In Vitro and in Vivo Evaluation of a Technetium-99m-Labeled Cyclic RGD Peptide as a Specific Marker of α_Vβ₃ Integrin for Tumor Imaging

Abstract

Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(V)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(V)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for 99mTc radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both 99mTc peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/micromol was achieved. Both 99mTc complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degrees C and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of 99mTc-labeled RGD to cells. The binding percentage of 99mTc-labeled RGD to purified alpha(V)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while 99mTc-labeled RGE showed no binding. The association constant for 99mTc-RGD was modest at 7 x 10(6) M(-)(1). In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of 99mTc-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers of alpha(V)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.

Prodrug Strategies in Anticancer Chemotherapy

“…[59] Angiogenesis can be inhibited by targeting integrins with peptides containing an RGD sequence (Arg-Gly-Asp) that is present in extracellular matrix proteins. [60] In subsequent work, a number of RGD-drug conjugates with cytostatic and diagnostic agents [61][62][63][64] were developed, and a proof of concept has been obtained for several candidates (reviewed in references [65][66][67][68][69]). …”

Quantifying the relation between adhesion ligand–receptor bond formation and cell phenotype

“…It is unclear whether a subset or all of these variables alter the cellular response by simply altering bond number, but this assumption underlies many aspects of current biomaterials design. One can analyze receptor-ligand bonding with radioactive molecules (17), fluorescent molecules (18), and chemical assays (19), but these methods have been limited to analyzing cells suspended in medium or adherent to 2D substrates. Encapsulation of cells in 3D better reflects in vivo cell adhesion to ECM (20) and leads to distinct patterns of gene expression (21,22) but is not amenable to the application of existing methods to quantify bond formation.…”

Molecular imaging: New applications for biochemistry

“…A cyclic pentapeptide cyclo (-Arg-Gly-Asp-DPhe-Val-) has been identified as having a high affinity (Kd < 10 nmole/L) binding to the aVb3 integrin. This has been modified to enable labeling with iodine or Fluorine-18 enabling in vivo imaging [Haubner et al, 2001a,b;Su et al, 2002]. Other androgenesis factors have been imaged which include the insulin growth factor (IGF) receptors IGF-1 and IGF-2 [Sun et al, 1997].…”