CN114163478B - 锝-99m标记含D-脯氨酸修饰的FAPI衍生物及制备方法和应用 - Google Patents
锝-99m标记含D-脯氨酸修饰的FAPI衍生物及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种结构通式为99mTc(HYNIC‑DP‑FAPI)(tricine/TPPTS)的锝‑99m标记含D‑脯氨酸修饰的FAPI衍生物及制备方法和应用。通过配体HYNIC‑DP‑FAPI的合成以及99mTc(HYNIC‑DP‑FAPI)(tricine/TPPTS)的制备两个步骤，得到99mTc(HYNIC‑DP‑FAPI)(tricine/TPPTS)配合物。该配合物制备简便，放射化学纯度高，稳定性好，在U87MG荷瘤小鼠肿瘤部位有很高的摄取和靶与非靶比值，且在肿瘤中与FAP有特异性结合，是一种有临床应用价值的新型肿瘤放射性药物。
Description
技术领域
本发明属于放射性药物领域，特别涉及锝-99m标记含D-脯氨酸修饰的FAPI衍生物及制备方法和应用。
背景技术
成纤维细胞激活蛋白(FAP)是肿瘤检测和治疗潜在的靶点，通过放射性核素标记成纤维细胞激活蛋白抑制剂(FAPI)及其衍生物用作肿瘤显像剂成为当前国际放射性药物研究的一大热点。本课题组在2020年研制出一种锝-99m标记含异腈的FAPI衍生物(专利号：ZL202011382815.2)，但是该药物存在肝、肾等非靶器官摄取高且肿瘤摄取值也有待提高等不足，因此研制性能更加优良的99mTc标记的FAPI肿瘤显像剂具有重要的现实意义。
连接剂(linker)连接了靶向基团和和与放射性核素相连的螯合基团，对于调节放射性药物的亲和性和在生物体内的生物分布性能发挥重要作用。本发明使用D-脯氨酸作为连接剂，通过改善配合物的亲水性，可以改变放射性药物的代谢途径，改善药代动力学性质，降低肝肠等非靶脏器摄取，提高其在肿瘤等靶器官的摄取。
肼基尼古酰胺(HYNIC)作为一种常用双功能联接剂，其可以与三羟甲基甘氨酸(tricine)和三苯基膦三磺酸钠(TPPTS)作为协同配体来制备稳定的99mTc标记的HYNIC配合物。基于以上背景，本发明以FAPI原料，对其进行结构修饰，转化为以D-脯氨酸修饰的含HYNIC基团的FAPI衍生物(简称为：HYNIC-DP-FAPI)，然后和协同配体tricine/TPPTS与99mTc配位形成稳定的99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物来探求新型肿瘤显像剂，具有重要的科学意义和广阔的应用前景。
发明内容
本发明的目的是提供锝-99m标记含D-脯氨酸修饰的FAPI衍生物及制备方法和应用。
为了实现上述目的，本发明提供的锝-99m标记含D-脯氨酸修饰的FAPI衍生物99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)，其结构如式(I)所示：
该结构式中：HYNIC-DP-FAPI分子中肼基上的氮原子、共配体TPPTS中的磷原子以及tricine中的氧原子和氮原子与99mTc配位得到99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物。
锝-99m标记含D-脯氨酸修饰的FAPI衍生物的制备方法，其制备步骤如下：
a:配体HYNIC-DP-FAPI的合成:
称取适量FAPI于圆底烧瓶中，加入适量DMF溶解，然后加入适量三乙胺和化合物1，60℃反应6小时。反应结束后减压蒸馏除去溶剂，柱层析纯化(二氯甲烷-甲醇)得到配体HYNIC-DP-FAPI。
具体合成路线为：
b:99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物的制备：
称取适量的tricine、TPPTS、HYNIC-DP-FAPI配体溶于生理盐水中，调节溶液pH为5.0，向其中加入适量的新鲜淋洗的Na99mTcO4，沸水浴加热30min即可得到所述的99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物。
通过上述方法制备的99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物的放射化学纯度大于95％，为亲水性物质，体外稳定性良好。其在荷瘤小鼠肿瘤部位有很高的摄取与良好的滞留，注射FAP抑制剂进行抑制后，肿瘤摄取显著性降低(由16.26±2.71％ID/g降低到1.93±0.35％ID/g)，表明其在肿瘤中的摄取与FAP具有特异性。与锝-99m标记的含异腈基的FAPI衍生物[99mTc-(CN-FAPI)6]+相比，在注射1小时后，该药物在U87 MG肿瘤的摄取显著性提高(由1.34±0.13％ID/g提高到16.26±2.71％ID/g)，且在肝、肾等非靶部位摄取明显降低(分别由11.57±1.38％ID/g、29.11±4.62％ID/g降低到2.12±0.25％ID/g、4.49±0.96％ID/g)，达到预期效果。显像结果表明其在肿瘤部位有明显浓集，非靶组织摄取低，且在肿瘤中的摄取可以被FAP抑制剂显著地抑制，是性能优良的可用于肿瘤显像的新型SPECT分子探针。
具体实施方式
下面通过实施例详述本发明：一种锝-99m标记以锝-99m标记含D-脯氨酸修饰的FAPI衍生物，结构通式为99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)，其结构式如下：
该结构式中：HYNIC-DP-FAPI分子中肼基上的氮原子、共配体TPPTS中的磷原子以及tricine中的氧原子和氮原子与99mTc配位得到99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物。
99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物的制备方法，其制备步骤如下：
a.HYNIC-DP-FAPI的合成
称取33mg(0.068mmol)FAPI于圆底烧瓶中，加入1mL DMF溶解，然后加入0.120mL(0.90mmol)三乙胺和45mg(0.084mmol)化合物1，60℃反应6小时。反应结束后减压蒸馏除去溶剂，柱层析纯化(二氯甲烷-甲醇＝3:1)得到配体HYNIC-DP-FAPI 20mg，产率32％。13C NMR(101MHz,DMSO-d6)δ177.36,170.52,168.61,168.05,166.52,158.50,157.42,148.39,148.09,146.55,144.69,141.51,141.31,137.93,132.60,131.32,129.21,128.13,127.32,125.93,125.68,125.34,123.08,119.79,118.39,105.21,88.38,82.07,77.80,70.94,66.39,54.26,51.78,50.35,44.82,41.93,36.93,29.27,28.87,25.82,22.99,20.59；1HNMR(600MHz,Methanol-d4)δ8.96–8.86(m,1H),8.72(dd,J＝15.8,4.4Hz,1H),8.39(d,J＝2.2Hz,1H),8.24–8.14(m,1H),8.01–7.86(m,4H),7.63–7.51(m,1H),7.45(d,J＝8.3Hz,2H),7.38–7.22(m,3H),5.20–4.98(m,2H),4.37–4.19(m,5H),4.14–3.94(m,2H),3.86–3.50(m,5H),3.05–2.66(m,7H),2.36(s,1H),2.26(d,J＝7.4Hz,2H),2.06–1.84(m,4H)；HR-MS(ESI)for C42H43N10O8F2S[M-Na]-:found 885.2965,calcd 885.2959。
b.99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物的制备
称取1mg tricine和2mg TPPTS溶于0.5mL生理盐水中，加入pH为5.0的琥珀酸盐缓冲液调节溶液pH为5.0，向其中依次加入5μg配体HYNIC-DP-FAPI和0.5mL新鲜淋洗的Na99mTcO4(约370MBq)，沸水浴加热30min即可得到所述的99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物。
本发明99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物的性能测定：
1.配合物的鉴定
99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)采用高效液相色谱(HPLC)法鉴定：用C18反向柱，SCL-10AVP型高压液相色谱仪，A相为水(含0.1％三氟乙酸)，B相为乙腈(含0.1％三氟乙酸)，梯度为0-2min B相为10％，2-10min B相由10％变为90％，10-15min B相为90％，15-20min B相由90％变为10％，20-30min B相为10％。进样量为20μL，流速为1mL/min。测定99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)保留时间(Rt)为：10.11min。
2.配合物的脂水分配系数的测定
取0.9mL pH 7.4的磷酸盐缓冲液(0.025mol/L)于5mL离心试管中，在离心试管中加入1mL正辛醇和0.1mL99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)溶液，盖上塞子，充分摇匀，离心5min(5000r/min)。然后分别从有机相和水相中取出3×0.1mL，测定二相的放射性计数，并计算其分配系数P(P＝有机相的放射性活度/水相的放射性活度)，重复五组。测得99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物的脂水分配系数(logP)为-3.32±0.05，表明其为亲水性物质。
3.配合物的稳定性测定
将配合物分别在室温下放置和37℃小鼠血清中放置4小时后测定其放射化学纯度，结果表明99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物在室温下和37℃小鼠血清中放置4小时后放射化学纯度均大于90％，说明其体外稳定性良好。
4.配合物在荷瘤小鼠中的生物分布实验
从荷U87MG肿瘤Balb/c模型小鼠的尾静脉注射0.10mL99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)标记液(约3.7×105Bq)，注射后1h和4h时断头处死小鼠。此外，使用FAPI对99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)进行小鼠体内抑制实验，方法如下：将100μL含有50μg FAPI的生理盐水溶液尾静脉注射小鼠体内，30min后注射0.10mL99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)标记液(约3.7×105Bq)，1h后断头处死小白鼠。取其心、肝、肺、肾、脾、骨、小肠、胃、肌肉、血、肿瘤等有关组织和器官，擦净后称重，并在γ-Counter上测其放射性计数，计算各组织的每克百分注射剂量(％ID/g)。每个时项的小鼠数为3只。结果见表1。
表1 99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)在荷U87MG肿瘤Balb/c裸鼠的生物分布
5.配合物在荷瘤小鼠的SPECT显像
从荷U87MG肿瘤Balb/c模型小鼠的尾静脉注射99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)溶液0.2mL(约37MBq)，1小时后，使用异氟烷气体麻醉。抑制组需要提前30分钟注射100μL含有50μg FAPI的生理盐水溶液，然后注射99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物0.2mL(约37MBq)，1小时后，使用异氟烷气体麻醉。将小鼠俯卧固定，使用SPECT/CT进行显像。SPECT显像结果表明在实验组中99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)在肿瘤中浓集明显，而在抑制组中肿瘤的摄取明显降低，进一步说明其在肿瘤中的摄取具有特异性，表明其可作为亲肿瘤性能优良的新型SPECT分子探针。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，利用L-脯氨酸作为连接剂进行结构修饰后得到的配体经放射性核素标记后得到的放射性制剂均属于本发明要求保护的范围。
Claims (3)
1.一种锝-99m标记含D-脯氨酸修饰的FAPI衍生物，结构通式为99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)，其结构如下式所示：
该结构式中：HYNIC-DP-FAPI分子中肼基上的氮原子、共配体TPPTS中的磷原子以及tricine中的氧原子和氮原子与99mTc配位得到99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物。
2.一种制备方法，其用于获得权利要求1所述的锝-99m标记含D-脯氨酸修饰的FAPI衍生物，其特征在于，其工艺步骤如下：
a:配体HYNIC-DP-FAPI的合成:
称取适量FAPI于圆底烧瓶中，加入适量DMF溶解，然后加入适量三乙胺和化合物1，60℃反应6小时，反应结束后减压蒸馏除去溶剂，柱层析纯化得到配体HYNIC-DP-FAPI；
具体合成路线为：
b:99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物的制备：
称取适量的N-三(羟甲基)甲基甘氨酸、三苯基膦三间磺酸钠、HYNIC-DP-FAPI配体溶于生理盐水中，调节溶液pH为5.0，向其中加入适量的新鲜淋洗的Na99mTcO4，沸水浴加热30min即可得到所述的99mTc(HYNIC-DP-FAPI)(tricine/TPPTS)配合物。
3.如权利要求1所述的锝-99m标记含D-脯氨酸修饰的FAPI衍生物在制备肿瘤显像剂中的应用。
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