CN114767659A - 一种靶向pitpnm3的药物制剂及其制备方法和应用 - Google Patents
一种靶向pitpnm3的药物制剂及其制备方法和应用 Download PDFInfo
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- CN114767659A CN114767659A CN202210453561.1A CN202210453561A CN114767659A CN 114767659 A CN114767659 A CN 114767659A CN 202210453561 A CN202210453561 A CN 202210453561A CN 114767659 A CN114767659 A CN 114767659A
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Abstract
本发明公开了一种靶向PITPNM3的药物制剂及其制备方法和应用;所述药物制剂使用PEG‑PLGA纳米微球包裹靶向PITPNM3小分子抑制剂。本发明的药物制剂的制备工艺简单,大小均一,稳定性好,包封量较高,分散性好,并且能够显著延长靶向PITPNM3小分子抑制剂在体内半衰期延长,具有靶向性好、不易被清除的特点,还具备良好的转染肿瘤细胞并递送靶向PITPNM3小分子抑制剂的能力,肿瘤细胞摄取效率可以达到92.4%,而且主要在肿瘤组织内蓄积,对治疗肿瘤转移具有非常重要的意义;可用于治疗乳腺癌远处转移,应用前景广阔。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种靶向PITPNM3的药物制剂及其制备方法和应用。
背景技术
PITPNM3由17号染色体的PITPNM3基因编码,是一个跨膜受体。PITPNM3已被证实在多种肿瘤细胞中高表达,包括乳腺癌、肝细胞肝癌、胰腺导管癌、肺癌肿瘤细胞等多种恶性肿瘤。PITPNM3在这些肿瘤的远处转移中发挥了重要的作用。我们前期研究证实PITPNM3激活后能促进下游转移相关通路磷酸化,包括:PYK2、Src、FAK等磷酸化,从而促进肿瘤细胞转移相关蛋白和上皮-间质转化相关蛋白的表达,最终导致乳腺癌的远处转移。因此,PITPNM3在肿瘤转移具有非常重要的作用。
靶向PITPNM3的小分子抑制剂是一类以PITPNM3为靶点的小分子抑制剂,这一类小分子抑制剂结构各异,分子量从200到900不等,并且其中一些是脂溶性的抑制剂,一些则是水溶性抑制剂。前期研究证实这些小分子抑制剂能够靶向PITPNM3,抑制PITPNM3下游通路激活,从而起到抑制肿瘤的转移的作用,因此这类小分子抑制剂可以用来治疗肿瘤,并抑制肿瘤的远处转移。但这些小分子抑制剂具有各自的理化性质及分子结构,在体内的代谢情况及药代动力学又不近相同,达不到很好的治疗效果。
纳米剂型作为近年来发展起来的一种新剂型得到了重视,例如:纳米脂质体紫杉醇是将紫杉醇与类脂质双分子层包封而形成的纳米级的微型泡囊体,药物包裹在脂质体微粒中从而实现提高药物的治疗指数,减少药物的治疗剂量和降低药物的毒性,提高患者耐受性的效果。因此急需一种可以有效递送靶向PITPNM3小分子抑制剂的新剂型。
发明内容
本发明的第一个目的,在于提供一种纳米组合物。
本发明的第二个目的,在于提供上述纳米组合物的制备方法。
本发明的第三个目的,在于提供上述纳米组合物在制备给药系统中的应用。
本发明的第四个目的,在于提供上述纳米组合物在制备抗肿瘤药物或抑制肿瘤转移药物中的应用。
本发明的第五个目的,在于提供一种药物。
本发明所采取的技术方案是:
本发明的第一方面,提供一种纳米组合物,所述纳米组合物的制备原料包含PITPNM3小分子抑制剂和载体;所述载体为PLA、PEG-PLA、PGA、PLGA、PEG-PLGA、PCL、PCLA和PMMA中的一种或多种。优选地,所述载体为PEG-PLGA。PEG-PLGA是一种效用准确,无毒性,无致敏性,无致畸作用,也无致癌作用的功能性高分子有机材料。目前PEG-PLGA已经通过美国的食品药品监督管理局(FDA)认证,已被广泛用于制备人工导管,药物缓释载体,埋置剂等。
在本发明的一些实施方式中,所述PITPNM3小分子抑制剂为以下结构的小分子抑制剂或其水合物或药学上可接受的盐、酯或其衍生物:
在本发明的一些实施方式中,所述小分子抑制剂和载体的质量比为1:(1.25~20)。
在本发明的一些优选实施方式中,所述小分子抑制剂和载体的质量比为1:(5~10)。
在本发明的一些实施方式中,所述纳米组合物的纳米粒径为30~400nm。
在本发明的一些实施方式中,所述纳米组合物的包封率按所述组合物的重量计为5%~100%。
在本发明的一些实施方式中,所述组合物适于肌内、静脉内、皮下或皮肤注射给药。
本发明的第二方面,提供本发明第一方面所述纳米组合物的制备方法,包含以下步骤:
S1:将PITPNM3小分子抑制剂溶液加入载体溶液中,超声制备得到初溶液。
S2:将初溶液加入水相形成复溶液,然后离心、清洗、过滤、离心洗涤。
在本发明的一些实施方式中,所述载体是通过有机溶剂进行溶解。所述小分子抑制剂是溶解于有机溶剂或去离子水中。
在本发明的一些实施方式中,所述溶解载体的有机溶剂为二甲基甲酰胺(Dimethyl formamide,DMF);所述溶解小分子抑制剂的拥挤溶剂为二甲基亚砜(Dimethylsulfoxide,DMSO)。
在本发明的一些实施方式中,所述小分子抑制剂和载体的质量比为1:(1.25~20)。
在本发明的一些优选实施方式中,所述小分子抑制剂和载体的质量比为1:(5~10)。
在本发明的一些实施方式中,所述小分子抑制剂溶液的浓度为1mg/ml~100mg/ml。
在本发明的一些优选实施方式中,所述小分子抑制剂溶液的浓度为20mg/ml~60mg/ml
在本发明的一些实施方式在,所述载体溶液的浓度为1mg/ml~50mg/ml。
在本发明的一些实施方式中,所述初溶液与水相的比例为1:20~1:30。
在本发明的一些实施方式中,当将初溶液加入水相时采用缓慢滴加的方式,所述滴加的速度为10-200μL/每分钟。
在本发明的一些实施方式中,所述初溶液加入水相前需要进行搅拌,所述搅拌的速度为800r/min~1200r/min。
本发明的第三方面,提供本发明第一方面所述组合物在制备给药系统中的应用。
本发明的第四方面,本发明还提供本发明第一方面所述组合物在制备抗肿瘤药物中的应用。
在本发明的一些实施方式中,所述抗肿瘤为抑制癌转移,具体为抑制PITPNM3或CCL18-PITPNM3介导的肿瘤转移。
在本发明的一些实施方式中,所述肿瘤为乳腺癌、肝细胞癌、胰腺导管癌、肺癌中的任一种。
本发明的第五方面,提供一种药物,所述药物包含本发明第一方面所述组合物。
在本发明的一些实施方式中,所述药物为抗肿瘤药物。
在本发明的一些实施方式中,所述抗肿瘤为抑制癌转移,具体为抑制PITPNM3或CCL18-PITPNM3介导的肿瘤转移。
在本发明的一些实施方式中,所述肿瘤为乳腺癌、肝细胞癌、胰腺导管癌、肺癌中的任一种。
本发明的有益效果是:
本发明公开了一种靶向PITPNM3小分子抑制剂的药物新制剂。该剂型使用PEG-PLGA纳米微球包裹靶向PITPNM3小分子抑制剂,该纳米材料由聚乳酸-羟基乙酸-聚乙二醇共聚物(mPEG5K-b-PLGA5K,缩写为PEG-PLGA)纳米粒包载靶向PITPNM3的小分子抑制剂后得到的纳米微球。
本发明的新剂型,制备工艺简单,大小均一,稳定性好,包封量较高,分散性好,并且能够显著延长靶向PITPNM3小分子抑制剂在体内半衰期延长,具有靶向性好、不易被清除的特点,还具备良好的转染肿瘤细胞并递送靶向PITPNM3小分子抑制剂的能力,肿瘤细胞摄取效率可以达到92.4%,而且主要在肿瘤组织内蓄积,从而实现小分子抑制剂更佳的药代动力学参数及靶向递送到肿瘤的特点。对治疗肿瘤转移具有非常重要的意义;可用于治疗乳腺癌远处转移,应用前景广阔。
附图说明
图1为实施例1中所使用的化合物荧光光谱图。其中图1A为不同浓度NO.1化合物在340nm波长激发光激发下所产生不同波长荧光强度图;图1B为不同浓度NO.1化合物在不同波长激发光下吸光度图;图1C为NO.1化合物在340nm激发光404nm荧光下化合物质量与荧光强度拟合回归曲线图。
图2为纳米微球剂型的包封率统计图。图2A为化合物在不同浓度下的包封率;图2B为化合物和PEG-PLGA在不同质量比下的包封率;图2C为小分子化合物溶液与PEG-PLGA溶液在不同体积比下的包封率。
图3为实施例2制备得到的最佳的纳米粒的粒径分布图与电势分布图。
图4为实施例2制备得到的最佳的纳米粒肿瘤细胞摄取效率图。
图5为transwell法检测实施例2制备的最佳的纳米粒体外乳腺癌细胞侵袭及迁移代表图及三次独立重复试验统计图。
图6为实施例2制备得到的最佳的纳米粒纳米微球剂型及原剂型在体内的药代动力学参数及体内分布情况。图6A为药代动力学参数;图B为体内分布情况。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
实施例1靶向PITPNM3小分子抑制剂的PEG-PLGA纳米微球剂型的包封率测定
(1)使用DMSO配置NO.1号化合物,使其浓度为0mM、10mM、20mM、40mM和80mM浓度,在康宁96孔底透板,每个孔分别加入不同浓度的NO.1号化合物溶液60μL,使用TECANSpark10M检测NO.1号化合物特异的激发光(excitation wave)和发射光(emission wave)光谱,以及NO.1号化合物特异的吸收光谱(absorbance wave),结果如图1A-图1B所示,NO.1号化合物在340nm激发光及404nm发射光处,有一特异性的发射光;而吸收光谱却无特异性的吸收峰。因此以NO.1号化合物质量为横坐标,以NO.1号化合物在340nm激发光及404nm发射光处荧光值做一曲线拟合NO.1号化合物质量-荧光值拟合曲线,该拟合曲线公式为:y=-5214x2+20428x+8066(其中y:荧光值,x:NO.1号化合质量),拟合系数R2=0.995,拟合度较高,因此该拟合曲线能够用于估算NO.1化合物荧光值与NO.1号化合物质量之间的关系(图1C)。
(2)包封率的测定
将包载靶向PITPNM3小分子抑制剂NO.1号化合物的PEG-PLGA纳米微球,进行低温冻干后,使用60μl DMSO进行溶解,加入康宁96孔底透板,在TECAN Spark10M中使用340nm激发光及404nm发射光检测,计算得到NO.1号化合物质量。按照以下公式计算NO.1号化合物包封率。
包封率(%)=(微球中药物的含量/投药量)×100%。
实施例2靶向PITPNM3小分子抑制剂的PEG-PLGA纳米微球剂型的制备及理化性质测定
1、称取聚乳酸-羟基乙酸-聚乙二醇共聚物(缩写为PEG-PLGA,CAS号:34346-01-5,分子量:10000)并溶解于二甲基甲酰胺(Dimethyl formamide,DMF)中,得到20mg/mL的PEG-PLGA溶液;使用二甲基亚砜(Dimethyl sulfoxide,DMSO)溶解NO.1小分子抑制剂,溶解浓度为40mg/ml。按照小分子化合物与PEG-PLGA质量比为1:1.25,1:2.5,1:5,1:10和1:20;将小分子抑制剂溶液加入200μL PEG-PLGA溶液中,水浴超声,0.6Hz,1分钟,制备得到初溶液。在无菌去离子水中,室温下搅拌,用注射器吸取小分子抑制剂溶液与PEG-PLGA溶液的混合初溶液中,深入水相液面下,缓慢加入形成复溶液。将复溶液高速离心2800rpm,10分钟,使用去离子水反复清洗,将洗涤液加入100000MWCO的超滤管,2800rpm/s,离心10min,弃去离心管下液体,保留超滤柱中液体,并再加入无菌去离子水,反复洗涤离心3次,得到包载靶向PITPNM3小分子抑制剂PEG-PLGA纳米微球,进行包封率检测。结果见图2B,当小分子化合物与载体的质量比高于1:10时,包封率下降并且制备小分子纳米粒中使用材料及成本升高,当小分子化合与载体质量比低于1:10时,则包封率不够稳定。
2、称取聚乳酸-羟基乙酸-聚乙二醇共聚物(缩写为PEG-PLGA)并溶解于二甲基甲酰胺(Dimethyl formamide,DMF)中,得到20mg/mL的PEG-PLGA溶液;使用二甲基亚砜(Dimethyl sulfoxide,DMSO)溶解NO.1小分子抑制剂,溶解浓度为20mg/ml、40mg/ml、60mg/ml;并按照NO.1小分子化合物与PEG-PLGA体积比为1:20将小分子抑制剂溶液加入200μLPEG-PLGA溶液中,水浴超声,0.6Hz,1分钟,制备得到初溶液。在无菌去离子水中,室温下搅拌,用注射器吸取小分子抑制剂溶液与PEG-PLGA溶液的混合初溶液中,深入水相液面下,缓慢加入形成复溶液。将复溶液高速离心2800rpm,10分钟,使用去离子水反复清洗,将洗涤液加入100000MWCO的超滤管,2800rpm/s,离心10min,弃去离心管下液体,保留超滤柱中液体,并再加入无菌去离子水,反复洗涤离心3次,得到包载靶向PITPNM3小分子抑制剂PEG-PLGA纳米微球,进行包封率检测。结果见图2A,可以看出不同浓度之间差异不大,因此选择小分子化合物溶液浓度为40mg/ml进行后续实验。
3、称取聚乳酸-羟基乙酸-聚乙二醇共聚物(缩写为PEG-PLGA)并溶解于二甲基甲酰胺(Dimethyl formamide,DMF)中,得到20mg/mL的PEG-PLGA溶液;使用二甲基亚砜(Dimethyl sulfoxide,DMSO)溶解NO.1小分子抑制剂,溶解浓度为40mg/ml。按照小分子化合物溶液与PEG-PLGA溶液体积比为1:20和1:10;将小分子抑制剂溶液加入200μL PEG-PLGA溶液中,水浴超声,0.6Hz,1分钟,制备得到初溶液。在无菌去离子水中,室温下搅拌,用注射器吸取小分子抑制剂溶液与PEG-PLGA溶液的混合初溶液中,深入水相液面下,缓慢加入形成复溶液。将复溶液高速离心2800rpm,10分钟,使用去离子水反复清洗,将洗涤液加入100000MWCO的超滤管,2800rpm/s,离心10min,弃去离心管下液体,保留超滤柱中液体,并再加入无菌去离子水,反复洗涤离心3次,得到包载靶向PITPNM3小分子抑制剂PEG-PLGA纳米微球,进行包封率检测。结果见图2C,可以看出小分子化合物溶液与PEG-PLGA溶液体积比为1:20时,所得到的纳米微球包封率最佳。
综上,申请人确定小分子化合物浓度为40mg/mL且小分子化合物与PEG-PLGA质量比为1:10与体积比为1:20获得最佳的纳米粒,并将此纳米粒用于后续的实验,后续实验所指的实施例2中的纳米微球均指该纳米粒。
4、使用马尔文ZS90粒度分析仪测量小分子化合物浓度为40mg/mL,质量比1:10,体积比1:20得到纳米微球的性质。结果显示包载靶向PITPNM3小分子抑制剂NO.1号化合物的PEG-PLGA纳米微球,最佳平均粒径为83.38nm,最佳粒径代表图见图3;最佳平均zeta电位为-15.97mV,最佳zeta电位代表图见图3,能够满足我们使用包载靶向PITPNM3小分子抑制剂PEG-PLGA纳米微球转染肿瘤细胞的需求。
实施例3靶向PITPNM3小分子抑制剂的PEG-PLGA纳米微球剂型的生物学效应检测
1、NO.1化合物PEG-PLGA纳米微球吞噬效率检测:
在37℃、5%CO2条件下,用含有10%胎牛血清的DMEM培养基传代及培养MDA-MB-231细胞,分别将50000个MDA-MB-231细胞接种于6孔板中,使用实施例2中得到的NO.1号化合物的PEG-PLGA纳米微球分别加入每一个孔中,使用BD FACSCelesta检测不同时间点0h、1h、6h、12h、24h乳腺癌MDA-MB-231细胞系对纳米微球的吞噬效率,如图4所示,结果显示,在24h时MDA-MB-231吞噬纳米微球的效率能够达到92.4%。
2、transwell法检测靶向PITPNM3小分子抑制剂的PEG-PLGA纳米微球剂型的生物学效应:
transwell侵袭实验是检测细胞侵袭能力的实验方法。其实验原理为:利用一层膜(基质胶)将高营养的培养液和低营养的培养液隔开,细胞放在低营养的培养液里,为了寻找营养,细胞会往高营养的培养液里面迁移,用于模拟细胞分泌金属蛋白酶消化细胞外基质,转移至其他组织的过程。
本实施例利用transwell试验对上述实施例2中获得的靶向PITPNM3小分子抑制剂新剂型,进一步实验的具体过程如下:在37℃、5%CO2和10%胎牛血清的DMEM培养基条件下使用不同浓度的NO.1号化合物的PEG-PLGA纳米微球预先处理MDA-MB-231细胞24h,阴性对照组为纳米粒对照;之后消化细胞,分别使用含有实施例2中得到的NO.1号化合物的PEG-PLGA纳米微球的无血清DMEM重悬预先处理的细胞,并将105个细胞铺于8μm的transwell的上室,在下室中使用含有1%胎牛血清及50ng/mL的CCL18的DMEM培养12小时后,擦去上室细胞,使用4%多聚甲醛固定后,甲紫进行染色并计数,从上室成功迁移的细胞将不会被棉球擦除,通过甲紫染色能够确定迁移到下室的细胞数,以此判断实施例1中得到的NO.1号化合物的PEG-PLGA纳米微球抑制细胞的侵袭迁移能力改变,MDA-MB-231细胞transwell迁移代表图如图5所示。
由图5细胞迁移代表图可以看出,实施例2中得到的NO.1号化合物的PEG-PLGA纳米微球有一定程度抑制细胞transwell迁移和侵袭能力的作用。
实施例4靶向PITPNM3小分子抑制剂的PEG-PLGA纳米微球剂型的体内代谢
1、使用SPF级别的SD雌性大鼠,正常饲养,随机分组,每组3只大鼠,将分别溶解靶向PITPNM3的NO.1化合物的PEG-PLGA纳米微球剂型及溶解于PBS中的化合物干粉原剂型于PBS中,通过单次静脉注射给予大鼠受试物化合物不同剂型后,于不同时间点5min,15min,30min,1h,2h,4h,6h,8h和24h采集血样,经颈静脉方式采血,每个样品采集约0.20mL,EDTA-K2抗凝,采集后放置冰上。血液样本采集后置于冰上,并于1小时之内离心分离血浆(离心条件:6800g,6分钟,2-8℃)。血浆样本在分析前存放时则放于-80℃冰箱内。使用LC-MS/MS测定给予受试物后大鼠血浆中受试物的浓度并计算相关参数(图6)。
结果如图6A所示,靶向PITPNM3小分子抑制剂的PEG-PLGA纳米微球剂型相对于原剂型半衰期延长至原来的4倍。
2、使用SPF级别的balb/c nu-nu裸鼠,正常饲养,随机分组,每组6只,将分别溶解靶向PITPNM3的NO.1化合物的PEG-PLGA纳米微球剂型及原剂型于PBS中,通过单次静脉注射给予裸鼠鼠受试物化合物不同剂型后,于同一时间处死裸鼠,分离裸鼠的心、肝、脾、肺、肾及肿瘤,每个样品采集后进行称重,使用组织匀浆机进行匀浆,再加入PBS使所有组织达到同一体积,然后放置冰上。样本在分析前存放时则放于-80℃冰箱内。使用LC-MS/MS测定不同组织中受试物的浓度并计算相关参数(图6B)。如图6B所示,可以看出靶向PITPNM3小分子抑制剂的PEG-PLGA纳米微球剂型相对于原剂型能够在肿瘤组织内显著蓄积。
上述具体实施方式对本发明作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种纳米组合物,所述纳米组合物的制备原料包含PITPNM3小分子抑制剂和载体,所述载体为PLA、PEG-PLA、PGA、PLGA、PEG-PLGA、PCL、PCLA和PMMA中的一种或多种;所述载体优选为PEG-PLGA。
3.根据权利要求1所述的纳米组合物,其特征在于,所述PITPNM3小分子抑制剂和载体的质量比为1:(1.25~20)。
4.根据权利要求1所述的纳米组合物,其特征在于,所述纳米组合物的纳米粒径为30~400nm。
5.一种权利要求1~4任一项所述的纳米组合物的制备方法,包含以下步骤:
S1:将PITPNM3小分子抑制剂溶液加入载体溶液中,超声制备得到初溶液;
S2:将初溶液加入水相形成复溶液,然后离心、清洗、过滤、离心洗涤。
6.根据权利要求5所述的制备方法,其特征在于:所述PITPNM3小分子抑制剂溶液的浓度为1mg/ml~100mg/ml。
7.根据权利要求5所述的制备方法,其特征在于,所述载体溶液的浓度为1mg/ml~50mg/ml。
8.权利要求1~4任一项所述的组合物在制备给药系统中的应用。
9.权利要求1~4任一项所述的组合物在制备抗肿瘤或抑制肿瘤转移药物中的应用。
10.一种药物,所述药物包含权利要求1~4任一项所述的组合物。
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