Background

[PubMed]

⁶⁴Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-PEGylated cyclic arginine-glycine-aspartic acid peptide [⁶⁴Cu-DOTA-PEG-c(RGDyK)] is an integrin-targeted molecular imaging agent developed for positron emission tomography (PET) of tumor vasculature and tumor angiogenesis (1). ⁶⁴Cu is a positron emitter with a physical half-life (t_½) of 12.8 h.

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 α_vβ₃, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin α_vβ₃ receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin α_vβ₃ 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 that carry the RGD motif that binds to the integrin α_vβ₃ can be used to image tumor vasculature and evaluate angiogenic responses to tumor therapy (6, 7). Various RGD peptides in both linear and cyclic forms (RGDfK or RGDyK) have been developed for in vivo binding to integrin α_vβ₃ (8). Chen et al (9). evaluated a cyclic RGD D-Tyr analog peptide [c(RGDyK)] labeled with ⁶⁴Cu or ¹⁸F in nude mice bearing breast tumor. Chen et al. used 1,4,7,10-tetrazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) for c(RGDyK) conjugation with ⁶⁴Cu. ⁶⁴Cu-DOTA-c(RGDyK) showed prolonged tumor radioactivity retention but persistent liver radioactivity. To improve the pharmacokinetics and tumor retention of the radiolabeled peptide, Chen et al. (10) modified c(RGDyK) with monofunctional methoxy-polyethylene glycol (PEG; molecular weight = 2,000) and showed that the modified PEGylated RGD peptide had faster blood clearance, lower kidney uptake, and prolonged tumor uptake. Using the same strategy, Chen et al. (1) inserted a heterobifunctional PEG linker (molecular weight = 3,400) between DOTA and c(RGDyK) to produce ⁶⁴Cu-DOTA-PEG-c(RGDyK). The PEG moiety appeared to improve the in vivo kinetics of this PET radioligand.

Synthesis

[PubMed]

Chen et al. (1) reported the synthesis of ⁶⁴Cu-DOTA-PEG-c(RGDyK). The c(RGDyK) peptide was first prepared via solution cyclization of fully protected linear pentapeptide H-Gly-Asp(OtBu)-D-Tyr(OtBu)-Lys(Boc)-Arg(Pbf)-OH, followed by trifluoroacetic acid (TFA) deprotection in the presence of the free radical scavenger triisopropylsilane. The c(RGDyK)-PEG conjugate was prepared by coupling c(RGDyK) with t-butoxycarbonyl (t-Boc)-protected PEG-succinimidyl ester (t-Boc-NH-PEG-CO₂Su). The t-Boc protecting group was unblocked by TFA cleavage. DOTA was obtained commercially. The DOTA-PEG-c(RGDyk) conjugate was prepared by in situ activation of DOTA by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and N-hydroxysulfonosuccinimide (SNHS) in a molar ratio of 10:5:4 (DOTA:EDC:SNHS).The c(RGDyk)-PEG conjugate in water at 4ºC was added to the resultant DOTA-sulfosuccinimydyl ester (OSSu), and the pH was adjusted to 8.5. The reaction mixture was incubated overnight at 4ºC. The DOTA-PEG-c(RGDyk) conjugate was then purified by semipreparative high-performance liquid chromatography (HPLC). The molecular weight of the DOTA-PEG-c(RGDyK) conjugate was determined to be 4,400 by mass spectrometry, which appeared to agree with the theoretical value. The purified product was labeled with ⁶⁴Cu by addition of ⁶⁴Cu in the ratio of 5–25 μg peptide per 37 MBq (1 mCi) ⁶⁴Cu in 0.1-N sodium acetate buffer (pH 5.5). This was followed by incubation at 50ºC for 45 minutes. The reaction was terminated by addition of ethylenediaminetetraacetic acid (EDTA). ⁶⁴Cu-DOTA-PEG-c(RGDyK) was purified with a C-18 Sep-Pak cartridge that used 85% ethanol as the elution solvent. The radiochemical purities were 50%, 80%, 95%, and ≥98% for 5, 10, 15, and 20 μg peptide, respectively. The specific activity of ⁶⁴Cu-DOTA-PEG-c(RGDyK) ranged from 14,800 to 29,600 GBq/mmol (400 to 800 Ci/mmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Solid-phase receptor-binding assays of unlabeled DOTA-PEG-c(RGDyK) were conducted by competitive displacement of ¹²⁵I-labeled echistatin binding to purified human α_vβ₃ integrin (1). The inhibition concentration (IC₅₀) was 67.5 ± 10.5 nM. In comparison, the IC₅₀ for unpegylated DOTA-c(RGDyK) was 31.2 ± 5.8 nM. In a hydrophilicity study, the octanol-water partition coefficient (LogP_OCT) for ⁶⁴Cu-DOTA-PEG-c(RGDyK) was determined to be −2.97 ± 0.30 (n = 3). The LogP_OCT for ⁶⁴Cu-DOTA-c(RGDyK) was −2.8 ± 0.04.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

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2.

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]

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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]

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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]

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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]

9.

Chen X. , Park R. , Tohme M. , Shahinian A.H. , Bading J.R. , Conti P.S. MicroPET and autoradiographic imaging of breast cancer alpha v-integrin expression using 18F- and 64Cu-labeled RGD peptide. Bioconjug Chem. 2004; 15 (1):41–9. [PubMed: 14733582]

10.

Chen X. , Park R. , Shahinian A.H. , Bading J.R. , Conti P.S. Pharmacokinetics and tumor retention of 125I-labeled RGD peptide are improved by PEGylation. Nucl Med Biol. 2004; 31 (1):11–9. [PubMed: 14741566]