Synthesis and characterization of αvβ3-targeting peptidomimetic chelate conjugates for PET and SPECT imaging

Integrins are a family of transmembrane glycoproteins with associated α and β subunits forming 25 unique heterodimers that facilitate adhesion and migration of cells on the extracellular matrix proteins found in intercellular spaces and basement membranes.1 One of these integrins, αvβ3 integrin, interacts with vitronectin, fibronectin, fibrinogen, thrombospondin, collagen, laminin and von Willebrand factor. This integrin is over-expressed in tumor induced angiogenic vessels and in various human tumors, but is found at low levels on epithelial and endothelial cells. It is therefore a widely recognized target for the development of molecular probes for imaging angiogenesis and cancer therapy. Towards this end, the tumor imaging capability of several RGD peptides that act as αvβ3 integrin antagonists has been demonstrated by several research groups. Additionally, several of these peptides have been shown to inhibit tumor angiogenesis and interrupt metastasis in many models.2–4 There is growing interest in peptidomimetic αvβ3 integrin antagonists composed of a stable core scaffold with basic and acidic groups that mimic the guanidine and carboxylate pharmacophore of RGD peptides. Peptidomimetics tend to have higher activity, specificity and longer duration of action compared to the peptides. One such peptidomimetic αvβ3 integrin antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-aminoethylsulfonyl-amino-β-alanine (IA) was synthesized by Hood et al.5 Subsequent, modification of IA to the corresponding carbamate derivatives by the Danthi group resulted in 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonyl-amino-β-alanine (IAC), with a binding affinity 20 times greater than that of IA.6 A SPECT (single photon emission computed tomography) imaging study with 111In-DOTA-Bz-SCN- IAC was also performed and tumor was clearly visualized at 4 h p.i.7111In-DOTA-Bz-SCN- IAC was prepared using the bifunctional chelate DOTA-Bz-SCN which differs from the DOTA-GA described in this study. Clinically, SPECT and PET (positron emission tomography) play significant roles allowing noninvasive imaging of internal physiological and biochemical function and pathologies in vivo. While PET is more expensive, it has significant advantages over SPECT with respect to its ability to better quantify images. Of metallic radionuclides currently being investigated for PET applications, gallium-68 (68Ga) has grown in popularity.8, 9 The popularity of 68Ga stems from the ease of on site production from a long lived generator system (68Ge/68Ga) rather than a cyclotron, and automation for incorporation into radiolabeled compounds.10 The 67.7 min half-life of 68Ga is an appropriate match to the biological half-lives of peptides. Gallium(III) typically binds with chelates possessing multiple anionic oxygen donors preferring a coordination number of six in an octahedral geometry. Fitting these preferences, the macrocyclic ligand, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), is well established as forming very stable complexes with a wide variety of metals, Ga(III) being one of them.11 Several NOTA derivatives have been reported (Figure 1) for use in the radiolabeling of proteins and peptides. Recently, Knetsch et al reported a 68Ga-labeled NODA-GA-conjugated RGD peptide ([68Ga]NODAGA-RGD) that showed better tumor to blood ratio in vivo than the corresponding [68Ga]DOTA-RGD derivative.12 Figure 1 Structures of NOTA derivatives. 7-(5-Maleimido-1-ethoxycarbonylphenyl)-1,4,7-triazacyclononane-1,4-diylacetic acid (1), 2-(4-Aminobutyl)-1,4,7-triazacyclononane-1,4,7-triyltriacetic acid (2), nNOTA (3), p-SCN-Bn-NOTA (4), NETA (5), and NODA-GA(tBu)3 ( ... While interest in 68Ga for PET imaging is currently significant, there are Pb(II) isotopes that have also been of interest for biomedical applications. Specifically, 203Pb and 212Pb are radiometals possessing favorable properties for use in nuclear medicine for potential diagnostic and therapeutic applications, respectively.13–21203Lead (t1/2 = 51.9 h) emits a γ-ray (279 keV) that is ideal for single photon emission computed tomography (SPECT) imaging and is suitable for pharmacokinetic and pharmacodynamic tracer studies. In addition, 203Pb can serve as one half of a potential matched-pair of radioisotopes when combined with 212Pb for therapeutic applications.212 Lead (t1/2 = 10.6 h) has been studied as an `in vivo generator' of 212Bi (t1/2 = 60 min) to overcome the short half-life of that daughter isotope. The macrocyclic polyaminocarboxylate chelate DOTA, (1,4,7,10-tetraazacyclododecane-N,N',N”,N”'-tetraacetic acid labeled with 212Pb provides a complex that is adequately stable in vivo to sequester the radionuclide.17 The Macke group in Switzerland have synthesized a DOTA derivative analogous to NOTA-GA, , 1-(1-carboxy-3-carbotertbutoxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA-GA(tBu)4).22 The DOTA-GA(tBu)4 affords four intact carboxylic acid functional groups with a free carboxylate side chain ready for conjugation to the N-terminus of peptides which makes it useful for biomedical applications. In the present study, the objective was to move beyond the use of RGD peptides as delivery vectors to the various integrin targets and explore the utility of IAC for such applications. 68Gallium labeling was investigated for PET applications using NODA-GA and 67Ga as a surrogate for 68Ga, and 203Pb for SPECT imaging using DOTA-GA. To this end, IAC was successfully conjugated to NODAGA (Scheme 1) and DOTA-GA (Scheme 2) and the conjugates were radiolabeled with 111In or 67Ga for the NODA-GA conjugate and 203Pb for the DOTA-GA conjugate. In brief, NODA-GA(tBu)3 or DOTAGA(tBu)4, N-hydroxysuccinimide, and EDC were dissolved in dichloromethane and the reaction mixture was stirred for 24 h. The mixture was extracted with saturated NaCl solution, 5% NaHCO3, and saturated NaCl again. The organic layer was dried over MgSO4, filtered, and dried under vacuum resulting in the formation of yellowish oils 2 or 5. The IAC and 2 or 5 were combined in anhydrous DMF and diisopropylethylamine was added to the mixture which was then stirred overnight at room temperature. Reverse-phase HPLC purification followed by TFA deprotection yielded 1 or 4, respectively.23, 24 To evaluate the radiolabeling efficiency of the NODA-GA and DOTA-GA conjugates,111In, 67Ga and 203Pb were employed to demonstrate facile formation of complexes with these radionuclides. The NODA-GA conjugate 1 was efficiently radiolabeled (> 90 %) with 111In and 67Ga within 30 min (Fig. 2A and 2B, respectively). The radiolabeling of the DOTA-GA conjugate 4 with 203Pb was equally efficient (Fig. 2C). Non-radioactive Ga(III)-1 and Pb(II)-4 were also synthesized in order to characterize the radiolabeled 67Ga and 203Pb complexes.25, 26 Figures 3 and ​and44 demonstrate HPLC profiles of the mixture containing both Ga(III)-1 and 67Ga-1; and Pb(II)-4 and 203Pb-4, respectively. Figure 2 Radio-HPLC profiles of 111In-1, A; 67Ga-1, B; and 203Pb-4, C and Figure 3 HPLC profiles of Ga(III)-1 (top) and 67Ga-1 (bottom) Figure 4 HPLC profiles of Pb(II)-4 (top) and 203Pb-4 (bottom). Scheme 1 Synthesis of compound 1. Scheme 2 Synthesis of compound 4. A radioimmunoassay was performed to assess the binding ability of the radiolabeled NODA-GA and DOTA-GA conjugates with αvβ3 integrin. The 111In-labeled 1 (2 × 106 cpm, 0.47 μM), 67Ga-labeled 1 (5 × 105 cpm, 0.45 μM) or 203Pb-labeled 4 (3 × 105 cpm, 0.5 μM) was incubated with 0, 0.5, 1.0 and 2.0 μM of purified human αvβ3 integrin (MW 237,000) in a total volume of 25 μL PBS for 3 h at 37 °C. For non-specific binding, excess IAC (20 μM) was added to the reaction mixture to block binding. The reaction mixture was then separated on a 10 mL Sephadex G50 column using PBS as eluent. Fractions (0.5 mL) were collected and subsequently counted in a γ-counter. As indicated in Table 1, the labeled conjugates bound the integrin to varying degrees. The binding of 111In-1 was greatest followed by 67Ga-1 and then 203Pb-4. In addition, binding was blocked ~95% by the addition of a 10 to 20-fold molar excess of the cold IAC to the reaction solution indicating specific binding of the labeled conjugates. Furthermore, it is worth noting that the reactivity of the 111In-1 with αvβ3 integrin (88 %) is higher than that reported for 111In-DOTA-IAC (72 %).7 Table 1 Binding of 111In-1, 67Ga-1 and 203Pb-4 to purified αvβ3 integrin. In conclusion, the peptidomimetic αvβ3 integrin antagonist (IAC) was conjugated to NODA-GA and DOTA-GA and successfully radiolabeled with 111In, 67Ga and 203Pb. This promising preliminary data is fueling further investigation of NODA-GA-IAC and DOTA-GA-IAC conjugates for targeting tumor associated angiogenesis and αvβ3 integrin positive tumors using PET and SPECT imaging. Other potential applications include the use of radionuclides such as 90Y, 177Lu and 212Pb for radiotherapy.