Development of a GRPR-Targeted ^99mTc/¹⁸⁸Re Theranostic Pair Based on NOTA-P2-RM26

Background: Gastrin-releasing peptide receptor (GRPR)-targeted radioligands are promising tools for imaging and treatment of GRPR-expressing malignancies. This study aimed to develop a NOTA-P2-RM26-based ^99mTc/¹⁸⁸Re theranostic pair using the fac-[M(CO)₃]⁺ core, with fac-[^99mTc][Tc(CO)₃(NOTA-P2-RM26)], ^99mTc2, as the diagnostic SPECT analogue and fac-[¹⁸⁸Re][Re(CO)₃(NOTA-P2-RM26)], ¹⁸⁸Re2, as the potential therapeutic counterpart. Methods: NOTA-P2-RM26 was radiolabeled with the fac-[^99mTc(CO)₃(H₂O)₃]⁺, ^99mTc1, precursor after systematic optimization of peptide amount, pH, buffer, temperature, and reaction time. The radioconjugate was purified by HPLC, reformulated by solid-phase extraction, and evaluated by radio-HPLC and radio-TLC. We assessed stability in the formulation, human serum, cysteine and histidine over 6 h. Lipophilicity was determined as logD_7.4. We evaluated in vitro specificity, cell association, and internalization in PC-3 cells. We performed biodistribution, blocking, dynamic imaging, and SPECT/CT imaging in PC-3 xenograft-bearing mice. The corresponding ¹⁸⁸Re analog was prepared under analogous conditions and evaluated for physicochemical matching. Results: Optimized labeling afforded ^99mTc2 with approximately 85% radiochemical conversion and >97% radiochemical purity after purification. The compound remained stable in formulation, with 93% of the tracer intact after 6 h, and exhibited a hydrophilic profile (logD_7.4 = -1.5 ± 0.2). In PC-3 cells, uptake reached 20.0 ± 1.0% of added activity and was reduced to 1.4 ± 0.3% under blocking conditions. In vivo, tumor uptake was 15.0 ± 1.0 %IA/g at 30 min and 17.0 ± 2.2 %IA/g at 1 h p.i. Blocking reduced tumor uptake to 0.84 ± 0.12 %IA/g, corresponding to approximately 95% inhibition. Pancreatic uptake decreased from 8.0 ± 0.4 %IA/g at 30 min to 3.0 ± 0.35 %IA/g at 1 h and was reduced to 0.20 ± 0.08 %IA/g after blocking. At 1 h p.i., tumor-to-blood, tumor-to-muscle, tumor-to-pancreas, and tumor-to-kidney ratios were approximately 21.3, 130.8, 5.7, and 3.6, respectively. SPECT/CT imaging confirmed clear, blockable visualization of the PC-3 tumor. The ¹⁸⁸Re analog was obtained with approximately 80% radiochemical conversion and >95% radiochemical purity post purification. It showed similar stability values to ^99mTc2 under the tested conditions and a logD_7.4 of -1.6 ± 0.2. Conclusions: Complex ^99mTc2 demonstrated efficient preparation, high stability, GRPR-mediated tumor uptake, favorable tumor-to-background ratios, and clear SPECT/CT visualization of PC-3 xenografts. The corresponding ¹⁸⁸Re analog supports the feasibility of a chemically matched ^99mTc/¹⁸⁸Re NOTA-P2-RM26 theranostic platform and warrants further evaluation of ¹⁸⁸Re biodistribution, dosimetry, and therapeutic efficacy.