Synthesis, Characterization, and Biological Evaluation of 99m Tc(CO) 3 -Labeled Peptides for Potential Use as Tumor Targeted Radiopharmaceuticals Rinku Baishya 1 , Dipak K. Nayak 1 , Nabanita Chatterjee 2 , Kamal K. Halder 1 , Sanmoy Karmakar 3 and Mita C. Debnath 1, * 1 Nuclear Medicine Department, Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India 2 Cancer Biology and Inflammatory Disorder Division, CSIR- Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India 3 Department of Pharmaceutical Technology, Jadavpur University, Kolkata, 700 032, India *Corresponding author: Mita C. Debnath, mitacd@iicb.res.in, mita_chdebnath@yahoo.com During the past decade, several peptides containing Arg-Gly-Asp sequence have been conjugated with dif- ferent chelating agents for labeling with various radio- nuclides 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 hexa- peptide [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 [ 99m Tc(CO) 3 (H 2 O) 3 ] + intermediate. A complex- ation yield of over 97% was achieved under mild condi- tions even at low ligand concentrations of 10 À2 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 attain- ing maximum value of about 40–60% after 2–4 h incubation). A good percentage of quick internalization was also observed in a v b 3 -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. 99m Tc(CO) 3 -tetra-Pep2 exhibited remarkable localiza- tion 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, 99m Tc(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. Key words: 99m Tc(CO) 3 -labeled peptides, internalization, pep- tide synthesis, tumor imaging Received 10 January 2013, revised 8 March 2013 and accepted for publication 3 May 2013 Technetium-99m is the most widely used radioisotope in diagnostic nuclear medicine owing to its ideal physical characteristics (t 1/2 6 h, photon energy 140 KeV, no cor- puscular radiation) and commercial availability from 99 Mo/ 99m Tc generator (1). The potential to incorporate this radionuclide into different targeting vectors has been the foremost consideration in developing diagnostic radiophar- maceuticals. In the past few years, 99m Tc-labeled, biologi- cally active, receptor-specific peptides have aroused increasing interest for imaging tumors, inflammations, and sites of infection (2–4). Angiogenesis, the formation of new blood vessels from an existing vasculature is a complex biochemical process and plays a prominent role in tumor growth and metastasis (5). This vascularization is a complex phenomenon and guided by various factors among them the interaction of a v b 3 inte- grin, the heterodimeric cell surface receptor with triplet peptides Arg-Gly-Asp (RGD) sequenced in the proteins of the extracellular matrix play an important role (6). Radiola- beled peptides containing the RGD amino acid sequence have been studied extensively to develop site-directed tar- geting vectors for integrin receptors upregulated on tumor cells and neovasculature. Different linear and cyclic pep- tides bearing RGD tri-peptide sequence were developed and labeled with different radioisotopes, for example, 64 Cu, 18 F, 99m Tc, 125 I, 188 Re, 111 In, and 90 Y for imaging and therapeutic purposes (7–9). 64 Cu- and 18 F-labeled tet- rameric and octameric cyclic RGD peptides were reported for tumor imaging by PET. 58 ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12166 Chem Biol Drug Des 2014; 83: 58–70 Research Article