Pyridyl-Ala in the third position of radiolabeled somatostatin antagonists: the effect of regioisomeric substitution.

Abstract

Background: The radiolabeled somatostatin receptor subtype 2 (SST₂) antagonists LM3 (Phe(4-Cl)¹-c(DCys²-Tyr³-DAph(Cbm)⁴-Lys⁵-Thr⁶-Cys⁷)-DTyr⁸-NH₂) and JR11 (Phe(4-Cl)¹-c(DCys²-Aph(Hor)³-DAph(Cbm)⁴-Lys⁵-Thr⁶-Cys⁷)-DTyr⁸-NH₂) are under clinical evaluation for imaging and treatment of neuroendocrine tumors. These peptides differ at position 3, where LM3 contains Tyr³, while JR11 incorporates Aph(Hor)³. The amino acid at this position is crucial in the design of somatostatin ligands, agonists and antagonists, influencing affinity and receptor subtype specificity. Pyridylalanine, a nonnatural amino acid, presents three regioisomers 2-pyridylalanine (2Pal), 3-pyridylalanine (3Pal), and 4-pyridylalanine (4Pal), which differ only in the nitrogen atom's position in the aromatic ring, allowing minimal chemical modification. We investigated whether the new somatostatin antagonists DOTA-[2Pal³]-LM3, DOTA-[3Pal³]-LM3 and DOTA-[4Pal³]-LM3, radiolabeled with Lu-177, differ among them and how they compare with the clinically used [¹⁷⁷Lu]Lu-DOTA-LM3.

Results: The synthesis of the DOTA-[2Pal³]-LM3 resulted in the formation of two diastereomers, with the D2Pal derivative lacking receptor recognition and affinity, contrary to the enantiomer L (L2Pal) derivative. The hydrophilicity of [¹⁷⁷Lu]Lu-DOTA-[xPal³]-LM3 increased in the order of L2Pal < 3Pal < 4Pal (logD = -2.3 ± 0.1 -2.5 ± 0.1 and -2.6 ± 0.1, respectively), being similar or significantly higher than [¹⁷⁷Lu]Lu-DOTA-LM3 (logD = -2.3 ± 0.1). Saturation binding studies indicated a trend of affinity improvement by L2Pal < 3Pal < 4Pal (K_D = 0.18 ± 0.02, 0.15 ± 0.01 and 0.11 ± 0.01 nM, respectively), which is similar to [¹⁷⁷Lu]Lu-DOTA-LM3 (K_D = 0.09 ± 0.02 nM). Surprisingly, despite similar accumulation in SST₂-positive tumors, differences were observed in the body distribution. The hydrophilicity of the Pal amino acids is likely responsible for the higher kidney uptake of the three ¹⁷⁷Lu-Pal-radioligands when compared to [¹⁷⁷Lu]Lu-DOTA-LM3. In particular, [¹⁷⁷Lu]Lu-DOTA-[3Pal³]-LM3 is characterized by high uptake and long retention in kidneys, probably due to its high stability in renal tissue. Chromatographic analysis of kidney homogenates revealed that more than 60% of peptide remained intact 1 h after injection.

Conclusions: Our study revealed that the replacement of Tyr³ with Pal³ isomers does not impact on SST₂ affinity, but chirality at this position is critical, as the D2Pal³ derivative loses binding. More interestingly, we demonstrated how the nitrogen's position in the pyridylalanine regioisomers influences the properties of the corresponding radioligand. The polar nature of the 3Pal, due to its electronic density dissymmetry, likely enhances the peptide interaction with specific kidney transporters explaining its high uptake and prolonged retention in renal tissue.