Integrin α_vβ₃ is a heterodimeric transmembrane glycoprotein and is overexpressed in tumor induced angiogenic vessels and in various malignant human tumors. The selective targeting of this receptor with radioligands may enable an assessment of the angiogenesis and receptor status in tumors. Arginylglycylaspartic acid (RGD) peptides and peptidomimetic antagonists specific for a α_vβ₃ receptor have recently been labeled with various gamma and positron emitters for scintigraphic detection and beta emitters for the radiotherapy of tumors (1-8). Steady progress has been reported in optimizing the labeling methodologies for an increase in tumor to non-tumor tissue ratios, especially a tumor to liver ratio by increasing the hydrophilicity of the product through glycosylation and PEGylation (9-12).

In this research, a peptidomimetic integrin α_vβ₃ antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl- 2-(S)-aminoethylsulfonyl-amino-β-alanine (PIA), as an integrin-receptor mediated tumor targeting agent was studied. PIA was previously coupled to a cationic polymerized lipid-based nanoparticle, and was successfully used to deliver a gene to the neovasculature for a tumor regression (13). PIA was also modified with carbamate linkers to conjugate to fluorescein isothiocyanate, an optical probe, for the optical imaging of tumors (14). In this study, PIA was labeled with ^99mTc using organometallic ^99mTc(I) aqua ions, [^99mTc(CO)₃ (H₂O)₃]⁺, as a ^99mTc(I) labeling reagent. Recently, [^99mTc(CO)₃ (H₂O)₃]⁺ has been intensively investigated to incorporate a small [^99mTc(CO)₃]⁺ moiety to biomolecules with various bidentate and tridentate ligands (15-23). To label PIA with [^99mTc(CO)₃]⁺, an aminodiacetic acid (ADA) moiety was conjugated to PIA by reacting it with tris-succinimidyl aminotriacetate (TSAT). The tridentate aminodiacetic acid moiety of the PIA conjugates was facially coordinated to ^99mTc(I) of [^99mTc(CO)₃]⁺ by displacing the tris-aqua molecules. The resulting ^99mTc(CO)₃-ADA-PIA was subjected to a biodistribution in nude mice bearing receptor-positive M- 21 human melanoma xenografts.

The aim of this research was to label PIA with ^99mTc and test its receptor-targeting properties in nude mice bearing a receptor-positive tumor.

Synthesis of PIA-COCH₂N(CH₂CO₂H)₂. We optimized the reaction conditions to acylate the amino group of PIA with one of the three activated esters of TSAT and to hydrolyze the remaining two esters to produce an aminodiacetic acid moiety: PIA (25 mM) was reacted with TSAT at a PIA-to-TSAT molar ratio ranging from 1.2 : 1 to 5 : 1 at pH 8.5 for 1 hr at room temperature (Fig. 1).

Fig. 1.

Preparation of fac-[^99mTc(CO)₃-ADA-PIA]. The resulting product PIA-COCH₂N(CH₂CO₂H)₂ (PIA-ADA) was reacted with [^99mTc(CO)₃(H₂O)₃]⁺¹ at pH 4.5 for 45 min at 70℃ to label the tridentate aminodiacetic acid moiety of the products with [^99mTc(CO)₃]⁺¹ by displacing the tris-aqua groups of [^99mTc(CO)₃(H₂O)₃]⁺¹(1314). The labeled product was purified sequentially on Sep-Pak C-18 and Sep-Pak QMA anion exchange cartridges. The radiochemical purity (> 95%) was confirmed by gradient C-18 reverse-phase HPLC (Ace 5 C18 column, 4.6 × 100 mm, 5 mm, MAC-MOD Analytical Inc.). HPLC solvents consisted of a 0.05 M triethylammonium phosphate (TEAP) buffer, pH 2.25 (solvent A), and acetonitrile (solvent B). The applied gradient was 0 to 2 min, 100% A; 2 to 5 min, from 100 to 80% A; 5 to 12 min, from 80 to 65% A; 12 to 20 min, from 35 to 100% B; and 20 to 25 min, 100% B. The HPLC showed one peak with a retention time of 10.5 min for ^99mTc(CO)₃ADA-PIA (Fig. 2).

Stability study. The stability of ^99mTc(CO)₃ADA-PIA was tested in a 0.15 M sodium phosphate buffer (PB) at pH 7.2 and against two more common trans-chelating reagents, histidine (20 mM in PB at pH 7.2) and cysteine (20 mM in PB at pH 7.2) at 200-times molar excess to the ^99mTc label at 37℃. The solutions were analyzed by RP HPLC as described above.

Fig. 2.

Biodistribution study. Biodistribution was performed in nude mice (n = 5 per time point) bearing receptor-positive M-21 human melanoma xenografts. The mice received intravenous ^99mTc(CO)₃-ADA-PIA (5 μCi/< 0.1 μg). The animals were euthanized at 0.33, 1, and 2 hrs after injection for the biodistribution study. In a separate experiment, a group of mice were co-injected with ^99mTc(CO)₃-ADA-PIA with 200 μg of PIA and euthanized at 1 hr to test if the tumor uptake was blocked with an excess amount of PIA.

Fig. 3.

PIA was conjugated with TSAT to generate an aminodiacetic acid moiety as iminodiacetic acid is one of the smallest tridentates that can complex with [^99mTc(CO)₃]⁺. The conjugation reaction of PIA to TSAT at a molar ratio of 1.2 : 1 produced a higher yield of mono PIA conjugated to TSAT as compared to the reaction at a PIA-to-TSAT molar ratio of 5 : 1. This result was also reflected in the radiolabeling yield; the product from a molar ratio of 1.2 : 1 resulted in a 78% labeling yield, whereas the remaining activity was complexed to nitirilotriacetic acid, the hydrolyzed product of TSAT. ^99mTc(CO)₃-ADA-PIA was stable in PB for 21 hrs, but lost 7.9, 11.5, and 25.4% of the radioactivity to histidine at 3, 6, and 21 hrs. It did not lose ^99mTc to cysteine but produced two polar degraded products composed of 14.6, 37.5, and 100% of the total at 3, 6, and 21 hrs, respectively. The result indicates that cysteine, a reducing agent, is harmful to this product. Since the labeled product was quite stable for 3 hrs under harsh conditions, a biodistribution study followed. In the tumor bearing mice, ^99mTc(CO)₃-ADA-PIA accumulated rapidly in receptor-positive tumors with a peak uptake of 1.2 ± 0.4% ID/g at 20 min, while being cleared rapidly from blood primarily through the hepatobiliary system with 6.5 ± 0.8 and 25.4 ± 2.8% ID/g localized in the liver and intestine, respectively, at that time (Fig. 3). At 20 min, the tumor-to-organ ratios were 1.84, 0.18, 0.69, 1.01, 2.89, 0.22, 0.04, 2.05, and 4.10 for the blood, liver, kidney, lung, heart, stomach, intestine, bone, and muscle, respectively. At 1 hr, the tumor uptake was 0.47 ± 0.15% ID/g, but decreased to 0.12 ± 0.02% ID/g when co-injected with an excess amount of PIA.

The goal of this research was to synthesize ^99mTc labeled peptidomimetic integrin α_vβ₃ antagonists that would target α_vβ₃ receptor-positive tumors and produce high target-tonontarget radioactivity ratio at an early time after injection. In this research, the effect of the receptor binding affinity of the ^99mTc labeled peptidomimetic antagonist on the in vivo kinetics in the tumor was investigated. TSAT was used as a bifunctional chelator. The conjugation reaction was optimized at a PIA-to-TAST molar ratio of 1.2.

The results of a biodistribution and blocking study with an excess amount of PIA indicated that ^99mTc(CO)₃-ADAPIA was accumulated through a receptor mediated uptake. The tumor-to-organ ratios did not improve much over time because the labeled PIA was not retained well in the tumor, which is perhaps due to its low affinity to the receptor.

In conclusion, a peptidomimetic antagonist to integrin was successfully labeled with ^99mTc. It accumulated in the tumor, mediated through specific receptor binding. However, the uptake in the tumor was very low because of its low blood concentration that is ascibed to its rapid excretion into the hepatobiliary system. This study suggests that to be useful as a tumor detection agent, it is necessary to improve the receptor-binding affinity by synthesizing the oligomers of PIA and increase the hydrophilicity of the product by glycosylation and PEGylation to minimize the rapid uptake into the hepatobiliary system.