不同水温下氟苯尼考及其代谢物氟苯尼考胺在鲫鱼体内的生理药动学模型研究

Because of the extensive and long term application of antimicrobials in fish farms, drug residue is easy to occur in aquatic products. Antimicrobial residue may directly endanger the health of consumers or potentially lead to the emergence of resistant bacteria. In order to ensure animal-derived food safety, reasonable withdrawal times (WTs) are required for the antimicrobials. Our country is vast in territory, and water temperatures are widely different with distinct seasons and different regions. Because the antimicrobial dispositions in fishes are markedly temperature-dependent, the WTs of antimicrobials may vary at different water temperatures. Therefore, it is important to reasonably estimate the WTs for antimicrobials used in fish farms at different water temperatures. Florfenicol is a broad-spectrum antimicrobial belonging to the chloramphenicol family. And it is widely used in crucian farms. Florfenicol is metabolized into its marker residue (florfenicol amine) by the liver enzyme (Cytochrome P450) whose activity is closely related to water temperature. The physiologically based pharmacokinetics (PBPK) model is a mathematical modeling technique to simulate blood flow in body circulatory system and to predict the drug concentrations in different tissues. In the present project, the temperature-dependent PBPK models will be developed to simulate the concentrations of florfenicol and florfenicol amine in crucian carp after multiple oral and intramuscular doses, and to predict the WTs of florfenicol in crucian carp at different water temperatures.

渔业养殖中，抗微生物药（Antimicrobials，As）用量大、应用时间长，鱼肉中易残留。As残留不但会影响消费者健康，还会诱发细菌耐药性。为了保证动物性食品安全，As使用过程中必须制定合理的休药期（WT）。我国幅员辽阔，四季分明，各地、各季节水温差别很大，而药物处置受水温影响巨大，不同水温下药物的WT会有差别。因此，合理制定不同水温下药物在鱼体内的WT意义重大。氟苯尼考（FLO）属第二代酰胺醇类As，在鲫鱼养殖过程中广泛应用。FLO在肝细胞色素P450酶的催化下转化为FLO的残留标示物——氟苯尼考胺，而酶的催化活性与水温密切相关。生理药动学（PBPK）模型是一种通过血液循环将各器官联结起来而建成的整体模型，可以用它模拟药物或代谢物在各组织中的浓度。本项目拟建立水温相关的PBPK模型，模拟连续肌内注射和灌服后FLO及氟苯尼考胺在鲫鱼体内的浓度，预测不同水温下FLO在鲫鱼体内的WT。

本研究首先使用差速离心法制备了鲫鱼肝微粒体，并利用考马斯亮蓝法检测了其内蛋白含量为（6.312±0.421）mg/g肝组织，用CO还原差示光谱法测定了其内CYP450含量为（0.385±0.047）nmol/mg蛋白；利用CYP450不同亚型的特异性抑制剂开展了体外共孵育实验，证实了CYP3A参与了体外催化氟苯尼考转化为氟苯尼考胺的过程；随后开展了不同温度下氟苯尼考在鲫鱼肝微粒体中代谢速率研究，计算出不同温度下氟苯尼考在鲫鱼肝微粒体中的固有清除率；此外还考察了底物浓度对氟苯尼考体外代谢的影响，证实了CYP450存在一定的饱和现象；接下来考察了水温对鲫鱼体内氟苯尼考肾清除率的影响，研究结果证实鲫鱼体内氟苯尼考的经肾排泄受水温的影响不大；随后开展了肌注和灌服两种给药途径下不同水温（10、20和25°C）条件下氟苯尼考及其代谢物氟苯尼考胺的药动学及残留消除规律研究，研究结果证实随着水温的升高，氟苯尼考及氟苯尼考胺的消除速率均有所增加：灌服给药后氟苯尼考的消除半衰期（t1/2λz）由24.318h（10°C）缩短到10.743 h（25°C），而氟苯尼考胺的t1/2λz则由29.407h（10°C）缩短为14.530h（25°C），而肌注给药后氟苯尼考的t1/2λz则由33.444h（10°C）缩短为18.433h（25°C），氟苯尼考胺的t1/2λz则由47.880h（10°C）缩短为25.891h（25°C）；项目组最后建立了不同给药途径下的生理药动学模型，并利用该模型成功模拟预测了不同水温下肌注或灌服给药后氟苯尼考及氟苯尼考胺的残留浓度，并在模型中加入了一个500次的蒙特卡洛模拟，预测得到了氟苯尼考的残留休药期如下：10、20和25°C水温下，连续5次灌胃给药（10mg/kg/day）后，休药期均为2天；而连续5次肌注给药后，休药期分别为3、3和2天。