CN116059388A - 一种多肽-化疗药物偶联物、其制备方法和应用 - Google Patents
一种多肽-化疗药物偶联物、其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种多肽‑化疗药物偶联物,所述多肽‑化疗药物偶联物由多肽与修饰后的化疗药物通过共价键连接而成,其中所述多肽为针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体。还提供了其制备方法和应用。本发明的CXCR4拮抗肽‑化疗药物偶联物既保留了拮抗肽对CXCR4跨膜受体的抑制功能,又保留了化疗药物自身对肿瘤细胞的杀伤效果,同时二者在生物学机制上存在协同增效的作用。体外实验表明,多肽‑化疗药物偶联物可以自组装成胶束结构,实现对肿瘤细胞的杀伤。体内实验表明,多肽‑化疗药物偶联物明显抑制转移器官对肿瘤细胞的募集和转移灶的生长,可用于临床转移性肿瘤的预防和治疗。
Description
技术领域
本发明属于药物工程技术领域,具体涉及一种多肽-化疗药物偶联物、其制备方法和应用。
背景技术
癌症转移涉及多个连续相互关联的生理过程,包括癌细胞从原发性病灶解离、外渗、扩散、定居并在远端部位生长。对于大多数实体瘤,大部分癌症相关死亡是由转移引起的。对于某些特定类型的肿瘤,它们倾向于转移到特定器官,例如,乳腺癌转移最常见于淋巴结、肺、肝和骨,这些转移灶的器官选择性模式很大程度上归因为趋化因子及其受体的调节和对循环肿瘤细胞的招募。
化学疗法是目前致力于源发和转移性癌症的主要治疗方式之一。紫杉烷类和蒽环类药物是广泛用于乳腺癌、前列腺癌、头颈癌等多种恶性肿瘤一线治疗的化疗药物。但由于他们的药物溶解度差且具有严重的辅料相关毒性(如中性粒细胞减少),临床应用受限。此外,紫杉烷类药物在抑制了原发性肿瘤的生长的同时,实际上促进了转移,这与治疗后肿瘤细胞中CXCR4表达水平升高有关。
C-X-C趋化因子受体4型(CXCR4)与其同源配体CXCL12的结合是诱导实现转移的关键途径,CXCL12对CXCR4的激活导致许多下游信号的激活,包括丝裂原活化蛋白激酶、磷脂酰肌醇3-激酶和钙动员。一些CXCR4抑制剂,如CTCE 9908在临床前研究中显示出抗肿瘤转移的效果,并且CXCR4拮抗剂与包括紫杉烷类在内的某些化疗药物具有协同作用,具有提高其抗肿瘤效果的潜力。
目前对于化疗药物偶联,常通过缀合抗体的形式来实现靶向和增效,但是由于其相对分子量大,难以有效渗透到病灶内部,对肿瘤的治疗效果有限。此外,由于抗体制备困难,成本较昂贵,特异性偶联化学毒性药物更是提高了制备成本,同时,抗体偶联药物后可能会导致其产生免疫原性,使其易被机体清除,临床应用受到限制。因此,利用小分子或者多肽片段作为识别肿瘤细胞表面受体的分子,同时偶联具有细胞毒性的药物,从而构建类似于ADC结构的小分子药物,具有显著的临床应用价值。
发明内容
因此,本发明的目的在于克服现有技术中的缺陷,提供一种多肽-化疗药物偶联物、其制备方法和应用。本发明旨在构建一种可广泛应用于各种由CXCL12募集导致的远端器官转移治疗的多肽-化疗药物偶联物,该偶联物同时具备两种分子单独的特性,CXCR4拮抗肽抑制CXCL12诱导的肿瘤细胞迁移和侵袭,同时增强化疗敏感性,而化疗成分则有效抑制肿瘤生长。同时两亲性偶联物可以自组装成纳米颗粒,从而避免具有生理刺激性和毒性的增溶剂和辅料的参与,扩展化学杀伤药物的临床适用性和提高病人的依从性。
在阐述本发明内容之前,定义本文中所使用的术语如下:
术语“CXCR4”是指:C-X-C趋化因子受体4型。
术语“CXCL12”是指:C-X-C趋化因子受体4型的同源配体。
术语“两亲性”是指:描述一个分子既有亲水性基团又有疏水性基团的特性。
为实现上述目的,本发明的第一方面提供了一种多肽-化疗药物偶联物,所述多肽-化疗药物偶联物由多肽与化疗药物或修饰后的化疗药物通过共价键偶联而成;其中,
所述多肽为针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体,所述多肽-化疗药物偶联物具有以难溶性化疗药物为疏水内核,以针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体为外层的胶束型结构。
根据本发明第一方面的多肽-化疗药物偶联物,其中,
所述多肽-化疗药物偶联物的胶束粒径为50~300nm,更优选为60~240nm,进一步优选为80~200nm;
优选地,所述胶束型结构由具有两亲性的分子自组装形成。
根据本发明第一方面的多肽-化疗药物偶联物,其中,所述多肽的序列为KGVSLSYRK(X1-X2-X3-Xn-C)RYSLSVGK;其中,
X选自以下一种或多种:丙氨酸Ala、缬氨酸Val、亮氨酸Leu、异亮氨酸ile、脯氨酸Pro、蛋氨酸Met、色氨酸Trp、苯丙氨酸Phe、谷氨酰胺Gln、丝氨酸Ser、苏氨酸Thr、半胱氨酸Cys、天冬酰胺Asn、酪氨酸Tyr、甘氨酸Gly、天冬氨酸Asp、谷氨酸Glu、赖氨酸Lys、精氨酸Arg、组氨酸His,优选为丙氨酸Ala、缬氨酸Val、亮氨酸Leu、异亮氨酸ile、脯氨酸Pro、蛋氨酸Met、色氨酸Trp、苯丙氨酸Phe、谷氨酰胺Gln、丝氨酸Ser、苏氨酸Thr、半胱氨酸Cys、天冬酰胺Asn、酪氨酸Tyr、甘氨酸Gly、天冬氨酸Asp、谷氨酸Glu、赖氨酸Lys;
n为2~8的整数,优选为2~7的整数,更优选为2~6的整数;
其中,X1、X2至Xn相同或不同,构成一系列可对一种或一类生物环境信号响应或非响应的序列;
优选地,所述对一种或一类生物环境信号响应或非响应的序列选自以下一种或多种:豆荚蛋白酶的底物序列、基质金属蛋白酶的底物序列、柔性非响应片段;其中,所述豆荚蛋白酶的底物序列更优选为AAN;所述基质金属蛋白酶的底物序列更优选为PLGLAG或PVGLIG;所述柔性非响应片段更优选为GG或GGS;
进一步优选地,所述多肽的序列选自以下一种或多种:SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5,最优选为SEQ ID NO:1。
根据本发明第一方面的多肽-化疗药物偶联物,其中,所述化疗药物为可与CXCR4拮抗剂产生协同作用的化疗药物分子;
优选地,所述化疗药物为水不溶性药物;
更优选地,所述化疗药物的分子结构中存在一个或多个活性修饰位点,可被交联剂修饰,且修饰后的结构仍具有抗肿瘤效果;
进一步优选地,所述交联剂为3-马来酰亚胺丙酸或3-马来酰亚胺基苯甲酸琥珀酰亚胺酯,最优选为3-马来酰亚胺丙酸。
根据本发明第一方面的多肽-化疗药物偶联物,其中,
所述化疗药物选自:紫杉烷类化疗药物和/或蒽环类化疗药物,优选为多西他赛、紫杉醇、阿霉素,更优选为多西他赛或紫杉醇;最优选为多西他赛;和/或
所述化疗药物的每个药物分子连接1条针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体。
本发明的第二方面提供了制备第一方面所述的多肽-化疗药物偶联物的方法,所述多肽-化疗药物偶联物由亲水性拮抗肽及其变体侧链半胱氨酸残基上的巯基与化疗药物分子上的活性羟基或氨基通过交联剂共价键连接而成。
根据本发明第二方面所述的方法,其中,所述多肽-化疗药物偶联物的制备方法包括以下步骤:
(1)化疗药物与交联剂在活化剂的参与下搅拌反应,形成交联剂修饰的化疗药物;
(2)将步骤(1)制备的交联剂修饰的化疗药物与多肽在混合溶液中搅拌,即得所述多肽-化疗药物偶联物;
优选地,所有反应均在氮气保护下进行。
根据本发明第二方面所述的方法,其中,所述步骤(1)中还包括:将生成的交联剂修饰的化疗药物通过制备型薄层色谱或液相色谱除去未反应的交联剂;
优选地,所述化疗药物与所述交联剂的摩尔比为0.020~0.5:1,更优选为0.025~0.5:1,进一步优选为0.030~0.5:1;
优选地,所述活化剂为二甲氨基吡啶或碳二亚胺盐酸盐;
优选地,所述搅拌反应的温度为4~8℃,更优选为4~6℃,最优选为4℃;和/或
优选地,所述搅拌反应的时间为8~12h,更优选为10~12h,最优选为12h。
根据本发明第二方面所述的方法,其中,所述步骤(2)中还包括:将制备的多肽-化疗药物偶联物用超滤管除去未反应的交联剂修饰的化疗药物;
优选地,所述超滤管为截留分子量1KDa的超滤管;
优选地,所述多肽与所述交联剂修饰的化疗药物的摩尔比为0.1~1:1,更优选为0.2~0.8:1,进一步优选为0.3~0.6:1;
优选地,所述混合溶液选自以下一种或多种:水、乙醇、乙腈、二甲基亚砜、N,N-二甲基甲酰胺;
优选地,所述搅拌的温度为20~30℃,更优选为22~28℃,进一步优选为24~26℃;和/或
优选地,所述搅拌的时间为12~36h,更优选为12~24h,最优选为24h。
本发明的第三方面提供了第一方面所述的多肽-化疗药物偶联物或按照第二方面所述的方法制备的多肽-化疗药物偶联物在制备用于治疗癌症的药物中的应用;
优选地,所述癌症选自以下一种或多种:乳腺癌、前列腺癌、头颈部癌、胃癌、肝癌、肺癌及他们的转移病灶;
优选地,所述癌症包括原位病灶和/或转移病灶。
本发明的多肽序列如SEQ ID NO.1所示:
SEQ ID NO:1:KGVSLSYRK(GGC)RYSLSVGK(由于多肽列表计算机可读载体无法识别(),SEQ ID NO.1以说明书此处记载的序列为准)。
SEQ ID NO:2:KGVSLSYRK(AANC)RYSLSVGK(由于多肽列表计算机可读载体无法识别(),SEQ ID NO.2以说明书此处记载的序列为准)。
SEQ ID NO:3:KGVSLSYRK(PLGLAGC)RYSLSVGK(由于多肽列表计算机可读载体无法识别(),SEQ ID NO.3以说明书此处记载的序列为准)。
SEQ ID NO:4:KGVSLSYRK(PVGLIGC)RYSLSVGK(由于多肽列表计算机可读载体无法识别(),SEQ ID NO.4以说明书此处记载的序列为准)。
SEQ ID NO:5:KGVSLSYRK(GGSC)RYSLSVGK(由于多肽列表计算机可读载体无法识别(),SEQ ID NO.5以说明书此处记载的序列为准)。
根据一个具体的实施例,本发明还提供了一种多肽-化疗药物偶联物的制备方法,该方法步骤简单,可重复性强,可用于工业化大量生产。
本发明还提供了一种多肽-化疗药物偶联物在制备抗癌药物中的应用,去除了商业化制剂中具有刺激性和毒性的辅料的参与,显著降低了药物在体内外水平对机体产生的毒副作用。
为了实现本发明目的,本发明的多肽-化疗药物偶联物,所述偶联物由多肽与修饰后的化疗药物通过共价键连接而成,其中所述多肽为针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体。
所述多肽的序列如下:KGVSLSYRK(X1-X2-X3-Xn-C)RYSLSVGK,其中X1、X2至Xn相同或不同,构成一系列可对一种或一类生物环境信号响应的序列,也可为非响应性序列,n为2-6的自然数。
本发明的偶联物由亲水性拮抗肽及其变体与药物通过3-马来酰亚胺丙酸共价键连接而成,即所述拮抗肽侧链半胱氨酸残基上的巯基与药物分子上的活性羟基或氨基通过3-马来酰亚胺丙酸共价交联而成。
本发明所述化疗药物为可与CXCR4拮抗剂产生协同作用的化疗药物分子,为水不溶性药物,分子结构中具有一个或多个活性修饰位点,可被3-马来酰亚胺丙酸修饰,且修饰后的结构仍具有抗肿瘤效果,包括紫杉醇、多西他赛等紫杉烷类化疗药物以及阿霉素等蒽环类药物。
所述多肽-化疗药物偶联物的多肽-化疗药物偶联物自组装形成的胶束结构不需要高分子或脂质及表面活性剂材料作为载体。
本发明的多肽-化疗药物偶联物,可按照如下方法制得,包括步骤:
S1、化疗药物与交联剂3-马来酰亚胺丙酸在二氯甲烷溶液中,在活化剂的参与下,-15℃搅拌反应8-12h(优选12h),形成马来酰亚胺修饰的化疗药物;
S2、将步骤S1所得产物与所述多肽在水、乙醇、乙腈的混合溶液中室温搅拌12-36h(优选24h),即得所述多肽-化疗药物偶联物。
其中,化疗药物和交联剂的摩尔比为0.025-0.5:1,多肽与马来酰亚胺修饰的化疗药物的摩尔比为0.1-1:1。
前述的方法,步骤S1还包括将生成的马来酰亚胺修饰的化疗药物通过制备型薄层色谱或液相色谱除去未反应的交联剂。
前述的方法,步骤S2还包括将生成的多肽-化疗药物偶联物用截留分子量1KDa的Amicon ultra超滤管除去未反应的马来酰亚胺修饰的化疗药物的步骤,所得产物通过质谱确定。
本发明多肽-化疗药物偶联物的制备方法中,反应所使用的缓冲液均通氮气充分排除氧气,所有反应均在氮气保护下进行。
本发明的目的是提供一种广泛应用于肿瘤转移灶的可产生协同作用的多肽-化疗药物偶联物,提高了抗肿瘤药物在水中的溶解性,扩大药物的临床应用范围,并提高其对肿瘤转移灶的抑制和杀伤效果。
本发明的多肽-化疗药物偶联物可以具有但不限于以下有益效果:
1、本发明成功解决了提高疏水性化疗药物溶解度的问题,同时无需典型的有毒辅料的掺杂,降低对机体的毒副作用。
2、本发明多肽-化疗药物偶联物的合成方法简单,可重复性强,可用于工业化大量生产。
3、该偶联体系提高了肿瘤细胞对化疗药物的化学敏感性同时可以靶向和治疗CXCL12诱导的转移。
4.本发明的CXCR4拮抗肽-化疗药物偶联物既保留了拮抗肽对CXCR4跨膜受体的拮抗功能,又保留了化疗药物自身对肿瘤细胞的杀伤效果,同时二者在生物学机制上存在协同增效的作用。偶联物在水中具有良好的溶解性,可直接静脉注射或加工成其他剂型。体外实验表明,多肽-化疗药物偶联物可以自组装成胶束结构,实现对肿瘤细胞的靶向和杀伤。体内实验表明,多肽-化疗药物偶联物明显抑制转移器官对肿瘤细胞的募集和转移灶的生长,可用于临床转移性肿瘤的预防和治疗。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1示出了本发明实施例1中CXCR4拮抗肽-多西他赛偶联物的合成过程。
图2示出了本发明实施例2中通过透射电镜观察CXCR4拮抗肽-多西他赛偶联物胶束的形貌图;其中,白色为标尺,标尺的长度代表200nm。
图3示出了本发明实施例1中CXCR4拮抗肽-多西他赛偶联物的质谱表征。
图4示出了本发明实施例3中CXCR4拮抗肽-多西他赛偶联物对细胞毒性的评价结果。
图5示出了本发明实施例4中CXCR4拮抗肽-多西他赛偶联物对小鼠三阴性乳腺癌细胞系4T1骨转移灶的抑制效果。
具体实施方式
下面通过具体的实施例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细具体地说明之用,而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚,在上下文中,如果未特别说明,本发明所用材料和操作方法是本领域公知的。
以下实施例中使用的试剂和仪器如下:
试剂:
CCK-8试剂盒,购自日本同仁化学公司。
PBS,购自北京索莱宝有限责任公司。
二氯甲烷、3-马来酰亚胺丙酸、二甲氨基吡啶、碳二亚胺盐酸盐、多西他赛,均购自上海毕得药业有限责任公司。
仪器:
IVIS成像,购自珀金埃尔默公司公司、型号IVIS Spectrum。
透射电镜,购自日立公司、型号HT7700。
实施例1
本实施例用来说明本发明的多肽-化疗药物偶联物的制备方法。
本实施例中CXCR4拮抗肽的序列为SEQ ID NO:1:KGVSLSYRK(GGC)RYSLSVGK,同时选择多西他赛作为模型化疗药物进行实施例1-4的制备和验证。
图1示出了本发明实施例1中CXCR4拮抗肽-多西他赛偶联物的合成过程。图3示出了本发明实施例1中CXCR4拮抗肽-多西他赛偶联物的质谱表征。
(1)3-马来酰亚胺丙酸对多西他赛的共价修饰
在室温25摄氏度下搅拌,向二氯甲烷中加入多西他赛(2.0g,2.5mmol),然后向溶液中加入3-马来酰亚胺基丙酸(628mg,3.7mmol),并搅拌混合物15分钟。将烧瓶冷却至4℃,然后将两倍摩尔量的二甲氨基吡啶和碳二亚胺盐酸盐加入反应混合物中作为活化剂,在4℃搅拌12小时(通过薄层色谱监测),反应瓶充氮气保护。反应混合物用碳酸钠溶液和水洗涤3次,有机相用无水硫酸钠干燥并蒸发至干。粗产物通过制备型TLC纯化,得到马来酰亚胺修饰的多西他赛,为白色固体。
(2)将步骤(1)所得产物取2mg(0.002mmol)溶解在无水乙醇与乙腈(1:1,v/v)的混合体系中,另取5mg多肽(0.002mmol)溶于水相,室温氮气保护搅拌反应24h,将生成的多肽-化疗药物偶联物用截留分子量1KDa的Amicon ultra超滤管除去未反应的马来酰亚胺修饰的多西他赛,所得产物通过质谱确定,为本发明的多肽-化疗药物偶联物:CXCR4拮抗肽-多西他赛偶联物单体,结果见图3。
实施例2
本实施例用来说明本发明的多肽-化疗药物偶联物的形貌。
取实施例1制备的CXCR4拮抗肽-多西他赛偶联物单体,使用三次水溶解,超声15min,通过醋酸双氧铀进行染色,在透射电镜下观测制备得到的胶束结构,如图2所示。
图2示出了本发明实施例2中通过透射电镜观察CXCR4拮抗肽-多西他赛偶联物胶束的形貌图;其中,标尺为200nm。
实施例3
本实施例用来说明本发明的多肽-化疗药物偶联物对细胞毒性的评价结果。
本实施例使用的是实施例1制备的多肽-化疗药物偶联物。
本实施例使用CCK-8试剂盒进行细胞活力分析,将4T1细胞接种到96孔板中并培养至50%融合度,然后用含有不同浓度梯度的药物(0,5,25,50,100,200,400,800nM)的新鲜培养基处理细胞72小时,最后用含有10%CCK-8试剂的新鲜培养基培养1h,在450nm波长处测定吸光度,计算细胞活力,见图4。
图4示出了本发明实施例3中CXCR4拮抗肽-多西他赛偶联物对细胞毒性的评价结果。结果表明,与纯多西他赛相比,CXCR4拮抗肽-多西他赛显著提高了对肿瘤细胞的毒性作用。
实施例4
本实施例用来说明本发明的多肽-化疗药物偶联物对小鼠骨转移灶的治疗效果。
本实施例使用的是实施例1制备的多肽-化疗药物偶联物。
本实施例用体重为16-18g的雌性Balb/c小鼠,随机分为四组,将与不同药物混合的1×103个4T1-luci细胞悬浮在100ul PBS中,并在短时间内(<3s)用注入尾动脉。一周后,每三天通过IVIS成像(Perkin Elmer,USA)监测小鼠,并在第3、6、9、12、15、18天给予常规给药,肿瘤组织的生长情况通过检测腿骨部位的生物发光信号进行相对定量。实验结果见图5。
图5示出了本发明实施例4中CXCR4拮抗肽-多西他赛偶联物对小鼠三阴性乳腺癌细胞系4T1骨转移灶的抑制效果。由图5可知,深色区域为肿瘤的荧光信号,肿瘤组织越大则区域越大,可知,CXCR4拮抗肽-多西他赛偶联物对于小鼠肿瘤骨转移灶具有显著的抑制和杀伤效果。
尽管本发明已进行了一定程度的描述,明显地,在不脱离本发明的精神和范围的条件下,可进行各个条件的适当变化。可以理解,本发明不限于所述实施方案,而归于权利要求的范围,其包括所述每个因素的等同替换。
Claims (10)
1.一种多肽-化疗药物偶联物,其特征在于,所述多肽-化疗药物偶联物由多肽与化疗药物或修饰后的化疗药物通过共价键偶联而成;其中,
所述多肽为针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体,所述多肽-化疗药物偶联物具有以难溶性化疗药物为疏水内核,以针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体为外层的胶束型结构。
2.根据权利要求1所述的多肽-化疗药物偶联物,其特征在于:
所述多肽-化疗药物偶联物的胶束粒径为50~300nm,更优选为60~240nm,进一步优选为80~200nm;
优选地,所述胶束型结构由具有两亲性的分子自组装形成。
3.根据权利要求1或2所述的多肽-化疗药物偶联物,其特征在于:所述多肽的序列为KGVSLSYRK(X1-X2-X3-Xn-C)RYSLSVGK;其中,
X选自以下一种或多种:丙氨酸Ala、缬氨酸Val、亮氨酸Leu、异亮氨酸ile、脯氨酸Pro、蛋氨酸Met、色氨酸Trp、苯丙氨酸Phe、谷氨酰胺Gln、丝氨酸Ser、苏氨酸Thr、半胱氨酸Cys、天冬酰胺Asn、酪氨酸Tyr、甘氨酸Gly、天冬氨酸Asp、谷氨酸Glu、赖氨酸Lys、精氨酸Arg、组氨酸His,优选为丙氨酸Ala、缬氨酸Val、亮氨酸Leu、异亮氨酸ile、脯氨酸Pro、蛋氨酸Met、色氨酸Trp、苯丙氨酸Phe、谷氨酰胺Gln、丝氨酸Ser、苏氨酸Thr、半胱氨酸Cys、天冬酰胺Asn、酪氨酸Tyr、甘氨酸Gly、天冬氨酸Asp、谷氨酸Glu、赖氨酸Lys;
n为2~8的整数,优选为2~7的整数,更优选为2~6的整数;
其中,X1、X2至Xn相同或不同,构成一系列可对一种或一类生物环境信号响应或非响应的序列;
优选地,所述对一种或一类生物环境信号响应或非响应的序列选自以下一种或多种:豆荚蛋白酶的底物序列、基质金属蛋白酶的底物序列、柔性非响应片段;其中,所述豆荚蛋白酶的底物序列更优选为AAN;所述基质金属蛋白酶的底物序列更优选为PLGLAG或PVGLIG;所述柔性非响应片段更优选为GG或GGS;
进一步优选地,所述多肽的序列选自以下一种或多种:SEQ ID NO:1、SEQ ID NO:2、SEQID NO:3、SEQ ID NO:4、SEQ ID NO:5,最优选为SEQ ID NO:1。
4.根据权利要求1至3中所述的多肽-化疗药物偶联物,其特征在于:所述化疗药物为可与CXCR4拮抗剂产生协同作用的化疗药物分子;
优选地,所述化疗药物为水不溶性药物;
更优选地,所述化疗药物的分子结构中存在一个或多个活性修饰位点,可被交联剂修饰,且修饰后的结构仍具有抗肿瘤效果;
进一步优选地,所述交联剂为3-马来酰亚胺丙酸或3-马来酰亚胺基苯甲酸琥珀酰亚胺酯,最优选为3-马来酰亚胺丙酸。
5.根据权利要求1至4中任一项所述的多肽-化疗药物偶联物,其特征在于:
所述化疗药物选自:紫杉烷类化疗药物和/或蒽环类化疗药物,优选为多西他赛、紫杉醇、阿霉素,更优选为多西他赛或紫杉醇;最优选为多西他赛;和/或
所述化疗药物的每个药物分子连接1条针对肿瘤细胞CXCR4跨膜受体的亲水性拮抗肽及其变体。
6.制备权利要求1至5中任一项所述的多肽-化疗药物偶联物的方法,其特征在于:所述多肽-化疗药物偶联物由亲水性拮抗肽及其变体侧链半胱氨酸残基上的巯基与化疗药物分子上的活性羟基或氨基通过交联剂共价键连接而成。
7.根据权利要求6所述的方法,其特征在于,所述多肽-化疗药物偶联物的制备方法包括以下步骤:
(1)化疗药物与交联剂在活化剂的参与下搅拌反应,形成交联剂修饰的化疗药物;
(2)将步骤(1)制备的交联剂修饰的化疗药物与多肽在混合溶液中搅拌,即得所述多肽-化疗药物偶联物;
优选地,所有反应均在氮气保护下进行。
8.根据权利要求6或7所述的方法,其特征在于,所述步骤(1)中还包括:将生成的交联剂修饰的化疗药物通过制备型薄层色谱或液相色谱除去未反应的交联剂;
优选地,所述化疗药物与所述交联剂的摩尔比为0.020~0.5:1,更优选为0.025~0.5:1,进一步优选为0.030~0.5:1;
优选地,所述活化剂选为二甲氨基吡啶或碳二亚胺盐酸盐;
优选地,所述搅拌反应的温度为4~8℃,更优选为4~6℃,最优选为4℃;和/或
优选地,所述搅拌反应的时间为8~12h,更优选为10~12h,最优选为12h。
9.根据权利要求6至8中任一项所述的方法,其特征在于,所述步骤(2)中还包括:将制备的多肽-化疗药物偶联物用超滤管除去未反应的交联剂修饰的化疗药物;
优选地,所述超滤管为截留分子量1KDa的超滤管;
优选地,所述多肽与所述交联剂修饰的化疗药物的摩尔比为0.1~1:1,更优选为0.2~0.8:1,进一步优选为0.3~0.6:1;
优选地,所述混合溶液选自以下一种或多种:水、乙醇、乙腈、二甲基亚砜、N,N-二甲基甲酰胺;
优选地,所述搅拌的温度为20~30℃,更优选为22~28℃,进一步优选为24~26℃;和/或
优选地,所述搅拌的时间为12~36h,更优选为12~24h,最优选为24h。
10.权利要求1至5中任一项所述的多肽-化疗药物偶联物或按照权利要求6至9中任一项所述的方法制备的多肽-化疗药物偶联物在制备用于治疗癌症的药物中的应用;
优选地,所述癌症选自以下一种或多种:乳腺癌、前列腺癌、头颈部癌、胃癌、肝癌、肺癌;
优选地,所述癌症包括原位病灶和/或转移病灶。
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