RGD conjugates of the H2dedpa scaffold: synthesis, labeling and imaging with 68Ga

https://doi.org/10.1016/j.nucmedbio.2012.01.003Get rights and content

Abstract

Introduction

The rekindled interest in the 68Ga generator as an attractive positron emission tomography generator system has led us and others to investigate novel chelate systems for 68Ga. We have previously reported our findings with the acyclic, rapidly coordinating chelate H2dedpa and its model derivatives.

Methods

In this report, we describe the synthesis of the corresponding bifunctional chelate scaffolds (H2dp-bb-NCS and H2dp-N-NCS) as well as the radiolabeling properties, transferrin stability, binding to the target using in vitro cell models and in vivo behavior the corresponding conjugates with the αvβ3 targeting cyclic pentapeptide cRGDyK (monomeric H2RGD-1 and dimeric H2RGD-2).

Results

The ability of the conjugated ligands to coordinate Ga isotopes within 10 min at room temperature at concentrations of 1 nmol was confirmed. Complex [67Ga(RGD-1)]+ was more stable (92% after 2 h) than [67Ga(RGD-2)]+ (73% after 2 h) in a transferrin challenge experiment. IC50 values for both conjugates (H2RGD-1 and H2RGD-2) and nonconjugated RGD were determined in a cell-based competitive binding assay with 125I-echistatin using U87MG cells, where enhanced specific binding was observed for the multivalent H2RGD-2 conjugate compared to the monovalent H2RGD-1 and nonconjugated cRGDyK. The U87MG cell line was also used to generate subcutaneous xenograft tumors on RAG2M mice, which were used to evaluate the in vivo properties of [68Ga(RGD-1)]+ and [68Ga(RGD-2)]+. After 2 h of dynamic imaging, both block and nonblock mice were sacrificed to collect select organs at the 2-h time point. Although the uptake is specific, as judged from the ratios of nonblock to block (2.36 with [67Ga(RGD-1)]+, 1.46 with [67Ga(RGD-2)]+), both conjugates display high uptake in blood.

Conclusions

We have successfully synthesized and applied the first bifunctional versions of H2dedpa for conjugation to a targeting vector and subsequent imaging of the corresponding conjugates.

Introduction

The radionuclides of Ga, Cu, Zr, In and Y have sparked great interest recently because of their increased availability from small biomedical cyclotrons [1]. Among these radiometals, 68Ga is of particular interest because it has outstanding properties for nuclear medicine applications. 68Ga has suitable properties for high-quality positron emission tomography (PET) imaging, including a short half-life (t1/2=68 min), decay by 89% positron emission and a maximum positron energy of 1.899 keV [1], [2], [3], [4]. While Ga(I) is only stable in nonaqueous media under nonoxidizing conditions, under physiological conditions Ga(III) is the only stable and relevant oxidation state. The availability of the 68Ge/68Ga generator system also provides imaging centers with a PET nuclide that is routinely available in a manner similar to the generator-produced single-photon emission computed tomography nuclide 99mTc.

One of the most sophisticated commercially available 68Ge/68Ga generator systems is based on titanium dioxide (Eckhard & Ziegler) and is currently used for research and clinical trials worldwide. The advantages of this particular generator include nontoxic packing materials and, most importantly, the elution of free, cationic 68Ga with an acid concentration low at 0.1 M HCl, allowing universal application for radiopharmaceutical preparations. Other advantages include low 68Ge breakthrough (between 0.001% and 0.005%) [4].

The ideal bifunctional chelate (BFC) for 68Ga would be one that was simply functionalized to enable easy conjugation to targeting vectors such as peptides, rapidly coordinated Ga(III) under mild conditions (pH>4, room temperature, labeling within 15 min) and formed a complex with high kinetic inertness (inert versus apo-transferrin challenge and in vivo conditions). A thermodynamic stability constant log KML above the value found for the main in vivo competitor, transferrin (log KML=20.3) [5], is desired; however, a kinetically inert formed radiochemical complex is key. The 3+ charge is preferentially reduced to 1+, 1− or neutral in order to avoid high kidney uptake and accelerated excretion. Mäcke and coworkers identified one of the most promising candidates, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), as well as its corresponding bifunctional version, 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), almost one decade ago for radiolabeling with 68Ga [6], and these chelates still prevail in various applications [7], [8]. Most recent developments in chelate design include macrocyclic chelates, such as the NOTA-type chelate 1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid, [9] or 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid, a pyridine-containing ligand system suitable for rapid coordination of both Ga and Cu-radionuclides [10], [11], [12]. Acyclic chelate systems such as a tris(hydroxypyridinone) ligand system (CP256) [13] and the chelate H2dedpa and its model derivatives H2dp-bb-NO2 and H2dp-N-NO2 developed by our group have also been studied [14].

Herein, we report the synthesis of the corresponding isothiocyanato derivatives for conjugation to terminal amines as the first true BFCs based on the H2dedpa scaffold. We used these novel BFCs to conjugate, as proof of principle, the widely investigated small cyclic peptide c(RGDyK) affording monomeric H2RGD-1 and dimeric H2RGD-2 (Fig. 1).This pentapeptide binds to the αvβ3 integrin, a transmembrane cellular protein that recognizes an open RGD frame in various components of the extracellular matrix. The expression of αvβ3 integrin has been detected on blood vessels undergoing angiogenesis (e.g., tumors, inflammation or wound healing [15]) and has also been linked to certain tumors with high metastatic potential [16]. Since c(RGDyK) localizes fast to its target and has excellent organ clearance, it is well suited for a shorter-lived radioisotope such as 68Ga and serves as a convenient tool to investigate chelate-induced effects such as clearance properties and nonspecific uptake. We aimed to investigate both the impact of our BFCs on the clearance and localization profile, as well as the possibility of the introduction of multivalency, similar to previously documented approaches with NODAGA- [8] and CB-TE2A-type systems [17].

Section snippets

Materials and methods

The analytical thin-layer chromatography (TLC) plates were aluminum-backed ultrapure silica gel 250 μm; the flash column silica gel (standard grade, 60 Å, 32–63 mm) was acquired from Silicycle. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on Bruker AV300, AV400 or AV600 instruments at ambient temperature; the NMR spectra are expressed on the δ scale and referenced to residual solvent peaks or internal tetramethylsilane. Electrospray ionization mass spectrometry (ESI-MS)

Organic synthesis and cold coordination chemistry

For the N-derivative, which has a lack of secondary amines and an inherent C2 symmetry (Scheme 1), the aniline was introduced via alkylation of Me2dedpa with Boc-protected p-anilino fragment to form 1. 1 that can be subsequently deprotected to afford 2. In order to furnish the corresponding isothiocyanato derivative, however, t-Boc deprotection and extraction workup was avoided since a large amount of the diamine 2 would be lost to the aqueous phase. Instead, an alternative sequence of

Conclusions

For the first time, we have successfully synthesized isothiocyanato derivatives of the H2dedpa scaffold. The ease of coupling to primary amines by the ready-to-couple BFCs was demonstrated by conjugation to the widely investigated cyclic pentapeptide c(RGDyK), which displays high affinity to αvβ3 integrin. The obtained conjugates H2RGD-1 and H2RGD-2 were able to maintain the labeling properties (10 min, RT, pH 4.5 10 mM NaOAc buffer) reported for model derivatives with 67/68Ga. The predicted

Acknowledgments

The authors acknowledge the University of British Columbia for a 4-year fellowship (E.B.), NORDION (Canada) and the Natural Sciences and Engineering Research Council (NSERC) of Canada for grant support, and NSERC for a postgraduate scholarship (E.W.P.).

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