[68Ga]Ga-PentixaFor-PET的临床成功的非侵入性成像结果证实了该靶向概念在采用低成本且广泛可用的常规闪烁显像/SPECT方面的扩展的合理性。为了实现这一目标，我们开发并比较评估了一系列基于PentixaFor基架的99mTc标记环状五肽。通过标准固相合成方法合成了六个mas3-结合的CPCR4类似物，这些类似物具有不同的4-氨基苯甲酸（Abz）-D-Ala-D-Arg-aa3连接剂（L1-L6），以及对应的L6-CPCR4的HYNIC-和N4-类似物。使用Jurkat T细胞淋巴瘤细胞和[125I]FC-131放射配基进行了竞争性结合研究（IC50和IC50inv）。利用hCXCR4过表达的Chem-1细胞研究了内化动力学。使用携带Jurkat异种移植瘤的CB17/SCID小鼠进行了生物分布和小动物SPECT/CT成像（1小时后注射）。根据临床前结果，[99mTc]Tc-N4-L6-CPCR4（[99mTc]Tc-PentixaTec）被选定用于早期进入人体实验。五名血液恶性肿瘤患者接受了[99mTc]Tc-N4-L6-CPCR4 SPECT/平面成像和个体剂量测定。 六个mas3-结合肽中，mas3-L6-CPCR4（mas3-dap-r-a-Abz-CPCR4）显示出最高的CXCR4亲和力（IC50 = 5.0 ± 1.3 nM）。与N4结合（N4-L6-CPCR4）进一步改善了对hCXCR4的亲和力，达到0.6 ± 0.1 nM。[99mTc]Tc-N4-L6-CPCR4也显示出最高的内化效率（2小时时细胞内总活性的97%）和化合物中最高的肿瘤富集（8.6 ± 1.3% iD/g，1小时后注射）。因此，[99mTc]Tc-N4-L6-CPCR4（命名为[99mTc]Tc-PentixaTec）被选定用于首次应用于人体。[99mTc]Tc-PentixaTec耐受性良好，在SPECT和平面成像中显示出良好的生物分布和剂量学特征（每500 MBq的剂量为2.1-3.4 mSv），以及优秀的肿瘤/背景比。成功优化连接剂结构的氨基酸组成和N-末端99mTc标记策略（mas3 vs HYNIC vs N4），提供了[99mTc]Tc-PentixaTec作为一种新型、极具潜力的针对CXCR4的SPECT成像剂，可用于临床应用。由于其优异的CXCR4亲和力、高效的内化、在CXCR4表达组织中的高摄取率、合适的清除/生物分布特性和良好的人体剂量学特征，它具有巨大的临床潜力。© 2023. 作者。

The clinical success non-invasive imaging of CXCR4 expression using [68 Ga]Ga-PentixaFor-PET warrants an expansion of the targeting concept towards conventional scintigraphy/SPECT with their lower cost and general availability. To this aim, we developed and comparatively evaluated a series of 99mTc-labeled cyclic pentapeptides based on the PentixaFor scaffold.Six mas3-conjugated CPCR4 analogs with different 4-aminobenzoic acid (Abz)-D-Ala-D-Arg-aa3 linkers (L1-L6) as well as the corresponding HYNIC- and N4-analogs of L6-CPCR4 were synthesized via standard SPPS. Competitive binding studies (IC50 and IC50inv) were carried out using Jurkat T cell lymphoma cells and [125I]FC-131 as radioligand. Internalization kinetics were investigated using hCXCR4-overexpressing Chem-1 cells. Biodistribution studies and small animal SPECT/CT imaging (1 h p.i.) were carried out using Jurkat xenograft bearing CB17/SCID mice. Based on the preclinical results, [99mTc]Tc-N4-L6-CPCR4 ([99mTc]Tc-PentixaTec) was selected for an early translation to the human setting. Five patients with hematologic malignancies underwent [99mTc]Tc-N4-L6-CPCR4 SPECT/planar imaging with individual dosimetry.Of the six mas3-conjugated peptides, mas3-L6-CPCR4 (mas3-dap-r-a-Abz-CPCR4) showed the highest CXCR4 affinity (IC50 = 5.0 ± 1.3 nM). Conjugation with N4 (N4-L6-CPCR4) further improved hCXCR4 affinity to 0.6 ± 0.1 nM. [99mTc]Tc-N4-L6-CPCR4 also showed the most efficient internalization (97% of total cellular activity at 2 h) and the highest tumor accumulation (8.6 ± 1.3% iD/g, 1 h p.i.) of the compounds investigated. Therefore, [99mTc]Tc-N4-L6-CPCR4 (termed [99mTc]Tc-PentixaTec) was selected for first-in-human application. [99mTc]Tc-PentixaTec was well tolerated, exhibits a favorable biodistribution and dosimetry profile (2.1-3.4 mSv per 500 MBq) and excellent tumor/background ratios in SPECT and planar imaging.The successive optimization of the amino acid composition of the linker structure and the N-terminal 99mTc-labeling strategies (mas3 vs HYNIC vs N4) has provided [99mTc]Tc-PentixaTec as a novel, highly promising CXCR4-targeted SPECT agent for clinical application. With its excellent CXCR4 affinity, efficient internalization, high uptake in CXCR4-expressing tissues, suitable clearance/biodistribution characteristics, and favorable human dosimetry, it holds great potential for further clinical use.© 2023. The Author(s).