Background

[PubMed]

⁶⁴Cu-1,4,7,10-Tetraazacyclodecane-N,N',N'',N'''-tetraacetic acid (DOTA)-vascular endothelial growth factor (⁶⁴Cu-DOTA-VEGF) is a radiolabeled molecular imaging agent developed for positron emission tomography (PET) imaging of tumor angiogenesis and guiding antiangiogenic treatment (1). ⁶⁴Cu is a positron emitter with a half-life (t_½) of 12.7 h.

Angiogenesis is a process of development and growth of new blood vessels from pre-existing vessels (2). Tumor growth depends on the formation of new blood vessels from this process. Normal angiogenesis is orderly and highly regulated, whereas tumor angiogenesis is chaotic and irregular. Abnormal angiogenesis is important in the carcinogenesis, growth and progression of human solid and hematologic tumors (3). VEGF is both a vascular growth factor and a vascular permeability factor, and it plays a central and critical role in the regulation of angiogenesis (4). VEGF induces proliferation, sprouting, migration, and tube formation of endothelial cells (5). Different forms of VEGF bind to receptors that exhibit a tyrosine-kinase activity (2). VEGF and VEGF receptors (VEGFR) have been shown to be upregulated in many tumors.

VEGF is a dimeric glycoprotein of 36−46 kDa that has been implicated in several steps throughout the process of angiogenesis (3). The VEGF family currently consists of 6 growth factors and 3 high-affinity tyrosine-kinase receptors. The specific action of VEGF on the endothelial cells is mainly regulated by VEGFR1 and VEGFR2 (2). VEGF-A is the most well characterized member of the VEGF family and is composed of at least 7 isoforms due to alternative gene splicing. Located at chromosome 6p21.3, VEGF-A is encoded by 8 exons separated by 7 introns. These forms differ mainly in their bioavailability and can be characterized by heparin and heparin-sulfate binding domains encoded by exons 6 and 7. VEGF₁₂₁ (121 amino acids) and VEGF₁₆₅ (165 amino acids) isoforms have been shown to induce mitogenic and permeability-enhancing activity on endothelial cells. VEGF gene expression is upregulated by many stimuli and hormones. In cancer, VEGF is upregulated by the genetic events leading to malignant transformation. VEGF and its receptors are overexpressed in a variety of solid tumors (1).

Molecular imaging of angiogenesis offers serial non-invasive evaluation of various tumor parameters (6) PET imaging with the appropriate radiolabeled tracer targeted to angiogenic signaling pathways may allow the evaluation of specific aspects of tumor vascular biology. VEGF has been labeled with ¹¹¹In, ¹²³I and ^99mTc as potential Single Photon Emission Tomography imaging agents (7). Cai et al. (1) prepared the first PET agent for imaging VEGF by labeling VEGF₁₂₁ with ⁶⁴Cu.

Synthesis

[PubMed]

Cai et al. (1) cloned and sequenced gene for VEGF₁₂₁ by polymerase chain reaction from human umbilical vein endothelial cells. The gene was inserted into the pET-32 vector downstream from the cleavable His₆ tag sequence. Bacterial cells were transformed and positive clones were selected for optimal protein expression. The protein was isolated and the His₆ tag was cleaved from VEGF₁₂₁. The final protein was purified and concentrated.

⁶⁴Cu labeling of VEGF₁₂₁ was performed through DOTA chelation (1, 8). DOTA was obtained commercially and activated by 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC) and N-hydroxysulfonosuccimide (SNHS) at pH 5.5 for 30 min with a molar ratio of 10:5:4 (DOTA/EDC/SNHS). Without purification, the DOTA-hydroxysulfosuccinimidyl was cooled to 4ºC and added to VEGF₁₂₁. The reaction mixture was adjusted to pH 8.5 with 0.1 N of sodium hydroxide and allowed to incubate overnight at 4º C. The DOTA-VEGF₁₂₁ conjugate was purified by a PD-10 column and concentrated. The final concentration was determined by ultraviolet absorbance at 280 nm. The number of DOTA molecules per VEGF₁₂₁ molecule was 4.3 ± 0.2 (n = 3). For radiolabeling, about 20 μg of this conjugate was added to 74 MBq (2.47 mCi) of ⁶⁴Cu chloride diluted in 300 μl of 0.1mol/L sodium acetate buffer. The reaction mixture was incubated for 1 h at 40ºC with constant shaking. ⁶⁴Cu-DOTA-VEGF₁₂₁ was purified by chromatography. The ⁶⁴Cu labeling time, including the final purification was 90 ± 10 min (n = 5), and the radiolabeling yield was 87.4 ± 3.2%. The specific activity was 3.2 ± 0.1 GBq/mg (86.5 ± 2.7 mCi/mg). The radiochemical purity was >98%.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Cai et al. (1) used ¹²⁵I-VEGF₁₆₅ as the radioligand to perform cell binding assays of VEGF₁₂₁ and DOTA-VEGF₁₂₁ to PAE/KDR endothelial cells expressing VEGFR2. The 50% inhibitory concentrations (IC₅₀) were 1.02 nmol/L and 1.66 nmol/L for VEGF₁₂₁ and DOTA-VEGF₁₂₁, respectively_. Stability studies of incubating ⁶⁴Cu-DOTA-VEGF₁₂₁ in complete mouse serum at 37ºC showed no significant metabolite peak by high-performance liquid chromatography up to 24 h.

Animal Studies

Human Studies

[PubMed]

On the basis of microPET quantification data of biodistribution and pharmacokinetics of ⁶⁴Cu-DOTA-VEGF₁₂₁ in rats, Cai et al. (1) estimated the radiation dosimetry in normal human organs. The radioactivity distribution in rats was similar to that in mice. ⁶⁴Cu-DOTA-VEGF₁₂₁ was primarily excreted by the liver. Only the blood, liver, and kidneys exhibited high levels of radioactivity. No appreciable activity was observed in the urinary bladder at any time point. This suggested that ⁶⁴Cu-DOTA-VEGF₁₂₁ had a very slow renal clearance, and that the radioactivity bound in the kidney was not excreted. The kidney was the critical organ with the highest estimated radiation-absorbed dose of 1.05 ± 0.27 mGy/MBq (3.87 ± 1.01 rad/mCi). The liver dose was estimated to be 0.12 ± 0.02 mGy/MBq (0.43 ± 0.07 rad/mCi). The whole-body absorbed dose was estimated to be 0.05 ± 0.01 mGy/MBq (0.19 ± 0.02 rad/mCi).

NIH Support

NIH NIBIB R21 EB001785, NCI R21 CA102123, NCI In Vivo Cellular Molecular Imaging Center Grant P50 CA114747, NCI Small Animal Imaging Resource Program Grant R24 CA93862, NCI Centers of Cancer Nanotechnology Excellence Grant 1U54CA119367-01.

References

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2.

Shinkaruk S. , Bayle M. , Lain G. , Deleris G. Vascular endothelial cell growth factor (VEGF), an emerging target for cancer chemotherapy. Curr Med Chem Anticancer Agents. 2003; 3 (2):95–117. [PubMed: 12678905]

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Ranieri G. , Patruno R. , Ruggieri E. , Montemurro S. , Valerio P. , Ribatti D. Vascular endothelial growth factor (VEGF) as a target of bevacizumab in cancer: from the biology to the clinic. Curr Med Chem. 2006; 13 (16):1845–57. [PubMed: 16842197]

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Jain R.K. Tumor angiogenesis and accessibility: role of vascular endothelial growth factor Semin Oncol 200229Suppl 166:3–9. [PubMed: 12516032]

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Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004; 25 (4):581–611. [PubMed: 15294883]

6. Laking, G.R. and P.M. Price, Positron emission tomographic imaging of angiogenesis and vascular function. Br J Radiol, 2003. 76 Spec No 1: p. S50-9. [PubMed: 15456714]

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Blankenberg F.G. , Mandl S. , Cao Y.A. , O'Connell-Rodwell C. , Contag C. , Mari C. , Gaynutdinov T.I. , Vanderheyden J.L. , Backer M.V. , Backer J.M. Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor. J Nucl Med. 2004; 45 (8):1373–80. [PubMed: 15299064]

8.

Cai W. , Wu Y. , Chen K. , Cao Q. , Tice D.A. , Chen X. In vitro and In vivo Characterization of 64Cu-Labeled AbegrinTM, a Humanized Monoclonal Antibody against Integrin {alpha}v{beta}3. Cancer Res. 2006; 66 (19):9673–81. [PubMed: 17018625]