【作者】 胡会青；

【导师】 许彦芳；

【作者基本信息】 河北医科大学 ， 药理学， 2009， 博士

【摘要】 双苯氟嗪是河北医科大学开发的哌嗪类钙通道拮抗剂,以往的研究表明双苯氟嗪能选择性地扩张椎动脉、基底动脉和冠状动脉,对大鼠脑水肿有保护作用,对局灶性脑缺血和全脑缺血再灌注损伤均具有保护作用。体外实验表明双苯氟嗪具有抗血小板聚集和预防血栓形成的作用。在对抗5-羟色胺诱导的猪基底动脉收缩实验中双苯氟嗪的药理作用强于同类药氟桂利嗪和桂利嗪。因此,双苯氟嗪有望开发成为治疗脑血管疾病的一类新药。考虑临床缺血性脑卒中病人常用的用药途径,新近我们将难溶于水的双苯氟嗪制成盐酸双苯氟嗪,大大提高了其水溶性,其注射液可血管内给药。本实验建立了RP-HPLC法测定血浆中盐酸双苯氟嗪的浓度,并系统研究了盐酸双苯氟嗪在Beagle犬体内的药代动力学、盐酸双苯氟嗪在大鼠的急性毒性和毒代动力学、盐酸双苯氟嗪在Beagle犬的毒代动力学以及盐酸双苯氟嗪在大鼠和犬肝微粒体代谢产物的测定,以揭示盐酸双苯氟嗪在动物体内的动态变化规律,阐明盐酸双苯氟嗪的毒性表现和特征。上述非临床评价资料将为进一步开发盐酸双苯氟嗪提供重要的实验依据。第一部分盐酸双苯氟嗪在Beagle犬的药代动力学研究目的:研究盐酸双苯氟嗪在Beagle犬体内的药代动力学,了解盐酸双苯氟嗪在体内的动态变化规律。方法:Beagle犬18只,随机分为低、中、高三个剂量组,每组6只,雌雄各半,三个剂量组分别单次股静脉注射1.5、3.0和6.0 mg·kg-1的盐酸双苯氟嗪溶液,分别于给药后0、1、3、5、10、15、30 min, 1、2、4、8、12和24 h自后肢股静脉取血,9000×g离心10min分离血浆。Zorbax C8色谱柱为分析柱,流动相水相A为0.2%甲酸溶液,有机相B为甲醇/乙腈/甲酸(60:40:0.2),采用梯度洗脱方式,在36min内,B液含量从0升至65%;流速1ml·min-1;柱温40℃,紫外检测波长为254nm,氟桂利嗪为内标,应用3P97软件计算主要药代动力学参数。结果:在所建立的RP-HPLC方法下,双苯氟嗪和氟桂利嗪的保留时间分别为32 min和34 min,标准曲线为Y=0.2225X-0.0037 (r=0.9999),在0.2-25 mg·L-1的血浆浓度范围内呈良好的线性关系;血浆中最低检测限、最低定量限、提取回收率、日内和日间精密度均符合药代动力学分析方法的要求。在室温和冷冻-解冻实验中,双苯氟嗪均具有良好的稳定性。按低、中、高三剂量单次静脉注射给药后,双苯氟嗪在Beagle犬体内的药代动力学过程均符合开放二房室模型,低、中、高三个剂量的主要药代动力学参数分别为:T1/2β分别为24.7、24.2和29.6 h; AUC分别为0.44、1.12和2.86 mg·min·ml-1;Vc分别为1.30、1.22和1.28 L·kg-1;CL分别为3.4×10-3、2.7×10-3和2.1×10-3 L·kg-1·min-1。结论:本研究建立的RP-HPLC法能够满足盐酸双苯氟嗪药代动力学研究的要求,静脉注射盐酸双苯氟嗪在犬体内消除过程属于两相消除, AUC与剂量呈线性相关。第二部分盐酸双苯氟嗪在大鼠的急性毒性和毒代动力学研究目的:研究大鼠单次静脉注射盐酸双苯氟嗪的急性毒性反应和死亡情况,以了解其毒性作用的靶器官,同时研究其毒代动力学为毒性反应发生提供依据。方法:Sprague-Dawley大鼠,每组6只,雌雄各半,分别以5、6、10、15、25、30、35、40mg·kg-1的剂量尾静脉注射盐酸双苯氟嗪溶液。根据前期实验,选择安全剂量5mg·kg-1为起始剂量。对照组静脉注射溶剂。持续观察大鼠给药后2h的一般行为、临床表现以及死亡率,2h后每4h观察一次直至给药后24h,然后每天观察一次直至给药后14天。所有动物分别于给药前、给药后2、4、8、15天称量体重。死亡动物立即进行剖检,存活动物于末次观察后,腹腔注射戊巴比妥麻醉后腹主动脉放血后剖检。异常组织保存在10%的中性福尔马林溶液中,然后浸入石蜡油中,切片,苏木精和伊红染色,显微镜下检测组织切片。另取6只大鼠,雌雄各半,以最大耐受剂量30mg·kg-1静脉给药后,于给药后1h和24h内眦静脉取血后测定血液生化学指标,包括谷氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、磷酸酶(ALP)、总胆红素(T-BIL)、尿素氮(BUN)、肌酸酐(CRE)、总胆固醇(TCHO)、葡萄糖(GLU)、白蛋白(ALB)、总蛋白(TP)、甘油酸酯(TG)、γ-谷氨酰(GGT)和肌酸激酶(CK)。144只大鼠,随机分为3组,每组48只,雌雄各半。三组大鼠分别以5、15、30 mg·kg-1的剂量尾静脉注射给予盐酸双苯氟嗪溶液。分别在给药后的0.08、0.16、0.25、0.5、1.0、2.0、8.0和24 h麻醉动物,腔静脉取血,分离血浆,-20℃保存备用。分离组织包括心、肝、脾、肺、肾、胰、脑、生殖器官,用生理盐水洗净表面血污,滤纸拭干,称重,用甲醇/水(1:1)制成1.0 g·ml-1匀浆,-20℃保存备用。RP-HPLC法测定血浆和组织匀浆中盐酸双苯氟嗪的浓度。结果:大鼠单次静脉注射盐酸双苯氟嗪的无毒反应剂量为5 mg·kg-1,最小毒性反应剂量为6 mg·kg-1,毒性反应症状于给药后立即出现,包括竖毛、全身颤抖、抽搐、眼充血、泡沫性痰、呼吸困难。在6~30 mg·kg-1剂量范围内,毒性反应症状在给药后20~60 min内逐渐减轻直至消失。在35 mg·kg-1,实验动物出现严重的毒性反应,6只受试动物中2只死亡,其它动物在给药1h后恢复正常。因此,最大耐受剂量为30 mg·kg-1,而最小致死剂量为35 mg·kg-1。在40 mg·kg-1剂量组,6只受试动物中3只在给药后20 min内死亡。所有存活动物在剩余的观察期限内均未表现出异常。实验过程中所有动物的体重未发生明显改变。给药后1h,30 mg·kg-1剂量组大鼠血液的AST、ALT、ALP、GLU、CK水平与对照组相比显著增加,其它参数包括T-BIL、BUN、CRE、TCHO、ALB、TP、TG、GGT没有显著差别。给药后24h,所有参数与对照组相比都没有差异。尸检结果显示死亡动物出现明显的肺充血现象,但存活动物并未观察到其它异常现象。组织病理学结果进一步显示出肺充血现象,大量血细胞浸入到肺泡壁中。毒代动力学实验结果表明静脉注射盐酸双苯氟嗪后,药时曲线显示出两相消除,拟合的药-时曲线符合静脉给药开放二房室模型。三个剂量的AUC分别为2.9、10.9、29.4μg?h?ml-1,AUC和剂量之间呈线性关系(r=0.9939)。T1/2α分别为14.5、36.0、23.8 min, T1/2β分别为11.2、11.6、23.3 h。T1/2β在30 mg·kg-1时显著增加。表观分布容积分别为3.34、3.54、1.96 L·kg-1,表明双苯氟嗪易分布到各组织中。所有组织的AUC与剂量均呈线性关系。肺中AUC水平最高,肺中三个剂量的AUC分别为8.9、26.2、52.9μg·h·ml-1 ,显著高于血浆中三个剂量的AUC水平(p<0.01)。脑、肾、胰的AUC和血浆AUC接近,而心、肝、脾、生殖AUC低于血浆AUC水平。三个剂量的肺、肾、脑的T1/2β均比同等剂量的血浆T1/2β长,在15和30mg·kg-1剂量组,肝和胰的T1/2β也长于血浆T1/2β。结论:静脉注射双苯氟嗪后,最大无毒剂量、最小毒性剂量、最大耐受剂量和最小致死剂量分别是5、6、30和35mg·kg-1。毒代动力学结果提示毒性症状严重程度与药物在体内的暴露呈线性关系。暴露水平最高的肺出现肺淤血现象。肾、脑、肝、胰的半衰期较长提示长期给药时这些组织可能成为药物蓄积的器官。临床生化学指标显示大剂量双苯氟嗪对肝脏和心脏有可逆性损伤。这些结果为设计长期毒性实验提供了参考信息。第三部分盐酸双苯氟嗪在Beagle犬体内的毒代动力学研究目的:研究盐酸双苯氟嗪在犬长期毒性实验的相伴毒代动力学,以了解大剂量连续给药情况下药物剂量与全身暴露量的关系以及盐酸双苯氟嗪在犬体内的可能蓄积的情况。方法:24只健康Beagle犬,雌雄兼用,体重8-10 kg,随机分为3组,每组雌雄各4只。给药前各取血制备空白血浆。三组动物分别按2.5、5.0和10.0 mg·kg-1三个剂量静脉滴注盐酸双苯氟嗪注射液(2ml·min-1),每天一次,连续给药4周。分别于首次(第一天)和末次给药(第27天)后0、5、10、15、30 min, 1、2、8和24 h自后肢静脉取血3ml,在9000×g离心10min分离血浆,保存在-20℃冰箱中。用HPLC方法测定血浆中盐酸双苯氟嗪的浓度。结果:Beagle犬分别以2.5、5.0、10mg·kg-1静脉滴注盐酸双苯氟嗪后,所得药时曲线符合开放二室模型。第一次取血后,三个剂量的CL分别为5.6、4.0、5.3 mL·kg-1·min-1,Vc分别为0.79、1.14、1.15 L·kg-1。三个剂量之间的CL和Vc没有显著差别。三个剂量的T1/2β分别为7.8、12.8、18.2 h,T1/2β随着剂量的增加而增加。三个剂量的AUC分别为0.47、1.06、2.38 mg·min·ml-1,AUC和剂量呈线性关系(r=0.9996)。静脉滴注给药4周(第27天)后,三个剂量的CL分别为4.5、4.0、2.9 mL·kg-1·min-1,高剂量组的CL比低、中剂量组的CL略低,但没有统计学意义。三个剂量的Vc值分别为0.97、0.85、0.88 L·kg-1,没有显著性差别;三个剂量的T1/2β分别为11.7、14.2、18.0 h。T1/2β随着剂量增加而增加。三个剂量的AUC分别为0.61、1.42、3.74 mg·min·ml-1, AUC和剂量呈非线性关系。和第一次给药后相比,重复给药后高剂量组的AUC增加,Vc和CL降低,三个剂量组的T1/2β均没有明显变化。结论:毒代动力学结果表明单次静脉滴注盐酸双苯氟嗪后,在2.5、5.0、10 mg·kg-1剂量下,体内暴露量与用药剂量呈线性关系,而连续用药4周后,高剂量组体内暴露量与用药剂量呈非线性关系。造成非线性的主要原因是高剂量AUC的增加超过剂量增加的比例。可能是代谢发生饱和现象,长期给药后药物在体内蓄积,导致药物在体内暴露量增加。第四部分盐酸双苯氟嗪在大鼠和犬体内、体外代谢研究目的:比较盐酸双苯氟嗪在大鼠和犬肝微粒体的代谢情况,并研究其在犬体内的代谢产物,了解盐酸双苯氟嗪可能的代谢种属差异。方法:差速离心法分离肝微粒体,考马斯亮蓝法测定大鼠和犬微粒体蛋白的浓度,牛血清白蛋白为标准测定蛋白。盐酸双苯氟嗪用甲醇溶解,分别加入大鼠或犬肝微粒体中,混合物在37℃水浴箱中振荡孵育3min后,加入β-NADPH反应系统启动反应。反应液总体积为1 ml,盐酸双苯氟嗪、NADPH和微粒体蛋白的终浓度分别为1 mmol·L-1、1 mmol·L-1和1 mg·ml-1,反应液中甲醇的比例小于1%。反应时间为30 min。冰裕终止反应。空白对照样品中加入甲醇代替盐酸双苯氟嗪溶液。所有样品制备三个复管。以2.5 mg·kg-1的剂量静脉注射给予Beagle犬盐酸双苯氟嗪溶液,并于给药后30 min和1 h分别自后肢静脉取血,分离血浆,-20℃保存备用。收集给药后0-24 h的尿液,离心取上清,-20℃保存备用。LC-MS/MS测定盐酸双苯氟嗪的代谢产物。结果:反应体系孵育30 min后,盐酸双苯氟嗪在不同种属肝微粒体中代谢未见有性别差异。盐酸双苯氟嗪在大鼠肝微粒体中共生成8个代谢产物,分别为1-(4-氟苯基)-4-哌嗪基丁酮(M1)、4-羟基二苯甲酮(M2)、4-氟-γ-羟基苯丁酸(M3)、二苯甲醇(M4)、二苯甲酮(M5)、1-羟基二苯甲基-4-[3-(4-氟苯基)]-哌嗪(M6)、1-羟基二苯甲基-4-[3-(4-氟苯羟基)]-哌嗪(M7)、1-二苯甲基-4-[3-(4-氟苯甲基)]-哌嗪(M8);盐酸双苯氟嗪在犬肝微粒体中也生成8个代谢产物,除M1、M2、M3、M4、M5、M6、M8同大鼠微粒体中代谢产物外,另有1-羟基二苯甲基-4-[3-(4-氟苯甲基)]-哌嗪(M9),在大鼠微粒体中未检测到M9,而在犬微粒体中也未见M7生成。犬血浆中除原型药双苯氟嗪外,代谢产物为M1、M2、M3、M4、M5、M6,主要代谢产物为M1、M4和M6;尿液中检测到的代谢产物和血浆中相同,但尿液中主要代谢产物为M1、M2和M4。血浆和尿液中均未检测到M8和M9。结论:根据生成的代谢产物判断,盐酸双苯氟嗪在大鼠和犬的代谢途径为1-和4-氮脱烷基代谢以及苯环氧化和/或烷基化代谢。大鼠和犬共同的代谢产物为M1、M2、M3、M4、M5、M6和M8,不同的是大鼠肝微粒体中的M7而犬肝微粒体中为M9。大鼠微粒体中M6的量显著高于犬微粒体中M6的量,这可能是由于大鼠体内氧化酶的活性高于犬,氧化酶活性的差别也可以由M7存在于大鼠微粒体中而犬微粒体中未见。本研究提示盐酸双苯氟嗪在大鼠和犬微粒体中代谢具有种属差异。更多还原

【Abstract】 Dipfluzine, a novel diphenylpiperazine calcium channel blocker, was first synthetized by Hebei Medical University. The previous studies have demonstrated that Dip is a high selective cerebral vasodilator. Experimental studies have shown that Dip exerts the protective effects against focal or whole cerebral ischemic injury via multiple mechanisms. Dip also possesses the effects of anti-aggregation of platelet in vitro and prevention of thrombus formation in vivo. In vivo and in vitro evidence from those studies have revealed that pharmacological effects of Dip are more potent than its analogues, cinnarizine or flunarizine, marketed calcium channel blockers. Dip, therefore, is a promising candidate drug to treat cerebral vascular diseases. However, Dip is poorly water soluble. Considered of parenteral administration of the drug in ischemic stroke patients, we recently developed a preparation of water-soluble Dip, dipfluzine hydrochloride. In this study, a RP-HPLC method was developed to determine the concentration of dipfluzine hydrochloride in plasma and tissues. We studied the pharmacokinetics of dipfluzine hydrochloride in Beagle dogs by using this method, the acute toxicity and toxicokinetics of dipfluzine hydrochloride in rats and the toxicokinetics of dipfluzine hydrochloride in Beagle dogs. Metabolites of dipfluzine hydrochloride in rat and dog liver microsomes were determined. The study would provide nonclinical information to the further development of dipfluzine hydrochloride as a new drug.Part1 The pharmacokinetics of dipfluzine hydrochloride in Beagle dogs. Aim: To study the pharmacokinetics character of dipfluzine hydrochloride in vivo by determining the plasma concentration after iv administration in Beagle dog.Methods: 18 Beagle dogs were distributed into three groups with 6 animals in each group (3 female and 3 male). Dogs were respectively given a single intravenous injection of 1.5, 3.0 and 6.0 mg·kg-1 dipfluzine hydrochloride for each group. Blood samples were collected via the femoral vein at 0, 1, 3, 5, 10, 15, 30 min, 1, 2, 4, 8, 12 and 24h after administration of dipfluzine hydrochloride. Plasma was separated by centrifugation at approximately 9000×g for 10 min and stored at -20℃until analyzed. A Zorbax C8 reversed-phase column was used as the analytic column. The mobile phase was developed by using 0.2% formic acid as aqueous phase and methanol/acetonitrile/ formic acid (60:40:0.2, v/v/v) as organic phase. The manner of gradient elution was achieved by a gradual increase of organic phase from 0% to 65% within 36 minutes. The flow-rate was maintained at 1 ml/min, and the detection was performed at a wavelength of 254 nm under constant column temperature of 40℃. Flunarizine was choosen as the inner standard. The pharmacokinetic parameters were calculated by 3P97 software.Results: Under the RP-HPLC method, the retention time of dipfluzine hydrochloride and flunarizine was 32 min and 34 min, respectively. Calibration curves was Y=0.2225X-0.0037 (r=0.9999). Results for the method were linear over the calibration range of 0.2-25 mg·L-1. The specificity, lowest limit of detection and quantification, extraction recoveries, the precision of intra- and inter-day were qualified to the pharmacokinetic study. Dipfluzine hydrochloride was found to be stable after three cycles of freeze and thaw ( room temperature), and no signs of degradation were found under the freeze condition. After intravenous administration of three doses of dipfluzine hydrochloride, the concentration-time courses of dipfluzine hydrochloride were best fitted to a two-compartment open model. The main pharmacokinetic parameters at three d?oses were ?24.7, 24.2 and 29.6 h for T1/2 , 0.44, 1.12 and 2.86 mg·min·ml-1 for AUC, 1.30, 1.22 and 1.28 L·kg-1 for Vc, and 3.4×10-3, 2.7×10-3 and 2.1×10-3 L·kg-1·min-1 for CL, respectively.Conclusion: The developed RP-HPLC method for determination of dipfluzine hydrochloride in plasma can satisfy the requirement of pharmacokinetic study after intravenous administration of dipfluzine hydrochloride. Analysis of plasma concentration-time curves indicated a biphasic decrease. There was a linear relationship between AUC and dose. Part2 The acute toxicity and toxicokinetics of dipfluzine hydrochloride.Aim: To find the potential toxic target-organs by observing the symptom of acute toxicity and its expiration after a single intravenous administration of dipfluzine hydrochloride, and simultaneously study the toxicokinetics of dipfluzine hydrochloride to provide explanation for the toxicity.Methods: Dipfluzine hydrochloride was administered by intravenous injection to groups of rats (3 males and 3 females for each group) at doses of 5, 6, 10, 15, 25, 30, 35, and 40 mg·kg-1 body weight. The starting dose of 5 mg·kg-1 was a safety dose based on a preliminary study. The control group received the vehicle only. The general demeanor, clinical signs, and mortality of rats were continuously observed for 2 h after injection, and then once every 4 h for 24 h and thereafter once a day for 14 d. The body weights of all animals were measured before dosing and on days 2, 4, 8, and 15. All dead rats were immediately subjected necropsy and surviving animals were euthanized after the final observation (day 15) by exsanguination from the abdominal aorta under intraperitoneal pentobarbital and subjected to gross necropsy. The abnormal organ detected was preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissue sections were microscopically examined.Another 6 rats (3 males and 3 females) were used for blood chemistry. Blood samples were collected via angular vein at 1 and 24 h after intravenous dosing at 30 mg·kg-1, the maximal tolerance dose. Clinical chemistry parameters were determined by automatic biomedical detector with assay kits and included aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirebin (T-BIL), urea nitrogen (BUN), creatinine (CRE), total cholesterol (TCHO), glucose (GLU), albumin (ALB), total protein (TP), triglyceride (TG), gamma-glutamyl transferase (GGT) and creatinkinase (CK).A total 144 rats were used for toxicokinetic study, which were distributed into three groups with 48 animals in each group. Rats were respectively given a single intravenous injection of 5, 15 and 30 mg·kg-1 dipfluzine hydrochloride for each group. Six rats (three males and three females) at each time-point were sacrificed under intraperitoneal pentobarbital at 0.08, 0.16, 0.25, 0.5, 1.0, 2.0, 8.0, and 24 h after administration of dipfluzine hydrochloride. Blood samples were collected via the posterior vena cava and then the following organs were taken: heart, liver, spleen, lung, kidney, brain, pancreas and reproductive tissues. Each tissue sample was rapidly weighed and rinsed with 0.9% NaCl to remove the blood or content, and then homogenized in methanol/water (1:1, v/v) solution to obtain the concentration of 1.0 g·ml-1. The obtained tissue homogenates were centrifuged at approximately 9000×g for 10 min and the supernatants were stored at -20℃until analyzed. Blood and tissue samples were subjected to the determination of dipfluzine hydrochloride concentration by RP-HPLC.Results: The no-observed-adverse-effect level (NOAEL) was 5 mg·kg-1, whereas the lowest-observed-adverse-effect level (LOAEL) was 6 mg·kg-1. The clinical signs of toxicity were immediately evident after the intravenous administration of dipfluzine hydrochloride and included hair-upright, whole body tremor or convulsion, eye congestion, frothy sputum, dyspnea. The toxic symptoms were gradually reduced and disappeared within 20-60 min post-dose from 6 to 30 mg·kg-1. At 35 mg·kg-1, rats showed the severe symptoms, and two of six test rats died within 30 min and the activity of others was fully recovered within 1 h. Thus, maximal tolerance dose (MTD) was 30 mg·kg-1, and minimal lethal dose (MLD) was around 35 mg·kg-1. At 40 mg·kg-1, three of six test rats died within 20 min. All surviving rats showed no any abnormal signs during the following test period. The body weight of rats was not significantly changed during the period of the test.At 1 h after dosing, AST, ALT, ALP, GLU and CK levels in 30 mg·kg-1 dose group were significant higher compared to the control group. There were no difference in other parameters including T-BIL, BUN, CRE, TCHO, ALB, TP, TG, and GGT. By 24 h all of clinical chemistry parameters in rats administrated with dipfluzine hydrochloride were not different from the control levels. From the autopsies of test mice, obvious lung congestion was found in dead mice, but the organs of surviving rat lacked any other abnormal indications. The histological analysis further demonstrated the pulmonary congestion, which characterized by an infiltration of lots of blood cells into alveolar wall.Following i.v. administration, analysis of plasma concentration-time curves indicated a biphasic decrease. A good fit of the observed data for a two-compartment model was obtained. The results showed that AUC in plasma were respectively 2.9, 10.9, 29.4μg?h·ml-1 at 5, 15 and 30 mg·kg-1. A good linear correlation was obtained in correlation and regression analysis of AUC-dosage plots (r=0.9939). The T1/2αwere respectively 14.5, 36.0, and 23.8 min, and the T1/2βwere respectively 11.2, 11.6 and 23.3 h. The T1/2βwas significantly increased at 30 mg·kg-1. The apparent volumes of distribution were respectively 3.3, 3.5 and 2.0 L·kg-1, suggesting that Dip easily penetrated all tissues. A good linear correlation was obtained in correlation and regression analysis of AUC-dosage plots in all of detected tissues. The highest AUC level was found in lung and it was respectively 8.9, 26.2 and 52.9μg?h/ml after iv administration of three doses of dipfluzine hydrochloride, which were significantly higher than those in plasma (p<0.01). AUCs in brain, kidney, and pancreas were similar to those in plasma, whereas in heart, liver, spleen, and reproductive tissues were smaller than those in plasma. The T1/2βwas much longer in lung, kidney and brain than that in plasma at all of three doses. The longer T1/2βwas also found in liver and pancreas at doses of 15 and 30 mg·kg-1.Conclusion: After intravenous administration of dipfluzine hydrochloride, the acute toxicity study demonstrated that NOAEL, LOAEL and MTD were 5, 6, 30 mg·kg-1, respectively. The toxicokinetic result indicated that there was a linear relationship between the symptom of toxicity and the exposure of dipfluzine hydrochloride in rat. Congestion was found in lung which was also the tissue with the highest dipfluzine hydrochloride concentration. The longer T1/2βin kidney, brain, liver and pancreas revealed that dipfluzine hydrochloride probably accumulated in these tissues after repeated administration. The clinical chemistry results revealed that there were temporary impairment in liver and heart. These results provide evidence for long-term toxicity study of dipfluzine hydrochloride. Part3 The toxicokinetics of dipfluzine hydrochloride in Beagle dogs.Aim: To study the relationship between doses and exposure and the possible accumulation of dipfluzine hydrochloride in dogs after repeated intravenous infusion by investigating the toxicokinetics of dipfluzine hydrochloride in Beagle dogs in the long-term toxicity study.Methods: 24 Beagle dogs were randomly distributed into three groups with 8 animals in each group (4 male and 4 female per group) and were respectively given intravenous infusion of 2.5, 5.0 and 10 mg·kg-1 dipfluzine hydrochloride (2ml·min-1 ) for each group once a day for a 4-week period. The blood was collected at 0, 5, 15, 30 min and 1, 2, 4, 8 and 24 h on the first day and the 27th day immediately after intravenous administration. Plasma was separated by centrifugation at approximately 9000×g for 10 min and stored at -20℃until analyzed. The plasma samples were subjected to the concentration determination of dipfluzine hydrochloride by HPLC.Results: A good fit of the observed data for a two-compartment model was obtained for intravenous infusion of dipfluzine hydrochloride at three doses of 2.5, 5.0 and 10 mg·kg-1. After a single (on the 1st day) intravenous infusion administration, the CL for three doses was 5.6, 4.0 and 5.3 mL·kg-1·min-1 and the Vc was 0.79, 1.14 and 1.15 L·kg-1, respectively. The CL and Vc were not significantly different among three doses. The T1/2βfor three doses were 7.8, 12.8 and 18.2 h, The T1/2βincreased with the dose. The AUC for three doses were respectively 0.47, 1.06 and 2.38 mg·min·ml-1, and there was linear relationship between AUC and dose. After 4 weeks (on the 27th day) of intravenous infusion administration, the CL for three doses was 4.5, 4.0 and 2.9 mL·kg-1·min-1. CL in high group was a little lower than that in low and medium group, but there was no statistic difference. The Vc was 0.97, 0.85 and 0.88 L·kg-1 respectively. Vc were not significantly different among three doses. The T1/2βfor three doses was 11.7, 14.2 and 18.0 h, The T1/2βincreased with dose. The AUC for three doses were respectively 0.61, 1.42 and 3.74 mg·min·mL-1, and there was nonlinear relationship between AUC and dose. When compared with those in the first single administration, the repeated administration resulted in an increase of Vc in low and medium group and AUC in the high group, and reduction in Vc and CL in high group. There was no difference in T1/2βin the three groups.Conclusion: After a single (on the 1st day) intravenous infusion administration of 2.5, 5.0 and 10 mg·kg-1, there was linear relationship between AUC and dose. However, after 4 weeks (on the 27th day) of repeated administration, AUC was not varied with dose in a linear relationship. This non-linear kinetics in vivo may result from the metabolism saturation in the high dose and thus the increment in AUC was not proportional to the dose. Our study suggested that repeated-administration may result in accumulation of dipfluzine hydrochloride in the body.Part4 The metabolic research of dipfluzine hydrochloride in rat and dog. Aim: To investigate the metabolites of dipfluzine hydrochloride in vitro and in vivo in rat and dog.Methods: The liver microsomes was prepared by differencial centrifugation method, the rat and dog microsomal protein contents were determined by Bradford assay method, with BSA as the standard protein. Dipfluzine hydrochloride stock solution in methanol was added to rat or dog liver microsomes respectively. The mixture was shaken for 3 min for equilibration in a shaking water bath at 37℃. The incubation was then initiated by addingβ-NADPH solution. The final concentrations of dipfluzine hydrochloride, NADPH and the microsomal protein were 1 mmol·L-1, 1 mmol·L-1 and 1 mg·ml-1 , respectively in a typical incubation mixture (1 ml) for metabolite identification study. The percentage of methanol in the incubation mixture was kept less than 1% (v/v). For metabolite identification study, samples were incubated for 30 min. The reaction was terminated with a ice bath. Negative controls were prepared with methanol added substituting the dipfluzine hydrochloride. All experiments were carried out in triplicate.Beagle dogs were intravenously given of 2.5 mg/kg dipfluzine hydrochloride. The blood was collected at 30 min and 1 h after administration of dipfluzine hydrochloride and plasma was separated by centrifugation at approximately 9000×g for 10 min and stored at -20℃until analyzed. The urine was collected for 0-24 h after administration of dipfluzine hydrochloride. After centrifugation, the supernatant was stored at -20℃until analyzed. The metabolites of dipfluzine hydrochloride were analyzed by LC-MS/MS.Results: After incubation for 30 min, metabolite profiles in females were found similar as their corresponding males for the same species. There were eight metabolites of dipfluzine hydrochloride generated in rat microsomes, which were respectively 1-(4-fluoro-benzene)-4- piperazine-butanone (M1), 4-OH-benzophenone (M2), 4-fluoro-γ-OH- phenylbutyric acid (M3), benzhydrol (M4), benzophenone (M5), 1-OH- diphenylmethyl-4-[3-(4-fluoro-benzene)]-piperazine (M6), 1-OH- diphenylmethyl-4-[3-(4-OH-fluoro-benzene)]-piperazine (M7) and 1- diphenylmethyl -4-[3-(4- fluoro-benzene-methyl)]- piperazine (M8). A new metabolite 1-OH-diphenylmethyl -4-[3-(4- fluoro-benzene-methyl)]- piperazine (M9) was found in microsomes in dog in addition to M1, M2, M3, M4, M5, M6 and M8. While the M7 existed in rat microsomes was not found in dog microsomes.In the dog plasma at 30 min and 1 h after administration of dipfluzine hydrochloride, the metabolites were respectively M1, M2, M3, M4, M5 and M6, and the main metabolites were M1, M4 and M6. The metabolites detected in dog urine were almost the same as that in plasma, with the exception of M1, M2 and M4 as the main metabolites. Both of M8 and M9 were not found in plasma and urine.Conclusion: Based on the metabolite profiling, we proposed that the primary metabolic pathways of dipfluzine hydrochloride in rat and dog were 1,4-N- dealkylation, hydroxylation and/or methylation on benzene ring. The identical metabolites in rat and dog were M1, M2, M3, M4, M5, M6 and M8. M7 existed only in rat and M9 only in dog. The content of M6 in rat microsomes was much higher than that of in dog. The result indicated that the activity of hydroxylase in rat was higher than that in dog, which was also manifested by the different existence of M7 (only in rat and not in dog). The study suggested that there was species-difference for the metabolism of dipfluzine hydrochloride.更多还原