【摘要】目的明确幼年动物对氨基糖甙类抗生素耳毒性有无更高敏感性。方法用扫描电镜及光镜观察以等效人类治疗剂量下丁胺卡那霉素（AMK）注射的10只早产、31只新生、31只幼年及31只成年豚鼠毛细胞，并用TDx法研究新生、幼年及成年组各16只豚鼠药代动力学特征。结果(1)单剂给药证实各年龄组均为二室开放性模型，峰浓度及达峰时间差异无显著性，但新生组清除率较其他组明显降低［分别为(0.004 4±0.001 1)、(0.009 7±0.004 2)和(0.008 8±0.001 4) L/(min·kg),均P＜0.01］，清除半衰期明显延长，［分别为(71±13)、(53±11)和(43±8) min,分别P＜0.05、P＜0.01］，曲线下面积明显增大分别为［(11 462±431 7)、(8 349±147 0)和(8 166±321 3)，均P＜0.01］；幼年、成年组无差异。多次给药新生组血药浓度明显高于同时点幼年、成年组，新生及幼年组存在明显体内药物蓄积。(2) 早产、新生组损伤出现早且严重，幼年组较成年组重；停药后毛细胞缺失数继续增多。结论即使等效人类治疗剂量的AMK对幼年（尤其早产和新生）豚鼠耳蜗均有不同程度损害，并随用药时间延长而加重。幼年动物这种高敏感性与该药从体内排出慢、易蓄积有关。

Experimental study on the ototoxicity of amikacin at therapeutic doses in infant guinea pig

XU Hongmei，WANG Shaoying（Department of Infectious and Digestive Diseases. Children's Hospital, Chongqing University of Medical Sciences, Chongqing 400014, China）

【Abstract】ObjectiveTo clarify the possibility of higher sensitivity to ototoxicity of aminoglycosides and its causes in infant guinea pig by comparing the differences of ototoxicity and pharmacokinetics among different age groups of guinea pig (including premature and neonatal animals) given amikacin with therapeutic doses equal to clinical doses. MethodsThere were 31 guinea pigs in each of neonatal, infant and adult groups and 10 in premature group. Six guinea pigs in each group were used as control. Amikacin was intramuscularly injected at a dose of 65 mg/kg (which is equal to the clinical therapeutic dose) once a day for 14 days. Five to 8 guinea pigs in each group were sacrificed for histological and scanning electron-microscopic examination of cochlea on the day after the 3rd, 5th, 7th, 10th and 14th day of drug administration, as well as on the 28th day. Pharmacokinetic parameters were investigated in all but premature group with 16 guinea pigs from each by TDx system.Results(1) The Pharmacokinetic parameters were compatible with the model of two compartments in all age groups. There were no significant differences in the peak level and peak time among the groups. The neonatal group had lower plasma clearance than the infant and adult groups ［(0.004 4±0.001 1), (0.009 7±0.004 2) and (0.008 8±0.001 4) L/(min·kg), P＜0.01 for all］; the half-life for elimination was longer［(71±13), (53±11) and (43±8) min, P＜0.05 and P＜0.01 respectively］; the AUC was larger［(11 462±4 317), (8 349±1 470) and (8 166±3 213), P＜0.01 for all］. There was no statistically significant difference between the infant and adult groups in single-dose pharmacokinetics. The blood levels of amikacin after daily administration in neonatal guinea pigs were predominantly higher than those in infant and adult groups, and were increased markedly since the 10th day. It did so in infant group since 14th day. These results indicated that there was an increased accumulation of amikacin in neonatal and infant guinea pigs. (2) On histological examinations of cochlea, many stereocilia were found lain down or disappeared as early as on the 3rd day and a number of outer hair cells were found disappeared on the 5th day in either premature or neonatal group on scanning electron-microscopy. The change of stereocilia at 5th day in infant and adult groups was similar to that in premature and neonatal groups. Microscopic observation showed that more hair cells were absent in neonatal group than in infant and adult groups after the 7th day (P＜0.05 orP＜0.01). The hair cell absence was more obvious in premature group than in neonatal group.The hair cell absence in infant group was more severe than in adult group on 28th day (P＜0.05). The severity of cochlea damage became worse with a prolonged course of amikacin administration. Continuous damage was observed within 2 weeks after the drug administration was stopped in all groups. ConclusionsSignificant ototoxicity of amikacin to cochlea was well observed in infant guinea pigs with various severity, particularly in premature and neonatal animals at a dose equal to clinical therapeutic dose. The severity of cochlear damage became worse with a prolonged course of the administration. The ototoxicity occurred much earlier and more severe in premature and neonatal animals. The damage to cochlea could continue for quite a longer period after the drug administration was discontinued. The higher sensitivity to ototoxicity of amikacin in young guinea pigs seemed to be related to their much slow clearance and accumulation of amikacin. The results suggest that close attention should be paid to the danger of ototoxicity caused by aminoglycosides in children (especially in premature, neonate or infant) even they are administrated at a dose recommended by pharmacopeia and for a regular course. Auditory electrophysiological eximation should be used to monitor the audition during aminoglycosides administration. Aminoglycosides should be avoided in children as long as possible, esp. in those from families with higher sensitivity to ototoxicity of aminoglycosides.

【Key words】Amikacin;Dose-response relationship, drug;Guinea pigs;Pharmacokinetics

氨基糖甙类抗生素耳毒性损害已成为目前我国儿童听力语言残疾的主要因素。本研究以耳蜗发育类似人类的豚鼠为对象、研究等效人类治疗剂量丁胺卡那霉素(AMK)在不同年龄组的药代动力学与耳蜗基底膜毛细胞形态学改变的关系，为深入探讨儿科合理使用该类药物提供理论依据。

材料与方法

一、实验材料

1.实验动物：耳蜗反射阳性、无中耳炎及外耳道炎的健康杂毛豚鼠（重庆医科大学实验动物中心提供），雌雄各半。31只成年豚鼠出生后5个月，体重500～600 g、31只幼年豚鼠出生后1～3周，体重150～250 g、31只新生豚鼠（孕龄65～70 d足月出生的豚鼠）、10只早产豚鼠（足月出生前1周剖宫产的豚鼠）。

2.AMK：苏州第六制药厂生产，批号：971222，豚鼠剂量按人［15 mg/(kg*d)］与动物等效剂量折算公式^［1］计算为65 mg/(kg*d)。

3.试剂：AMK荧光偏振免疫测定试剂盒（南京军区南京总医院临床药理研究室提供，批号：981204），0.5%的硝酸银，10 %的甲醛，0.1 mol的磷酸缓冲液，4%的戊二醛。

二、研究方法

1.药代动力学：（1）单剂给药：新生组、幼年组、成年组各6只豚鼠，上午9时大腿内侧肌肉注射，单剂给予豚鼠AMK 65 mg/kg，给药后5、10、20、30、40、60、120、180、240 min割耳缘静脉各取血0.5 ml，分离血清，TDx仪测定AMK浓度，3P87程序求出每组各个动物药代动力学参数。（2）多次给药：新生组、幼年组、成年组各10只豚鼠（AMK剂量、途径同上），每日上午9时给药×14 d，实验第3、5、7、10、14 d给药后、各组达峰时间割耳缘静脉取血0.5 ml，测定AMK浓度。

2.耳蜗基底膜毛细胞形态学：（1）分组：实验组：新生组、幼年组、成年组各31只，早产组10只，处理方法同多次给药药代动力学；对照组：对照组各组同期饲养未给予任何处理的豚鼠各6只。（2）耳蜗标本收集时间：新生组、幼年组、成年组：第3、5天给药次日各组处死1只豚鼠取一侧耳蜗作扫描电镜标本，第7、10、14天给药次日及第28天分别处死7～8只豚鼠作光镜标本；早产组：第3、5天给药次日各处死一只豚鼠作扫描电镜标本，第28天处死8只豚鼠作光镜标本；对照组：各组同期饲养6只豚鼠第28天并处死作光镜检查(由于实验期间环境噪音有可能致少数毛细胞缺失，故选用与实验组环境相同、最长实验时间内未给予处理的豚鼠为对照组)。(3)光镜标本制备及观察：采用全内耳硝酸银染色法^［2］制备标本及改良 Stockwell等^［3］法(每圈基底膜随机取5个视野)计算耳蜗基底膜毛细胞缺失总数。(4)扫描电镜标本制备：断头处死豚鼠，2 min内用4%的戊二醛灌注固定耳蜗，0.1 mol磷酸缓冲液漂洗，锇酸固定、乙醇脱水、叔丁醇干