【作者】 梁骏；

【导师】 韩国柱；

【作者基本信息】 大连医科大学 ， 药理学， 2008， 硕士

【摘要】 目的:通过比较头孢拉定（Cephradine,CED）在糖尿病大鼠和正常大鼠体内药代动力学行为的差异,研究糖尿病状态对CED药代动力学的影响,为糖尿病患者临床合理使用CED提供实验依据。方法:采用静脉注射四氧嘧啶（60mg/kg）建立糖尿病大鼠模型,取血糖高于16mmol/L的大鼠作为糖尿病组用于研究,同时观察并记录大鼠“三多一少”症状。实验分为两大组,即正常对照（CTL）组和糖尿病（DM）组,每大组又按给药途径分为静脉给药组和口服给药组,每组再按CED给药量分为高剂量（180mg/kg）和低剂量（90mg/kg）两小组。静脉给药各动物于给药前及给药后5、15、30、50、80、120、180、240、360、450、600min,口服给药各动物于给药前和给药后15、30、45、60、75、105、150、240、330、450、600min自眼球后静脉丛收集0.4mL血样,置于肝素化离心管中,3000r/min×15min离心,分离得血浆样品。血浆样品中CED采用反相HPLC法测定,其色谱条件为:固定相采用Kromasil C₁₈色谱柱（4.6mm×250mmID, 5μm） ,并装配以Kromasil C₁₈保护柱（4.6×20mmID,10μm）,流动相为0.025mol/L KH₂PO₄-MeOH-CH₃CN（87:6:7 v/v）,流速1.0mL/min,UV检测波长261nm。200μL血浆样品,精密加入400μg/mL的内标头孢氨苄（Cephalexin,CEX）10μL,蒸馏水10μL, 10%三氯醋酸80μL,旋混0.5分钟,12000r/min×10min离心,取上清液50μL进样。以峰面积内标法定量。方法学验证包括特异性、精密度、回收率、线性范围和最低定量限,按常规方法进行。稳定性考察包括CED血浆样品-20℃冰冻保存稳定性、CED血浆样品室温保存稳定性、CED标准品及内标CEX储备液-20℃冰冻保存稳定性、CED血浆样品加三氯醋酸后2～4℃存放稳定性。大鼠血样采集结束后,立即处死取出肾脏,精确称量肾脏重量（KW）,并计算肾脏指数（肾重/体重,KW/BW）。结果:CED和CEX的保留时间（t_r）分别为16.38min和11.50min,两者之间达基线分离;空白血浆中杂质峰均在此之前出现,不干扰CED的测定;给药后之血浆样品的HPLC图谱中未见代谢物干扰,血浆样品中CED和CEX的tr均与标准品相同。CED和CEX峰面积之比与CED浓度呈线性相关（r>0.9999）,血浆标准曲线线性范围为1～100μg/mL,线性方程为Y=0.0325X +0.0096（r=0.9999）,最低定量限分别为1μg/mL,能满足测定5个半衰期的血药浓度;方法学验证表明日内及日间精密度RSD均小于9.40%,方法回收率达93.23%～100.33%,萃取回收率达83.98%～87.88%。CED血浆样品-20℃保存和室温放置分别至少一个月和8小时内稳定;CED标准品及CEX储备液-20°C保存至少一个月内稳定;血浆样品加三氯醋酸后2～4°C保存至少8小时内稳定。CTL组和DM组大鼠静脉注射CED后的血药浓度时程均符合二室模型、线性动力学过程。高、低剂量组间AUC随剂量成比例增加（P<0.05）,t_1/2β、CL_t、MRT不随剂量明显改变（P>0.05）。与CTL组比较,DM组t_1/2β、MRT明显缩短（P<0.05）,CL_t显著增加（P<0.05）,说明DM组CED消除加快。两组大鼠口服CED后的血药浓度时程均符合口服一室模型、线性动力学过程。高、低剂量组间AUC随剂量成比例增加（P<0.05）,t_1/2k、CL_t不随剂量明显改变（P>0.05）。与CTL组比较,DM组口服CED后t_max、t_1/2k显著缩短（P<0.05） ,t_1/2k_a呈缩短趋势,C_max、CL_t明显增加（P<0.05）,说明DM组CED吸收和消除均加快。CED在大鼠体内的口服生物利用度（F）,在DM组与CTL组间无显著性差异（P>0.05）。DM组大鼠KW、KW/BW显著增加（P<0.05）。结论:本文采用的HPLC法符合CED血浆药动学研究的方法学要求;血药浓度数据及药动学参数表明CTL组和DM组大鼠经静脉或口服本研究确定之高、低剂量CED后,血药浓度时程均呈现线性动力学;糖尿病状态下CED静脉及口服给药后大鼠体内消除明显加快,表现为t_1/2β、t_1/2k、MRT缩短,CL_t增加;DM大鼠口服CED后t_max显著缩短,C_max明显增加,t_1/2k_a缩短,提示糖尿病时CED吸收加快,但F与CTL相比无显著性差异;DM组大鼠肾KW、KW/BW增加,提示糖尿病早期肾脏出现代偿性肥大继而功能亢进,这也许是DM大鼠CED消除加快的原因之一。更多还原

【Abstract】 Objective: To study effects of diabetes mellitus on pharmacokinetics of cephradine （CED） by comparing the difference in pharmacokinetics （PK） behaviour of CED between diabetic and normal rats in order to provide experiment basis for clinical rational usages of CED in patients with diabetes mellitus.Methods: Diabetes was induced by a single injection of freshly prepared Alloxan solution, 60 mg/kg body weight through the tail vein. Rats whose blood glucose was over 16mmol/l were taken as diabetes mellitus group and the consumption of food and water, as well as body weight, urine output of rats were measured at same time. Experiment rats were divided into two large groups: normal control （CTL） large group and diabetes mellitus （DM） large group. Each of large groups was subdivided into 2 groups, based on the different routes of administration, namely, intravenous （iv） dosing group and intragastric （ig） dosing group, each group including high dose （180mg/kg） and low dose （90mg/kg） small groups. In iv group, blood samples （0.4ml） were collected from angular vein at time zero and 5,15,30,50,80,120,180,240,360,450 and 600 min with heparinized Eppendorff tubes. In ig group, blood samples （0.4ml） were collected similarly at time zero and 15,30,45,60,75,105,150,240,330,450 and 600 min and subsequently centrifuged at 3000rpm for 15 min （4℃） to yield plasma samples. CED concentration in plasma was determined by a reversed phase HPLC. Chromatographic separation was achived on a Kromasil C₁₈ column （250×4.6mm ID, 5μm） equipped with a Kromasil C₁₈ pre-column （20×4.6mm ID, 10μm）, using a mobile phase composed of 0.025mol/L KH₂PO₄-MeOH-CH₃CN （87:6:7 v/v） running at a flow rate of 1.0mL/min. The UV detector was set at 261nm. 200μL of plasma sample was added with internal standard cephalexin （CEX） 10μL（400μg/mL）, 10μL water and 80μL of 10% Trichloracetic acid （TCA） solution and vortexed for 0.5min, and then centrifuged at 12000rpm for 10 min （4℃）. The resultant 50μL supernatant was injected into the column. The validation of the method including specificity, precision, recovery, linearity and detection limit was conducted by routine procedures. The stability test included the stability of CED plasma samples kept frozen at–20℃and at room temperature, the stability of CED standard stock solution and CEX stock solution stored frozen at–20℃, the stability of the TCA-deproteinized plasma samples at 2-4℃. At the end of the experiment, the kidneys were excised and weighed as wet weight （KW） for calculation of ratio of kidney weight to body weight （KW/BW）.Results: The calibration curve of CED had a good linearity over the range from 1μg/mL to 100μg/mL （r>0.9999）. The quantification limit was 1μg/mL. The method developed in present paper showed a good precision of RSD<9.40 %, a high method recovery of 96.00%～100.33% and a high extraction recovery of 83.98%～87.88%. It had been demonstrated that CED plasma samples kept frozen at–20℃and at room temperature were stable for at least 1 month and 8h, respectively. CED standard stock solution and CEX stock solution stored frozen at–20℃was also stable for at least 1 month, the TCA-deproteinized plasma samples at 2-4℃were stable for at least 8h. The PK behaviour of CED in plasma after iv administration could be described by two-compartment model and first-order kinetics. Compared with the CTL groups, t_1/2β and MRT were decreased remarkably and CL_t was increased significantly in DM groups （p<0.05）. These results indicate that the elimination of CED in DM groups was faster than that in the CTL groups. After oral administration, the PK behaviour of CED in plasma could be described by one-compartment model and first-order kinetics. Comparing with CTL groups, a significant decrease in t_1/2k and t_max and a remarkable increase in CL_t and C_max were observed for DM groups （p<0.05）. These results demonstrated that the absorption and elimination of CED in DM groups were faster than those in the CTL groups. There was a decreased trend of t_1/2ka in DM rats, in spite of p>0.05 vs CTL rats. No significant difference in the bioavailability （F） between the two groups was observed （p>0.05）. KW and KW/BW in DM groups were increased remarkably compared with in CTL groups （p<0.05）.Conclusion: The method developed by us is validated to fully meet requirements for CED PK study in rats. Diabetic rats showed a decreased t_1/2β and MRT and an increased CL_t when CED was iv administered, and a decreased t_max, increased C_max and almost identical F when CED was orally administered in relation to CTL group. These results indicate that in DM pathological status, CED eliminates from body more quickly than in normal status and intestinal absorption of CED in DM groups was faster than that in the CTL groups. The KW and KW/BW were increased remarkably, suggesting that kidney become compensatory hypertrophy and hyperfunction in early-stage diabetes. This might constitute one of causes of quick elimination of CED in diabetic rats.更多还原