選擇性TNFR1拮抗肽Hydrostatin-SN10治療炎癥性腸病的初步藥效學(xué)研究
發(fā)布時間：2018-09-17 17:55
【摘要】：炎癥性腸病(inflammatory bowel disease,IBD)是一種累及回腸、直腸和結(jié)腸的特發(fā)性炎癥性腸道疾病,臨床表現(xiàn)為反復(fù)腹痛、腹瀉、腹部包塊、黏液血便、腸梗阻、腸穿孔、體重減輕等,此外患者還表現(xiàn)出各種不同程度的全身癥狀。IBD過去多見于西方發(fā)達(dá)國家,近幾年來包括中國在內(nèi)的亞洲地區(qū)的發(fā)病率呈直線上升趨勢,嚴(yán)重影響了患者的生活質(zhì)量,關(guān)于IBD治療的研究已成為消化系統(tǒng)疾病領(lǐng)域的研究熱點之一。然而到目前為止,臨床上尚未出現(xiàn)能夠有效治療IBD的藥物。本課題組前期獲得了一條抗炎活性肽Hydrostatin-SN10(10AA),該活性肽只與TNFR1結(jié)合,不與TNNF-α和TNFR2結(jié)合,具有專一選擇性�；诖搜芯炕A(chǔ),本課題通過將m PEG2000的羧基與Hydrostatin-SN10肽鏈N-端天冬氨酸的游離氨基共價連接實現(xiàn)選擇性修飾,得到PEG-SN10。本研究采用葡聚糖硫酸鈉(dextran sulphate sodium salt,DSS,26~50KDa)和VA唑酮(oxazolone,OXZ)誘導(dǎo)的小鼠急性結(jié)腸炎模型,探討了候選藥物選擇性TNFR1拮抗肽Hydrostatin-SN10治療炎癥性腸病的初步藥效學(xué)研究。本文研究發(fā)現(xiàn),在DSS誘導(dǎo)的小鼠急性結(jié)腸炎模型中,模型對照組小鼠體重明顯減輕,毛色暗淡,精神萎靡,肉眼可見血便,腹瀉嚴(yán)重,與空白對照組小鼠疾病活動指數(shù)(disease activity index,DAI)評分相比,模型組小鼠DAI評分明顯升高;體重明顯減輕;頸椎脫臼法處死全部小鼠并將其解剖,取出完整結(jié)腸,觀察并測量發(fā)現(xiàn):模型組小鼠結(jié)腸長度明顯縮短(P0.001);脾臟指數(shù)增加(P0.001);小鼠血清中促炎因子(TNF-α、IL-6、IL-1β、IFN-γ)含量顯著升高,抑炎因子(IL-10)含量顯著降低(P0.01);通過收集小鼠結(jié)腸組織勻漿液,測得小鼠結(jié)腸組織中髓過氧化物酶(myeloperoxidase,MPO)活性顯著升高(P0.001);提取小鼠結(jié)腸組織RNA,TNF-α、IL-6、IL-1β、IFN-γmRNA相對表達(dá)量明顯升高,IL-10 mRNA相對表達(dá)量明顯降低(P0.05)。小鼠腹腔注射Hydrostatin-SN10和PEG-SN10后,上述病癥均得到顯著緩解。與DSS模型組小鼠相比,Hydrostatin-SN10和PEG-SN10治療組小鼠體重下降減緩,腹瀉次數(shù)減少,結(jié)腸長度縮短趨勢得到有效控制(P0.001),脾臟指數(shù)、MPO活性均下降(p0.001),說明Hydrostatin-SN10和PEG-SN10可以明顯改善DSS誘導(dǎo)的小鼠急性結(jié)腸炎癥狀。在OXZ誘導(dǎo)的小鼠急性結(jié)腸炎模型中,Hydrostatin-SN10及PEG-SN10治療組的小鼠各項疾病癥狀均明顯好于模型組,比如小鼠體重變化情況、結(jié)腸炎相關(guān)疾病活動指數(shù)等,Hydrostatin-SN10和PEG-SN10給藥組小鼠的體重下降趨勢明顯得到抑制,DAI指數(shù)也低于模型組小鼠,治療效果與兩種陽性對照藥SASP和IFX等同,且陰性對照組(隨機(jī)肽組和PEG組)并無疾病緩解癥狀;同樣,Hydrostatin-SN10及PEG-SN10治療組小鼠的結(jié)腸長度縮短、脾腫脹情況也有明顯的緩解現(xiàn)象(P0.001);MPO活性測定結(jié)果表明,Hydrostatin-SN10及PEG-SN10能顯著減少小鼠結(jié)腸病變組織的中性粒細(xì)胞數(shù)量(P0.001);蘇木精-伊紅(hematoxylin eosin,HE)染色結(jié)果表明,Hydrostatin-SN10及PEG-SN10能顯著改善結(jié)腸的炎癥程度和病變范圍;實時熒光定量PCR(qRT-PCR)結(jié)果證實Hydrostatin-SN10及PEG-SN10對小鼠結(jié)腸組織中相關(guān)促炎因子(TNF-α、IL-6、IL-1β、IFN-γ)的表達(dá)具有抑制效果,而對結(jié)腸組織中相關(guān)抑炎因子(IL-10)的表達(dá)具有促進(jìn)作用(P0.05),因此Hydrostatin-SN10及PEG-SN10對于OXZ誘導(dǎo)的小鼠急性結(jié)腸炎也具有明顯的治療作用。初步藥代動力學(xué)研究表明:單劑量腹腔注射給藥1mg/kg Hydrostatin-SN10、5mg/kg PEG-SN10后測得的6只成年、健康SD大鼠體內(nèi)Hydrostatin-SN10的藥物濃度-時間曲線變化規(guī)律并不嚴(yán)格符合典型的房室模型,以非房室模型(統(tǒng)計矩)法計算的藥代動力學(xué)參數(shù)反映SD大鼠體內(nèi)藥代動力學(xué)的消除過程基本呈線性動力學(xué)特征。單劑量腹腔注射給藥1mg/kg Hydrostatin-SN10、5mg/kg PEG-SN10后測得的6只成年、健康SD大鼠體內(nèi)Hydrostatin-SN10所對應(yīng)的藥代動力學(xué)藥時曲線下面積(AUC0-t)與給藥劑量基本呈正相關(guān),相關(guān)系數(shù)分別為r2=0.9936和r2=0.9921。Hydrostatin-SN10及PEG-SN10在SD大鼠體內(nèi)均具有較好的藥代動力學(xué)性質(zhì),SD大鼠腹腔注射給藥1mg/kg Hydrostatin-SN10和5mg/kg PEG-SN10后,注射給藥的藥峰濃度Cmax分別為86.92±4.584μg/L和33.071±1.697μg/L,達(dá)峰時間Tmax分別為0.833h和0.833h,末端消除半衰期t1/2分別為2.18±0.654h和3.789±1.368h,注射暴露量AUC(0-t)分別為74.043±17.065ug/L*h和32.087±6.317ug/L*h。本課題完成了TNFR1專一選擇性拮抗肽Hydrostatin-SN10以及基于mPEG2000修飾的PEG-SN10治療IBD的初步藥效學(xué)研究和初步藥代動力學(xué)研究,為后續(xù)篩選治療IBD的專一選擇性新藥提供了理論和實驗依據(jù)。
[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.
【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R574
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