CN102219812B - 一种肿瘤靶向的脱氧葡萄糖复合药物及其制备方法 - Google Patents
一种肿瘤靶向的脱氧葡萄糖复合药物及其制备方法 Download PDFInfo
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Abstract
本发明涉及药物合成及应用领域。涉及具有一种肿瘤靶向的脱氧葡萄糖复合药物及其制备方法。该药物可提高肿瘤部位的有效药物浓度并能降低全身毒副作用。将具有靶向作用和一定抗肿瘤作用的脱氧葡萄糖修饰阿霉素或尼莫司汀的氨基,从而达到靶向给药,降低阿霉素和尼莫司汀的毒副作用,提高肿瘤治疗作用等效果,从而起到协同抗肿瘤作用。本发明以脱氧葡萄糖和阿霉素、尼莫司汀为主要原料,以丁二酸酐为链接体,采用共价连接法制备脱氧葡萄糖-阿霉素、脱氧葡萄糖-尼莫司汀抗肿瘤复合药物,使其对肿瘤具有靶向治疗作用。
Description
技术领域
本发明涉及药物合成及应用领域。涉及具有肿瘤靶向的脱氧葡萄糖与抗肿瘤药物的反应连接,构建一种肿瘤靶向的脱氧葡萄糖-抗肿瘤复合药物的制备方法和体内外药效实验。
背景技术
目前,肿瘤是威胁人类健康的重大疾病之一。现今治疗肿瘤的方法主要包括放疗、化疗、手术疗法和基因治疗等。其中,放疗和化疗是极其重要的非手术疗法,但是放疗和化疗在杀死肿瘤细胞的同时也对机体正常细胞产生严重损伤。尤其是化疗,由于药物本身缺乏靶向性,因而使肿瘤部位有效药物浓度降低,全身毒副作用增强,出现治愈率低、多药耐药性、毒副作用大等治疗问题。
葡萄糖(glucose)是自然界分布最广且最为重要的一种单糖,它是一种多羟基醛。纯净的葡萄糖为无色晶体,有甜味,宜溶于水,微溶于乙醇,不溶于乙醚。水溶液旋光向右,故亦称“右旋糖”。葡萄糖在生物学领域具有重要地位,是生物体内新陈代谢不可缺少的能源物质,是活细胞的能量来源和新陈代谢的中间产物,在医药领域有着广泛应用。葡萄糖进入血液循环后,在体内代谢首先需要进入细胞,这是依赖葡萄糖转运体(glucose transporter,GLUT)而实现的。葡萄糖代谢在不同类型细胞中的代谢途径有所不同,其分解代谢方式在很大程度上受供氧状况的影响。在缺氧情况下,葡萄糖经糖酵解途径分解成丙酮酸,然后将丙酮酸转变为乳酸。在有氧情况下,葡萄糖分解产生的丙酮酸氧化脱羧生成乙酰辅酶A,并进入三羧酸循环而氧化成水和二氧化碳并释放出能量。
肿瘤细胞的能量代谢异常,代谢旺盛,对葡萄糖的代谢率增高,需求量增加,与正常细胞相比,肿瘤细胞GLUT的表达和活性明显增强。Warburg发现即使在有氧条件下肿瘤细胞也能大量摄取葡萄糖并产生乳酸,从氧化磷酸化转变为无氧酵解,被称为“Warburg效应”。
目前,用于抗肿瘤的化疗药物种类繁多,其中阿霉素类药物与其他抗肿瘤化疗药物相比,其优点在于抗瘤谱广,对多数肿瘤具有治疗作用,并且抗肿瘤效果明显。但其也有一些缺点,主要表现为毒副作用明显,尤其是致癌毒副作用,并且本身不具有肿瘤特异靶向性,致使肿瘤部位药物有效浓度降低,全身毒副作用明显。因此,研究者开始致力于阿霉素类药物的修饰。CN101234205公开了具有靶向功能的高分子阿霉素键合药纳米胶囊及其制备方法。CN1606450公开了多肽、其与阿霉素的结合物和基于结合物的药物组合物。CN101274100公开了一种阿霉素-二肽复合物的制备方法和应用。CN101353361公开了一种阿霉素前体药物的制备方法及其应用,将阿霉素与α-亚麻酸连接合成前体药物,从而增强阿霉素抗肿瘤作用,降低药物毒副作用。CN101624443公开了一种阿霉素前体药物的制备方法及其应用,合成了阿霉素-聚乙二醇二酸腙键连接物,从而提高靶向性,增强阿霉素的抗肿瘤作用,降低药物毒副作用。CN101914138A公开了一种用偶联剂制得的肝癌靶向肽-阿霉素及合成方法。Turdi S等将阿霉素共价连接酸酐并进行毒性实验比较,显示阿霉素的氨基被修饰成酰胺键后致癌毒副作用明显减小(Toxicol Lett,2008;178(3):197-201)。Cai S等将阿霉素连接透明质酸制成纳米粒,发现其对乳腺癌具有更好的疗效,并且降低了阿霉素的毒副作用(J Control Release,2010;146(2):212-8)。Boddu SH等以叶酸为靶向物质,以阿霉素为治疗药物制成靶向微粒,通过体外实验与阿霉素比较发现复合微粒被肿瘤细胞更多的摄取,具有更好的抗肿瘤作用(JOcul Pharmacol Ther,2010;26(5):459-68)。
目前尚无将尼莫司汀与靶向物质连接用于肿瘤靶向治疗的报道。
发明内容
目前,各种靶向机制为指导的抗肿瘤药物已经逐渐成为更精确有效地用于肿瘤治疗研究;其中,以代谢为靶向机制,以脱氧葡萄糖为靶向物质的抗肿瘤药物还没有被报道。本发明将氨基葡萄糖通过链接体丁二酸酐与阿霉素和尼莫司汀共价连接,即葡萄糖第二位碳原子上的羟基被取代。本发明中的脱氧葡萄糖-阿霉素复合药物、脱氧葡萄糖-尼莫司汀复合药物,其中脱氧葡萄糖起到了靶向物质作用,使药物特异进入肿瘤部位,增加肿瘤部位有效浓度,降低全身毒副作用;同时药物与脱氧葡萄糖的抗肿瘤作用起到协同治疗作用;且阿霉素的氨基被修饰后致癌毒副作用降低,起到增效减毒作用。
本发明的脱氧葡萄糖-阿霉素复合药物(I)、脱氧葡萄糖-尼莫司汀复合药物(II)结构式如下:
本发明的结构式(I)或(II)的氨基葡萄糖复合物可用下列方法制备:
D-氨基葡萄糖盐酸盐溶于三乙胺溶液,搅拌至溶解;滴加丁二酸酐的丙酮溶液,搅拌反应,反应液减压浓缩,加入冰乙酸,静置,析出白色沉淀;白色沉淀抽滤,乙醇和乙醚洗涤,乙醇纯化,真空干燥,得白色粉末颗粒;白色粉末颗粒溶于蒸馏水,搅拌溶解,加1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺,搅拌反应,活化羧基;将阿霉素盐酸盐或尼莫司汀溶于蒸馏水后滴加入活化液中,避光搅拌反应;反应液干燥,纯化,即得。
D-氨基葡萄糖盐酸盐和丁二酸酐的投料摩尔比优选1∶1~1∶10。
白色粉末颗粒、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、阿霉素盐酸盐或尼莫司汀的投料摩尔比优选1~30∶1~20∶1~40∶1~35。
本发明的氨基葡萄糖复合物可以和药学上可接受的载体混合制备成各种药物制剂。
本发明的脱氧葡萄糖-阿霉素复合药物(I)制备中,先将脱氧葡萄糖通过形成酰胺键连接丁二酸酐后,形成中间产物,质谱鉴定C10H17NO8[MH]-:278.1,见图1。后将阿霉素通过形成酰胺键连接中间产物得到终产物脱氧葡萄糖-阿霉素复合药物,经HPLC分离纯化后,质谱鉴定C37H44N2O18[MH]-:803.1,见图2。进行紫外吸收和荧光发射光谱鉴定,显示脱氧葡萄糖-阿霉素复合药物具有稳定的光学性质。紫外吸收光谱特征峰位是:210、239、252、288、482、494、530nm,见图3;荧光发射光谱特征峰位是:564、593、656nm,见图4。通过体外药效实验显示脱氧葡萄糖-阿霉素复合药物与单用阿霉素比较,具有更好的抗肿瘤作用,肿瘤抑制率增高,且在此浓度范围,两者的抗肿瘤作用均与浓度呈正相关,见图5。通过体内药效实验显示脱氧葡萄糖-阿霉素复合药物与对照组和单用阿霉素比较,瘤重和肿瘤体积均明显降低,肿瘤生长抑制率明显增高,见表1。
将尼莫司汀用与阿霉素相同的方法,将其与脱氧葡萄糖连接丁二酸酐后的中间产物反应,得到脱氧葡萄糖-尼莫司汀复合药物。经HPLC分离纯化后,质谱鉴定C15H24ClN7O6[MH]-:432.6,见图6。通过体外药效实验显示脱氧葡萄糖-尼莫司汀复合药物与单用尼莫司汀比较,具有更好的抗肿瘤作用,肿瘤抑制率增高,且在此浓度范围,肿瘤抑制率与浓度呈正相关,见图7。通过体内药效实验显示脱氧葡萄糖-尼莫司汀复合药物与对照组和单用尼莫司汀比较,瘤重和肿瘤体积均明显降低,肿瘤生长抑制率明显增高,见表2。
本发明的该复合药物(I)或(II)具有很好的水溶性,因此可被生物体高效吸收。复合药物中脱氧葡萄糖的靶向作用和一定的抗肿瘤作用,使其能够靶向协同增效减毒给药。
附图说明
图1是脱氧葡萄糖连接丁二酸酐质谱
图2是脱氧葡萄糖-阿霉素复合药物质谱
图3是脱氧葡萄糖-阿霉素复合药物的紫外吸收光谱
图4是脱氧葡萄糖-阿霉素复合药物的荧光发射光谱
图5是脱氧葡萄糖-阿霉素复合药物和阿霉素体外作用后抑制率随药物浓度的变化
图6是脱氧葡萄糖-尼莫司汀复合药物质谱
图7是脱氧葡萄糖-尼莫司汀复合药物和尼莫司汀体外作用后抑制率随药物浓度的变化
具体实施方式
实施例1
脱氧葡萄糖连接丁二酸酐的制备
称取5gD-氨基葡萄糖盐酸盐,溶于3.4ml三乙胺和7.5ml蒸馏水,常温搅拌至D-氨基葡萄糖盐酸盐全部溶解。滴加2.5g丁二酸酐的18.75ml丙酮溶液至D-氨基葡萄糖溶液,室温搅拌反应4h。将得到的混合溶液在45℃减压浓缩,得到褐色粘稠母液。将4倍浓缩母液体积的冰乙酸加入至褐色粘稠母液,室温静置,析出白色沉淀。将白色沉淀抽滤,并用乙醇和乙醚洗涤,乙醇纯化,真空干燥,得白色粉末颗粒即。质谱鉴定C10H17NO8[MH]-:278.1见图1。
脱氧葡萄糖-阿霉素复合药物的制备
称取脱氧葡萄糖连接丁二酸酐后的白色粉末颗粒4.81mg溶于0.5ml蒸馏水,搅拌溶解,加入4.96mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)和2.98mg N-羟基琥珀酰亚胺(NHS),室温搅拌反应3h,活化羧基。将10mg阿霉素盐酸盐溶于1.0ml蒸馏水,滴加入羧基活化液,室温避光搅拌反应过夜。将反应混合液挥发干燥,用HPLC进行分离纯化,得到脱氧葡萄糖-阿霉素复合药物,挥发干燥得到橙红色粉末。质谱鉴定C37H44N2O18[MH]-:803.1,见图2。
将脱氧葡萄糖-阿霉素复合药物溶于蒸馏水中进行紫外吸收光谱鉴定,特征峰位是:210、239、252、288、482、494、530nm,见图3。将脱氧葡萄糖-阿霉素复合药物溶于蒸馏水中进行荧光发射光谱鉴定,特征峰位是:564、593、656nm,见图4。结果显示脱氧葡萄糖-阿霉素复合药物具有稳定的光学性质。
实施例2
脱氧葡萄糖-阿霉素复合药物(I)的体外药效实验1
将肿瘤细胞MCF-7以5×107/L接种于96孔培养板,每孔100μl,在37℃,5%CO2条件下培养24h。分别加入不同浓度0.25、0.5、1、2、4μg/ml的脱氧葡萄糖-阿霉素复合药物(I)和阿霉素,调零组和对照组加相应体积的培养液,每组设5个平行孔。培养48h后,每孔加5g/L的MTT 20μl(调零组除外),培养4h,倾去培养液,加150μl/孔DMSO,待甲臢完全溶解后,用酶标仪在波长570nm处调零后读取吸光度(A)。取5孔A值的均数,按公式:抑制率=(对照组OD值-实验组OD值)/对照组OD值,计算药物对肿瘤细胞的抑制率。以上实验重复3次。MTT比色法显示在0.25、0.5、1、2、4μg/ml浓度作用下,脱氧葡萄糖-阿霉素复合药物(I)较阿霉素具有更好的抗肿瘤作用,且在此浓度范围,两者的抗肿瘤作用均与浓度呈正相关,见图5。
脱氧葡萄糖-阿霉素复合药物(I)的体内药效实验2
取裸鼠30只(4~6周龄,雌性,体重18~22g),于SPF级无菌饲养室饲养。取指数生长期的MCF-7乳腺癌细胞,加入胰蛋白酶和EDTA混和消化液消化,加入1640培养液吹打、离心、洗涤,制成单细胞悬液,将单细胞悬液调整为细胞浓度2.5×107/mL。每只裸鼠于一侧背部皮下接种该单细胞悬液0.2mL,待肿瘤直径超过0.5cm(7~14天),致瘤率达100%后用于实验。
取裸鼠30只,称重后随机分成3组,每组10只:(1)对照组:腹腔注射等容积生理盐水,每7天注射1次,共3次;(2)阿霉素组:按6mg/kg腹腔注射,每7天注射1次,共3次;(3)脱氧葡萄糖-阿霉素复合药物(I)组:按6mg/kg腹腔注射,每7天注射1次,共3次。
每3天观察裸鼠肿瘤的成瘤和生长情况及体质量变化,根据公式V=a×b2/2计算肿瘤体积(a长轴、b短轴)。治疗3周后处死裸鼠,取出肿瘤,称重,计算肿瘤生长抑制率,抑制率=(对照组瘤重-治疗组瘤重)/对照组瘤重×100%。
经过3周的治疗,脱氧葡萄糖-阿霉素复合药物(I)组治疗的裸鼠肿瘤重量及体积明显小于阿霉素组及对照组,差异具有显著性。对照组裸鼠肿瘤生长最迅速;阿霉素组和脱氧葡萄糖-阿霉素复合药物组肿瘤生长较对照组减慢;脱氧葡萄糖-阿霉素复合药物组肿瘤生长较阿霉素组缓慢。裸鼠在3周处死时均存活,见表1。
实施例3
脱氧葡萄糖-尼莫司汀复合药物(II)的制备
称取脱氧葡萄糖连接丁二酸酐后的白色粉末颗粒4.81mg溶于0.5ml蒸馏水,搅拌溶解,加入4.96mg 1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)和2.98mg N-羟基琥珀酰亚胺(NHS),室温搅拌反应3h,活化羧基。将5.33mg尼莫司汀盐酸盐溶于0.5ml蒸馏水,滴加入羧基活化液,室温避光搅拌反应过夜。将反应混合液挥发干燥,用HPLC进行分离纯化,得到脱氧葡萄糖-尼莫司汀复合药物(II),挥发干燥。质谱鉴定C15H24ClN7O6[MH]-:432.6,见图6。
实施例4
脱氧葡萄糖-尼莫司汀复合药物(II)的体外药效实验1
将U87MG胶质瘤细胞以5×107/L接种于96孔培养板,每孔100μl,在37℃,5%CO2条件下培养24h。分别加入不同浓度2.5、5、10、20、40μg/ml的脱氧葡萄糖-尼莫司汀复合药物(II)和尼莫司汀,调零组和对照组加相应体积的培养液,每组设5个平行孔。培养48h后,每孔加5g/L的MTT 20μl(调零组除外),培养4h,倾去培养液,加150μl/孔DMSO,待甲臢完全溶解后,用酶标仪在波长570nm处调零后读取吸光度(A)。取5孔A值的均数,按公式:抑制率=(对照组OD值-实验组OD值)/对照组OD值,计算药物对肿瘤细胞的抑制率。以上实验重复3次。MTT比色法显示在2.5、5、10、20、40μg/ml浓度作用下,脱氧葡萄糖-尼莫司汀复合药物(II)较尼莫司汀具有更好的抗肿瘤作用,且在此浓度范围,两者的抗肿瘤作用均与浓度呈正相关,见图7。
脱氧葡萄糖-尼莫司汀复合药物的体内药效实验2
取裸鼠30只(4~6周龄,体重18~22g),于SPF级无菌饲养室饲养。取指数生长期的U87MG胶质瘤细胞,加入胰蛋白酶和EDTA混和消化液消化,加入DMEM培养液吹打、离心、洗涤,制成单细胞悬液,将单细胞悬液调整为细胞浓度2.5×107/mL。每只裸鼠于一侧背部皮下接种该单细胞悬液0.2mL,待肿瘤直径超过0.5cm(7~10天),致瘤率达100%后用于实验。
取裸鼠30只,称重后随机分成3组,每组10只:(1)对照组:腹腔注射等容积生理盐水,每7天注射1次,共3次;(2)尼莫司汀组:按30mg/kg腹腔注射,每7天注射1次,共3次;(3)脱氧葡萄糖-尼莫司汀复合药物(II)组:按30mg/kg腹腔注射,每7天注射1次,共3次。
每3天观察裸鼠肿瘤的成瘤和生长情况及体质量变化,根据公式V=a×b2/2计算肿瘤体积(a长轴、b短轴)。治疗3周后处死裸鼠,取出肿瘤,称重,计算肿瘤生长抑制率,抑制率=(对照组瘤重-治疗组瘤重)/对照组瘤重×100%。
经过3周的治疗,脱氧葡萄糖-尼莫司汀复合药物(II)组治疗的裸鼠肿瘤重量及体积明显小于尼莫司汀组及对照组,差异具有显著性。对照组裸鼠肿瘤生长最迅速;尼莫司汀组和脱氧葡萄糖-尼莫司汀复合药物组肿瘤生长较对照组减慢;脱氧葡萄糖-尼莫司汀复合药物组肿瘤生长较尼莫司汀组缓慢。裸鼠在3周处死时均存活,见表2。
Claims (3)
1.结构式(I)或(II)的氨基葡萄糖复合物的制备方法,包括:
D-氨基葡萄糖盐酸盐溶于三乙胺溶液,搅拌至溶解;滴加丁二酸酐的丙酮溶液,搅拌反应,反应液减压浓缩,加入冰乙酸,静置,析出白色沉淀;白色沉淀抽滤,乙醇和乙醚洗涤,乙醇纯化,真空干燥,得白色粉末颗粒;白色粉末颗粒溶于蒸馏水,搅拌溶解,加1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和N-羟基琥珀酰亚胺,搅拌反应,活化羧基;将阿霉素盐酸盐或尼莫司汀溶于蒸馏水后滴加入活化液中,避光搅拌反应;反应液干燥,纯化,即得。
2.权利要求1的制备方法,其中D-氨基葡萄糖盐酸盐和丁二酸酐的投料摩尔比是1:1~1:10。
3.权利要求1的制备方法,其中白色粉末颗粒、1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐、N-羟基琥珀酰亚胺、阿霉素盐酸盐或尼莫司汀的投料摩尔比是1~30:1~20:1~40:1~35。
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Non-Patent Citations (4)
Title |
---|
Therapeutics》.2009,第121卷(第1期),29-40. * |
Vadivel Ganapathy,等.Nutrient transporters in cancer: Relevance to Warburg hypothesis and beyond.《Pharmacology & Therapeutics》.2009,第121卷(第1期),29-40. |
Vadivel Ganapathy,等.Nutrient transporters in cancer: Relevance to Warburg hypothesis and beyond.《Pharmacology & * |
乔岩,等.2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖的合成.《化学试剂》.2004,第26卷(第2期),107-108. * |
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