Radiolabeled α(ν)β₃ integrin antagonists: A new class of tracers for tumor targeting

The α(ν)β₃ integrins play an important role during tumor metastasis and tumor-induced angiogenesis. Targeting of this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Cyclo(-Arg-Gly-Asp-D-Phe-Val-) has been shown to be a selective c{ν)β₃ integrin antagonist with high affinity. In this study we describe the synthesis and biological evaluation of [¹²⁵I]-3-iodo-D- Tyr⁴-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) ([¹²⁵I]P2), [¹²⁵I]-3-iodo-Tyr⁵- cyclo(-Arg-Gly-Asp-D-Phe-Tyr-) ([¹²⁵I]P4) and the negative control peptide [¹²⁵I]-3-iodo-D-Tyr⁴-cyclo(-Arg-D-Ala-Asp-Tyr-Val-) ([¹²⁵I]P6). Methods: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl amino acid coupling protocols. Radioiodination was performed using the iodogen method. The in vitro binding assays were performed using isolated, immobilized α(IIb)β₃ and α(ν)β₃ integrins. Expression of the α(ν)β₃ receptor on the different tumors was validated by immunohistochemical methods using α(ν) and α(ν)β₃ specific antibodies. For biodistribution studies, nude mice with melanoma M21 or mammary carcinoma MaCaF and BALB/c mice with osteosarcoma were used. Results: The in vitro binding assays demonstrate that the introduction of tyrosine and subsequent iodination have no influence on the high affinity and selectivity for α(ν)β₃. Immunohistochemical staining clearly indicates the presence of the α(ν)β₃ integrins on the tumor tissue of the melanoma and the osteosarcoma. Pretreatment and displacement studies show specific binding of [¹²⁵I]P2 on melanoma M21-bearing nude mice and osteosarcoma-bearing BALB/c mice but less specific binding on mammary carcinomas. [¹²⁵I]P2 exhibits fast elimination kinetics. The accumulation in the tumor 10 min postinjection is 2.07 ± 0.32 %ID/g for the melanoma M21 and 3.50 ± 0.49 %ID/g for the osteosarcoma and decreases to 1.30 ± 0.13 %ID/g and 2.03 ± 0.49 %ID/g 60 min postinjection, respectively. [¹²⁵I]P4 shows even faster elimination kinetics, resulting in a tumor accumulation of 0.40 ± 0.10 %ID/g 60 min postinjection for the osteosarcoma-bearing BALB/c mice. Both peptides reveal predominately hepatobiliary excretion. For [¹²⁵I]P2, this also is confirmed by autoradiography. The negative control peptide [¹²⁵I]P6 shows no specific activity accumulation. Conclusion: [¹²⁵I]P2 exhibits high affinity and selectivity for the α(ν)β₃ integrin in vitro and in vivo and, thus, represents the first radiolabeled α(ν)β₃ antagonist for the investigation of angiogenesis and metastasis in vivo.