[99mTc][Tc(N)PNP]-tagging of the small RGDfK peptide: impact of different PNP structures on the labeling and pharmacokinetics of the radioconjugates

Introduction. The [99mTc][Tc(N)(PNP)]-framework (PNP = bisphosphinoamine) is an effective system for the development of tumor ‘receptor-specific' radiotracers. The peculiarity of this technology is the high chemical flexibility, which permits fine-tuning of the physical and chemical properties of the final compounds through the independent variation in the PNP substituents and/or of the coligand. Therefore, chelation systems can be rigorously selected to obtain the right balance between stability, charge, and lipophilicity of complexes, thus improving their pharmacokinetics and making possible modulation of their biological properties [1]. Hence, to select the best performing framework useful for the preparation of [99mTc][Tc(N)(PNP)]-tagged molecular targeting agents, in this study, we compared the reactivity and impact of three chemically different [Tc(N)(PNP)]-synthons (PNP = N-Methoxyethyl-N,N-bis(di-methoxypropyphosphonoethyl)amine, PNP3, NMethoxyethyl- N,N-bis(di-methylphosphonoethyl)amine, PNP43 or N,N-Bis(dihydroxymethylenphosphinoethyl) methoxyethylamine, PNP3OH) on the stability, receptor targeting properties, biodistribution, and metabolism of the corresponding [99mTc][Tc(N)(PNP)]-labeled RGDfK pentapeptide. Methods. RGDfK was first derivatized with a terminal Cys residue and labeled with the [Tc(N)(PNP)]-synthons. Radiolabeled peptides were evaluated for their lipophilicity, stability, in vitro and in vivo targeting properties, and performance. Results. All compounds were equally easy to synthesize and purify (RCP ≥ 95%). Notably, the use of PNP3OH allows the labeling of the peptide at room temperature without significantly reducing the labeling efficiency or stability of the final compound. The main influences of the synthon on the radioconjugate were observed in the in vitro cell binding and in vivo performances. In healthy and xenograft animal models, different pharmacokinetics and tumor accumulation were observed as a function of lipophilicity and sterical hindrance of the [99mTc][Tc(N)(PNP)]-framework [2]. Conclusions. The variation in the substituents on the phosphorus atoms of the PNP lets fine modulation of the biological properties of the radioconjugates. ws[99mTc][Tc(N)(PNP3OH)]– and [99mTc][Tc(N)(PNP3)]- are the better performing synthons in terms of labeling efficiency and in vivo performance than the [99mTc][Tc(N)(PNP43)] framework and are therefore more suitable for further radiopharmaceutical purposes. Furthermore, the good labeling properties of the ws[99mTc][Tc(N)(PNP3OH)]–synthon can be exploited to extend this technology to the labeling of temperature sensitive biomolecules suitable for SPECT imaging. Funding: Associazione Italiana per la Ricerca sul Cancro: AIRC, IG 2020 ID 24528 and Bracco Imaging SpA REFERENCES 1. Bolzati, C.; Salvarese, N.; Spolaore, B.; Vittadini, A.; Forrer, D.; Brunello, S.; Ghiani, S.; Maiocchi, A. Water-Soluble [Tc(N)(PNP)] Moiety for Room-Temperature 99mTc Labeling of Sensitive Target Vectors. Mol. Pharm. 2022, doi:10.1021/acs.molpharmaceut.1c00816. 2. Salvarese, N.; Carpanese, D.; Meléndez-Alafort, L.; De Nardo, L.; Calderan, A.; Biondi, B.; Ruzza, P.; Rosato, A.; Bolzati, C. Impact of Different [Tc(N)PNP]-Scaffolds on the Biological Properties of the Small CRGDfK Peptide: Synthesis, In Vitro and In Vivo Evaluations. Molecules 2022, 27, 2548, doi:10.3390/molecules27082548.