Radiolabeled high affinity peptidomimetic antagonist selectively targets alpha(v)beta(3) receptor-positive tumor in mice.

OBJECTIVES: The aim of this research was to synthesize radiolabeled peptidomimetic integrin alpha(v)beta(3) antagonists that selectively target integrin alpha(v)beta(3) receptor and clear rapidly from the whole body. METHODS: Integrin alpha(v)beta(3) antagonists, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-beta-alanine (IA) and 4-[2-(3,4,5,6-tetrahydro-pyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-beta-alanine hydrochloride (IAC), a hydrophobic carbamate derivative of IA, were conjugated with 2-p-isothiocyanatobenzyl-DOTA at the amino terminus and labeled with (111)In. The (111)In labeled IA and IAC were subjected to in vitro receptor binding, biodistribution and imaging studies using nude mice bearing the receptor-positive M21 human melanoma xenografts. RESULTS: The (111)In-labeled IA (40%) and -IAC (72%) specifically bound in vitro to alpha(v)beta(3) (0.8 microM) at a molar excess. This receptor binding was completely blocked by a molar excess of cold IA to alpha(v)beta(3). The higher receptor-binding affinity of the (111)In-labeled IAC was reflected in higher tumor uptake and retention: 5.6+/-1.4 and 4.5+/-0.7 %ID/g vs. 3.8+/-0.9 and 2.0+/-0.3 %ID/g for the (111)In-labeled IA at 0.33 and 2 h. The tumor uptakes were inhibited by the co-injection of 200 microg of IA, indicating that the uptake was receptor mediated. These antagonists were excreted primarily via the renal system. The (111)In activity retained in the whole body was quite comparable between the (111)In-labeled IA (24% ID) and the (111)In-labeled IAC (33% ID) at 2 h. The higher peak tumor uptake and longer retention resulted in higher tumor-to-background ratios for the (111)In-labeled IAC at 2 h with 9.7, 2.3, 0.8, 1.9, 7.1, 2.2, 0.9, 3.7 and 9.9 for blood, liver, kidney, lung, heart, stomach, intestine, bone and muscle, respectively. The imaging studies with the (111)In-labeled IAC also clearly visualized the receptor-positive tumor at 4 h. CONCLUSIONS: The (111)In-labeled IAC showed an improve tumor targeting kinetics with rapid accumulation and prolonged retention in the alpha(v)beta(3) receptor-positive tumor. This together with the rapid whole-body clearance pharmacokinetics warrants further studies on this IAC analog for molecular imaging of tumor-induced angiogenic vessels and various malignant human tumors expressing the receptor.

A novel antiangiogenesis therapy using an integrin antagonist or anti-Flk-1 antibody coated 90Y-labeled nanoparticles.

PURPOSE: Integrin alpha(v)beta(3) and vascular endothelial growth factor receptor 2 (Flk-1) have been shown to be involved in tumor-induced angiogenesis. Selective targeting of upregulated alpha(v)beta(3) and Flk-1 on the neovasculature of tumors is a novel antiangiogenesis strategy for treating a wide variety of solid tumors. In the studies described here, we investigated the potential therapeutic efficacy of two three-component treatment regimens using two murine tumor models. METHODS AND MATERIALS: The treatment regimens used nanoparticle (NP) based targeting agents radiolabeled with (90)Y. The small molecule integrin antagonist (IA) 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethoxy]-benzoyl-2-(5)-aminoethylsulfonylamino-beta-alanine, which binds to the integrin alpha(v)beta(3), and a monoclonal antibody against murine Flk-1 (anti-Flk-1 MAb) were used to target the NPs. Murine tumor models K1735-M2 (melanoma) and CT-26 (colon adenocarcinoma) were used to evaluate the treatment efficacy. RESULTS: In K1735-M2 and CT-26 tumors, a single treatment with IA-NP-(90)Y (14.2 microg/g IA, 5 or 6 microCi/g (90)Y) caused a significant tumor growth delay compared to untreated control tumors, as well as tumors treated with IA, IA-NP, and NP-(90)Y, respectively (p < 0.025, Wilcoxon test). In K1735-M2 tumors, a single treatment with anti-Flk-1 MAb-NP-(90)Y (0.36 microg/g anti-Flk-1 MAb, 5 microCi/g (90)Y) also caused a significant tumor growth delay (p < 0.05, Wilcoxon test) compared to untreated tumors, as well as tumors treated with anti-Flk-1 MAb, anti-Flk-1 MAb-NP, and conventional radioimmunotherapy with (90)Y-labeled anti-Flk mAb. Anti-CD31 staining showed a marked decrease in vessel density in tumors treated with anti-Flk-1 MAb-NP-(90)Y, which was associated with a high level of apoptotic death in these tumors, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. CONCLUSIONS: The present studies provide proof of principle that targeted radiotherapy works using different targeting agents on a nanoparticle, to target both the integrin alpha(v)beta(3) and the vascular endothelial growth factor receptor. These encouraging results demonstrate the potential therapeutic efficacy of the IA-NP-(90)Y and anti-Flk-1 MAb-NP-(90)Y complexes as novel therapeutic agents for the treatment of a variety of tumor types.

Tc-labeling of Peptidomimetic Antagonist to Selectively Target alpha(v)beta(3) Receptor-Positive Tumor: Comparison of PDA and EDDA as co-Ligands.

OBJECTIVES: The aim of this research was to synthesize radiolabeled peptidomimetic integrin alpha(v)beta(3) antagonist with (99m)Tc for rapid targeting of integrin alpha(v)beta(3) receptors in tumor to produce a high tumor to background ratio. METHODS: The amino terminus of 4-[2-(3,4,5,6-tetra-hydropyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-beta-alanine hydrochloride (IAC) was conjugated with N-hydroxysuccinimide ester of HYNIC and labeled with (99m)Tc using tricine with either 1,5-pyridinedicarboxylic acid (PDA) or ethylenediamine-N,N'-diacetic acid (EDDA) as the co-ligand. The products, (99m)Tc EDDA(2)/HYNIC-IAC (P1) and (99m)Tc PDA (tricin)/HYNIC-IAC (P2) were subjected to in vitro serum stability, receptor-binding, biodistribution and imaging studies. RESULTS: P1 and P2 were synthesized with an overall yield of >80%. P1 was slightly more stable than P2 when incubated in serum at 37 degrees C for 18 hrs (84 vs 77% intact). The In vitro receptor-binding of P1 was higher than that of P2 (78.02 +/- 13.48 vs 51.05 +/- 14.05%) when incubated with alpha(v)beta(3) at a molar excess (0.8 muM). This receptor binding was completely blocked by a molar excess of an unlabeled peptidomimetic antagonist. Their differences shown in serum stability and the receptor-binding appeared to be related to their biological behaviors in tumor uptake and retention; the 1 h tumor uptakes of P1 and P2 were 3.17+/-0.52 and 2.13+/-0.17 % ID/g, respectively. P1 was retained in the tumor longer than P2. P1 was excreted primarily through the renal system whereas P2 complex was excreted equally via both renal and hepatobiliary systems. Thus, P1 was retained in the whole-body with 27.25 +/- 3.67% ID at 4 h whereas 54.04 +/- 3.57% ID of P2 remained in the whole-body at 4 h. This higher whole-body retention of P2 appeared to be resulted from a higher amount of radioactivity retained in liver and intestine. These findings were supported by imaging studies showing higher tumor-to-abdominal contrast for P1 than for P2 at 3 h postinjection. CONCLUSIONS: P1 showed good tumor targeting properties with a rapid tumor uptake, prolonged tumor retention and fast whole-body clearance kinetics. These findings warrant further investigation of the HYNIC method of (99m)Tc labeling of other peptidomimetic antagonists using EDDA as a coligand.

Use of antibody as carrier of oligomers of peptidomimetic alphavbeta3 antagonist to target tumor-induced neovasculature.

Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin alphavbeta3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino-beta-alanine (IA) to monoclonal antibody (MoAb) to increase integrin alphavbeta3 receptor-binding avidity. To generate sulfhydryl groups, N-succinimidyl-S-acetylthioacetate (SATA) was conjugated to both MoAb and IA. Sulfhydryl groups were then generated upon the deacetylation of the protecting acetyl group from the S-acetylthioacetato (ATA) moiety of MoAb-(ATA)n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The major focus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels of IA-(A-SH) to MoAb-(A-SH)n without causing the inter- and intramolecular cross-linking of MoAb. Stepwise reactions of MoAb-(A-SH)n (15 microM MoAb) with a homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM[PEO]2) at a >50x molar excess to the -SH, followed by the reaction of the purified product MoAb-(A-S-succinimidomaleimido-[PEO]2)n with IA-(A-SH) at pH 7.2 afforded monomeric MoAb-(A-S-succinimido-[PEO]2-succinimido-S-A-IA)n with <10% high molecular weight oligomeric MoAb. Monomeric MoAb-(A-S-S-[PEO]2-S-S-A-IA)10 (MoAb-IA10) radiolabeled with 111In using 2-(p-isothiocyanatobenzyl)cyclohexyl-DTPA and with 125I using the Iodogen method showed >70% bindability to 0.4 microM alphavbeta3. When injected iv to nude mice with the receptor-positive M21 tumor, MoAb-IA10 radiolabeled with both 111In and 125I accumulated rapidly and was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the blood, thereby resulting in increasing tumor-to-blood ratios over time. The tumor uptake was similar between the 125I label and the 111In label for a 44 h period. In contrast, the blood radioactivity was lower, but liver and other organ uptakes were much higher for the 111In label than for the 125I. The 111In label produced higher tumor-to-blood ratios but much lower tumor-to-organ ratios than the 125I. The rapid blood clearance, a short peak tumor uptake time, and a low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesis that MoAb-IA10 primarily binds to alphavbeta3 receptors on angiogenic vessels, but not on the tumor. This hypothesis was substantiated by the fluorescence microscopic analysis of FITC-MoAb-IA10, which showed that FITC-MoAb-IA10 outlined neovasculatures but not tumor cells at 4 and 21 h ex vivo. Additional proof was observed when blood vessels outlined with rhodamine-lectin, which specifically binds to blood vessels, were superimposable on neovasculatures outlined with FITC-MoAb-IA10.

Synthesis, in vitro, and in vivo characterization of an integrin alpha(v)beta(3)-targeted molecular probe for optical imaging of tumor.

Integrin alpha(v)beta(3) is a widely-recognized target for the development of targeted molecular probes for imaging pathological conditions. alpha(v)beta(3) is a cell-surface receptor protein that is upregulated in various pathological conditions including osteoporosis, rheumatoid arthritis, macular degeneration, and cancer. The synthesis of an alpha(v)beta(3)-targeted optical probe 7 from compound 1, and its in vitro and in vivo characterization is described. A series of aliphatic carbamate derivatives of the potent non-peptide integrin antagonist 1 was synthesized and the binding affinity to alpha(v)beta(3) was determined in both enzyme linked immunosorbent assay (ELISA) and cell adhesion inhibition assays. The hydrophobic carbamate-linked appendages improved the binding affinity of the parent compound for alpha(v)beta(3) by 2-20 times. A Boc-protected neopentyl derivative in the series is shown to have the best binding affinity to alpha(v)beta(3) (IC(50)=0.72 nM) when compared to compound 1 as well as to c-RGDfV. Optical probe 7 utilizes the neopentyl linker and demonstrates increased binding affinity and significant tumor cell uptake in vitro as well as specific tumor accumulation and retention in vivo. These results illustrate the potential of employing integrin-targeted molecular probes based on 1 to image a multitude of diseases associated with alpha(v)beta(3) overexpression.