RP-HPLC法测定人尿中甲苯磺丁脲及其代谢产物的浓度

目的:建立以反相高效液相色谱法测定人尿中甲苯磺丁脲及其代谢产物羧基甲苯磺丁脲和羟基甲苯磺丁脲浓度的方法。方法:以氯磺丁脲为内标,尿样经酸化后用异丙醚萃取,并进样分析;色谱柱为DiamonsilTM C18,流动相为乙腈-0.05 mol.L-1NaH2PO4(pH4.0,梯度洗脱),流速为1.0 mL.min-1,检测波长为230 nm,柱温为40℃。结果:甲苯磺丁脲、羧基甲苯磺丁脲、羟基甲苯磺丁脲尿药浓度均在0.3～10μg.mL-1范围内线性关系良好(r分别为0.999 9、1.000 0、0.999 9),最低检测浓度均为0.1μg.mL-1。各样本低、中、高浓度的方法回收率在95.00%～105.88%之间,日内、日间RSD均<6%。结论:本方法简便、准确、灵敏、重复性好,适用于细胞色素P4502C9表型分析及甲苯磺丁脲与其代谢产物的人体药动学研究。     

中国健康人群P450 2C9基因多态性与甲苯磺丁脲代谢的关系

目的 研究甲苯磺丁脲人体代谢过程与中国健康志愿者P450 2C9基因多态性的关系。方法 不同P450 2C9＊1／＊1（n=53）、＊1／＊3（n=9）及＊3／＊3（n=1）基因型的健康志愿者单次口服甲苯磺丁脲500mg后，用HPLC方法测定血和尿中的甲苯磺丁脲及其代谢产物浓度，研究甲苯磺丁脲的药代过程。结果 P450 2C9＊1／＊3与P450 2C9＊3／＊3基因型的受试者与P450 2C9＊1／＊1基因型相比，AUC（0-∞）增加20％和116％，t1/2增加60％和813％，CL0为68％和11％，CLform仅为39％和3％。结论 P450 2C9＊1／＊3对甲苯磺丁脲的代谢活性较P4502C9＊1／＊1显著降低，P4502C9＊3／＊342谢活性更低，并呈现基因剂量关系。     

细胞色素P450 CYP2C9基因多态性对甲苯磺丁脲代谢动力学的影响

目的研究细胞色素P450 CYP2C9基因多态性对甲苯磺丁脲代谢动力学的影响。方法用基因芯片对137名健康志愿者进行CYP2C9基因多态性检测，将受试者分为CYP2C9野生型、杂合突变型和纯合突变型3组，用高效液相色谱法检测甲苯磺丁脲在受试者体内的药物代谢动力学参数，统计分析各组间药代动力学性质差异。结果在137名受试者中发现了9个CYP2C9*1/*3杂合型突变体和1个CYP2C9*3/*3纯合型突变体，其余为野生型个体。将9名CYP2C9*1/*3，1名CYP2C9*3/*3以及随机抽取的10名野生型个体分组，以甲苯磺丁脲为探药进行药物代谢动力学研究。结果在杂合型突变个体组以及纯合型突变个体组中，甲苯磺丁脲的代谢率显著低于对照的野生型个体组。结论CYP2C9基因多态性对甲苯磺丁脲代谢具有显著影响并呈基因剂量效应，检测突变型个体对指导临床合理用药和个体化医疗具有重要意义。     

甲苯磺丁脲与其他药物的相互作用

目的：了解甲苯磺丁脲在治疗Ⅱ型糖尿病及其并发症时,与其他药物配伍时降血糖作用的变化规律。方法：对近年来物相关文献资料进行分析、归纳。结果：二甲双胍等１６种（类）药物皆能不同程度地影响甲苯磺丁脲的降血糖作用。结论：二甲双胍、拜糖平和胰岛素等与甲苯磺丁脲具协同降糖作用;哌唑嗪、苯那普利、前列地尔、普瑞博思、血脂康、复方磺胺甲恶唑和肝药酶抑制剂氯霉素等能增强甲苯磺丁脲的降血糖作用;而普萘洛尔、氢氯噻嗪、乙醇、赛庚啶和雄性激素等将减弱其作用。     

细胞色素P450 2C9酶活性测定探针的分析

P450 2C9是人体中重要的药物代谢酶.P450 2C9基因编码区的多态性造成氨基酸序列的变化,主要包括P450 2C9*2和P450 2C9*3两种突变体,变异纯合子对P450 2C9底物的代谢能力明显降低.目前常用的P450 2C9的探药包括甲苯磺丁脲、华法令和洛沙坦等,各种探药在检测方法、不良反应和灵敏度方面各有优缺点.     

盐酸沙格雷酯对大鼠体内细胞色素P4502D1/2的影响

目的观察盐酸沙格雷酯对大鼠细胞色素P4502D1/2(CYP2D1/2)的底物右美沙芬药动学的影响。方法♂SD大鼠,随机分成2组,对照组按右美沙芬组10 mg·kg-1灌胃给药,盐酸沙格雷酯组按右美沙芬和盐酸沙格雷酯均10 mg·kg-1同时灌胃给药,按不同时间从大鼠眼底静脉丛取血,血样处理后,用LC-MS/MS法测定大鼠血浆中右美沙芬的浓度,用DAS 2.0软件进行分析,求出其主要药代动力学参数。结果盐酸沙格雷酯组与对照组比较,右美沙芬的T12明显延长(2.49 h±0.93 h vs 1.47 h±0.20 h,P<0.05),Cmax明显升高(325.7μg·L-1±133.2μg·L-1vs 104.5μg·L-1±52.4μg·L-1,P<0.05),AUC0-t(785.5μg·L-1·h±451.9μg·L-1·h vs 244.8μg·L-1·h±168.3μg·L-1·h,P<0.05)和AUC0-∞(804.7μg·L-1·h±445.6μg·L-1·h vs 251.4μg·L-1·h±173.4μg·L-1·h,P<0.05)明显增大。结论盐酸沙格雷酯在大鼠体内对右美沙芬的代谢有抑制作用,能降低其消除过程。     

细胞色素P4502C9基因多态性对健康人体内格列喹酮药代动力学的影响

目的研究细胞色素CYP2C9基因多态性对中国人体内格列喹酮药物代谢动力学的影响。方法用RELP-PCR方法对38名健康受试者进行CYP2C9基因多态性检测,并按不同CYP2C9基因型进行分组,用高效液相色谱-荧光检测法测定受试者体内格列喹酮的血药浓度并计算相应的药代动力学参数,比较不同基因型受试者间药代动力学参数的差异。结果在38名健康受试者中,发现3名CYP2C9*1/*3杂合突变型个体与35名CYP2C9*1/*1野生型个体;未发现CYP2C9*3/*3纯合突变型个体。随机抽取的17名野生型个体与3名杂合突变型个体在给予格列喹酮60 mg后,发现杂合突变型个体AUC0-t、AUC0-∞、CL/F分别相当于野生型个体的1.854,1.787,0.554倍。结论 CYP2C9基因多态性对格列喹酮的药代动力学有显著影响,CYP2C9*1/*3突变型个体临床应适当调整给药剂量。     

高效液相色谱法测定甲苯磺丁脲片的含量

目的 建立了高效液相色谱法测定甲苯磺丁脲片中甲苯磺丁脲含量的方法.方法 主要参数为:色谱柱为KromasilC18(250mm×4.6mm,5μm),流动相为:甲醇-0.02mol/L的乙酸铵溶液-0.1%醋酸溶液(20:20:60),检测波长为235nm,流速为lmL/min.该方法 时甲苯磺丁脲的平均回收率为99.10%.RSD为0.62%(n=5).本法与标准法比较,没有显著性差别.结果 方法 简便,结果 准确,专属性强.结论 可用于含量的测定.     

HPLC法测定甲苯磺丁脲片的含量

目的：建立HPLC法测定甲苯磺丁脲片的含量。方法：色谱柱为VP—ODSC18（250mm×4．6mm,5μm）,甲醇一磷酸二氢铵溶液（pH：3．5）（65：35）为流动相,流速为1．0ml·min^-1,检测波长为254nm,柱温：室温,进样量：20μl。结果：甲苯磺丁脲在0．08—1．20mg·ml^-1浓度范围内与峰面积呈良好的线性（r=0．9998）。平均回收率为99．61％（RSD=0．8％,n=9）。结论：本法定量准确,可用于甲苯磺丁睬片的含量测定。     

浙江沿海汉族人群CYP2C9基因多态性与甲苯磺丁脲代谢活性关系

目的探讨浙江沿海地区汉族人群CYP2C9基因多态性与甲苯磺丁脲代谢关系。方法采用寡核苷酸基因芯片,对浙江沿海地区汉族人群137名健康志愿者进行基因分型;选择含有突变型等位基因CYP2C9*3志愿者和部分野生型志愿者共20名参加甲苯磺丁脲药代动力学试验;统计分析不同CYP2C9基因型个体的代谢参数。结果在137名健康受试者DNA标本中发现CYP2C9*1/*3杂合突变型个体9名,CYP2C9*3/*3纯合突变个体1名,未发现CYP2C9*2、*4和*53种突变型等位基因,其中本研究受试人群中CYP2C9等位基因频率CYP2C9*1/*1占92.7%,CYP2C9*1/*3占6.6%,CYP2C9*3/*3占0.7%。药物代谢显示,CYP2C9*3纯合子个体的药物清除率显著低于CYP2C9*1纯合子个体。CYP2C9*1/*3杂合突变型个体组及1名CYP2C9*3/*3纯合型突变个体组,甲苯磺丁脲的代谢显著慢于野生型个体组。野生型(*1/*1)个体组、*1/*3杂合型突变个体组及1名*3/*3纯合型突变个体组,甲苯磺丁脲的药物代谢动力学参数差异与基因分型结果一致。结论CYP2C9多态性是甲苯磺丁脲药物代谢临床表现复杂性的主要因素,建立高效可靠的药代酶基因分型方法对CYP2C9代谢相关药物的临床个体化医疗具有一定的指导作用。     

Limited value of the urinary phenytoin metabolic ratio for the assessment of cytochrome P4502C9 activity in vivo

Relationships between the ratio of p -hydroxyphenytoin (p-HPPH), the major metabolite of phenytoin, to unchanged phenytoin excreted in urine (the urinary metabolic ratio or MR) were compared with a number of indices of the metabolic clearances of phenytoin and tolbutamide published previously for seventeen subjects separately administered these known cytochrome P4502C9 (CYP2C9) substrates. Significant correlations ( r _s=0.50–0.60, P <0.05) were observed between the phenytoin MR, derived from either 0–24 or 24–48  h urine collections, and inverse areas under the plasma unbound concentration-time curves (measured over various time intervals) of phenytoin and with plasma unbound tolbutamide clearance. Significant correlations ( r _s =0.59–0.74) were also observed between the phenytoin MRs and metabolic unbound clearances for p -hydroxyphenytoin formation. Despite the significant correlations, variability in tolbutamide and phenytoin metabolic clearance parameters tended to account for <50% of the variability in phenytoin MR. Correlations between the renal clearance of phenytoin and the phenytoin MRs suggest that variability in the renal clearance of unchanged drug limits the usefulness of the phenytoin MR for the investigation of factors influencing CYP2C9 activity in vivo .     

金雀异黄酮在体内对CYP3A活性的影响

目的:阐明金雀异黄酮在体内对细胞色素氧化酶P-4503A(CYP3A)活性的影响。方法:本试验采用双周期交叉临床试验。临床试验按照两阶段交叉方案进行。第一阶段随机分成的2组受试者随机给予金雀异黄酮片或安慰剂处理14 d,第15天给所有受试者米哒唑仑探针药,经过14 d洗脱期后交叉重复试验,分别以0~36 h血浆中米哒唑仑的血浆曲线下面积、半衰期作为CYP3A的活性指标。HPLC测定其活性。服药后0~36 h血中咪达唑仑以及1’-羟基咪达唑仑用HPLC-MS/MS方法测定。结果:经金雀异黄酮处理后,咪达唑仑AUC0-36 h明显降低[(144±55)ng.mL-1.hvs(126±40)ng.mL-1.h,P<0.05],AUC0-∞显著降低[(209±57)vs(181±43)ng.mL-1.h,P<0.05],Cmax显著降低[(49±20)vs(36±14),P<0.05]。结论:金雀异黄酮对体内CYP3A酶活性具有显著的诱导作用,应当关注经CYP3A代谢的药物与金雀异黄酮合用时可能发生的潜在的药物相互作用。     

拉米夫定片人体生物等效性评价

目的评价拉米夫定片在健康人体的生物等效性。方法健康志愿者22例,随机2×2交叉,单剂量口服受试和参比制剂拉米夫定片100mg,洗脱期为1周。采用LC．MS／MS法测定血药浓度,并用DAS药动学软件进行参数计算及评价2种制剂的生物等效性。结果单剂量口服受试和参比制剂后,血浆中拉米夫定的主要药动学参数：pmax分别为（881．44-236．8）和（985．0±292．4）μg·L-1,tmax分别为（O．989±0．323）和（1．0344-0．651）h,t1／2分别为（2．889±0．622）和（2．820±0．368）h,AUc-t分别为（3435±627．7）和（3504±506．7）μg·h·L-1,AUC0→∞分别为（3529±630．9）和（3592±519．4）μg·h·L-1,受试制剂与参比制剂的人体相对生物利用度（F）是（98．45±13．50）％。结论拉米夫定片受试制剂与参比制剂具有生物等效性。     

Selective inhibition of human cytochrome P4502C8 by montelukast.

The leukotriene receptor antagonist montelukast was examined for its inhibition of the human drug-metabolizing enzyme cytochrome P4502C8 (CYP2C8). Montelukast was demonstrated to be a potent inhibitor of CYP2C8-catalyzed amodiaquine N-deethylase, rosiglitazone N-demethylase, and paclitaxel 6alpha-hydroxylase in human liver microsomes. Inhibition was also observed when the reaction was catalyzed by recombinant heterologously expressed CYP2C8. The mechanism of inhibition was competitive, with K(i) values ranging from 0.0092 to 0.15 microM. Inhibition potency was highly dependent on the microsomal protein concentration. Increasing the microsomal protein concentration by 80-fold yielded a 100-fold decrease in inhibition potency. Preincubation of montelukast with human liver microsomes and NADPH did not alter the inhibition potency, suggesting that montelukast is not a mechanism-based inactivator. Montelukast was a selective inhibitor for human CYP2C8; inhibition of other human cytochrome P450 enzymes was substantially less. These in vitro data support the use of montelukast as a selective CYP2C8 inhibitor that could be used to determine the contribution of this enzyme to drug metabolism reactions. These data also raise the possibility that montelukast could have an effect on the metabolic clearance of drugs possessing CYP2C8-catalyzed metabolism as a major clearance pathway, thereby eliciting pharmacokinetic drug-drug interactions.     

Evaluation of cytochrome P450 2C9 activity in normal,healthy, adult Western Indian population by both phenotyping and genotyping

Objectives:Cytochrome P450 2C9 (CYP2C9) is a member of cytochrome P450 (CYP) family that accounts for nearly 18% of the total CYP protein content in the human liver microsomes and catalyzes almost 15–20% of the drugs. Considering the paucity of data on the polymorphisms of CYP2C9 in Western Indian population, the present study was conducted to evaluate the prevalence of CYP2C9 polymorphisms (*1, *2 and *3) and correlate it with the activity using flurbiprofen (FLB) as a probe drug.Results:Of the total 298 participants, phenotype was assessable in 288 and genotype was performed in 289 participants. The median (range) MR of the study population was 6.6 (1.65–66.05). Five participants were found to be PMs by phenotype. Of the total 289 participants, 209 (72.3%) (66.7, 77.2) had CYP2C9*1/*1, 25 (8.7%) (5.8, 12.7) with CYP2C9*1/*2, 55 (19%) (14.8, 24.1) had CYP2C9*1/*3, 3 (1%) (0.3, 3.3) had CYP2C9*2/*3 genotype. A significant association between phenotype and genotype was observed.Conclusion:To conclude, the present study found significant association of CYP2C9 activity by both phenotype and genotype and these findings have to be corroborated in different kinds of patients.KEY WORDS: Flurbiprofen, genotyping, phenotyping, polymorphism, probe drug     

Cytochrome P4502C9: an enzyme of major importance in human drug metabolism

Accumulating evidence indicates that CYP2C9 ranks amongst the most important drug metabolizing enzymes in humans. Substrates for CYP2C9 include fluoxetine, losartan, phenytoin, tolbutamide, torsemide, S-warfarin, and numerous NSAIDs. CYP2C9 activity in vivo is inducible by rifampicin. Evidence suggests that CYP2C9 substrates may also be induced variably by carbamazepine, ethanol and phenobarbitone. Apart from the mutual competitive inhibition which may occur between alternate substrates, numerous other drugs have been shown to inhibit CYP2C9 activity in vivo and/or in vitro . Clinically significant inhibition may occur with coadministration of amiodarone, fluconazole, phenylbutazone, sulphinpyrazone, sulphaphenazole and certain other sulphonamides. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino acid residues 144 (Arg144Cys) and 359 (Ile359Leu) of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, although the frequency of this allele is relatively low. Consistent with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. Individualisation of dose is essential for those CYP2C9 substrates with a narrow therapeutic index.     

Inhibition of tolbutamide 4-methylhydroxylation by a series of non-steroidal anti-inflammatory drugs in V79-NH cells expressing human cytochrome P4502C10

1. To study the role of cytochrome P4502C10 in the metabolism of the non-steroidal antiinflammatory drugs (NSAIDs) diclofenac, phenylbutazone, fenoprofen, ibuprofen, flurbiprofen, ketoprofen and naproxen, a cell line was developed stably expressing CYP2C10 cDNA. A retroviral vector construct, containing a human CYP2C10 cDNA, was transfected in V79-NH Chinese hamster lung cells by calcium phosphate co-precipitation. Sublines stably expressing human cytochrome P450 cDNA were established by selection with the neomycin analogue G418.

2. Enzymatic activity of CYP2C10 was detected by 4-methylhydroxylation of tolbutamide. This activity was inhibited to background levels by preincubation with the CYP2C9/10 inhibitor sulphaphenazole.

3. Preincubations with the NSAIDs ketoprofen, phenylbutazone, flu-biprofen and diclofenac (all 250 μM) caused a decrease in 4-methylhydroxylation of tolbutamide (500 μM), significantly different from control values (p < 0.05). Inhibition of this activity was not seen in preincubations with the NSAIDs fenoprofen, ibuprofen and naproxen (250 μM).

4. The V79-NH CYP2C10 cell line we have developed has been shown to be a useful tool to predict drug-drug interactions.     

Roles of two allelic variants (Arg144Cys and Ile359Leu) of cytochrome P4502C9 in the oxidation of tolbutamide and warfarin by human liver microsomes

1. Tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities were determined in liver microsomes of 39 Japanese and 45 Caucasians genotyped for the cytochrome P450 (P450 or CYP) 2C9 gene into three groups, namely the wild-type (Arg144·Ile359), and two heterozygous Cys allele (Cys144·Ile359) and Leu allele (Arg144·Leu359) variants. 2. Good correlations were found between tolbutamide methyl hydroxylation and racemic warfarin 7-hydroxylation activities in liver microsomes of Japanese and Caucasians. Humans with the Cys allele CYP2C9 variant, which was detected in 22% of Caucasians, were found to have similar catalytic rates to those of the wild-type in the oxidations of tolbutamide and racemic warfarin, whereas humans with the Leu allele, which was detected in 8% Japanese and 7% Caucasian samples, had lower catalytic rates than those of other two groups. 3. The rates of 6- and 7-hydroxylation of racemic warfarin were correlated well with those of S-warfarin, but not R-warfarin, in human liver microsomes. 4. Both human liver microsomes and recombinant CYP2C9 catalysed 7-hydroxylation of S-warfarin more extensively than those of R-warfarin. K_m's for the 7-hydroxylation of S-warfarin were not very different in liver microsomes of humans with these three genotypes. Anti-CYP2C9 antibodies and sulphaphenazole inhibited the 6- and 7- hydroxylation of S-warfarin, but not R-warfarin,by > 90% and the methyl hydroxylation of tolbutamide by about 50%. 5. These results suggest that humans with Leu allele of CYP2C9 have lower V_max's for S-warfarin 7-hydroxylation and tolbutamide methyl hydroxylation than those with wildtype and Cys allele CYP2C9, although the K_m's are not very different in liver microsomes m of these three groups of humans. R-warfarin hydroxylation may be catalysed by P450 enzymes other than CYP2C9 in man.     

Inhibitory effect of valproic acid on cytochrome P450 2C9 activity in epilepsy patients

Drug interactions constitute a major problem in the treatment of epilepsy because drug combinations are so common. Valproic acid is a widely used anticonvulsant drug with a broad therapeutic spectrum. Case reports suggest interaction between valproic acid and other drugs metabolized mainly by cytochrome P450 isoforms. The aim of this study was to evaluate the inhibitory effect of valproic acid on cytochrome P450 2C9 (CYP2C9) activity by using losartan oxidation as a probe in epilepsy patients. Patients were prescribed sodium valproate (mean 200 mg/day for the first week and 400 mg/day in the following period) according to their clinical need. A single oral dose of 25 mg losartan was given to patients before and after the first dose, first week and 4 weeks of valproic acid treatment. Losartan and E3174, the CYP2C9-derived carboxylic acid metabolite of losartan in 8 hr urine were assayed by using high pressure liquid chromatography. Urinary losartan/E3174 ratio did not change significantly on the first day (0.9, 0.3-3.5; median, range), and first week (0.6, 0.2-3.8; median, range), while a significant increase was observed after 4 weeks of valproic acid treatment (1.1, 0.3-5.7; median, range) as compared to that of measured before valproic acid administration (0.6, 0.1-2.1; median, range) (P = 0.039). The degree of inhibition was correlated with the steady-state plasma concentrations of valproic acid (r(2) = 0.70, P = 0.04). The results suggest an inhibitory effect of valproic acid on CYP2C9 enzyme activity in epilepsy patients at steady state. The risk of pharmacokinetic drug-drug interactions should be taken into account during concomitant use of valproic acid and CYP2C9 substrates.