【作者】 张玲玲；

【导师】 江善祥；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2010， 博士

【摘要】 本研究测定离子载体类抗生素对鸡CYP1A和3A的诱导作用及作用机制；氟喹诺酮类药物对CYP1A和3A的抑制作用及作用机制；经典抑制剂对鸡CYP1A和3A亚型的抑制作用,旨在探讨药物对鸡CYP4501A和3A的影响及影响机制,为临床合理用药提供理论依据。具体研究内容分为以下五部分：1.研究三种离子载体类抗生素对鸡CYP1A与3A活性的影响。将120 mg/kg的莫能菌素,60mg/kg的盐霉素和5mg/kg的马杜拉霉素分别添加到28日龄的雄性AA肉鸡饲料中,连续14d,通过体内探针实验和体外微粒体孵育实验观察CYP1A和3A活性的变化。体内探针采用的是咖啡因与氨苯砜,体外孵育实验采用的是0.5-20mM的非那西丁与0.125-5mM的咪达唑仑作为底物。体内探针实验测得三种离子载体类抗生素不影响咖啡因代谢的药动学参数(P>0.05)；使氨苯砜的药时曲线下面积AUC,消除半衰期t1/2有不同程度的降低,清除率Cl有不同程度的增加,体内实验结果表明离子载体类抗生素可诱导CYP3A的活性,盐霉素的诱导能力>莫能菌素>马杜拉霉素。体外微粒体孵育实验测得：三种离子载体抗生素组与对照组相比对乙酰氨基酚生成的最大反应速率Vmax及米氏常数Km均差异不显著(P>0.05)；莫能菌素极显著的加快1-羟基咪达唑仑的生成最大反应速率Vmax(P<0.01)；盐霉素组与马杜拉霉素组差异不显著(P>0.05)；三个处理组的米氏常数Km差异不显著(P>0.05)。体内体外结果表明饲料中连续14d添加莫能菌素、盐霉素、马杜拉霉素对鸡肝CYP3A的活性存在不同程度的诱导作用,其中马杜拉霉素的诱导作用最弱,而对CYP1A活性没有诱导作用。2.研究三种离子载体类抗生素对鸡CYP450与b5含量的影响,及对CYP1A与3A蛋白表达与mRNA表达的影响,以期了解离子载体类抗生素对CYP450活性的诱导机制。采用了分光光度计测定CYP450与b5含量；荧光定量PCR测定鸡CYP1A4,1A5与3A37mRNA的表达；免疫印迹测定CYP1A蛋白与3A蛋白的表达。结果表明CYP450与b5的含量各组间没有显著差异(P>0.05)；荧光定量PCR检测显示各组间CYP1A4,1A5与3A37mRNA表达没有显著差异(P>0.05)；免疫印迹结果显示莫能菌素组与盐霉素组的CYP3A蛋白表达增加,差异显著(P<0.05),而马杜拉霉素对CYP3A蛋白表达影响差异不显著(P>0.05)；三者对CYP1A蛋白的表达没有影响(P>0.05)。结果提示莫能菌素、盐霉素、马杜拉霉素不同程度地诱导鸡肝CYP3A的活性,而这种诱导作用通过转录后机制来调节。3.研究三种氟喹诺酮类药物对雄性AA肉鸡CYP1A与3A活性的影响.本实验给予恩诺沙星、沙拉沙星与麻保沙星的剂量分别为20,8,5.5 mg/kg BW,连续7d。通过体内探针实验和体外微粒体孵育实验观察对CYP1A和3A活性的抑制作用。体内探针实验测得恩诺沙星与麻保沙星显著增加了咖啡因的消除半衰期t1/2<0.05)；同时麻保沙星显著增加了咖啡因代谢的AUC,降低了Cl(P<0.05)；恩诺沙星显著增加了氨苯砜代谢的AUC和t1/2(P<0.05),极显著降低了Cl(P<0.01)。体外微粒体孵育实验表明,三种氟喹诺酮类药物组使对乙酰氨基酚生成的最大反应速率Vmax显著降低(P<0.05)；恩诺沙星组使1-羟基咪达唑仑的最大生成速率Vmax极显著的降低(P<0.01);三个药物处理组的米氏常数Km均有所下降,恩诺沙星组差异极显著(P<0.01)。体内体外结果表明高剂量恩诺沙星、沙拉沙星与麻保沙星均对鸡的CYP1A活性存在着不同程度的抑制作用,麻保沙星的抑制能力>恩诺沙星>沙拉沙星,同时高剂量的恩诺沙星抑制着CYP3A的活性,此剂量的麻保沙星与沙拉沙星尚未表现出对CYP3A的抑制作用。4.研究三种氟喹诺酮类药物对鸡CYP450与b5含量的影响,及对鸡CYP1A与3A蛋白表达与mRNA表达的影响。恩诺沙星、沙拉沙星、麻保沙星高剂量给药,连续7d,采用了分光光度计测定对CYP450与b5含量的影响；荧光定量PCR测定对鸡CYP1A4,1A5与3A37mRNA表达的影响；免疫印迹测定对CYP1A蛋白与3A蛋白表达的影响。结果表明,恩诺沙星与麻保沙星使CYP450的含量显著降低(P<0.05),沙拉沙星对其没有影响；各组间b5的含量没有显著差异；荧光定量PCR检测显示各组间CYP1A4,1A5与3A37mRNA表达没有显著差异；免疫印迹结果显示恩诺沙星使CYP1A和3A蛋白表达减少,差异显著(P<0.05),而麻保沙星对CYP1A蛋白表达呈现抑制的作用,差异显著(P<0.05),沙拉沙星对CYP1A与3A蛋白的表达没有影响。结果提示高剂量的恩诺沙星抑制雄性肉鸡CYP1A和3A的活性和蛋白表达,麻保沙星抑制CYP1A的活性和蛋白表达。5.比较鸡与大鼠肝微粒体代谢非那西丁与咪达唑仑的活性,研究经典抑制剂α萘黄酮和酮康唑及三种氟喹诺酮类药物恩诺沙星、沙拉沙星、麻保沙星对鸡非那西丁O-脱乙基化与咪达唑仑1-羟基化活性的抑制程度及抑制机制。取肝微粒体混合样,建立体外孵育体系,以对乙酰氨基酚和1-羟基咪达唑仑的生成速率反映CYP1A与3A的活性。鸡与大鼠代谢非那西丁与咪达唑仑的过程均符合米曼氏动力学特征,代谢非那西丁的Vmax分别为0.089±0.053和0.671±0.288nmol/mg/min, Km分别为78.45±48.10和107.1±45.5μM,代谢咪达唑仑的Vmax分别为0.081±0.022和0.781±0.275nmol/mg/min, Km分别为5.33±2.56和1.85±0.67μM；α萘黄酮对鸡非那西丁的代谢呈现非竞争性抑制作用,IC50=0.1304μM,ki=0.580μM,而麻保沙星的ICso值为93.79μM, Ki=77.12μM,为竞争性与非竞争性混合型抑制作用,未检测到恩诺沙星与沙拉沙星对非那西丁代谢的抑制作用。酮康唑对咪达唑仑1-羟基化活性的可逆抑制机理与恩诺沙星、沙拉沙星和麻保沙星相同,均为非竞争性与反竞争性混合型抑制,IC50值分别为30.6,63.93、145.4和42.52μM, Ki值分别为93.01,70.76、150.84与64.24μM；体外检测到恩诺沙星、沙拉沙星和麻保沙星均对非那西丁和咪达唑仑的代谢呈现时间依赖性的抑制作用。结果表明与大鼠相比,鸡的CYP1A和3A的活性均很低；a萘黄酮对鸡CYP1A的抑制作用很强；CYP3A的经典抑制剂酮康唑对鸡咪达唑仑1-羟基化活性的抑制作用并不强,,而与恩诺沙星、沙拉沙星与麻保沙星相当；时间依赖性的不可逆抑制作用可能是导致CYP450活性降低和蛋白表达减少的原因,最终会导致体内药物的相互作用。更多还原

【Abstract】 This research determined the induction effects and mechanism of ionophores, the inhibitory effects and mechanism of fluoquinolones and the inhibitory effects of classic inhibitors on CYPIA and 3A in broiler chicks. The purpose of research was to investigate pharmacological mechanism of clinical drug on chicken CYPIA and 3A enzyme, thus to provide a theoretical basis for clinical application. The details are divided into five parts as follows:1. To investigate the effects of three ionophore antibiotics on the enzyme activities of CYP1A and 3A in broiler chicks. Monensin, salinomycin and maduramycin at the dosage of 120,60 and 5 mg/kg were administrated in feed for 14 days. CYP1A and 3A activities were quantitated using cocktail probe drug including caffeine and dapsone in vivo and 0.5-20 mM phenacetin and 0.125-5 mM midazolam as substrates of microsome incubation in vitro. The results in. vivo indicated that three ionophores did not change the pharmacokinetics parameters of caffeine (P>0.05), but did decrease the AUC and t1/2 of dapsone and increase the total clearance, compared with control group, and the induction ability on dapsone metabolism is salinomycin> monensin> maduramycin. The results of microsome incubation trials indicated that three ionophores didn’t change the Vmax of acetaminophen production (P>0.05). Michaelis constant Km showed no significant difference with control group (P>0.05). Monensin significantly increased the production velocity of 1-hydroxy midazolam (P<0.01), while salinomycin and maduramycin increasd it with no significant difference (P>0.05). Three ionophore-treated groups had the similar Km value as control group (P>0.05). The similar results in vivo and in vitro suggest that monensin, salinomycin and maduramycin in feed for 14 days may have an induction effect on CYP3A enzyme activity of male AA broilers and maduramycin has the least induction effect, while they all don’t induce the CYP1A enzyme activity.2. To know the induction mechanism, the effect of three ionophore antibiotics on the CYP450, b5 content and the mRNA expression of hepatic CYP1A4,1A5 and 3A37 mRNA and the protein expression of CYP1A and 3A were investigated in broiler chicks. Content of CYP450 and b5 was measured by spectrophotometry. The mRNA levels of CYP1A4, CYP1A5 and CYP3A37 was detected by Real-time reverse transcriptase-polymerase chain reaction (Real-time PCR). The protein expression of CYP1A and CYP3A was detected by Western blot. No change was found in the CYP450 and b5 content, and CYP1A4, CYP1A5 and CYP3A37 mRNA expression in ionophore-treated groups compared with control group. Monensin and salinomycin increased the CYP3A protein expression with significant difference (P<0.05). However, maduramycin increased it with no significance difference (P>0.05). No change was observed in CYP1A protein expression. It is suggested that ionophore antibiotics have an induction effect on CYP3A expression and enzyme activity and such effects might be related to the post-transcriptional regulation of its protein expression.3. To investigate the inhibitory effects of three fluoquinolone antibiotics on the enzyme activities of CYP1A and 3A in broiler chicks. Enrofloxacin, sarafloxacin and marbofloxacin at the dosage of 20,8 and 5.5 mg/kg body weight were administrated for 7 days. Inhibitory effects of fluoquinolones on CYP1A and 3A activities were quantitated using cocktail probe drugs in vivo and microsome incubation in vitro. The results in vivo indicated that enrofloxacin and marbofloxacin increased the t1/2 of caffeine (P<0.05), and marbofloxacin increased the AUC, decreased the total clearance, compared with control group(p<0.05). Enrofloxacin increased AUC and t1/2 of dapsone metabolism (P<0.05), and decreased the total clearance (P<0.01). The results of microsome incubation trials indicated that all of three fluoquinolones decreased the Vmax of acetaminophen production (P<0.05). Enrofloxacin significantly decreased the production velocity of 1-hydroxy midazolam and the Km value (P<0.01), while sarafloxacin and marbofloxacin groups had the smaller Km value with no significant difference (P>0.05). The results in vivo and in vitro suggest that high dosage of enrofloxacin, sarafloxacin and marbofloxacin may have an inhibitory effects on CYP1A enzyme activity of broilers (marbofloxacin> enrofloxacin> sarafloxacin). Meanwhile, CYP3A activity could be inhibited by high dosage of enrofloxacin, but not sarafloxacin and marbofloxacin.4. To investigate the effect of three fluoquinolone antibiotics on the CYP450, b5 content, the CYP450 mRNA expression and CYPIA and 3A protein expression in broiler chicks. Enrofloxacin, sarafloxacin and marbofloxacin at twice the recommended dosage were administrated for 7 days. Content of cytochrome P450 and b5 was measured by spectrophotometry. The mRNA levels of CYP1A4, CYP1A5 and CYP3A37 was detected by Real-time PCR. The protein expressions of CYP1A and CYP3A was detected by Western blot. The results indicated that enrofloxacin and marbofloxacin decreased CYP450 content (P<0.05), while sarafloxacin didn’t change it. No change was found in the b5 content, and CYP1A4, CYP1A5 and CYP3A37 mRNA expression in fluquinolone-treated groups compared with control group. Enrofloxacin and marbofloxacin decreased the CYP1A protein expression with significant difference (P<0.05), meanwhile, enrofloxacin decreased the CYP3A protein expression (P<0.05). However, this high dosage of sarafloxacin did no effect on CYP1A and 3A protein expression. It is suggested that high dosage of enrofloxacin may inhibit CYPIA and 3A activities and protein expression, while marbofloxacin inhibit CYPIA activity and protein expression.5. Phenacetin O-deethylation and midazolam 1-hydroxylation activities were measured in chickens and rats. The inhibitory effects and mechanism of classic inhibitor a-napthtoflavone, ketoconazole and three fluoquinolone antibiotics were examined on phenacetin O-deethylation and midazolam 1-hydroxylation activities in chickens. We used mixed microsome incubation in vitro, and the formation velocity of acetaminophen and 1-hydroxy midazolam were referred as CYPIA and 3A activities. Both chicks and rats demonstrated Michaelis-Menten kinetics for the phenactin and midazolam metabolite. The Vmax for phenactin of chicks and rats was 0.089±0.053,0.671±0.288 nmol/mg/min and Km was 78.45±48.10,107.1±45.5μM, respectively. The Vmax for midazolam was 0.081±0.022,0.781±0.275μmol/mg/min and Km 5.33±2.56,1.85±0.67μM, respectively. a-napthtoflavone inhibited the phenacetin metabolism in a noncompetitive manner and IC50 = 0.1304μM, Ki= 0.580μM, while marbofloxacin with IC50= 93.79μM, Ki= 77.12μM, in a competitive-noncompetitive mixed manner. No inhibitory effect was examined of enrofloxacin and sarafloxacin. Ketoconazole inhibited the midazolam metabolism in the noncompetitive-uncompetitive mixed manner, as the same like three fluoquinolones. The IC50 value was 30.6,63.93,145.4 and 42.52μM, and Ki value was 93.01,70.76,150.84 and 64.24μM, respectively. Enrofloxacin, sarafloxacin and marbofloxacin showed time-dependent irreversible inhibitory effects on both phenacetin and midazolam metabolism. The result showed that the CYP1 A and 3A activities were markedly lower in chickens than in rats. a-napthtoflavone had strong.inhibitory effect on chicken CYP1 A activity, while ketoconazole had only weak inhibitory effect on chicken CYP3A activity like enrofloxacin, sarafloxacin and marbofloxacin. The time-dependent inhibitory may be the reason for the decreased CYP1A and 3A enzyme activities and protein expression, and the irreversible inhibitory would result in substantial drug-drug interaction in vivo.更多还原