬右爪的时间长度作为疼痛反应的指标。观察并记录0～5min，15～30min反应时间。抑制率（％）=（阴性对照组平均反应时间-给药组平均反应时间）／阴性对照组平均反应时间&;#215;100％。注射甲醛4h后，将小鼠断头处死，之后自膝关节切脚，称重，比较左右足差异，以此代表炎症程度。③坐骨神经慢性轻度结扎损伤实验：80只小鼠分为阴性对照组、阳性对照组（吗啡，5mg／kg）、单用河豚毒素组（0．19，0．39，0．79μg／kg）、联合给药组[河豚毒素（0．19μg／kg）＋indoxacarb（1．4，2．8．5．6mg／kg）]。75mg／kg戊巴比妥钠腹腔注射麻醉小鼠。用细铜丝在坐骨神经上结扎2次。在术前及术后第1，3，5，7，9，11，13天测定热板潜伏期。第15天给药后15，30，45，60min测定小鼠热板潜伏期。结果：纳入动物240只，均进入结果分析。①单独使用河豚毒素（0．19，0．39，0．79μg／kg），扭体抑制率分别为22．2％，3113％，39．2％，其抑制率与浓度存在剂量依赖关系。将河豚毒素（0．19μg／ks）与不同浓度的indoxacarb联合给药发现，扭体抑制率均显示升高趋势，达到38．6％，46．1％，50．6％，65．3％。②在早期（0～5min），单独使用河豚毒素（0．19μg／kg）能够减少小鼠舔脚时间，河豚毒素与indoxcarb（1．4，2．8mg／kg）两者联合给药显著减少小鼠舔脚时间，抑制率达到49.3％，50．2％，与吗啡的镇痛强度相差不多（抑制率为57．5％）。在晚期（15～30min），单独使用河豚毒素（0．19μg／kg）能够显著减少小鼠舔脚时间，将河豚毒素与indoxacarb（1．4，2．8mg／kg）两者联合给药，其作用极其显著，抑制率高达97．2％，92．4％，远远超过吗啡的镇痛强度（抑制率为79．7％）。③单独使用河豚毒素（0．19μg／kg）能够显著增强抗炎活性，抑制率为66．3％；将河豚毒素（0．19μg／kg）＋indoxacarb（1．4，2．8mg／kg）两者联合用药，其作用极其显著，抑制率高达75_2％，124．2％，远远超过吗啡的抗炎活性（抑制率仅为28．7％），而吗啡与阴性对照组相比差异无显著性意义。④单独使用河豚毒素（0．19μg／kg）能够显著增强热板潜伏期的变化率（P〈0．05），能显著缓解结扎坐骨神经引起的小鼠痛觉过敏现象。将河豚毒素（0．19μg／kg）＋indoxacarb（1．4，2．8，5．6mg／kg）两者联合用药，未看到显著的热板潜伏期的变化。结论：河豚毒素具有一定镇痛作用，能够抑制甲醛引起炎症性疼痛和水肿；河豚毒素联合小剂量indoxacarb具有协同镇痛抗炎作用。     

铅中毒大鼠抗氧化酶活性的改变及核酸的干预效应

目的：观察染铅大鼠抗氧化系统的改变及核酸对其的影响。 方法：实验于2003-03／05在哈尔滨医科大学公共卫生学院完成。选择Wistar大鼠32只，随机分为4组，即空白组、模型组、200mg／kg和700mg／kg核酸组，每组8只。空白组大鼠正常饮水，其余3组饮（8g／L）醋酸铅水溶液。另外空白组、模型组大鼠灌蒸馏水，200mg／kg和700mg／kg核酸组大鼠分别灌胃核酸200，700mg／kg，5周后麻醉处死动物，测定大鼠血清中超氧化物歧化酶、谷胱甘肽过氧化物酶、过氧化氢酶活性、丙二醛、谷胱甘肽含量及总抗氧化能力水平。 结果：纳入32只大鼠全部进入结果分析，无脱失。①各组大鼠血清超氧化物歧化酶、谷胱苷肽过氧化物酶活性及丙二醛含量比较：模型组血清超氧化物歧化酶、谷胱苷肽过氧化物酶活性显著低于空白组[（5374．57&;#177;819．00，3516．37&;#177;597．62；7088．75&;#177;668．63，4677．10&;#177;856．84）μkag/L（q=4．02，6．03，P〈0．05-0．01）]，而丙二醛含量则显著高于空白组[（8．63&;#177;1.47，6．64&;#177;1．06）μmol／L（q=4．52，P〈0．05）]。200，700mg／kg核酸组血清超氧化物歧化酶、谷胱苷肽过氧化物酶活性显著高于模型组，而丙二醛含量显著低于模型组（q=4．01～4．91，P〈0．01）。②各组大鼠血清中谷胱甘肽含量、过氧化氢酶活性、总抗氧化能力水平比较：模型组血清谷胱甘肽含量、总抗氧化能力、过氧化氢酶活性均显著低于空白组（q=3．64～7．53，P〈0．05-0．01）。200，700mg／kg核酸组血清总抗氧化能力、过氧化氢酶活性显著高于模型组（q=3．49-5．74，P〈0．05～0．01）。 结论：铅染毒能够造成实验大鼠机体氧化损伤，核酸能够提高机体抗氧化酶的活性，减轻铅中毒引起的过氧化损伤。     

黄芩茎叶总黄酮对U937细胞增殖的影响

目的研究黄芩茎叶总黄酮（TF）对人类单核肿瘤细胞U937增殖的影响。方法U937细胞以5×10^4个／ml的浓度，培养于含胎牛血清（FCS）10％的RPMI 1640全培养液中，TF浓度分别为0、25、50、100、150、2130mg／L组，每组8孔接种于24孔培养板中孵育7d。每天计数各孔细胞密度，第7天测定细胞悬液中乳酸脱氢酶（LDH）活性。结果TF 25～200mg／L组U937细胞密度均低于TF 0mg／L组，浓度越高细胞密度越低（P〈0．05）。TF 0～150mg／L组细胞悬液LDL活性差别无统计学意义（P〉0．05），TF 200mg／L组LDL与其他5组相比均具有统计学意义（均P〈0．05）。结论TF具有抑制U937细胞增殖的作用，高浓度的TF（〉1200mg／L）对体外培养的U937细胞具有毒性作用。     

不同剂量氯胺酮对体外循环心脏手术患者CRP、IL-6及IL-8的影响

【目的】探讨不同小剂量氯胺酮对体外循环（Cardiopumonary bypass，CPB）瓣膜替换手术患者超敏C反应蛋白（hypersensitive C-reactive protein，hs—CRP）、白细胞介素-6（IL-6）和白细胞介素-8（IL-8）的影响。【方法】30例择期瓣膜替换术病人，随机分成氯胺酮Ⅰ组（n=10）、氯胺酮Ⅱ组（n=10）和对照组（n=10）。氯胺酮Ⅰ、Ⅱ组于麻醉诱导开始时分别按0．25mg／kg和0．5mg／kg静注氯胺酮一次，对照组则采用等量生理盐水注射。三组分别于麻醉诱导前（T1）、术后1h（T2）及术后24h（T3）三个时间点测定动脉血中hs—CRP、IL6和IL-8的水平。【结果】三组hs-CRP的水平在L均较T1有明显升高（P〈0．05）；IL-6和IL-8的水平在T2、T3较T1有明显升高（P〈0．05）。氯胺酮Ⅱ组hs-CRP的水平在T3较对照组明显降低（P〈0．05），且氯胺酮Ⅰ,Ⅱ组间hPCRP的水平差异有显著性（P〈0．05）；氯胺酮Ⅰ、Ⅱ组IL6、IL-8的水平在T2、T3较对照组均明显降低（P〈0．05），但氯胺酮两组间差异无显著性（P〉0．05）。【结论】小剂量氯胺酮能有效抑制CPB诱发的CRP、IL-6和IL-8水平升高，0．5mg／kg剂量氯胺酮比0．25mg／kg剂量似乎更有效。     

Hepatoprotective Activity of Licorice Water Extract against Cadmium-induced Toxicity in Rats

Licorice is commonly used as a cure for digestive disorders and as a detoxification agent in East Asia. This study investigated the protective effect of licorice water extract against cadmium (CdCl(2), Cd)-induced liver toxicity in rats. To induce acute toxicity, Cd (4 mg/kg body weight) was dissolved in normal saline and intravenously (i.v.) injected into rats. The rats then received either a vehicle or licorice water extract (50, 100 mg/kg/day) for 3 days, and were subsequently exposed to a single injection of Cd 24 h after the last licorice/vehicle treatment. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were significantly increased by Cd treatment. In contrast, pretreatment with licorice reduced ALT, AST and LDH. In histopathological analysis, licorice decreased the central necrosis around central veins, the peripheral hemorrhage around portal triads, the percentage of degenerative hepatic regions (%/mm(2) hepatic parenchyma) and the number of degenerative hepatic cells (N/100 hepatic cells). Licorice also inhibited the increment of Bad (a BH3 domain-containing protein) translocation by Cd in liver cells. These results demonstrate that licorice could have a hepatoprotective effect by inhibiting the translocation of Bad to the mitochondria in Cd-intoxificated rats.     

朱砂、雄黄对感染性脑损伤大鼠乳酸脱氢酶及其同工酶的影响

背景要提高含重金属和砷化物矿物药(如朱砂、雄黄)中药制剂的用药安全和排除该类药物的出口障碍,有必要对朱砂、雄黄进行有效性和安全性评价.目前,对复方中朱砂、雄黄的药效作用机制尚不清楚.目的研究生理、病理状态下安宫牛黄丸中的朱砂、雄黄对机体作用的差异,探讨其药理作用机制.设计随机对照研究.单位一所大学的临床药理研究所.材料实验于2003-01在广州中医药大学临床药理研究所完成.选用第一军医大学实验动物中心提供的体质量为250～300 g SD雄性大鼠51只.方法SD大鼠随机分成6组(8～10只/组)正常组,正常+安宫牛黄散(下简称整方)组(278 mg/kg);正常+除去朱砂、雄黄的安宫牛黄散(下简称拆方)组(222.7 mg/kg);脑水肿模型组(一侧大鼠颈总动脉注射百日咳杆菌250亿/kg);模型+整方组(造模前1 h给药278 mg/kg);模型+拆方组(造模前1 h给药222.7 mg/kg).一次给药后5 h(模型组注菌后4 h)采血、制备脑匀浆.主要观察指标脑组织、血清中乳酸脱氢酶(LDH)总活力,血清中乳酸脱氢酶同工酶LDH1-5百分酶活力.结果与正常组比较,正常+整方组、正常+拆方组LDH总活力显著升高32.4%～38.4%(P＜0.05),LDH1 2百分酶活力均显著升高(P＜0.01),LDH4,5百分酶活力下降(P＜0.01),但除LDH5外,两组同工酶酶活间无显著差异.与模型组比较,模型+整方组、模型+拆方组LDH总活力显著下降23.4%～38.5%(P＜0.01),LDH5百分酶活力显著升高(P＜0.01),但两组间无显著差异;模型+整方组LDH2,3百分酶活力显著下降(P＜0.01),LDH1,4百分酶活力无显著变化;模型+拆方组LDH1,4百分酶活力显著下降(P＜0.05,0.01),而LDH2,3百分酶活力变化不显著.结论正常生理状态下服用安宫牛黄散,对心肌、肾等有一定的损伤作用.感染性脑水肿病理状态下,整方和拆方均可抑制被过度激活的LDH酶,两者之间无显著差异;复方中的朱砂、雄黄对LDH同工酶水平有不同程度的影响.     

E-4695, a new C-7 azetidinyl fluoronaphthyridine with enhanced activity against gram-positive and anaerobic pathogens.

E-4695, (-)-7-[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-1-cyclopropyl-1,4- dihydro-6-fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid, is a new fluorinated naphthyridine with an azetidine moiety. The MICs of E-4695 at which 90% of the isolates were inhibited (MIC90s) were 0.06 to 0.5 microgram/ml for gram-positive cocci, including species of the genera Staphylococcus, Streptococcus, and Enterococcus, and the MIC90s against gram-negative pathogens such as members of the family Enterobacteriaceae (with the exception of Providencia spp. [MIC90, 8 micrograms/ml]) and Pseudomonas aeruginosa were 0.015 to 0.5 microgram/ml. E-4695 inhibited 90% of the Clostridium perfringens and Bacteroides fragilis isolates at 0.25 and 4 micrograms/ml, respectively. Against gram-positive cocci the potency of E-4695 was 2- to 8-fold higher than that of ciprofloxacin, 4- to 8-fold higher than that of ofloxacin, and 8- to 16-fold higher than that of fleroxacin. Against enteric bacteria and P. aeruginosa the potency of E-4695 was, in general, similar to that of ciprofloxacin and eightfold higher than those of ofloxacin and fleroxacin. E-4695 was four- and eightfold more potent than ciprofloxacin against C. perfringens and B. fragilis isolates, respectively. E-4695 and ciprofloxacin showed similar properties when the effects of pH or magnesium concentration were tested on them. E-4695 and ciprofloxacin had substantial reductions of activity only when pH decreased below 4.8. E-4695 and ciprofloxacin activities were not markedly affected by the presence of 5 or 10 mM Mg2+. The presence of serum and human urine at pH 7.2 decreased the activity of E-4695 between two- and fourfold. After an oral dose of 50 mg/kg of body weight, the maximum level in serum, the biological half-life, and the area under the concentration-time curve from 0 to 10 h for E-4695 were 13.2 microgram/ml, 3.3 h, and 45.6 microgram . h/ml, respectively. The area under the concentration-time curve from 0 to 4 h for ciprofloxacin was 2.3 microgram . h/ml at the same dose. Fifty-percent effective doses (ED50S) against Staphylococcus aureus HS-93 infections in mice were 4.5 mg/kg with E-4695 and 37.6 mg/kg with ciprofloxacin. Infection with Streptococcus pneumoniae 29206 was more effectively treated with E-4695 (ED50, 41,2 mg/kg) than with ciprofloxacin (ED50, 200 mg/kg). The ED50 of E-4695 for infections with Streptococcus pneumoniae 1625 was 132.2 mg/kg; ciprofloxacin was ineffective at 400 mg/kg against this strain. E-4695 was also more potent than ciprofloxacin in treatment of infections caused by gram-negative organisms such as Escherichia coli HM-42 (ED50S, 1.0 and 3.9 mg/kg, respectively). The ED50S of E-4695 and ciprofloxacin were 33.0 and 145.5 mg/kg against P. aeruginosa HS-116 and 9.6 and 18.9 mg/kg against P. aeruginosa B-120, respectively. The therapeutic efficacy of E-4695 may depend not only on its in vitro activity but also on its improved pharmacokinetic properties.     

Evaluation of daptomycin activity against Staphylococcus aureus in an in vitro pharmacodynamic model under normal and simulated impaired renal function

OBJECTIVES: Daptomycin is a lipopeptide antimicrobial that is primarily excreted by the kidney. We examined daptomycin bactericidal activity in an in vitro pharmacodynamic model (IVPM) under normal and simulated impaired renal function against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). METHODS: Two clinical strains MSSA-1199 and MRSA-494 were used in an IVPM. MICs and MBCs were determined according to CLSI. Daptomycin free concentrations were simulated that corresponded to dose regimens of 4, 6 and 8 mg/kg every 24 h at 8 h t(1/2) (Scheme I) and every 48 h at 30 h t(1/2) (Scheme II). In addition, we simulated daptomycin free concentrations corresponding to fractional dose regimens of 2, 3 and 4 mg/kg every 24 h at 30 h t(1/2) (Scheme III). The targeted C(max free)/MIC for 2, 3, 4, 6 and 8 mg/kg against MSSA-1199 ranged from 5.2 to 21.2. The targeted C(max free)/MIC for 2, 3, 4, 6 and 8 mg/kg against MRSA-494 ranged from 10.4 to 42.2. The targeted AUC(free)/MIC for Schemes I, II and III against MSSA-1199 ranged from 94 to 392. The targeted AUC(free)/MIC for Schemes I, II and III against MRSA-494 ranged from 188 to 581. Bactericidal activity and the potential for resistance were determined over 96 h. All models were completed in triplicate. RESULTS: Daptomycin MICs (MBCs) for MSSA-1199 and MRSA-494 were 0.5 (1.0) mg/L and 0.25 (0.25) mg/L, respectively. Daptomycin 6 and 8 mg/kg at both 8 and 30 h t(1/2) achieved 99.9% kill as early as 1 h. Daptomycin 4 mg/kg achieved 99.9% kill as early as 1 h when given at 8 and 30 h t(1/2) but was not maintained to an endpoint of 96 h (P > 0.05). CONCLUSIONS: Overall, there was no difference in kill noted for daptomycin regimens at 4, 6 and 8 mg/kg every 24 h at 8 h t(1/2) versus every 48 h at 30 h t(1/2). Fractional doses of daptomycin at 30 h t(1/2) were inferior to daptomycin regimens of 4, 6 and 8 mg/kg administered every 48 h (P = 0.03).     

7-But-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one is a novel competitive and selective inhibitor of dipeptidyl peptidase IV with an antihyperglycemic activity

7-But-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one (ER-319711) is a novel dipeptidyl peptidase (DPP)-IV inhibitor discovered in our laboratories. In this study, we have characterized this DPP-IV inhibitor in vitro and in vivo as an antidiabetic agent. The trifluoroacetate salt form of ER-319711, ER-319711-15, inhibited human DPP-IV with an IC(50) value of 0.089 microM, whereas its IC(50) values toward human DPP8 and DPP9 were >100 microM. Inhibition kinetic pattern analysis indicated that ER-319711-15 inhibited DPP-IV in a competitive manner. ER-319711-15 (1 mg/kg) reduced glucose excursion in an oral glucose tolerance test (OGTT) using Zucker fa/fa rats, with significant increases in plasma insulin and active glucagon-like peptide-1 levels. In an OGTT using mice fed a high-fat diet in which ER-319711-15 (0.1-10 mg/kg) was orally administered at 0 h, and glucose was loaded at 0 and 5 h, this compound improved glucose tolerance dose dependently at both 0- and 5-h glucose loading. Next, we compared efficacy of ER-319711-15, E3024, a competitive DPP-IV inhibitor having an imidazopyridazinone structure, or vildagliptin, a slow-binding and long-acting DPP-IV inhibitor, at the same dose, 10 mg/kg, in the same procedures. At the first glucose challenge, all compounds lowered area under the curve (AUC) values of delta blood glucose between 0 and 2 h significantly to the same degree. At the second glucose load, the AUC values between 5 and 7 h were significantly decreased by ER-319711-15 and vildagliptin, but not by E3024. Therefore, ER-319711 might be a potent, competitive, and selective DPP-IV inhibitor with an antihyperglycemic activity.     

Effect of melatonin on cadmium-induced hepatotoxicity in male Sprague-Dawley rats.

Effect of melatonin on toxicity of cadmium (Cd) was studied in male SD rats co-administered daily Cd (1 mg/kg b.w., s.c.) with melatonin (10 mg/kg b.w., i.p.) for 15 days. Cd alone injection decreased GSH concentrations in the liver and RBC by 35% and 43% compared with those in saline-treatment group, but not in the kidney and whole brain. The activity of GSSG-reductase was significantly decreased in the liver of Cd alone injected rats, while melatonin given in combination with Cd failed to prevent the Cd-induced decreased activity of hepatic GSSG-reductase. However, the hepatic GSH concentration decreased by Cd alone was restored by melatonin treatment, and the melatonin also ameliorated Cd-induced histopathological changes in the liver. Therefore, data indicate that melatonin restores the reduction of hepatic GSH level induced with Cd regardless of GSSG-reductase activity, and suggests that melatonin may ameliorate Cd-induced hepatotoxicity.     

NSAID-induced cyclooxygenase inhibition differentially depresses long-lasting versus brief synaptically-elicited responses of rat spinal dorsal horn neurons in vivo.

This electrophysiological study examined the effects of NSAID administration on synaptically-elicited responses of rat single spinal dorsal horn neurons to natural stimulation of peripheral receptive fields. Nociceptive responses consisted of a fast initial discharge during the stimulus followed by a slowly-decaying afterdischarge. The cyclooxygenase inhibitor, indomethacin (2.0-8.0 mg/kg, i.v.), was without effect on the on-going rate of discharge but dose-dependently inhibited synaptically-elicited responses to noxious cutaneous mechanical stimulation (fast initial discharge: n = 3/3 with 2 mg/kg, 5/8 with 4 mg/kg, 5/6 with 8 mg/kg; slowly-decaying afterdischarge: n = 3/3 with 2 mg/kg, 6/8 with 4 mg/kg, 6/6 with 8 mg/kg) and thermal (fast initial discharge: n = 7/9 with 8 mg/kg; slowly-decaying afterdischarge: n = 3/4 with 4 mg/kg, n = 7/9 with 8 mg/kg). The inhibitory effect of indomethacin started within 2-4 min and lasted up to 120 min. To eliminate any effect of indomethacin via cutaneous sensory receptors it was tested on the responses of some neurons to high intensity electrical stimulation of the sciatic nerve; indomethacin depressed these evoked responses (fast initial discharge: n = 5/6 with 2 mg/kg, n = 7/7 with 4 mg/kg; slowly-decaying afterdischarge: n = 6/6 with 2 mg/kg, n = 7/7 with 4 mg/kg). The brief excitatory responses to innocuous pressure (fast initial discharge: n = 2/3 with 2 mg/kg, n = 6/8 with 4 mg/kg, n = 4/6 with 8 mg/kg) and hair (n = 2/7 with 2 and 4 mg/kg, respectively) stimulation in both non-nociceptive and wide dynamic range neurons were also depressed but to a lesser extent. However, the prolonged excitation of three wide dynamic range neurons to continuous hair stimulation was almost entirely inhibited by indomethacin. Overall, inhibition of the afterdischarge and the excitatory effect of long-lasting synaptic input were greater than inhibition of the fast synaptic input-evoked initial discharge. The evidence supports the suggestion that systemically-administered indomethacin has an effect in the spinal cord and demonstrates an action specifically in the dorsal horn. The data are interpreted to suggest that sensory inputs are more involved than input-independent excitation of dorsal horn neurons in leading to de novo synthesis of eicosanoids and that the time course of this synthesis brings the levels to a point where COX inhibition can have an observable effect during prolonged excitation. Although the data suggest that COX inhibition differentially inhibits nociceptive versus non-nociceptive mechanisms at the cellular level, irrespective of the modality of the stimulus, this is the first direct demonstration that prolonged activation of synaptic mechanisms are preferentially inhibited. According to this it would be predictable that NSAIDs would be more effective on nociceptive types of pain characterized by time or prolonged inputs of primary afferents.     

Glucuronidation of monohydroxylated warfarin metabolites by human liver microsomes and human recombinant UDP-glucuronosyltransferases

Our understanding of human phase II metabolic pathways which facilitate detoxification and excretion of warfarin (Coumadin) is limited. The goal of this study was to test the hypothesis that there are specific human hepatic and extrahepatic UDP-glucuronosyltransferase (UGT) isozymes, which are responsible for conjugating warfarin and hydroxylated metabolites of warfarin. Glucuronidation activity of human liver microsomes (HLMs) and eight human recombinant UGTs toward (R)- and (S)-warfarin, racemic warfarin, and major cytochrome P450 metabolites of warfarin (4'-, 6-, 7-, 8-, and 10-hydroxywarfarin) has been assessed. HLMs, UGT1A1, 1A8, 1A9, and 1A10 showed glucuronidation activity toward 4'-, 6-, 7-, and/or 8-hydroxywarfarin with K(m) values ranging from 59 to 480 microM and V(max) values ranging from 0.03 to 0.78 microM/min/mg protein. Tandem mass spectrometry studies and structure comparisons suggested glucuronidation was occurring at the C4'-, C6-, C7-, and C8-positions. Of the hepatic UGT isozymes tested, UGT1A9 exclusively metabolized 8-hydroxywarfarin, whereas UGT1A1 metabolized 6-, 7-, and 8-hydroxywarfarin. Studies with extrahepatic UGT isoforms showed that UGT1A8 metabolized 7- and 8-hydroxywarfarin and that UGT1A10 glucuronidated 4'-, 6-, 7-, and 8-hydroxywarfarin. UGT1A4, 1A6, 1A7, and 2B7 did not have activity with any substrate, and none of the UGT isozymes evaluated catalyzed reactions with (R)- and (S)-warfarin, racemic warfarin, or 10-hydroxywarfarin. This is the first study identifying and characterizing specific human UGT isozymes, which glucuronidate major cytochrome P450 metabolites of warfarin with similar metabolic rates known to be associated with warfarin metabolism. Continued characterization of these pathways may enhance our ability to reduce life-threatening and costly complications associated with warfarin therapy.     

Cadmium-Metallothionein nephropathy: relationships between ultrastructural/biochemical alterations and intracellular cadmium binding

Cadmium metallothionein (CdMT) nephrotoxicity was studied in rats injected i.p. with a single nonlethal dose of CdMT (0.6 mg of Cd per kg). Within 8 hr of CdMT injection, urine volume and urine sodium excretion were increased and sodium dodecyl sulfate gel electrophoresis of urine proteins showed that elevated levels of low molecular weight proteins were present in the urines of CdMT-treated rats. Urine RNAase activity was also elevated, approximately 7-fold, by CdMT but not by zinc metallothionein (ZnMT) or lysozyme at equivalent protein doses, demonstrating that a proteinuria indicative of proximal tubule cell dysfunction develops as an early response to CdMT exposure. Ultrastructural alterations were also present in animals injected with CdMT but not ZnMT or lysozyme. The earliest alterations occurred in the lysosome compartment of the cell. By 1 hr, the number of small lysosomes in renal proximal convoluted tubule cells increased significantly with no changes in other organelle compartments. By 4 and 8 hr, there was a further increase in lysosome number with a concomitant decrease in size and a marked increase in the number of small clear apical vacuoles. Lysosomal cathepsin D activity was decreased at 4 and 8 hr after CdMT injection, and in vitro studies indicated that this effect was not due to a direct inhibition of the enzyme by Cd++ or CdMT. Thus, both lysosome size and protease activity were rapidly altered by CdMT exposure. Studies of Cd binding in the kidney suggest that non-MT-bound Cd is an important factor in CdMT-associated toxicity. Approximately 97% of the Cd present in the cytoplasm at 1 hr was non-MT-bound. Prior induction of renal MT by treatment with zinc (20 mg of Zn per kg as ZnSO4, i.p. 16 hr before CdMT injection) markedly reduced non-MT binding of Cd++ in kidneys of treated animals and inhibited the alterations in urine volume and low molecular weight protein reabsorption induced by CdMT. These data suggest that acute CdMT exposure provides an excellent system for studying the mechanism of cadmium tubular proteinuria and that the intracellular renal MT pool plays a key role in regulating this process.