南京农业大学学报  2015, Vol. 38 Issue (3): 459-463   PDF    
http://dx.doi.org/10.7685/j.issn.1000-2030.2015.03.016
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文章信息

徐曹燕, 汤春莲, 阮祥春, 李琳, 曾明华. 2015.
XU Caoyan, TANG Chunlian, RUAN Xiangchun, LI Lin, ZENG Minghua. 2015.
泰拉霉素在鸡体内的药物代谢动力学研究
Pharmacokinetic and bioavailability study of tulathromycin in chickens
南京农业大学学报, 38(3): 459-463
Journal of Nanjing Agricultural University, 38(3): 459-463.
http://dx.doi.org/10.7685/j.issn.1000-2030.2015.03.016

文章历史

收稿日期：2014-05-17
引用本文
徐曹燕, 汤春莲, 阮祥春, 李琳, 曾明华. 2015. 泰拉霉素在鸡体内的药物代谢动力学研究[J]. 南京农业大学学报, 38(3): 459-463.
XU Caoyan, TANG Chunlian, RUAN Xiangchun, LI Lin, ZENG Minghua. Pharmacokinetic and bioavailability study of tulathromycin in chickens[J]. Journal of Nanjing Agricultural University, 38(3): 459-463.  DOI: 10.7685/j.issn.1000-2030.2015.03.016
泰拉霉素在鸡体内的药物代谢动力学研究
徐曹燕1, 汤春莲2, 阮祥春1, 李琳1, 曾明华1     
1. 安徽农业大学动物科技学院, 安徽 合肥 230036;
2. 安徽省兽药饲料监察所, 安徽 合肥 230039
收稿日期：2014-05-17
基金项目：安徽省教育厅重点项目(KJ2012A111)
作者简介：徐曹燕,硕士研究生。
通迅作者：曾明华,副教授,主要从事兽医药理学和毒理学研究,E-mail:zmhzmh@ahau.edu.cn。
摘要：[目的] 探讨泰拉霉素在鸡体内的药物代谢动力学过程和特征及其生物利用度。[方法] 给受试健康鸡分别进行静脉注射、肌肉注射及口服泰拉霉素,给药后在120 h内分点心脏采血,血浆样品经乙腈-0.1%甲酸溶液(体积比为95:5)沉淀,离心去蛋白,以HPLC-ESI-MS/MS法检测鸡血浆中泰拉霉素含量,最后采用药动学软件Winnonlin的非房室模型进行数据分析。[结果] 受试鸡经静脉注射、肌肉注射和口服给药后药时数据均适合非房室模型。静脉注射给药后,血浆清除率(CL)为17.71 mL·kg-1·h-1,稳态表观分布容积(Vss)为77.15 L·kg-1,消除半衰期(t1/2)为21.65 h。肌肉注射和口服泰拉霉素后,在0.25和0.5 h时,血浆浓度(Cmax)达峰值,分别为0.83和0.69 ng·mL-1,血浆中t1/2平均分别为20.96和24.33 h;生物利用度分别为92.59%和62.38%,说明肌肉注射给药较口服吸收完全,生物利用度高。[结论] 泰拉霉素肌肉注射或口服在健康鸡体内表观分布容积大,吸收和分布迅速,达峰时间短,消除缓慢,生物利用度高。
关键词： 泰拉霉素     药代动力学     生物利用度     鸡    
Pharmacokinetic and bioavailability study of tulathromycin in chickens
XU Caoyan1, TANG Chunlian2, RUAN Xiangchun1, LI Lin1, ZENG Minghua1     
1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China;
2. Anhui Institute of Vetenary Drug and Feed Control, Hefei 230039, China
Abstract: [Objectives] Pharmacokinetics characteristics and bioavailability of the triamilide antimicrobial, tulathromycin were investigated in healthy chickens. [Methods] A total of 306 healthy chickens were allotted to 3 groups at 102 chickens per group and received tulathromycin following single intravenous, intramuscular and oral administration at the dose of 20 mg·kg-1 of body weight respectively. Blood samples were collected at different time points in 120 h after administration, then treated with acetonitrile and centrifuged to removal proteins and tulathromycin concentrations were determined by high-performance liquid chromatography(HPLC)with tandem mass pectrometry detection(LC-MS/MS)using ESI with a limit of detection of 1.67 ng·mL-1 and a limit of quantitation of 2.96 ng·mL-1. Pharmacokinetic parameters were estimated using the Winnonlin software package. [Results] The tulathromycin concentration-time data were all fitted to noncompartment model after single intravenous, intramuscular and oral administration to the healthy chickens. There is summary of the mean plasma pharmacokinetic parameters in chickens with a single intramuscular, intravenous and oral administration of tulathromycin at 20 mg·kg-1. The mean pharmacokinetic parameters of tulathromycin after intravenous administration in chicken were as follows:area under the curve(AUClast)was 5.34 μg·h·mL-1;cloral(CL)was 17.71 mL·kg-1·h-1;the volume of distribution at steady-state(Vss)was 77.15 L·kg-1 and the elimination t1/2 was 21.65 h. The mean pharmacokinetic parameters of tulathromycin after intramuscular administration in chickens were as follows:t1/2 20.96 h;AUClast 4.93 μg·h·mL-1;tmax was 0.25 h;Cmax was 0.83 μg·mL-1. The mean pharmacokinetic parameters of tulathromycin after oral administration in chickens were as follows:t1/2 was 24.33 h;AUClast was 3.43 μg·h·mL-1;tmax was 0.50 h;Cmax was 0.69 μg·mL-1. The bioavailability of tulathromycin after single intramuscular and oral administration were 92.59% and 62.38%, respectively. Tulathromycin has shown higher efficacy by intramuscular than oral administration. The results of studies showed that pharmacokinetics characteristics of tulathromycin in healthy chickens were rapidly absorption and wide distribution and slow elimination after single intravenous, intramuscular and oral administration in healthy chickens. The drug exhibited large volume of distribution and was absorbed completely from the injection site and had high bioavailability after single intramuscular administration compared with oral administration in chickens. The bioavailability of tulathromycin by intramuscular was higher than oral administration obviously. [Conclusions] Tulathromycin is a novel triamilide antimicrobial that has been approved for use in the treatment and prevention of bovine and swine respiratory diseases. No one discovers any reports concerning tulathromycin about chickens at the present time. The study explores the pharmacokinetics characteristics and bioavailability of tulathromycin in chickens following single intravenous, intramuscular and oral administration for the first time, which will provide reasonable reference for veterinary clinical study.
Keywords: tulathromycin     pharmacokinetic     bioavailability     chicken    

泰拉霉素是一种动物专用的新型大环内酯类广谱抗生素,分子式为C41H79N3O12,相对分子质量806.23,由10%的13元氮杂内酯环和90%的15元氮杂内酯环2个同分异构体组成。目前,泰拉霉素主要用于胸膜肺炎放线杆菌、支原体、巴氏杆菌、副嗜血杆菌、支气管败血性博德特菌等引起的猪和牛呼吸系统疾病的防治[1, 2]。在我国,用于治疗畜禽呼吸道疾病的药物有很多,这些药物通常以拌料或饮水的给药方式,重复多次给药且随着使用时间的延长,我国很多地区出现了不同程度的耐药性,传统药物的药效逐渐降低。因此,市场上急需治疗呼吸道感染的高效、安全、广谱、低残留的新型抗菌药物[3]。泰拉霉素作为新型抗菌药物,单次肌肉注射或皮下给药即能提供全程的治疗。因其具有吸收迅速、达峰时间短、生物利用度高,在肺中可达到很高的组织浓度、消除半衰期长、药效持久等优点,治疗效果明显优于其他大环内酯类抗生素,故将会有很广阔的应用前景[4, 5, 6]。目前,国内外关于泰拉霉素的药物动力学研究已在牛、猪、马等动物体内进行,但在鸡体内的药物代谢动力学研究国内未见报道。本研究通过对鸡采用3种不同给药途径用药,探讨泰拉霉素在鸡体内的药物代谢动力学特征,为临床合理用药提供参考。 1 材料与方法

1.1 试验材料

1.1.1 动物

306只1日龄罗曼蛋鸡购于安徽省长丰县安禽禽业有限公司,饲养于25 ℃左右的试验动物房,饲喂不含任何药物的饲料,自由采食饮水,饲养至21日龄。试验前,临床观察1周,所有供试鸡均表现健康。试验前16 h内禁食,仅自由饮水。试验时随机分为3组,每组102只分别进行静脉注射、肌肉注射及口服给药,每组再随机分成17组,每组6只,分别在给药前以及给药后的不同时间点采集血液。 1.1.2 药品和试剂

泰拉霉素标准品(纯度大于96%)购自南京森贝伽生物科技有限公司;瑞可新注射液(含0.1 g · mL-1泰拉霉素,批号:A280910),由辉瑞动物保健品公司法国Amboise生产。甲醇、乙腈、甲酸均为国产分析纯。超纯水:DZG-303A纯水仪制备,水质18.3 MΩ · cm-1。1.1.3 主要仪器

液质联用仪由Agilent1100液相系统和API4000三重四极杆质谱串联组成。NEVAPTM111氮气吹干仪购于美国Organomation公司。高速冷冻离心机购于日本Hitachi公司。

1.2 给药和采集血样

将受试鸡称质量后,按体质量给药,剂量均为20 mg · kg-1,静脉注射试验动物侧翼下静脉;肌肉注射部位为试验动物腿部肌肉;灌胃组将药物用胃管直接注入嗉囊给药。给药后分别于0、5、15和30 min及1、2、4、6、8、10、12、24、48、72、96和120 h心脏采血。采集的血样置于含肝素的离心管中,混匀,4 000 r · min-1离心10 min,分离血浆,-20 ℃冰箱保存待测。 1.3 血浆样品预处理

从冰箱中取出血浆,自然解冻,摇匀,取0.5 mL血浆置于2.0 mL的离心管中,加入0.5 mL乙腈-0.1%甲酸溶液(体积比为95 ∶ 5),于旋涡混合器上混合2 min后以14 000 r · min-1高速离心5 min,吸取上清液,过0.22 μm纤维滤膜至进样瓶,供高效液相色谱串联质谱(HPLC-MS/MS)分析。 1.4 血浆中泰拉霉素质量浓度的测定

采用HPLC-MS/MS进行血浆中泰拉霉素浓度的测定,其色谱和质谱条件:色谱柱为Luna C18(150 mm× 2.00 mm,5 μm)反相柱;流动相A相为50%甲醇甲酸溶液、B相为0.1%甲酸水溶液,梯度洗脱,流速为300 L · min-1;柱温40 ℃;进样量10 μL。采用电喷雾离子源(ESI),正离子扫描,多反应监测(MRM),扫描时间200 ms,电喷雾电压(IS)为5 500 V,离子源温度500 ℃,气帘气流速(CUR)为25 L · min-1,雾化气流速(GS1)为50 L · min-1,辅助气流速(GS2)为50 L · min-1,聚焦电压(FP)为400 V,碰撞室射出电压(CXP)为13.0 V,碰撞气(CAD)为高纯氮气,压力为68.95 kPa。

在上述条件下测定,泰拉霉素的检测限为1.67 ng · mL-1,定量限为2.96 ng · mL-1。血浆中泰拉霉素的相对回收率为89.3%~102.1%,泰拉霉素在2~200 ng · ml-1条件下,呈良好的线性关系,相关系数(r)为0.997 7~0.999 1。批内变异系数为5.2%~7.6%,批间变异系数为3.1%~9.7%。 1.5 数据处理

采用美国Pharsigh公司药动学软件Winnonlin 5.2.1的非房室模型处理药代动力学数据,计算每只鸡的有关药物代谢动力学参数,进一步求出各参数的平均值±标准差(x±SD)。同时以血药浓度平均值对时间作血药浓度-时间曲线图。 2 结果与分析

试验鸡单剂量静脉注射、肌肉注射和口服泰拉霉素后不同时间的血药浓度值测定结果见表 1。药代动力学数据用3P97药动学程序软件进行处理,对药时数据进行房室模型的拟合,用马夸德(Marquardt)法对一、二、三房室分别以权重1、1/C、1/C2三种情况进行计算,根据残差平方和(WSS)和AIC(Akaike′s informatinn criterion)值最小、拟合度(R2)最大判断最适药代动力学模型。如果WWS和R2的值相近,AIC值愈小,则拟合愈好[7]。结果发现:泰拉霉素注射液血药浓度数据均不能拟合出合适的生理模型,因此采用非房室模型的分析方法,计算出相关药物动力学参数。静脉注射、肌肉注射和口服泰拉霉素后药代动力学参数见表 2。

表 1 鸡静脉注射、肌肉注射及口服泰拉霉素(20 mg · kg-1)后的血药浓度(x±SD,n=6) Table 1 The mean plasma concentration in chickens administered a single intravenous,intramuscular and oral administration of tulathromycin at 20 mg · kg-1 body weight
t/h 血药浓度/(ng·mL -1) Plasma concentration
静脉注射Intravenous 肌肉注射Intramuscular 口服Oral administration
0 0 0 0
0.0833 973.60±106.18 185.77±49.57 151.30±51.17
0.25 822.42±88.03 828.29±73.18 261.04±50.50
0.5 701.97±63.27 678.15±58.03 687.18±102.71
1 619.37±92.60 524.24±42.04 343.16±100.02
2 513.45±96.81 379.26±53.37 203.20±57.33
4 375.17±41.42 280.91±71.00 99.07±9.52
6 237.91±41.22 195.95±37.74 79.05±15.96
8 99.29±7.27 96.22±8.60 70.15±12.47
10 82.63±6.20 89.36±13.10 54.61±12.68
12 51.58±9.01 66.10±6.55 46.90±9.70
24 38.16±8.17 46.90±11.24 37.86±6.91
36 24.13±6.85 22.92±4.86 25.42±4.27
48 15.53±3.44 14.68±2.85 16.78±2.69
72 11.17±2.48 9.10±2.27 10.81±1.51
96 7.94±1.19 6.37±2.09 8.36±1.96
120 4.31±0.85 4.46±1.10 6.67±0.78
表选项

表 2 鸡静脉注射、肌肉注射以及口服泰拉霉素的药代动力学参数(x±SD,n=6) Table 2 The pharmacokinetic parameters in chickens administered a single intravenous,intramuscular and oral administration of tulathromycin at 20 mg · kg-1 body weight
参数
Parameters		 给药方式Route of administration
静脉注射Intravenous 肌肉注射Intramuscular 口服Oral administration
T last/h 120 120 120
AUC last/(μg·h·mL -1) 5.34±0.23 4.93±0.19 3.43±0.30
AUC inf/(μg·h·mL -1 5.44±0.22 5.07±0.19 3.66±0.30
T max/h NA 0.25±0.00 0.50±0.00
C max/(μg·mL -1) NA 0.83±0.07 0.69±0.10
C 0/(μg·mL -1) 1.06±0.13 NA NA
k el/h 0.032 1±0.001 4 0.033 2±0.002 1 0.028 6±0.001 8
t 1/2/h 21.65±0.98 20.96±1.39 24.33±1.52
CL/(mL·kg -1·h -1) 17.71±2.46 NA NA
MRT/h 3.67±0.15 20.17±2.02 27.29±3.06
V ss/(L·kg -1) 77.15±9.64 NA NA
F/% NA 92.59±6.18 62.38±7.48
注:NA:未检测到Not detected
表选项

从表 2可以看出,试验鸡肌肉注射和口服泰拉霉素后,两者的平均最大血药浓度(Cmax)分别为0.83和0.69 μg · mL-1,达峰时间Tmax为0.25和0.5 h,药时曲线下面积(AUClast)分别为4.93和3.43 μg · h · mL-1,血浆平均半衰期(t1/2)分别为20.96和24.33 h。2种给药方式的生物利用度(F)分别为92.59%和62.38%。表明肌肉注射给药效果明显高于口服给药。 3 讨论 3.1 泰拉霉素在鸡体内的药物代谢动力学特征

受试鸡静脉注射、肌肉注射以及口服泰拉霉素(20 mg · kg-1)后,在鸡体内获得的血药浓度-时间数据均符合非房室模型。静脉注射泰拉霉素后鸡体内血浆清除率为17.71 mL · kg-1 · h-1,稳态表观分布容积为77.15 L · kg-1,说明静脉注射给药后,表观分布容积大,半衰期长,吸收迅速,分布广泛,消除缓慢。这有利于提高药物的生物利用度,增加其抗菌效果。但目前泰拉霉素不用静脉注射给药,因为静脉注射给药不仅会有一定的刺激性且可能会产生中度的毒性。肌肉注射和口服泰拉霉素后0.25和0.5 h时的Cmax分别为0.83和0.69 μg · mL-1,血浆中平均消除半衰期分别为20.96和24.33 h,生物利用度分别为92.59%和62.38%,说明肌肉注射给药比口服吸收迅速且较完全,生物利用度高。这可能是由于泰拉霉素含有弱碱性叔胺基团,口服时会和酸性物质发生中和,从而降低药效。给药方式的不同可对药代动力学特性产生直接影响。另外,泰拉霉素的靶器官是肺,肺的药物浓度总是很高,其对特定组织的亲和性决定动物给药后的血药浓度,这只能间接反映其体内药物的抗菌活性。 3.2 种属间药物代谢动力学差异

经肌肉注射后泰拉霉素在鸡体内吸收迅速,达峰时间与猪经肌肉注射的达峰时间(0.25 h)[5]一致,低 于驹的3.89 h[5],其在鸡体内的Cmax为0.83 g · mL-1,与猪的(0.84 g · mL-1)[8]近似,高于驹的410 ng · mL-1[5]。 经肌肉注射后泰拉霉素在鸡体内的生物利用度(92.59%)较在猪体内(112%)[8]低。口服给药后泰拉霉素在鸡体内的达峰时间(0.5 h)比替米考星(2.5 h)[9]快,Cmax为0.69 g · mL-1,生物利用度为64.38%,表明口服给药时,泰拉霉素较替米考星吸收迅速,但利用不完全。口服给药简单方便,但会出现低度急性毒性[3],因此,使用泰拉霉素需在兽医指导下进行。

泰拉霉素主要用于预防和治疗动物呼吸道疾病,所以本试验选取了抵抗力较弱的21日龄左右的雏鸡。由于试验采用心脏采血,考虑到试验动物日龄较小,血量较少,且重复采血会出现溶血现象,同时为了避免个体差异所引起的误差,因此本试验采用同一时间点采集多个平行动物血样。

总之,泰拉霉素在鸡体内的药代动力学特征与该药在猪[10]、牛[1]、羊[11, 12, 13]、兔[14]体内的药代动力学特征相似:具有吸收迅速、达峰时间短、分布广泛、峰浓度高、表观分布容积大等特点。鸡经静脉注射、肌肉注射泰拉霉素后,其血浆半衰期、血浆清除率和生物利用度都低于猪[8],表明该药物在猪、鸡体内的药物代谢动力学过程存在种属差异。肌肉注射泰拉霉素的生物利用度明显高于口服泰拉霉素的生物利用度。

安徽省兽药饲料监察所陶小平所长和汤春莲主任在本研究中给予了帮助,谨致谢意。

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