本文選題：尼達尼布 + 雙胍��； 參考：《江蘇大學》2017年碩士論文

【摘要】：雙胍類是常用的治療糖尿病藥物之一,近年該類藥物具有抗癌療效常有報道,其結構中的雙胍基被認為是一種具有潛在抗腫瘤作用的化學基團,同時該基團對磷酸根骨架的核酸分子具有高親合性,且對磷脂雙分子層結構的細胞膜具有較強穿透能力。尼達尼布(Nintedanib,NDNB)是用于特發(fā)性肺纖維化治療的小分子化學藥物,作為一種可抑制血管生成和組織纖維化的三重受體酪氨酸激酶抑制劑,大量臨床前及臨床研究已表明其具有較好的抗腫瘤作用。本課題先以雙氰胺為底物合成了含雙胍基的中間體,再采用混合酸酐法將其與牛血清白蛋白偶聯,以其作為材料制備納米粒運載藥物NDNB,并對其體內外藥動藥效進行研究。課題旨在構建偶聯改性的白蛋白納米載體,增強NDNB抗腫瘤作用。論文分為以下幾部分:第一部分綜述本部分主要對雙胍基的抗腫瘤作用的研究、偶聯/修飾白蛋白載體的研究、NDNB的抗腫瘤作用研究進行介紹。了解雙胍類藥物及雙胍基抗腫瘤的研究前沿,蛋白偶聯/修飾方法及設計思路,NDNB的性質、藥理作用及藥動學性質等,為后續(xù)制劑研究進行理論準備。第二部分處方前研究本部分建立了紫外分光光度法作為體外藥物含量檢測的分析方法。通過紫外可見全波長掃描確定藥物最大吸收波長為385nm,以此波長作為藥物檢測波長。配制相應不同濃度的藥物溶液建立標準曲線,并對建立的含量測定方法進行方法學驗證考察。實驗表明所建立的體外藥物含量測定方法合理可靠,符合方法學要求。對NDNB的釋放介質中溶解度的考察結果表明NDNB在純水中溶解度大,在磷酸鹽溶液中的溶解度非常小,加入表面活性劑吐溫80可增加其溶解度。第三部分雙胍基偶聯牛血清白蛋白的制備本部分先以雙氰胺與對氨基苯甲酸為底物,反應合成具有潛在抗腫瘤活性的雙胍基中間體對雙胍基苯甲酸(DP),再采用混合酸酐法將其與牛血清白蛋白偶聯(DP-BSA),采用薄層色譜法、紅外光譜、核磁譜、質譜、紫外掃描、高效液相色譜法對合成中間體的結構及純度進行表征,采用紫外及凝膠電泳對蛋白偶聯產物DP-BSA進行確認表征,采用基體輔助激光解吸電離飛行時間質譜(MALDI-TOF-MS)和紫外掃描對DP-BSA的偶聯度進行分析估算。經投料比、反應時間、反應pH環(huán)境單因素考察,確認最佳工藝處方,所得DP-BSA的偶聯度為42.19。另外,還對DP-BSA溶解性等性質進行了初步考察。第四部分載藥納米載體的制備及體外釋放本部分使用雙胍基偶聯白蛋白為載體材料制備雙胍基偶聯白蛋白納米粒DP-BSANPs運載抗腫瘤模型藥物NDNB,并通過使用激光粒徑分析儀、透射電鏡及紫外分光光度法對尼達尼布載藥納米粒(NDNB-DP-BSANPs)溶液的粒徑分布、包封率、載藥濃度進行檢測表征。以粒徑分布、形態(tài)、包封率、載藥濃度為指標對處方及制備工藝進行單因素考察以選擇孵化法最佳處方及工藝。結果顯示,所制納米粒平均粒徑為47.18nm,多分散系數為0.15,平均載藥濃度為1.586mg/mL,48h內含藥量穩(wěn)定。此外,以紫外分光光度法作為藥物體外釋放檢測方法,通過透析法對于NDNB原料藥溶液及載藥納米粒溶液在PBS(0.5%Tween80)溶液中的釋放進行考察,體外藥物釋放結果表明載藥納米粒具有緩釋作用。第五部分體內藥物動力學考察本部分建立高效液相色譜法進行血藥濃度檢測,SD大鼠尾靜脈給藥進行體內藥動學考察。方法學驗證結果表明,所建立的方法專屬性強、精密度高、回收率高、穩(wěn)定性良好。體內藥動學實驗結果顯示,NDNB在大鼠體內的藥物動力學過程符合雙室模型,給藥后載藥納米粒組各時間點血藥濃度均高于原料藥組,原料藥組半衰期為2.526h,載藥納米粒組半衰期4.588h;原料藥組平均滯留時間為2.914h,明顯低于載藥納米粒組為5.905h,載藥納米粒組相對生物利用度為162.96%,表明載藥納米粒可以增加藥物的半衰期和生物利用度。第六部分體內抗腫瘤模型藥效學研究及細胞實驗本部分通過動物抗腫瘤模型給藥實驗及細胞實驗對合成的偶聯蛋白納米載體及其NDNB載藥納米制劑抗腫瘤效果進行研究�？鼓[瘤藥效實驗和細胞實驗結果顯示,雙胍基偶聯白蛋白載體對于腫瘤細胞有一定的抑制作用,載藥納米粒在兩個實驗中表現出較好的腫瘤生長抑制作用。
[Abstract]:Metformin is one of the commonly used drugs for the treatment of diabetes. In recent years, the antitumor effect of this kind of drugs is often reported. The metformin in its structure is considered as a chemical group with potential antitumor effect. At the same time, the group has high affinity to the nucleic acid molecule of the phosphate skeleton, and it has the cell membrane of the phospholipid bimolecular layer structure. Strong penetration. Neda Knibb (Nintedanib, NDNB) is a small molecular chemical used for the treatment of idiopathic pulmonary fibrosis. As a three receptor tyrosine kinase inhibitor that inhibits angiogenesis and tissue fibrosis, a large number of preclinical and clinical studies have shown that it has better anti-tumor effects. The intermediates containing bis guanidine were synthesized for the substrate, and then they were coupled with bovine serum albumin by the mixed anhydride method. The nanoscale carrier drug NDNB was prepared as a material, and the pharmacokinetics of the nanoparticles were studied. The aim of the study is to construct the modified albumin nanoscale and enhance the anti-tumor effect of NDNB. The paper is divided into the following parts Part 1: the first part is a summary of the research on the antitumor effect of metformin, the study of coupling / modified albumin carrier, the research on the anti-tumor effect of NDNB. The research frontier of the Antitumor of guanidine and metformin, the method of protein coupling / modification and the thinking, the properties of NDNB, the pharmacological action and the pharmacokinetic properties of the metformin In the second part, the second part of the study set up an analytical method for the determination of the drug content in vitro by UV spectrophotometry. The maximum absorption wavelength of the drug was determined by UV visible full wavelength scanning, and the wavelength was used as the detection wavelength of the drug. The standard curve of the solution was established and the method of determination was verified. The experiment showed that the method of determination of drug content in vitro was reasonable and reliable. The results of solubility in the release medium of NDNB showed that the solubility of NDNB in pure water was large and the solubility in phosphate solution was very good. Small, adding surface active agent Twain 80 can increase its solubility. Third the preparation of bis guanidine conjugated bovine serum albumin (BSA) in this part first reaction with dicyandiamide and p-aminophenic acid as substrate, the synthesis of dianidine intermediate with potential antitumor activity against guanidine benzoic acid (DP), and then mixed anhydride with bovine blood white eggs DP-BSA, the structure and purity of the synthetic intermediates were characterized by TLC, IR, NMR, MS, UV, and high performance liquid chromatography. The protein coupling product DP-BSA was characterized by UV and gel electrophoresis, and the base assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was used. The coupling degree of DP-BSA was analyzed and estimated by UV scanning. The optimum process was confirmed by the ratio of feeding, reaction time and reaction of pH environment. The coupling degree of DP-BSA was 42.19., and the solubility of DP-BSA was preliminarily investigated. The preparation of the fourth part of drug carrying nanoscale and the release of this part in vitro were used in two parts. Guanidine coupling albumin was used as carrier material to prepare antitumor model drug NDNB, which was loaded with guanidine coupling albumin nanoparticles DP-BSANPs. The particle size distribution, encapsulation efficiency and drug concentration of Neda Knibb loaded nanoparticles (NDNB-DP-BSANPs) solution were detected by laser particle size analyzer, transmission electron microscope and ultraviolet spectrophotometry. The optimum formulation and process of the formulation and preparation process were selected by the particle size distribution, morphology, encapsulation efficiency and drug loading. The results showed that the average particle size of the nanoparticles was 47.18nm, the polydispersity coefficient was 0.15, the average drug concentration was 1.586mg/ mL, and the content of the 48h was stable. In addition, UV spectrophotometry was used. As a method of drug release detection in vitro, the release of NDNB drug solution and drug loaded nanoparticles solution in PBS (0.5%Tween80) solution was investigated by dialysis. The drug release in vitro showed that the drug loaded nanoparticles had sustained release effect. The fifth part of the pharmacokinetic study in vivo was established by high performance liquid chromatography The pharmacokinetic study of the tail vein of SD rats was conducted in vivo. The results showed that the established method was highly exclusive, high precision, high recovery and good stability. The pharmacokinetic experiment in vivo showed that the pharmacokinetics of NDNB in rats was in accordance with the double chamber model, and the drug loaded nanoparticles were given each time after the drug was given. The concentration of blood drug was higher than that of the drug group, the half-life of the drug group was 2.526h, the half life of the drug loaded nanoparticles group was 4.588h, the average retention time of the drug drug group was 2.914h, which was significantly lower than the drug loaded nanoparticles group was 5.905h, and the relative bioavailability of the drug loaded nanoparticles group was 162.96%, indicating that the drug loaded nanoparticles could increase the half-life and biological benefits of the drug loaded nanoparticles. Sixth part of the antitumor model in vivo and cell experiment in this part, the anti tumor effect of the synthesized coupling protein nanoscale carrier and its NDNB drug loaded nanoscale preparation was studied by the animal antitumor model administration and cell experiment. The antitumor effect experiment and the cell experiment result showed that the double guanidine coupling albumin was found. The carrier has a certain inhibitory effect on tumor cells. Drug loaded nanoparticles show a good inhibitory effect on tumor growth in two experiments.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TB383.1;TQ460.1

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