Cheng KT, Paik CH, Danthi N.

Publication Details



In vitro Rodents



4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyloxy]benzoyl-2-(S)-[N-3-amino-(SCN-Bz-DOTA-111In)-neopenta-1-carbamyl)]-aminoethylsulfonylamino-β-alanine (IAC-SCN-Bz-DOTA-111In) is an integrin-targeted molecular imaging agent conjugated with 111In that was developed for single-photon emission computed tomography (SPECT) or planar gamma imaging of tumor vasculature and tumor angiogenesis (1). 111In is a gamma emitter with a physical half-life (t½) of 2.8 days.

Cellular survival, invasion, and migration control embryonic development, angiogenesis, tumor metastasis, and other physiological processes (2, 3). Among the molecules that regulate angiogenesis are integrins, which comprise a superfamily of cell adhesion proteins that form heterodimeric receptors for extracellular matrix (ECM) molecules (4, 5). These transmembrane glycoproteins consist of two noncovalently associated subunits, α and β (18 α- and 8 β-subunits in mammals), which are assembled into at least 24 α/β pairs. Several integrins, such as integrin αvβ3, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin αvβ3 receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin αvβ3 receptor is generally not found in normal tissue, but it is strongly expressed in vessels with increased angiogenesis, such as tumor vasculature. It is significantly upregulated in certain types of tumor cells and in almost all tumor vasculature. Molecular imaging probes carrying the RGD motif that binds to the integrin αvβ3 can be used to image tumor vasculature and evaluate angiogenic response to tumor therapy (6, 7).

Various RGD peptides in both linear and cyclic forms have been developed for in vivo binding to integrin αvβ3 (8). Non-peptide integrin αvβ3–targeted molecular probes have also been investigated (9-11). Duggan et al. (10) modified a potent fibrinogen receptor antagonist from the sulfonamide exo-site class to generate non-peptide compounds with high affinity for the integrin αvβ3 receptor. The centrally -constrained benzoylamino-3-propionic acid scaffold appeared to provide optimum spacing between the acidic and basic portions of these ligands for binding. Based on the potent αvβ3 inhibitor 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-β-alanine (IA) which has a 50% inhibitory concentration (IC50) of 0.04 μM and was used by Hood et al. (11) to covalently couple to a cationic nanoparticle for targeted gene therapy, Burnett et al. (9) designed and synthesized a series of aliphatic carbamate derivatives with enhanced binding affinity. The peptidomimetic 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-β-alanine hydrochloride (IAC) ligand was conjugated to a fluorescence label (FITC-IAC) as an integrin αvβ3 molecular probe for optical imaging of αvβ3-expressing tumors. To develop a radioligand for SPECT imaging, Jang et al. (1) successfully prepared IAC-SCN-Bz-DOTA-111In for studies in nude mice bearing the human M21 melanoma tumor.



Burnett et al. (9) reported the synthesis of IAC from commercially available amino alcohol. The amine was first protected as the t-butoxycarbonyl (t-Boc) derivative and then activated as 3-(Boc-amino)neopentyl-1-O-carbonylimidazole. The activated alcohol was then coupled to the free amine of IA to provide the Boc carbamate. The Boc carbamate was deprotected with anhydrous hydrochloric acid in dioxane to yield IAC. For radiolabeling, a bifunctional chelate was first conjugated to the amino terminus of IAC. Commercially obtained 2-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (SCN-Bz-DOTA) was reacted with IAC in 0.1 M sodium bicarbonate (pH 8.4) for 2 days at room temperature (1). A 10:1 molar ratio of IAC to SCN-Bz-DOTA appeared to give a quantitative yield of the IAC-SCN-Bz-DOTA conjugate. The authors suggested that the product formed was a 1:1 conjugate because the primary amine was the only reactive functional group at pH 8.4. This conjugate was purified with a Sep-Pak C-18 cartridge and then incubated with 111In-chloride in an aqueous solution (pH 5) of 0.23 M sodium acetate and 0.015 M sodium ascorbate at 75ºC for 1 h. The final radioligand was purified with a Sep-Pak C-18 cartridge. The labeling yield was >80%. The specific activity was calculated to be >7.5 kBq/pmol (202.7 Ci/mmol). Preparations with >95% radiochemical purity were used for in vitro and in vivo studies.

In Vitro Studies: Testing in Cells and Tissues


Burnett et al. (9) used the enzyme-linked immunosorbent assay to determine the IC50 of unlabeled IAC to be 2.94 nM. In vitro binding assays of IAC-SCN-Bz-DOTA-111In were performed with purified human αvβ3 protein (molecular weight = 237,000) (1). The binding was assessed by size-exclusion high-performance liquid chromatography to detect the shift to a higher molecular weight peak that was identical to the integrin αvβ3 protein. The percent bound of IAC-SCN-Bz-DOTA-111In was 6%, 40%, and 72% when incubated with 0.08 μM, 0.4 μM, and 0.8 μM integrin αvβ3, respectively. This binding was completely blocked by a 25-fold molar excess of unlabeled IA. The authors reported that IAC-SCN-Bz-DOTA-111In was stable in serum for 18 h at 37ºC.

Animal Studies



The in vivo tumor accumulation, distribution, and gamma imaging of IAC-SCN-Bz-DOTA-111In were studied in nude mice bearing s.c. human M21 melanomas (~0.5 cm3) in the right flanks (1). Unlabeled IAC was added to the radioligand to achieve the specific activity of 85 kBq/<160 pmol (2.30 μCi/<160 pmol) so that each mouse received 160 pmol of total IAC. The radioligand was rapidly cleared from the circulation via the renal system. The tumor radioactivity levels (n = 4–5) were 5.58 ± 1.43% injected dose per gram (ID/g) (20 min), 5.34 ± 0.60% ID/g (60 min), and 4.47 ± 0.66% ID/g (120 min). In most major organs (except the lung and kidney), the tumor/organ ratios at 20 min were all >1. The tumor/blood ratios were 2.36 ± 0.64 (20 min), 4.69 ± 1.30 (60 min), and 9.74 ± 1.00 (120 min). The tumor/muscle ratios were 4.99 ± 1.46 (20 min), 7.07 ± 1.48 (60 min), and 9.87 ± 1.22 (120 min). The tumor/kidney ratios were 0.19 ± 0.07 (20 min), 0.37 ± 0.07 (60 min), and 0.78 ± 0.12 (120 min). The tumor/liver ratios were 1.58 ± 0.60 (20 min), 1.79 ± 0.31 (60 min), and 2.26 ± 0.35 (120 min). The tumor/intestine ratios were 1.01 ± 0.38 (20 min), 1.00 ± 0.15 (60 min), and 0.92 ± 0.13 (120 min). In a blocking study, 200 μg of unlabeled IA was co-injected with 111In-SCN-Bz-DOTA-IAC. At 60 min after injection, the tumor radioactivity level was decreased from 5.3 ± 0.6% ID/g to 1.4 ± 0.3% ID/g. The radioactivity levels in most other organs were also significantly decreased. Co-injection of 30 mg l-lysine did not decrease either the tumor or kidney radioactivity level. The static posterior gamma imaging of the mice at 4 h after injection clearly visualized the tumor, and this visualization remained for 72 h. The abdomen and bladder areas were visualized at 4 h but gradually cleared out. With co-injection of 200 μg unlabeled IA, the tumor was not distinguished from the surrounding background radioactivity.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

NIH Intramural Support.


Jang B.S. , Lim E. , Hee Park S. , Shin I.S. , Danthi S.N. , Hwang I.S. , Le N. , Yu S. , Xie J. , Li K.C. , Carrasquillo J.A. , Paik C.H. Radiolabeled high affinity peptidomimetic antagonist selectively targets alpha(v)beta(3) receptor-positive tumor in mice. Nucl Med Biol. 2007;34(4):363–70. [PubMed: 17499725]
Jin H. , Varner J. Integrins: roles in cancer development and as treatment targets. Br J Cancer. 2004;90(3):561–5. [PMC free article: PMC2410157] [PubMed: 14760364]
Paulhe F. , Manenti S. , Ysebaert L. , Betous R. , Sultan P. , Racaud-Sultan C. Integrin function and signaling as pharmacological targets in cardiovascular diseases and in cancer. Curr Pharm Des. 2005;11(16):2119–34. [PubMed: 15974963]
Hood J.D. , Cheresh D.A. Role of integrins in cell invasion and migration. Nat Rev Cancer. 2002;2(2):91–100. [PubMed: 12635172]
Hwang R. , Varner J. The role of integrins in tumor angiogenesis. Hematol Oncol Clin North Am. 2004;18(5):991–1006. [PubMed: 15474331]
Cai W. , Shin D.W. , Chen K. , Gheysens O. , Cao Q. , Wang S.X. , Gambhir S.S. , Chen X. Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects. Nano Lett. 2006;6(4):669–76. [PubMed: 16608262]
Massoud T.F. , Gambhir S.S. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev. 2003;17(5):545–80. [PubMed: 12629038]
Haubner R. , Wester H.J. Radiolabeled tracers for imaging of tumor angiogenesis and evaluation of anti-angiogenic therapies. Curr Pharm Des. 2004;10(13):1439–55. [PubMed: 15134568]
Burnett C.A. , Xie J. , Quijano J. , Shen Z. , Hunter F. , Bur M. , Li K.C. , Danthi S.N. Synthesis, in vitro, and in vivo characterization of an integrin alpha(v)beta(3)-targeted molecular probe for optical imaging of tumor. Bioorg Med Chem. 2005;13(11):3763–71. [PubMed: 15863003]
Duggan M.E. , Duong L.T. , Fisher J.E. , Hamill T.G. , Hoffman W.F. , Huff J.R. , Ihle N.C. , Leu C.T. , Nagy R.M. , Perkins J.J. , Rodan S.B. , Wesolowski G. , Whitman D.B. , Zartman A.E. , Rodan G.A. , Hartman G.D. Nonpeptide alpha(v)beta(3) antagonists. 1. Transformation of a potent, integrin-selective alpha(IIb)beta(3) antagonist into a potent alpha(v)beta(3) antagonist. J Med Chem. 2000;43(20):3736–45. [PubMed: 11020288]
Hood J.D. , Bednarski M. , Frausto R. , Guccione S. , Reisfeld R.A. , Xiang R. , Cheresh D.A. Tumor regression by targeted gene delivery to the neovasculature. Science. 2002;296(5577):2404–7. [PubMed: 12089446]

This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.