CN111333692A - 一种白桦脂酸衍生物及其制备方法和应用 - Google Patents
一种白桦脂酸衍生物及其制备方法和应用 Download PDFInfo
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- CN111333692A CN111333692A CN202010136699.XA CN202010136699A CN111333692A CN 111333692 A CN111333692 A CN 111333692A CN 202010136699 A CN202010136699 A CN 202010136699A CN 111333692 A CN111333692 A CN 111333692A
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- biotin
- betulinic acid
- carboxylic acid
- glycol carboxylic
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Abstract
本发明属于生物医药技术领域,公开了一种白桦脂酸衍生物及其制备方法和应用。制备方法包括以下步骤:制备生物素酯化偶联低聚化乙二醇羧酸或生物素酰胺偶联低聚化乙二醇羧酸;将白桦脂酸溶于溶剂中,在脱水剂以及催化剂作用下,0℃搅拌反应1~6h;然后按白桦脂酸摩尔比1:1‑3倍量加入生物素或生物素酯化偶联低聚化乙二醇羧酸或生物素酰胺偶联低聚化乙二醇羧酸,从冰浴中升高温度至室温,避光搅拌过夜;过滤液浓缩,冰乙醚或异丙醇重结晶,层析或制备液相纯化,冻干即可获得白桦脂酸衍生物。该白桦脂酸衍生物及其药学上可接受的盐、同位素标记物可应用于制备治疗抗癌药物、治疗肥胖或非酒精性脂肪肝药物。
Description
技术领域
本发明属于生物医药技术领域,特别涉及一种白桦脂酸衍生物及其制备方法和应用。
背景技术
白桦脂酸(Betulinic acid,BA)是一种五环三萜类化合物,属五萜类有机酸,不溶于水,微溶于甲醇、乙醇、丙酮,易溶于四氢呋喃、吡啶,广泛存在于多种植物药材中,如中国油茶、白桦树、夏枯草、木瓜、迷迭香、夹竹桃等多种植物中,尤其是申请人项目组新发现BA在油茶饼中高含量富集存在,具有深入开发价值。具有抗肿瘤(Cancer Res.,2011;71(15):5182-93.)、抗艾滋病病毒(Med.Chem.Res.,2011;20:1247-59.;Proc Natl Acad Sci U SA.1994;91(9):3564-8.)、抗炎、免疫调节、肝保护、抗氧化应激、抗菌、抗寄生虫、抗疟疾及抗溃疡等多种活性(中草药,2014;45(14):2118-24.),而且毒性很小,是最有前途的抗癌药物前体,为治疗艾滋病、肿瘤的新物质(Nat.Med.1995;1(10):1046-51.)。同时,美国国家癌症研究所(National Cancer Institute,NCI)发现白桦脂酸可以选择性地杀伤黑色素瘤细胞,而对正常细胞无明显毒性,对癌细胞的敏感强度约是正常细胞的10倍,不易产生耐药性,不存在药物残留等优点,且具有高剂量安全性(高达500mg/kg)。后续的研究发现白桦脂酸对卵巢癌、宫颈癌、前列腺癌、乳腺癌、结肠癌、白血病等肿瘤细胞株均具有较好的抗癌活性。白桦脂酸作为一种天然产物因其新颖的抗肿瘤机制和显著的抗肿瘤活性,正日益显示出其良好的单独或与传统抗肿瘤手段、药物联用的开发前景。由于其生物利用度低、疏水性强、细胞内累积量不足,限制其成为潜在治疗药物。因此,改善这类化合物的药代动力学特性十分必要。因此,通过有机合成方法合成其天然产物类似物,进行结构优化,构建产量更高,抗肿瘤活性更好的新型白桦脂酸衍生物,并用于抗肿瘤药物的开发研究具有重要意义。
随着对桦木酸药理活性的认识不断加深,不少研究者尝试以桦木酸为母核进行结构修饰,以求提高其溶解度,增加其生物利用度,降低其毒性。对桦木酸的结构修饰主要集中在三个位置:C-3位、C-20位和C-28位;其中,对3位羟基的改造一般是以吡啶作溶剂,与各种环状二酸酐反应,合成末端带羧酸基的酯;该类型的酰基可能增强抗HIV活性,这一类型的改造还是比较成功的。提示发挥活性时关键的氢键反应可能与3β位的氧相关。其中,化合物3(DSB,YK-FH312),尤其引人注目。28位羧基是当前桦木酸的修饰热点,对其修饰得到的衍生物种类繁多。对28位羧基的结构改造以生物活性为指导,逐渐集中于将羧酸基转变成各种酰胺,而且在这些酰胺类衍生物中大多数的支链末端都保留了羧酸基。研究表明,28-COOH是影响该类化合物抗肿瘤活性的主要部位(中国药学杂志,2014;49(14):1200-3.)。对C-3位羟基和C-28位羧基的改造已取得一定进展,但C-20位的结构修饰和改造尚缺乏令人满意的结果,其它位置取代的桦木酸衍生物则罕见报道。至今为止,桦木酸母体修饰的衍生物,如RPR103611和PA457(Bevirimat)已经处于抗HIV的临床实验阶段,NVX-207在进行抗肿瘤的临床实验;但是由于其在水中的溶解性太差,导致生物利用度低及体内传输、代谢等方面的缺点。
生物素(Biotin,B)因其结构简单,分子量低,高肿瘤特异性及其生物素受体在肿瘤组织膜表面广泛高表达而被作为一种靶向配体广泛应用于各种抗癌药物的靶向递送运输。生物素又称维生素H、辅酶R,是水溶性维生素,也属于维生素B族,分子量为244.31,基本结构为双环结构:I环为咪唑酮环,是与亲和素结合的部位;II为噻吩环,含一个戊酸侧链,其末端羧基可与生物大分子连接,形成生物素标记抗原、抗体、酶等。其化学结构中有咪唑酮环,可与亲和素(avidin,AV)和链霉亲和素(streptavidin,SA)特异性结合。生物素与亲和素之间结合的亲和力高(结合常数约为1×10-15mol/L,且可视为不可逆结合)、特异性强,各自均可以与各型大小分子结合,一分子链霉亲合蛋白可与四分子生物素结合,二者“链霉亲和素-生物素”结合反应时具有多级放大作用等优越性,其相关技术被广泛应用在各种标记免疫分析技术和肿瘤靶向治疗药物领域中。为基于B构建疾病组织靶向成像和靶向药物于疾病靶向精准治疗奠定了良好的分子基础。
最为关键的是,迄今为止,尚未见生物素修饰的白桦脂酸衍生物,也未见将生物素修饰的白桦脂酸与亲和素特异结合构建成纳米药物系统及生物医药应用。
发明内容
为了克服现有技术中存在的缺点和不足,本发明的首要目的在于提供一种白桦脂酸衍生物。
本发明的再一目的在于提供一种上述白桦脂酸衍生物的制备方法。
本发明的又一目的在于提供上述白桦脂酸衍生物的应用。
本发明的又一目的在于提供一种由上述的白桦脂酸衍生物与亲和素组装成的纳米颗粒。
本发明的又一目的在于提供上述纳米颗粒的应用。
本发明的目的通过下述技术方案实现:
一种白桦脂酸衍生物,该衍生物具有如下式所示的结构:
其中n为1~10之间的自然数。
上述的一种白桦脂酸衍生物的制备方法,包括以下操作步骤:
(1)取生物素溶于溶剂中,在脱水剂N,N'-二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,以及催化剂对二甲氨基吡啶作用下,0℃搅拌反应1~6h;然后与生物素等摩尔比加入低聚化乙二醇羧酸,从冰浴中升高温度至室温,避光搅拌过夜;过滤除去副产物,滤液浓缩,冰乙醚沉淀重结晶或制备液相纯化,冻干即得生物素酯化偶联低聚化乙二醇羧酸;
或者,取生物素的活性酯溶解于无水DMSO/TEA中,加入与生物素的活性酯等摩尔的氨基化低聚化乙二醇羧酸,无水条件下反应过夜,真空冻干分离或色谱纯化,即得生物素酰胺偶联低聚化乙二醇羧酸;
所述低聚化乙二醇羧酸为HO(CH2CH2O)nCH2COOH,所述氨基低聚化乙二醇羧酸为NH2(CH2CH2O)nCH2COOH),其中n为1~10之间的自然数;
(2)将白桦脂酸溶于溶剂中,在脱水剂N,N'-二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,以及催化剂对二甲氨基吡啶作用下,0℃搅拌反应1~6h;然后按白桦脂酸摩尔比1:1-3倍量加入生物素或步骤(1)所得生物素酯化偶联低聚化乙二醇羧酸或生物素酰胺偶联低聚化乙二醇羧酸,从冰浴中升高温度至室温,避光搅拌过夜;过滤液浓缩,冰乙醚或异丙醇重结晶,层析或制备液相纯化,冻干即可获得白桦脂酸衍生物。
步骤(1)所述溶剂为CH2Cl2、DMSO或DMF;所述搅拌反应的时间为2.5h;所述室温为25℃;所述无水DMSO/TEA中DMSO和TEA的体积比为2:1;所述生物素、脱水剂和催化剂的摩尔比为1:1:1~1:30:30。
步骤(2)所述溶剂为CH2Cl2、DMSO或DMF;所述搅拌反应的时间为2.5h;所述室温为25℃;所述白桦脂酸、脱水剂、催化剂的摩尔比为1:1:1~1:25:25。
上述的一种白桦脂醇衍生物及其药学上可接受的盐、同位素标记物在制备治疗抗癌药物中的应用,所述抗癌药物包括抗宫颈癌药物、抗肝癌药物和卵巢癌药物。
上述的一种白桦脂醇衍生物在制备抗病毒药物、治疗肥胖或非酒精性脂肪肝(NAFLD)、抗肾纤维化/肾损伤炎症、抗腹泻制剂、改善睡眠药物或保健品中的应用。
一种由上述的白桦脂酸衍生物与亲和素组装成的纳米颗粒。
上述的纳米颗粒在制备治疗抗癌药物中的应用,所述抗癌药物包括抗宫颈癌药物、抗肝癌和卵巢癌药物。
上述的纳米颗粒在制备治疗肥胖或非酒精性脂肪肝(NAFLD)、抗肾纤维化/肾损伤炎症、抗病毒药物、抗腹泻制剂、改善睡眠药物或保健品中的用途。
上述药物还包括药学上可接受的载体。
所述药物可以为片剂、胶囊、粉剂、颗粒剂、口服液、丸剂、散剂、缓释制剂、溶液剂、悬浮液、注射剂、微针、软膏、乳膏或栓剂。
白桦脂醇衍生物的合成示意图如图1~3所示。
本申请所述“药学上可接受的盐”是指保留了指定化合物的游离酸和游离碱的生物效力,并且在生物学或其他方面没有不良作用的盐。本申请中的盐指用有机酸/无机酸形成的酸式盐,以及用有机碱/无机碱形成的碱式盐。
本申请所述“同位素标记物”是指由同位素标记的本申请化合物。例如本申请的化合物中的同位素包括H、C、O的各种同位素,如2H,3H,13C,14C,18O,17O。
本发明的原理是:
本发明基于生物素和其与亲和素组装纳米药物及介导疾病细胞富集原理,以生物素修饰白桦脂酸解决其成药溶解性问题,同时生物素和连接基团低聚合度的PEG衍生物(HO(CH2CH2O)nCH2COOH或NH2(CH2CH2O)nCH2COOH,其中n=1~10)具有很好的水溶性、生物相容性和无免疫源性等优点,可以获得具有疾病富集趋势作用的生物素偶联白桦脂酸药物,提高药效,增加疾病细胞选择性,降低毒副作用,更适合临床使用。
本发明人在设计和实验发明中发现通过生物素化学修饰的白桦脂酸衍生物,显著增加了疾病细胞选择性,提高了抗癌活性,大大降低了原型药物的毒性,与白桦脂酸钠盐比较(小鼠静脉注射的LD50=42.6mg/kg),部分生物素修饰白桦脂酸类药物的LD50显著增加(B-BA、B-COOCH2CH2OCH2COO-BA和B-CONHCH2CH2OCH2COO-BA纳米药物的小鼠静脉注射LD50分别为74.3、84.6和92.7mg/kg,以白桦脂酸值换算)。
2%兔红细胞溶血试验表明本发明的生物素修饰的白桦脂酸衍生物在4h内未见红细胞溶血聚集,等价白桦脂酸剂量>40mg。家兔耳缘静脉刺激性实验表明本发明的生物素修饰白桦脂酸衍生物溶解在生理盐水中静脉滴注无血管刺激性。
经过细胞毒实验MTT法测试表明,本发明衍生物对具有叶酸受体阳性表达的肿瘤细胞(宫颈癌HeLa和肝癌HepG2等)体外生长抑制作用IC50比原型药物(白桦脂酸为例)显著提高4-6倍。对正常肝脏细胞LO2毒性数据表明本发明衍生物与原型药物具有类似的高度安全性,提示生物素修饰药物具有更低的毒性和更好的肿瘤靶向性。溶解度实验表明,与原型相比(白桦脂酸不溶于水,微溶于甲醇、乙醇、丙酮),修饰后的白桦脂酸衍生物水溶性明显提高(约为2.7-20mg/ml),尤其是与亲和素组装成纳米制剂后,具有良好的水溶性,对药物剂型制备及血管给药等更加方便。
本发明相对于现有技术,具有如下的优点及有益效果:
本发明生物素修饰的白桦脂酸衍生物,以及将生物素修饰的白桦脂酸衍生物与亲和素特异结合构建成纳米药物系统,极大地解决了白桦脂酸溶解性不好、代谢快、靶向性缺乏等技术问题,具有良好的临床应用前景。
附图说明
图1为B-BA合成示意图。
图2为B-COOCH2CH2OCH2COO-BA合成示意图。
图3为B-CONHCH2CH2OCH2COO-BA合成示意图。
图4为白桦脂酸衍生物与亲和素组装成纳米颗粒的示意图。
图5为B-CONHCH2CH2OCH2COO-BA与亲和素组装成纳米颗粒的投射电镜图。
图6为白桦脂酸衍生物B-BA对裸鼠皮下移植瘤的生长和其体重影响图。
图7为白桦脂酸衍生物B-BA对高脂饮食诱导的肥胖小鼠体重的影响图。
图8为白桦脂酸衍生物B-BA对高脂小鼠肝脏组织和血清生化的影响图,其中A)为肝脏指数影响图;B)为肝脏组织和血清水平的丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)改变图;C)为肝脏组织中甘油三酯(Triglyceride,TG)和总胆固醇(Serumtotal cholesterol,TC)的差异图。
具体实施方法
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1白桦脂酸衍生物(生物素-白桦脂酸)的制备
生物素-白桦脂酸的制备:取白桦脂酸(0.15mmol)溶于60ml的DMSO中,在脱水剂DCC(3.0mmol)和催化剂DMAP(3.0mmol)作用下,0℃搅拌6h;然后按白桦脂酸摩尔比1:3倍加入生物素(B),从冰浴中升高温度至室温25℃,避光搅拌过夜。过滤液浓缩,冰乙醚或异丙醇重结晶,层析或制备液相纯化,冻干即可获得白桦脂酸衍生物(B-BA)(收率约67%),质谱鉴定离子峰[M+H]+为:683.6。
1H-NMR(400MHz,DMSO):生物素的特征峰:10.76-10.82(NH,2H,宽峰),4.47-4.65(2C-H,2H),2.80-3.10(CH2,2H),3.24(CH,1H),2.32(CH2,2H),1.23-1.68(3CH2,6H);白桦脂酸的特征峰:0.85-1.05(2CH2,6CH,10H),1.34-1.78(11CH2,CH,19H),2.03(CH,1H),4.35(CH,1H),4.88-5.06(2CH,2H),12.01(OH,1H)。
IR:红外光谱中明显增加了1726cm-1的酯羰基峰,说明白桦脂酸和生物素通过乙二醇的酯键连接上。
上述质谱数据证明本实施例所得白桦脂酸衍生物具有如下所示结构:
实施例2白桦脂酸衍生物(生物素-乙二醇-白桦脂酸)的制备
1)生物素-乙二醇羧酸的制备:取生物素(0.57g,2.3mmol)溶于40ml的DMSO中,在脱水剂(N,N'-二环己基碳二亚胺,DCC,0.47g,2.3mmol)和催化剂(对二甲氨基吡啶,DMAP,0.29g,2.3mmol)作用下,0℃搅拌4h;然后按生物素等摩尔比加入乙二醇羧酸(HOCH2CH2OCH2COOH),从冰浴中升高温度至室温25℃,避光搅拌过夜24h;过滤除副产物,滤液浓缩,冰乙醚沉淀重结晶或制备液相纯化,冻干即得生物素-乙二醇羧酸偶联产物(B-COOCH2CH2OCH2COOH)。
2)生物素-乙二醇-白桦脂酸的制备:取白桦脂酸(0.15mmol)溶于60ml的DMSO中,在脱水剂DCC(3.0mmol)和催化剂DMAP(3.0mmol)作用下,0℃搅拌6h;然后按白桦脂酸摩尔比1:3倍加入生物素-乙二醇羧酸偶联产物(B-COOCH2CH2OCH2COOH),从冰浴中升高温度至室温25℃,避光搅拌过夜。过滤液浓缩,冰乙醚或异丙醇重结晶,层析或制备液相纯化,冻干即可获得白桦脂酸衍生物(B-COOCH2CH2OCH2COO-BA)(收率约60%),质谱鉴定离子峰[M+H]+为:786.7。
1H-NMR(400MHz,DMSO):3.6和4.2左右为乙二醇羧酸上氢的峰(3CH2,6H),生物素的特征峰:10.72-10.81(NH,2H,宽峰),4.45-4.62(2CH,2H),2.80-3.13(CH2,2H),3.26(CH,1H),2.31(CH2,2H),1.25-1.69(3CH2,6H);白桦脂酸的特征峰:0.84-1.07(2CH2,6CH,10H),1.33-1.79(11CH2,CH,19H),2.05(CH,1H),4.37(CH,1H),4.89-5.07(2CH,2H),12.00(OH,1H)。IR:红外光谱中明显增加了1729cm-1的酯羰基峰,说明白桦脂酸和生物素通过乙二醇的酯键连接上。
上述质谱数据证明本实施例所得白桦脂酸衍生物具有如下所示结构:
实施例3生物素-氨基乙二醇-白桦脂酸的合成及亲和素复合纳米制备
1)生物素活性酯(B-NHS)的合成:取生物素(0.57g,2.3mmol)溶解于40ml无水DMSO中,加入0.5ml TEA三乙胺混合并于无水环境下室温避光搅拌过夜;然后混合0.47g(2.3mmol)的DCC和0.26g(2.3mmol)NHS,避光搅拌24h,过滤除去副产物二环己脲,低温真空干燥除去DMSO和TEA,得到生物素活性酯。
2)生物素-氨基乙二醇羧酸的合成:将真空干脆的生物素活性酯溶解于1.5ml的无水DMSO/TEA(体积比2:1)混合溶液中,取与生物素活性酯等摩尔的NH2-乙二醇羧酸加入混合溶液中于无水条件下反应过夜,真空干脆反应液即得生物素偶联乙二醇羧酸(B-CONHCH2CH2OCH2COOH)。
3)生物素-氨基乙二醇-白桦脂酸的合成:取白桦脂酸(0.15mmol)溶于60ml的无水DMSO中,在脱水剂DCC(3.0mmol)和催化剂DMAP(3.0mmol)作用下,0℃搅拌6h;然后按白桦脂酸摩尔比1:3倍加入生物素偶联乙二醇羧酸(B-CONHCH2CH2OCH2COOH),从冰浴中升高温度至室温25℃,避光搅拌过夜。过滤液浓缩,冰乙醚或异丙醇重结晶,层析或制备液相纯化,冻干即可获得白桦脂酸衍生物(B-CONHCH2CH2OCH2COO-BA),质谱鉴定离子峰[M+H]+为:785.3。
1HNMR(400MHz,DMSO):3.6和4.2左右为氨基乙二醇羧酸上3个-CH2-上氢的峰(3CH2,6H),8.07(NH,1H,宽峰);生物素的特征峰:10.71-10.85(NH,2H,宽峰),4.42-4.64(2CH,2H),2.81-3.14(CH2,2H),3.25(CH,1H),2.34(CH2,2H),1.27-1.71(3CH2,6H);白桦脂酸的特征峰:0.84-1.07(2CH2,6CH,10H),1.33-1.79(11CH2,CH,19H),2.04(CH,1H),4.35(CH,1H),4.88-5.08(2CH,2H),12.02(OH,1H)。
IR:红外光谱中明显增加了1500-1650cm-1的酰胺峰和1728cm-1的酯羰基峰,说明生物素和白桦脂酸通过氨基乙二醇羧酸的酰胺键和酯键连接成功。
上述质谱数据证明本实施例所得白桦脂酸衍生物具有如下所示结构:
4)生物素-氨基乙二醇-白桦脂酸亲和素纳米制剂的制备:将上述所得白桦脂酸衍生物B-CONHCH2CH2OCH2COO-BA(0.5mmol)溶于少量DMSO或乙醇中,再与亲和素或链霉亲和素按摩尔比1~4:1(如图4所示)溶于60ml的无菌生理盐水中,室温混匀,经过即可得B-CONHCH2CH2OCH2COO-BA纳米药物,经过透射电镜表征,纳米尺寸约为50nm(纳米颗粒的投射电镜图如图5所示)。
实施例4生物素化白桦脂酸注射液的制备
取等量于白桦脂酸20g的经由实施例2制备的白桦脂酸衍生物,溶解于注射用水中,加入氯化钠5.0g搅拌均匀,稀盐酸调节pH=5.0,然后加入0.5%的注射用活性炭,60℃恒温30min,脱炭后,滤液加注射用水至1000ml,0.22μm的无菌滤膜过滤,2ml/支分装于玻璃曲颈安瓿中,熔封,100℃流通蒸汽湿热灭菌40min,然后贴标签保存。
实施例5生物素化白桦脂酸亲和素纳米注射液的制备
取等量于白桦脂酸20g的经由实施例2制备的白桦脂酸衍生物,溶解于注射用水中,加入氯化钠5.0g和亲和素或链霉亲和素0.2g搅拌均匀,稀盐酸调节pH=5.0,然后加入0.5%的注射用活性炭,60℃恒温30min,脱炭后,滤液加注射用水至1000ml,0.22μm的无菌滤膜过滤,2ml/支分装于玻璃曲颈安瓿中,熔封,然后贴标签保存。
实施例6生物素化白桦脂酸亲和素纳米注射液的制备
取等量于白桦脂酸20g的经由实施例3制备的白桦脂酸衍生物,溶解于注射用水中,加入氯化钠5.0g和亲和素或链霉亲和素0.1g搅拌均匀,稀盐酸调节pH=5.0,然后加入0.5%的注射用活性炭,60℃恒温30min,脱炭后,滤液加注射用水至1000ml,0.22μm的无菌滤膜过滤,2ml/支分装于玻璃曲颈安瓿中,熔封,然后贴标签保存。
实施例7生物素化白桦脂酸冻干粉的制备
取等量于白桦脂酸20g的经由实施例3制备的白桦脂酸衍生物粉末,加入注射用葡萄糖粉末25g搅拌均匀,按40mg/瓶无菌分装于玻璃曲颈安瓿中,冷冻干燥,钴60辐射灭菌,密封然后贴标签保存。
实施例8本发明白桦脂酸衍生物的血管刺激性试验
取用注射用水50ml稀释经过微量DMSO溶解好的实施例2制备的白桦脂酸衍生物20mg,实验家兔6只,随机3组(白桦脂酸衍生物药物组、白桦脂醇组及生理盐水组),其中生理盐水作对照,给家兔左耳缘静脉滴注生物素-白桦脂酸注射液,右耳缘静脉滴注同体积生理盐水,3h内滴注完成。滴注完成后取注射点下端1cm处的血管及心、肝、脾、肺、胰腺、肾和脑组织进行固定福尔马林溶液,石蜡包埋切片,HE染色,数字病理学分析评价。
经观察给药后每日家兔的饮食、毛发、肛门、呼吸、中枢神经系统、四肢活动状态等均正常,无中毒表现。至约48h左右,处死的动物,观察直肠粘膜光滑平整,无异常;其余留存家兔逐日监测,无异常出现。到第七天处死动物,观察体重和参照《新药研究指南》对血管刺激进行分级。家兔血管刺激性试验的病理组织学检查结果为耳廓、表皮无异常,真皮血管内皮细胞无肿胀,毛细血管管壁无出血、坏死或炎性细胞浸润,软骨层、软骨细胞无增生或坏死,软骨细胞排列整齐;肝脏、心肌组织、脑组织、肺部、肾脏和胰腺组织均无异常。生理盐水对照组耳廓表皮无异常,真皮血管内皮细胞无肿胀,毛细血管壁无出血、坏死或炎性细胞浸润,软骨细胞排列整齐,无增生或坏死,软骨层、软骨细胞无增生或坏死。白桦脂酸衍生物药物组与白桦脂酸及生理盐水组在病理学组织上无明显差异,但溶解性显著变好。
实施例9细胞毒检测评价实验
BA和B及其实施例1~3制备的白桦脂酸衍生物(B-BA、B-COOCH2CH2OCH2COO-BA、B-CONHCH2CH2OCH2COO-BA)的体外抗癌效应评价。鉴于生物素与叶酸受体特异表达的疾病细胞有很强的特异性,本实施例中采用多种癌组织来源的叶酸受体阳性表达的肿瘤细胞(HeLa、HepG2和SK-OV-3)对实施例1~3制备的相应偶联产物B-BA、B-COOCH2CH2OCH2COO-BA、B-CONHCH2CH2OCH2COO-BA及其原型化合物BA和B进行药效评价,同时LO2肝脏细胞对其进行正常细胞的毒实验测试。
取对数生长期的细胞,根据细胞的大小接种2~10×103个于96孔板上,待生长24h后,弃上清,然后按以下分组给药:癌细胞设不加药组和加药组(浓度0.1~50μM对癌细胞,浓度0.5~200μM对LO2细胞),顺铂(Cisplatin)为阳性药物参照,B和BA作对照比较,对实施例1~3制备的相应偶联产物B-BA、B-COOCH2CH2OCH2COO-BA、B-CONHCH2CH2OCH2COO-BA进行细胞毒性检测。每组设4~6个复孔,培养72h后,弃上清,加入100μl含0.5mg/ml的MTT(四氮唑盐)无血清培养液培养4h,加入100μl DMSO(二甲亚砜),放置于微型振荡仪上振荡10min,再置于酶标仪上570nm处检测OD值。正常人细胞系LO2做对照。每次实验均重复3次。
结果显示,随着药物浓度增加,与相应不加药对照组比较,细胞增殖活性分别下降,说明白桦脂酸衍生物呈浓度依赖性抑制癌细胞的生长增殖。而对正常肝细胞系LO2细胞的增殖活性未有明显变化,显示出该白桦脂酸衍生物对正常细胞具有低毒特性(表1)。
表1不同细胞的IC50值(72h)及不同化合物IC50比值
实施例10本发明白桦脂酸衍生物的LD50试验
取18-22g的健康小鼠40只,10只/组,雌雄各半,实验前禁食12h,自由饮水。按等摩尔量的白桦脂酸对不同白桦脂酸衍生物组药物(实施例1制备得到的B-BA、实施例2制备得到的B-COOCH2CH2OCH2COO-BA和实施例3制备得到的B-CONHCH2CH2OCH2COO-BA纳米药物组)进行小鼠腹腔注射给药,按0.2ml/10g体重给药,溶解溶媒为阴性参照进行等体积给药,白桦脂酸钠盐组为原药对照,观察各组小鼠出现的反应症状、死亡时间并记录死亡数目数,首24h内多次观察,后续每天观察3次,连续观察12天,计算LD50的大概数值。实验结果表明,生物素偶联的白桦脂酸大大降低了原型药物的毒性,与白桦脂酸钠盐比较(小鼠腹腔注射的LD50=42.6mg/kg),部分生物素修饰白桦脂酸类化合物的LD50显著增加(B-BA、B-COOCH2CH2OCH2COO-BA和B-CONHCH2CH2OCH2COO-BA纳米药物的小鼠腹腔注射LD50分别为74.3、84.6和92.7mg/kg,以白桦脂酸值换算)。
实施例11白桦脂酸衍生物(B-BA)抗裸鼠移植瘤的体内抗肿瘤药效学评价
将实施例1制备的B-BA、B和BA先用DMSO配置成储备液,然后按需溶解在PEG200和10%乙醇的混合溶媒(1:5,v/v)中,最后用生盐水稀释配制成注射液。雌性BALB/c裸鼠(初始重量18~20g,湖南省实验动物实验中心提供),接种8×106/只裸鼠的MDA-MB-231细胞后形成实体瘤,待长成50-100mm3后分组,按50mg/kg白桦脂酸等剂量按3天/次进行腹腔注射给药6次,末次给药后继续观察6天进行裸鼠牺牲,采用游标卡尺测定瘤体尺寸,并计算瘤体大小。
研究结果表明(图6所示),于生理盐水对照组相比,B-BA和BA注射液均有统计学差异抗肿瘤效应。与BA和B组相比,注射B-BA组的抗肿瘤效应更显著(##,p<0.01),与BA治疗组比较亦具有显著性统计差异(**,p<0.01);且BA和B-BA处理组的裸鼠体重与生理盐水对照组具有显著统计差异(##,p<0.01),相对BA处理组体重而言,B-BA组裸鼠体重具有一定的统计学优异性(*,p<0.05),提示B-BA具有很高的安全性,甚至且比BA更好。
实施例12:实施例1所得白桦脂酸衍生物(B-BA)对高脂食物饮食诱导的肥胖小鼠体重的作用
将6周龄大小的健康SPF雄性C57BL/6J小鼠在SPF动物房适应性喂养一周后,随机分为三组,每组12只,三组小鼠平均初始体重基本相同,初始体重无差异。分别自由摄食普通食物(Negative control)、高脂饲料(Control;60%普通饲料粉、15%猪油、10%蛋黄粉、10%奶粉、3%蔗糖、2%胆固醇、鱼肝油1mL/kg)和高脂饲料+灌胃白桦脂酸衍生物(B-BA,250mg/kg),喂养近40天,自由饮水;每三天对小鼠体重进行称量。在给药第6天时,高脂小鼠和高脂加BE-CLA的体重开始出现差异,高脂加B-BA体重低于高脂组(*,p<0.05;);在给药12天后,高脂食物+灌胃B-BA小鼠组的体重明显低于高脂组(**,p<0.01;如图7所示)。小鼠干预实验结果表明,B-BA能够显著抑制高脂饮食小鼠的体重增长,治疗肥胖。
实施例13:实施例1所得白桦脂酸衍生物(B-BA)对高脂小鼠肝脏组织的影响
现代临床研究表明,甘油三酯是肝内酯类沉积引起非酒精性脂肪肝(NAFLD)的重要因素。在实施例12末次灌胃给药结束时,禁食不禁水12h,10%水合氯醛麻醉,眼球取血,静置30min,3500r/min离心10min,血清于-20℃低温保存,用于检测小鼠血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、甘油三酯(Triglyceride,TG)和总胆固醇(Serumtotal cholesterol,)生化指标。同时,取小鼠肝脏,分别称取湿重,取一片较大肝叶置4%福尔马林固定,HE染色,切片,显微镜观察(200×)病理变化;按肝脏湿重量/小鼠体重量=肝脏指数进行计算。同时,取小鼠肝脏组织于冰浴匀浆后,再进行肝脏测定。具体为将约30mg肝脏放入1.5mL的EP管中,加入500μL的5%的NP40,于冰浴上用研磨仪研磨制成匀浆。然后,将上清全部转移至微离心管,接着将1.5mL的EP管95℃水浴5min形成白色沉淀;室温放置10min;再95℃水浴5min,再室温放置10min;室温,最大转速13000×g,离心3min;转移上清液约100μL到新的0.5mL的EP管内,按甘油磷酸氧化酶法测TG。设置空白管三个,其中加入2μL双蒸水,设置校准管单个,其中中加入2μL校准品,样本管中加入2μL血清样本,每个酶标孔中加入125μL R1,混匀后37℃水浴5min;接着在各孔中加入62.5μL R2,混匀,37℃水浴5min。其中TG于主波长550nm,副波长660nm处比色;TC于主波长546nm,副波长660nm处比色。计算时用双蒸水校零,读取各管吸光度。按TG(mmol/L)=(样本管吸光度-空白管吸光度)/(校准管吸光度-空白管吸光度)×2.26(校准品浓度)进行计算;TC(mmol/L)=(样本管吸光度-空白管吸光度)/(校准管吸光度-空白管吸光度)×5.17(校准品浓度)。
HE染色观察表明,模型组小鼠多数肝小叶结构轮廓消失,肝细胞形态不规则,多数肿胀,内含大空泡状脂滴把核挤到一侧,甚者相间气球样变,肝窦不清晰,部分肝细胞出现点状或片状坏死,可见炎症细胞浸润;高脂食物+生物素-白桦脂酸灌胃组小鼠肝细胞大小均匀,形态正常,少数肝细胞肿胀或含脂滴,肝窦清晰,肝脏脂质侵润被显著改善。小鼠肝脏指数差异统计表明,与高脂模型小鼠相比,高脂食物+高脂食物+白桦脂酸衍生物(B-BA)灌胃组小鼠肝脏指数显著下降(图8中的A));生化酶指标检测表明,与普通高脂小鼠相比,高脂食物+白桦脂酸衍生物(B-BA)灌胃组小鼠肝脏TG和TC显著下降(图8中的C)),AST和ALT水平显著回升上调(图8中的B)),说明白桦脂酸衍生物(B-BA)可以显著降低肝脏脂质沉积(与模型组比较,**,p<0.01;);小鼠肝脏指数,血清中AST和ALT均具有统计学意义(**,p<0.01;如图8中的B)所示)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
2.根据权利要求1所述的一种白桦脂酸衍生物的制备方法,其特征在于包括以下操作步骤:
(1)取生物素溶于溶剂中,在脱水剂N,N'-二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,以及催化剂对二甲氨基吡啶作用下,0℃搅拌反应1~6h;然后与生物素等摩尔比加入低聚化乙二醇羧酸,从冰浴中升高温度至室温,避光搅拌过夜;过滤除去副产物,滤液浓缩,冰乙醚沉淀重结晶或制备液相纯化,冻干即得生物素酯化偶联低聚化乙二醇羧酸;
或者,取生物素的活性酯溶解于无水DMSO/TEA中,加入与生物素的活性酯等摩尔的氨基化低聚化乙二醇羧酸,无水条件下反应过夜,真空冻干分离或色谱纯化,即得生物素酰胺偶联低聚化乙二醇羧酸;
所述低聚化乙二醇羧酸为HO(CH2CH2O)nCH2COOH,所述氨基低聚化乙二醇羧酸为NH2(CH2CH2O)nCH2COOH),其中n为1~10之间的自然数;
(2)将白桦脂酸溶于溶剂中,在脱水剂N,N'-二环己基碳二亚胺或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,以及催化剂对二甲氨基吡啶作用下,0℃搅拌反应1~6h;然后按白桦脂酸摩尔比1:1-3倍量加入生物素或步骤(1)所得生物素酯化偶联低聚化乙二醇羧酸或生物素酰胺偶联低聚化乙二醇羧酸,从冰浴中升高温度至室温,避光搅拌过夜;过滤液浓缩,冰乙醚或异丙醇重结晶,层析或制备液相纯化,冻干即可获得白桦脂酸衍生物。
3.根据权利要求2所述的制备方法,其特征在于:步骤(1)所述溶剂为CH2Cl2、DMSO或DMF;所述搅拌反应的时间为2.5h;所述室温为25℃;所述无水DMSO/TEA中DMSO和TEA的体积比为2:1;所述生物素、脱水剂和催化剂的摩尔比为1:1:1~1:30:30。
4.根据权利要求2所述的制备方法,其特征在于:步骤(2)所述溶剂为CH2Cl2、DMSO或DMF;所述搅拌反应的时间为2.5h;所述室温为25℃;所述白桦脂酸、脱水剂、催化剂的摩尔比为1:1:1~1:25:25。
5.根据权利要求1所述的一种白桦脂醇衍生物及其药学上可接受的盐、同位素标记物在制备治疗抗癌药物中的应用,其特征在于:所述抗癌药物包括抗宫颈癌药物、抗肝癌和卵巢癌药物。
6.根据权利要求1所述的一种白桦脂醇衍生物在制备治疗肥胖或非酒精性脂肪肝药物中的应用。
7.一种由权利要求1所述的白桦脂酸衍生物与亲和素组装成的纳米颗粒。
8.根据权利要求1所述的纳米颗粒在制备治疗抗癌药物中的应用,其特征在于:所述抗癌药物包括抗宫颈癌药物、抗肝癌和卵巢癌药物。
9.根据权利要求1所述的纳米颗粒在制备治疗肥胖或非酒精性脂肪肝药物中的应用。
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