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

Molecular Imaging Laboratory, Clinical Center, National Institutes of Allergies and Infectious Diseases, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA.
Bioorganic & Medicinal Chemistry (Impact Factor: 2.79). 07/2005; 13(11):3763-71. DOI: 10.1016/j.bmc.2005.03.024
Source: PubMed


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.

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    • "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. "
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    ABSTRACT: The selective targeting of an integrin αvβ3 receptor using radioligands may enable the assessment of angiogenesis and integrin αvβ3 receptor status in tumors. The aim of this research was to label a peptidomimetic integrin αvβ3 antagonist (PIA) with (99m)Tc(CO)3 and to test its receptor targeting properties in nude mice bearing receptor-positive tumors. PIA was reacted with tris-succinimidyl aminotriacetate (TSAT) (20 mM) as a PIA per TSAT. The product, PIA-aminodiacetic acid (ADA), was radiolabeled with [(99m)Tc(CO)3(H2O)3](+1), and purified sequentially on a Sep-Pak C-18 cartridge followed by a Sep-Pak QMA anion exchange cartridge. Using gradient C-18 reverse-phase HPLC, the radiochemical purity of (99m)Tc(CO)3-ADA-PIA (retention time, 10.5 min) was confirmed to be > 95%. Biodistribution analysis was performed in nude mice (n = 5 per time point) bearing receptor-positive M21 human melanoma xenografts. The mice were administered (99m)Tc(CO)3-ADA-PIA intravenously. The animals were euthanized at 0.33, 1, and 2 hr after injection for the biodistribution study. A separate group of mice were also co-injected with 200 μg of PIA and euthanized at 1 hr to quantify tumor uptake. (99m)Tc(CO)3-ADA-PIA was stable in phosphate buffer for 21 hr, but at 3 and 6 hr, 7.9 and 11.5% of the radioactivity was lost as histidine, respectively. In tumor bearing mice, (99m)Tc(CO)3-ADA-PIA accumulated rapidly in a receptor-positive tumor with a peak uptake at 20 min, and rapid clearance from blood occurring primarily through the hepatobiliary system. At 20 min, the tumor-toblood ratio was 1.8. At 1 hr, the tumor uptake was 0.47% injected dose (ID)/g, but decreased to 0.12% ID/g when co-injected with an excess amount of PIA, indicating that accumulation was receptor mediated. These results demonstrate successful (99m)Tc labeling of a peptidomimetic integrin antagonist that accumulated in a tumor via receptor-specific binding. However, tumor uptake was very low because of low blood concentrations that likely resulted from rapid uptake of the agent into the hepatobiliary system. This study suggests that for (99m)Tc(CO)3-ADA-PIA to be useful as a tumor detection agent, it will be necessary to improve receptor binding affinity and increase the hydrophilicity of the product to minimize rapid hepatobiliary uptake.
    Toxicological Research 03/2013; 29(1):21-25. DOI:10.5487/TR.2013.29.1.021
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    • "After obtaining the curve fittings by SigmaPlot (Figure 2A), the calculated IC50 concentration of inhibitors RGDfK and IAC for vitronectin binding to integrin αvβ3 were 4.05 nM and 3.38 nM, respectively. These results confirmed the relatively comparable binding affinities of IAC and RGDfK to integrin αvβ3 protein and closely matched the reported IC50 of 2.94 ± 0.19 nM for IAC by Burnett et al.27 The lower IC50 of RGDfK than that noted by Burnett et al for cyclo-( RGDfV) (6.41 ± 0.49 nM) might be due to the different amino acids in the two peptides valine (V) and lysine (K), with the amine group in lysine potentially affording greater protein binding. "
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    ABSTRACT: Gold nanoshells (NSs) have already shown great promise as photothermal actuators for cancer therapy. Integrin αvβ3 is a marker that is specifically and preferentially overexpressed on multiple tumor types and on angiogenic tumor neovasculature. Active targeting of NSs to integrin αvβ3 offers the potential to increase accumulation preferentially in tumors and thereby enhance therapy efficacy. Enzyme-linked immunosorbent assay (ELISA) and cell binding assay were used to study the in vitro binding affinities of the targeted nanoconjugate NS-RGDfK. In vivo biodistribution and tumor specificity were analyzed using 64Cu-radiolabeled untargeted and targeted NSs in live nude rats bearing head and neck squamous cell carcinoma (HNSCC) xenografts. The potential thermal therapy applications of NS-RGDfK were evaluated by subablative thermal therapy of tumor xenografts using untargeted and targeted NSs. ELISA and cell binding assay confirmed the binding affinity of NS-RGDfK to integrin αvβ3. Positron emission tomography/computed tomography imaging suggested that tumor targeting is improved by conjugation of NSs to cyclo(RGDfK) and peaks at ~20 hours postinjection. In the subablative thermal therapy study, greater biological effectiveness of targeted NSs was implied by the greater degree of tumor necrosis. The results presented in this paper set the stage for the advancement of integrin αvβ3-targeted NSs as therapeutic nanoconstructs for effective cancer therapy.
    International Journal of Nanomedicine 01/2011; 6:259-69. DOI:10.2147/IJN.S15479 · 4.38 Impact Factor
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    • "These particles also demonstrated angiogenesis targeting in murine tumor models. We have developed a novel integrin antagonist which has more than 20 times better binding affi nity to α v β 3 compared to the one that was used by Hood et al (Duggan et al 2000; Burnett et al 2005). Here we report a novel ITNP system that has this high affi nity integrin antagonist compound as the targeting agent and the subsequent studies involving the in vivo tumor angiogenesis targeting. "
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    ABSTRACT: Angiogenesis is an important process in cancer growth and metastasis. During the tumor angiogenic process, endothelial cells express various cell surface receptors which can be utilized for molecular imaging and targeted drug delivery. One such protein receptor of interest is the integrin alphav beta3. Our group is involved in the development of molecular imaging probes and drug delivery systems targeting alphav beta3. Based on extensive lead optimization study with the integrin antagonist compounds, we have developed a new generation of integrin alphav beta3 compound (IA) which has superior binding affinity to alphav beta3. Utilizing this IA as a targeting agent, we have developed a novel integrin-targeted nanoparticle (ITNP) system for targ alphav beta3 was observed. These ITNPs also were rapidly taken up by cells that express alphav beta3. The ITNPs accumulated in the angiogenic vessels, after systemic administration in a murine squamous cell carcinoma model. This novel intergrin targeted ITNP platform will likely have an application in targeted delivery of drugs and genes in vivo and can also be used for molecular imaging.
    International Journal of Nanomedicine 02/2007; 2(3):479-85. · 4.38 Impact Factor
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