高效液相色谱法同时测定血清中咖啡因和氯唑沙宗的浓度

目的:建立同时测定血清中两种探针药物(咖啡因、氯唑沙宗)浓度的高效液相色谱法.方法:采用迪马C18色谱柱(250 mm×4.6 mm,5 μm);流动相:乙腈-0.02 mol·L-1 磷酸二氢钾溶液(1∶3,v/v),含0.01 mol·L-1三乙胺;流速:1.5 mL·min-1;紫外检测波长:280 nm;柱温:30℃.结果: 咖啡因在2.5～12.5 μg·mL-1、氯唑沙宗在2.5～12.5 μg·mL-1的浓度范围内线性关系良好,最低检测限分别为0.1 μg·mL-1、0.4 μg·mL-1,回收率分别为103.70%±4.36%、102.82%±4.39%.结论: 本法操作简便,灵敏度高,快速可靠,可用于血清中咖啡因和氯唑沙宗的浓度测定.     

HPLC法测定宗胺因片中氯唑沙宗、乙水杨胺和咖啡因的含量

目的：建立高效液相色谱法，测定复方制剂宗胺因片中氯唑沙宗、乙水扬胺和咖啡因的含量。方法：采用C18柱，以甲醇-水（48：52）为流动相，检测波长：280nm。结果：线性范围为氯唑沙宗0．0825～0．742ug（r=0．9999）；乙水扬胺0．165～0．823ug（r=1.0000）；咖啡因0．0263-0．131ug（r=0．9999），平均回收率（n=9）为氯唑沙宗100．1％（RSD％=1．01％）；乙水扬胺99．7％（RSD％=0．74％）；咖啡因99．3％（RSD％=0．57％）。结论：本方法简便、快速，专属性强，可有效的控制宗胺因片中氯唑沙宗、乙水扬胺和咖啡因的含量。     

Stimulatory effects of chlorzoxazone,a centrally acting muscle relaxant,on large conductance calcium-activated potassium channels in pituitary GH3 cells

Chlorzoxazone, a centrally acting muscle relaxant, has been used as a marker for hepatic CYP2E1 activity. However, little is known about the mechanism of chlorzoxazone actions on ion currents in neurons or neuroendocrine cells. We thus investigated its effects on ion currents in GH(3) lactotrophs. Chlorzoxazone reversibly increased Ca(2+)-activated K(+) current (I(K(Ca))) in a concentration-dependent manner with an EC(50) value of 30 microM. The chlorzoxazone-stimulated I(K(Ca)) was inhibited by iberitoxin (200 nM) or clotrimazole (10 microM), but not by glibenclamide (10 microM) or apamin (200 nM). Chlorzoxazone (30 microM) suppressed voltage-dependent L-type Ca(2+) current. In the inside-out configuration, chlorzoxazone applied to the intracellular side of the patch did not modify single-channel conductance of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, but did increase channel activity by increasing mean open time and decreasing mean closed time. Chlorzoxazone also caused a left shift in the activation curve of BK(Ca) channels. However, Ca(2+)-sensitivity of these channels was unaffected by chlorzoxazone. 1-Ethyl-2-benzimidazolinone (30 microM), 2-amino-5-chlorobenzoxazole (30 microM) or chlormezanone (30 microM) enhanced BK(Ca) channel activity, while 6-hydroxychlorzoxazone (30 microM) slightly increased it; however, chlorphenesin carbamate (30 microM) had no effect on it. Under the current-clamp condition, chlorzoxazone (10 microM) reduced the firing rate of action potentials. In neuroblastoma IMR-32 cells, chlorzoxazone (30 microM) also stimulated BK(Ca) channel activity. The stimulatory effects of chlorzoxazone on these channels may be responsible for the underlying mechanism of chlorzoxazone actions on neurons and neuroendocrine cells.     

细胞色素P450 2E1活性的测定方法

细胞色素P450(cytochmme P450)是主要的肝细胞Ⅰ相代谢酶之一,其亚家族细胞色素：P450 2E1(CYP2E1)在外来化学物的代谢活化中起重要作用,CYP2E1活性的高低与毒物对机体的最终毒性大小直接相关。多年来,体内CYP2E1活性的测定一直是国内外学者颇感棘手的问题。本文从体外、体内以及淋巴细胞中CYP2E1活性的测定等几方面对该问题进行综述。     

Kinetic study of alkaline induced hydrolysis of the skeletal muscle relaxant chlorzoxazone using ratio spectra first derivative spectrophotometry

2-Amino-4-chlorophenol was found to be the alkaline induced degradation product and the synthetic precursor of chlorzoxazone. The aim of this work is to study different factors affecting the degradation process due to the high toxicity of 2-amino-4-chlorophenol. Chlorzoxazone was found to follow pseudofirst order kinetics. Ratio spectra first derivative spectrophotometry (DR(1)) was developed for monitoring the change in chlorzoxazone concentration during the degradation process. Kinetic parameters (rate constant (K) and half-life (t(0.5))) were calculated at different temperatures (40-120 degrees C) and different sodium hydroxide concentrations (3-10 M). Activation energy at 3 and 8 M sodium hydroxide concentration and alkaline induced catalysis constant at 60, 70 and 80 degrees C were also calculated.     

Metabolism of benzene in human liver microsomes: individual variations in relation to CYP2E1 expression

In human liver microsomes the oxidations of benzene, chlorzoxazone, aniline, dimethylformamide, and 4-nitrophenol were significantly correlated with each other and with the level of cytochrome P450 (CYP) 2E1 estimated by immunoblotting. Moreover, benzene oxidation to water-soluble metabolites was suppressed by 0.1 mM diethyldithiocarbamate, supposedly a specific inhibitor of CYP2E1 at this level. None of these metabolic rates correlated with immunochemically determined levels of CYP1A2, 2C9, and 3A4 nor oxidation of 7-ethoxyresorufin, tolbutamide, and nifedipine. Benzene oxidation to water-soluble metabolites was characterized by typical Michaelis-Menten kinetics. The different benzene K _m values seen in individual human microsomal samples were not correlated with the level or activity of CYP1A2, 2C9, 2E1, and 3A4 but could be due to CYP2E1 microheterogeneity. The lowest K _m for benzene oxidation could be related to C/D and/or c1/c2 polymorphism of CYP2E1 gene. Covalent binding of benzene reactive metabolites to microsomal proteins was also correlated with the CYP2E1 metabolic rates and immunochemical levels. At high concentrations of benzene covalent binding was inversely related to benzene concentrations (as well as to formation of water-soluble metabolites) in agreement with the view that secondary metabolites, mainly benzoquinone, are responsible for the covalent binding. Received: 8 September 1998 / Accepted: 24 November 1998     

利用细菌/杆状病毒系统在昆虫细胞中表达人CYP2E1

目的获得人CYP2E1重组酶,并用该重组酶的特征性探针底物对其进行代谢活性研究.方法以人肝组织RNA为模板,通过RT-PCR得到CYP2E1 cDNA片断,然后与pFastBac质粒连接,得到pFastBac-CYP2E1重组质粒,将其转化E.coli DH 10Bac大肠杆菌,通过转座作用,获得重组Bacmid-CYP2E1,将其转染草地夜蛾细胞(Sf9) 后,产生重组杆状病毒.将该病毒以及分别含有人CYPOR和人CYPb5的病毒共同感染Sf9细胞,收集共表达蛋白,以氯唑沙宗为底物鉴定重组酶的活性.结果利用细菌/杆状病毒系统得到重组人CYP2E1的表达,其对氯唑沙宗的Km值为(72.4±8.7)μmol·L-1,Vmax值为(2.41±0.10)μmol·min-11·g-1蛋白.结论利用杆状病毒系统成功表达了有催化活性的人CYP2E1重组酶,其活性与文献报道值相似.     

Cocktail法研究脉络宁注射液对大鼠CYP1A2、CYP2E1和CYP3A4活性的影响

目的：研究脉络宁注射液对大鼠CYP1A2、CYP2E1和CYP3A4活性的影响。方法：14只大鼠随机均分成临床等效剂量组和高剂量组,连续2周静脉给予脉络宁注射液（临床等效剂量纽,2mL／kg;高剂量组,4mL／kg）前后,均同时灌胃给予3个探针底物（茶碱,30mg／kg;氯唑沙宗,50rag／kg;氨苯砜,20mg/kg）,进行采血试验。用HPLC法同时测定大鼠体内各探针的血药浓度,DAS1．0软件计算药动学参数,并以配对t检验对各组大鼠前后两轮主要药动学参数进行差异性比较。结果：在1个给药疗程（14d）内,临床等效剂量组大鼠用药前后,3个探针的药动学参数均无显著性变化（P〉0．05）;高剂量组大鼠用药后,与用药前相比,茶碱的药动学参数没有显著变化（P〉0．05）;氨苯砜和氯唑沙宗的AUC0-24h均有升高趋势（P〈0．05）,给药后分别是给药前的1．44倍和1．28倍,同时氯唑沙宗的CL显著降低（P〈0．05）。结论：临床等效剂量脉络宁对大鼠CYP1A2、CYP2E1和CYP3A4活性均无显著影响,而高剂量脉络宁对大鼠CYP2E1和CYP3A4均有弱抑制作用。     

体内代谢法研究新药对细胞色素P450 1A2和2E1的影响

目的研究新药对大鼠肝药酶的影响及其性别差异,从而对这些新药进行安全性评价。方法通过研究肝药酶细胞色素P450（CYP）1A2和CYP2E1的专属探针药物咖啡因和氯唑沙宗在对照组与给药组的体内代谢过程的变化,判断药物对这些酶有无诱导或抑制作用。结果新药SPMG对CYP1A2和CYP2E1均无影响,AOSC对CYP1A2无影响,但对雄性大鼠的CYP2E1有诱导作用。GC对雌性大鼠的CYP1A2有抑制作用而对雄性大鼠的CYP2E1有明显的诱导作用。结论药物对CYP各亚酶的影响存在明显的性别差异,AOSC和GC在与各种与CYP 1A2和/或CYP 2E1代谢有关的药物合用时,应充分考虑其在不同性别间的差异,以避免潜在的毒性或不良反应。而SPMG在此情况下则相对安全。     

Chlorzoxazone exhibits neuroprotection against Alzheimer’s disease by attenuating neuroinflammation and neurodegeneration in vitro and in vivo

Alzheimer’s disease (AD), a complex and an age-related brain disease, is induced by the accumulation of amyloid beta (Aβ) and neuroinflammation. Chlorzoxazone (CZ) is a classical FDA-approved drug, and shows anti-inflammatory effects. However, up until now, its regulatory role in AD has not been investigated. Therefore, in this study we attempted to explore if CZ could be an effective therapeutic strategy for AD treatment. At first, the in vitro study was performed to mimic AD using Aβ. We found that Aβ caused p65 nuclear translocation in both primary microglial cells and astrocytes, which were, however, restrained by CZ treatments. Meanwhile, CZ incubation markedly decreased the expression of pro-inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β). Aβ deposition was also markedly reduced in glial cells treated with CZ. Importantly, we found that glial activation and its-related pro-inflammation induced by Aβ led to obvious neurodegeneration and neuroinflammation, which were effectively attenuated by CZ pre-treatment in the isolated primary cortical neurons. Then, the in vivo study was performed using APP/PS1 mice with AD. Behavior tests showed that CZ administration effectively improved cognitive deficits in AD mice. Neuron death in hippocampus of AD mice was also inhibited by CZ. Aβ accumulation in brain was markedly decreased in CZ-treated AD mice. We finally found that hippocampal glial activation in AD mice was obviously blocked by CZ supplementation, along with remarkable decreases in TNF-α, IL-1β and p65 nuclear translocation. Together, these findings above demonstrated that CZ could inhibit glial activation and inflammatory response, contributing to the suppression of neurodegeneration and neuroinflammation. Therefore, CZ may be an effective therapeutic strategy for AD treatment.