【作者】 李文；

【导师】 胡晓；

【作者基本信息】 南昌大学 ， 药理学， 2011， 硕士

【摘要】 背景羟基脲(Hydroxyurea, HU)为核糖核苷酸抑制剂类抗肿瘤药物,是骨髓增生异常综合征如慢性粒细胞性白血病和真性红细胞增多症的经典化疗药物。但其不良反应较严重,使其在临床上的应用受到限制。1-(4-溴苄基)-1-(4-溴苄氧基)脲(1-(4-bromobenzyl)-1-(4-bromobenzyloxy)-urea, HY-D11)是对HU进行结构改造所获得的系列衍生物中最有前景的化合物之一,在动物体内外实验中呈现较强的抗肿瘤作用,且毒性反应很低。药代动力学研究在新药研发中占有重要的地位,探明HY-D11体内过程,研究其代谢及其机制,对于HY-D11能否成为抗肿瘤创制新药有着重要的意义。目的通过体外大鼠肝微粒体酶抑制实验,探明HY-D11在大鼠肝脏中的代谢途径及机制。研究HY-D11在大鼠体内药代动力学过程,为HY-D11作为抗肿瘤重大创制新药研发提供理论和实验依据。方法①建立反相高效液相-紫外检测方法,测定大鼠血浆及肝微粒体系温孵液中HY-D11的浓度。②通过考察温孵时间、肝微粒体蛋白浓度、底物浓度对HY-D11在大鼠肝微粒体温孵液中代谢的影响,研究HY-D11在大鼠肝微粒体温孵液中的酶促动力学特征；分别研究CYP1A抑制剂α-萘黄酮(naphthoflavone)、CYP2B抑制剂舍曲林(sertraline)、CYP2C抑制剂槲皮素(quercetin)、CYP2D抑制剂育亨宾(yohimbine)、CYP2E抑制剂双硫仑(disulfiram)、CYP3A抑制剂醋竹桃霉素(troleandomycin)对HY-D11在大鼠肝微粒体温孵液中代谢的影响。③SD大鼠6只,按66.7 mg.kg-1的剂量灌胃给予HY-D11混悬液,于给药前和给药后不同时间点采集系列血样,RP-HPLC检测血浆中HY-D11浓度,运用DAS2.1软件求算主要药动学参数。结果①HY-D11的HPLC检测方法为流动相：乙腈-10mM醋酸铵水溶液(65：35,V：V)；流速：1.0 mL.min-1;检测波长：225nm；灵敏度：0.004 Aufs;柱温：室温。②HY-D11在大鼠肝微粒体温孵液中经37℃空气浴振荡器振荡孵育15-60min；呈时间依赖性近似线性消除,当孵育时间超过60 min时,HY-D11的代谢速率减慢,故选择60 min为实验孵育时间；HY-D11的消除随着肝微粒体蛋白浓度的增加而增加,在0.25-1.5 mg.mL-1范围内呈近似线性消除,故选择1.0mg.mL-1作为实验用肝微粒体蛋白浓度；在已经确定的实验条件下,HY-D11在大鼠肝微粒体温孵液中的消除呈现明显的酶促动力学特征：当HY-D11的浓度在0-16μM范围内,HY-D11的代谢呈浓度依赖性,代谢消除量随底物浓度增加呈近似线性增加,而当浓度超过16μM, HY-D11代谢消除量不再随着底物浓度的增加而增加,呈现饱和现象。③在酶抑制实验中,CYP1A抑制剂α-萘黄酮(a-nNap)、CYP2B抑制剂舍曲林(Ser)、CYP2C抑制剂槲皮素(Que)、CYP2D抑制剂育亨宾(Yoh)、CYP2E抑制剂双硫仑(Dis)对HY-D11在肝微粒体中的代谢消除率没有影响,与对照组相比无显著差异(P>0.05)。而在CYP3A抑制剂醋竹桃霉素(Tro)共孵育的肝微粒体温孵液中HY-D11的代谢消除率减少,与对照组相比则具有显著性差异(P<0.01)。④HY-D11在大鼠体内的药代动力学过程符合二室模型。主要的药代动力学参数：Tmax为3.67±0.52 h, Cmax为1610.35±130.28 ng·mL-1, Vz/F为9540.42±1530.62 mL·kg-1, tl/2z为3.66±0.25 h, MRT(0-∞)为7.05±0.29 h。结论本实验建立HY-D11 HPLC检测方法灵敏、可靠。大鼠肝微粒体中HY-D11线性范围为0.25-32μM,大鼠血浆HY-D11的线性范围为50-2400 ng·mL-1,生物样本中HY-D11低、中、高各浓度方法回收率在85%-115%之间,低、中、高三个浓度批内及批间变异RSD均<15%,符合生物样本分析的要求。HY-D11在大鼠肝微粒体温孵液代谢实验研究的结果提示HY-D11的代谢具有酶促动力学代谢特征。CYP1A抑制剂α-萘黄酮(a-Nap)、CYP2B抑制剂舍曲林(Ser)、CYP2C抑制剂槲皮素(Que)、CYP2D抑制剂育亨宾(Yoh)、CYP2E抑制剂双硫仑(Dis)对HY-D11在大鼠肝微粒体温孵液中的代谢无抑制作用,表明CYP1A、CYP2B、CYP2C、CYP2D、CYP2E不参与大鼠肝脏HY-D11的代谢。CYP3A抑制剂醋竹桃霉素(Tro)对HY-D11的代谢有明显抑制作用,提示CYP3A可能参与了HY-D11在大鼠肝微粒中的代谢。灌胃给予HY-D11 (66.7mg.kg-1)后,HY-D11在大鼠体内的药代动力学过程符合二室模型。其主要动力学参数：Tmax为3.67±0.52 h,表明灌胃给药后HY-D11在大鼠体内吸收迅速；Cmax为1610.35±130.28ng·mL-1,该值远远高于HY-D11体外最低有效抑瘤浓度；Vz/F为9540.42±1530.62 mL-kg-1,表明HY-D11在大鼠体内分布较广泛；t1/2z和MRT(0-∞)分别为3.66±0.25 h、7.05±0.29 h,表明HY-D11在大鼠体内消除速率较快。更多还原

【Abstract】 BackgroundHydroxyurea is antitumor drug of DNA nucleotide inhibitors, Once was applied for blood disease malignancy, sickle cell anemia and myeloproliferative syndrome.But it is so serious aderse effects that clinical application of it is limited. (1-(4-bromobenzyl)-1-(4-bromobenzyloxy)urea,HY-D11) is one of the most promising series of derivatives which were come from HU. In vivo and vitro tests on animals it presented strong antitumor effect, and low toxicity.Pharmacokinetic studies is very important in new drug development,and investigated the process of HY-D11 in vivo, and the metabolism is important significance for HY-D11 as an new anti-tumor drug.ObjectivesThe aim of this study is to investigate the pharmacokinetic of 1-(4-bromobenzyl)-1-(4-bromobenzyloxy)urea profiles in rats and metabolism of 1-(4-bromobenzyl)-1-(4-bromobenzyloxy)urea in rat liver microsome incubation system,and provide theoretical and experimental basis of research and development for the HY-D11 as new antineoplastic drug.Methods①Seting up the HPLC analyse method of HY-D11 to detect the concentration of HY-D11 in rat plasma and live microsomal.②Investigate the effect of incubation time,substrate concentration and microsomal protein concentration on the enzyme kinetics of the metabolism of HY-D11 in rat live microsome incubation solution.Various selective CYP inhibitors (CYP1A inhibitor:α-naphthoflavone,CYP2B inhibitor:sertraline,CYP2C inhibitor: quercetin,CYP2D inhibitor:yohimbine, CYP2E inhibitor:disulfiram and CYP3A inhibitor:troleandomycin) were used to investigate their inhibitory effects on the metabolism of HY-D11. Oral administration of HY-D11 to six SD rats with 66.7mg/kg, collecting plasma samples before and after administration of HY-D11 in different time. Detecting the concentration of HY-D11 in plasma with RT-HPLC.Acquained the pharmacokinetic parameters by using the DAS2.0 software.③Oral administration of HY-D11 to six SD rats with 66.7mg/kg, collecting plasma samples before and after administration of HY-D11 in different time. Detecting the concentration of HY-D11 in plasma with RT-HPLC.Acquained the pharmacokinetic parameters by using the DAS2.1 software.Results①The HPLC detection method of HY-D11:mobile phase:cetonitrile-10mM ammonium acetate(65:35,V:V); Velocity:1.0 ml/min; detected wavelength:225nm; sensitivity:0.004Aufs; Column temperature:room temperature.②The metabolism of HY-D11 in live microsome incubation solution was nearly linear time dependence, When put HY-D11 in live microsome incubation solution in 37℃from 0 to 60 min. When incubation time is more than 60min, HY-D11 metabolic rates was decreased, so choose 60 min as experimental incubation time. The elimination of HY-D11 was increased with the increasing protein concentration. The elimination of HY-D11 was linear increase when the protein concentration was 0.25 to 1.5 mg/ml,so we choosed the 1.0 mg/mL liver microsomal protein.The metabolism of HY-D11 in live microsome incubation solution showed the characteristic of enzyme kinetics:In the concentration range of 0-16μM, the disappeared rate of Ost in live microsome incubation solution all linearly increase, when the concentration above 16μM, the metabolism of HY-D11 showed saturation. When the live microsome incubation solution maintained sealed in air at 37℃in a shaking bath for 0-90 min, the disappeared rate of Ost all linearly increased.③a-naphthoflavone(an inhibitor of CYP1A),sertraline (an inhibitor of CYP2B),quercetin (an inhibitor of CYP2C),yohimbine (an inhibitor of CYP2D),disulfiram (an inhibitor of CYP2E) did not have effect on the HY-D11 metabolism in live microsome incubation solution(P>0.05).troleandomycin,an inhibitor of CYP3A,reduced HY-D11 metabolism in live microsome incubation solution. (P<0.05).④The pharmacokinetic profiles of HY-D11 is described by two compartment model legitimately after oral administration of HY-D11 (66.7 mg/kg) in rats. Tmax is 3.67±0.52 h, Cmax is 1610.35±130.28 ng-mL-1, Vz/F is 9540.42±1530.62 mL-kg-1.tl/2z is 3.66±0.25 h,MRT(0-∞) is 7.05±0.29 h.ConclusionsThe HPLC detection method of HY-D11 we have established are Sensitivity and reliability.The linear range of HY-D11 in rat plasma was 50 to 2400 ng·mL-1. The linear range of HY-D11 in rat live microsome incubation was 0.25 to 32μM. The recovery of HY-D11 in low.medium and high concentration were between 85%and 115%, RSD all were less than 15%. It could meet nalysis requirements.The metabolism of HY-D11 in live microsome incubation solution showed the characteristic of enzyme kinetics,a-naphthoflavone (an inhibitor of CYP1A),sertraline (an inhibitor of CYP2B),quercetin (an inhibitor of CYP2C),yohimbine (an inhibitor of CYP2D),disulfiram (an inhibitor of CYP2E) did not have effect on HY-D11 metabolism in live microsome incubation solution(P>0.05),It Shows that CYP2B, CYP2C, CYP1A, CYP2D, CYP2E don’t participate in HY-D11 metabolism in rat liver. Troleandomycin, an inhibitor of CYP3A, reduced HY-D11 metabolism in live microsome incubation solution. (P<0.05).It shows that the metabolism of HY-D11 in rats may be mediated by CYP3A.The pharmacokinetic profiles of HY-D11 were described by two compartment model legitimately after oral administration of HY-D1166.7mg/kg in rats.main dynamics parameters:Tmax is 3.67±0.52 h, it means the absorbtion of HY-D11 is very fast; Cmax is 1610.35±130.28 ng-mL-1,it was much larger the concentration of Lowest effectie antibacterial concentrations in vitro; Vz/F is 9540.42±1530.62 mL-kg-1,it means that the distribution of HY-D11 is very aboad;1/2z and MRT(O-∞)) are is 3.66±0.25 h,7.05±0.29 h,respectively,it demonstrated that the elimination of HY-D11 is fast.更多还原