CN116354968A - 与内源性脂蛋白结合的那伏莫德前药及纳米粒 - Google Patents
与内源性脂蛋白结合的那伏莫德前药及纳米粒 Download PDFInfo
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Abstract
Description
技术领域
本发明属于生物医药领域,尤其是涉及一种与内源性脂蛋白结合的那伏莫德前药及纳米粒。
背景技术
免疫检查点蛋白吲哚胺2,3-双加氧酶在肿瘤中高度表达,作为催化色氨酸分解代谢生成犬尿氨酸的主要酶,在抑制效应T细胞增殖以及诱导调节T细胞扩增方面起到重要作用并促进肿瘤进展,是缓解免疫抑制微环境、提高免疫治疗效率的理想靶点。近几十年来,新兴的吲哚胺2,3-双加氧酶抑制剂NLG919(那伏莫德)可以有效缓解免疫逃避,被认为是肿瘤免疫治疗的重大突破。然而,NLG919表现出较差的水溶性,需要极高的剂量才能有效,这会导致严重的不良反应,如腹痛、低钾血症和疲劳,这些问题导致其在临床应用中受到限制。因此,如何提高其生物利用度并降低其毒性是亟待解决的问题。
研究发现,通过靶向药物递送系统,即在纳米载体表面修饰特定配体,可实现肿瘤高效识别和靶向,提高药物在肿瘤组织的积累并减少副作用以达到更好的治疗效果,是解决上述问题的一种有效策略。目前常用的配体有抗体、适配体和肽。然而,上述配体作为外源蛋白修饰在纳米载体表面后,通过静脉注射将在纳米载体表面形成蛋白冠,导致不利的生物分布和免疫原性变化;同时,复杂的制备工艺导致较高的成产成本。体内变化的不确定性以及高昂的治疗费用使得靶向给药系统的临床转化过程缓慢。
因此,迫切需要探索新的靶向药物递送平台来克服这些挑战,以求在避免引入外源蛋白的条件下实现高效靶向,还可以简化制备工艺降低成本。
发明内容
本发明的目的是克服现有技术的不足,提供一种与内源性脂蛋白结合的那伏莫德前药。
本发明的第二个目的是提供一种与内源性脂蛋白结合的那伏莫德前药的制备方法。
本发明的第三个目的是提供一种与内源性脂蛋白结合的那伏莫德前药的纳米粒。
本发明的第四个目的是提供一种与内源性脂蛋白结合的那伏莫德前药的纳米粒的制备方法。
本发明的第五个目的是提供一种与内源性脂蛋白结合的那伏莫德前药在制备免疫调节剂中的应用。
本发明的技术方案概述如下:
与内源性脂蛋白结合的那伏莫德前药,其结构式如式I所示:
简称P-NLG919。
上述与内源性脂蛋白结合的那伏莫德前药的制备方法,包括如下步骤:将2-正十八烷氧基烯丙醚和NLG919溶于超干二氯甲烷中,加入超干二氯乙酸,室温下反应;检测反应完成,加入纯水淬灭反应,二氯甲烷萃取后得到与内源性脂蛋白结合的那伏莫德前药,如式I所示,所述NLG919为那伏莫德的简称。
优选地,2-正十八烷氧基烯丙醚和NLG919的摩尔比为4:1。
与内源性脂蛋白结合的那伏莫德前药的纳米粒的制备方法,包括如下步骤:将上述与内源性脂蛋白结合的那伏莫德前药溶于体积浓度为0.1%的三乙胺的丙酮溶液中,加入聚山梨酯-80,超声混合;滴入持续搅拌、pH=8.0、6.7mM PBS中,得到与内源性脂蛋白结合的那伏莫德前药的纳米粒。
优选地,与内源性脂蛋白结合的那伏莫德前药与聚山梨酯-80的质量比为1:10。
搅拌的转速是500rpm,搅拌30分钟。
上述制备方法制备的与内源性脂蛋白结合的那伏莫德前药的纳米粒。
上述与内源性脂蛋白结合的那伏莫德前药在制备免疫调节剂中的应用。
本发明的优点:
本发明的与内源性脂蛋白结合的那伏莫德前药为酸敏感前药,不依赖机体内酶的激活,肿瘤部位的酸性微环境可以持续触发与内源性脂蛋白结合的那伏莫德前药中缩丙酮键的水解,可实现免疫调节剂的长效释放。
本发明与内源性脂蛋白结合的那伏莫德前药可与内源性低密度脂蛋白结合,利用肿瘤细胞低密度脂蛋白受体的高表达,实现肿瘤靶向治疗,提高免疫治疗的效果。
本发明与内源性脂蛋白结合的那伏莫德前药和纳米粒制备条件温和,工艺简单,重复性高,利于实现大规模生产。
附图说明
图1为P-NLG919的核磁共振氢谱图;
图2为P-NLG919的核磁共振碳谱图;
图3为P-NLG919在pH 5.0缓冲液中的水解率-时间曲线;
图4为P-NLG919在pH 5.0缓冲液中的水解速率图,其水解动力学方程为y=–12.36x+4.2293(R2=0.9933);
图5为P-NLG919在pH 7.4缓冲液中的水解率-时间曲线;
图6为P-NLG919在pH 7.4缓冲液中的水解速率图,其水解动力学方程为y=–0.2899x+4.3843(R2=0.9869);
图7为NLM的扫描电镜图;
图8为NLM的粒径和Zeta电位变化图;
图9为NLM的对Panc02细胞的细胞毒性;
图10为NLM对低密度脂蛋白的结合情况;
图11为NLM给药后小鼠第28天血常规评价图;
图12为2-正十八烷氧基烯丙醚的核磁共振氢谱图。
图中NLM为P-NLG919纳米粒的简写。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明创造所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
2-正十八烷氧基烯丙醚的制备,参见中国专利CN202211046523.0一种帕利哌酮前药、制剂及其制备方法和应用。
具体步骤为:取250mL圆底烧瓶,向其中加入2-甲氧基丙烯(147.87mmol,13.85mL,16.0eq),硬脂醇(9.24mmol,2.5g,1.0eq)和对甲苯磺酸2,6-二甲基吡啶鎓盐(0.0462mmol,13.03mg,0.005eq),随后加入100mL四氢呋喃溶解,室温下反应5h,向其中加入500μL三乙胺终止反应,旋蒸浓缩后用含1(V/V)%三乙胺的石油醚/乙酸乙酯(石油醚/乙酸乙酯的体积比为1:4)进行硅胶柱层析分离,得到产物,产物采用核磁共振技术鉴定表征,见图12:分子式II如下式所示,产率53%。
实施例1
与内源性脂蛋白结合的那伏莫德前药的制备方法,包括如下步骤:
取10mL干燥的Schlenk管,向其中加入NLG919(0.18mmol,50mg,1.0eq),分子式II所示分子2-正十八烷氧基烯丙醚(0.72mmol,0.345g,4.0eq)溶于3.5mL超干二氯甲烷中,加入7.5μL超干二氯乙酸作催化剂,室温下反应,TCL检测,反应12h完成,反应结束后向其中加入5mL纯水淬灭反应,水相用二氯甲烷萃取,合并有机相,无水硫酸钠干燥,过滤,浓缩,得到与内源性脂蛋白结合的那伏莫德前药(P-NLG919),如式I所示,所述NLG919为那伏莫德的简称,产率25%。
P-NLG919分子采用核磁共振技术鉴定表征,具体结果如下:
1H NMR(400MHz,Chloroform-d)δ7.75(s,1H),7.52(d,J=7.6Hz,1H),7.41(d,J=7.6Hz,1H),7.34(t,J=7.4Hz,1H),7.22(t,J=7.4Hz,1H),7.17(s,1H),5.23(t,J=5.9Hz,1H),3.93(td,J=6.4,3.2Hz,1H),3.50–3.28(m,2H),2.12(t,J=6.2Hz,2H),1.84–1.53(m,7H),1.36–1.08(m,42H),0.88(t,J=6.8Hz,3H).见图1。
13C NMR(100MHz,Chloroform-d)δ145.81,137.46,132.14,130.88,130.05,129.50,128.24,126.26,123.55,119.94,119.84,118.35,100.97,77.29,72.75,72.59,67.78,62.35,62.02,58.54,58.46,42.60,42.09,37.69,31.95,30.12,29.73,29.70,29.68,29.64,29.61,29.58,29.54,29.47,29.39,29.35,29.02,27.42,26.75,26.69,26.65,26.48,26.24,26.18,25.74,25.62,25.49,23.01,22.71,14.13,14.06,见图2。
氢谱(图1)和碳谱(图2)分别在δ1.4ppm(s,6H)和δ101.80ppm显示了P-NLG919的特征峰,说明顺利得到了目标产物,化合物I。
简称P-NLG919。
实验例1
P-NLG919的酸响应水解测试
pH为5.0的乙酸缓冲液的制备:将乙酸/乙酸钠缓冲液(50mM,pH 3.7)和乙腈按体积比为2:3的比例混合;
pH为7.4的磷酸缓冲液的制备:将磷酸缓冲液(50mM,pH 6.4)和乙腈按体积比为2:3的比例混合;
称取P-NLG919并溶于体积浓度为0.1%的三乙胺甲醇溶液中,制备P-NLG919储备液(3mM)。
取3mL 37℃、pH为5.0的乙酸缓冲液与20μL P-NLG919储备液混合,得到第一种水解体系。
取3mL 37℃、pH为7.4的磷酸缓冲液与20μL P-NLG919储备液混合,得到第二种水解体系。
将上述两种水解体系置于37℃恒温培养振荡器中以100rpm的转速震荡。在预定时间(0.5h、1h、2h、4h、8h、12h)各取200μL,并加入等体积水解终止液(体积比40%磷酸缓冲液(100mM,pH 10.0)和60%乙腈(v/v))终止酸解。使用涡旋混匀器均匀混合,以12000rpm的转速离心5分钟并取100μL上清液至进样瓶中,按下述色谱条件检测NLG919及P-NLG919浓度,绘制并拟合水解曲线。
选用安捷伦C8色谱柱,5μm,150×4.6mm;柱温30℃;流速1.2mL/min;紫外检测波长250nm;进样量20μL。流动相甲醇(A)-水(B),梯度洗脱:0-12.00分钟(A:80%-80%),NLG919在3.4分钟出峰,P-NLG919在10.0分钟出峰。经高效液相色谱测定纯度达标,满足后续实验要求。
水解率计算公式如下:
按照一级动力学方程绘制水解曲线,得到水解速率常数k,并按照如下公式计算t1//2。
P-NLG919在pH=5.0及7.4下的水解率-时间曲线如图3和图5所示,pH=5.0及7.4下水解动力学如图4和图6所示。
P-NLG919水解表现出准一级动力学,在pH 5.0和7.4下的水解半衰期分别为0.056h和2.39h。以上结果说明P-NLG919分子在酸性条件水解速率更快。因此证明,与内源性脂蛋白结合的那伏莫德前药可以做为免疫调节剂应用。
实施例2
与内源性脂蛋白结合的那伏莫德前药的纳米粒的制备方法,包括如下步骤
称取实施例1制备的P-NLG919 3mg,溶于100μL体积浓度为0.1%的三乙胺的丙酮溶液中,加入30mg聚山梨酯-80,超声混合,得混合溶液,采用纳米沉淀法制备纳米粒,即在室温条件下,将上述混合溶液滴加至持续搅拌,1mL pH=8.0,6.7mM PBS中,滴加过程保持500rpm的转速并持续搅拌30分钟。随后使用旋转蒸发仪去除丙酮,0.22μm过滤除菌,4℃备用,得到与内源性脂蛋白结合的那伏莫德前药的纳米粒(命名为NLM)。
NLM采用透射电子显微镜进行鉴定表征,如图7所示,从图中可以看出,制备的纳米粒呈均一球形,粒径为8-10nm。
实验例2
NLM的稳定性评价
将实施例2获得的NLM 4℃条件保存,连续一周使用动态光散射仪测定纳米粒的粒径和Zeta电位,用于评估NLM纳米粒的稳定性。如图8所示,NLM的Zeta电位在一周的存储过程中维持在-3.9mV到-4.2mV,粒径维持在7.9-8.2nm,没有发生明显的变化,说明NLM纳米粒能够在储存过程中保持较好的稳定性。
实验例3
NLM的细胞毒性评价
选择对数生长期的Panc02小鼠胰腺癌细胞,调整细胞数为3×104个/mL,接种于96孔板中,每孔100μL,细胞贴壁生长24h后,加入由培养基稀释的不同浓度的NLM与Panc02细胞共同培养,继续培养48h后,弃去旧培养基,每孔加入含有10μL CCK8的新鲜培养基100μL后继续孵育1.5h,使用酶标仪测定各孔在450nm处的吸光度(OD值),计算细胞活力。细胞存活率(Cell viability,%)=(ODsample-ODblank)/(ODcontrol-ODblank)×100。结果如图9所示,NLM未见明显细胞毒性。
实验例4
以实施例2获得的NLM与内源性低密度脂蛋白结合能力的测试:
将NLM作为配体,内源性低密度脂蛋白作为受体蛋白,使用微量热泳动仪,检测解离常数。以聚山梨酯-80纳米粒作为空白对照。
图10为NLM与内源性低密度脂蛋白结合情况的结果,可以看出NLM与内源性低密度脂蛋白结合的解离常数为16μM,这种结合能力与聚山梨酯-80无关,说明NLM在与蛋白孵育过程中释放出P-NLG919前药,并与内源性低密度脂蛋白高效结合。
实验例5
进行NLM安全性评价:
雌性C57bl/6j小鼠(20±2g)用异氟烷麻醉,按照10mg NLG919每千克的给药剂量,静脉注射100μLNLM,然后于给药后第28天进行血生化评价。
图11为第28天的血生化结果,可以看出肌酐、尿素氮、谷丙转氨酶和谷草转氨酶含量与未给药组相比,没有明显区别。
通过以上实施例和实验例可以说明,本发明制备的与内源性脂蛋白结合的那伏莫德前药和其纳米粒具有结构明确、制备方法和过程简单高效、优良的酸响应性、优异的内源性低密度脂蛋白结合能力以及生物安全性的特点。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
2.权利要求1的与内源性脂蛋白结合的那伏莫德前药的制备方法,其特征是包括如下步骤:
将2-正十八烷氧基烯丙醚和NLG919溶于超干二氯甲烷中,加入超干二氯乙酸,室温下反应;反应完成,加入纯水淬灭反应,二氯甲烷萃取后得到与内源性脂蛋白结合的那伏莫德前药,如式(I)所示,所述NLG919为那伏莫德的简称。
3.根据权利要求2所述的制备方法,其特征是所述2-正十八烷氧基烯丙醚和NLG919的摩尔比为4:1。
4.与内源性脂蛋白结合的那伏莫德前药的纳米粒的制备方法,其特征是包括如下步骤:将权利要求1的与内源性脂蛋白结合的那伏莫德前药溶于体积浓度为0.1%的三乙胺的丙酮溶液中,加入聚山梨酯-80,超声混合;滴入持续搅拌、pH=8.0、6.7mM PBS中,得到与内源性脂蛋白结合的那伏莫德前药的纳米粒。
5.根据权利要求4所述的制备方法,其特征是与内源性脂蛋白结合的那伏莫德前药与聚山梨酯-80的质量比为1:10。
6.根据权利要求4所述的制备方法,其特征是所述搅拌的转速是500rpm,搅拌30分钟。
7.权利要求4、5、6中任一制备方法制备的与内源性脂蛋白结合的那伏莫德前药的纳米粒。
8.权利要求1的与内源性脂蛋白结合的那伏莫德前药在制备免疫调节剂中的应用。
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