选择性TNFR1拮抗肽Hydrostatin-SN10治疗炎症性肠病的初步药效学研究
发布时间：2018-09-17 17:55
【摘要】：炎症性肠病(inflammatory bowel disease,IBD)是一种累及回肠、直肠和结肠的特发性炎症性肠道疾病,临床表现为反复腹痛、腹泻、腹部包块、黏液血便、肠梗阻、肠穿孔、体重减轻等,此外患者还表现出各种不同程度的全身症状。IBD过去多见于西方发达国家,近几年来包括中国在内的亚洲地区的发病率呈直线上升趋势,严重影响了患者的生活质量,关于IBD治疗的研究已成为消化系统疾病领域的研究热点之一。然而到目前为止,临床上尚未出现能够有效治疗IBD的药物。本课题组前期获得了一条抗炎活性肽Hydrostatin-SN10(10AA),该活性肽只与TNFR1结合,不与TNNF-α和TNFR2结合,具有专一选择性。基于此研究基础,本课题通过将m PEG2000的羧基与Hydrostatin-SN10肽链N-端天冬氨酸的游离氨基共价连接实现选择性修饰,得到PEG-SN10。本研究采用葡聚糖硫酸钠(dextran sulphate sodium salt,DSS,26~50KDa)和VA唑酮(oxazolone,OXZ)诱导的小鼠急性结肠炎模型,探讨了候选药物选择性TNFR1拮抗肽Hydrostatin-SN10治疗炎症性肠病的初步药效学研究。本文研究发现,在DSS诱导的小鼠急性结肠炎模型中,模型对照组小鼠体重明显减轻,毛色暗淡,精神萎靡,肉眼可见血便,腹泻严重,与空白对照组小鼠疾病活动指数(disease activity index,DAI)评分相比,模型组小鼠DAI评分明显升高;体重明显减轻;颈椎脱臼法处死全部小鼠并将其解剖,取出完整结肠,观察并测量发现:模型组小鼠结肠长度明显缩短(P0.001);脾脏指数增加(P0.001);小鼠血清中促炎因子(TNF-α、IL-6、IL-1β、IFN-γ)含量显著升高,抑炎因子(IL-10)含量显著降低(P0.01);通过收集小鼠结肠组织匀浆液,测得小鼠结肠组织中髓过氧化物酶(myeloperoxidase,MPO)活性显著升高(P0.001);提取小鼠结肠组织RNA,TNF-α、IL-6、IL-1β、IFN-γmRNA相对表达量明显升高,IL-10 mRNA相对表达量明显降低(P0.05)。小鼠腹腔注射Hydrostatin-SN10和PEG-SN10后,上述病症均得到显著缓解。与DSS模型组小鼠相比,Hydrostatin-SN10和PEG-SN10治疗组小鼠体重下降减缓,腹泻次数减少,结肠长度缩短趋势得到有效控制(P0.001),脾脏指数、MPO活性均下降(p0.001),说明Hydrostatin-SN10和PEG-SN10可以明显改善DSS诱导的小鼠急性结肠炎症状。在OXZ诱导的小鼠急性结肠炎模型中,Hydrostatin-SN10及PEG-SN10治疗组的小鼠各项疾病症状均明显好于模型组,比如小鼠体重变化情况、结肠炎相关疾病活动指数等,Hydrostatin-SN10和PEG-SN10给药组小鼠的体重下降趋势明显得到抑制,DAI指数也低于模型组小鼠,治疗效果与两种阳性对照药SASP和IFX等同,且阴性对照组(随机肽组和PEG组)并无疾病缓解症状;同样,Hydrostatin-SN10及PEG-SN10治疗组小鼠的结肠长度缩短、脾肿胀情况也有明显的缓解现象(P0.001);MPO活性测定结果表明,Hydrostatin-SN10及PEG-SN10能显著减少小鼠结肠病变组织的中性粒细胞数量(P0.001);苏木精-伊红(hematoxylin eosin,HE)染色结果表明,Hydrostatin-SN10及PEG-SN10能显著改善结肠的炎症程度和病变范围;实时荧光定量PCR(qRT-PCR)结果证实Hydrostatin-SN10及PEG-SN10对小鼠结肠组织中相关促炎因子(TNF-α、IL-6、IL-1β、IFN-γ)的表达具有抑制效果,而对结肠组织中相关抑炎因子(IL-10)的表达具有促进作用(P0.05),因此Hydrostatin-SN10及PEG-SN10对于OXZ诱导的小鼠急性结肠炎也具有明显的治疗作用。初步药代动力学研究表明:单剂量腹腔注射给药1mg/kg Hydrostatin-SN10、5mg/kg PEG-SN10后测得的6只成年、健康SD大鼠体内Hydrostatin-SN10的药物浓度-时间曲线变化规律并不严格符合典型的房室模型,以非房室模型(统计矩)法计算的药代动力学参数反映SD大鼠体内药代动力学的消除过程基本呈线性动力学特征。单剂量腹腔注射给药1mg/kg Hydrostatin-SN10、5mg/kg PEG-SN10后测得的6只成年、健康SD大鼠体内Hydrostatin-SN10所对应的药代动力学药时曲线下面积(AUC0-t)与给药剂量基本呈正相关,相关系数分别为r2=0.9936和r2=0.9921。Hydrostatin-SN10及PEG-SN10在SD大鼠体内均具有较好的药代动力学性质,SD大鼠腹腔注射给药1mg/kg Hydrostatin-SN10和5mg/kg PEG-SN10后,注射给药的药峰浓度Cmax分别为86.92±4.584μg/L和33.071±1.697μg/L,达峰时间Tmax分别为0.833h和0.833h,末端消除半衰期t1/2分别为2.18±0.654h和3.789±1.368h,注射暴露量AUC(0-t)分别为74.043±17.065ug/L*h和32.087±6.317ug/L*h。本课题完成了TNFR1专一选择性拮抗肽Hydrostatin-SN10以及基于mPEG2000修饰的PEG-SN10治疗IBD的初步药效学研究和初步药代动力学研究,为后续筛选治疗IBD的专一选择性新药提供了理论和实验依据。
[Abstract]:Inflammatory bowel disease (IBD) is a kind of idiopathic inflammatory bowel disease involving ileum, rectum and colon. Its clinical manifestations are recurrent abdominal pain, diarrhea, abdominal mass, mucous bloody stool, intestinal obstruction, intestinal perforation, weight loss, and so on. IBD was common in the West in the past. In developed countries, the incidence of IBD in Asia, including China, has risen sharply in recent years, seriously affecting the quality of life of patients. Research on the treatment of IBD has become one of the hotspots in the field of digestive diseases. However, so far, there is no effective drug for the treatment of IBD. An anti-inflammatory peptide, Hydrostatin-SN10 (10AA), was synthesized. The peptide only binds to TNFR1, but does not bind to TNNF-a and TNFR2. Based on this study, the selective modification of PEG-SN10 was achieved by covalently linking the carboxyl group of M PEG2000 with the free amino group of N-terminal aspartic acid of the peptide chain of Hydrostatin-SN10. In this study, dextran sulphate sodium salt (DSS, 26-50KDa) and VAazolone (OXZ) were used to induce acute colitis in mice, and the preliminary pharmacodynamic study of the selective TNFR1 antagonist peptide Hydrostatin-SN10 in the treatment of inflammatory bowel disease in mice induced by DSS was carried out. In the inflammation model, the mice in the model control group were significantly lighter in weight, dim in color, listless in spirit, bloody stool and severe diarrhea. Compared with the disease activity index (DAI) score in the blank control group, the DAI score in the model group was significantly higher; the weight of the mice was significantly lighter; all the mice were killed by cervical dislocation and dissected. The length of colon was significantly shortened (P 0.001), spleen index was increased (P 0.001), serum levels of pro-inflammatory factors (TNF-a, IL-6, IL-1beta, IFN-gamma) were significantly increased, and the levels of anti-inflammatory factors (IL-10) were significantly decreased (P 0.01) in the model group. The activity of myeloperoxidase (MPO) was significantly increased (P 0.001), the relative expression of RNA, TNF-a, IL-6, IL-1beta, IFN-gamma mRNA in colon tissue was significantly increased, and the relative expression of IL-10 mRNA was significantly decreased (P 0.05). Compared with mice treated with Hydrostatin-SN10 and PEG-SN10, the weight loss, diarrhea frequency, colon length, spleen index and MPO activity of mice treated with Hydrostatin-SN10 and PEG-SN10 decreased significantly (P 0.001), indicating that Hydrostatin-SN10 and PEG-SN10 could significantly improve the symptoms of DSS-induced acute colitis in mice treated with OXZ. In the model of sexual colitis, the symptoms of various diseases in the treatment group of Hydrostatin-SN10 and PEG-SN10 were significantly better than those in the model group, such as the change of body weight, the activity index of colitis-related diseases, etc. The decline trend of body weight in the treatment group of Hydrostatin-SN10 and PEG-SN10 was obviously inhibited, and the DAI index was also lower than that in the model group. Similarly, the colon length of the mice treated with Hydrostatin-SN10 and PEG-SN10 was shortened, and the spleen swelling was also significantly alleviated (P 0.001); MPO activity assay showed that Hydrostatin-SN10 and PEG-SN10 could significantly alleviate the disease. The results of hematoxylin eosin (HE) staining showed that Hydrostatin-SN10 and PEG-SN10 could significantly improve the degree and extent of colonic inflammation; Real-time fluorescence quantitative PCR (qRT-PCR) results confirmed that Hydrostatin-SN10 and PEG-SN10 could significantly improve the degree and extent of colonic inflammation in mice. The expression of TNF-alpha, IL-6, IL-1beta and IFN-gamma was inhibited, while the expression of IL-10 in colon tissue was promoted (P 0.05). Therefore, Hydrostatin-SN10 and PEG-SN10 had obvious therapeutic effects on OXZ-induced acute colitis in mice. The concentration-time curves of Hydrostatin-SN10 in six adult SD rats after intraperitoneal injection of 1 mg/kg Hydrostatin-SN 10,5 mg/kg PEG-SN 10 were not strictly consistent with the typical atrioventricular model. The pharmacokinetic parameters calculated by non-compartment model (statistical moment) method reflected the pharmacokinetic disappearance in SD rats. The area under the pharmacokinetic time curve (AUC0-t) of hydrostatin-SN10 in healthy SD rats was positively correlated with the dosage, and the correlation coefficients were R2 = 0.9936 and R2 = 0.9921.H, respectively. Both ydrostatin-SN10 and PEG-SN10 showed good pharmacokinetic properties in SD rats. After intraperitoneal injection of 1 mg/kg Hydrostatin-SN10 and 5 mg/kg PEG-SN10 into SD rats, the peak concentrations of Cmax were 86.92 (+ 4.584) ug/L and 33.071 (+ 1.697) ug/L, respectively. The peak time of Tmax was 0.833 h and 0.833 h, and the terminal elimination half-life of t1/2. The doses of AUC (0-t) were 74.043 (+ 17.065 ug/L * h) and 32.087 (+ 6.317 ug/L * h) respectively. The preliminary pharmacodynamic and pharmacokinetic studies of TNFR1-specific selective antagonistic peptide Hydrostatin-SN10 and PEG-SN10 modified by mPEG2000 for IBD were completed. BD provides a theoretical and experimental basis for specific selective new drugs.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574
[Abstract]:Inflammatory bowel disease (IBD) is a kind of idiopathic inflammatory bowel disease involving ileum, rectum and colon. Its clinical manifestations are recurrent abdominal pain, diarrhea, abdominal mass, mucous bloody stool, intestinal obstruction, intestinal perforation, weight loss, and so on. IBD was common in the West in the past. In developed countries, the incidence of IBD in Asia, including China, has risen sharply in recent years, seriously affecting the quality of life of patients. Research on the treatment of IBD has become one of the hotspots in the field of digestive diseases. However, so far, there is no effective drug for the treatment of IBD. An anti-inflammatory peptide, Hydrostatin-SN10 (10AA), was synthesized. The peptide only binds to TNFR1, but does not bind to TNNF-a and TNFR2. Based on this study, the selective modification of PEG-SN10 was achieved by covalently linking the carboxyl group of M PEG2000 with the free amino group of N-terminal aspartic acid of the peptide chain of Hydrostatin-SN10. In this study, dextran sulphate sodium salt (DSS, 26-50KDa) and VAazolone (OXZ) were used to induce acute colitis in mice, and the preliminary pharmacodynamic study of the selective TNFR1 antagonist peptide Hydrostatin-SN10 in the treatment of inflammatory bowel disease in mice induced by DSS was carried out. In the inflammation model, the mice in the model control group were significantly lighter in weight, dim in color, listless in spirit, bloody stool and severe diarrhea. Compared with the disease activity index (DAI) score in the blank control group, the DAI score in the model group was significantly higher; the weight of the mice was significantly lighter; all the mice were killed by cervical dislocation and dissected. The length of colon was significantly shortened (P 0.001), spleen index was increased (P 0.001), serum levels of pro-inflammatory factors (TNF-a, IL-6, IL-1beta, IFN-gamma) were significantly increased, and the levels of anti-inflammatory factors (IL-10) were significantly decreased (P 0.01) in the model group. The activity of myeloperoxidase (MPO) was significantly increased (P 0.001), the relative expression of RNA, TNF-a, IL-6, IL-1beta, IFN-gamma mRNA in colon tissue was significantly increased, and the relative expression of IL-10 mRNA was significantly decreased (P 0.05). Compared with mice treated with Hydrostatin-SN10 and PEG-SN10, the weight loss, diarrhea frequency, colon length, spleen index and MPO activity of mice treated with Hydrostatin-SN10 and PEG-SN10 decreased significantly (P 0.001), indicating that Hydrostatin-SN10 and PEG-SN10 could significantly improve the symptoms of DSS-induced acute colitis in mice treated with OXZ. In the model of sexual colitis, the symptoms of various diseases in the treatment group of Hydrostatin-SN10 and PEG-SN10 were significantly better than those in the model group, such as the change of body weight, the activity index of colitis-related diseases, etc. The decline trend of body weight in the treatment group of Hydrostatin-SN10 and PEG-SN10 was obviously inhibited, and the DAI index was also lower than that in the model group. Similarly, the colon length of the mice treated with Hydrostatin-SN10 and PEG-SN10 was shortened, and the spleen swelling was also significantly alleviated (P 0.001); MPO activity assay showed that Hydrostatin-SN10 and PEG-SN10 could significantly alleviate the disease. The results of hematoxylin eosin (HE) staining showed that Hydrostatin-SN10 and PEG-SN10 could significantly improve the degree and extent of colonic inflammation; Real-time fluorescence quantitative PCR (qRT-PCR) results confirmed that Hydrostatin-SN10 and PEG-SN10 could significantly improve the degree and extent of colonic inflammation in mice. The expression of TNF-alpha, IL-6, IL-1beta and IFN-gamma was inhibited, while the expression of IL-10 in colon tissue was promoted (P 0.05). Therefore, Hydrostatin-SN10 and PEG-SN10 had obvious therapeutic effects on OXZ-induced acute colitis in mice. The concentration-time curves of Hydrostatin-SN10 in six adult SD rats after intraperitoneal injection of 1 mg/kg Hydrostatin-SN 10,5 mg/kg PEG-SN 10 were not strictly consistent with the typical atrioventricular model. The pharmacokinetic parameters calculated by non-compartment model (statistical moment) method reflected the pharmacokinetic disappearance in SD rats. The area under the pharmacokinetic time curve (AUC0-t) of hydrostatin-SN10 in healthy SD rats was positively correlated with the dosage, and the correlation coefficients were R2 = 0.9936 and R2 = 0.9921.H, respectively. Both ydrostatin-SN10 and PEG-SN10 showed good pharmacokinetic properties in SD rats. After intraperitoneal injection of 1 mg/kg Hydrostatin-SN10 and 5 mg/kg PEG-SN10 into SD rats, the peak concentrations of Cmax were 86.92 (+ 4.584) ug/L and 33.071 (+ 1.697) ug/L, respectively. The peak time of Tmax was 0.833 h and 0.833 h, and the terminal elimination half-life of t1/2. The doses of AUC (0-t) were 74.043 (+ 17.065 ug/L * h) and 32.087 (+ 6.317 ug/L * h) respectively. The preliminary pharmacodynamic and pharmacokinetic studies of TNFR1-specific selective antagonistic peptide Hydrostatin-SN10 and PEG-SN10 modified by mPEG2000 for IBD were completed. BD provides a theoretical and experimental basis for specific selective new drugs.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574
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