背景:半胱氨酸-天冬氨酸蛋白酶caspase-3被认为是响应于特定的外在和内在刺激的细胞中凋亡的主要执行者。Caspase-3代表了一种有趣的生物标志物来评估治疗反应,因为许多癌症疗法通过诱导肿瘤细胞死亡发挥其作用。以前开发的caspase-3PET示踪剂由于肿瘤摄取低或缺乏靶标选择性而无法达到常规临床应用。这是癌症患者有效治疗反应评估的两个重要要求。因此,本研究的目的是开发和临床前评估用于细胞凋亡成像的新型caspase-3选择性活性探针(ABP).
结果:开发了caspase-3选择性ABP文库用于肿瘤凋亡检测。在第一次尝试中,抑制剂Ac-DW3-KE(Ac-3Al-Asp-βhLeu-Phe-Asp-KE)在N-末端被18F标记以产生不能充分检测体内细胞凋亡增加的放射性示踪剂。体内无法有效检测活性caspase-3可能归因于缓慢的结合,如体外抑制动力学所示。因此,基于Ac-ATS010-KE(Ac-3Al-Asp-Phe(F5)-Phe-Asp-KE)开发了第二代caspase-3选择性ABP,其结合动力学大大改善了Ac-DW3-KE。我们的基于Ac-ATS010-KE的探针是通过用6种不同的接头修饰N端来制备的。所有的接头修饰对结合动力学的影响有限,目标选择性,和健康小鼠的药代动力学特征。在体外凋亡模型中,与对照组相比,亲水性最低的示踪剂[18F]MICA-316显示凋亡细胞的摄取增加。最后,[18F]MICA-316在体内结直肠癌模型中进行了测试,它显示出有限的肿瘤摄取,并且无法区分未治疗组的肿瘤,尽管证明放射性示踪剂能够在体外结合复杂混合物中的caspase-3。相比之下,磷脂酰乙醇胺(PE)结合放射性示踪剂[99mTc]Tc-杜拉霉素能够识别疾病模型中增加的细胞死亡,使其成为迄今为止开发的性能最佳的治疗反应评估示踪剂。
结论:结论:我们开发了一种新的caspase-3结合型PET示踪剂文库,其保留了与原始抑制剂相似的结合动力学.最有前途的示踪剂,[18F]MICA-316在体外凋亡模型中显示出增加的摄取,并且能够选择性地结合凋亡肿瘤细胞中的胱天蛋白酶-3。为了区分治疗反应性和无反应性肿瘤,下一代caspase-3-选择性ABP将被开发具有更高的肿瘤积累和体内稳定性。
BACKGROUND: The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging.
RESULTS: A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-βhLeu-Phe-Asp-KE) was 18F-labelled on the N-terminus to generate a
radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5)-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the
radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding
radiotracer [99mTc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far.
CONCLUSIONS: In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability.