Radiolabeled Cyclic RGD Peptide Bioconjugates as Radiotracers Targeting Multiple Integrins

Abstract

Angiogenesis is a requirement for tumor growth and metastasis. The angiogenic process depends on vascular endothelial cell migration and invasion, and is regulated by various cell adhesion receptors. Integrins are such a family of receptors that facilitate the cellular adhesion to and migration on extracellular matrix proteins in the intercellular spaces and basement membranes. Among 24 members of the integrin family, α_vβ₃ is studied most extensively for its role in tumor angiogenesis and metastasis. The α_vβ₃ is expressed at relatively low levels on epithelial cells and mature endothelial cells, but it is highly expressed on the activated endothelial cells of tumor neovasculature and some tumor cells. This restricted expression makes α_vβ₃ an excellent target to develop antiangiogenic drugs and diagnostic molecular imaging probes. Since α_vβ₃ is a receptor for extracellular matrix proteins with one or more RGD tripeptide sequence, many radiolabeled cyclic RGD peptides have been evaluated as “α_vβ₃–targeted” radiotracers for tumor imaging over the last decade. This article will use the dimeric and tetrameric cyclic RGD peptides developed in our laboratories as examples to illustrate basic principles for development of α_vβ₃–targeted radiotracers. It will focus on different approaches to maximize the radiotracer tumor uptake and tumor/background ratios. This article will also discuss some important assays for pre-clinical evaluations of integrin–targeted radiotracers. In general, multimerization of cyclic RGD peptides increases their integrin binding affinity and the tumor uptake and retention times of their radiotracers. Regardless of their multiplicity, the capability of cyclic RGD peptides to bind other integrins (namely α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆) is expected to enhance the radiotracer tumor uptake due to the increased integrin population. The result from preclinical and clinical studies clearly show that radiolabeled cyclic RGD peptides (such as ^99mTc-3P-RGD₂, ¹⁸F-Alfatide-I and ¹⁸F-Alfatide-II) are useful as the molecular imaging probes for early cancer detection and noninvasive monitoring of the tumor response to antiangiogenic therapy.

Graphical Abstract

Introduction

Cancer is the second leading cause of death worldwide.¹ Most cancer patients will survive if it can be detected at the early stage. The sooner cancer is diagnosed and treated, the better chance a cancer patient will have for a full recovery. Thus, early detection is of great clinical importance for implementation of a therapeutic regimen before primary tumors become widely spread. In fact, early detection is the best option to reduce deaths from cancer substantially.

There are several imaging modalities available for diagnosis of cancer, including X-ray computed tomography (CT), ultrasound (US), nuclear magnetic resonance imaging (MRI), positron emission tomography (PET) and single photon emission computed tomography (SPECT). While CT, US and MRI techniques are better suited for anatomic analysis of solid tumors, it is difficult to use them for evaluation of biochemical changes in tumor tissues due to the fact that they require a high concentration of contrast agent in order to achieve sufficient contrast. The specificity and sensitivity of CT and US for high-incidence tumors (e.g. breast, colorectal, lung and prostate) are relatively low.² In contrast, PET or SPECT offers significant advantages with respect to specificity and sensitivity (~10⁻¹⁰ M for SPECT and 10⁻¹⁰ - 10⁻¹² M for PET).^2–4 Both modalities are able to provide the detailed information related to biochemical changes in tumor tissues at the cellular and molecular levels.

Nuclear imaging with PET or SPECT requires administration of radiopharmaceuticals (also called radiotracers), which are drugs containing a radionuclide for routine diagnosis of diseases. According to their biodistribution characteristics, diagnostic radiotracers can be divided into two classes: those whose biodistribution is determined almost exclusively by their chemical and physical properties; and those whose ultimate distribution properties are determined by their receptor binding affinity and receptor population in the diseased tissue. The latter ones are often called target-specific radiotracers. A number of target-specific radiotracers have been developed to target the receptors overexpressed on tumor cells and/or tumor vasculature.^2–10 In many cases, small peptides are used as targeting biomolecules (BM) for receptor binding in order to achieve high tumor specificity and selectivity.

Many radiolabeled cyclic RGD peptides have been evaluated as SPECT and PET radiotracers for tumor imaging.^11–50 A number of review articles have appeared to cover their nuclear medicine applications.^51–65 Instead of being an exhaustive review of current literature on radiolabeled cyclic RGD peptides, this article will use the dimeric and tetrameric cyclic RGD peptides developed in our laboratories as examples to illustrate the basic principles for development of α_vβ₃-targeted radiotracers. It will focus on different approaches to maximize radiotracer tumor uptake and tumor/background (T/B) ratios. It will also discuss some important biological assays for evaluations of α_vβ₃-targeted radiotracers, and their potential as molecular imaging probes for noninvasive monitoring of tumor metastasis and early detection of tumor response to treatment (chemotherapy, antiangiogenic therapy, radiation therapy or combination thereof). The same basic principles illustrated in this review article may also apply to the radiotracers based on other receptors. Whenever possible, it will use the references published over the last 10 years. The author would apologize to those whose work has not been cited in this article.

Radiotracer Design

Requirements for New Radiotracers

For a new radiotracer to be successful, it must show clinical indications for several of high-incidence tumor types (namely breast, lung and prostate cancers). Its localization of in tumors has to be sufficient so that clinically useful diagnostic images can be obtained within hours after intravenous administration of the radiotracer. Since most of high-incidence tumor types occur in the torso (e.g. lung, colorectal and breast cancers), renal excretion without significant kidney retention is necessary in order to maximize the T/B ratios. The main objective of tumor imaging is to achieve one or more of the following goals: (1) to detect the presence of tumor at early stage, (2) to distinguish between benign and malignant tumors, (3) to follow the tumor growth and tumor response to a specific therapy (chemotherapy, antiangiogenic therapy, radiation therapy or combination thereof), (4) to predict the success or failure of a specific therapeutic regimen, and (5) to assess the prognosis of a particular tumor in a specific cancer patient.

Basics of Receptor Binding

Receptor–ligand binding is defined by non-covalent interactions between a receptor and its ligand. These include hydrogen-bonding, lipophilic interactions, and static interactions between negative and positive charges. Using steady-state approximation, the association (K_a) and dissociation (K_d) constants could be calculated according to the equations illustrated in Chart I. Frequently the dissociation constant K_d is preferred as the receptor binding affinity of a specific receptor ligand. K₁ and K₂ are association and dissociation rates, respectively. For receptor-based radiotracers, R_o is the total receptor population (bound and unbound), [B] is the concentration of receptor-bound radiotracer, and [F] is the unbound free radiotracer in blood circulation. If the total receptor population is much higher than the number of radiotracer molecules (radiolabeled receptor ligand), the target/background ratio will be directly proportional to K_a and R_o (Chart I). Both binding affinity of the targeting biomolecule and receptor population are important to the tumor uptake and T/B ratios of a receptor-based radiotracer. Ligand binding is characterized in terms of the concentration of ligand at which half of the receptor binding sites are occupied, known as the IC₅₀, which is related to but different from the association and dissociation constants. If two ligands were present at the same time, more of the higher-affinity ligand would be bound to the available receptor binding sites. Binding affinity is most commonly determined using a radiolabeled ligand, known as hot ligand. Competitive binding experiments involve binding-site competition between a hot ligand and a cold ligand (untagged ligand). Non-labelled methods such as surface plasmon resonance and dual polarization interferometry can also quantify the affinity from concentration based assays but also from the kinetics of association and dissociation.⁶⁶ Microscale thermophoresis (MST) is a method that allows the determination of binding affinity without any limitation to the ligand’s molecular weight.⁶⁷

Chart 1.

Schematic Presentation of Receptor Binding.

Integrin-Targeted Radiotracer

Figure 1 shows the schematic illustration of an integrin-targeted radiotracer. A cyclic RGD peptide serves as the targeting biomolecule to carry an isotope to integrins expressed on tumor cells and activated endothelial cells of tumor neovasculature. BFC is a bifunctional coupling agent to attach the isotope to cyclic RGD peptide.⁶⁸ PKM is the pharmacokinetic modifying linker which is often used to improve the excretion kinetics of radiotracers.^68–70

Figure 1.

Schematic presentation of integrin-targeted radiotracers. The cyclic RGD peptide (monomeric, dimeric or multimeric) serves as the targeting biomolecule to carry an isotope to the integrins (particularly α_vβ₃ and α_vβ₅). The bifunctional coupling agent (BFC) is used to attach the isotope to the targeting biomolecule. The PKM linker is often utilized to modify its pharmacokinetics.

Radionuclide

The choice of radionuclide depends on the imaging modality (SPECT vs. PET). More than 80% of radiotracers for SPECT in nuclear medicine are ^99mTc compounds due to the optimal nuclear properties of ^99mTc and its easy availability at low cost.^68–70 The 6 h half-life is long enough to allow radiopharmacists to carry out radiosynthesis and for physicians to collect clinically useful images. At the same time, it is short enough to permit administration of 20 – 30 mCi of ^99mTc without imposing a significant radiation dose to the cancer patient. The most clinically relevant PET isotopes are ¹⁸F, ⁶⁴Cu and ⁶⁸Ga. ¹⁸F is a cyclotron-produced isotope. Despite its short half-life (t_1/2 = 110 min), the commercial availability of preparative modules makes ¹⁸F radiotracers more accessible to clinicians. ⁶⁴Cu is another PET isotope to develop target-specific radiotracers. It has a half-life of 12.7 h and a β⁺ emission (abundance: 18%; and E_max = 0.655 MeV). Despite poor nuclear properties, its long half-life makes it feasible to prepare, transport and deliver ⁶⁴Cu radiotracers for clinical applications.⁷¹ The breakthroughs in the production of ⁶⁴Cu with high specific activity have made it more available to research institutions without the on-site cyclotron facilities.^{7,71 64}Cu is a viable alternative to ¹⁸F for research programs that wish to incorporate high resolution and sensitivity of PET, but cannot afford to maintain the expensive isotope production infrastructure.^{71 68}Ga is generator-produced PET isotope with a half-life of 68 min. The ⁶⁸Ge-⁶⁸Ga generator can be used for more than a year, allowing PET studies without the on-site cyclotron. If the radiotracer is properly designed, ⁶⁸Ga can be as useful for PET as ^99mTc for SPECT.⁷² The ⁶⁸Ga-labeled somatostatin analogs have been studied for PET imaging of somatostatin-positive tumors in pre-clinical animal models and cancer patients.^72–75 Gallium chemistry and related nuclear medicine applications have been reviewed recently.⁷²

Bifunctional Coupling Agent (BFC)

The choice of BFC depends on radionuclide. Among various BFCs (Figure 2), HYNIC is of great interest due to its high ^99mTc-labeling efficiency, the high solution stability of its ^99mTc complexes, and the use of co-ligands to modify biodistribution properties of ^99mTc radiotracers.^{46,47,68–70} HYNIC and related radiochemistry have been reviewed.⁷⁰ In contrast, DOTA and NOTA derivatives (Figure 2) have been widely used for ⁶⁸Ga and ⁶⁴Cu-labeling of biomolecules due to the high hydrophilicity and high stability of their ⁶⁸Ga/⁶⁴Cu chelates.^7,71,72 Because of the short half-life of ⁶⁸Ga (t_1/2 = 68 min), fast and efficient radiolabeling is important for ⁶⁸Ga radiotracers. Organic prosthetic groups (Figure 2: 4-FB, 4-FBz, 2-FP and 2-FDG) are needed for ¹⁸F-labeling.^76–89 Because of aromatic rings, 4-FB, 4-FBz and 2-FP groups often have relatively high lipophilicity, which might lead to more hepatobiliary excretion. The results from recent studies indicate that Al(NOTA) (Figure 2) is highly efficient for routine radiosynthesis of ¹⁸F-labeled small biomolecule radiotracers using the kit formulation.^90–97 The Al(NOTA) chelate offers significant advantages over organic prosthetic groups (e.g. 4-FB, 4-FBz and 2-FP) with respect to the ¹⁸F-labeling efficiency and hydrophilicity, which is important for rapid renal excretion of ¹⁸F radiotracers and minimization of their accumulation in normal organs, such as liver and lungs.

Figure 2.

Examples of BFCs useful for radiolabeling of small biomolecules, such as cyclic RGD peptides. HYNIC and MAG₂ are useful for ^99mTc-labeling. DOTA, NOTA and their derivatives are better suited for chelation of ⁶⁴Cu, ⁶⁸Ga and ¹¹¹In. For ¹⁸F-labeling, 4-FB, 4-FBz, 2-FP and 2-FDG are often used as prosthetic groups. The Al(NOTA) chelate is highly efficient for routine radiosynthesis of ¹⁸F radiotracers using a kit formulation.

Integrins as Molecular Targets for Tumor Imaging

Tumors produce many angiogenic factors, which are able to activate endothelial cells in the established blood vessels and induce endothelial proliferation, migration, and new vessel formation (angiogenesis) through a series of sequential but partially overlapping steps. Angiogenesis is a requirement for tumor growth and metastasis.^98–101 The angiogenic process depends on the vascular endothelial cell migration and invasion, and is regulated by various cell adhesion receptors. Integrins are such a family of receptors that facilitate cellular adhesion to and migration on extracellular matrix proteins in the intercellular spaces and basement membranes, and regulate the entry and withdraw from the cell cycle. The integrin family comprises 24 transmembrane receptors (Figure 3).^98–101 Integrins possess redundancy in ligand recognition, adhesion and signaling. Their main function is to integrate cell adhesion and interaction with the extracellular microenvironment with the intracellular signaling and cytoskeletal rearrangement through transmitting signals across the cell membrane upon ligand binding. Many integrins are crucial to tumor growth and metastasis. They also contribute to the pathological events such as thrombosis,¹⁰² atherosclerosis,¹⁰³ infection caused by pathogenic microorganisms,^104,105 and immune dysfunction.¹⁰⁶ Among the 24 members of integrin family, the α_vβ₃ is studied most extensively for its role in tumor angiogenesis and metastasis.^{98–101,107,108} It is not surprising that radiolabeled cyclic RGD peptides are often called “α_vβ₃–targeted” radiotracers in most of the current literature.^11–65

Figure 3.

Top: Combinations of integrin subunits that form the 24 human receptors. Bottom: Natural integrin ligands and their corresponding recognition peptide sequences. Both the schematic illustration and table were adapted from reference 98.

The changes in the α_vβ₃ expression levels and activation state have been well-documented during tumor growth and metastasis.^{98–101,107,108} The α_vβ₃ is expressed at relatively low levels on the epithelial cells and mature endothelial cells, but it is highly expressed in solid tumors, including osteosarcomas, glioblastoma, melanomas, and carcinomas of lung and breast.^107–124 Studies show that the α_vβ₃ is over-expressed on both tumor cells and activated endothelial cells of tumor neovasculature.^44,47,115 It is believed that the α_vβ₃ on endothelial cells modulate the cell adhesion and migration during tumor angiogenesis, while the α_vβ₃ on carcinoma cells potentiate metastasis by facilitating the invasion and movement of tumor cells across blood vessels.^119–124 It has also been shown that the α_vβ₃ expression levels correlate well with the metastatic potential and aggressiveness of solid tumors.^{106–108,122–124} The α_vβ₃ is an important biological target for development of antiangiogenic drugs,^125–135 and molecular imaging probes for diagnosis of α_vβ₃-positive tumors.^11–65

Cyclic RGD Peptides as Targeting Biomolecules

The α_vβ₃ is a receptor for extracellular matrix proteins with the exposed RGD tripeptide sequence. Theoretically, both linear and cyclic RGD peptides can be used as targeting biomolecules to develop α_vβ₃–targeted radiotracers. The drawback associated with the linear RGD peptides is their low binding affinity (IC₅₀ > 100 nM), lack of specificity (α_vβ₃ vs. α_IIBβ₃, α_vβ₅, α₅β₁, α₆β₄, α₄β₁ or α_vβ₆), and rapid degradation by proteases in serum.^130,134 It was shown that cyclization of RGD peptides via linkers, such as S-S disulfide, thioether, and aromatic rings, leads to the increased integrin binding affinity. The integrin selectivity (α_vβ₃ vs. α_IIbβ₃) could be achieved by altering the cyclic RGD peptide backbone (Figure 4). Incorporation of the RGD tripeptide sequence into a cyclic pentapeptide (Figure 4: c(RGDfV) and EMD121974 or Cilengitide) significantly increases the binding affinity and selectivity of α_vβ₃/α_vβ₅ over α_IIbβ₃.^128–134 Many cyclic RGD peptides have been evaluated as α_vβ₃/α_vβ₅ antagonists for treatment of cancer. The results from structure-activity studies indicate that the amino acid residue in position 5 has little impact on the α_vβ₃/α_vβ₅ binding affinity.^128–134 The valine (V) residue in c(RGDfV) can be replaced by lysine (K) or glutamic acid (E) to afford c(RGDfK) and c(RGDfE), respectively, without significantly changing their α_vβ₃/α_vβ₅ binding affinity. Cilengitide is currently under phase III clinical investigations as an “orphan drug” for treatment of glioblastoma and other cancer types either stand-alone or in combination with radiation therapy.^135–142 It seems that the α_IIBβ₃ is less sensitive to variations in the peptide backbone and can accommodate a larger distance than both α_vβ₃ and α_vβ₅.^66,129 The addition of a rigid aromatic ring into a cyclic peptide structure (Figure 4: DMP728 and DMP757) enhance the binding affinity and selectivity of α_IIBβ₃ over α_vβ₃/α_vβ₅. DMP728 and DMP757 were originally developed by DuPont Pharma as anti-thrombosis agents.^143–145 We have been using the ^99mTc-labeled DMP757 derivatives as SPECT radiotracers for thrombosis imaging.^146–154

Figure 4.

Examples of cyclic RGD peptides and key features at the binding site between the cyclic RGD peptide and integrins (particularly α_vβ₃, α_vβ₅ and α_IIbβ₃). The integrin selectivity can be achieved by altering the linker group in the cyclic peptide backbone. Cyclic pentapeptides are highly selective for α_vβ₃ and α_vβ₅ whereas the cyclic peptides with rigid aromatic rings show very high selectivity for α_IIBβ₃ over α_vβ₃, α_vβ₅ and α₅β₁.

Figure 5 displays examples of monomeric cyclic RGD peptides. Among the radiotracers evaluated in various pre-clinical tumor-bearing animal models, ¹⁸F-Galacto-RGD was the first PET radiotracer under clinical investigation for visualizing the α_vβ₃ expression in cancer patients.[¹⁸F]AH111585 is another α_vβ₃-targeted PET radiotracer under clinical evaluation for tumor imaging.¹⁵⁹ The imaging studies in cancer patients showed that there was sufficient α_vβ₃ expression for PET imaging, and the tumor uptake of ¹⁸F-Galacto-RGD correlates well with the α_vβ₃ expression level.^157,158 However, the radiotracers derived from monomeric cyclic RGD peptides all have relatively low tumor uptake and T/B ratios because of their low α_vβ₃ binding affinity and fast dissociation kinetics.

Figure 5.

Examples of the radiolabeled monomeric cyclic RGD peptides as radiotracers. The D₃ (Asp-Asp-Asp), S₃ (Ser-Ser-Ser), PEG (polyethylene glycol) and sugar linkers are used to enhance radiotracer excretion kinetics from non-cancerous organs. [¹⁸F]Galacto-RGD was the first PET radiotracer under clinical investigation for visualization of α_vβ₃ expression in cancer patients. [¹⁸F]AH111585 was developed as a new PET radiotracer for tumor imaging.

It is important to note that cyclic RGD peptides bind not only α_vβ₃ but also α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆ integrins regardless of their multiplicity because they all share similar features at the RGD-binding sites (Figure 4). While α_vβ₃ plays a pivotal role in tumor growth, progression and metastasis, α_IIBβ₃ is critical for platelet aggregation during thrombosis formation. It was believed that the interaction between α_vβ₃ and α_IIbβ₃ facilitates the adhesion of tumor cells to tumor vasculature, which often leads to metastasis.^160,161 The α_vβ₅ is very similar to α_vβ₃ in the ligand binding region (Figure 4), and they share a similar expression pattern and biological function. Both α_vβ₅ and α_vβ₃ are highly expressed on the activated endothelial cells and have similar roles in tumor angiogenesis, promoting angiogenic response to growth factors. The α_vβ₅ is overexpressed on many tumor types in both cell lines and clinical material.¹⁶² A number of tumors co-express α_vβ₃ and α_vβ₅,^163–169 because they both engage the same extracellular matrix ligands and activate cell signaling pathways to promote tumor progression.^168,169 It was reported that the expression of α_vβ₃, α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆ on the tumor cells is well correlated with the tumor progression.^98,164,165 Structures of other RGD-binding integrins (e.g. α_vβ₆, α_vβ₈, α_vβ₁ and α₈β₁) have not yet been studied in details. However, it is well-known that the α₄β₁, α₉β₁, α₄β₇, α_Eβ₇, α_Lβ₂, α_Mβ₂, α_Xβ₂ and α_Dβ₂ integrins all recognize the LDV, LDT and IDS tripeptide sequences.⁹⁸ In contrast the collagen and laminin-binding integrins (α₁β₁, α₂β₁, α₁₀β₁ and α₁₁β₁) recognize the GFOGER hexapeptide sequence.¹⁶⁹

There has been a continuing debate on whether one should separate α_vβ₃ from α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆. The answer to this question lies in the purpose of PET or SPECT imaging studies. If the purpose is to detect the presence of tumor or to distinguish between the benign and malignant tumors, there will be no need to separate them. The presence of multiple integrins results in a larger “receptor population”. Actually, the capability of radiolabeled cyclic RGD peptides to target multiple integrins is expected to improve their tumor uptake because larger receptor population than α_vβ₃ alone. Studies on the compounds highly active against α_vβ₃ reveals that they also possess similar activity against α_vβ₅.¹⁷⁰ Dual antagonists have been deliberately prepared and were found to have very similar affinity for α_vβ₃ and α_vβ₅.^170,171 As long as the biomolecule contains one or more RGD tripeptide sequence, it will bind α_vβ₃, α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆ regardless of peptide multiplicity. If the purpose of PET or SPECT study is to screen the appropriate patients for a specific therapy with α_vβ₃ antibodies or small-molecule α_vβ₃ antagonists, the separation of α_vβ₃ from α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆ may become necessary because the α_vβ₃-specificity is more important in this situation. However, radiolabeled cyclic RGD peptides may still be useful for this purpose if the expression of α_vβ₃ is linearly correlated to the expression levels of α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and/or α_vβ₆.

Maximizing Binding Affinity via Multimerization

Multimerization of Cyclic RGD Peptides

Multivalent interactions have been used in such a way that the weak interactions may become biological relevant.^172–174 The multivalence concept has been used to develop α_vβ₃-targted radiotracers.^11–65 It is believed that multimeric RGD peptides could provide more potent antagonists with better tumor-targeting capability. RGD₂ (Figure 6) was the first cyclic RGD dimer for development of diagnostic (^99mTc and ⁶⁴Cu) and therapeutic (⁹⁰Y and ¹⁷⁷Lu) radiotracers.^175–179 RGD₄ (Figure 6) was also used to develop SPECT and PET radiotracers.^19,21,30 The in vitro assays and biodistribution data clearly showed that the radiolabeled dimeric and tetrameric cyclic RGD peptides have higher α_vβ₃ binding affinity and better tumor uptake than their corresponding monomeric analogs.^{19,21,30,34–50}

Figure 6.

Top: Schematic illustration of the interactions between a dimeric cyclic RGD peptide and α_vβ₃. A: The distance between two RGD motifs is long due to the presence of two linkers (L). As a result, the cyclic RGD dimer is able to bind α_vβ₃ in a “bivalent” fashion. B: The distance between two RGD motifs is not long enough for simultaneous α_vβ₃ binding. However, the RGD concentration is “locally enriched” in the vicinity of neighboring α_vβ₃ sites once the first RGD motif is bound. In both cases, the end-result would be higher α_vβ₃ binding affinity for dimeric cyclic RGD peptides. Bottom: Examples of cyclic RGD dimers and tetramers for development of α_vβ₃–targeted radiotracers. The D₃, G₃, PEG₂, PEG₄ and sugar linkers are used to increase the distance between two cyclic RGD motifs and to improve radiotracer excretion kinetics from non-cancerous organs.

Bivalency and Locally Enhanced RGD Concentration

Figure 6 illustrates the interactions between a dimeric RGD peptide and α_vβ₃. Two important factors (Figure 6A: bivalency and enhanced RGD concentration) contribute to the high α_vβ₃ binding affinity of multimeric cyclic RGD peptides (Table 1).^52,55,61 The concentration factor exists in all multimeric cyclic RGD peptides regardless of the linker length between two RGD motifs. The key to achieve bivalency is the distance between two cyclic RGD motifs. Given the short distance (6 bonds excluding the side-arms of K-residues) between two RGD motifs in RGD₂ it is unlikely that they bind to two adjacent α_vβ₃ sites simultaneously. However, the binding of one RGD motif to α_vβ₃ will increase the local concentration of second RGD motif in the vicinity of neighboring α_vβ₃ sites (Figure 6A). The concentration factor may explain the higher tumor uptake of radiolabeled RGD₂ than that of its monomeric derivatives (Table 1).^175–179 The distance between two cyclic RGD motifs is 38 bonds in 3P-RGD₂, and 26 bonds 3G-RGD₂, which are definitely long enough for 3P-RGD₂ and 3G-RGD₂ to achieve bivalency. As a result, HYNIC-3P-RGD₂ (IC₅₀ = 60 ± 3 nM) and HYNIC-3G-RGD₂ (IC₅₀ = 59 ± 3 nM) have higher α_vβ₃ binding affinity than HYNIC-P-RGD₂ (IC₅₀ = 89 ± 7 nM).^{34,35 99m}Tc-3P-RGD₂ and ^99mTc-3G-RGD₂ had higher tumor uptake than ^99mTc-P-RGD₂ (Figure 7). The concentration factor is likely responsible for the higher α_vβ₃ binding affinity of HYNIC-RGD₄ (IC₅₀ = 7 ± 2 nM) than that of HYNIC-3P-RGD₂ and HYNIC-3G-RGD₂. The fact that the tumor uptake of ^99mTc-3G-RGD₂ and ^99mTc-3P-RGD₂ is well-comparable to that of ^99mTc-RGD₄ (Figure 7) within experimental errors suggests that multimeric cyclic RGD peptides are not necessarily multivalent in binding to integrins, and the contribution from the concentration factor is not as much as that from the bivalency factor.^39,41 In addition, the capability of a multimeric cyclic RGD peptide to achieve bivalency also depends on the α_vβ₃ density in tumor tissues. If the tumor α_vβ₃ density is high, the distance between two neighboring α_vβ₃ sites will be short. As a result, it is easier for the multimeric cyclic RGD peptide to achieve bivalency. If the tumor α_vβ₃ density is low, the distance between two neighboring α_vβ₃ sites will be long. In this case, it will be much more difficult for the same multimeric cyclic RGD peptide to achieve simultaneous α_vβ₃ binding.

Table 1.

The IC₅₀ values of selected cyclic peptides and their corresponding DOTA, FITC, HYNIC and NOTA conjugates.

Peptide/Conjugate	IC50 (nM)*	Radiotracer	Ref.
c(RGDyK)	458 ± 45		34, 35
HYNIC-G-RGD	358 ± 8	99mTc-G-RGD	34
HYNIC-P-RGD	452 ± 11	99mTc-P-RGD	35
HYNIC-RGD2	112 ± 21	99mTc-RGD2	35
HYNIC-P-RGD2	84 ± 7	99mTc-P-RGD2	35
HYNIC-2G-RGD2	60 ± 4	99mTc-2G-RGD2	34
HYNIC-2P-RGD2	52 ± 7	99mTc-2P-RGD2	35
HYNIC-P2D-RGD2	61 ± 2	99mTc-P2D-RGD2	49
HYNIC-P2G-RGD2	62 ± 5	99mTc-P2G-RGD2	49
HYNIC-3G-RGD2	59 ± 3	99mTc-3G-RGD2	34
HYNIC-3P-RGD2	60 ± 3	99mTc-3P-RGD2	35
HYNIC-Galacto-RGD2	29 ± 5	99mTc-Galacto-RGD2	48
HYNIC-RGD4	7 ± 2	99mTc-RGD4	35
DOTA-RGD2	102 ± 5	64Cu-RGD2/111In-RGD2	33,39, 41, 42
DOTA-3G-RGD2	74 ± 3	64Cu-3G-RGD2/111In-3G-RGD2	37,39, 41
DOTA-3P-RGD2	62 ± 6	64Cu-3P-RGD2/111In-3P-RGD2	37,39, 42
DOTA-3P-RGK2	596 ± 48	111In-Galacto-RGD2	50
DOTA-Galacto-RGD2	27 ± 2	111In-Galacto-RGD2	50
DOTA-RGD4	10 ± 2	64Cu-RGD4/111In-RGD4	20
NOTA-RGD2	100 ± 3	68Ga(NOTA-RGD2)	23
NOTA-2G3-RGD2	66 ± 4	68Ga(NOTA-2G-RGD2)	23
NOTA-2P-RGD2	54 ± 2	68Ga(NOTA-2P-RGD2)	23
FITC-RGD2	89 ± 17		183
FITC-3P-RGD2	32 ± 7		183
FITC-Galacto-RGD2	28 ± 8		183
FITC-3P-RGK2	589 ± 73		183

^*The IC₅₀ values depend largely on the radioligand and tumor cell lines. To keep the consistency and reproducibility, U87MG glioma cells (high α_vβ₃ and α_vβ₅ expression) have been used as the “host cells” and ¹²⁵I-echistatin as the radioligand. Since the whole-cell displacement assay is operator-dependent, caution should be taken when comparing their IC₅₀ values reported in the literature.

Figure 7.

Direct comparison of tumor uptake for ^99mTc-P-RGD₂, ^99mTc-3G-RGD₂, ^99mTc-3P-RGD₂ and ^99mTc-RGD₄ in athymic nude mice bearing the MDA-MB-435 human breast cancer xenografts. The biodistribution data were from references 30, 34 and 35.

Multimerization on Radiotracer Tumor Retention Time

We examined the impact of multiplicity on the retention time of ¹¹¹In-labeled cyclic RGD peptide DOTA conjugates (Figure 8) in the U87MG tumors.^39,41,42 It was found that the glioma uptake (% ID/g at < 24 h p.i.) follow the general ranking order of ¹¹¹In-6G-RGD₄ ~ ¹¹¹In-6P-RGD₄ ~ ¹¹¹In-3P-RGD₂ ≫ ¹¹¹In-P-RGD₂ > ¹¹¹In-P-RGD (Figure 9). Multimerization of significantly enhances the radiotracer tumor uptake. The tumor retention times follow the ranking order of ¹¹¹In-6G-RGD₄ (T_1/2 > 72 h) > ¹¹¹In-6P-RGD₄ (T_1/2 ~ 35 h) > ¹¹¹In-3P-RGD₂ > (T_1/2 ~ 30 h) > ¹¹¹In-P-RGD₂ (T_1/2 ~ 24 h) > ¹¹¹In-P-RGD (T_1/2 ~ 10 h).⁴¹ As a result of the increased peptide multiplicity, the tumor retention time of ¹¹¹In-labeled cyclic RGD peptides increases and their dissociation rate from the tumor tissue decreases. The biodistribution data were consistent with the results from the whole-body planar imaging studies.^39,41 The bivalency factor most likely contributes to the higher tumor uptake and longer tumor retention time of ¹¹¹In-3P-RGD₂ than that of ¹¹¹In-P-RGD₂. ¹¹¹In-3P-RGD₂ and ¹¹¹In-6P-RGD₄ share very similar (not identical) glioma tumor uptake values at <24 h p.i. Even though ¹¹¹In-6P-RGD₆ has a longer tumor retention time (as indicated by its higher tumor uptake at 72 h p.i.) than ¹¹¹In-3P-RGD₂, the tumor/liver ratios of ¹¹¹In-3P-RGD₂ are better than those of ¹¹¹In-6P-RGD₄.^39,41 There is always a subtle balance between peptide multiplicity and tumor/background ratios. The combination of high tumor uptake and high tumor/liver ratios makes ¹¹¹In-3P-RGD₂ better suited for imaging than ¹¹¹In-P-RGD, ¹¹¹In-P-RGD₂ and ¹¹¹In-6P-RGD₄.

Figure 8.

Chemdraw structures of ¹¹¹In-labeled cyclic RGD peptide DOTA-conjugates.

Figure 9.

Comparison of the glioma uptake (Top) and tumor/liver ratios (Bottom) of ¹¹¹In-labeled cyclic RGD peptides in athymic nude mice bearing U87MG glioma xenografts at 0.5, 1, 4, 24 and 72 h after administration of ¹¹¹In radiotracer. The biodistribution data were from references 41 and 42.

Impact of Linker Groups

As illustrated in Figure 6, the linker groups are used for two purposes: increasing the distance between two RGD motifs so that they are able to the α_vβ₃ in a bivalent fashion, and improving the radiotracer excretion kinetics. Different linkers (Figure 6) have been proposed to increase the distance between two RGD motifs and improve pharmacokinetics of radiotracers.^{38–42,47–50} The results from in vitro and in vivo assays showed that the linkers (G₃ vs. D₃ and PEG₄ or PEG₄ vs. SAA, 1,2,3-triazole and PEG₂ moieties) have little impact on the α_vβ₃ binding affinity of HYNIC-conjugated dimeric cyclic RGD peptides (Table 1) as long as they are long enough for bivalency. However, the overall molecular charges may have significant impact on the radiotracer uptake in both tumors and normal organs. For example, HYNIC-3G-RGD₂ and HYNIC-3P-RGD₂ share almost identical α_vβ₃ binding affinity (Table 1). Since the G₃ and PEG₄ linkers are neutral, their corresponding radiotracers ^99mTc-3G-RGD₂ and ^99mTc-3P-RGD₂ have very similar glioma tumor uptake (Figure 7) and excretion kinetics from normal organs.^34,35 Similar comparison can be made between ^99mTc-3P-RGD₂ and ^99mTc-Galactor-RGD₂.⁴⁸ In contrast, the D₃ linker is triply charged under physiological conditions. Even though HYNIC-P2D-RGD₂ and HYNIC-P2G-RGD₂ share almost identical α_vβ₃ binding affinity,⁴⁹ the tumor uptake of ^99mTc-P2D-RGD₂ (2.20±0.42, 2.85±0.55, 3.11±0.47 and 2.45±0.90 %ID/g at 5, 30, 60 and 120 min p.i., respectively) was significantly lower (p < 0.01) than that of ^99mTc-P2G-RGD₂ (9.27±0.72, 8.85±0.67, 8.17±1.10 and 7.82±0.76 %ID/g at 5, 30, 60 and 120 min p.i., respectively) over the 2 h study period.⁴⁹ In contrast, ^99mTc-P2G-RGD₂ and ^99mTc-3P-RGD₂ shared similar tumor uptake values (7.82 – 9.27 %ID/g for ^99mTc-P2G-RGD₂; and 7.24 – 8.72 %ID/g for ^99mTc-3P-RGD₂).⁴⁹ However, ^99mTc-P2D-RGD₂ had the intestine uptake values of 5.86±1.37, 6.58±0.88, 7.08±0.92 and 4.74±0.33 %ID/g at 5, 30, 60 and 120 min p.i., respectively, which were much lower than those of ^99mTc-P2G-RGD₂ (11.72±2.01, 9.27±1.15, 6.17±1.55 and 4.74±1.09 %ID/g at 5, 30, 60 and 120 min p.i., respectively) over the 2 h study period. Therefore, the linker groups between the two cyclic RGD moieties have a significant impact on the blood clearance, tumor uptake and biodistribution properties of ^99mTc-labeled dimeric cyclic RGD peptides.

Impact of ^99mTc Chelates

Figure 10 compares the 60-min biodistribution data of ^99mTc-3P-RGD₂, ^99mTc-3P-RGD₂-A and ^99mTc-3P-RGD₂-B in the U87MG glioma model.^46,47 It was found that replacing the highly charged and bulky [^99mTc(HYNIC)(tricine)(TPPTS)] (MW ~ 970 Daltons) with a smaller [^99mTc(HYNIC-K(NIC))(tricine)] (MW ~ 650 Daltons) or [^99mTc(K(HYNIC)₂)(tricine)] (MW ~ 670 Daltons) had little impact on the tumor uptake,^46,47 suggesting that changing the ^99mTc chelates had no adverse effect on their tumor-targeting capability. However, the change in ^99mTc chelates resulted in significant uptake differences in normal organs, (e.g. intestines, kidneys, lungs and spleen).^46,47 Since [^99mTc(HYNIC-K(NIC))(tricine)] is structurally similar to [^99mTc(K(HYNIC)₂)(tricine)] (Figure 10), it was not surprising that ^99mTc-3P-RGD₂-A and ^99mTc-3P-RGD₂-B shared almost identical biodistribution properties.⁴⁷ A major advantage of using K(HYNIC)₂ and HYNIC-K(NIC) as BFCs is the use of SnCl₂ as a reducing agent during ^99mTc-labeling because TPPTS can easily reduce the S-S disulfide bond, which is often important for small biomolecules to maintain their biological activity and tumor-targeting capability. The disadvantage of K(HYNIC)₂ and HYNIC-K(NIC) is that there are more than two isomers in [^99mTc(HYNIC-K(NIC))(tricine)] and [^99mTc(K(HYNIC)₂)(tricine)].^46,47

Figure 10.

Direct comparison of the 60-min biodistribution data for ^99mTc-3P-RGD₂, ^99mTc-3P-RGD₂-A and ^99mTc-3P-RGD₂-B in the athymic nude mice bearing U87MG human glioma xenografts to show the impact of ^99mTc chelates on biodistribution properties of ^99mTc radiotracers. The biodistribution data were from references 46 and 47.

Impact of Radiometal Chelates

¹¹¹In-3P-RGD₂ and ⁶⁴Cu-3P-RGD₂ share the same DOTA-3P-RGD₂ conjugate. In spite of the difference in their structures and overall molecular charge, the tumor uptake of ¹¹¹In-3P-RGD₂ (10.89 ± 2.55 and 7.65 ± 3.17 %ID/g at 30 and 240 min p.i., respectively) was very close to that of ⁶⁴Cu-3P-RGD₂ (8.23 ± 1.97 and 6.43 ± 1.22 %ID/g at 30 and 240 min p.i., respectively).^37,39–42 They also shared very similar uptake in normal organs. For example, the kidney uptake of ¹¹¹In-3P-RGD₂ was 5.80 ± 0.95 at 30 min p.i. and 2.78 ± 0.20 %ID/g at 240 min p.i., and was well comparable to that of ⁶⁴Cu-3P-RGD₂ (6.59 ± 0.93 %ID/g at 30 min p.i. and 2.81 ± 0.36 % ID/g at 240 min p.i.). The liver uptake of ¹¹¹In-3P-RGD₂ is 2.52 ± 0.57 %ID/g at 30 min and 1.61 ± 0.06 %ID/g at 240 min p.i. while ⁶⁴Cu-3P-RGD₂ has the liver uptake of 2.80 ± 0.35 %ID/g at 30 min p.i. and 1.87 ± 0.51 %ID/g at 240 min p.i.^37,39–42 Changing from ¹¹¹In(DOTA) to ⁶⁴Cu(DOTA) has minimal impact on the tumor uptake and excretion kinetics of the corresponding radiotracers. Similar conclusion can be made by comparing biodistribution properties of ⁶⁴Cu-3P-RGD₂ and ¹¹¹In-3G-RGD₂.^37,41,42

Maximizing Radiotracer Uptake by Targeting Multiple Receptors

Both receptor binding affinity and receptor population are important for the tumor selectivity and tumor uptake of radiolabeled cyclic RGD peptides. There are two general approaches to maximize the “receptor population”. The first approach (Figure 11A) involves the use of same cyclic RGD peptide (monomeric and multimeric) to target two or more integrins (particularly α_vβ₃ and α_vβ₅) overexpressed on both tumor cells and activated endothelial cells of tumor neovasculature. Radiolabeled multimeric cyclic RGD peptides have advantages over those targeting only α_vβ₃ or α_vβ₅ because of their capability to target multiple integrins (e.g. α_vβ₃, α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆). Another approach involves the use of a bifunctional peptide (Figure 11B) that is able to target two different receptors, such as α_vβ₃ and gastrin-releasing peptide receptor (GRPR). By targeting two different receptors, the “bifunctional” radiotracer will have more opportunities to localize in the tumor tissue (including the tumor cells and tumor neovasculature) and are expected to have a slower dissociation rate from the tumor.

Figure 11.

A: Schematic presentation of a dimeric cyclic RGD peptide targeting two or more integrins (e.g. α_vβ₃, α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆). B: Schematic illustration of a bifunctional peptide targeting two different receptors (e.g. α_vβ₃ and GRPR). By targeting two different receptors, the radiotracer will have more opportunities to localize in tumor due to the increased receptor population. The two targeted receptors (e.g. α_vβ₃/α_vβ₅ or α_vβ₃/GRPR) must be co-localized and the distance between them must be short for the bifunctional radiotracer to achieve simultaneous receptor binding. Bottom: Examples of bifunctional peptides containing c(RGDfK)/c(RGDyK) and Aca-BBN(7–14)NH₂ (ε-aminocaproic acid- Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂).

Bivalent heterodimers containing BBN(7-14) and c(RGDfK) or c(RGDyK) (Figure 11) have been used to target the α_vβ₃ and GRPR simultaneously.^180–182 The xenografted PC-3 and MDA-MB-435 tumor-bearing animal models were used to evaluate their tumor-targeting capability. However, there is lack of concrete in vivo evidence to prove if c(RGDfK)-BBN(7-14) and c(RGDyK)-BBN(7-14) are indeed “bivalent”, and whether there is a “synergetic effect” between the cyclic RGD and BBN(7-14) peptides. It is also unknown about which receptor binding unit actually contributes to the tumor uptake of “bifunctional radiotracers”. Since the PC-3 tumors have low α_vβ₃ expression and the MDA-MB-435 tumors have little or no expression of GRPR,^{40,47,181,182} the xenografted PC-3 and MDA-MB-435 tumor-bearing models are not appropriate to prove the concept of “bivalent heterodimers” because the α_vβ₃ and GRPR receptors must be co-localized and the distance between them must be short in order for the bifunctional radiotracer to achieve bivalency. Otherwise, it is very difficult to observe the “synergetic effect” even if they are able to target individual receptors.

Important Biological Assays

Integrin Binding Assays

The integrin binding affinity of a cyclic RGD peptide can be determined using the immobilized α_vβ₃ assay or the whole-cell competitive displacement assay with ¹²⁵I-echistatin or ¹²⁵I-c(RGDyK) as the radioligand. The immobilized α_vβ₃ assay can provide the α_vβ₃ binding affinity of a cyclic RGD peptide with high specificity and selectivity. However, the IC₅₀ (the concentration to achieve 50% displacement of the radioligand or fluorescent probe) values from the immobilized α_vβ₃ assays don’t reflect the contributions from its binding to other integrins (namely α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆), and are not reliable to predict the tumor uptake of radiolabeled cyclic RGD peptides since the α_vβ₃ density would never be as high as that used in the immobilized α_vβ₃ assay. In contrast, the whole-cell displacement assay will provide a measure of the capability of a specific cyclic RGD peptide in binding to all integrins (α_vβ₃, α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆) on tumor cells. However, the IC₅₀ values depends largely on the radioligand (¹²⁵I-c(RGDyK) vs. ¹²⁵I-echistatin) and tumor cell lines (U87MG vs. MDA-MB-435) used in this assay. The IC₅₀ values using ¹²⁵I-echistatin as the radioligand are much higher than those with ¹²⁵I-c(RGDyK) because of the higher integrin binding affinity of ¹²⁵I-echistatin. To keep the consistency and reproducibility, we have been using U87MG glioma cells (high α_vβ₃ and α_vβ₅ expression) as the “host cells” and ¹²⁵I-echistatin as the radioligand.^34–50 Since the whole-cell displacement assay is operator-dependent, caution should be taken when comparing their IC₅₀ values with those reported in the literature. Whenever possible, a “control compound”, such as c(RGDfK) or c(RGDyK) should be used in each experiment.

Blocking Experiments to Demonstrate Integrin Specificity

Blocking experiment (Chart II) is commonly used to demonstrate the α_vβ₃ specificity of radiolabeled cyclic RGD peptides with a known α_vβ₃ antagonist (e.g. c(RGDfK) or RGD₂) as the blocking agent. The blocking experiment is often performed using the in vitro tissue (Figure 12A) and cellular (Figure 12B) IHC staining assays, or by exvivo biodistribution (Figure 12C) and/or in vivo imaging (Figure 12D). The blocking agent can be administered before injection of the radiotracer or co-administered with the radiotracer. Co-injection of excess blocking agent (e.g. c(RGDfK) or RGD₂) will result in partial or complete blockage of the radiotracer tumor uptake (Figure 12C). There is also a significant reduction in radiotracer uptake in the α_vβ₃-positive organs (e.g. eyes, intestine, kidneys, lungs, liver, muscle and spleen).⁴¹

Chart 2.

Schematic Illustration of Blocking Experiments.

Figure 12.

A: Selected microscopic images (Magnification: 400×) of living U87MG glioma cells stained with FITC-Galacto-RGD₂ in the absence (left) and presence (right) of excess RGD₂. B: Microscopic images (Magnification: 200×) of a tumor slice stained with FITC-Galacto-RGD₂ in the absence (left) and presence (right) of excess RGD₂. The cellular and tumor staining data were from reference 183. C: Comparison of organ uptake (%ID/g) for ^99mTc-2P-RGD₂ in the absence or presence of excess RGD₂ at 60 min p.i. D: The 60-min planar images of the tumor-bearing mice administered with ^99mTc-3P-RGD₂ in the absence/presence of RGD₂. Co-injection of excess RGD₂ resulted in significant reduction in the uptake of ^99mTc-3P-RGD₂ in both tumor and normal organs. The biodistribution and imaging data were obtained from reference 35.

Nonsense Peptide to Demonstrate RGD Specificity

There are several ways to determine the RGD-specificity of cyclic RGD peptides. These include: (1) in vitro α_vβ₃ binding assay, (2) tissue or cellular IHC staining assay, (3) PET or SPECT imaging, and (4) ex-vivo biodistribution. In all cases, a “nonsense peptide” with the “scrambled sequence” will be used for compassion purposes. For example, 3P-RGK₂ is a nonsense peptide with the chemical composition identical to that of 3P-RGD₂. The α_vβ₃ binding affinity of DOTA-3P-RGK₂ (IC₅₀ = 596±48 nM) was >20x lower than that of DOTA-3P-RGD₂ (IC₅₀ = 29±4 nM). Similar results were also observed with FITC-3P-RGK₂ (IC₅₀ = 589±73 nM) and FITC-3P-RGD₂ (IC₅₀ = 32±7 nM). Due to the low α_vβ₃ affinity of DOTA-3P-RGK₂,^{41,42 111}In-3P-RGK₂ had much lower uptake than ¹¹¹In-3P-RGD₂ in tumors and the α_vβ₃–positive organs, such as intestine, liver, lungs and spleen (Figure 13A).^41,42 The U87MG glioma cells (Figure 13B) and tumor tissue (Figure 13C) could be stained with FITC-3P-RGD₂, but not with FITC-3P-RGK₂ under the same conditions.¹⁸³ These results clearly show that the radiotracer uptake in tumors indeed is RGD–specific. It must be noted that using the “scrambled” peptides (e.g. 3P-RGK₂) can significantly reduce the α_vβ₃ binding affinity, but this may not totally eliminate their capability for α_vβ₃ binding because the α_vβ₃ binding involves all three amino acid residues (Figure 4) in order to achieve maximal binding capability. Even if the α_vβ₃ binding capability can be totally eliminated, the scrambled peptides can still bind to other integrins (α_vβ₅, α₅β₁, α₆β₄, α₄β₁ and α_vβ₆) with low affinity.

Figure 13.

A: comparison of the 60-min biodistribution data of ^99mTc-3P-RGD₂ and ^99mTc-3P-RGK₂ in athymic nude mice bearing U87MG glioma xenografts. The low tumor uptake of ^99mTc-3P-RGK₂ indicates that the α_vβ₃-binding of radiolabeled dimeric cyclic peptides are RGD-specific. B: Selected microscopic images (magnification: 400x) of the acetone-fixed U87MG glioma cells stained with FITC-3P-RGD₂ and FITC-3P-RGK₂. C: Microscopic images (magnification: 200×) of the xenografted U87MG glioma tissue stained with FITC-3P-RGD₂ and FITC-3P-RGK₂. Blue color indicates the presence of nuclei stained with DAPI. The cellular and tissue staining data were from reference 183.

Pharmacokinetics and Metabolism

Pharmacokinetics describes how the body affects a specific drug after administration through the mechanisms of absorption and distribution, as well as the chemical changes of drug substance in the body. There are two important biological interactions (Chart III: receptor binding and protein binding) once the radiotracer is injected into blood circulation. Receptor binding is necessary for the radiotracer to localize in tumor selectively. Higher binding affinity will lead to more radiotracer tumor uptake.⁴⁹ Protein bonding is generally detrimental because it will reduce the number of radiotracer molecules available for receptor binding, and result in more blood radioactivity (Chart III).⁴⁹ Therefore, the protein bonding should be minimized. In addition, hydrophilic radiotracers tend to fast renal excretion, which will lead to lower background radioactivity in the blood pool and normal organs (such as liver, lungs and muscle) with better T/B ratios. In contrast, lipophilic radiotracers tend to excreted through hepatobiliary route with a higher degree of metabolism. High metabolic instability may result in lower tumor uptake, and higher background activity if the metabolite has longer body retention, which will definitely lead to poorer target-to-background ratios.

Chart 3.

Schematic Illustration of Biological Interactions, Elimination Routes and Metabolism of Target-Specific Radiotracers.

The tumor uptake and distribution properties of are normally determined by biodistribution at different time points after administration of the radiotracer. Even though PET has the capacity to quantify absolute radioactivity, the CT component must be included and co-registered during image acquisition and data processing. Otherwise, it would be very difficult to determine the volume and radioactivity concentration in each organ. Both PET and SPECT have the capability to determine the tumor uptake and washout kinetics via dynamic planar imaging. The tumor uptake is often expressed as the percentage of initial uptake. The blood clearance kinetics of a radiotracer can be measured by collecting blood samples at specific time points over a specific period of time.⁴⁹ The blood radioactivity usually expressed as the percentage of its initial radioactivity will be plotted against time. The most important parameter for kinetic studies is the rate of changes in the radiotracer organ uptake. The metabolic stability is determined by analyzing both urine and feces samples at a specified time point. In certain situations, tissue samples are harvested to determine the in vivo stability of radiotracers in the tumor, kidneys, liver and lungs. It is important to note that the radiotracer uptake in a specific organ represents only a small portion of the total radioactivity injected into each animal. A major portion of the injected radiotracer and its metabolites has been excreted via both renal and hepatobiliary routes. Thus, the assays of radioactivity in urine and fasces samples are more reliable for determination of the metabolic stability of the radiotracer.

Monitoring Tumor Response to Antiangiogenic Therapy

Clinical Need for Biomarkers to Monitor Antiangiogenic Therapy

Inhibiting angiogenesis is a promising strategy for cancer therapy. As antiangiogenic therapies have become a common practice in clinics, finding suitable biomarkers for modulation of the tumor vasculature has become important. An ideal biomarker for monitoring antiangiogenic therapy should have the capacity to track biological changes in the tumor vasculature during and after antiangiogenic treatment. Microvessel density has been proposed as the prognostic indicator of the progression, overall survival, and disease-free survival in cancer patients. Evaluation of microvessel density is often performed by immunostaining endothelial cells in the tumor tissues and counting the number of vessels. However, this approach is not practical for routine monitoring of antiangiogenic therapy mainly due to invasive nature of the biopsy procedure. DCE-MRI were used to measure the tumor perfusion properties;^184–187 but it is technically challenging and standardization is very complicated. The MRI techniques have little or no capability to monitor biological changes in the tumor vasculature during and after antiangiogenic treatment. ¹⁸F-FDG PET has been used to monitor the antiangiogenic linifanib therapy by determining the reduced metabolic activity in the treated tumors.¹⁸⁸ However, glucose metabolism may not be an ideal biomarker for monitoring antiangiogenic therapy because most of the metabolic activities take place in tumor cells rather than tumor vasculature.

Linear Relationship between Tumor Uptake and α_vβ₃ Expression

It is well-established that the total α_vβ₃ expression on both tumor cells and activated endothelial cells of the tumor neovasculature contributes to the tumor uptake of a α_vβ₃-targeted radiotracer regardless of peptide multiplicity.^44,115 If the α_vβ₃–targeted radiotracer is used for accurate measurement of α_vβ₃ expression, there must be a linear relationship between its tumor uptake and α_vβ₃ expression levels. However, there were a few reports to describe this relationship in the literature.^{20,155–158 99m}Tc-3P-RGD₂ was studied for its capability to monitor the α_vβ₃ expression levels.⁴⁴ IHC was performed to determine the β₃ levels in the xenografted U87MG, MDA-MB-435, A549, HT29 and PC-3 tumors.⁴⁴ An excellent relationship (Figure 14A) was observed between the tumor uptake of ^99mTc-3P-RGD₂ and the α_vβ₃ expression levels.⁴⁴ There is also an excellent relationship between its tumor uptake and CD31 expression levels (Figure 14B). These linear relationships suggest that ^99mTc-3P-RGD₂ is useful to monitor the α_vβ₃ and CD31 expression in cancer patients, to select most appropriate cancer patients who will benefit most from antiangiogenic therapy, and to monitor the tumor response to the antiangiogenic treatment.

Figure 14.

Relationship between the tumor uptake (%ID/g: radioactivity density) and relative β₃ (top) CD31 (bottom) expression levels (fluorescence density) in five different xenografted tumors (U87MG, MDA-MB-435, A549, HT29 and PC-3). The total β₃ expression was represented by the percentage of red area over total area in each slice of tumor tissue. Each data point was derived from at least 15 different areas of same tissue (100X magnification). The experimental data were from reference 40.

Monitoring Antiangiogenic Therapy with α_vβ₃–Targeted Radiotracers

The cross-talk between integrins and receptor tyrosine kinases (e.g. VEGFR and PDGFR) is crucial for many cellular functions.^189–194 The α_vβ₃ and PDGFR-β co-localize and PDGFR activation increases the endothelial cell migration and proliferation. The functional association between α_vβ₃ and VEGFR2/PDGFR is of reciprocal nature since each is able to promote the activation of its counterpart. This mutually beneficial relationship provides the conceptual basis to use radiolabeled cyclic RGD peptides for monitoring antiangiogenic therapy.^195–198 It was reported that the tumor uptake of [¹⁸F]AH111585 was significantly reduced 2 days after sunitinib treatment, and [¹⁸F]AH111585 was better than ¹⁸F-FDG for monitoring angiogenesis therapy.^{196 64}Cu-DOTA-RGD was used to monitor dasatinib therapy.¹⁹⁸ It was found that dasatinib treatment significantly reduced the ⁶⁴Cu-DOTA-RGD uptake in treated tumors. A significant challenge for ¹⁸F PET radiotracers, such as [¹⁸F]-Galacto-RGD and [¹⁸F]AH111585, to assume widespread clinical utility is their poor availability at high cost.

^99mTc-3P-RGD₂ was used to monitor the tumor response to linifanib treatment.^199,200 Linifanib a multi-targeted receptor tyrosine kinase inhibitor targeting VEGF and PDGF receptors.^201–206 It was found that there was a significant decrease in its tumor uptake and T/M ratios in the xenografted U87MG model while no significant changes in tumor uptake of ^99mTc-3P-RGD₂ were seen in the PC-3 model after linifanib therapy.¹⁹⁹ The uptake changes in MDA-MB-435 tumors were between those observed in the U87MG and PC-3 models (Figure 15).²⁰⁰ This is consistent with the α_vβ₃ expression levels on three xenografted tumors.^44,47 Highly vascularized U87MG tumors with high levels of α_vβ₃ and CD31 have better response to linifanib therapy than poorly vascularized PC-3 tumors with low levels of α_vβ₃ and CD31 (Figure 15). Thus, ^99mTc-3P-RGD₂ has the potential as the pre-treatment screening tool to select appropriate patients who will benefits most the antiangiogenic treatment. If the tumor in cancer patient has a high α_vβ₃ expression, as indicated by high tumor uptake of ^99mTc-3P-RGD₂ in SPECT/CT images at the time of diagnosis, antiangiogenic therapy would more likely be effective. If the tumor has little α_vβ₃ expression, as indicated by the low tumor uptake of ^99mTc-3P-RGD₂, antiangiogenic therapy would not be effective regardless the amount of antiangiogenic drug administered into each patient.

Figure 15.

The transverse views of representative SPECT/CT images for athymic nude mice bearing U87MG glioma (top), MDA-MB-435 breast cancer (middle) and PC-3 prostate (bottom) cancer xenografts at −1, 1, 4, 11 and 18 days for the vehicle and linifanib-treated groups. The imaging data were from references 199 and 217.

Monitoring Tumor Metastasis

Recently, we used ^99mTc-3P-RGD₂ for noninvasive monitoring of tumor growth and progression of breast cancer metastasis.^207,208 As illustrated in Figure 16A, there were no metastatic tumors detectable in the lungs at week 4 after tumor cell inoculation. By week 6, small lesions started to appear in the mediastinum and lungs. At week 8, SPECT/CT images revealed many metastatic tumors in both lungs. Figure 16B compares the %ID (left) and %ID/cm³ (right) uptake values of ^99mTc-3P-RGD₂ in the lungs. Even though the lung uptake of ^99mTc-3P-RGD₂ (0.41 ± 0.05 %ID) at week 4 slightly higher than that in the control animals (0.36 ± 0.06 %ID), this difference was not significant (p > 0.05) within experimental errors. At week 6, the tumor burden in lungs became significant (Figure 16B). The lung uptake of ^99mTc-3P-RGD₂ was higher (0.89 ± 0.12 %ID; p < 0.01) than that in the control group.²⁰⁸ By week 8, the lung uptake of ^99mTc-3P-RGD₂ increased to 1.40 ± 0.42 %ID. In all cases, the lung size remained relatively unchanged (1.21 – 1.32 cm³) during the 8-week study period. It is clear that ^99mTc-3P-RGD₂ SPECT is an excellent molecular imaging tool to monitor the tumor micrometastasis and metastatic tumor burden in a noninvasive fashion.

Figure 16.

Top: The 3D views of SPECT/CT images of an athymic nude mouse at week 4, 6 and 8 after tail-vein injection of 1.0 × 10⁶ MDA-MB-231 cells. Bottom: The %ID (left) and %ID/cm³ (right) uptake values of ^99mTc-3P-RGD₂ in the lungs obtained from SPECT/CT quantification in the athymic nude mice (n = 8) at week 4, 6 and 8 after tail-vein injection of 1.0 × 10⁶ MDA-MB-231 cells. Normal animals (n = 4) were used in the control group. †: p < 0.05, significantly different from the control group; *: p > 0.05, no significant difference from the control group. The imaging and SPECT quantification data were from reference 200.

Clinical Experiences with ^99mTc-3P-RGD₂

^99mTc-3P-RGD₂ is under clinical investigation as a new radiotracer for tumor imaging. In the first-in-human study, ^99mTc-3P-RGD₂ was investigated for its capability to differentiate solitary pulmonary nodules (SPNs).²⁰⁹ Among the 21 patients with SPNs, 15 (71%) were diagnosed as malignant, and 6 (29%) were benign. The sensitivities for CT and ^99mTc-3P-RGD₂ SPECT were 80% and 100%, respectively. All SPNs undetected by CT can be accurately diagnosed by ^99mTc-3P-RGD₂ SPECT. These results demonstrated the utility of ^99mTc-3P-RGD₂ SPECT in differentiating SPNs.²⁰⁹ A multicenter study was performed in 70 patients with suspected lung cancer.²¹⁰ It was found that ^99mTc-3P-RGD₂ SPECT was effective in detecting lung malignancies. Planar imaging and chest SPECT are complementary for detection of primary tumors and metastasis.²¹⁰ In a recently study, ^99mTc-3P-RGD₂ SPECT was used to detect the radioactive iodine-refractory differentiated thyroid carcinoma (DTC).²¹¹ It was found that ^99mTc-3P-RGD₂ SPECT was able to identify all the DTC lesions. There was a significant correlation between the T/B ratios and growth rates of DTC lesions. It was concluded that ^99mTc-3P-RGD₂ SPECT is useful for the diagnosis of DTC lesions.^{211 99m}Tc-3P-RGD₂ SPECT was also evaluated for its capability to assess the breast cancer lessons.²¹² It was found that the mean target/non-target ratio of ^99mTc-3P-RGD₂ in malignant lesions was significantly higher than that in benign lesions (3.54±1.51 vs. 1.83±0.98, p < 0.001). The sensitivity, specificity, and accuracy of ^99mTc-3P-RGD₂ were 89.3%, 90.9% and 89.7%, respectively, with the target/non-target cut-off value of 2.40.²¹³ The mean target/non-target ratio of ^99mTc-MIBI in malignant lesions was also significantly higher than that in the benign lesions (2.86±0.99 vs. 1.51±0.61, p < 0.001). The sensitivity, specificity and accuracy of ^99mTc-MIBI were 87.5%, 72.7% and 82.1%, respectively, with the target/non-target cut-off value of 1.45. The area under the curve for ^99mTc-3P-RGD₂ was higher than that for ^99mTc-MIBI, but this difference was not statistically significant, most likely because of the limited number of patients.

Clinical Experiences with ¹⁸F-Alfatide-I

[¹⁸F]AlF(NOTA-P-RGD₂) (denoted as ¹⁸F-Alfatide-I) is the ¹⁸F-labeled cyclic RGD dimer P-RGD₂, the ^99mTc and ¹¹¹In analogs of which have been evaluated as SPECT radiotracers for tumor imaging.^38,41 The Al(NOTA) chelate was used platform for ¹⁸F-labeling of P-RGD₂ to avoid the multistep and time-consuming radiosynthesis.²¹⁴ Under optimized conditions, ¹⁸F-Alfatide-I was prepared in high yield (~42%) with >95% radiochemical purity. However, chromatographic purification is still needed after ¹⁸F-labeling. It took about 20 min to complete both radiosynthesis and post-labeling filtration. Nine patients with lung cancer were examined with ¹⁸F-Alfatide-I PET, and 1 tuberculosis patient was also investigated using ¹⁸F-Alfatide-I and ¹⁸F-FDG PET. It was found that ¹⁸F-Alfatide-I PET could identify all the tumors with the mean uptake of 2.90 ± 0.10. The tumor/muscle and tumor/blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. It was concluded that ¹⁸F-Alfatide-I PET allows specific imaging of the α_vβ₃ expression in lung cancer patients.^{214 18}F-Alfatide-I was also compared to ¹⁸F-FDG in detection of DTC lymph node metastasis.²¹⁵ Twenty DTC patients with presumptive lymph node metastasis were examined with ¹⁸F-Alfatide-I and ¹⁸F-FDG PET/CT. Sixteen patients were evaluated by cytology results. A total of 39 presumptive lymph node metastases were clearly visualized on PET/CT images, and 35 lesions were confirmed as malignant tumor by biopsy and other clinical findings. It was also found that 15 DTC lesions with the diameter >1.5 cm had higher ¹⁸F-Alfatide-I uptake than the lesions <1.5 cm. Although most DTC lymph node metastases showed abnormal uptake of ¹⁸F-Alfatide-I, its diagnostic value was not as good as that of ¹⁸F-FDG.²¹⁵

Clinical Experiences with ¹⁸F-Alfatide-II

[¹⁸F]AlF(NOTA-2P-RGD₂) (denoted as ¹⁸F-Alfatide-II) is another ¹⁸F-labeled dimeric cyclic RGD peptide 2P-RGD₂, which was first developed by Dr. Liu’s group at Purdue University for preparation of ^99mTc-2P-RGD₂.^35,41 Dr. Chen’s group at the National Institute of Biomedical Imaging and Bioengineering (NIBIB) recently reported the use of ¹⁸F-Alfatide-II and ¹⁸F-FDG for monitoring the tumor responses to doxorubicin therapy in xenografted U87MG and MDA-MB-435 models.²¹⁶ It was found that there were substantial differences in the ¹⁸F-Alfatide-II binding potential and ¹⁸F-FDG influx rate 3 days after doxorubicin treatment.²¹⁶ It was also found that injection of [¹⁸F]Alfatide-II was well tolerated in all healthy volunteers.^{217 18}F-Alfatide-II showed a rapid clearance from the blood pool and kidneys. Nine patients with 20 brain metastatic lesions identified by MRI and/or CT were enrolled in this study. All 20 brain lesions were visualized by ¹⁸F-Alfatide-II PET, while only ten lesions were visualized by ¹⁸ F-FDG, and 13 by CT.²¹⁷ It was concluded that ¹⁸F-Alfatide-II is valuable biomarker of angiogenesis in finding brain metastases of different cancers.

New Opportunities and Challenges

Advantages of Multimeric Cyclic RGD Peptides

Tumor imaging with radiolabeled cyclic RGD peptides depends on their α_vβ₃ binding affinity and the total α_vβ₃ population. The advantage of multimeric cyclic RGD peptides is their higher α_vβ₃ binding affinity than that of their monomeric analogs, and their capability to target multiple integrins in tumor tissues. The multi-integrin targeting capability may contribute to the fact that they are able to localize in human carcinomas of different origin. Our studies show that radiolabeled tetrameric cyclic RGD peptides have higher uptake than their dimeric analogs in both tumors and α_vβ₃-positive organs.^33,50 High tumor uptake is important for the sensitivity, but their potential as tumor imaging agents will rely upon the contrast between tumor and normal tissues. There is always a subtle balance between the tumor uptake and T/B ratios.

Other Applications and Limitations

The success of radiolabeled cyclic RGD peptides as PET or SPECT radiotracers can be attributed to their capability to target a large population of cancer patients with carcinomas of breast, lung and prostate. It is important to note that α_vβ₃ is also overexpressed on activated endothelial cells during wound healing and post-infarct remodeling, in rheumatoid arthritis and atherosclerotic plaque.^218–221 Thus, α_vβ₃ is a biomarker for inflammation and angiogenesis.²²¹ The α_vβ₃–targeted radiotracers have been used for imaging myocardial angiogenesis,^222,223 inflammatory diseases,²²⁴ hindlimb ischemia,²²⁵ and plaque vulnerability.^226,227 It was reported that the ¹¹¹In-labeled α_vβ₃ antagonist was able to image angiogenesis after myocardial infarction.^{222 18}F-Galacto-RGD has been used to distinguish between the acute and chronic phases of T-cell mediated immune responses,²²⁸ and to image the α_vβ₃ expression in human carotid atherosclerosis.²²⁹ These promising results suggest that α_vβ₃–targeted radiotracers might be valuable for imaging angiogenesis after ischemic injury, myocardial infarction and inflammation. These broad applications of α_vβ₃–targeted radiotracers also raise significant questions related to their tumor specificity. It important to note that radiolabeled RGD peptides are α_vβ₃–targeted radiotracers. As long as the diseased tissue has the α_vβ₃ over-expression, it will have radiotracer uptake. In this situation, the differentiation between tumors and other diseased tissues will depend on their difference in α_vβ₃ expression levels. For example, the mean T/B ratio of ^99mTc-3P-RGD₂ in malignant breast cancer lesions was 3.54±1.51, which was significantly (p < 0.001) higher than that in the inflammatory benign lesions (1.83±0.98).²¹³ In this respect, radiolabeled cyclic RGD peptides are very similar to ¹⁸F-FDG since the metabolic activity is elevated during tumor growth, wound healing, post-infarct remodeling, and in rheumatoid arthritis and atherosclerotic plaque.

Integrin α_vβ₃ Expression Heterogeneity

Because of variations in the tumor vasculature, different tumor tissues often have a large difference in α_vβ₃ expression levels. Recently, we evaluated FITC-labeled dimeric RGD peptides (e.g. FITC-Galacto-RGD₂) for their potential as fluorescent probes to quantify the total α_vβ₃ expression levels in both the xenografted tumors and human carcinoma tissues.¹⁸³ It was found that the total α_vβ₃ levels followed a general order: colon cancer > pancreatic cancer > lung adenocarcinoma ≈ squamous cell lung cancer ≫ gastric cancer ≈ esophageal cancer. The same conclusion was also made using the fluorescence-labeled integrin β₃ antibody. For the xenografted tumors (cancer cells of human origin and tumor vasculature of murine origin), the α_vβ₃ expression levels followed the general trend: U87MG (high α_vβ₃ expression on both glioma cells and tumor vasculature) > MDA-MB-435 (moderately high α_vβ₃ expression on breast tumor cells and tumor vasculature) ~ A549 (moderately high α_vβ₃ expression on lung tumor cells and tumor vasculature) ~ HT29 (high α_vβ₃ expression on the tumor vasculature, but low α_vβ₃ expression on tumor cells) > PC-3 (low α_vβ₃ expression on both prostate cancer cells and tumor vasculature).^44,46,183 It has been estimated that the percentage of contribution from the tumor neovasculature to the total α_vβ₃ expression level and tumor uptake of ^99mTc-3P-RGD₂ is ~60% in the xenografted U87MG glioma model.¹⁹⁹ In case of the xenografted HT29 tumors, the main contribution to the tumor uptake of ^99mTc-3P-RGD₂ is from the α_vβ₃ on neovasculature.^44,46 Furthermore, the α_vβ₃ expression level may change with tumor metastatic status. For example, the α_vβ₃ expression level may be low in primary tumors. However, it could be very high once the α_vβ₃ is activated and the tumor becomes highly metastatic. Therefore, caution must be taken in generalizing the α_vβ₃-expression levels in different cancer types.

Comparison with ¹⁸F-FDG

¹⁸F-FDG is the gold-standard in diagnostic oncology. It is not surprising that ¹⁸F-FDG is often utilized as the positive control in evaluations of α_vβ₃–targeted radiotracers. However, this comparison may not be fair because they are targeting two different biological processes. ¹⁸F-FDG is used to detect the metabolic activity while the RGD-based radiotracers target tumor α_vβ₃ expression. Clinical data from cancer patients suggest that perfusion, α_vβ₃ expression and glucose metabolism in active tumor areas are independent variables of the tumor biology.^230,231 There is no direct relationship between the tumor metabolism and α_vβ₃ expression.^230,231 Molecular imaging with ¹⁸F-FDG and α_vβ₃-targeted radiotracers can provide complementary information and might help to evaluate both tumor metabolism and angiogenesis in-vivo in its full complexity.

Challenges in Radiotracer Development

The discovery of highly novel targeting biomolecules (such as multimeric cyclic RGD peptides) requires intensive efforts from researchers and resources from their institutions. It is easy to use known cyclic RGD peptides from the literature to develop α_vβ₃–targeted radiotracers by simply changing the radioisotope (^99mTc vs. ¹⁸F or ⁶⁸Ga) or radiometal chelate. However, the novelty of radiotracers developed from this approach is highly questionable. No industrial partners would commit millions of dollars to develop a new radiotracer without the intellectual proprietary position for commercialization. It must be emphasized that discovery of new multimeric cyclic RGD peptides is only the first step of a long process of radiotracer development. For diagnostic radiopharmaceuticals, the development times are generally 8 – 10 years and the total development costs are between $100 million and $200 million.²³² For research purposes, it is common to use post-labeling chromatography to improve radiotracer purity and specific activity before being used for pre-clinical animal studies. In clinical settings, chromatographic purification is not practical. Thus, development of efficient radiolabeling techniques is very important regardless of radioisotope. HYNIC and MAG₂ are useful for routine ^99mTc-labeling of small biomolecules using kit formulation,^{46,47,68–70} while DOTA and NOTA derivatives are better suited for chelation of ⁶⁴Cu and ⁶⁸Ga.^{7,67,68,71,72} Successful development of Al(NOTA) as a prosthetic group for ¹⁸F-labeling of biomolecules represents a milestone for routine radiosynthesis of receptor-based target-specific ¹⁸F radiotracers.^90–97 Another significant challenge that many academic researchers have to face is how to translate the results from bench discovery and preclinical evaluations in small animal models into actual clinical practice. Academic researchers often consider clinical translation to be the first use of a new radiotracer in humans.²³² From the industry point of view, clinical translation means sustained sales and an impact of a new radiotracer on patient care.²³² The costs to develop a new radiotracer into a successful “diagnostic drug” routinely used in nuclear medicine clinics are much more than those to test a new radiotracer in the physician-sponsored clinical trials. Successful development of a new radiotracer requires teamwork from several different disciplines and strong commitment from radiopharmaceutical and pharmaceutical industry. Otherwise, clinical translation will remain as “an elusive goal” for many, including academic researchers, industrial drug developer, clinicians and cancer patients.

Potential Impact of Future SPECT Scanners

The success of a new SPECT radiotracer also depends on availability of high quality SPECT scanners. It is well-accepted that conventional dual-head SPECT scanners currently available in nuclear medicine clinics have significant drawbacks in radioactivity quantification, speed of dynamic imaging, spatial resolution and tissue attenuation. If the industry is willing to devote the resources for development of new SPECT cameras as much as those for PET cameras, most of these challenges can be overcome. The development of ultrafast SPECT scanners (e.g. D-SPECT from Spectrum Dynamics, IQ SPECT® developed by Siemens Medical Solutions and CardiArc® manufactured by CardiArc Inc.) represents an excellent examples of future SPECT cameras with the capability for quantification of organ uptake and fast speed of dynamic imaging with good spatial resolution.^233–240 The larger radiation collection angles along with the use of many stationary solid state CZT (cadmium zinc telluride) detectors in a D-SPECT scanner provide 10x more efficient photon collection, leading to improved count statistics and higher quality images. Furthermore, development of the CZT-based whole-body SPECT/CT scanners and new computer software with the capability for attenuation and photon-scattering correction will help to achieve full clinical potentials of the α_vβ₃–targeted SPECT radiotracers.

Conclusions

Radiolabeled cyclic RGD peptides are called “α_vβ₃–targeted” radiotracers because α_vβ₃ is studied most extensively for its role in tumor angiogenesis and metastasis. The capability of radiolabeled cyclic RGD peptides to target multiple integrins will improve their tumor uptake due to the increased receptor population. Multimerization of cyclic RGD peptides increases the tumor uptake and retention times of their radiotracers. Two most important factors (bivalency and locally enhanced RGD concentration) contribute to the higher α_vβ₃ binding affinity of multimeric cyclic RGD peptides than their corresponding monomeric analogs. The concentration factor exists in all multimeric cyclic RGD peptides regardless of the linker length between two cyclic RGD motifs. The key to achieve bivalency is the distance between two cyclic RGD motifs. Among the dimeric and tetrameric cyclic RGD peptides evaluated in our laboratories, 2P-RGD₂, 3P-RGD_2, 2G-RGD₂, 3G-RGD₂ and Galacto-RGD₂ show the most promising results with respect to T/B ratios of their radiotracers.^{23,24,34–50} In the literature, a few ⁶⁸Ga-labeled trimeric RGD peptides were used as PET radiotracers.^11,14 It would be interesting to compare them with their dimeric and tetrameric analogs with respect to the tumor uptake and T/B ratios of ^99mTc, ¹¹¹In, ⁶⁴Cu and ⁶⁸Ga radiotracers in the same model, and determine the optimal multiplicity for development of α_vβ₃-targeted cyclic RGD peptides.

^99mTc-3P-RGD₂, ¹⁸F-Alfatide-I and ¹⁸F-Alfatide-II are currently evaluated as new radiotracers for early cancer detection in clinics. Since ^99mTc-3P-RGD₂ is prepared in >95% purity without purification, it offers significant advantages over the corresponding ¹⁸F radiotracers, which are often requires post-labeling chromatographic purification. It would be difficult for ¹⁸F radiotracers to assume widespread clinical acceptance if they have a poor clinical availability. Their high cost may also prove to be too much for a large population of cancer patients to afford the prescribed procedures. The beauty of new radiotracer development lies in science; but the success of new radiotracer relies on its clinical availability at low cost and the easiness of its routine radiosynthesis. The ultimate goal is to improve the quality of life for cancer patients with α_vβ₃–targeted radiotracers.

ABBREVIATIONS

General terms

BFC

bifunctional coupling agent

CT

X-ray computed tomography

DCE-MRI

dynamic contrast-enhanced magnetic resonance imaging

dasatinib

N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide

¹⁸F-FDG

2-deoxy-2-(¹⁸F)fluoro-D-glucose

FITC

Fluorescein isothiocyanate isomer I

IHC

immunohistochemistry

MRI

magnetic resonance imaging

MW

molecular weight

PET

positron emission tomography

PDGFR

Platelet-derived growth factor receptors

RGD

arginine-glycine-aspartic (Arg-Gly-Asp)

SPECT

single photon emission computed tomography

sunitinib

hydroxy-(2S)-compound with N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro1,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide; and

VEGFR

vascular endothelial growth factor receptors

Chelators

DOTA

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid

HYNIC

6-hydazinonicotinic acid; and

NOTA

1,4,7-tritazacyclononane-1,4,7-triacetic acid

Cyclic Peptides

P-RGD

PEG₄-c(RGDfK) = cyclo(Arg-Gly-Asp-D-Phe-Lys(PEG₄)) (PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid)

RGD₂

E[c(RGDfK)]₂ = Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]₂

Galacto-RGD₂

Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(SAA-PEG₂-(1,2,3-triazole)-1-yl-4-methylamide)]]₂ (SAA = 7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxyheptanamide, and PEG₂ = 3,6-dioxaoctanoic acid)

P-RGD₂

PEG₄-E[c(RGDfK)]₂ = PEG₄-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]₂

P2D-RGD₂

PEG₄-E[D₃-c(RGDfK)]₂ = PEG₄-Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(D₃)]]₂ (D₃ = Asp-Asp-Asp)

P2G-RGD₂

PEG₄-E[G₃-c(RGDfK)]₂ = PEG₄-Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(G₃)]]₂ (G₃ = Gly-Gly-Gly)

2G-RGD₂

E[G₃-c(RGDfK)]₂ = Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(G₃)]]₂

2P-RGD₂

E[PEG₄-c(RGDfK)]₂ = Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]]₂

3G-RGD₂

G₃-E[G₃-c(RGDfK)]₂ = G₃-Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(G₃)]]₂

3P-RGD₂

PEG₄-E[PEG₄-c(RGDfK)]₂ = PEG₄-Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG₄)]]₂

3P-RGK₂

PEG₄-E[PEG₄-c(RGKfD)]₂ = PEG₄-Glu[cyclo[Arg-Gly-Lys(PEG₄)-D-Phe-Asp]]₂)

RGD₄

E{E[c(RGDfK)]₂}₂ = Glu{Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]₂}₂

6G-RGD₄

E{G₃-E[G₃-c(RGDfK)]₂}₂ = Glu{G₃-Glu[cyclo(Lys(G₃)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(G₃)-Arg-Gly-Asp-D-Phe)}-{PEG₄-Glu[cyclo(Lys(G₃)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(G₃)-Arg-Gly-Asp-D-Phe)}; and

6P-RGD₄

E{PEG₄-E[PEG₄-c(RGDfK)]₂}₂ = Glu{PEG₄-Glu[cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)}-{PEG₄-Glu[cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)]-cyclo(Lys(PEG₄)-Arg-Gly-Asp-D-Phe)}

Cyclic Peptide Bioconjugates

DOTA-RGD

DOTA-c(RGDfK)

DOTA-P-RGD

DOTA-PEG₄-c(RGDfK)

DOTA-RGD₂

DOTA-E[c(RGDfK)]₂

DOTA-P-RGD₂

DOTA-PEG₄-E[c(RGDfK)]₂

DOTA-2G-RGD₂

DOTA-E[G₃-c(RGDfK)]₂

DOTA-2P-RGD₂

DOTA-E[PEG₄-c(RGDfK)]₂

DOTA-3G-RGD₂

DOTA-G₃-E[G₃-c(RGDfK)]₂

DOTA-3P-RGD₂

DOTA-PEG₄-E[PEG₄-c(RGDfK)]₂

DOTA-3P-RGK₂

DOTA-PEG₄-E[PEG₄-c(RGKfD)]₂

DOTA-Galacto-RGD₂

DOTA-Glu[c[RGDfK(SAA-PEG₂-(1,2,3-triazole)-1-yl-4-methylamide)]]₂

DOTA-RGD₄

DOTA-E{E[c(RGDfK)]₂}₂

DOTA-6G-RGD₄

DOTA- E{G₃-E[G₃-c(RGDfK)]₂}₂

DOTA-6P-RGD₄

E{PEG₄-E[PEG₄-c(RGDfK)]₂}₂

FITC-3P-RGD₂

FITC-PEG₄-E[PEG₄-c(RGDfK)]₂

FITC-3P-RGK₂

FITC-PEG₄-E[PEG₄-c(RGKfD)]₂

FITC-Galacto-RGD₂

FITC-Glu[c[RGDfK(SAA-PEG₂-(1,2,3-triazole)-1-yl-4-methylamide)]]₂

HYNIC-RGD₂

HYNIC-E[c(RGDfK)]₂

HYNIC-P-RGD₂

HYNIC-PEG₄-E[c(RGDfK)]₂

HYNIC-P2D-RGD₂

HYNIC-PEG₄-E[D₃-c(RGDfK)]₂

HYNIC-P2G-RGD₂

HYNIC-PEG₄-E[G₃-c(RGDfK)]₂

HYNIC-2G-RGD₂

HYNIC-E[G₃-c(RGDfK)]₂

HYNIC-2P-RGD₂

HYNIC-E[PEG₄-c(RGDfK)]₂

HYNIC-3G-RGD₂

HYNIC-G₃-E[G₃-c(RGDfK)]₂

HYNIC-3P-RGD₂

HYNIC-PEG₄-E[PEG₄-c(RGDfK)]₂

HYNIC-K(NIC)-3P-RGD₂

HYNIC-K(NIC)-PEG₄-E[PEG₄-c(RGDfK)]₂ (NIC = nicotinyl)

K(HYNIC)₂-3P-RGD₂

K(HYNIC)₂-PEG₄-E[PEG₄-c(RGDfK)]₂

HYNIC-Galacto-RGD₂

HYNIC-E[c[RGDfK(SAA-PEG₂-(1,2,3-triazole)-1-yl-4-methylamide)]]₂

HYNIC-RGD₄

HYNIC-E{E[c(RGDfK)]₂}₂

NOTA-P-RGD₂

NOTA-PEG₄-E[c(RGDfK)]₂

NOTA-2G-RGD₂

NOTA-E[G₃-c(RGDfK)]₂

NOTA-2P-RGD₂

NOTA-E[PEG₄-c(RGDfK)]₂

NOTA-3G-RGD₂

NOTA-G₃-E[G₃-c(RGDfK)]₂; and

NOTA-3P-RGD₂

NOTA-PEG₄-E[PEG₄-c(RGDfK)]₂

Radiolabeled Cyclic RGD Peptides

¹⁸F-Alfatide-I

[¹⁸F]AlF(NOTA-P-RGD₂)

¹⁸F-Alfatide-II

[¹⁸F]AlF(NOTA-2P-RGD₂)

¹⁸F-Galacto-RGD

(2-[¹⁸F]fluoropropanamide c(RGDfK(SAA))

⁶⁴Cu-P-RGD₂

⁶⁴Cu(DOTA-P-RGD₂)

⁶⁴Cu-2G-RGD₂

⁶⁴Cu(DOTA-2G-RGD₂)

⁶⁴Cu-2P-RGD₂

⁶⁴Cu(DOTA-2P-RGD₂)

⁶⁴Cu-3G-RGD₂

⁶⁴Cu(DOTA-3G-RGD₂)

⁶⁴Cu-3P-RGD₂

⁶⁴Cu(DOTA-3P-RGD₂)

⁶⁸Ga-3G-RGD₂

⁶⁸Ga(DOTA-3G-RGD₂)

⁶⁸Ga-3P-RGD₂

⁶⁸Ga(DOTA-3P-RGD₂)

¹¹¹In-P-RGD

¹¹¹In(DOTA-P-RGD)

¹¹¹In-P-RGD₂

¹¹¹In(DOTA-P-RGD₂)

¹¹¹In-3G-RGD₂

¹¹¹In(DOTA-3G-RGD₂)

¹¹¹In-3P-RGD₂

¹¹¹In(DOTA-3P-RGD₂)

¹¹¹In-Galacto-RGD₂

¹¹¹In(DOTA-Galacto-RGD₂)

¹¹¹In-6G-RGD₄

¹¹¹In(DOTA-6G-RGD₄)

¹¹¹In-6P-RGD₄

¹¹¹In(DOTA-6P-RGD₄)

^99mTc-Galacto-RGD₂

[^99mTc(HYNIC-Galacto-RGD₂)(tricine)(TPPTS)])

^99mTc-RGD₂

[^99mTc(HYNIC-RGD₂)(tricine)(TPPTS)])

^99mTc-P-RGD₂

[^99mTc(HYNIC-P-RGD₂)(tricine)(TPPTS)]

^99mTc-P2D-RGD₂

[^99mTc(HYNIC-P2D-RGD₂)(tricine)(TPPTS)]

^99mTc-P2G-RGD₂

[^99mTc(HYNIC-P2G-RGD₂)(tricine)(TPPTS)]

^99mTc-2G-RGD₂

[^99mTc(HYNIC-2G-RGD₂)(tricine)(TPPTS)]

^99mTc-2P-RGD₂

[^99mTc(HYNIC-2P-RGD₂)(tricine)(TPPTS)]

^99mTc-3G-RGD₂

[^99mTc(HYNIC-3G-RGD₂)(tricine)(TPPTS)]

^99mTc-3P-RGD₂

[^99mTc(HYNIC-3P-RGD₂)(tricine)(TPPTS)]

^99mTc-3P-RGD₂-A

[^99mTc(HYNIC-K(NIC)-3P-RGD₂)(tricine)]

^99mTc-3P-RGD₂-B

[^99mTc(K(HYNIC)₂-3P-RGD₂)(tricine)]; and

^99mTc-RGD₄

[^99mTc(HYNIC-RGD₄)(tricine)(TPPTS)])