低剂量格列本脲在大鼠的药代动力学研究及其在重症脑血管病患者的小样本临床研究

发布时间：2018-05-09 08:41

本文选题：格列本脲 + 药代动力学　；参考：《南方医科大学》2016年硕士论文

【摘要】：格列本脲,自1969年起作为一种降糖药用于Ⅱ型糖尿病的治疗,作用机制为促进胰岛素的释放从而降血糖。近年来,低剂量格列本脲在各种神经系统疾病中展示出多样的脑保护作用,这引起了研究者们极大的兴趣。格列本脲被证实在缺血性卒中、创伤性脑损伤、脊髓损伤等多种疾病的动物模型中有神经保护作用。此外,回顾性研究也显示,格列本脲可以减轻缺血性卒中后的脑水肿。国外针对格列本脲脑保护作用的研究,在大鼠使用腹腔注射的给药方式,在临床试验中使用静脉持续泵注的给药方式。而格列本脲在我国作为一种口服降糖药,具有价格低廉,口服吸收好等明显的优势。国外研究所使用的静脉注射剂型的格列本脲(RP-1127, Remedy Pharmaceuticals)有很好的安全性且很可能有临床治疗价值,但该剂型从临床试验到上市必然仍要经过漫长的时间。相比之下,口服制剂的格列本脲已经作为一种相对廉价的降糖药物在临床上有数十年的使用历史,其安全性已经得到了广泛的证实。值得注意的是,相比于用于2型糖尿病的降糖治疗,格列本脲在神经保护作用方面所需要的剂量更低[2],因此将具有更好的安全性。既往针对格列本脲的药代动力学研究,所使用的剂量均为针对治疗糖尿病,产生降血糖所用的相对高剂量,而针对格列本脲的脑保护作用,文献中所涉及的剂量均低于格列本脲作为口服降糖药所需要使用的剂量。低剂量格列本脲的药代动力学尚未有研究,我们的研究将为下一步临床试验做好准备。我们进行了如下实验研究：第一章低剂量格列本脲在大鼠的药代动力学研究第一节低剂量格列本脲口服给药与腹腔注射给药在正常大鼠体内的药代动力学对比研究实验目的：研究低剂量格列本脲口服给药与腹腔注射给药在正常大鼠体内的药代动力学特性。实验方法：20只大鼠随机分为口服组和腹腔注射组(n=10)。口服组大鼠灌胃给予格列本脲混悬液1 mg/kg。腹腔注射组给予格列本脲10μg/kg。两组大鼠分别于给药后5、15、30、60、90、 120、 150、 180、360、720 min采血,以LC-MS/MS法测定格列本脲在大鼠血浆中的浓度,并监测血糖。WinNonlin药代动力学分析软件计算药代动力学参数。实验结果：格列本脲的线性回归方程y=1.1731x-0.0127。线性范围1.02～204 ng/mL,相关系数R20.9996。所测定的格列本脲的高、中、低浓度的日内、日间精密度的变异系数(CV)值均小于5%。高、中、低浓度的格列本脲在大鼠血浆中的平均回收率分别为(99.37±7.22)%、(101.63±5.91)%、(95.19±2.44)%。口服组大鼠血浆中格列本脲的药时曲线较腹腔注射组右移,即达峰时间延长,两组格列本脲峰浓度均在20-25 ng/mL之间。口服给药1 mg/kg与腹腔注射给药10μg/kg相比,格列本脲在大鼠体内的药物峰浓度(Cmax)、半衰期(t1/2)、药时曲线下面积(AUClast)无显著性差异(P0.05)。口服给药组达峰时间(Tmax)较腹腔注射给药组明显延长,差异具有统计学意义(P0.05)。给药前两组大鼠基础血糖水平在6 mmol/L左右,在12h的血糖监测过程中,口服组大鼠血糖最低至4.9 mmol/L,腹腔注射组大鼠血糖最低至4.7mmol/L。两组大鼠均未出现低血糖的情况。实验结论：本实验建立了一种精确简便的低剂量格列本脲在大鼠的血药浓度测定方法。口服1 mg/kg格列本脲,可以在大鼠体内产生与腹腔注射给药10 μg/kg相近的血药浓度,两种给药方式并具有相似的药时曲线下面积。低剂量的格列本脲在口服给药及腹腔注射给药时都是安全的,12h监测过程中并未有低血糖的发生。第二节低剂量格列本脲口服给药与腹腔注射给药在MCAO大鼠体内的药代动力学对比研究实验目的：研究低剂量格列本脲口服给药与腹腔注射给药在MCAO大鼠体内的药代动力学实验方法：20只SD大鼠随机分为两组,MCAO口服组及MCAO腹腔注射组,n=10。两组大鼠分别进行MCAO模型后股静脉插管。拔出线栓口立即给予相应剂量的格列本脲,MCAO口服组大鼠灌胃给予格列本脲混悬液1mg/kg, MCAO腹腔注射组大鼠给予格列本脲10μg/kg。两组大鼠分别于给药后5、15、30、60、90、120、150、180、360、720 min,采血,以LC-MS/MS法测定格列本脲在大鼠血浆中的浓度,并监测血糖。WinNonlin药代动力学分析软件计算药代动力学参数。实验结果：所测定的格列本脲的高、中、低浓度的日内、日间精密度的变异系数(CV)值均小于5%。高、中、低浓度的格列本脲在大鼠血浆中的平均回收率分别为(98.66±3.12)%、(103.19±6.72)%、(99.37±5.44)%。MCAO口服组药时曲线较MCAO腹腔注射组右移,即达峰时间延长,两组格列本脲峰浓度均在20-25 ng/mL之间。口服给药1 mg/kg与腹腔注射给药10μg/kg相比,格列本脲在MCAO大鼠体内的药物峰浓度(Cmax)、半衰期(t1/2)、药时曲线下面积(AUClast)无显著性差异(P0.05)。MCAO口服给药组达峰时间(Tmax)较MCAO腹腔注射给药组明显延长,差异具有统计学意义(P0.05)。与第一节中口服给药组大鼠相比,格列本脲在MCAO口服给药组大鼠体内的药物峰浓度(Cmax)、达峰时间(Tmax)、半衰期(t1/2)、药时曲线下面积(AUClast)无显著性差异(P0.05)。与第一节中腹腔注射组大鼠相比,格列本脲在MCAO腹腔注射组大鼠体内的药物峰浓度(Cmax)、达峰时间(Tmax)、半衰期(t1/2)、药时曲线下面积(AUClast)无显著性差异(P0.05)。实验中两组大鼠均未出现低血糖的情况。实验结论：口服1 mg/kg格列本脲,可以在MCAO大鼠体内产生与腹腔注射给药10μg/kg相近的血药浓度,两种给药方式并具有相似的药时曲线下面积。大鼠在2 h MCAO模型中,与未造模生理状态下相比,低剂量格列本脲口服给药与腹腔注射给药均有相似的药代动力学特性。低剂量的格列本脲在口服给药及腹腔注射给药时都是安全的,监测过程中并未有低血糖的发生。第三节亚低温下低剂量格列本脲在大鼠的药代动力学研究背景与目的：临床研究提示低剂量格列本脲在多种神经系统疾病中通过抑制SUR1-TRPM4通道发挥多效性保护作用。我们的研究数据表明在体内及体外格列本脲均增强亚低温的有效性。但是亚低温条件下低剂量的格列本脲药代动力学尚不明确。我们假设亚低温条件下低剂量的格列本脲药物代谢动力学变化源于CYP2C9活性的改变,CYP2C9活性通过双氯芬酸作为探针药物去检测。本研究的目的是评估老鼠亚低温状态对低剂量格列本脲药物代谢动力学的影响并初步阐述其可能的机制。实验方法：雄性SD大鼠被随机分为常温组与低温组。两组分别注射33ug/kg格列本脲及10 mg/kg双氯芬酸钠。在9个不同时间点采集血标本。LC-MS/MS检测大鼠血浆格列本脲及双氯芬酸钠浓度,并通过WinNonlin软件计算二者药代动力学参数。实验结果：亚低温条件下格列本脲的总体清除率有显著下降(下降58%；16.00±4.1 to 6.72±2.1 mL/min/Kg; p 0.001)。两组半衰期改变没有显著差异(亚低温组2.71±1.7h,正常体温组1.64±0.34h; p=0.157)。AUClast在亚低温组(77.8±18 h*ng/ml)较正常体温组(33.2±11 h*ng/ml)显著升高(p0.001)。血糖水平均高于70mg/dL。双氯芬酸总体清除率从正常体温组的10.33±1.53mL/min/Kg下降至亚低温组的7.20±1.66mL/min/Kg。(p0.01)。实验结论：亚低温改变低剂量格列本脲药代动力学参数,使格列本脲在大鼠的血浆清除率下降,血药浓度升高。亚低温状态下CYP2C9的活性降低。第二章低剂量格列本脲在重症脑血管病患者的小样本临床研究研究目的：初步验证口服低剂量格列本脲在重症脑血管病患者的安全性,并测定格列本脲血药浓度。资料和方法：前瞻性研究2015年06月至2015年12月因重症脑血管病入住南方医科大学南方医院神经危重症监护病房的患者。纳入条件：1)临床和影像学诊断为脑梗塞或脑出血；2)年龄为17-75岁；3)起病72小时内入院；4)入院后空腹血糖7.0 mmol/L。受试者经鼻饲管或口服给药,首次给药负荷剂量1mg,8小时后追加剂量0.5mg,之后每8小时追加0.5mg,至首剂72h后停药。首次给药后2h、4h、6h、8h、10h、16h、18h、24h、48h、72h测定格列本脲血药浓度,血糖,血清胰岛素水平。结果：格列本脲血药浓度最高达56 ng/mL,于给药后4h测得。24h后,格列本脲的血药浓度波动在20-40 ng/mL之间。没有患者出现低血糖的临床表现,血糖值最低4.2 mmol/L。患者给药前的血清胰岛素为25.72±12.31 pmol/L,给药后最高达67.82 pmol/L。结论：低剂量口服格列本脲在重症脑血管病患者具有良好的安全性,不伴有严重不良反应。
[Abstract]:Glibenclamide, which has been used as a hypoglycemic agent in the treatment of type 2 diabetes since 1969, plays a role in promoting insulin release and reducing blood sugar. In recent years, low dose glibenclamide has shown a variety of brain protection in various nervous system diseases, which has aroused great interest in the researchers. There are neuroprotective effects in animal models of a variety of diseases such as sexual apoplexy, traumatic brain injury, and spinal cord injury. In addition, a retrospective study has also shown that glibenclamide can reduce cerebral edema after ischemic stroke. Study on the protective effect of glibenclamide abroad and the use of intraperitoneal injection in rats and use in clinical trials in rats As an oral hypoglycemic agent, glibenclamide has the advantages of low price and good oral absorption in our country. The intravenous injection of RP-1127 (Remedy Pharmaceuticals) used by foreign research institutes has good safety and is likely to be of clinical value, but the dosage form is from Clinical trials are bound to take a long time to go on the market. In comparison, the oral preparation of glibenclamide has been used as a relatively cheap antidiabetic drug for decades, and its safety has been widely confirmed. It is worth noting that glibenclamide is compared to the hypoglycemic treatment for type 2 diabetes. The dose required for the neuroprotective effect of a lower dose of [2] is better. The pharmacokinetic study of glibenclamide has been used for a relatively high dose for the treatment of diabetes and the production of hypoglycemia, while the brain protection against glibenclamide is low in the literature. The pharmacokinetics of glibenclamide as oral hypoglycemic agents. Pharmacokinetics of low dose glibenclamide have not yet been studied. Our study will prepare for further clinical trials. We have conducted the following experimental studies: the first of the low dose glibenclamide pharmacokinetics in rats, the first low dose glenben The pharmacokinetic study of oral administration of urea and intraperitoneal injection in normal rats: the pharmacokinetic characteristics of low dose of glibenclamide oral administration and intraperitoneal injection in normal rats. Experimental methods: 20 rats were randomly divided into oral and intraperitoneal injection group (n=10). Oral administration of rats was administered to rats. Glipebenclamide suspension was given to the stomach for 1 mg/kg. intraperitoneal injection to give glipezourea 10 g/kg. two groups of rats in the group of rats after administration of 5,15,30,60,90, 120, 150, 180360720 min. The concentration of glibenclamide in rat plasma was measured by LC-MS/MS, and the pharmacokinetic parameters of blood glucose.WinNonlin pharmacokinetic analysis software were monitored. The linear regression equation y=1.1731x-0.0127. of glibenclamide was 1.02 ~ 204 ng/mL, and the coefficient of variation (CV) of daytime precision was less than 5%. in high, medium and low concentration of R20.9996., and the average recovery rate of glibenclamide in rat plasma was (99.), respectively. 37 + 7.22)%, (101.63 + 5.91)%, (95.19 + 2.44)%. The plasma concentration of glibenclamide in the oral group was longer than that in the intraperitoneal injection group, that is, the peak time was prolonged and the peak concentration of the two groups of glibenclamide was 20-25 ng/mL. The drug peak concentration of glibenclamide in rats was compared with the oral administration of 1 mg/kg and the intraperitoneal injection of 10 u g/ kg (Cmax). There was no significant difference in the area (AUClast) under the curve of the drug time (t1/2). The peak time (Tmax) in the oral administration group was significantly longer than that in the intraperitoneal injection group (P0.05). The basal blood glucose level of the two groups of rats before the administration was 6 mmol/L left right, and the blood sugar of the oral group was the lowest to 4.9 during the 12h monitoring of blood glucose. Mmol/L, the blood glucose of the rats in the group of intraperitoneal injection group was the lowest to 4.7mmol/L. two rats. The experimental conclusion: a simple and simple method for measuring the blood concentration of low dose glibenclamide in rats was established. Oral 1 mg/kg glibenclamide, which could be produced in rats, was similar to that of the intraperitoneal injection of 10 mu g/kg. The concentration of blood, two ways of administration and the area under the curve of similar drug. Low dose of glibenclamide is safe in oral administration and intraperitoneal injection. There is no hypoglycemia in the 12h monitoring process. The pharmacokinetics of second segments of low dose glibenclamide oral administration and intraperitoneal injection in MCAO rats Objective: To study the pharmacokinetics of low dose of glibenclamide oral administration and intraperitoneal injection in MCAO rats: 20 SD rats were randomly divided into two groups, MCAO oral group and MCAO intraperitoneal injection group, and n=10. two groups of rats respectively after MCAO model femoral venous intubation. The dose of glibenclamide, MCAO oral group rats were given gastric perfusion of glibenclamide suspension 1mg/kg, MCAO intraperitoneal injection group rats were given glipenourea 10 g/kg. two groups of rats after the administration of 5,15,30,60,90120150180360720 min, blood, LC-MS/MS method to determine the concentration of glibenclamide in rat plasma, and monitoring the blood sugar.WinNonlin medicine The kinetic analysis software was used to calculate the pharmacokinetic parameters. Experimental results: the variation coefficient (CV) of daytime precision was less than 5%. in high, medium and low concentrations, and the average recovery of glibenclamide in rat plasma was (98.66 + 3.12)%, (103.19 + 6.72)%, (99.37 + 5.44)%.MCAO in the middle and low concentration of rat plasma. The curve of the oral group was more than that of the MCAO group, that is, the peak time was prolonged, and the peak concentration of the two groups was 20-25 ng/mL. The drug peak concentration (Cmax), the half life (t1/2) and the area under the curve (AUClast) in the MCAO rats were no significant difference compared with the oral administration of 1 mg/kg and the intraperitoneal injection 10 mu g/kg (t1/2). P0.05) the peak time of.MCAO oral administration group (Tmax) was significantly longer than that of MCAO intraperitoneal injection group, and the difference was statistically significant (P0.05). Compared with the first oral administration group, the peak concentration (Cmax), peak time (Tmax), half life (t1/2), and the area under the curve of drug time (AUClast) in the group of MCAO oral administration rats were compared with the first oral administration group. There was no significant difference (P0.05). Compared with the first abdominal injection group, the drug peak concentration (Cmax), peak time (Tmax), half life (t1/2), and the area under the curve (AUClast) were no significant difference (P0.05) in the rats of MCAO intraperitoneal injection group (P0.05). No hypoglycemia was found in the two groups of rats in the experiment. Experimental conclusions: 1 mg/kg glibenclamide was taken orally in MCAO rats to produce a blood drug concentration similar to that of the intraperitoneal injection of 10 u g/kg, the two kinds of administration and the area under the curve of similar drug. In the 2 h MCAO model, the low dose glibenclamide oral administration was similar to that of the intraperitoneal injection. Pharmacokinetic characteristics. Low dose of glibenclamide is safe in oral administration and intraperitoneal injection. There is no hypoglycemia in the monitoring process. Research background and purpose of pharmacokinetics of low dose glibenclamide at third sub hypothermia in rats: clinical study suggests that low dose glibenclamide is in a variety of nervous system diseases. Our data show that both in vivo and in vitro glibenclamide enhance the effectiveness of mild hypothermia. However, low dose of glibenclamide pharmacokinetics under mild hypothermia is not clear. We hypothesized that low dose of glibenclamide metabolizing power under mild hypothermia conditions The changes in CYP2C9 activity and CYP2C9 activity were detected by diclofenac as a probe drug. The purpose of this study was to assess the effects of hypothermia on the pharmacokinetics of low dose glibenclamide and to preliminarily describe the possible mechanisms. Experimental methods: male SD rats were randomly divided into normal and low temperature groups. The two groups were injected with 33ug/kg glibenclamide and 10 mg/kg diclofenac sodium respectively. Blood samples were collected at 9 different time points to detect the concentration of glibenclamide and diclofenac sodium in the plasma of rats, and the pharmacokinetic parameters were calculated by the WinNonlin software. The results were as follows: the total clearance rate of glenepourea under mild hypothermia was significantly lower. Drop (58%; 16 + 4.1 to 6.72 + 2.1 mL/min/Kg; P 0.001). There was no significant difference in the half life change of the two group (mild hypothermia group 2.71 + 1.7h, normal body temperature 1.64 + 0.34h; p=0.157).AUClast in mild hypothermia group (77.8 + 18 h*ng/ml) significantly higher than normal body temperature group (33.2 + 11 h*ng/ml) (p0.001). The average blood sugar water was higher than that of 70mg/dL. diclofenac The total clearance rate decreased from 10.33 + 1.53mL/min/Kg in the normal temperature group to 7.20 1.66mL/min/Kg. (P0.01) in the sub hypothermia group. Experimental conclusion: low hypothermia changed the pharmacokinetic parameters of low dose glibenclamide, which made the plasma clearance rate of glibenclamide decreased and the blood concentration increased. The activity of CYP2C9 decreased under the mild hypothermia state. Second chapters A small sample of low dose glibenclamide in patients with severe cerebrovascular disease: a preliminary study of the safety of oral low dose glibenclamide in patients with severe cerebrovascular disease and the determination of serum concentration of glibenclamide. Data and methods: a prospective study from 06 months to December 2015 2015 for severe cerebrovascular disease in southern medicine Patients in the neurocritical care unit of the Southern Hospital of the University were included in the conditions: 1) clinical and imaging diagnosis of cerebral infarction or cerebral hemorrhage; 2) age 17-75 years old; 3) onset within 72 hours of onset; 4) after admission to the hospital, the fasting blood glucose 7 mmol/L. was administered by nasal feeding tube or oral administration, for the first time the dose was 1mg, and the additional dose of 0.5mg after 8 hours. After the first dose of 0.5mg, after the first dose of 72h, 2h, 4h, 6h, 8h, 10h, 16h, 18h, 24h, 48h were first given, and 72h measured the blood concentration of glibenclamide, blood glucose and serum insulin. Results: the blood concentration of glibenclamide was up to 56. After the administration, the blood concentration of glybenclamide fluctuated between 20-40. There was a clinical manifestation of hypoglycemia in the patients. The serum insulin was 25.72 + 12.31 pmol/L before the 4.2 mmol/L. patients, and the highest level of 67.82 pmol/L. after administration: low dose of glibenclamide in patients with severe cerebrovascular disease had good safety and no serious adverse reaction.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R743

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