单点采血反映口服CYP3A探针咪达唑仑的代谢清除率

目的:研究中国男性健康受试者口服咪达唑仑后其1＇-羟化代谢的药代动力学规律,并寻找适合的单个采血点血浆中1＇-羟化咪达唑仑/咪达唑仑的浓度比值来反映咪达唑仑的血浆清除率。方法:10名受试者禁食8小时后清晨空腹口服7.5mg咪达唑仑,利用非房室模型计算药代动力学参数。结果:咪达唑仑药代动力学参数C_(max)为(191±17)nmol/L,t_(max)为(1.01±0.14)h,t_(1/2)为(3.2±0.4)h,AUC_(0→∞)为(681±43)nmol·h·L~(-1),Cl_(oral)为(0.54±0.04)L/(h·kg),K_e为(0.2415±0.0021)h~(-1),K_α为(0.82±0.18)h~(-1)。1小时血浆中咪达唑仑与其代谢产物1＇-羟化咪达唑仑的比值与其清除率的相关性统计学上具有显著意义(r=0.7,P<0.05,n=10)。结论:可用口服咪达唑仑后1小时单个采血点的代谢产物1＇-羟化咪达唑仑与咪达唑仑浓度的比值来反映其血浆清除率,应用于CYP3A活性测定的人群试验。     

Cocktail探针药物法评价威麦宁胶囊对大鼠体内CYP450活性的影响

目的采用Cocktail探针药物法评价威麦宁胶囊对大鼠体内6种CYP450亚型酶活性的影响。方法分别选用甲苯磺丁脲、氯唑沙宗、茶碱、咪达唑仑、奥美拉唑和右美沙芬作为CYP2C6、CYP2E1、CYP1A2、CYP3A2、CYP2D1和CYP2D2的探针底物。大鼠每日灌胃威麦宁胶囊1.6 g·kg-1,采用LC-MS/MS测定给药前后大鼠体内6种混合探针的血药浓度,计算药动学参数。结果威麦宁胶囊连续给药2周后,与给药前相比,奥美拉唑ρmax、AUC0-t、tmax及AUC0-∞显著升高(P<0.05);咪达唑仑ρmax、AUC0-t和AUC0-∞显著升高(P<0.01或P<0.05);右美沙芬ρmax、AUC0-t升高(P<0.05);甲苯磺丁脲t1/2、AUC0-∞和tmax显著升高(P<0.01或P<0.05),而CL/F显著降低(P<0.01),ρmax、AUC0-t无显著改变(P>0.05);氯唑沙宗ρmax升高(P<0.05),CL/F降低(P<0.05),AUC0-t升高但无显著差异(P>0.05);茶碱ρmax、AUC0-t升高但无显著差异(P>0.05)。表明奥美拉唑、咪达唑仑和右美沙芬代谢明显减慢(均P<0.05),茶碱、甲苯磺丁脲、氯唑沙宗的代谢无显著差异;威麦宁胶囊对大鼠体内CYP2D1、CYP3A2、CYP2D2酶有抑制作用,对CYP1A2、CYP2C6、CYP2E1酶的活性无显著影响。结论当威麦宁胶囊与CYP2C19、CYP3A4、CYP2D6酶的底物药物合用时,需要调整给药剂量,避免因药物相互作用使体内血药浓度过高产生毒副作用。     

酮康唑对大鼠鼻腔和灌胃给予咪达唑仑及其代谢产物药动学差异的影响

目的在系统比较大鼠鼻腔和灌胃给予咪达唑仑药动学特征的基础上,进一步比较酮康唑对2种给药途径药动学的影响。方法 24只大鼠随机分为4组,每组6只。其中2组分别经鼻腔或灌胃只给予咪达唑仑(1 mg?kg~(- 1)),另外2组联用细胞色素P450酶3A(CYP3A)抑制剂酮康唑(30 mg?kg~(- 1))后再分别经鼻腔或灌胃给予咪达唑仑,不同时间点采集血样,测定咪达唑仑和1′-羟基咪达唑仑浓度,计算药代动力学参数,并进行统计学分析。结果大鼠单独经鼻腔和灌胃给予咪达唑仑后,原型药物的达峰时间(T_(max))分别约为2和25 min,药时曲线下面积(AUC)分别为296和179μg?L~(-1)?h。合用酮康唑后,在鼻腔和灌胃给药条件下,咪达唑仑原型药物在大鼠体内的AUC分别增加到原来的2.1和3.3倍。但是,酮康唑不改变咪达唑仑鼻腔给药的T_(max),而合用酮康唑后,咪达唑仑灌胃给药的T_(max)延长至1.14h。结论咪达唑仑经鼻腔给药与经口服给药相比,吸收迅速、药物暴露量大,更适合于临床急救。咪达唑仑经鼻腔给药合用酮康唑后,不改变吸收速度,但抑制其代谢转化,药物体内驻留时间明显延长;咪达唑仑口服给药合并给予酮康唑后,吸收减慢,抑制代谢转化,体内药物暴露量明显增加。由于咪达唑仑的中枢镇静作用,2种途径合用酮康唑时均应考虑适当减少给药剂量或延长给药间隔。     

六神丸对CYP活性的诱导作用研究

目的:研究六神丸对CYP活性的诱导作用。方法:体外传代培养人肝癌Hep G2细胞。以3、1、0.5μg/ml(以丸质量计,下同)六神丸培养细胞48 h后,采用实时荧光聚合酶链反应(RT-PCR)法测定细胞CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP2E1、CYP3A4 m RNA的表达;采用Western blot法测定细胞CYP3A4、CYP1A2、CYP2C19、CYP2C9蛋白的表达,采用高效液相质谱法测定咪达唑仑代谢产物1-羟基咪达唑仑的含量,以咪达唑仑生成速率代表CYP3A4活性。上述试验均设阴性对照(0.3%二甲基亚砜)组、阳性对照(30μmol/L利福平、100μmol/L地塞米松)组。将18只Wistar大鼠随机均分为空白对照[等容羧甲基纤维素钠(CMC-Na)]组、六神丸(1.613 mg/kg)组、地塞米松(50 mg/kg)组,连续6 d ig给药后测定大鼠肝微粒1-羟基咪达唑仑生成速率以判定CYP3A1、CYP3A2活性;采用RT-PCR法测定CYP3A m RNA的表达。结果:3、1、0.5μg/ml六神丸培养细胞48 h后,与阴性对照比较,Hep G2细胞中CYP2C9、CYP2C19、CYP2E1、CYP3A4 m RNA表达增强,CYP3A4蛋白表达增强,CYP3A4活性增强,差异均有统计学意义(P<0.05)。与空白对照组比较,大鼠肝微粒体CYP3A1、CYP3A2活性增强,CYP3A1、CYP3A2 m RNA表达增强,差异均有统计学意义(P<0.05)。结论:六神丸对CYP具有一定的诱导作用,其机制可能与增强CYP m RNA的表达作用有关。     

五味子甲素对大鼠咪达唑仑及其代谢物1′-羟基咪达唑仑药动学影响

目的:探讨五味子甲素(SchA)是否影响CYP3A底物-咪达唑仑在大鼠体内的代谢。方法:不同剂量的五味子甲素或酮康唑75mg/kg连续灌胃3d,采用单次十二指肠给药及腹股沟动脉插管,以CYP3A抑制剂(酮康唑)作为阳性对照,研究咪达唑仑在大鼠体内的代谢。高效液相色谱法(HPLC)测定血浆中咪达唑仑及其代谢物的浓度。结果:口服不同药物后咪达唑仑的主要药动学参数为:AUC(0-t)(mg.L-1.h)分别为:(1.08±0.29)(阴性对照)、(1.58±0.58)(SchA 8mg/kg)、(2.02±1.29)(SchA16mg/kg)、(2.22±1.25)(SchA 32mg/kg)、(3.34±2.25)(酮康唑75mg/kg);Cmax(mg/L)分别为:(1.6±0.6)(阴性对照)、(1.8±0.8)(SchA 8mg/kg)、(2.2±1.2)(SchA16mg/kg)、(2.2±0.7)(SchA 32mg/kg)、(2.9±1.1)(酮康唑75mg/kg)。1′-羟基咪达唑仑的主要药动学参数如下:AUC(0-t)(mg.L-1.h)分别为:(0.61±0.17)(阴性对照)、(0.40±0.15)(SchA 8mg/kg)、(0.39±0.20)(SchA16mg/kg)、(0.40±0.14)(SchA 32mg/kg)、(0.35±0.09)(酮康唑75mg/kg);Cmax(mg/L)分别为:(0.54±0.13)(阴性对照)、(0.42±0.15)(SchA 8mg/kg)、(0.39±0.16)(SchA 16 mg/kg)、(0.36±0.16)(SchA 32mg/kg)、(0.35±0.12)(酮康唑75mg/kg)。结论:结果表明,五味子甲素可以显著抑制咪达唑仑的代谢。     

高效液相色谱法测定大鼠肝微粒体CYP3A酶的活性

目的 以咪达唑仑为探针药用HPLC法测定大鼠肝微粒体CYP3A酶活性.方法 用ZORBAX SB-C18色谱柱分离;流动相为乙腈-水-0.1％三氟乙酸;流速为1 mL· min-1;柱温40℃;检测波长为230 nm.肝微粒体加入咪达唑仑孵育5 min后,用乙腈终止反应,加入内标溶液50 μL,混匀后离心10 min,取上清进行HPLC分析;计算Km和Vmax.结果 1-羟基咪达唑仑浓度在0.06～3.00 mg· L-1内线性关系良好;日内、日间精密度均＜10％,回收率＞75％.咪达唑仑羟化反应的Km =7.32μmol· L-,Vmax=0.59 nmol·min-1·mg-1 pro.结论 本方法稳定可靠,能准确反映CYP3A酶的活性.     

雷公藤多苷对大鼠与人肝微粒体CYP3A酶活性的体外抑制作用

目的:建立液相色谱-串联质谱(LC-MS/MS)法测定大鼠和人肝微粒体中1′-羟基咪达唑仑的含量,研究雷公藤多苷对体外大鼠与人肝微粒体细胞色素P45(0CYP)3A酶活性的抑制作用。方法:将不同质量浓度的雷公藤多苷(0.5、1.0、2.0、5.0、10.0、50.0、100.0μg/ml)分别与大鼠和人肝微粒体(0.4 mg/ml)共同孵育20 min后终止反应,采用LC-MS/MS法测定孵育液中咪达唑仑的代谢产物1′-羟基咪达唑仑的浓度以评价CYP3A酶活性并计算酶活性抑制率,采用Graph Pad Prism 5.0软件进行拟合并计算半数抑制浓度(IC50)。色谱柱为Agilent Eclipse XBD-C18,流动相为水-甲醇(梯度洗脱),流速为0.3 ml/min,柱温为30℃,进样量为2μl。采用电喷雾电离(ESI),定量离子为m/z 342.01→324.1(1′-羟基咪达唑仑)、m/z 383.01→337.2(内标氯雷他定)。结果:1′-羟基咪达唑仑检测浓度在0.01~3μmol/L范围内与其和内标物峰面积比值呈良好线性关系;精密度试验RSD均小于10%,提取回收率为97.92%~101.65%,方法回收率为96.70%~104.10%,稳定性试验RSD均小于10%。雷公藤多苷对大鼠和人微粒体中CYP3A的IC50分别为(48.610±1.32)μg/ml和(4.754±1.12)μg/ml;在0.5~100.0μg/ml范围内雷公藤多苷对CYP3A酶活性有抑制作用,且与质量浓度呈正相关。结论:所建立的LC-MS/MS法可用于检测大鼠和人肝微粒体中1′-羟基咪达唑仑的含量。雷公藤多苷对体外大鼠和人肝微粒体药物代谢酶CYP3A均存在抑制作用,且对人肝微粒体CYP3A的抑制作用明显强于大鼠。     

评价HPPH对细胞色素P450酶体外代谢活性的影响

目的:评价HPPH在体外对大鼠及人肝微粒CYP450酶的6种亚型酶活性的影响,预测使用HPPH可能出现的药物相互作用。方法:将注射用HPPH与CYP450酶的6种亚型的特异性探针底物非那西汀(CYP1A2)、甲苯磺丁脲(CYP2C9)、S-美芬妥因(CYP2C19)、右美沙芬(CYP2D6)、氯唑沙宗(CYP2E1)、咪达唑仑(CYP3A4)和睾酮(CYP3A4)与大鼠及人肝微粒进行孵育反应,采用HPLC-MS/MS法测定对应的7种代谢产物(对乙酰氨基酚、羟基甲苯磺丁脲、4-羟基美芬妥因、O-去甲基右美沙芬、6-羟基氯唑沙宗、1′-羟基咪达唑仑、6β-羟基睾酮)的浓度。结果:在本实验条件下,HPPH浓度为1.00~50.00μmol·L-1时,未发现其对大鼠的CYP1A2、CYP2C9、CYP2D6、CYP2E1、CYP3A4产生抑制作用。在本实验条件下,HPPH浓度为0.50~10.00μmol·L-1时,未发现其对人CYP1A2产生抑制作用;但对人CYP2C9、CYP2C19、CYP2D6、CYP2E1均存在抑制作用;对人CYP3A4,对底物咪达唑仑存在抑制作用,对底物睾酮未发现抑制作用。结论:HPPH对CYP450酶的抑制作用存在种属差异,在人体内HPPH与CYP450酶作用有待体内实验进一步证明。     

液-质联用法测定中年妇科病人咪达唑仑及其代谢物的血药浓度并研究其细胞色素P450 3A4酶活性分布特征

目的 研究中年妇科手术病人应用咪哒唑仑(抗焦虑药)及其CYP3A_4酶活性分布特征.方法 以咪哒唑仑作为探针药物,用液-质联用法测定血桨中咪达唑仑、1'-羟基咪达唑仑的单点药物浓度,以1'-羟基咪达唑仑/咪达唑仑的比值评估CYP3A4酶活性.结果 260例妇科手术病人CYP3A4酶的活性为0.46±0.14,推测中年妇科手术病人CYP3A4酶活性的正常值范围为0.18～0.73.结论 CYP3A4酶活性在中年妇科手术病人中呈正态分布,酶活性的个体差异较小,对术后的个体化药物治疗影响不大.     

慢性不可预见性温和应激模型大鼠的肝脏药物代谢酶活性变化研究

采用鸡尾酒(cocktail)探针药物法研究慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)造成的抑郁状态对大鼠体内6种CYP450亚酶活性的影响。根据Katz法建立CUMS大鼠抑郁模型;分别选用甲苯磺丁脲、氯唑沙宗、茶碱、咪达唑仑、奥美拉唑及右美沙芬作为大鼠CYP2C6、CYP2E1、CYP1A2、CYP3A2、CYP2D1、CYP2D2的探针底物,采用液质联用法(LC-MS/MS)测定对照组与模型组大鼠体内6种混合探针的血药浓度,计算药动学参数。结果表明,茶碱和氯唑沙宗代谢显著加快(P<0.01),甲苯磺丁脲、右美沙芬、奥美拉唑、咪达唑仑的代谢无显著差异。结果说明,慢性不可预见性温和应激造成的抑郁状态对CYP1A2有强诱导、对CYP2E1有中强诱导作用。     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Gender-related symptoms and correlates of alcohol dependence among men and women with a lifetime diagnosis of alcohol use disorders

This study explored gender-related symptoms and correlates of alcohol dependence in a crosssectional study of 150 men and 150 women with a lifetime diagnosis of alcohol use disorders (AUD). Participants were recruited in equal numbers from treatment settings, correctional centres and the general community. Standardized measures were used to determine participants' use of substances, history of psychiatric disorders and psychosocial stress, their sensation seeking and family history of substance use and mental health disorders. Multivariate analyses were used to detect patterns of variables associated with gender and the lifetime severity of AUD. Men had a longer history of severe AUD than women. Women had similar levels of alcohol dependence and medical and psychological sequelae as men, despite 6 fewer years of AUD. More women than men had a history of severe psychosocial stress, severe dependence on other substances and antecedent mental health problems, especially mood and anxiety disorders. There were differences in family history of alcohol-related problems approximating same-gender aggregation. The severity of a lifetime AUD was predicted by its earlier age at onset and the occurrence of other disorders, especially anxiety, among both men and women. The limitations in the generalizability of these findings due to sample idiosyncrasies are discussed.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.