Human Hepatic Alcohol Dehydrogenase and Human Erythrocyte Catalase Do Not Metabolize the Cytochrome P-4502E1 Substrate, Chlorzoxazone

Studies of cytochrome P-4502E1 (CYP2E1)-mediated oxidation of ethanol have been hampered by the lack of a suitable probe for in vivo human studies. Chlorzoxazone, a prescribed skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone by CYP2E1 and has been advocated as a specific probe of this enzyme on the basis of microsomal studies. The applications of this probe may include delineating the contribution of CYP2E1 to in vivo human ethanol metabolism. However, the activity of nonmicrosomal enzymes in metabolizing chlorzoxazone is unknown. Alcohol dehydrogenase (ADH), predominantly a hepatic cytosolic enzyme, may be more important than CYP2E1 in the oxidation of ethanol to acetaldehyde. The contribution of catalase in the in vivo oxidation of ethanol to acetaldehyde is controversial. To determine if either of these enzymes metabolizes chlorzoxazone and whether ethanol oxidation by either enzyme is inhibited by chlorzoxazone or its metabolite, multiple in vitro studies were performed. ADH enzyme kinetics were performed with human recombinant β₁β₁ and β₃β₃ ADH with ethanol and chlorzoxazone (0.5 to 2.5 mM). Neither ADH isoenzyme exhibited NAD⁺-dependent oxidation of chlorzoxazone, but displayed Michaelis-Menten kinetics for ethanol with K_m values of 89 μM and 34 mM, for β₁β₁, and β₃β₃, respectively. Typical in vivo concentrations of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone, did not alter β₁β₁, or β₃β₃ ADH-mediated oxidation of ethanol to acetaldehyde. Studies of human hepatic nonmicrosomal enzyme activity were expanded to include all nonmicrosomal NAD⁺-dependent hepatic enzymes by starch gel electrophoresis assessment. Human hepatic enzymatic activity in the presence of chlorzoxazone was similar to that observed in the control sample (no added substrate), suggesting a lack of metabolism by NAD⁺-dependent enzymes. Similarly, human erythrocyte catalase, in the presence of a hydrogen peroxide generating system, did not metabolize chlorzoxazone. Furthermore, neither chlorzoxazone nor 6-hydroxychlorzoxazone altered the catalase-induced formation of acetaldehyde from ethanol. These data are consistent with chlorzoxazone as a specific probe of CYP2E1 that may be useful to alcohol researchers.     

氯唑沙宗残留溶剂检测方法研究

目的：建立统一的、适合于不同生产企业的氯唑沙宗残留溶剂检测方法。方法：采用两种极性的毛细管色谱柱（SPB-1和HP—FFAP）和两种温度系统，对来自于不同企业的氯唑沙宗样品进行残留溶剂筛选考查，结合生产企业工艺信息确定被测残留溶剂种类；采用聚乙二醇（Supelco—Wax）色谱柱，以N，N-二甲基甲酰胺为溶解介质、正丙醇为内标，顶空进样法检测氯唑沙宗中的甲醇、乙醇、醋酸丁酯和氯苯。结果：经筛选考查实验，供试样品中筛查出甲醇和乙醇两种残留溶剂。4种被测残留组分的线性关系良好（r=0．99995～0．99999，n=8）；3种浓度的平均加样回收率（n=13）为93．8—102．4％（RSD为0．5～2．3％）；4种残留溶剂的最低检测限为0．00015～0．00027％；本方法的日间重复性良好，三种浓度的对照品溶液3d重复进样，峰面积比值的RSD（n=9）为0．65—2．93％。结论：筛选考查实验结果为被测残留溶剂种类的确定提供了可靠的依据；所建立的残留溶剂检测方法结果准确、灵敏度高、重现性好，并适合于不同生产企业的质量控制。     

复方氯唑沙宗乳膏治疗颞下颌关节紊乱的研究

目的：研究复方氯唑沙宗乳膏透皮制剂对不同类型颞下颌关节紊乱病（TMJD）的疗效。以确定此制剂的最佳适应证。方法：TMJD68例，局部使用复方氯唑沙宗乳膏，观察其疗效。结果：此乳膏对咀嚼肌紊乱类的TMJD患者效果最好。有效率达94.88%，对其它类型的TMJD患者效果不理想。结论：复方氯唑沙宗乳膏可缓解TMJD患者疼痛和肌肉痉挛等临床症状。用以治疗咀嚼肌群疼痛和痉挛效果较好，对关节内紊乱及器质性损害所致的疼痛疗效较差。     

氯唑沙宗及其代谢物的HPLC测定方法和药代动力学研究

目的旨在建立氯唑沙宗及其代谢物的方法。应用高效液相色谱法,内标物为5-fluorobenzoxazolone,经乙酸乙酯提取,紫外检测波长为287nm。结果表明,6-羟氯唑沙宗、内标物及氯唑沙宗的保留时间分别为6.12,10.47和18.65min。6-羟氯唑沙宗及氯唑沙宗在0.5～20μg·ml^-1血浆浓度范围内线性关系良好,回收率均在82.80%～100.76%之间,当日及日间相对标准差分别小于8%和11%。两药的最低检测浓度分别为0.2和0.5μg·ml^-1。多种常用药物对样品的色谱峰无干扰。曾对8名健康受试者单次口服氯唑沙宗400mg的药代动力学进行了观察。提示此方法可用于氯唑沙宗的体内氧化代谢研究。     

振源胶囊对细胞色素P_(450)酶CYP1A2、CYP3A4、CYP2E1的影响

目的:研究振源胶囊对细胞色素P450酶CYP1A2、CYP3A4、CYP2E1的影响。方法:用Cocktail探针药物法,将Wistar大鼠随机分组,灌胃给予振源胶囊溶液,以生理盐水组为空白对照,诱导10d,于股动脉插管,注射给予3种探针药物咖啡因、氨苯砜、氯唑沙宗,通过高效液相色谱法检测各探针药物的代谢率来评价各组CYP1A2、CYP3A4、CYP2E1亚型酶的活性;药动学计算采用DAS2.0软件完成。结果:给予振源胶囊的大鼠,咖啡因代谢加快,半衰期缩短;氨苯砜代谢减慢,半衰期延长;氯唑沙宗半衰期与空白对照组比较无显著差异(P>0.05)。结论:振源胶囊对大鼠CYP1A2有诱导作用,对CYP3A4有抑制作用,对CYP2E1的作用不明显。     

高效液相色谱法测定血清中氯唑沙宗的浓度

目的建立测定血清中氯唑沙宗浓度的高效液相色谱法。方法采用迪马C18色谱柱(250×4.6mm,5μm);流动相:乙腈-0.02MKH2PO4(pH6.3)=9∶11(V/V),含0.16%三乙胺;流速:1.0mL.min-1;紫外检测波长:280nm;柱温:30℃。结果氯唑沙宗在2.5~12.5μg.mL-1的浓度范围内线性关系良好,最低检测限为0.4μg.mL-1,回收率为102.82±4.39%。结论本法操作简便,灵敏度高,快速可靠,可用于血清中氯唑沙宗的浓度测定。     

Effect of piperine on CYP2E1 enzyme activity of chlorzoxazone in healthy volunteers

1.?The purpose of the present study was to investigate the effect of piperine (PIP) on CYP2E1 enzyme activity and pharmacokinetics of chlorzoxazone (CHZ) in healthy volunteers.

2.?An open-label, two period, sequential study was conducted in 12 healthy volunteers. A single dose of PIP 20?mg was administered daily for 10 days during treatment phase. A single dose of CHZ 250?mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected at predetermined time intervals after CHZ dosing and analyzed by HPLC.

3.?Treatment with PIP significantly enhanced maximum plasma concentration (C_max) (3.14–4.96?μg/mL)_, area under the curve (AUC) (10.46–17.78?μg h/mL), half life (T_1/2) (1.26–1.82?h) and significantly decreased elimination rate constant (K_el) (0.57–0.41?h?^??¹), apparent oral clearance (CL/F) (24.76–13.65?L/h) of CHZ when compared to control. In addition, treatment with PIP significantly decreased C_max (0.22–0.15?μg/mL), AUC (0.94–0.68?μg h/mL), T_1/2 (2.54–1.68?h) and significantly increased K_el (0.32–0.43?h?^??¹) of 6-hydroxychlorzoxazone (6-OHCHZ) as compared to control. Furthermore, treatment with PIP significantly decreased metabolite to parent (6-OHCHZ/CHZ) ratios of C_max, AUC, T_1/2 and significantly increased K_el ratio of 6-OHCHZ/CHZ, which indicate the decreased formation of CHZ to 6-OHCHZ.

4.?The results suggest that altered pharmacokinetics of CHZ might be attributed to PIP mediated inhibition of CYP2E1 enzyme, which indicate significant pharmacokinetic interaction present between PIP and CHZ. The inhibition of CYP2E1 by PIP may represent a novel therapeutic benefit for minimizing ethanol induced CYP2E1 enzyme activity and results in reduced hepatotoxicity of ethanol.     

Circadian rhythm of cytochrome P4502E1 and its effect on disposition kinetics of chlorzoxazone in rats

The aim of this report is to study the circadian rhythm of cytochrome P4502E1 (CYP2E1) and its effect on the disposition kinetics of chlorzoxazone in male Wistar rats. The rats were housed under a 12-h light/dark cycle (lights from 9:00 to 21:00) with food and water ad libitum for 3 months. It was found that the expression of microsomal CYP2E1 mRNA in the liver during the dark phase was significantly lower than during the light phase, whereas the content of CYP2E1 protein and its hydroxylation activity were significantly higher. Therefore, chlorzoxazone 20 mg/kg was intravenously administered at 12:00 (light phase group) or 24:00 (dark phase group) to determine the effect on the disposition kinetics. The value of the area under the plasma concentration-time curve from 0 to 8 h (AUC(0-8 h)) of chlorzoxazone showed no significant difference between the two groups. However, the value of chlorzoxazone half-life in plasma of the light phase group was significant longer than the dark phase group. The AUC(0-8 h) of 6-hydroxychlorzoxazone, a metabolite formed from chlorzoxazone mainly by CYP2E1, was significantly higher in the dark phase than in the light phase. In conclusion, microsomal CYP2E1 shows a substantial circadian variation in rats, and this was associated with a decrease of chlorzoxazone half life, and an increase of 6-hydroxychlorzoxazone production. Therefore, the temporal variations of therapeutic response and toxicological effects may have to be taken into consideration for other xenobiotics that are predominantly metabolized by CYP2E1, particularly those with a short half-life.