CN114989256A - 一种放射性同位素标记的分子探针及其制备方法与应用 - Google Patents
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Abstract
本发明涉及高分子多肽化合物与核医学显像技术领域,具体提供一种放射性同位素标记的分子探针及其制备方法与应用,本发明提供的T细胞免疫球蛋白黏蛋白‑3(TIM‑3)靶向的多肽类PET分子探针,该分子探针对于对靶标TIM‑3具有较好的亲和力,对多种肿瘤微环境中的TIM‑3表达水平进行高效、准确的活体显像,具有较高的区分度,活体显示肿瘤微环境中的TIM‑3表达水平不仅可为以TIM‑3为靶点的抑制药物的患者选择提供重要参考,还可以实时检测体内TIM‑3的表达情况,以便于临床可及时调整肿瘤的免疫治疗方案;同时,该小分子探针选择性的靶向靶标TIM‑3,也即是能准确靶向肿瘤部位,能够对肿瘤的部位进行精准的PET显像,具有较好的应用前景。
Description
技术领域
本发明涉及高分子多肽化合物与核医学显像技术领域,具体涉及一种放射性同位素标记的分子探针及其制备方法与应用。
背景技术
正电子发射型计算机断层显像(Positron Emission Computed Tomography,PET)是唯一可在活体上显示生物分子代谢、受体及神经介质活动的新型影像技术,现已广泛用于多种疾病的诊断与鉴别诊断、病情判断、疗效评价、脏器功能研究和新药开发等方面。PET检查主要是用于肿瘤的检查,因为绝大部分恶性肿瘤葡萄糖代谢高,FDG作为与葡萄糖结构相似的化合物,静脉注射后会在恶性肿瘤细胞内积聚起来,所以PET能够鉴别恶性肿瘤与良性肿瘤及正常组织,同时也可对复发的肿瘤与周围坏死及瘢痕组织加以区分,现多用于肺癌、乳腺癌、大肠癌、卵巢癌、淋巴瘤,黑色素瘤等的检查,其诊断准确率在90%以上。
T淋巴细胞免疫球蛋白黏蛋白3(T cell immunoglobulin domain and mucindomain-3,TIM-3)又称HAVCR2,属于TIM基因家族,是一种Ⅰ型膜蛋白,由281个氨基酸组成。TIM-3作为一种负调控的免疫检查点,存在于不同类型的免疫细胞中,包括T细胞、调节性T细胞(Tregs)、树突状细胞(DC)、B细胞、巨噬细胞、自然杀伤细胞(NK)和肥大细胞。在肿瘤免疫过程中,TIM-3通过介导T细胞耗竭抑制抗肿瘤免疫。TIM-3阳性的CD8+T细胞存在Stat5和p38信号通路受损。阻断TIM-3途径可增强肿瘤细胞免疫功能,促进T细胞产生干扰素-γ(IFN-γ)。在体内外模型中,TIM-3阳性CD8+T细胞的表达与PD-1的表达呈正相关(P<0.05)。同时TIM-3可抑制IL-2等细胞因子产物的增殖及其效应。PD-1和TIM-3阳性CD8+T细胞产生的IFN-γ低于TIM-3阴性的CD8+T细胞,抗TIM-3抗体也能增加外周NK细胞的IFN-γ。表达TIM-3的肥大细胞可通过含有ITAM的IgE受体(FcepsilonRI)被激活,其信号通路类似于T细胞中的信号通路。TIM-3作用于受体近端位点,增强Lyn激酶依赖的信号通路,调节FcepsilonRI连接的速发期脱颗粒及晚期细胞因子的产生。在非小细胞肺癌(NSCLC)、肝细胞癌(HCC)、大肠癌、宫颈癌、卵巢癌、头颈部癌等组织中均可检测到TIM-3的表达,而且越来越多的临床前研究表明,TIM-3可以提高癌症免疫治疗的疗效。
目前对于肿瘤微环境中TIM -3表达水平的高效活体显像检测未见报道,开发一种靶向TIM-3的PET分子探针是本领域技术人员亟需解决的关键技术问题。
发明内容
为解决上述技术问题,本发明提出了一种放射性同位素标记的分子探针及其制备方法与应用,其目的在于提供一种高效选择性靶向TIM-3的分子探针,能够对多种肿瘤微环境中的TIM-3表达水平进行高效、准确活体显像。
为了实现上述目的,本发明首先提供了一种放射性同位素标记的分子探针,其化学结构式如下式I所示:
式I;
所述式I中R为螯合基团、X为放射性同位素。
作为优选,所述螯合基团为NOTA、DOTA和DTPA中的一种,所述放射性同位素为68Ga、Al18F、177Lu、89Zr、64Cu、225AC中的一种,其中螯合基团NOTA、DOTA和DTPA的结构式分别如下式II、式III和式IV所示:
式II;
式III;
式IV。
作为优选,所述分子探针由式V所示的分子探针前体为原料制备而成:
式V。
作为优选,所述螯合基团为NOTA,所述放射性同位素为68Ga,所述分子探针化学结构式如下式VI所示:
式VI。
基于一个总的发明构思,本发明还提供一种所述放射性同位素标记的分子探针的制备方法,包括以下步骤:
S1、将分子探针前体与NaOAc水溶液混和均匀并转移至反应管中;
S2、用HCl将放射性同位素淋洗至反应管中,90 ℃的条件下反应10 min,加入去离子水淬灭反应;
S3、将步骤S2中反应管中溶液过 C18 Plus柱进行富集,并用去离子水清洗C18Plus柱,然后依次用乙醇和生理盐水将产物淋洗至装有滤膜产品瓶中,制得放射性同位素标记的分子探针。
作为优选,所述步骤S1中分子探针前体的浓度为10~30 nM。
作为优选,所述步骤S2中HCL淋洗放射性同位素至淋洗液放射性活度为30-35mCi。
作为优选,所述步骤S3中乙醇和生理盐水的体积比为1:10,所述制得的放射性同位素标记的分子探针放射性活度为20~25mCi。
基于一个总的发明构思,本发明还提供一种所述放射性同位素标记的分子探针在制备T细胞免疫球蛋白黏蛋白-3靶向的PET显像剂中的应用。
基于一个总的发明构思,本发明还提供一种所述放射性同位素标记的分子探针在制备肿瘤靶向的PET显像剂中的应用。
与现有技术相比,本发明具有以下有益效果:
1、本发明提供了一种放射性同位素标记的分子探针,该分子探针对于对靶标T细胞免疫球蛋白黏蛋白-3(TIM-3)具有较好的亲和力,对多种肿瘤微环境中的TIM-3表达水平进行高效、准确的活体显像,具有较高的区分度,活体显示肿瘤微环境中的TIM-3表达水平可为以TIM-3为靶点的抑制药物的患者选择提供重要参考,还可以实时检测体内TIM-3的表达情况,以便于临床可及时调整肿瘤的免疫治疗方案;
2、该小分子探针选择性的靶向靶标TIM-3,而靶标TIM-3特异性的在肿瘤部位进行富集,小分子探针靶向并活体显示出TIM-3便可准确对肿瘤部位进行活体显像,为肿瘤的PET 显像提供一种可靠的分子探针。
3、本发明提供的放射性同位素标记的分子探针制备过程简单,条件温和,制得的分子探针纯度接近100%,具有良好的体外稳定性,生物安全性高,利于市场大规模推广运用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实验例1中68Ga-NOTA-AI-11放射化学纯度HPLC图;
图2为实验例2中68Ga-NOTA-AI-11放置3h后的放射化学纯度HPLC图;
图3为实验例3中PBMC经PHA诱导上调之后对68Ga-NOTA-AI-11的摄取变化图;
图4为实验例4中68Ga-NOTA-AI-11分子探针的小鼠PET成像图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段;若未特别指明,实施例中所用试剂均为市售。
实施例1
制备放射性同位素标记的分子探针(68Ga-NOTA-AI-11)
1)向含有30 µg前体化合物(NOTA-AI-11)的EP管中加入1 mL 0.25M NaOAc水溶液,混匀;
2)NOTA-AI-11溶液转移至20 mL反应管中;
3)用4 mL 0. 05 M HCl将68Ga淋洗至反应管中,放射性活度为30mCi;
4)在反应管中,90 ℃的条件下反应10 min,反应式如下:
5)加入10 mL去离子水淬灭反应;
6)反应体系过 C18 Plus柱进行富集,并用10 mL去离子水清洗C18 Plus柱;
7)依次用1 mL乙醇和10 mL生理盐水将产物淋洗至装有滤膜产品瓶中,制得68Ga-NOTA-AI-11产品, 放射性活度为23.8 mCi 。
实验例1
68Ga-NOTA-AI-11产品进行HPLC纯度分析
流动相A为含0.1% TFA的蒸馏水,流动相B为含0.1% TFA 的乙腈,色谱柱为ZORBAXSB-C18。洗脱方式为梯度洗脱(0-2 mim:5%乙腈;3-15min:90% 乙腈)。
得到的PET分子探针的放化纯度HPLC结果如图1所示。结果显示68Ga-NOTA-AI-11溶液中只有唯一放射性峰,表明溶液中68Ga-NOTA-AI-11的放化纯度接近100%,能够满足临床显像需求。
实验例2
68Ga-NOTA-AI-11体外稳定性
取适量68Ga-NOTA-AI-11溶液,常温静置3h后进行HPLC纯度分析,分析条件为:流动相A为含0.1% TFA的蒸馏水,流动相B为含0.1% TFA 的乙腈,色谱柱为ZORBAX SB-C18。洗脱方式为梯度洗脱(0-2 mim:5%乙腈;3-15min:90% 乙腈)。得到的PET分子探针的放化纯度HPLC结果如图2所示。结果显示68Ga-NOTA-AI-11溶液中依然只有唯一放射性峰,与实验例1进行的产品HPLC纯度分析图1比较没有明显变化,图2结果表明68Ga-NOTA-AI-11的体外稳定性好。
实验例3
68Ga-NOTA-AI-11细胞摄取实验
聚蔗糖-泛影葡胺(Ficoll-Hypaque)分离法提取的健康志愿者外周血单个核细胞(PBMC)均匀置于24孔板内,分为对照组和实验组。其中实验组在每孔内加入植物血凝素(PHA)用于T细胞,NK细胞等的活化。两组分别加入含有放射性药物的溶液,放置于细胞培养箱培养30min后去除培养液,用 PBS 冲洗两次后加入NaOH裂解细胞,收集细胞裂解液并用γ计数仪测量每孔细胞裂解液的放射性计数。实验结果如图3所示,表明PHA活化T细胞,NK细胞等之后能够明显增加此类细胞对探针的摄取能力。
实验例4
68Ga-NOTA-AI-11动物显像实验
1、购买40只C57小鼠,随机分为四组,每组10只小鼠,其中一组作为对照组,正常养殖,另外三组作为实验组,实验组分别构建皮下黑色素瘤B16模型,胰腺癌PANC02模型和结直肠癌MC38模型,肿瘤大小为0.8cm左右时作为模型鼠备用;
2、模型鼠尾静脉注射68Ga-NOTA-AI-11(0.1-0.2 mCi);
3、30min后进行小动物PET/CT显像,结果如图4所示,图4中圈内为肿瘤位置。
由图4结果可知,注射68Ga-NOTA-AI-1130min时即可清洗观察到小鼠的肿瘤位置,说明本发明所述的分子探针能够快速在肿瘤部分聚集,且对比度高,说明探针对肿瘤的靶向性较好。
分析获取肿瘤部位的标准摄取SUV值,其中68Ga-NOTA-AI-11探针在黑色素瘤B16模型,胰腺癌PANC02模型和结直肠癌MC38模型中肿瘤摄取SUV值分别为4.4±1.5、3.1±0.9和3.9±1.3,表明68Ga-NOTA-AI-11能较好的对多种肿瘤模型中的TIM-3表达量进行定量分析检测,且有较高的对比度。
以上所述实施例,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明的技术范围内,根据本发明的技术方案及其构思加以等同替换或改变,都应涵盖在本发明的保护范围内。
Claims (10)
2.根据权利要求1所述放射性同位素标记的分子探针,其特征在于,所述螯合基团为NOTA、DOTA和DTPA中的一种,所述放射性同位素为68Ga、Al18F、177Lu、89Zr、64Cu、225AC中的一种。
5.一种如权利要求1~4所述放射性同位素标记的分子探针的制备方法,其特征在于,包括以下步骤:
S1、将分子探针前体与NaOAc水溶液混和均匀并转移至反应管中;
S2、用HCl将放射性同位素淋洗至反应管中,90 ℃的条件下反应10 min,加入去离子水淬灭反应;
S3、将步骤S2中反应管中溶液过 C18 Plus柱进行富集,并用去离子水清洗C18 Plus柱,然后依次用乙醇和生理盐水将产物淋洗至装有滤膜产品瓶中,制得放射性同位素标记的分子探针。
6.根据权利要求5所述的制备方法,其特征在于,所述步骤S1中分子探针前体的浓度为10~30 nM。
7.根据权利要求5所述的制备方法,其特征在于,所述步骤S2中HCL淋洗放射性同位素至淋洗液放射性活度为30-35 mCi。
8.根据权利要求5所述的制备方法,其特征在于,所述步骤S3中乙醇和生理盐水的体积比为1:10,所述制得的放射性同位素标记的分子探针放射性活度为20~25mCi。
9.一种如权利要求1~4任一所述的放射性同位素标记的分子探针或权利要求5~8任一项所述制备方法制得的放射性同位素标记的分子探针在制备T细胞免疫球蛋白黏蛋白-3靶向的PET显像剂中的应用。
10.一种如权利要求1~4任一所述的放射性同位素标记的分子探针或权利要求5~8任一项所述制备方法制得的放射性同位素标记的分子探针在制备肿瘤靶向的PET显像剂中的应用。
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