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Abstract

Neurokinin 1 receptors (NK1R) are overexpressed on several types of important human cancer cells. Substance P (SP) is the most specific endogenous ligand known for NK1Rs. Accordingly,a new SP analogue was synthesized and evaluated for detection of NK1R positive tumors.[6-hydrazinopyridine-3-carboxylic acid (HYNIC)-Tyr8-Met(O)11-SP] was synthesized and radiolabeled with 99mTc using ethylenediamine-N,N'-diacetic acid (EDDA)and Tricine as coligands. Common physicochemical properties of radioconjugate were studied and in-vitro cell line biological tests were accomplished to determine the receptor mediated characteristics. In-vivo biodistribution in normal and tumor bearingnude mice was also assessed. The cold peptide was prepared in high purity (>99%) and radiolabeled with 99mTc at high specific activities (84-112GBq/µmol) with an acceptable labeling yield (>95%). The radioconjugate was stable in-vitro in the presence of human serum and showed 44% protein binding to human serumalbumin. In-vitro cell line studies on U373MG cells showed an acceptable uptake up to 4.91 ± 0.22% with the ratio of 60.21 ± 1.19% for its specific fraction and increasing specific internalization during 4 h. Receptor binding assays on U373MG cells indicated a mean Kd of 2.46 ± 0.43 nM and Bmax of 128925 ± 8145 sites/cell. In-vivo investigations determined the specific tumor uptake in 3.36 percent of injected dose per gram (%ID/g) for U373MG cells and noticeable accumulations of activity in the intestines and lung. Predominant renal excretion pathway was demonstrated.

Experimental

Labeling of HYNIC-peptide was performed as previously described (-) with 99mTc in the presence of ethylenediamine-N,N'-diacetic acid (EDDA) and tris(hydroxymethyl)methylglycine (Tricine) as coligands using an exchange labeling approach with both present together. In brief, the labeling process was done by adding 10.75 μL of the stock solution (20 μg of peptide), 15 mg of Tricine and 5 mg of EDDA as coligands in 0.5 mL of water. Then, 40 μg SnCl2 (10 μL of freshly prepared 4 mg/mL SnCl2in nitrogen-purged 0.1 M HCl) was added to this solution. Instantly, 30-35 mCi (1110-1295MBq) of freshly eluted sodium pertechnetate (Na99mTcO4) in 0.5 mL normal saline was added to the solution and incubated for 15 min at 95 °C in a shielded container. After cooling down to room temperature, the labeling yield was checked on RP-HPLC.