喹赛多在猪、鸡、鱼和大鼠体内代谢及分布研究
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摘要
喹赛多是一种喹噁啉类抗菌促生长动物专用药,对畜禽具有明显促生长和保健的作用,且副作用小、安全性高、用药后吸收快、消除迅速、残留期短,有望成为喹噁啉类高效安全的新药。在新药研究中要明确药物有效性和安全性,揭示药理活性成分和毒理作用靶点是关键,药物代谢和分布是基础。目前美国食品和药物管理局(FDA)和欧盟(EU)推荐采用放射性示踪法研究新药的代谢分布。放射性同位素作为示踪剂具有灵敏度高,测量方法简便易行,能准确地定位及符合所研究对象的生理条件等特点,已广泛应用于医药学领域,用以揭示药物的体内分布、代谢与排泄过程。本课题采用放射性示踪技术与液相色谱-离子阱-飞行时间质谱(LC/MS-IT-TOF)技术,开展喹赛多在猪、鸡、鱼和大鼠体内的代谢、分布及排泄研究,发现并鉴定喹赛多在各种动物的代谢物,分析各代谢物在动物体内的分布和消除规律,确定喹赛多在各个动物体内的残留靶组织和残留标示物,分析种属差异,为喹赛多食品安全性标准的制定和药理毒理作用机制的深入探讨提供依据。
前期研究发现喹赛多促生长机理和药理活性与肠道环境和代谢有关。喹赛多的生物利用度较低,在肠道中驻留,肠道中的大量的药物代谢酶和细菌代谢酶使喹赛多产生降解,然而代谢物及药理毒理活性尚不清楚。因此,本课题建立体外肠道代谢模型,采用LC/MS-ITTOF技术,开展喹赛多在猪、鸡、鱼和大鼠肠微粒体、肠黏膜和肠道细菌中代谢研究,发现鉴定喹赛多在动物肠道体外模型中代谢物,明确喹赛多在肠道中特有的代谢物分析代谢物不同的产生部位,阐明肠道代谢途径分析种属差异,为代谢物药理和毒理研究提供基础。
1喹赛多在动物体内的物料平衡和代谢研究
猪、鸡、鱼和大鼠各6头(只),分别一次性灌胃给药(3H喹赛多),给药后每24h收集粪便、尿液、血液和胆汁(鱼检测活动范围内的水),一部分样品消化之后经静态液闪仪测定放射性活度,直到排泄物中放射性检测不到为止,计算氚标喹赛多的排泄情况。一部分样品经提取之后,采用流动液闪仪和HPLC/MS-IT-TOF鉴定代谢产物。
猪给药后12天,放射性总共排出96.1%,尿中排出44.5%,粪便中排出51.6%。鸡给药后12天,放射性总共排出95.2%。鱼给药后12天,放射性总共排出97.4%。大鼠给药后12天,放射性总共排出94.5%,尿中排出28.3%,粪便排出66.2%。喹赛多在动物体内主要是通过粪便排出体外,排泄迅速,给药后3天放射性排出可达到90%以上。
在猪尿液中检测到原形(Cy0)和13种代谢物,脱氧代谢物Cy1、Cy2和Cy10,氢化代谢物Cy4和Cy9,羟化代谢物Cy7和Cy8,侧链断裂代谢物Cy5、Cy6、Cy11和Cy12,甘氨酸结合物Cy3,水解代谢物Cy13和Cy14；粪便中发现原形(Cy0)和10种代谢物(Cy1、Cy2、Cy3、Cy4、Cy5、Cy6、Cy7、Cy9、Cy11和Cy12);猪血液中检测到11种代谢物(Cy1、Cy2、Cy3、Cy4、Cy5、Cy6、Cy9、 Cy11、Cy12、Cy13和Cy14)；在猪的的胆汁中检测到13种代谢物(Cy1、Cy2、Cy3、Cy4、Cy5、Cy6、Cy7、Cy8、Cy9、Cy11、Cy12、Cy13和Cyl4)。
在鸡粪便中检测到原形(Cy0)和7种代谢物(Cy1、Cy2、Cy4、Cy5、Cy6、 Cy7和Cy15),鸡血液中检测到4种代谢物(Cy2、Cy4、Cy5和Cy6),鸡胆汁中检测到原形(Cy0)和7种代谢产物(Cy1、Cy2、Cy4、Cy5、Cy10、Cy11和Cy13)。在鱼的排泄物中发现了大量的原形(Cy0)和2种代谢物(Cy2和Cy13),鱼血液中检测到3种代谢物(Cy1、Cy2和Cy5)。在大鼠的尿液中检测到6种代谢物(Cy1、Cy4、Cy5、Cy6、Cy9和Cy11)；粪便中检测到原形(Cy0)和7种代谢产物(Cy1、Cy2、Cy4、Cy5、Cy6、Cy11和Cy15)；大鼠血液中检测到5种代谢物(Cy1、Cy4、 Cy5、Cy6和Cy11)。
喹赛多在四种动物体内的主要代谢途径为：一是N-O键还原生成脱氧代谢产物；二是发生侧链C=N键的断裂,发生羟化和氧化；三是发生C=N键的氢化；四是发生酰胺键的水解。Cy3(Cy6甘氨酸结合物)只在猪体内发现：在鸡和大鼠体内发现了Cy15,在猪和鱼中没有检测到。四种动物中,鱼的代谢能力最差,在排泄物中能够检测到大量的原形和两种代谢物。通过喹赛多在不同种属动物体内的代谢研究,分析种属差异,对药理活性成分和代谢物毒性研究具有重要的指导意义。
2喹赛多在动物体内的分布和残留消除
24头猪(36只大鼠)随机分为6组,30只鸡(30尾鱼)随机分为5组,连续7天灌胃给药(3H喹赛多),在给药后6小时(h)、1天(d)、3d、7d、14d和21d宰杀(鸡和鱼为6h、1d、3d、7d和14d),取血液、胆汁、肝脏、肺脏、脾脏、皮肤、胃、大肠、小肠、肾脏等组织,消化液(solvable)水浴消化,经液闪仪检测放射活度测定药物在体内各组织中的分布情况；利用乙腈、乙酸乙酯和碱解法对可食性组织样品进行提取、净化,测定提取液放射活度计算提取率,然后进行LC/MS-IT-TOF流动液闪进行代谢物分离鉴定和定量。
喹赛多在猪体内广泛分布。给药后6h,肝、胆汁、肾和消化道中药物浓度高,胆汁中浓度超过14mg/kg。脂肪中浓度最低,只有0.9mg/kg。24h,大多数组织的放射性活度只有6h的一半左右,3d,绝大多数组织的浓度已下降到0.5mg/kg以下。7d,大部分组织中的浓度接近或低于0.1mg/kg,一些组织已不能检出。14d,只有肝和肾中能检出少量药物。6h猪的肝中能检测到8种代谢物,分别为Cy1、Cy3、Cy4、Cy5、Cy6、Cy9、Cy11和Cy12,7d时只检测到了Cy1。6h猪肾中检测到8种代谢物,分别是Cy1、Cy3、Cy4、Cy5、Cy6、Cy9、Cy11和Cy12。7d时只能检出Cy1。6h猪肌肉能检出8种代谢物,分别是Cy1、Cy3、 Cy4、Cy5、Cy6、Cy9、Cy11和Cy12。3天也只检出Cy3。6h猪脂肪中可检出6种代谢物Cy1、Cy2、Cy3、Cy4、Cy5和Cy6。肌肉、脂肪、肠道和血液中药物消除最快,消除半衰期为0.5～0.6d。其次为脾、肺、胃和肾上腺,消除速度相近,消除半衰期分别为1.08d～1.53d。肝脏和肾脏中药物消除速度较慢,消除半衰期分别为2.39d和2.57d。总放射性检测结果显示,喹赛多及其代谢物在肾脏中残留量最高,持续时间最长,消除速度最慢,因此肾脏是残留靶组织。猪肾中Cyl消除最慢,消除半衰期2.67d,与总残留相近,总体消除趋势相似,因此Cy1被确定为残留标示物。
喹赛多在鸡组织中分布广泛。给药后6h,胆汁、肝脏、肾脏和消化道的药物浓度高,胆汁中浓度甚至超过15mg/kg。脂肪中浓度最低,只有1.0mg/kg。24h,大多数组织的药物浓度下降到0.4mg/kg以下,但胆汁中浓度高达2.56mg/kg。3d,除肾和小肠外,其他组织中浓度均在0.2mg/kg。7d,只有肝脏和肾脏中有少量药物检出。6h鸡肝中检出原形和6种代谢物,分别是Cy1、Cy2、Cy4、Cy5、Cy6和Cy15。7d只检测到Cyl。6h,鸡肾脏检测到原形和6种代谢物,分别是Cy1、 Cy2、Cy4、Cy5、Cy6和Cy15。7d也只检测到Cy1。6h,鸡肌肉中检出原形和4种代谢物,分别是Cy1、Cy4、Cy5和Cy6。肌肉、脂肪、大肠、肺、肌胃和肾上腺消除最快,消除半衰期在0.63-0.67天之间。脾、小肠和血液次之,消除半衰期在0.73-0.95d之间。以上组织消除半衰期比较接近,都没有超过1d。肝脏和肾脏中药物消除速度较慢,消除半衰期分别为1.39d和2.38d。喹赛多及其代谢物在肾脏中残留量最高,持续时间最长,消除速度最慢,因此肾脏是残留靶组织。鸡肾中,Cyl消除最慢,消除半衰期3.15d,与总残留相近,总体消除趋势相似,因此Cyl被确定为残留标示物。
喹赛多在鱼体内广泛分布。给药后6h,肝、脾、肾和消化道中药物浓度高。脂肪中浓度最低,只有0.32mg/kg。24h,大多数组织的放射性活度下降迅速,大部分都在1.0mg/kg以下,但肝、肾和消化道中的浓度仍然较高,肠中的浓度仍在1.6mg/kg以上。3d,绝大多数组织的浓度已下降到0.5mg/kg以下。7d,只有肝和肠道中能检出少量药物。6h,鱼肝中检出5种代谢物,分别为Cy1、Cy2、 Cy4、Cy5和Cy10,并检测到原形药物。7d只检测到Cy1。6h,鱼肾脏检测到了6种代谢物,分别为Cy1、Cy2、Cy4、Cy5、Cy6和Cy10,并检测到原形药物。3d只检测到Cyl。6h,鱼肌肉检测到4种代谢物,分别为Cy1、Cy2、Cy4和Cy5,并检测到原形药物。3d只检测到Cy4,7d检测不到代谢物。鱼肠道检测到6种代谢物,分别为Cy1、Cy2、Cy4、Cy5、Cy6和Cy10,并检测到原形药物。7d可以检测到Cyl。脾消除最快,消除半衰期只有0.44d。心、肾脏和皮肤次之,消除半衰期在0.51-0.78d之间。以上组织消除半衰期比较接近,都没有超过1d。肝脏和肠中药物消除速度较慢,消除半衰期分别为1.14d和1.24d。总放射性检测结果显示,喹赛多及其代谢物在肠中残留量最高,持续时间最长,消除速度最慢,因此肠是残留靶组织。鱼肠道中,Cyl消除最慢,消除半衰期1.82d,与总残留相近,总体消除趋势相似,Cyl被确定为残留标示物。
喹赛多在大鼠体内也广泛分布。给药后6h,肝脏、肾脏、膀胱、肾上腺和消化道中药物浓度高,脂肪最低(0.42mg/kg)。24h,大部分组织的药物浓度下降50%以上。3d,所有组织中的药物浓度均下降到0.5mg/kg以下。7d,大多数组织的浓度在0.1mg/kg以下,有些组织已不能测出。14d,只有肝脏和肾脏中有少量药物检出。6h,大鼠肝中检出6种代谢物,分别为Cy1、Cy4、Cy5、Cy6、Cy9和Cy11,但没有检出原形。7d只检测到Cy5。6h,大鼠肾脏检测到了6种代谢物,分别为Cy1、Cy2、Cy4、Cy5、Cy6和Cy9。7d只检测到Cy4。6h大鼠肌肉中检测到5种代谢物,分别为Cy1、Cy4、Cy5、Cy6和Cy9。3d检测到Cy4和Cy5。肌肉、脂肪和血液中药物消除最快,消除半衰期分别为1.0d、1.14d和1.26d。其次为小肠、大肠和肾上腺,消除速度相近,消除半衰期分别为1.61d、1.58d和1.44d。肝脏中药物消除速度较慢,消除半衰期为2.57d。肾脏中药物消除速度最慢,消除半衰期为4.33d。喹赛多及其代谢物在肾脏中残留量最高,持续时间见最长,消除速度最慢,因此肾脏是残留靶组织。大鼠肾脏中,Cy4消除最慢,消除半衰期2.04天,但是Cyl的消除半衰期与总残留最相近。
最终确定,肾脏是猪、鸡和大鼠的残留靶组织,肠道是鱼的残留靶组织。Cyl是猪、鸡和鱼的残留标示物。Cy1、Cy4和Cy5可能是喹赛多药理和毒理作用的主要化合物。
3喹赛多在动物体外肠道代谢研究
根据肠道代谢的特点本课题建立了肠微粒体、肠黏膜、结肠内容物和回肠内容代谢系统。制备猪、鸡、鱼和大鼠肠微粒体和肠道黏膜,与烟酰胺腺嘌呤二核苷酸磷酸(NADPH)再生系统进行孵育,采用细胞色素3A(CYP3A)的探针药物硝苯地平验证体系的活性,采用Brandford法测定蛋白浓度。结果表明,硝苯地平在肠微粒体和肠道黏膜中能够生成氧化的硝苯地平,表明肠黏膜和肠微粒体代谢酶活性良好,可以用于药物代谢研究。
在厌氧条件下,制备结肠内容物和回肠内容物体外孵育液,通过对肠道细菌总厌氧菌、总需氧菌、大肠杆菌、双歧杆菌、乳酸菌和脆弱拟杆菌进行计数,同时与探针酶底物p-硝基苯β-D-葡糖醛酸苷、p-硝基苯β-D-半乳糖苷和p-硝基苯β-D-半乳糖苷孵育验证体系的代谢活性。结果表明,细菌数量在0-48h内保持了良好的稳定性,酶活力稳定而且符合标注,可以用于代谢研究。
在猪、鸡、鱼和大鼠肠微粒体NADPH再生系统中只发现了一种代谢物(Cy2),在猪、鸡和大鼠肠黏膜NADPH再生系统中只发现了脱二氧代谢产物(Cy1)和脱一氧代谢物(Cy2和Cy10),在鱼肠黏膜中发现了Cyl和Cy2。
在猪、鸡和大鼠结肠内容物代谢体系中分别检测到了4种、4种和5种代谢产物,在鱼肠内容物中检测到了6种代谢物。猪和鸡中代谢物分别为Cy1、Cy2、Cy4和Cy5,大鼠中代谢物为Cy1、Cy2、Cy4、Cy5和Cy10,鱼中Cy1、Cy2、Cy9和Cy10。Cy1和Cy4是猪、鸡和大鼠结肠细菌代谢系统中的主要代谢产物,Cyl是鱼肠道细菌代谢系统中的主要代谢物。
在回肠内容物代谢体系中,猪、鸡和大鼠分别检测到5种代谢物。猪回肠内容物中检测到Cy1、Cy2、CylO、Cy13和Cy16,鸡和大鼠回肠内容物中检测到相同的代谢产物。
在动物的肠道肠黏膜代谢体系中,喹赛多主要发生了脱氧代谢。在肠道细菌代谢体系中,喹赛多主要发生了脱氧代谢,氢化反应,羟化反应,水解反应和侧链的断裂反应。羟化反应和水解反应只在小肠细菌中发生,氢化和断裂反应只在结肠体系中发生。在四种动物肠黏膜中,喹赛多代谢没有差异。在鸡回肠内容物中Cy2的比例最高,这可能与鸡肠道酸性有关系。在鱼肠道中发现了氢化代谢物,这可能与鱼类肠道细菌代谢有关。综合体内代谢和前期肝脏代谢发现,Cy4是在肠道细菌的作用下产生的。
综上所述,本课题首次采用放射性示踪和LC/MS-IT-TOF技术研究喹赛多在猪、鸡、鱼和大鼠体内的代谢、分布和排泄,分离并鉴定了猪、鸡、鱼和大鼠体内的所有代谢产物,并对可食性组织中代谢物进行了定量,阐明了总残留和代谢在各个器官中的消除特征,确定了喹赛多的残留靶组织和残留标示物。本研究是喹赛多毒副作用机制研究、药理活性和毒性化合物研究的基础；对将来体内残留分析方法建立,最大残留限量和休药期的制定具有重要的指导意义。
Cyadox (CYX),2-formylquinoxaline-1,4-dioxide cyanoacetylhydrazone, is an antimicrobial and growth-promoting feed additive of quindoxaline class for food-producing animals. CYX is an excellent antibacterial growth promoter and has a lot of good quality, such as lesser toxicity and side effects, high security, rapid elimination and short residue after treatment, which has been recognized to be a safe and effective candidate of feed additive. Drug efficacy and safety were important in new drug study. The pharmacological active ingredient and toxicity targets were found based on drug metabolism and distribution studies. The Food and Drug Administration (FDA) and the European Union (EU) recommended that radiotracer methodology was adopted in drug absorption, disposition, metabolism and depletion studies. Radioisotope measurement method is simple, accurate positioning with high sensitivity and corresponding to physiological conditions, which has been widely used in the field of medicine to reveal drug distribution, metabolism and excretion process.. This study was to investigated metabolism, distribution and excretion of cyadox in swine, broiler, carp and rats by radiotracer technique and high-performance liquid chromatography-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). All unknown metabolites were identified by those techniques. The residue target organ and residue marker were determined, the data of species differences were obtained based on analysis of distribution and depletion of cyadox in animals. It will provide the basis for determining the standards of food safety of cyadox and exploring the depth of the toxicological mechanism of caydox action.
The preliminary studied demonstrated that the growth promoting mechanism and pharmacological activity of cyadox were relative to cyadox metabolism in intestine. The bioavailability of cyadox was low, then a lot of cyadox remained in intestine, which was metabolized by drug metabolizing enzymes and bacterial metabolic enzymes in the gut. However, the metabolites and their toxicological activity were not clear. Hence, the intestinal metabolic systems of swine, broilers, carp and rats were established. The metabolism of cyadox was investigated in intestinal microsomes, intestinal mucosa and gut microflora using LC/MS-IT-TOF. All unknown metabolites were characterized to reveal the metabolic organ of cyadox. The whole metabolic pathways were obtained to analysis of species differences. It will provide a basis for pharmacological and toxicological studies of cyadox and its metabolites.
1Mass balance and metabolism of cyadox in vivo
Six animals (swine, broilers, carp and rats) for material balance test were administrated one single dose. Urine and feces were collected per24h (water was collected in carp). Blood and bile were also collected. The total of radioactivity was detected by liquid scintillation after digestion. The metabolites were identified by LC/MS-IT-TOF after extracted by agents.
The total radioactive recovery (TRR) was96.1%,44.5%in urine,51.6%in feces after12days post dose. TRR was95.2%in broilers after12days post dose. The excretion rate was97.4%in carp after12days. In rats, TRR was94.5%after12days,28.3%in urine,66.2%in feces. Those results demonstrated that the excretion rate of cyadox reached to more than90%at3days after administration, and feces was main route of excretion.
The parent drug (Cy0) and its thirteen metabolites were identified in swine urine, deoxidation metabolites (Cy1, Cy2and Cy10), hydrogenation metabolites (Cy4and Cy9), hydroxylation metabolites (Cy7and Cy8), side chain cleavage metabolites (Cy5, Cy6, Cy11and Cy12), glycine conjugate (Cy3), hydrolysis metabolites (Cy13and Cy14). The parent drug and ten metabolites (Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7, Cy9, Cy11and Cy12) were identified in feces. Eleven metabolites (Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy9, Cy11, Cy12, Cy13and Cyl4) were identified. Thirteen metabolites (Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7, Cy8, Cy9, Cy11, Cy12, Cy13and Cy14) were found in swine bile.
The parent drug and seven metabolites (Cy1, Cy2, Cy4, Cy5, Cy6, Cy7and Cy15) were detected in broilers feces. Four metabolites (Cy2, Cy4, Cy5and Cy6) were found in broilers blood. The parent drug and seven metabolites (Cy1, Cy2, Cy4, Cy5, Cy10, Cy11and Cy13) were detected in broilers bile. The parent drug and two metabolites(Cy2and Cy13) were identified in carp excreta. Three metabolites (Cy1, Cy2and Cy5)were found in carp blood. Six metabolites (Cy1, Cy4, Cy5, Cy6, Cy9and Cy11) were detected in rats urine. The parent drug and seven metabolites (Cy1, Cy2, Cy4, Cy5, Cy6, Cyll and Cy15) were identified in rats feces. Five metabolites (Cy1, Cy4, Cy5, Cy6and Cy11) were found in rats blood.
Four main metabolic pathways were found in four species, the reduction of N-O bond, the cleavage of C=N bond and oxidation, the hydrogenation of C=N bond, the hydrolysis of amid bond. Glycine conjugate of quinoxaline-2-carboxylic acid was found only in swine. Cy15was detected in broiler and rats, and not found in swine and carp. The metabolic ability of carp was the weakest in four species. A large number of parent drug and a few metabolites were detected in carp excreta. The metabolic study and analysis of species differences has important guiding significance for the study of pharmacologically active ingredient and metabolite toxicity.
2Disposition and depletion of cyadox in vivo
Twenty-four swine and thirty-six rats were divided into six groups (thirty carp and thirty broilers for five groups) for7consecutive days administration based on a feeding rate of body weight per day. Swine and rats were slaughtered at6h,1d,3d,7d,14d and21d after last dose (broilers and carp for6h,1d,3d,7d and14d). Blood, bile, liver, lung, spleen, skin, stomach, large intestine, small intestine, kidney et al were collected for determination of total radioactivity after digestion. The edible tissues were extracted by different agents (acetonitrile, ethyl acetate and alkaline hydrolysis) for determining the extraction efficiency. The metabolite quantitative was performed by flow liquid scintillation. The metabolites were identified by LC/MS-IT-TOF.
3H-Cyadox was widely distributed in swine. The concentrations of radioactivity in liver, bile, kidney and intestine were the highest at6h after continuous administration7days, and it was more than14mg/kg in bile. The concentration in fat was the lowest, only0.9mg/kg. The concentration of radioactivity in most organs were reduced to50%at24h; they were declined to less than0.5mg/kg at72h; they were reduced to under0.1mg/kg at7d; the radioactivity was only detected in liver and kidney at14d. Eight metabolites (Cy1, Cy3, Cy4, Cy5, Cy6, Cy9, Cy11and Cy12) were found in swine liver at6h post final dose. Cy1was only metabolite which can be detected at7days. Eight metabolites (Cy1, Cy3, Cy4, Cy5, Cy6, Cy9, Cy11and Cy12) were identified in swine kidney at6h, and Cyl can be detected at7days. The identical metabolites were found in swine muscle at6h, and Cy3can be detected at3days. Six metabolites (Cy1, Cy2, Cy3, Cy4, Cy5and Cy6) were identified in swine fat, and only Cy3was found at3days. The depletion speed of muscle, fat, intestine and blood was the fastest. Their elimination half-lives were between0.5d-0.6d. Followed by spleen, lung, stomach, and adrenal glands, their elimination half-lives were between1.08d and1.53d. The drug elimination in liver and kidney was the slowest, and half-lives were from2.39d to2.57d. The total radioactivity determining showed that the concentration of drug in kidney was the highest, the residue time was the longest. Therefore, kidney was determined as residue target tissues. In kidney, the speed elimination of Cy1was the slowest. Its elimination half-life (2.67d) was relative to total residue. The elimination trend of Cy1was similar to total residue. Hence, Cy1 was determined as residue marker.
3H-Cyadox was widely distributed in broilers. The concentrations of radioactivity in liver, bile, kidney and intestine were the highest at6h after continuous administration7days, and it was more than14mg/kg in bile. The concentration in fat was the lowest, only0.1mg/kg. The concentration of radioactivity in most organs were reduced to under0.4mg/kg at24h, but it was more than2.56mg/kg in bile; they were declined to less than0.4mg/kg at72h except kidney and small intestine; the radioactivity was only detected in liver and kidney at7d. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy15) were found in swine liver at6h post final dose. Cy1was only metabolite which can be detected at7days. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy15) were identified in swine kidney at6h, and Cy1can be detected at7days. Four metabolites (Cy1, Cy4, Cy5and Cy6) were identified in muscle. The depletion speed of muscle, fat, large intestine, lung, muscular stomach and adrenal glands was the fastest. Their elimination half-lives were between0.63d-0.67d. Followed by spleen, small intestine and blood, their elimination half-lives were between0.73d and0.95d. The drug elimination in liver and kidney was the slowest, and half-lives were from1.39d to2.38d. The total radioactivity determining showed that the concentration of drug in kidney was the highest, the residue time was the longest. Therefore, kidney was determined as residue target tissues. In kidney, the speed elimination of Cyl was the slowest. Its elimination half-life (3.15d) was relative to total residue. The elimination trend of Cyl was similar to total residue. Hence, Cy1was determined as residue marker.
3H-Cyadox was widely distributed in carp. The concentrations of radioactivity in liver, spleen, kidney and intestine were the highest at6h after continuous administration7days. The concentration in fat was the lowest, only0.32mg/kg. The concentration of radioactivity in most organs were reduced to under0.1mg/kg at24h, but it was high in liver, kidney and intestine; they were declined to less than0.5mg/kg at72h; the radioactivity was only detected in liver and intestine at7d. Five metabolites (Cy1, Cy2, Cy4, Cy5and Cy10) were found in carp liver at6h post final dose. Cy1was only metabolite which can be detected at7days. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy10) were identified in carp kidney at6h, and Cy1can be detected at3days. The parent drug and four metabolites (Cyl, Cy2, Cy4and Cy5) were identified in muscle. Cy4was detected at3d. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy10) were found in carp intestine. Cyl was detected at7d. The depletion speed of spleen was the fastest. Its elimination half-lives was0.44d. Followed by heart, kidney and skin, their elimination half-lives were between0.51d and0.78d. The drug elimination in liver and intestine was the slowest, and half-lives were from1.14d to1.24d. The total radioactivity determining showed that the concentration of drug in intestine was the highest, the residue time was the longest. Therefore, intestine was determined as residue target tissues. In intestine, the speed elimination of Cy1was the slowest. Its elimination half-life (1.82d) was relative to total residue (1.24d). The elimination trend of Cyl was similar to total residue. Hence, Cy1was determined as residue marker.
3H-Cyadox was widely distributed in rats. The concentrations of radioactivity in liver, kidney, bladder, adrenal gland and intestine were the highest at6h after continuous administration7days. The concentration in fat was the lowest, only0.42mg/kg. The concentration of radioactivity in most organs were reduced to more than50%at24h; they were declined to less than0.5mg/kg at72h; they were reduced to less than0.1mg/kg. The radioactivity was only detected in liver and kidney at14d. Six metabolites (Cy1, Cy4, Cy5, Cy6, Cy9and Cy11) were found in rats liver at6h post final dose. Cy5was only metabolite which can be detected at7days. Six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy9) were identified in rats kidney at6h, and Cy4can be detected at7days. Five metabolites (Cy1, Cy4, Cy5, Cy6and Cy9) were identified in muscle. Cy4and Cy5was detected at3d. The depletion speed of muscle, fat and blood was the fastest. Their elimination half-lives was1.00d,1.14d and1.16d. Followed by small intestine, large intestine and adrenal gland, their elimination half-lives were between1.44d and1.61d. The drug elimination in liver (2.57d) was slower. It in kidney was the slowest, and the half-life was4.33d. The total radioactivity determining showed that the concentration of drug in kidney was the highest, the residue time was the longest. Therefore, kidney was determined as residue target tissues. In kidney, the speed elimination of Cy4was the slowest, but the elimination half-life of Cyl was relative to total residue (4.33d). The elimination trend of Cyl was similar to total residue.
Finally, the kidney was determined as the residue target tissues in swine, broilers, and rats, the intestine was determined as the residue target tissues in carp. Cy1was the residue marker in swine, broilers and carp. Cy1, Cy4and Cy5may be relative to pharmacological and toxicological effects.
3Metabolism of cyadox in intestinal systems in vitro
According to the characteristics of the subject of intestinal metabolism, the metabolic systems of intestinal microsomes, intestinal mucosa, ileal and colonic microbiota have been established. Preparation of swine, broiler, carp and rat intestinal microsomes and intestinal mucosa, were incubated with NADPH regeneration system using the probe drug CYP3A, nifedipine, for verification system activity and using method of Brandford for determination protein concentration. The results show that oxidation of nifedipine can be generated in intestinal mucoca and microsomes, indicating that the metabolic activity of intestinal mucosa and microsomes exists which can be used for drug metabolism research.
Preparation of the ileal and colonic contents under anaerobic conditions, the metabolic system activity was verified by total anaerobic bacteria, total aerobic bacteria, Escherichia coli, Bifidobacterium, Lactobaccillus and Bacteroides fragilis counts and using probe enzyme substrates p-nitrophenyl P-D-glucuronide, p-nitrophenyl (3-D-galactoside and p-nitrophenyl β-D-glucoside incubation with the contents. Bacterial counts showed that the number of total bacteria maintained the normal level during0-48h. The concentration of enzyme maintained at a constant value during0-24h., indicating that bacteria in the system to maintained normal metabolic activity.
Only one metabolite (Cy2) was detected in swine, broiler, carp and rat intestinal microsomes NADPH regeneration system incubation with cyadox. Metabolites Cy1, Cy2and Cy10were found in incubation of swine, broiler and rat intestine homogenate. In carp intestine homogenate, Cy1and Cy2were detected.
Four metabolites (Cy1, Cy2, Cy4and Cy5) were found in swine and broiler colonic system, five metabolites (Cy1, Cy2, Cy4, Cy5and Cy10) in rat colonic flora, and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy9and Cy10) in carp intestinal flora. Cy1and Cy4were main metabolites in swine, broiler and rat colonic flora. Cyl was the major metabolite in carp intestinal flora.
In swine, broiler and rat ileal flora system, five metabolites (Cy1, Cy2, Cy10, Cy13and Cy16) were detected. The proportion of Cy2in chicken ileal flora was the highest than other species, which may be related to broiler intestinal environment.
In summary, this study illustrates that metabolism, distribution, and excretion of cyadox in swine, broilers, carp and rats using radiotracer methodology and LC/MS-IT-TOF technique. All metabolites were isolated and identified among four species. Cyadox and its metabolites were quantified in edible tissues for clearing the depletion regular in organs. The residue marker and target tissue of cyadox in animals were determined. The present study provides basis for the study of toxic mechanism and determining the pharmacological and toxicological compounds. It has important guiding significance for establishing the reasonable maximum residue limits and withdrawal period.
前期研究发现喹赛多促生长机理和药理活性与肠道环境和代谢有关。喹赛多的生物利用度较低,在肠道中驻留,肠道中的大量的药物代谢酶和细菌代谢酶使喹赛多产生降解,然而代谢物及药理毒理活性尚不清楚。因此,本课题建立体外肠道代谢模型,采用LC/MS-ITTOF技术,开展喹赛多在猪、鸡、鱼和大鼠肠微粒体、肠黏膜和肠道细菌中代谢研究,发现鉴定喹赛多在动物肠道体外模型中代谢物,明确喹赛多在肠道中特有的代谢物分析代谢物不同的产生部位,阐明肠道代谢途径分析种属差异,为代谢物药理和毒理研究提供基础。
1喹赛多在动物体内的物料平衡和代谢研究
猪、鸡、鱼和大鼠各6头(只),分别一次性灌胃给药(3H喹赛多),给药后每24h收集粪便、尿液、血液和胆汁(鱼检测活动范围内的水),一部分样品消化之后经静态液闪仪测定放射性活度,直到排泄物中放射性检测不到为止,计算氚标喹赛多的排泄情况。一部分样品经提取之后,采用流动液闪仪和HPLC/MS-IT-TOF鉴定代谢产物。
猪给药后12天,放射性总共排出96.1%,尿中排出44.5%,粪便中排出51.6%。鸡给药后12天,放射性总共排出95.2%。鱼给药后12天,放射性总共排出97.4%。大鼠给药后12天,放射性总共排出94.5%,尿中排出28.3%,粪便排出66.2%。喹赛多在动物体内主要是通过粪便排出体外,排泄迅速,给药后3天放射性排出可达到90%以上。
在猪尿液中检测到原形(Cy0)和13种代谢物,脱氧代谢物Cy1、Cy2和Cy10,氢化代谢物Cy4和Cy9,羟化代谢物Cy7和Cy8,侧链断裂代谢物Cy5、Cy6、Cy11和Cy12,甘氨酸结合物Cy3,水解代谢物Cy13和Cy14；粪便中发现原形(Cy0)和10种代谢物(Cy1、Cy2、Cy3、Cy4、Cy5、Cy6、Cy7、Cy9、Cy11和Cy12);猪血液中检测到11种代谢物(Cy1、Cy2、Cy3、Cy4、Cy5、Cy6、Cy9、 Cy11、Cy12、Cy13和Cy14)；在猪的的胆汁中检测到13种代谢物(Cy1、Cy2、Cy3、Cy4、Cy5、Cy6、Cy7、Cy8、Cy9、Cy11、Cy12、Cy13和Cyl4)。
在鸡粪便中检测到原形(Cy0)和7种代谢物(Cy1、Cy2、Cy4、Cy5、Cy6、 Cy7和Cy15),鸡血液中检测到4种代谢物(Cy2、Cy4、Cy5和Cy6),鸡胆汁中检测到原形(Cy0)和7种代谢产物(Cy1、Cy2、Cy4、Cy5、Cy10、Cy11和Cy13)。在鱼的排泄物中发现了大量的原形(Cy0)和2种代谢物(Cy2和Cy13),鱼血液中检测到3种代谢物(Cy1、Cy2和Cy5)。在大鼠的尿液中检测到6种代谢物(Cy1、Cy4、Cy5、Cy6、Cy9和Cy11)；粪便中检测到原形(Cy0)和7种代谢产物(Cy1、Cy2、Cy4、Cy5、Cy6、Cy11和Cy15)；大鼠血液中检测到5种代谢物(Cy1、Cy4、 Cy5、Cy6和Cy11)。
喹赛多在四种动物体内的主要代谢途径为：一是N-O键还原生成脱氧代谢产物；二是发生侧链C=N键的断裂,发生羟化和氧化；三是发生C=N键的氢化；四是发生酰胺键的水解。Cy3(Cy6甘氨酸结合物)只在猪体内发现：在鸡和大鼠体内发现了Cy15,在猪和鱼中没有检测到。四种动物中,鱼的代谢能力最差,在排泄物中能够检测到大量的原形和两种代谢物。通过喹赛多在不同种属动物体内的代谢研究,分析种属差异,对药理活性成分和代谢物毒性研究具有重要的指导意义。
2喹赛多在动物体内的分布和残留消除
24头猪(36只大鼠)随机分为6组,30只鸡(30尾鱼)随机分为5组,连续7天灌胃给药(3H喹赛多),在给药后6小时(h)、1天(d)、3d、7d、14d和21d宰杀(鸡和鱼为6h、1d、3d、7d和14d),取血液、胆汁、肝脏、肺脏、脾脏、皮肤、胃、大肠、小肠、肾脏等组织,消化液(solvable)水浴消化,经液闪仪检测放射活度测定药物在体内各组织中的分布情况；利用乙腈、乙酸乙酯和碱解法对可食性组织样品进行提取、净化,测定提取液放射活度计算提取率,然后进行LC/MS-IT-TOF流动液闪进行代谢物分离鉴定和定量。
喹赛多在猪体内广泛分布。给药后6h,肝、胆汁、肾和消化道中药物浓度高,胆汁中浓度超过14mg/kg。脂肪中浓度最低,只有0.9mg/kg。24h,大多数组织的放射性活度只有6h的一半左右,3d,绝大多数组织的浓度已下降到0.5mg/kg以下。7d,大部分组织中的浓度接近或低于0.1mg/kg,一些组织已不能检出。14d,只有肝和肾中能检出少量药物。6h猪的肝中能检测到8种代谢物,分别为Cy1、Cy3、Cy4、Cy5、Cy6、Cy9、Cy11和Cy12,7d时只检测到了Cy1。6h猪肾中检测到8种代谢物,分别是Cy1、Cy3、Cy4、Cy5、Cy6、Cy9、Cy11和Cy12。7d时只能检出Cy1。6h猪肌肉能检出8种代谢物,分别是Cy1、Cy3、 Cy4、Cy5、Cy6、Cy9、Cy11和Cy12。3天也只检出Cy3。6h猪脂肪中可检出6种代谢物Cy1、Cy2、Cy3、Cy4、Cy5和Cy6。肌肉、脂肪、肠道和血液中药物消除最快,消除半衰期为0.5～0.6d。其次为脾、肺、胃和肾上腺,消除速度相近,消除半衰期分别为1.08d～1.53d。肝脏和肾脏中药物消除速度较慢,消除半衰期分别为2.39d和2.57d。总放射性检测结果显示,喹赛多及其代谢物在肾脏中残留量最高,持续时间最长,消除速度最慢,因此肾脏是残留靶组织。猪肾中Cyl消除最慢,消除半衰期2.67d,与总残留相近,总体消除趋势相似,因此Cy1被确定为残留标示物。
喹赛多在鸡组织中分布广泛。给药后6h,胆汁、肝脏、肾脏和消化道的药物浓度高,胆汁中浓度甚至超过15mg/kg。脂肪中浓度最低,只有1.0mg/kg。24h,大多数组织的药物浓度下降到0.4mg/kg以下,但胆汁中浓度高达2.56mg/kg。3d,除肾和小肠外,其他组织中浓度均在0.2mg/kg。7d,只有肝脏和肾脏中有少量药物检出。6h鸡肝中检出原形和6种代谢物,分别是Cy1、Cy2、Cy4、Cy5、Cy6和Cy15。7d只检测到Cyl。6h,鸡肾脏检测到原形和6种代谢物,分别是Cy1、 Cy2、Cy4、Cy5、Cy6和Cy15。7d也只检测到Cy1。6h,鸡肌肉中检出原形和4种代谢物,分别是Cy1、Cy4、Cy5和Cy6。肌肉、脂肪、大肠、肺、肌胃和肾上腺消除最快,消除半衰期在0.63-0.67天之间。脾、小肠和血液次之,消除半衰期在0.73-0.95d之间。以上组织消除半衰期比较接近,都没有超过1d。肝脏和肾脏中药物消除速度较慢,消除半衰期分别为1.39d和2.38d。喹赛多及其代谢物在肾脏中残留量最高,持续时间最长,消除速度最慢,因此肾脏是残留靶组织。鸡肾中,Cyl消除最慢,消除半衰期3.15d,与总残留相近,总体消除趋势相似,因此Cyl被确定为残留标示物。
喹赛多在鱼体内广泛分布。给药后6h,肝、脾、肾和消化道中药物浓度高。脂肪中浓度最低,只有0.32mg/kg。24h,大多数组织的放射性活度下降迅速,大部分都在1.0mg/kg以下,但肝、肾和消化道中的浓度仍然较高,肠中的浓度仍在1.6mg/kg以上。3d,绝大多数组织的浓度已下降到0.5mg/kg以下。7d,只有肝和肠道中能检出少量药物。6h,鱼肝中检出5种代谢物,分别为Cy1、Cy2、 Cy4、Cy5和Cy10,并检测到原形药物。7d只检测到Cy1。6h,鱼肾脏检测到了6种代谢物,分别为Cy1、Cy2、Cy4、Cy5、Cy6和Cy10,并检测到原形药物。3d只检测到Cyl。6h,鱼肌肉检测到4种代谢物,分别为Cy1、Cy2、Cy4和Cy5,并检测到原形药物。3d只检测到Cy4,7d检测不到代谢物。鱼肠道检测到6种代谢物,分别为Cy1、Cy2、Cy4、Cy5、Cy6和Cy10,并检测到原形药物。7d可以检测到Cyl。脾消除最快,消除半衰期只有0.44d。心、肾脏和皮肤次之,消除半衰期在0.51-0.78d之间。以上组织消除半衰期比较接近,都没有超过1d。肝脏和肠中药物消除速度较慢,消除半衰期分别为1.14d和1.24d。总放射性检测结果显示,喹赛多及其代谢物在肠中残留量最高,持续时间最长,消除速度最慢,因此肠是残留靶组织。鱼肠道中,Cyl消除最慢,消除半衰期1.82d,与总残留相近,总体消除趋势相似,Cyl被确定为残留标示物。
喹赛多在大鼠体内也广泛分布。给药后6h,肝脏、肾脏、膀胱、肾上腺和消化道中药物浓度高,脂肪最低(0.42mg/kg)。24h,大部分组织的药物浓度下降50%以上。3d,所有组织中的药物浓度均下降到0.5mg/kg以下。7d,大多数组织的浓度在0.1mg/kg以下,有些组织已不能测出。14d,只有肝脏和肾脏中有少量药物检出。6h,大鼠肝中检出6种代谢物,分别为Cy1、Cy4、Cy5、Cy6、Cy9和Cy11,但没有检出原形。7d只检测到Cy5。6h,大鼠肾脏检测到了6种代谢物,分别为Cy1、Cy2、Cy4、Cy5、Cy6和Cy9。7d只检测到Cy4。6h大鼠肌肉中检测到5种代谢物,分别为Cy1、Cy4、Cy5、Cy6和Cy9。3d检测到Cy4和Cy5。肌肉、脂肪和血液中药物消除最快,消除半衰期分别为1.0d、1.14d和1.26d。其次为小肠、大肠和肾上腺,消除速度相近,消除半衰期分别为1.61d、1.58d和1.44d。肝脏中药物消除速度较慢,消除半衰期为2.57d。肾脏中药物消除速度最慢,消除半衰期为4.33d。喹赛多及其代谢物在肾脏中残留量最高,持续时间见最长,消除速度最慢,因此肾脏是残留靶组织。大鼠肾脏中,Cy4消除最慢,消除半衰期2.04天,但是Cyl的消除半衰期与总残留最相近。
最终确定,肾脏是猪、鸡和大鼠的残留靶组织,肠道是鱼的残留靶组织。Cyl是猪、鸡和鱼的残留标示物。Cy1、Cy4和Cy5可能是喹赛多药理和毒理作用的主要化合物。
3喹赛多在动物体外肠道代谢研究
根据肠道代谢的特点本课题建立了肠微粒体、肠黏膜、结肠内容物和回肠内容代谢系统。制备猪、鸡、鱼和大鼠肠微粒体和肠道黏膜,与烟酰胺腺嘌呤二核苷酸磷酸(NADPH)再生系统进行孵育,采用细胞色素3A(CYP3A)的探针药物硝苯地平验证体系的活性,采用Brandford法测定蛋白浓度。结果表明,硝苯地平在肠微粒体和肠道黏膜中能够生成氧化的硝苯地平,表明肠黏膜和肠微粒体代谢酶活性良好,可以用于药物代谢研究。
在厌氧条件下,制备结肠内容物和回肠内容物体外孵育液,通过对肠道细菌总厌氧菌、总需氧菌、大肠杆菌、双歧杆菌、乳酸菌和脆弱拟杆菌进行计数,同时与探针酶底物p-硝基苯β-D-葡糖醛酸苷、p-硝基苯β-D-半乳糖苷和p-硝基苯β-D-半乳糖苷孵育验证体系的代谢活性。结果表明,细菌数量在0-48h内保持了良好的稳定性,酶活力稳定而且符合标注,可以用于代谢研究。
在猪、鸡、鱼和大鼠肠微粒体NADPH再生系统中只发现了一种代谢物(Cy2),在猪、鸡和大鼠肠黏膜NADPH再生系统中只发现了脱二氧代谢产物(Cy1)和脱一氧代谢物(Cy2和Cy10),在鱼肠黏膜中发现了Cyl和Cy2。
在猪、鸡和大鼠结肠内容物代谢体系中分别检测到了4种、4种和5种代谢产物,在鱼肠内容物中检测到了6种代谢物。猪和鸡中代谢物分别为Cy1、Cy2、Cy4和Cy5,大鼠中代谢物为Cy1、Cy2、Cy4、Cy5和Cy10,鱼中Cy1、Cy2、Cy9和Cy10。Cy1和Cy4是猪、鸡和大鼠结肠细菌代谢系统中的主要代谢产物,Cyl是鱼肠道细菌代谢系统中的主要代谢物。
在回肠内容物代谢体系中,猪、鸡和大鼠分别检测到5种代谢物。猪回肠内容物中检测到Cy1、Cy2、CylO、Cy13和Cy16,鸡和大鼠回肠内容物中检测到相同的代谢产物。
在动物的肠道肠黏膜代谢体系中,喹赛多主要发生了脱氧代谢。在肠道细菌代谢体系中,喹赛多主要发生了脱氧代谢,氢化反应,羟化反应,水解反应和侧链的断裂反应。羟化反应和水解反应只在小肠细菌中发生,氢化和断裂反应只在结肠体系中发生。在四种动物肠黏膜中,喹赛多代谢没有差异。在鸡回肠内容物中Cy2的比例最高,这可能与鸡肠道酸性有关系。在鱼肠道中发现了氢化代谢物,这可能与鱼类肠道细菌代谢有关。综合体内代谢和前期肝脏代谢发现,Cy4是在肠道细菌的作用下产生的。
综上所述,本课题首次采用放射性示踪和LC/MS-IT-TOF技术研究喹赛多在猪、鸡、鱼和大鼠体内的代谢、分布和排泄,分离并鉴定了猪、鸡、鱼和大鼠体内的所有代谢产物,并对可食性组织中代谢物进行了定量,阐明了总残留和代谢在各个器官中的消除特征,确定了喹赛多的残留靶组织和残留标示物。本研究是喹赛多毒副作用机制研究、药理活性和毒性化合物研究的基础；对将来体内残留分析方法建立,最大残留限量和休药期的制定具有重要的指导意义。
Cyadox (CYX),2-formylquinoxaline-1,4-dioxide cyanoacetylhydrazone, is an antimicrobial and growth-promoting feed additive of quindoxaline class for food-producing animals. CYX is an excellent antibacterial growth promoter and has a lot of good quality, such as lesser toxicity and side effects, high security, rapid elimination and short residue after treatment, which has been recognized to be a safe and effective candidate of feed additive. Drug efficacy and safety were important in new drug study. The pharmacological active ingredient and toxicity targets were found based on drug metabolism and distribution studies. The Food and Drug Administration (FDA) and the European Union (EU) recommended that radiotracer methodology was adopted in drug absorption, disposition, metabolism and depletion studies. Radioisotope measurement method is simple, accurate positioning with high sensitivity and corresponding to physiological conditions, which has been widely used in the field of medicine to reveal drug distribution, metabolism and excretion process.. This study was to investigated metabolism, distribution and excretion of cyadox in swine, broiler, carp and rats by radiotracer technique and high-performance liquid chromatography-ion trap-time of flight mass spectrometry (LC/MS-IT-TOF). All unknown metabolites were identified by those techniques. The residue target organ and residue marker were determined, the data of species differences were obtained based on analysis of distribution and depletion of cyadox in animals. It will provide the basis for determining the standards of food safety of cyadox and exploring the depth of the toxicological mechanism of caydox action.
The preliminary studied demonstrated that the growth promoting mechanism and pharmacological activity of cyadox were relative to cyadox metabolism in intestine. The bioavailability of cyadox was low, then a lot of cyadox remained in intestine, which was metabolized by drug metabolizing enzymes and bacterial metabolic enzymes in the gut. However, the metabolites and their toxicological activity were not clear. Hence, the intestinal metabolic systems of swine, broilers, carp and rats were established. The metabolism of cyadox was investigated in intestinal microsomes, intestinal mucosa and gut microflora using LC/MS-IT-TOF. All unknown metabolites were characterized to reveal the metabolic organ of cyadox. The whole metabolic pathways were obtained to analysis of species differences. It will provide a basis for pharmacological and toxicological studies of cyadox and its metabolites.
1Mass balance and metabolism of cyadox in vivo
Six animals (swine, broilers, carp and rats) for material balance test were administrated one single dose. Urine and feces were collected per24h (water was collected in carp). Blood and bile were also collected. The total of radioactivity was detected by liquid scintillation after digestion. The metabolites were identified by LC/MS-IT-TOF after extracted by agents.
The total radioactive recovery (TRR) was96.1%,44.5%in urine,51.6%in feces after12days post dose. TRR was95.2%in broilers after12days post dose. The excretion rate was97.4%in carp after12days. In rats, TRR was94.5%after12days,28.3%in urine,66.2%in feces. Those results demonstrated that the excretion rate of cyadox reached to more than90%at3days after administration, and feces was main route of excretion.
The parent drug (Cy0) and its thirteen metabolites were identified in swine urine, deoxidation metabolites (Cy1, Cy2and Cy10), hydrogenation metabolites (Cy4and Cy9), hydroxylation metabolites (Cy7and Cy8), side chain cleavage metabolites (Cy5, Cy6, Cy11and Cy12), glycine conjugate (Cy3), hydrolysis metabolites (Cy13and Cy14). The parent drug and ten metabolites (Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7, Cy9, Cy11and Cy12) were identified in feces. Eleven metabolites (Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy9, Cy11, Cy12, Cy13and Cyl4) were identified. Thirteen metabolites (Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7, Cy8, Cy9, Cy11, Cy12, Cy13and Cy14) were found in swine bile.
The parent drug and seven metabolites (Cy1, Cy2, Cy4, Cy5, Cy6, Cy7and Cy15) were detected in broilers feces. Four metabolites (Cy2, Cy4, Cy5and Cy6) were found in broilers blood. The parent drug and seven metabolites (Cy1, Cy2, Cy4, Cy5, Cy10, Cy11and Cy13) were detected in broilers bile. The parent drug and two metabolites(Cy2and Cy13) were identified in carp excreta. Three metabolites (Cy1, Cy2and Cy5)were found in carp blood. Six metabolites (Cy1, Cy4, Cy5, Cy6, Cy9and Cy11) were detected in rats urine. The parent drug and seven metabolites (Cy1, Cy2, Cy4, Cy5, Cy6, Cyll and Cy15) were identified in rats feces. Five metabolites (Cy1, Cy4, Cy5, Cy6and Cy11) were found in rats blood.
Four main metabolic pathways were found in four species, the reduction of N-O bond, the cleavage of C=N bond and oxidation, the hydrogenation of C=N bond, the hydrolysis of amid bond. Glycine conjugate of quinoxaline-2-carboxylic acid was found only in swine. Cy15was detected in broiler and rats, and not found in swine and carp. The metabolic ability of carp was the weakest in four species. A large number of parent drug and a few metabolites were detected in carp excreta. The metabolic study and analysis of species differences has important guiding significance for the study of pharmacologically active ingredient and metabolite toxicity.
2Disposition and depletion of cyadox in vivo
Twenty-four swine and thirty-six rats were divided into six groups (thirty carp and thirty broilers for five groups) for7consecutive days administration based on a feeding rate of body weight per day. Swine and rats were slaughtered at6h,1d,3d,7d,14d and21d after last dose (broilers and carp for6h,1d,3d,7d and14d). Blood, bile, liver, lung, spleen, skin, stomach, large intestine, small intestine, kidney et al were collected for determination of total radioactivity after digestion. The edible tissues were extracted by different agents (acetonitrile, ethyl acetate and alkaline hydrolysis) for determining the extraction efficiency. The metabolite quantitative was performed by flow liquid scintillation. The metabolites were identified by LC/MS-IT-TOF.
3H-Cyadox was widely distributed in swine. The concentrations of radioactivity in liver, bile, kidney and intestine were the highest at6h after continuous administration7days, and it was more than14mg/kg in bile. The concentration in fat was the lowest, only0.9mg/kg. The concentration of radioactivity in most organs were reduced to50%at24h; they were declined to less than0.5mg/kg at72h; they were reduced to under0.1mg/kg at7d; the radioactivity was only detected in liver and kidney at14d. Eight metabolites (Cy1, Cy3, Cy4, Cy5, Cy6, Cy9, Cy11and Cy12) were found in swine liver at6h post final dose. Cy1was only metabolite which can be detected at7days. Eight metabolites (Cy1, Cy3, Cy4, Cy5, Cy6, Cy9, Cy11and Cy12) were identified in swine kidney at6h, and Cyl can be detected at7days. The identical metabolites were found in swine muscle at6h, and Cy3can be detected at3days. Six metabolites (Cy1, Cy2, Cy3, Cy4, Cy5and Cy6) were identified in swine fat, and only Cy3was found at3days. The depletion speed of muscle, fat, intestine and blood was the fastest. Their elimination half-lives were between0.5d-0.6d. Followed by spleen, lung, stomach, and adrenal glands, their elimination half-lives were between1.08d and1.53d. The drug elimination in liver and kidney was the slowest, and half-lives were from2.39d to2.57d. The total radioactivity determining showed that the concentration of drug in kidney was the highest, the residue time was the longest. Therefore, kidney was determined as residue target tissues. In kidney, the speed elimination of Cy1was the slowest. Its elimination half-life (2.67d) was relative to total residue. The elimination trend of Cy1was similar to total residue. Hence, Cy1 was determined as residue marker.
3H-Cyadox was widely distributed in broilers. The concentrations of radioactivity in liver, bile, kidney and intestine were the highest at6h after continuous administration7days, and it was more than14mg/kg in bile. The concentration in fat was the lowest, only0.1mg/kg. The concentration of radioactivity in most organs were reduced to under0.4mg/kg at24h, but it was more than2.56mg/kg in bile; they were declined to less than0.4mg/kg at72h except kidney and small intestine; the radioactivity was only detected in liver and kidney at7d. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy15) were found in swine liver at6h post final dose. Cy1was only metabolite which can be detected at7days. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy15) were identified in swine kidney at6h, and Cy1can be detected at7days. Four metabolites (Cy1, Cy4, Cy5and Cy6) were identified in muscle. The depletion speed of muscle, fat, large intestine, lung, muscular stomach and adrenal glands was the fastest. Their elimination half-lives were between0.63d-0.67d. Followed by spleen, small intestine and blood, their elimination half-lives were between0.73d and0.95d. The drug elimination in liver and kidney was the slowest, and half-lives were from1.39d to2.38d. The total radioactivity determining showed that the concentration of drug in kidney was the highest, the residue time was the longest. Therefore, kidney was determined as residue target tissues. In kidney, the speed elimination of Cyl was the slowest. Its elimination half-life (3.15d) was relative to total residue. The elimination trend of Cyl was similar to total residue. Hence, Cy1was determined as residue marker.
3H-Cyadox was widely distributed in carp. The concentrations of radioactivity in liver, spleen, kidney and intestine were the highest at6h after continuous administration7days. The concentration in fat was the lowest, only0.32mg/kg. The concentration of radioactivity in most organs were reduced to under0.1mg/kg at24h, but it was high in liver, kidney and intestine; they were declined to less than0.5mg/kg at72h; the radioactivity was only detected in liver and intestine at7d. Five metabolites (Cy1, Cy2, Cy4, Cy5and Cy10) were found in carp liver at6h post final dose. Cy1was only metabolite which can be detected at7days. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy10) were identified in carp kidney at6h, and Cy1can be detected at3days. The parent drug and four metabolites (Cyl, Cy2, Cy4and Cy5) were identified in muscle. Cy4was detected at3d. The parent drug and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy10) were found in carp intestine. Cyl was detected at7d. The depletion speed of spleen was the fastest. Its elimination half-lives was0.44d. Followed by heart, kidney and skin, their elimination half-lives were between0.51d and0.78d. The drug elimination in liver and intestine was the slowest, and half-lives were from1.14d to1.24d. The total radioactivity determining showed that the concentration of drug in intestine was the highest, the residue time was the longest. Therefore, intestine was determined as residue target tissues. In intestine, the speed elimination of Cy1was the slowest. Its elimination half-life (1.82d) was relative to total residue (1.24d). The elimination trend of Cyl was similar to total residue. Hence, Cy1was determined as residue marker.
3H-Cyadox was widely distributed in rats. The concentrations of radioactivity in liver, kidney, bladder, adrenal gland and intestine were the highest at6h after continuous administration7days. The concentration in fat was the lowest, only0.42mg/kg. The concentration of radioactivity in most organs were reduced to more than50%at24h; they were declined to less than0.5mg/kg at72h; they were reduced to less than0.1mg/kg. The radioactivity was only detected in liver and kidney at14d. Six metabolites (Cy1, Cy4, Cy5, Cy6, Cy9and Cy11) were found in rats liver at6h post final dose. Cy5was only metabolite which can be detected at7days. Six metabolites (Cy1, Cy2, Cy4, Cy5, Cy6and Cy9) were identified in rats kidney at6h, and Cy4can be detected at7days. Five metabolites (Cy1, Cy4, Cy5, Cy6and Cy9) were identified in muscle. Cy4and Cy5was detected at3d. The depletion speed of muscle, fat and blood was the fastest. Their elimination half-lives was1.00d,1.14d and1.16d. Followed by small intestine, large intestine and adrenal gland, their elimination half-lives were between1.44d and1.61d. The drug elimination in liver (2.57d) was slower. It in kidney was the slowest, and the half-life was4.33d. The total radioactivity determining showed that the concentration of drug in kidney was the highest, the residue time was the longest. Therefore, kidney was determined as residue target tissues. In kidney, the speed elimination of Cy4was the slowest, but the elimination half-life of Cyl was relative to total residue (4.33d). The elimination trend of Cyl was similar to total residue.
Finally, the kidney was determined as the residue target tissues in swine, broilers, and rats, the intestine was determined as the residue target tissues in carp. Cy1was the residue marker in swine, broilers and carp. Cy1, Cy4and Cy5may be relative to pharmacological and toxicological effects.
3Metabolism of cyadox in intestinal systems in vitro
According to the characteristics of the subject of intestinal metabolism, the metabolic systems of intestinal microsomes, intestinal mucosa, ileal and colonic microbiota have been established. Preparation of swine, broiler, carp and rat intestinal microsomes and intestinal mucosa, were incubated with NADPH regeneration system using the probe drug CYP3A, nifedipine, for verification system activity and using method of Brandford for determination protein concentration. The results show that oxidation of nifedipine can be generated in intestinal mucoca and microsomes, indicating that the metabolic activity of intestinal mucosa and microsomes exists which can be used for drug metabolism research.
Preparation of the ileal and colonic contents under anaerobic conditions, the metabolic system activity was verified by total anaerobic bacteria, total aerobic bacteria, Escherichia coli, Bifidobacterium, Lactobaccillus and Bacteroides fragilis counts and using probe enzyme substrates p-nitrophenyl P-D-glucuronide, p-nitrophenyl (3-D-galactoside and p-nitrophenyl β-D-glucoside incubation with the contents. Bacterial counts showed that the number of total bacteria maintained the normal level during0-48h. The concentration of enzyme maintained at a constant value during0-24h., indicating that bacteria in the system to maintained normal metabolic activity.
Only one metabolite (Cy2) was detected in swine, broiler, carp and rat intestinal microsomes NADPH regeneration system incubation with cyadox. Metabolites Cy1, Cy2and Cy10were found in incubation of swine, broiler and rat intestine homogenate. In carp intestine homogenate, Cy1and Cy2were detected.
Four metabolites (Cy1, Cy2, Cy4and Cy5) were found in swine and broiler colonic system, five metabolites (Cy1, Cy2, Cy4, Cy5and Cy10) in rat colonic flora, and six metabolites (Cy1, Cy2, Cy4, Cy5, Cy9and Cy10) in carp intestinal flora. Cy1and Cy4were main metabolites in swine, broiler and rat colonic flora. Cyl was the major metabolite in carp intestinal flora.
In swine, broiler and rat ileal flora system, five metabolites (Cy1, Cy2, Cy10, Cy13and Cy16) were detected. The proportion of Cy2in chicken ileal flora was the highest than other species, which may be related to broiler intestinal environment.
In summary, this study illustrates that metabolism, distribution, and excretion of cyadox in swine, broilers, carp and rats using radiotracer methodology and LC/MS-IT-TOF technique. All metabolites were isolated and identified among four species. Cyadox and its metabolites were quantified in edible tissues for clearing the depletion regular in organs. The residue marker and target tissue of cyadox in animals were determined. The present study provides basis for the study of toxic mechanism and determining the pharmacological and toxicological compounds. It has important guiding significance for establishing the reasonable maximum residue limits and withdrawal period.
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