Synthesis, characterization, and biological evaluation of ^99mTc(CO)₃-labeled peptides for potential use as tumor targeted radiopharmaceuticals

During the past decade, several peptides containing Arg-Gly-Asp sequence have been conjugated with different chelating agents for labeling with various radionuclides for the diagnosis of tumor development. In this study, we report the synthesis of two tetrapeptides (Asp-Gly-Arg-His and Asp-Gly-Arg-Cys) and one hexapeptide [Asp-Gly-Arg-D-Tyr-Lys-His] by changing the amino acid sequence of the Arg-Gly-Asp motif. Peptide synthesis was initiated from aspartic acid. Aspartic acid placed at C-terminal end of the peptide chain can be conjugated with different drug molecules facilitating their transport to the site of action. The peptides were synthesized in excellent yield and labeled using freshly prepared [^99mTc(CO)₃(H₂O) ₃]⁺ intermediate. A complexation yield of over 97% was achieved under mild conditions even at low ligand concentrations of 10 ^-2 m. Radiolabeled peptides were characterized by HPLC and were found to be substantially stable in saline, in His solution as well as in rat serum and tissue (kidney, liver) homogenates. Internalization studies using Ehrlich ascites carcinoma cell line showed rapid and significant internalization (30-35% at 30 min of incubation attaining maximum value of about 40-60% after 2-4 h incubation). A good percentage of quick internalization was also observed in α_vβ₃-receptor-positive B16F10 mouse melanoma cell line (14-16% after 30 min of incubation and 25-30% after 2-4 h incubation). Imaging and biodistribution studies were performed in Swiss albino mice bearing Ehrlich ascites tumor in right thigh. Radiolabeled peptides exhibited fast blood clearance and rapid elimination through the urinary systems. ^99mTc(CO)₃-tetra-Pep2 exhibited remarkable localization at tumor site (1.15%, 1.17%, and 1.37% ID/g at 2, 4, and 6 h p.i., respectively) which could be due to slow clearance of the radiolabeled peptide from blood in comparison with the other two radiolabeled peptides. However, ^99mTc(CO)₃-hexa-Pep exhibited the highest tumor to muscle and tumor to blood ratios among the three. The preliminary results with these amino acid-based peptides are encouraging enough to carry out further experiments for targeting tumor. Three linear peptides were synthesised by changing the amino acid sequence of RGD motif and radiolabeled with freshly prepared [^99mTc(CO)₃(H₂O)₃] ⁺ precursor (yield ~ 97%). Radiolabeled peptides were substantially stable up to 24 h when incubated with saline, serum and histidine solution (10^-2 m), also exhibited significant and rapid internalization in both EAC and α_vβ₃-positive B16F10 mouse melanoma cell line. Fast blood clearance, rapid urinary excretion and significantly high tumor to muscle ratios are encouraging enough to carry out further experiments for targeting tumor.