CN117503950A - 一种聚氨基酸肿瘤选择性前药、其制备方法和应用 - Google Patents
一种聚氨基酸肿瘤选择性前药、其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种聚氨基酸肿瘤选择性前药,具有式(I)所示结构。上述基于聚氨基酸的“凝血靶向”乏氧激活IMDQ前药。该前药利用高分子血管阻断剂治疗选择性地破坏肿瘤血管,使肿瘤出血‑凝血,阻断肿瘤养分、氧气供给,从而放大肿瘤组织与正常组织之间凝血和乏氧的差异;“凝血靶向”乏氧激活IMDQ前药对凝血靶向、乏氧响应,使IMDQ的瘤内分布具有主动肿瘤靶向与肿瘤选择性激活的双重效果。因此,本专利发明的“凝血靶向”乏氧激活IMDQ前药可极大的提高IMDQ的肿瘤选择性,并显著优于现有技术。
Description
技术领域
本发明涉及材料技术领域,尤其是涉及一种聚氨基酸肿瘤选择性前药、其制备方法和应用。
背景技术
近年来,癌症已经成为影响人类健康的第一杀手,严重威胁人类生命健康。癌症免疫疗法利用宿主免疫系统来识别和杀死原发性和转移性病变中的癌细胞,已成为最有前景的癌症治疗手段。尽管免疫检查点阻断(ICB)疗法和嵌合抗原受体T(CAR-T)细胞疗法在临床实践中取得成功,但只有一小部分癌症患者呈现阳性免疫反应,这可能归因于肿瘤处于免疫原性差、树突状细胞(DCs)的低效成熟、细胞毒性T淋巴细胞的活化和浸润差的免疫抑制微环境。进一步扩大免疫治疗领域的一种有希望的策略是通过激活巨噬细胞(macrophages)、树突状细胞(dendritic cells,DCs)、自然杀伤细胞(natural killercell,NK)和B淋巴细胞等免疫细胞上的Toll样受体(TLRs)信号,抑制促进肿瘤生长的免疫信号传导来调节肿瘤免疫微环境。
咪唑喹啉类(Imidazoquinolines,IMDQs)是一类合成的TLR7/8的激动剂,可通过刺激TLR7/8诱导抗原呈递细胞(如DCs)的成熟、促进细胞因子的释放,特别是可激活辅助性T细胞1(Th1)和细胞毒性T细胞,在对抗癌症方面发挥着关键作用。小分子IMDQs在系统给药后会引起全身炎性因子风暴,危及患者安全,限制了其临床尽可局部使用;对于肿瘤细胞已发生转移的患者无效,而90%以上的癌症患者都死于肿瘤转移。因此,有必要开发可系统给药且对肿瘤部位具有高选择性的IMDQs纳米药物,实现IMDQs在肿瘤部位的有效富集,提高治疗效果的同时减少因药物脱靶而带来的毒副作用。
现有的能降低IMDQs药物的全身毒性的策略主要有:1)肿瘤选择性药物激活策略,根据肿瘤组织与正常组织之间存在的酶、pH、谷胱甘肽、活性氧、氧分压(乏氧)差异,构建肿瘤微环境响应抗肿瘤前药,实现药物在肿瘤部位选择性激活;2)主动肿瘤靶向策略,通过在载体表面或药物上修饰靶头,它可与肿瘤细胞表面或肿瘤微环境高表达受体特异性结合,从而实现药物在肿瘤部位的主动富集。肿瘤选择性药物激活策略和主动靶向药物输送策略都受到肿瘤组织与正常组织之间差异性不足的限制,迄今为止,除了抗体药物偶联物(ADC药物)外,还没有采用这两种策略药物通过临床试验研究并上市。因此,开展新型高效的肿瘤选择性IMDQ前药具有重要意义。
发明内容
有鉴于此,本发明要解决的技术问题在于提供一种聚氨基酸肿瘤选择性前药,本发明提供的聚氨基酸肿瘤选择性前药可以有效地富集在肿瘤部位,在肿瘤部位选择性释放。该类前药和血管阻断剂联用可以有效地提高药物在肿瘤的分布浓度,在显著抑制肿瘤生长和转移的同时减少了药物脱靶带来的全身炎性反应副作用。
本发明提供了一种聚氨基酸肿瘤选择性前药,具有式(I)所示结构:
其中,R1选自C2-C10的直链烷基、C3-C10的支链烷基或C6~C20的芳基;
R2为肿瘤响应的咪唑喹啉类药物;
R3为H或阳离子;
R4为凝血靶向多肽;
L1、L2、L3选自-CH2-或-CH2CH2-;
L4选自-CH2O-、-CH2CH2O-或-CH2NH-、-CH2CH2NH-;
x,y,z为重复单元含量,0<x<1,0<y<1,0<z<1;n为聚合度,10≤n≤500。
优选的,所述R1为选自C2~C10的直链烷基、C3~C10的支链烷基或C6~C20的芳基;R3独立的或者组合的选自H、金属阳离子或有机阳离子。
优选的,所述R1为乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、正己基、正庚基、正辛基、苯基、萘基、联苯基或蒽;R1为H、钠离子、钾离子、铵离子或者带正电荷的氨基酸离子。
优选的,所述R2独立的或者组合的选自如下结构:
优选的,所述R4独立的或者组合的选自如下结构;
本发明提供了一种聚氨基酸肿瘤选择性前药的制备方法,包括如下步骤:
A)聚氨基酸、式(II)所示的Toll样受体激动剂叠氮前药、式(Ⅲ)所示的含马来酰亚胺基团的小分子进行缩合反应;
B)反应后,采用调节pH值后冻干,得到冻干样品;将冻干样品溶解后与凝血靶向肽反应,即得;
优选的,所述
步骤A)所述反应溶剂为二甲基甲酰胺、二甲基亚砜;所述Toll样受体激动剂叠氮前药的反应原料质量占聚氨基酸的质量比为1%~30%;
马来酰亚胺基团与聚氨基酸的摩尔比为(1~10):1;
反应温度为20℃~60℃;反应时间为24~72h;缩合反应的缩合剂为2,4,6-三氯苯甲酰氯,N,N-二异丙基碳二亚胺,二环己基碳二亚胺,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;
步骤B)所述凝血靶向肽的溶剂为水;反应温度为30℃~40℃;反应时间为4h~48h;凝血靶向肽与聚氨基酸的摩尔比为(1~10):1。
本发明提供了一种抗肿瘤组合物,包括上述技术方案任意一项所述的聚氨基酸肿瘤选择性前药和血管阻断剂。
本发明提供了上述技术方案所述的聚氨基酸肿瘤选择性前药在制备治疗癌症的药物中的应用。
优选的,所述癌症包括鼻腔及鼻窦恶性肿瘤、鼻咽癌、口腔癌、喉癌、颅内肿瘤、甲状腺癌、舌癌、肺癌、食管癌、乳腺癌、胃癌、大肠癌、乙状结肠和直肠癌、肝癌、胰腺癌与壶腹周围癌、胆道癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、子宫颈癌、子宫内膜癌、卵巢癌、纤维组织细胞癌、横纹肌肉癌、滑膜肉瘤、黑色素瘤、骨肉瘤、尤文氏肉瘤、白血病、淋巴瘤和多发性骨髓瘤中的一种或几种。
与现有技术相比,本发明提供了一种聚氨基酸肿瘤选择性前药,具有式(I)所示结构:其中,R1选自C2-C10的直链烷基、C3-C10的支链烷基或C6~C20的芳基;R2为肿瘤响应的咪唑喹啉类药物;R3为H或阳离子;R4为具有凝血靶向多肽;L1、L2、L3选自-CH2-或-CH2CH2-;L4选自-CH2O-、-CH2CH2O-或-CH2NH-、-CH2CH2NH-;x,y,z为重复单元含量,0<x<1,0<y<1,0<z<1;n为聚合度,10≤n≤500。上述基于聚氨基酸的“凝血靶向”乏氧激活IMDQ前药。该前药利用高分子血管阻断剂治疗选择性地破坏肿瘤血管,使肿瘤出血-凝血,阻断肿瘤养分、氧气供给,从而放大肿瘤组织与正常组织之间凝血和乏氧的差异;“凝血靶向”乏氧激活IMDQ前药对凝血靶向、乏氧响应,使IMDQ的瘤内分布具有主动肿瘤靶向与肿瘤选择性作用的双重效果。因此,本专利发明的“凝血靶向”乏氧激活IMDQ前药可极大的提高IMDQ的肿瘤选择性,并显著优于现有技术。
附图说明
图1为实施例1所述的肿瘤响应的咪唑喹啉类叠氮前药的合成与表征;其中,a为IMDQ-N3的制备示意图,b为核磁共振氢谱(1HNMR)图,c为质谱(ESI)表征图;
图2为实施例1所述的聚氨基酸肿瘤选择性前药的合成表征;其中,a为凝血靶向乏氧激活IMDQ纳米前药Apcitide-PLG-IMDQ-N3的制备;b为通过1HNMR对结构进行表征结果图;c为x射线光电子能谱,d为Apcitide-PLG-IMDQ-N3的粒径的表征结果图,e为酯键稳定性的表征结果图;
图3为实施例3聚氨基酸肿瘤选择性前药体外乏氧激活评估;其中a为HPLC分析在酶的作用下纳米前药Apcitide-PLG-IMDQ-N3的叠氮还原为氨基情况;b为HPLC分析纳米前药在4T1细胞水平叠氮还原为氨基的情况;c为纳米前药在体外乏氧激活DC细胞流式结果分析;d为纳米前药在体外乏氧条件下促进M1型巨噬细胞表型流式结果分析;
图4为实施例4聚氨基酸肿瘤选择性前药体内毒性评估;其中a为纳米原药的MTD实验小鼠体重变化图;b为纳米原药的MTD实验小鼠生存率图;c纳米前药的MTD实验小鼠体重变化图d纳米前药的MTD实验小鼠生存率图;e为纳米前药有效降低小鼠体内IL-6炎性因子的结果图;f为纳米前药有效降低小鼠体内IFN-γ炎性因子的结果图;
图5为实施例5聚氨基酸肿瘤选择性前药在体内的组织分布;其中a为血管阻断剂CA4-NPs作用后不同时间瘤内血小板上GPIIb-IIIa糖蛋白的表达水平分析;b为给药后小鼠瘤内IMDQ-N3的含量;c为给药后小鼠瘤内IMDQ和IMDQ-N3的总含量d为给药后小鼠瘤内有效药物IMDQ的含量;
图6为实施例6聚氨基酸肿瘤选择性前药小鼠4T1肿瘤模型抑瘤评估;其中a为抑瘤实验方案;b为小鼠的肿瘤体积变化图;c抑瘤结束后小鼠的肿瘤组织照片图;d小鼠的体重变化图;e小鼠的生存率图;
图7为实施例7聚氨基酸肿瘤选择性前药小鼠体内肿瘤免疫激活评估;其中a为瘤内活化的DC细胞含量图;b为瘤内CD8+T细胞含量图c瘤内CD4+T细胞含量图d瘤内MDSC细胞含量图e瘤内M1型巨噬细胞含量图f脾内CD8+T细胞含量图g脾内CD4+T细胞含量图;
图8为聚氨基酸肿瘤选择性前药抑制4T1肿瘤转移能力评估;其中a为小鼠肿瘤向肺转移照片b为小鼠的主要脏器HE染色图。
具体实施方式
本发明提供了一种聚氨基酸肿瘤选择性前药、其制备方法和应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都属于本发明保护的范围。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明提供了一种聚氨基酸肿瘤选择性前药,具有式(I)所示结构:
其中,R1选自C2-C10的直链烷基、C3-C10的支链烷基或C6~C20的芳基;优选的,所述R1为乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、正己基、正庚基、正辛基、苯基、萘基、联苯基或蒽;更优选的,R1为H、钠离子、钾离子、铵离子或者带正电荷的氨基酸离子。
R2为肿瘤响应的咪唑喹啉类药物;
优选的,所述R2独立的或者组合的选自如下结构:
R3为H或阳离子;优选的,R3独立的或者组合的选自H、金属阳离子或有机阳离子。
R4为具有凝血靶向多肽;
优选的,所述R4独立的或者组合的选自如下结构;
L1、L2、L3选自-CH2-或-CH2CH2-;
L4选自-CH2O-、-CH2CH2O-或-CH2NH-、-CH2CH2NH-;
x,y,z为重复单元含量,0<x<1,0<y<1,0<z<1;,优选的,0<x<0.3,0<y<1,0<z<0.3;n为聚合度,10≤n≤500;优选的,50≤n≤450。
本发明一种聚氨基酸肿瘤选择性前药具体结构可以为
本发明提供了一种聚氨基酸肿瘤选择性前药的制备方法,包括如下步骤:
A)式(II)所示的聚氨基酸、式(II)所示的Toll样受体激动剂叠氮前药、式(Ⅲ)所示的含马来酰亚胺基团的小分子进行缩合反应;
B)反应后,采用调节pH值后冻干,得到冻干样品;将冻干样品溶解后与凝血靶向肽反应,即得;
本发明提供的一种聚氨基酸肿瘤选择性前药的制备方法首先将聚氨基酸、式(III)所示的Toll样受体激动剂叠氮前药、式(IV)所示的含马来酰亚胺基团的小分子进行缩合反应。
本发明将聚氨基酸与Toll样受体7和8(TLR7/TLR8)的激动剂叠氮前药、含马来酰亚胺基团的小分子进行缩合反应;其中,聚氨基酸键合Toll样受体7和8(TLR7/TLR8)的激动剂叠氮前药、含马来酰亚胺基团的小分子可一锅反应,或分两次反应;溶剂优选二甲基甲酰胺、二甲基亚砜;Toll样受体7和8(TLR7/TLR8)的激动剂叠氮前药的反应原料质量占聚氨基酸的投料质量比优选为1%~30%;马来酰亚胺基团与聚氨基酸的摩尔比优选为(1~10):1,更优选为3:1;反应温度优选为20℃~60℃;缩合剂优选为2,4,6-三氯苯甲酰氯,N,N-二异丙基碳二亚胺,二环己基碳二亚胺,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,反应时间优选为24~72h。
反应后,采用调节pH值后冻干,得到冻干样品;反应结束后,优选用碳酸氢钠调水溶液呈碱性进行透析,冻干机冻干。
将冻干样品溶解后与凝血靶向肽反应,即得。冻干样品优选用弱碱性水溶解后与凝血靶向肽反应;其中,凝血靶向肽优选溶剂为水;反应温度优选为30℃~40℃;反应时间优选为4h~48h,更优选为24h;凝血靶向肽与聚氨基酸的摩尔比优选为(1~10):1,更优选为3:1。
本发明中,聚氨基酸侧链羧基接枝Toll样受体激动剂;聚氨基酸侧链羧基与Toll样受体激动剂上的氨基或者羟基在缩合剂的作用下反应制备;聚氨基酸侧链羧基接枝凝血靶向多肽;聚氨基酸侧链羧基与含马来酰亚胺的小分子上的氨基或羟基在缩合剂的作用下反应制备接枝马来酰亚胺的聚氨基酸。聚氨基酸侧链马来酰亚胺基团与凝血靶向肽上巯基进行点击化学反应制备。
本发明提供了一种抗肿瘤组合物,包括上述技术方案任意一项所述的聚氨基酸肿瘤选择性前药和血管阻断剂。
本发明提供的抗肿瘤组合物包括上述技术方案任意一项所述的聚氨基酸肿瘤选择性前药。本发明对于上述聚氨基酸肿瘤选择性前药上述已经有了清楚的描述,在此不再赘述。
该类前药可以有效地富集在肿瘤部位,在肿瘤部位选择性释放。该类前药和血管阻断剂联用可以有效地提高药物在肿瘤的分布浓度,在显著抑制肿瘤生长和转移的同时减少了药物脱靶带来的全身炎性反应副作用。
本发明提供的抗肿瘤组合物包括血管阻断剂。本发明对于所述血管阻断剂的具体种类和来源不进行限定,本领域技术人员熟知的即可。如CA4 NPs。
本发明提供了上述技术方案所述的聚氨基酸肿瘤选择性前药在制备治疗癌症的药物中的应用。
本发明所述癌症包括鼻腔及鼻窦恶性肿瘤、鼻咽癌、口腔癌、喉癌、颅内肿瘤、甲状腺癌、舌癌、肺癌、食管癌、乳腺癌、胃癌、大肠癌、乙状结肠和直肠癌、肝癌、胰腺癌与壶腹周围癌、胆道癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、子宫颈癌、子宫内膜癌、卵巢癌、纤维组织细胞癌、横纹肌肉癌、滑膜肉瘤、黑色素瘤、骨肉瘤、尤文氏肉瘤、白血病、淋巴瘤和多发性骨髓瘤中的一种或几种。
本发明提供了一种聚氨基酸肿瘤选择性前药,具有式(I)所示结构:其中,R1选自C2-C10的直链烷基、C3-C10的支链烷基或C6~C20的芳基;R2为肿瘤响应的咪唑喹啉类药物;R3为H或阳离子;R4为具有凝血靶向多肽;L1、L2、L3选自-CH2-或-CH2CH2-;L4选自-CH2O-、-CH2CH2O-或-CH2NH-、-CH2CH2NH-;x,y,z为重复单元含量,0<x<1,0<y<1,0<z<1;n为聚合度,10≤n≤500。上述基于聚氨基酸的“凝血靶向”乏氧激活IMDQ前药。该前药利用高分子血管阻断剂治疗选择性地破坏肿瘤血管,使肿瘤出血-凝血,阻断肿瘤养分、氧气供给,从而放大肿瘤组织与正常组织之间凝血和乏氧的差异;“凝血靶向”乏氧激活IMDQ前药对凝血靶向、乏氧响应,使IMDQ的瘤内分布具有主动肿瘤靶向与肿瘤选择性作用的双重效果。因此,本专利发明的“凝血靶向”乏氧激活IMDQ前药可极大的提高IMDQ的肿瘤选择性,并显著优于现有技术。
为了进一步说明本发明,以下结合实施例对本发明提供的一种聚氨基酸肿瘤选择性前药、其制备方法和应用进行详细描述。
实施例1:
IMDQ(4-amino-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol,CAS:339545-50-5)是可以被Toll样受体(TLR)7/8识别的小分子化合物。据报道,IMDQ具有刺激免疫和调节免疫抑制的功能。机体对IMDQ的识别导致树突状细胞(Dendritic Cells,DCs)的激活并诱导促炎性细胞因子和趋化因子的分泌。此外,IMDQ还可以调节免疫抑制细胞,例如肿瘤相关巨噬细胞(Tumor-Associated Macrophages,TAMs)和骨髓来源的免疫抑制细胞(Myeloid-Derived Suppressor Cells,MDSCs),IMDQ可以将MDSCs转换为抗原呈递细胞(Antigen Presenting Cells,APCs),例如DCs和TAMs,将TAMs从M2表型极化为M1表型。然而,临床试验和研究数据表明,无论是局部给药还是全身给药,IMDQ都会产生严重的全身炎症反应,这也是限制其给药剂量和临床应用的重要原因。本发明人通过将IMDQ的活性氨基更改为叠氮基(IMDQ-N3)进行修饰,来避免其全身性炎症反应,并将IMDQ键合到聚氨基酸上,制备IMDQ-N3纳米药物,最后再修饰上凝血靶向靶头,制备出凝血靶向IMDQ纳米前药,并且与发明人已经报道的高分子血管阻断剂康普瑞汀A4(Combretastatin A4Nanoparticles,CA4 NPs)进行联合治疗,CA4 NPs具有出色的血液循环时间,并且可以在实体肿瘤血管周围积聚以选择性地破坏肿瘤血管,使肿瘤内发生出血-凝血级联反应,产生大量新的凝血受体,增加带凝血靶头IMDQ纳米前药在肿瘤富集,血管破坏同时显著增加肿瘤部位的乏氧水平,提高肿瘤内CYP450酶的含量,催化IMDQ-N3还原为IMDQ活性药物。
IMDQ-N3的制备示意图如图1a所示。首先通过点击反应,利用FSO2N3与IMDQ进行反应(FSO2N3是根据文献报道制备的氟磺酰叠氮化物)。向50mL玻璃圆底烧瓶中依次添加IMDQ(1.0mmol,286.34mg,溶于2mL二甲基亚砜(DMSO)中,FSO2N3溶液(含1.0mmol FSO2N3,200mM,溶于DMSO/甲基叔丁基醚(MTBE)=1:1中,约5mL),并加入KHCO3水溶液(3.0M,1.33mL,含有4.0mmol KHCO3)。将反应混合物在30℃下搅拌5小时。反应完成后,加入乙酸乙酯(40mL),并依次用饱和食盐水(60mL×6),水(60mL×2)和饱和食盐水(60mL)洗涤混合物,经无水Na2SO4干燥,过滤后通过旋转蒸发浓缩并真空干燥。粗产物经硅胶层析(CH2Cl2/MeOH=100:2,v/v)进一步纯化得到IMDQ-N3。并使用DMSO作为溶剂通过1H NMR结构(图1b),还使用ESI-MS(ESI+)表征IMDQ-N3,[M+Na]+=335.4,[M+K]+=351.4(图1c)。
实施例2:
Apcitide是用于深静脉栓塞的阿西肽锝(99mTc)中的不含放射性元素的有效部分,可特异性结合活化血小板上的血小板糖蛋白(GP)IIb/IIIa受体。凝血靶向乏氧激活IMDQ纳米前药Apcitide-PLG-IMDQ-N3的制备如图2a所示。首先,将聚谷氨酸(PLG,1000.0mg)溶解到超干N,N-二甲基甲酰胺(DMF)中,然后加入4-二甲氨基吡啶(DMAP,24.4mg,0.2mmol),IMDQ-N3(200.0mg,0.64mmol),N-(2-羟乙基)马来酰亚胺(37.8mg,0.3mmol),N,N'-二异丙基碳二亚胺(DIC,168.4mg,1.3mmol)。在30℃油浴条件下,反应72h后,在过量乙醚中沉淀,过滤后抽干乙醚,再用DMF复溶后透析,冻干得到不含凝血靶头的IMDQ纳米前药PLG-IMDQ-N3。再将PLG-IMDQ-N3(420mg)溶解在PBS(pH=7.4)中,用氢氧化钠水溶液(0.01mM)调节pH=8,然后加入Apcitide(52.8mg),硫醇和马来酰亚胺间进行点击化学反应,在37℃条件下反应12h后,透析后冻干得到Apcitide-PLG-IMDQ-N3。并使用D2O和DMSO的混合液作为溶剂通过1H NMR对结构进行表征(图2b),还使用x射线光电子能谱(XPS)(图2c)表征Apcitide-PLG-IMDQ-N3的成功合成。同时也对Apcitide-PLG-IMDQ-N3的粒径(图2d)、酯键稳定性(图2e)进行了表征。
实施例3:
接下来检测了Apcitide-PLG-IMDQ-N3在体外和细胞内的还原效果。Apcitide-PLG-IMDQ-N3用PBS(pH=7.4)溶解至含IMDQ-N3浓度为50μM,取稀释后的Apcitide-PLG-IMDQ-N3溶液240μL与25μL的0.1mM NADPH(还原辅酶)和20μL的0.1mg/mL CYP2D6(CYP450酶亚型之一)反应。样品在反应前用氩气脱气,并密封以保持缺氧条件。对照实验中,将样品、CYP2D6和NADPH溶于pH=7.4的PBS中,不脱气。每个时间点样品独立反应,在37℃振荡箱中轻轻摇晃1小时、12小时和24小时,反应后每个反应加入195μL甲醇终止,吹干甲醇后加100μL注射用水溶解,再加入20μL浓度为1M的氢氧化钠水溶液,37℃碱解12h后,加入1.4M的磷酸水溶液20μL进行中和,最后加入360μL甲醇,通过紫外-高效液相色谱(HPLC)检测IMDQ、IMDQ-N3的含量(图3a)。HPLC检测用甲醇:水(50:50,v/v)和0.05%(vt%)乙酸洗脱,并在254nm处以1.0mL/min的流速进行检测。结果表明,在乏氧条件下,Apcitide-PLG-IMDQ-N3在反应24h后,92.5%的IMDQ-N3被还原为IMDQ,且所有样品的回收率均在90%以上。相反,Apcitide-PLG-IMDQ-N3在CYP450酶和NADPH的存在下常氧可以保持24小时稳定。
为了确定IMDQ-N3纳米药物是否可以在乏氧细胞中条件中还原,接下来评估了Apcitide-PLG-IMDQ-N3和PLG-IMDQ-N3在4T1肿瘤细胞中的生物还原性。在细胞培养液中加入含5μg/mL IMDQ-N3的纳米药物后,分别在常氧(21%O2)和低氧(0.1%O2)下培养,24小时后,将细胞裂解和碱解酯键后,通过HPLC分析胞内IMDQ的含量(图3b)。在4T1肿瘤细胞中,IMDQ-N3在乏氧条件下可还原为IMDQ,其还原超过90%。在常氧条件下,24小时后未观察明显的IMDQ的产生。还检测了Apcitide-PLG-IMDQ-N3在乏氧条件下对DCs的激活和对TAMs表型转变的影响,通过表面活化标记物的表达来研究Apcitide-PLG-IMDQ-N3对APCs的免疫刺激作用。发明人用Apcitide-PLG-IMDQ-N3处理骨髓来源的树突状细胞(bone marrowdendritic cells,BMDCs)和骨髓来源的巨噬细胞(bone marrow-derived macrophages,BMDMs),在细胞培养液中加入含有5μg/mL IMDQ-N3纳米药物后,分别在常氧和低氧条件下培养24小时,然后用流式细胞术检测DCs和TAMs表面标记物的表达。Apcitide-PLG-IMDQ-N3处理24小时后,申请人发现乏氧条件下DCs的CD80和MHCII表达明显高于常氧处理后(图3c),并且M1型巨噬细胞(F4/80+CD80+标记)的表达上调,M2型巨噬细胞(F4/80+CD206+标记)的表达下调(图3d)。以上说明在乏氧条件下,Apcitide-PLG-IMDQ-N3可被还原并显著激活DCs,增加M1型TAMs的表型。
实施例4:
限制IMDQ给药剂量和临床应用的重要原因是全身给药可引起严重的全身炎症,产生大量促炎细胞因子,如白介素-6(IL-6)和II型干扰素(IFN-γ)等。因此,接下来对其系统给药后的全身毒性进行了评估。PLG-IMDQ静脉给药后,给药剂量从1mg·kg-1到64mg·kg-1均显著降低健康BALB/c小鼠的体重,在IMDQ剂量达到64mg·kg-1时出现小鼠死亡(图4a和图4b)。与之相反,与注射相同剂量的PLG-IMDQ相比,健康小鼠注射PLG-IMDQ-N3后,即使在IMDQ-N3剂量高达128mg·kg-1时,也没观察到明显小鼠体重下降,和小鼠死亡(图4c和图4d)。在健康小鼠6小时内,PLG-IMDQ能引起血清IL-6和IFN-γ水平显著升高,从而引起全身性炎性反应,而PLG-IMDQ-N3和Apcitide-PLG-IMDQ-N3均没有引起明显的IFN-γ和IL-6水平升高(图4e和图4f)。这些结果证实了IMDQ-N3纳米药物作为免疫调节剂在避免全身炎症反应方面有巨大潜力。
实施例5:
接下来对靶向活化血小板的凝血靶向肽Apcitide对提升IMDQ在肿瘤部位的富集能力进行了评估。在移植4T1肿瘤的BALB/c荷瘤小鼠上,通过尾静脉注射CA4 NPs(15mg/kg)后,在给药前(0h)和给药后1h、3h、6h取小鼠肿瘤研磨后,利用小鼠GPIIbIIIa ELISA试剂盒评估了CA4-NPs引发凝血级联反应增加活化血小板的数量与时间的关系。发现尾静脉注射CA4-NPs后的第3h检测到的血小板活化标志物GPIIbIIIa最高,达到最大值,为0h的5.2倍(图5a)。接下来在荷瘤鼠上进行了组织分布实验,通过向移植4T1肿瘤的BALB/c荷瘤小鼠尾静脉注射IMDQ-N3纳米药物或IMDQ-N3纳米药物+CA4-NPs,IMDQ-N3纳米药物的给药剂量以IMDQ-N3计为4mg·kg-1,CA4-NPs以CA4计为15mg·kg-1。联合治疗组中,CA4 NPs注射两小时后,再注射IMDQ-N3纳米药物。在注射IMDQ-N3纳米药物后1h、4h、12h,杀掉小鼠取其肿瘤组织和心、肝、脾、肺、肾,称重,加注射用水(0.1g/mL)研磨后,取1mL,加入100μL浓度为1M的氢氧化钠水溶液,37℃碱解12h后,加入1.4M的磷酸水溶液100μL进行中和,吹干后,加200uL注射用水溶解后,加入200μL甲醇,用0.22μm有机滤头过滤后通过紫外-高效液相色谱(HPLC)检测IMDQ、IMDQ-N3的含量。结果表明,Apcitide凝血靶向肽能显著提高药物在肿瘤的富集,Apcitide-PLG-IMDQ-N3+CA4 NPs时,瘤内总的IMDQ+IMDQ-N3是不联合且无凝血靶头的PLG-IMDQ-N3的7.1倍(图5c),瘤内有效药物浓度IMDQ则是PLG-IMDQ-N3组的10.4倍(图5d)。以上结果表明,Apcitide-PLG-IMDQ-N3具有实现在肿瘤部位有效地富集,和在肿瘤部位高效地选择性还原激活能力。
实施例6:
接下来考察了该“凝血靶向”药物控释系统纳米药物在小鼠乳腺癌4T1肿瘤模型上的治疗效果。在4T1模型上给药方案如图6a所示,从小鼠的肿瘤体积随时间变化曲线(图6b)和治疗结束后肿瘤照片(图6c)可以发现,PBS组的小鼠肿瘤生长极为迅速,第12天治疗结束时大部分小鼠肿瘤体积已经接近2000mm3,经三次尾静脉给药后,治疗组CA4 NPs、PLG-IMDQ-N3、Apcitide-PLG-IMDQ-N3对肿瘤的生长有一定的抑制作用,PLG-IMDQ-N3+CA4 NPs联合治疗组治疗效果较为明显,但是其治疗效果还是远不如既有靶头又联合治疗组Apcitide-PLG-IMDQ-N3+CA4 NPs效果好,5只鼠中有4只鼠的生存期超过了60天,且有3只鼠的肿瘤彻底消失(图6e)。由于引入凝血靶头Apcitide,增加了药物在肿瘤内富集能力,联合CA4 NPs提升肿瘤内部乏氧,实现药物在瘤内的高效选择性还原,实现了对4T1肿瘤的有效治疗。
实施例7:
进一步利用流式细胞术评估不同治疗组肿瘤免疫微环境的变化(图7)。经Apcitide-PLG-IMDQ-N3+CA4 NPs治疗后,在肿瘤内检测到了更多的活化DCs(CD11c+CD80+cells),更多的CD8+T细胞和CD4+T细胞,这表明该组治疗有效激活了抗肿瘤免疫反应(图7a-c)。此外,肿瘤内部的免疫抑制微环境也发生了改变,与其他治疗组相比,Apcitide-PLG-IMDQ-N3+CA4 NPs治疗组MDSCs的比例明显降低(图7d),M1型巨噬细胞比例显著升高(图7e)。除了肿瘤中的免疫细胞外,发明人还用流式细胞术分析了脾脏中的免疫细胞。发明人发现,Apcitide-PLG-IMDQ-N3+CA4 NPs治疗组的CD8+T细胞比例(图7f)和CD4+T细胞比例(图7g)明显高于其他治疗组,由此可以发现脾脏内也形成了强烈的抗肿瘤免疫响应。
实施例8:
评价了Apcitide-PLG-IMDQ-N3+CA4 NPs的抗转移作用。如预期的那样,印度墨水检测显示(图8a)和苏木素伊红(hematoxylin and eosin,H&E)染色(图8b)显示,与PBS组或单药治疗组相比,联合治疗显著抑制了肺部转移。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (11)
1.一种聚氨基酸肿瘤选择性前药,其特征在于,具有式(I)所示结构:
其中,R1选自C2-C10的直链烷基、C3-C10的支链烷基或C6~C20的芳基;
R2为肿瘤响应的咪唑喹啉类药物;
R3为H或阳离子;
R4为凝血靶向多肽;
L1、L2、L3选自-CH2-或-CH2CH2-;
L4选自-CH2O-、-CH2CH2O-或-CH2NH-、-CH2CH2NH-;
x,y,z为重复单元含量,0<x<1,0<y<1,0<z<1;n为聚合度,10≤n≤500。
2.根据权利要求1所述的聚氨基酸肿瘤选择性前药,其特征在于,所述R1为选自C2~C10的直链烷基、C3~C10的支链烷基或C6~C20的芳基;R3独立的或者组合的选自H、金属阳离子或有机阳离子。
3.根据权利要求2所述的聚氨基酸肿瘤选择性前药,其特征在于,所述R1为乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、正己基、正庚基、正辛基、苯基、萘基、联苯基或蒽;R1为H、钠离子、钾离子、铵离子或者带正电荷的氨基酸离子。
4.根据权利要求1所述的聚氨基酸肿瘤选择性前药,其特征在于,所述的聚氨基选自聚谷氨酸或聚天冬氨酸。
5.根据权利要求1所述的聚氨基酸肿瘤选择性前药,其特征在于,所述R2独立的或者组合的选自如下结构:
6.根据权利要求1所述的聚氨基酸肿瘤选择性前药,其特征在于,所述R4独立的或者组合的选自如下结构;
7.一种聚氨基酸肿瘤选择性前药的制备方法,其特征在于,包括如下步骤:
A)聚氨基酸、式(Ⅱ)所示的Toll样受体激动剂叠氮前药、式(Ⅲ)所示的含马来酰亚胺基团的小分子进行缩合反应;
B)反应后,采用调节pH值后冻干,得到冻干样品;将冻干样品溶解后与凝血靶向肽反应,即得。
8.根据权利要求7所述的制备方法,其特征在于,所述
步骤A)所述反应溶剂为二甲基甲酰胺、二甲基亚砜;所述Toll样受体激动剂叠氮前药的反应原料质量占聚氨基酸的质量比为1%~30%;
马来酰亚胺基团与聚氨基酸的摩尔比为(1~10):1;
反应温度为20℃~60℃;反应时间为24~72h;缩合反应的缩合剂为2,4,6-三氯苯甲酰氯,N,N-二异丙基碳二亚胺,二环己基碳二亚胺,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;
步骤B)所述凝血靶向肽的溶剂为水;反应温度为30℃~40℃;反应时间为4h~48h;凝血靶向肽与聚氨基酸的摩尔比为(1~10):1。
9.一种抗肿瘤组合物,其特征在于,包括权利要求1~6任意一项所述的聚氨基酸肿瘤选择性前药和血管阻断剂。
10.权利要求1~6任意一项所述的聚氨基酸肿瘤选择性前药在制备治疗癌症的药物中的应用。
11.根据权利要求10所述的应用,其特征在于,所述癌症包括鼻腔及鼻窦恶性肿瘤、鼻咽癌、口腔癌、喉癌、颅内肿瘤、甲状腺癌、舌癌、肺癌、食管癌、乳腺癌、胃癌、大肠癌、乙状结肠和直肠癌、肝癌、胰腺癌与壶腹周围癌、胆道癌、肾癌、前列腺癌、膀胱癌、睾丸恶性肿瘤、阴茎癌、子宫颈癌、子宫内膜癌、卵巢癌、纤维组织细胞癌、横纹肌肉癌、滑膜肉瘤、黑色素瘤、骨肉瘤、尤文氏肉瘤、白血病、淋巴瘤和多发性骨髓瘤中的一种或几种。
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