Enhancing PET Signal at Target Tissue in Vivo: Dendritic and Multimeric Tris(hydroxypyridinone) Conjugates for Molecular Imaging of α_vβ₃ Integrin Expression with Gallium-68

Abstract

Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 (⁶⁸Ga) under mild conditions, and the resulting radiotracers can delineate peptide receptor expression at sites of diseased tissue in vivo. We have synthesized a dendritic bifunctional chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (SCN-HP₉) that can coordinate up to three Ga³⁺ ions. This derivative has been conjugated to a trimeric peptide (RGD₃) containing three peptide groups that target the α_vβ₃ integrin receptor. The resulting dendritic compound, HP₉-RGD₃, can be radiolabeled in 97% radiochemical yield at a 3-fold higher specific activity than its homologues HP₃-RGD and HP₃-RGD₃ that contain only a single metal binding site. PET scanning and biodistribution studies show that [⁶⁸Ga(HP₉-RGD₃)] demonstrates higher receptor-mediated tumor uptake in animals bearing U87MG tumors that overexpress α_vβ₃ integrin than [⁶⁸Ga(HP₃-RGD)] and [⁶⁸Ga(HP₃-RGD₃)]. However, concomitant nontarget organ retention of [⁶⁸Ga(HP₉-RGD₃)] results in low tumor to nontarget organ contrast in PET images. On the other hand, the trimeric peptide homologue containing a single tris(hydroxypyridinone) chelator, [⁶⁸Ga(HP₃-RGD₃)], clears nontarget organs and exhibits receptor-mediated uptake in mice bearing tumors and in mice with induced rheumatoid arthritis. PET imaging with [⁶⁸Ga(HP₃-RGD₃)] enables clear delineation of α_vβ₃ integrin receptor expression in vivo.

Introduction

Peptide-based imaging agents in nuclear medicine have tremendous utility in diagnosis, prognosis, and selection of therapeutic regimes for patients. Radiometals can be incorporated into clinically relevant peptides via a bifunctional chelator, providing effective and sensitive radiotracers that can be prepared conveniently in a radiopharmacy. The metallic isotope ⁶⁸Ga possesses decay properties that are suitable for positron emission tomography (PET) (68 min half-life, 1899 keV β⁺ emission with 88% abundance), and the availability of a pharmaceutical grade ⁶⁸Ge/⁶⁸Ga generator means that the number of molecular imaging agents based on ⁶⁸Ga is likely to increase in the coming years. The somatostatin receptor 2-targeted imaging agent, ⁶⁸Ga-DOTATATE for neuroendocrine tumors,¹⁻³ and more recently, the prostate specific membrane antigen targeted conjugate, ⁶⁸Ga-HBED-PSMA,^4,5 have demonstrated clinical utility in patient prognosis and management. Other chelator systems have exhibited efficient ⁶⁸Ga³⁺ radiolabeling properties, and the resulting radiolabeled conjugates are effective at delineating target tissue in vivo.⁶⁻¹² Among these are the tripodal tris(hydroxypyridinone) chelators containing three 1,6-dimethyl-3-hydroxypyridin-4-one groups that can coordinate radiometallic Ga³⁺ and Zr⁴⁺ ions,¹³⁻¹⁵ as well as Fe³⁺ and Al³⁺ with high affinity.^16,17 Upon deprotonation of hydroxyl groups, the hexadentate O₆ ligand, THP-Ac, can coordinate ⁶⁸Ga³⁺ at mild pH (pH 6.5–7.5) and low ligand concentrations (10 μM) in <5 min.¹³ Bifunctional derivatives of THP-Ac can be attached to peptides, with the resulting conjugates able to rapidly and quantitatively coordinate aqueous ⁶⁸Ga³⁺ at room temperature at pH 5–7.^18,19

Multimeric peptide-based imaging agents that incorporate more than one peptide targeting group (and in particular small peptides that target the α_vβ₃ integrin receptor) have demonstrated increased accumulation of activity at tissue targets in vivo relative to their monomeric analogues, and are effective contrast agents.^8,12,20−27 This increased accumulation of radiotracer in the case of multimeric compounds can be attributed to either (i) divalent binding, where two targeting peptides bind to two receptors simultaneously, requiring sufficient distance between the two intramolecular targeting ligands to form this bridge; or (ii) an increased local effective concentration of targeting peptide at the receptor site, resulting in a shift in equilibrium toward peptide receptor binding. In the case where the multimeric derivative has an increased persistence in the blood pool in vivo relative to the monomeric derivative, it is also possible that increased uptake is a result of increased bioavailability.

Dendritic and multimeric chelator systems for coordination of Gd³⁺ and lanthanides provide enhanced contrast as MRI agents compared to their single-chelator counterparts, in large part due to the increased number of metal complexes present.²⁸⁻³⁶ Multimeric constructs containing multiple chelating groups (including hydroxypyridinones) have also been synthesized for the purposes of sequestering trace metal ions.^17,37 A small body of work has been described in which the number of radiometal coordination sites on a radiolabeled protein/antibody has been increased by incorporation of either a dendrimer possessing multiple chelating groups,³⁸⁻⁴¹ or a pendant function (such as polylysine) on which multiple chelators are appended.⁴²⁻⁴⁴ In these cases, the functionalized/radiolabeled biomolecule retained affinity for receptor targets in vitro^40,41,44 and in vivo.^38,39,42,43 Additionally, when compared to bioconjugates containing fewer chelating groups, dendrimer or multichelator conjugated proteins demonstrated higher specific activity—that is, a higher number of radionuclides were bound per bioconjugate compared to homologues containing a single chelator.^38,41−43 In some cases, this resulted in higher concentrations of radioactivity at target tissue (tumors) in vivo.⁴²

We are interested in using tris(hydroxypyridinone) ligands to explore new ways of increasing accumulated radioactivity at diseased tissue where target receptors are localized in vivo. Here we aim to incorporate multiple in vivo targeting peptide groups and multiple chelator groups into a single molecule, thus increasing the affinity of the radiolabeled conjugate for receptors in vivo as well as increasing the specific activity of the radiolabeled species itself. This approach mimics that of multifunctionalized nanoparticles, where multiple copies of a targeting biomolecule/modifying agent and multiple chelating groups are appended to the surface of a nanoparticle.⁴⁵⁻⁴⁸ We have employed the cyclic pentapeptide, c(RGDfK), (Chart 1) that targets the endothelial α_vβ₃ integrin receptor expressed in angiogenesis, and is associated with metastatic disease in cancers. The biology of such conjugates is well understood, and derivatives of c(RGDfK) are useful model compounds for assessing novel approaches to molecular imaging using peptide-based agents.

Chart 1.

We have synthesized a novel bifunctional chelator construct that incorporates three hexadentate tris(hydroxypyridinone) metal binding sites for Ga³⁺ and an isothiocyanate group that can be utilized as an attachment point to lysine residues of the N-terminus of peptides. We have also employed a scaffold for facile attachment of three c(RGDfK) peptide targeting groups that contain available Lys or N-terminal primary amine groups, ultimately providing a trimeric targeting motif. Conjugating these two motifs together results in a dendritic compound where an aryl group links a trimeric peptide targeting group and three tris(hydroxypyridinone) (THP) groups. We have also synthesized homologues of this compound to investigate the effect of incorporating multiple copies of a peptide/chelating group on in vitro uptake and in vivo biodistribution and target tissue accumulation. Finally, we have chosen the radiotracer with the best in vivo tumor to nontarget organ contrast and assessed the radiotracer in a model of rheumatoid arthritis to further illustrate the utility of ⁶⁸Ga-labeled THP conjugates that target integrin receptors for molecular imaging of in vivo events.

Results

Synthesis

To synthesize a multidentate chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (as three tris(hydroxypyridinone) moieties), the benzyl-protected tris(hydroxypyridinone) (2) (3 equiv) was reacted with tricarboxylate (1), HATU, and diisopropylethylamine under microwave conditions (Scheme 1). The resulting compound (3) was purified using silica chromatography. Subsequent removal of the phthalimide group furnished 4, which possesses a primary amine that can be further functionalized for attachment to biomolecules. This species was converted to a phenyl isothiocyanate by reacting it with an excess of p-phenylene diisothiocyanate, resulting in 5, which was isolated via semipreparative reverse phase HPLC. Compound SCN-HP₉ was obtained upon deprotection of the benzyl-protected hydroxypyridinone groups by reaction of 5 with boron trichloride in dichloromethane, followed by addition of methanol.

Scheme 1. Synthesis of SCN-HP₉.

This study aimed to assess a trimeric scaffold in which all three peptide motifs are equidistant from the bifunctional chelator and chemically equivalent to each other, thus ensuring that all three motifs possess the same affinity for the target receptor. To achieve this, tricarboxylate 1 was converted to a N-hydroxysuccinimide-activated intermediate (6) which was reacted with 3 equiv of c(RGDfK), to provide the multimeric compound 7. Compound 7 was purified and isolated using RP-HPLC. The phthalimide group was removed by reaction with hydrazine in ethanol, followed by addition of HCl (Scheme 2). Subsequently, RGD₃, which contains three c(RGDfK) peptide groups and an apical primary amine, was isolated using RP-HPLC.

Scheme 2. Synthesis of RGD₃.

Reaction of RGD₃ with SCN-HP₉ at 120 °C at 300 W in a microwave reactor provided HP₉-RGD₃ (Chart 1) which was purified and isolated using RP-HPLC in ≥95% purity. HP₉-RGD₃ possesses three c(RGDfK) groups and three tris(hydroxypyridinone) groups, each attached through a tripodal scaffold to a central phenyl group via thiourea linkages. Similarly, the compound SCN-HP₃ (Chart 1), synthesized as previously described,¹⁸ was reacted with (i) RGD₃ and (ii) c(RGDfK) under the same microwave conditions. This provided the derivatives HP₃-RGD₃ and HP₃-RGD (Chart 1) which are trimeric and monomeric c(RGDfK) homologues of HP₉-RGD₃, the former two conjugates containing only a single tris(hydroxypyridinone) ligand for coordination of a single metal ion. These conjugates were also purified using RP-HPLC and isolated in ≥95% purity (as previously reported for HP₃-RGD¹⁸).

Reaction of Hydroxypyridinone Derivatives with ⁶⁸Ga³⁺/^natGa³⁺

Solutions of HP₉-RGD₃, HP₃-RGD₃, and HP₃-RGD were added to an excess of ^natGa(NO₃)₃. For each of these solutions, the reverse phase LCMS total ion chromatogram revealed Ga³⁺ complexation of the hydroxypyridinone conjugates (Figure 1). As predicted, the conjugate HP₉-RGD₃ that contains nine hydroxypyridinone groups (three hexadentate tris(hydroxypyridinone) groups) coordinated three Ga³⁺ metal ions, whereas the conjugates HP₃-RGD₃ and HP₃-RGD, each possessing only a single tris(hydroxypyridinone) chelator, were bound to only a single Ga³⁺ ion. In each case, the most intense set of peaks in the mass spectrum (Figure 1) corresponding to this chromatographic signal matched the stoichiometry of the complexes of formulas listed in Table 1.

Figure 1.

Radio-HPLC chromatogram of ⁶⁸Ga-labeled peptide conjugates (bottom, black traces), and total ion count LCMS chromatograms of ^natGa-complexed peptide conjugates acquired using the same mobile phase conditions (middle, blue traces); mass spectral signals associated with each LCMS chromatographic signal (top, black—experimental signal, red—calculated signal): (a) [Ga(HP₃-RGD)], (b) [Ga(HP₃-RGD₃)], (c) [Ga₃(HP₉-RGD₃)].

Table 1. Observed and Calculated m/z Values for Mass Spectral Signals of [Ga(HP₃-RGD)], [Ga(HP₃-RGD₃)], and [Ga₃(HP₉-RGD₃)], HPLC Retention Times of ^nat/68Ga Labeled Conjugates, and Log D values of ⁶⁸Ga Labeled Conjugates.

compound	stoichiometry of observed MS signal	observed (and calculated) m/z (monoisotopic)	LCMS retention time (min)	radio-HPLC retention time (min)	log DOCT/PBS (±SD)
[nat/68Ga(HP3-RGD)]	C72H94N19O17S2Ga + 2H+	815.80 (815.80)	9.00	8.38	–2.62 ± 0.05
[nat/68Ga(HP3-RGD3)]	C139H192N39O25S2Ga + 3H+	1034.45 (1034.45)	9.05	8.37	–2.23 ± 0.05
[natGa3/68Ga(HP9-RGD3)]	C226H306N57O59S2Ga3 + 4H+	1259.26 (1259.25)	8.88	8.32	–2.05 ± 0.19

Solutions of ⁶⁸Ga³⁺ produced from a ⁶⁸Ge/⁶⁸Ga generator (80–120 MBq mL^–1, 0.1 M HCl aqueous solution, 1 mL) were added to HP₉-RGD₃, HP₃-RGD₃, and HP₃-RGD and immediately adjusted to pH 6 by addition of ammonium acetate solution (1 M, 300 μL). As previously described,^18,19 these radiolabeling reactions proceeded rapidly at room temperature in less than 5 min, providing radiolabeled conjugates in >98% radiochemical yield. These mixtures were analyzed using the same HPLC conditions as those employed for the above LCMS studies. Each reaction solution gave rise to only a single signal in the radiochromatogram, each with a similar retention time to corresponding LCMS chromatogram signals (Figure 1, Table 1). Under these conditions, the maximum occupancy of bioconjugate molecules by ⁶⁸Ga³⁺ was low—for HP₉-RGD₃, maximum occupancy was <0.03%, and for HP₃-RGD₃ or HP₃-RGD, maximum occupancy was <0.01%.

To discern whether [Ga(HP₉-RGD₃)] could be chromatographically distinguished from [Ga₂(HP₉-RGD₃)] and [Ga₃(HP₉-RGD3)], solutions of HP₉-RGD₃ were added to aqueous solutions containing 1, 2, or 3 equiv of Ga³⁺, and the samples were analyzed by reverse phase LCMS coupled to a UV detector (Figure 2) using a less steep solvent gradient than that employed above. [Note: To avoid confusion over the oxidation state of the metal and the pH being monitored, we adopt normal convention in showing peptides as neutral or uncharged ligands either alone or in metal complexes, even though Arg/R side chains are protonated in water at pH < 9, and Asp/D side chains are deprotonated at pH > 5. Additionally, we have elected not to indicate the protonation state of HP groups in our nomenclature, even though at pH < 4.5 in water, HP groups are protonated bearing a single positive charge, and >8 HP groups are deprotonated bearing a single negative charge. At pH 5–7.5 in water, HP groups are neutral. Upon coordination to a metal ion, each bidentate HP group is deprotonated, formally bearing a single negative charge. Thus, HP₃ and HP₉ groups and trivalent metal complexes thereof are uncharged at pH 5–7.5 in aqueous solutions.] When an analytical HPLC column was utilized for LCMS analysis, the UV chromatogram of non-Ga-treated HP₉-RGD₃ revealed at least three species (Figure 2b). The mass spectrum indicated that these species corresponded to [Al(HP₉-RGD₃)], [AlFe(HP₉-RGD₃)], [Fe(HP₉-RGD₃)], and [Fe₂(HP₉-RGD₃)] (Figure 2b). It is likely that the metal ions that gave rise to these complexes came from storage vials and/or parts of the LCMS instrumentation, including the analytical reverse phase C18 column. Notably, samples of HP₉-RGD₃ isolated immediately after preparative HPLC that were not passed through an analytical HPLC column, nor lyophilized and stored for extended periods of time, contained fewer metal complex species, as measured by mass spectrometry (Figure 2a), although the [Al(HP₉-RGD₃)] complex was still prominent.

Figure 2.

(a) Mass spectrum (right) of HP₉-RGD₃ acquired immediately after preparative HPLC purification (without analytical HPLC separation). Analytical HPLC UV chromatograms (left) and mass spectra (right) of solutions of HP₉-RGD₃ with (b) no added Ga³⁺, (c) 1 equiv of Ga³⁺, (d) 2 equiv of Ga³⁺, (e) 3 equiv of Ga³⁺, (f) ⁶⁸Ga generator eluate; (g) HPLC radiochromatogram (left) of [⁶⁸Ga(HP₉-RGD₃)]. Charge of mass spectral ions (z) = 4+.

In solutions containing a single equivalent of Ga³⁺, the major Ga-containing species corresponded to [Ga(HP₉-RGD₃)] and [AlGa(HP₉-RGD₃)], and the corresponding UV chromatograms indicated the presence of multiple species (Figure 2c). In solutions containing 2 equiv of Ga³⁺, several Ga-containing species could be detected including [AlGa(HP₉-RGD₃)], [FeGa(HP₉-RGD₃)], [Ga₂(HP₉-RGD₃)], [AlFeGa(HP₉-RGD₃)], [AlGa₂(HP₉-RGD₃)], and [Fe₂Ga(HP₉-RGD₃)] (Figure 2d). Similarly, the corresponding UV chromatogram indicated the presence of multiple species. Lastly, when 3 equiv of Ga³⁺ were added to HP₉-RGD₃, only two Ga-containing species were observed: [FeGa₂(HP₉-RGD₃)] and [Ga₃(HP₉-RGD₃)] (Figure 2e). Only two significant signals were discernible in the UV chromatogram.

The same “slow” LCMS gradient coupled to UV detection was used to analyze a sample nominally described here as [⁶⁸Ga(HP₉-RGD₃)] (Figure 2f,g). The concentration of ⁶⁸Ga was too low to allow detection of ⁶⁸Ga-containing species using the mass spectrometer available in our laboratory. The species of lowest molecular weight most closely matched stoichiometries of [AlFe(HP₉-RGD₃)], [Fe₂(HP₉-RGD₃)], and [FeGa(HP₉-RGD₃)]. Unlike samples of HP₉-RGD₃, neither [Al(HP₉-RGD₃)] nor [Fe(HP₉-RGD₃)] was detected. Generator eluate is known to contain significant amounts of Fe³⁺,⁴⁹ and it is likely that HP₉-RGD₃ complexes this during the radiolabeling reaction. Indeed, unpublished data from our laboratory indicate that concentrations of Fe, Al, and ^natGa in generator eluate (0.1 M HCl, from an Eckert and Ziegler generator) are approximately 0.5 μM, 3 μM, and 1 μM, respectively. At these concentrations, Fe³⁺, Al³⁺, and ^natGa³⁺ will compete with ⁶⁸Ga³⁺ (present at picomolar concentrations) for ligand coordination, accounting for low percentage occupancy by ⁶⁸Ga³⁺ of all three conjugates. Most importantly, the radiolabeled species in solution, referred to as [⁶⁸Ga(HP₉-RGD₃)] for the purposes of simple nomenclature, most likely corresponds to [M₂⁶⁸Ga(HP₉-RGD₃)] or [M⁶⁸Ga(HP₉-RGD₃)] (M = Al, Fe, or ^natGa). Thus, in vivo studies, described herein, measure the biodistribution of ⁶⁸Ga-labeled HP₉-RGD₃ in which one or two tris(hydroxypyridinone) sites are occupied by nonradioactive metal ions. The resolution of the scintillation detector was lower than the resolution of the UV detector, and using the same gradient, the radiochromatogram of [⁶⁸Ga(HP₉-RGD₃)] gave rise to a single, very broad peak, coincident with the UV chromatogram signals.

Significantly higher amounts of ⁶⁸Ga³⁺ were utilized to assess the maximum specific activity that could be complexed quantitatively by the conjugates. In this procedure, ⁶⁸Ga³⁺ eluted from an iThemba generator was processed to preconcentrate the generator eluate and remove ⁶⁸Ge breakthrough.⁵⁰ The final ⁶⁸Ga³⁺ solution consisted of 700–1000 MBq ⁶⁸Ga³⁺ in H₂O/ethanol (82%/18%, with [HCl] = 0.18 M, total volume = 1 mL). The conjugates HP₉-RGD₃, HP₃-RGD₃, and HP₃-RGD were added to this solution followed by ammonium acetate and saline to obtain solutions (2.5 mL total volume) at pH 5.5–6.5 that were suitable for in vivo administration. Radiochemical yields of >97% were achieved using a minimum of 4 nmol of HP₉-RGD₃ and 12 nmol of either HP₃-RGD₃ or HP₃-RGD. Decreasing the amount of either HP₃-RGD₃ or HP₃-RGD to 4 nmol decreased the radiochemical yield (86% and 88%, respectively). Again, these reactions proceeded rapidly (<5 min) at room temperature, illustrating the utility of tris(hydroxypyridinone) chelators for applications requiring rapid, kit-based labeling. The maximum specific activity of ⁶⁸Ga-labeled HP₉-RGD₃ (180–240 MBq nmol^–1) compared with that of HP₃-RGD₃ or HP₃-RGD (60–80 MBq nmol^–1) was 3-fold higher. The maximum occupancy by ⁶⁸Ga³⁺ per molecule of bioconjugate was low in all cases. For HP₉-RGD₃, maximum occupancy was <0.3%, and for HP₃-RGD₃ or HP₃-RGD, maximum occupancy was <0.1%.

Hydrophilicity of ⁶⁸Ga-Radiolabeled Tracers, and Affinity for α_vβ₃ Integrin Receptor

The log D_OCT/PBS values of the ⁶⁸Ga-labeled conjugates indicated that all three radiotracers possess comparable hydrophilicity (Table 1), with log D values all in the range of −2.7 to −2.0. These data are consistent with the similar reverse-phase HPLC retention times observed for all three radiotracers.

The half-maximal inhibitory concentration (IC₅₀) values of c(RGDfK), [Ga(HP₃-RGD)], [Ga(HP₃-RGD₃)], and [Ga₃(HP₉-RGD₃)] were determined using a cell-free solid-phase competitive binding assay with ¹²⁵I-echistatin. Binding of ¹²⁵I-echistatin to α_vβ₃ integrin was inhibited by all compounds in a concentration-dependent manner broadly indicating that [Ga(HP₃-RGD)], [Ga(HP₃-RGD₃)], and [Ga₃(HP₉-RGD₃)] retained affinity for α_vβ₃ integrin (Table 2, Figure S1). The IC₅₀ value of [Ga(HP₃-RGD)] (46 ± 16 nM) was comparable to that of c(RGDfK) (28 ± 8 nM), demonstrating that conjugation did not markedly affect affinity for the α_vβ₃ integrin receptor. However, the IC₅₀ values of [Ga(HP₃-RGD₃)] (73 ± 22 nM) and [Ga₃(HP₉-RGD₃)] (79 ± 28 nM) were higher than those of c(RGDfK) and [Ga(HP₃-RGD)], suggesting that, in this series of compounds, the presence of three copies of c(RGDfK) peptide on a single molecule did not impart higher affinity toward α_vβ₃ integrin compared to the monomeric THP derivative. This was surprising given that it has been reported that other dimeric,^22,23 trimeric,²⁷ and multimeric⁵¹ RGD peptides demonstrate higher affinities for α_vβ₃ integrin than their monomeric homologues. The addition of the dendritic HP₉ chelator did not affect affinity of RGD₃ for α_vβ₃ integrin—IC₅₀ values of [Ga₃(HP₉-RGD₃)] and [Ga₃(HP₃-RGD₃)] were very similar.

Table 2. IC₅₀ Values for c(RGDfK), [Ga(HP₃-RGD)], [Ga(HP₃-RGD₃)], and [Ga₃(HP₉-RGD₃)], Determined Using a Solid-Phase Competitive Binding Assay with ¹²⁵I-Echistatin.

compound	IC50 (nM) (±standard error)	95% confidence interval (nM)
c(RGDfK)	28 ± 8	12–45
[Ga(HP3-RGD)]	46 ± 16	13–79
[Ga(HP3-RGD3)]	73 ± 22	30–116
[Ga3(HP9-RGD3)]	79 ± 28	23–134

Biodistribution of ⁶⁸Ga-Labeled RGD Conjugates

The biodistribution of [⁶⁸Ga(HP₉-RGD₃)], [⁶⁸Ga(HP₃-RGD₃)], and [⁶⁸Ga(HP₃-RGD)] was assessed in Balb/c nu/nu mice bearing α_vβ₃ integrin-positive glioblastoma U87MG tumors (n = 3). Each animal was administered 18–33 MBq of ⁶⁸Ga-labeled tracer and PET scanned at either 1 or 2 h post-injection (PI) for 10 min, followed immediately by euthanasia and organ harvesting for ex vivo radioactivity counting. To assess specificity of each radiotracer, a separate group of animals was coadministered c(RGDfK) (0.4 mg per animal) with the tracer, followed by scanning, euthanasia, and ex vivo organ counting 1 h PI.

Ex vivo biodistribution data (Figures 3 and S2, and Tables 3, 4, and S1) demonstrated that tumor uptake of dendritic [⁶⁸Ga(HP₉-RGD₃)] was 1.75-fold higher than that of monomeric [⁶⁸Ga(HP₃-RGD)] at 1 h PI. Tumor uptake of trimeric [⁶⁸Ga(HP₃-RGD₃)] was 1.49-fold higher than that of [⁶⁸Ga(HP₃-RGD)]. Tumor uptake of [⁶⁸Ga(HP₉-RGD₃)] was higher than that of [⁶⁸Ga(HP₃-RGD₃)], but this was not statistically significant. By 2 h PI, there were no differences in normalized tumor uptake between the three tracers.

Figure 3.

(a) Ex vivo biodistribution of mice bearing α_vβ₃ integrin-positive U87MG tumors administered (a-i) [⁶⁸Ga(HP₃-RGD)], (a-ii) [⁶⁸Ga(HP₃-RGD₃)], and (a-iii) [⁶⁸Ga(HP₉-RGD₃)], 1 and 2 h PI, and 1 h PI of coadministered c(RGDfK) (blocked); n = 3. Error bars correspond to standard error of the mean. (b) Representative PET maximum intensity projections of Balb/c nu/nu mice bearing a U87MG tumor on right flanks at 1 h PI of (b-i) [⁶⁸Ga(HP₃-RGD)], (b-ii) [⁶⁸Ga(HP₃-RGD)] + c(RGDfK), (b-iii) [⁶⁸Ga(HP₃-RGD₃)], (b-iv) [⁶⁸Ga(HP₃-RGD₃)] + c(RGDfK), (b-v) [⁶⁸Ga(HP₉-RGD₃)], and (b-vi) [⁶⁸Ga(HP₉-RGD₃)] + c(RGDfK). Red arrow indicates position of tumors on animals.

Table 3. α_vβ₃ Integrin-Positive U87MG Tumor Uptake as Measured by Ex Vivo Biodistribution, 1 h PI, and 1 h PI of Coadministered c(RGDfK) (Blocked); n = 3.

compound	1 h PI, %ID g-1 (±SEM)	with block, 1 h PI, %ID g-1 (±SEM)	mean difference (95% CI), %ID g-1	p value
[68Ga(HP3-RGD)]	2.86 ± 0.43	0.99 ± 0.12	1.88 (0.86–2.90)	0.0069
[68Ga(HP3-RGD3)]	4.28 ± 0.53	0.98 ± 0.14	3.29 (2.04–4.54)	0.0019
[68Ga(HP9-RGD3)]	4.69 ± 0.35	1.17 ± 0.11	3.52 (2.68–4.37)	0.0003

Table 4. Differences in Tumor Uptake of ⁶⁸Ga-Tracers 1 h PI, as Measured by Ex Vivo Biodistribution.

comparison	mean difference 1 h PI (95% CI), %ID g-1	p value
[68Ga(HP3-RGD)] and [68Ga(HP3-RGD3)]	1.42 (−0.14–2.97)	0.065
[68Ga(HP3-RGD)] and [68Ga(HP9-RGD3)]	1.83 (0.56–3.10)	0.016
[68Ga(HP3-RGD3)] and [68Ga(HP9-RGD3)]	0.41 (−1.04–1.86)	0.473

Animals administered [⁶⁸Ga(HP₉-RGD₃)] showed increased persistence of activity in blood, and increased liver, spleen, muscle, and lung accumulation compared to animals administered either [⁶⁸Ga(HP₃-RGD₃)] or [⁶⁸Ga(HP₃-RGD)] at both 1 and 2 h PI (Figure 3, Table S1). At both 1 and 2 h PI, the concentration of kidney activity for animals administered [⁶⁸Ga(HP₉-RGD₃)] (1 h PI, 28.32 ± 3.76%ID g^–1) was considerably higher than that of animals administered [⁶⁸Ga(HP₃-RGD₃)] (1 h PI, 10.73 ± 0.18%ID g^–1), which in turn was higher than that of animals administered [⁶⁸Ga(HP₃-RGD)] (1 h PI, 4.91 ± 0.74%ID g^–1).

Additionally, ex vivo biodistribution data (Figures 3 and S2, Table 3) for all three tracers demonstrated that tumor uptake was target-specific at 1 h PI, with significantly lower tumor uptake for animals coadministered c(RGDfK) (“blocked” groups). In the blocked groups for all three tracers, uptake in lungs, heart, spleen, and muscle was generally lower than that observed in “nonblocked” groups. This is consistent with previous reports of receptor-mediated uptake of α_vβ₃ integrin targeted radiotracers,^18,22,25,27 and with the expression of α_vβ₃ integrin at low levels in normal vasculature tissue.^52,53 For all three tracers, radioactivity concentration in the kidneys was higher for the blocked groups than for the nonblocked groups. Similar patterns have previously been described for RGD peptide conjugates,¹¹ and have been attributed to slower kidney filtration rates in the presence of larger amounts of peptide.

PET imaging data for all three tracers was consistent with ex vivo biodistribution data. In PET scans of all three radiotracers acquired 1 h PI, tumors could be delineated, whereas in PET scans of animals coadministered c(RGDfK), tumors could not be delineated (Figure 3). Concentration of radioactivity in kidneys and livers for animals administered [⁶⁸Ga(HP₉-RGD₃)] was significantly higher than that of animals administered either [⁶⁸Ga(HP₃-RGD₃)] or [⁶⁸Ga(HP₃-RGD)].

Thus, although tumor uptake of dendritic [⁶⁸Ga(HP₉-RGD₃)] was higher at 1 h PI than that of either [⁶⁸Ga(HP₃-RGD₃)] or [⁶⁸Ga(HP₃-RGD)], high nontarget organ uptake of [⁶⁸Ga(HP₉-RGD₃)] rendered tumor to nontarget organ ratios significantly lower than that of either [⁶⁸Ga(HP₃-RGD₃)] or [⁶⁸Ga(HP₃-RGD)] at 1 h PI. Contrast between tumor and nontarget organs was best achieved with [⁶⁸Ga(HP₃-RGD₃)] (Figure S3); therefore, further imaging studies assessing α_vβ₃ integrin expression in a mouse model of rheumatoid arthritis utilized this tracer.

Imaging Arthritis with [⁶⁸Ga(HP₃-RGD₃)]

To demonstrate the broad utility of tris(hydroxypyridinone) conjugates for molecular imaging of α_vβ₃ integrin receptor expression, [⁶⁸Ga(HP₃-RGD₃)] was used to image synovial inflammation in mice with induced rheumatoid arthritis. The α_vβ₃ integrin receptor is associated with cell types that are involved in rheumatoid arthritis at synovial joints, including bone resorbing osteoclasts, activated macrophages, and angiogenic endothelial cells.^53,54 Previous studies have demonstrated that radiolabeled RGD peptides target arthritic tissue⁵⁵⁻⁵⁷ as well as osteoclasts.⁵⁸

In these experiments, [⁶⁸Ga(HP₃-RGD₃)] (8–13 MBq) was administered to healthy mice (C57Bl/6 male 6–8 week mice), and mice with induced mild and severe rheumatoid arthritis.^59,60 A separate group of animals with severe rheumatoid arthritis was administered both [⁶⁸Ga(HP₃-RGD₃)] and an excess of c(RGDfK) to assess specificity of in vivo targeted accumulation. Prior to induction of arthritis and administration of radiotracer, the diameters of wrist and ankle joints of all animals were measured using calipers to score the severity of arthritis. At 1 h PI of radiotracer, animals were euthanized by CO₂ asphyxiation followed by PET scanning and organ collection for ex vivo biodistribution.

For all animals, radioactivity concentrations in nontarget tissues and organs, as determined by ex vivo biodistribution data (Figure S4), were consistent with results obtained from animals bearing U87MG tumors administered the same radiotracer. Blockade studies with c(RGDfK) in rheumatoid arthritis animals also indicated that some localization of [⁶⁸Ga(HP₃-RGD₃)] in all harvested organs except the kidneys was likely to be receptor-mediated (Table S2).

The ex vivo biodistribution data demonstrated that the amount of radioactivity in severely arthritic joints (increase in wrist and ankle diameter >0.5 mm) was higher than that in joints of healthy animals, or in joints of animals with mild arthritis (increase in wrist and ankle diameter <0.5 mm). In this analysis, joints were grouped according to their type (wrist or ankle) and the increase in swelling (caliper joint measurements) from point of disease induction to the point of imaging (8 days). Total %ID is reported rather than %ID g^–1, because of the large experimental error associated with dissecting and measuring the mass of mouse joint tissue. In severely arthritic joints, there was significantly higher radioactivity accumulation than in joints that were not classified as severely arthritic (including healthy animals and animals induced with mild arthritis) (Figure 4, Table 5). On the basis of %ID, severely arthritic joints could be differentiated from healthy joints, or joints that did not exhibit severe inflammation. Furthermore, blockade studies in severely arthritic animals demonstrated that uptake of [⁶⁸Ga(HP₃-RGD₃)] in inflamed arthritic joints was receptor-mediated (Figure 4, Table 5). Joint uptake in severely arthritic animals coadministered [⁶⁸Ga(HP₃-RGD₃)] and c(RGDfK) peptide was significantly lower than that of severely arthritic animals administered solely [⁶⁸Ga(HP₃-RGD₃)].

Figure 4.

Ex vivo biodistribution of arthritic joints of healthy mice (blue), mice with induced mild rheumatoid arthritis (red) and mice with induced severe rheumatoid arthritis (green), administered [⁶⁸Ga(HP₃-RGD₃)] (8–13 MBq). A blockade study was also undertaken in mice with induced severe rheumatoid arthritis, in which [⁶⁸Ga(HP₃-RGD₃)] was coadministered with cRGDfK (pink). Top: %ID in feet/ankle joints; bottom: %ID in hand/wrist joints. Increase in swelling of joints was measured as the increase from the point of induction of arthritis to PET scanning/biodistribution studies (8 days).

Table 5. Ex Vivo Biodistribution of Joints of Mice Administered [⁶⁸Ga(HP₃-RGD₃)].

group	%ID 1 h PI (±SEM)	mean difference (95% CI) compared to severely arthritic joints, %ID	p value
Wrists
severely arthritic (>0.5 mm increase)	0.26 ± 0.02	-	-
severely arthritic with block	0.08 ± 0.01	0.18 (0.16–0.20)	<0.0001
healthy or mildly arthritic (<0.5 mm increase)	0.14 ± 0.02	0.12 (0.09–0.14)	<0.0001
Ankles
severely arthritic (>0.5 mm increase)	0.68 ± 0.18	-	-
severely arthritic with block	0.24 ± 0.02	0.44 (0.27–0.63)	0.0005
healthy or mildly arthritic (<0.5 mm increase)	0.29 ± 0.03	0.39 (0.30–0.50)	<0.0001

When animals’ joints were grouped according to extent of disease (classified according to whether animals were induced with mild or severe arthritis rather than increase in joint swelling), there were no statistically significant differences in joint uptake between healthy mice and mice with mild arthritis. There were statistically significant differences between healthy mice and mice with severe arthritis, and between mice with mild arthritis and those with severe arthritis (Figure S5, Table S3).

PET scans were consistent with biodistribution data (Figure 5). In PET images acquired 1 h PI, severely arthritic joints could be distinguished from joints that were not inflamed, both between different animals (Figure 5a vs b) and within the same animal (Figure 5b). For example, within one animal with severe arthritis, the left wrist was severely inflamed (a 1 mm increase in swelling from the point of disease induction) but the right demonstrated no swelling throughout the disease induction period. In the PET/CT scan, higher radiotracer uptake was discernible in the left wrist compared to the right wrist (Figure 5b), consistent with ex vivo biodistribution data.

Figure 5.

PET/CT maximum intensity projections of mice administered [⁶⁸Ga(HP₃-RGD₃)] (8–13 MBq) 1 h PI (all PET images scaled from 0.08 to 8.0%ID g^–1): (a) a healthy C57Bl/6 mouse; (b) a C57Bl/6 mouse with severe rheumatoid arthritis; (c) a C57Bl/6 mouse with severe rheumatoid arthritis coadministered c(RGDfK). Arrows indicate joints with an increase in swelling >0.5 mm.

Discussion and Concluding Remarks

Multimeric receptor-targeted radiolabeled agents, in which each molecule contains either multiple copies of each targeting peptide, or multiple copies of a multidentate chelator,⁴² can deliver increased radioactive payloads to target tissue relative to monomeric homologues. Dimeric,^25,61−63 trimeric,^8,27 tetrameric,^64,65 and octameric⁶⁶ RGD conjugates radiolabeled with metallic PET isotopes have demonstrated efficacious α_vβ₃ integrin targeting in vivo. Herein, we have described the first radiolabeled molecular compound that integrates multiple copies of a chelator and multiple copies of peptide, in this case, α_vβ₃ integrin-targeted c(RGDfK). In contrast to previous work on multimeric RGD peptide conjugates, the new dendritic system possesses tris(hydroxypyridinone) chelators that coordinate ⁶⁸Ga³⁺ quantitatively at ambient temperature, near neutral pH, and micromolar concentrations of conjugate, allowing for rapid, one-step labeling.

Synthesis of HP₉-RGD₃, which consists of a bifunctional enneakis(hydroxypyridinone) ligand attached to a trimeric peptide targeting α_vβ₃ integrin receptors, enabled assessment of whether dendritic chelator systems could enhance specific activity in radiolabeled molecular imaging agents. Incorporation of multiple hexadentate tris(hydroxypyridinone) groups into a single construct, HP₉-RGD₃, increased specific activity relative to HP₃ derivatives. At concentrations of ∼1.6 μM, near-quantitative radiochemical yields (>97%) were achieved for HP₉-RGD₃, whereas for HP₃-RGD and HP₃-RGD₃, concentrations of 5 μM were required to achieve the same radiochemical yields. The maximum specific activity of ⁶⁸Ga-labeled HP₉-RGD₃ (180–240 MBq nmol^–1) was 3-fold higher when compared with that of HP₃-RGD₃ or HP₃-RGD (60–80 MBq nmol^–1).

Analytical HPLC using a UV detector (interfaced with a mass spectrometer and a gamma scintillation detector) could distinguish numerous signals arising from a sample of [⁶⁸Ga(HP₉-RGD₃)]. For the same sample, line widths of signals in the radiochromatogram and the TIC chromatograms were much broader, and could not be precisely correlated with individual signals in the UV chromatogram. Mass spectral analysis indicated that all tris(hydroxypyridinone) sites in [⁶⁸Ga(HP₉-RGD₃)] were likely occupied by trivalent metal ions. Hexadentate THP-Ac has a very high affinity for Fe³⁺, with log K₁ = 32.52 and pFe³⁺ = 28.47 at pH 7.4 ([Fe³⁺] = 10^–6 M, [Ligand] = 10^–5 M).¹⁷ Dendritic derivatives have similar affinities.¹⁷ While affinities of 3-hydroxy-4-pyridinones for Al³⁺ are normally several orders of magnitude lower than for Fe³⁺, they are still high—for example, for a series of tripodal tris(hydroxypyridinones), pAl³⁺ consistently measured 21–22 at pH 7.4.¹⁶ It is unsurprising that in ⁶⁸Ga-labeled HP₉-RGD₃, tris(hydroxypyridinone) sites unoccupied by ⁶⁸Ga³⁺ complex adventitious Fe³⁺, Al³⁺, and ^natGa³⁺.

It was hypothesized that the higher specific activity and the trimeric peptide nature of [⁶⁸Ga(HP₉-RGD₃)] might result in targeted tumor delivery of a comparatively higher radioactive payload per molecule of receptor-bound bioconjugate compared to [⁶⁸Ga(HP₃-RGD₃)] and [⁶⁸Ga(HP₃-RGD)].

Despite the lower IC₅₀ value of the monomeric [⁶⁸Ga(HP₃-RGD)] species, its in vivo normalized tumor accumulation (as measured by ex vivo biodistribution) was lower than that of trimeric [⁶⁸Ga(HP₃-RGD₃)] and dendritic [⁶⁸Ga(HP₉-RGD₃)] at 1 h PI (Figure 3). The radiotracers’ pharmacokinetics as well as α_vβ₃ integrin affinities are likely to be important in influencing their relative tumor uptake. It is possible that in vivo, blood clearance of [⁶⁸Ga(HP₃-RGD)] was faster than that of [⁶⁸Ga(HP₃-RGD₃)] and [⁶⁸Ga(HP₉-RGD₃)] at early time points (before 1 h PI), resulting in higher tumor bioavailability and thus higher uptake of [⁶⁸Ga(HP₃-RGD₃)] and [⁶⁸Ga(HP₉-RGD₃)] 1 h PI. Additionally, for HP₉-RGD₃, it is likely that there was comparatively lower occupancy of target α_vβ₃ integrin in vivo by unlabeled conjugate. Comparable amounts of radioactivity were administered to each mouse for the three different radiotracers, but in the case of HP₉-RGD₃, ∼0.15 nmol of conjugate was administered to each animal, compared to ∼0.5 nmol for HP₃-RGD₃ and HP₃-RGD.

There was an increase in radioactivity concentration in tumors at 1 h PI for [⁶⁸Ga(HP₉-RGD₃)] compared to [⁶⁸Ga(HP₃-RGD₃)] (mean difference = 0.41%ID g^–1), and for [⁶⁸Ga(HP₃-RGD₃)] compared to [⁶⁸Ga(HP₃-RGD)] (mean difference = 1.42%ID g^–1) (Tables 3, 4). There was a statistically significant increase (mean difference = 1.83%ID g^–1) in tumor accumulation at 1 h PI for [⁶⁸Ga(HP₉-RGD₃)] compared to [⁶⁸Ga(HP₃-RGD)]. Thus, the combination of increased specific activity and a multimeric effect could be responsible for this increase in radioactivity concentration in tumors of animals administered the dendritic radiotracer. Such an observation lends credence to the hypothesis that dendritic radiotracers that incorporate multiple chelating groups and copies of targeting peptides can increase radioactive payload at tissue, relative to monomeric homologues. The higher mean difference in tumor uptake between [⁶⁸Ga(HP₃-RGD₃)] and [⁶⁸Ga(HP₃-RGD)], compared to [⁶⁸Ga(HP₉-RGD₃)] and [⁶⁸Ga(HP₃-RGD₃)], suggests that the presence of a multimeric peptide is the more significant of the two factors in enhancing tumor uptake.

The distance between c(RGDfK) groups in HP₃-RGD₃ and HP₉-RGD₃ is not sufficient to allow simultaneous binding to two or more α_vβ₃ integrin receptors. It is possible that increasing this distance to allow simultaneous binding could result in higher uptake and retention in tissue that overexpresses α_vβ₃ integrin receptors.^62,63

We have previously demonstrated that THP peptide conjugates can rapidly and quantitatively complex ⁶⁸Ga³⁺ at near-neutral pH, ambient temperature, and low chelator concentrations, and that the resulting radiotracers can delineate tumor tissue in vivo.^18,19 [⁶⁸Ga(HP₃-RGD₃)], prepared using simple radiolabeling procedures, can image synovial inflammation in mice with induced rheumatoid arthritis, distinguishing severely arthritic joints from disease-free joints. Few data are available on α_vβ₃ integrin targeted radiotracers for rheumatoid arthritis,^55,56 and the mouse models employed in previously published studies differ from the model utilized here. However, we note that [⁶⁸Ga(HP₃-RGD₃)] uptake in severely arthritic synovial joints of mice in a serum transfer-induced arthritis model (0.26 ± 0.02%ID in wrists and 0.68 ± 0.18%ID in ankles 1 h PI) is comparable to ¹¹¹In-DOTA-E(RGDfK)₂ uptake in mice in a collagen-induced arthritis model (0.2 ± 0.1%ID in arthritic joints at 1 h PI).⁵⁵

Rheumatoid arthritis is currently diagnosed using information from physical examination (signs and symptoms), blood tests, X-rays, and MRI. Ideally, it is diagnosed early so that treatment that slows or halts disease progression can begin. Current early diagnostic methods detect anatomical changes in patients’ joints and so arthritic sites in patients that are not symptomatic or visually identifiable are missed.^67,68 PET/CT imaging of α_vβ₃ integrin expression using kit-based radiopharmaceuticals could provide whole-body diagnosis at early stages of disease before the manifestation of debilitating symptoms, as well as monitor patient response throughout treatment, providing greater effectiveness of treatment at early stages of disease. Such radiotracers could also be used to predict whether an individual patient will respond to a particular molecular therapeutic, based on the presence of a therapeutic receptor target. The availability of ⁶⁸Ga from a benchtop generator, coupled with a chelator conjugate that enables one-step, kit-based syntheses, which are not readily achievable with other chelators, brings about the possibility of widely available whole body PET molecular imaging of rheumatoid arthritis without the need for a local cyclotron or automated radiosynthesis equipment.

Experimental Section

Materials and Instrumentation

Chemicals and reagents were obtained from Sigma-Aldrich (Dorset, UK) unless otherwise specified. High-performance liquid chromatography (HPLC) analysis was carried out using an Agilent 1200 LC system with in-line UV and gamma detection (Flow-Count, LabLogic). NMR spectra were acquired on either a Bruker Avance 400 or 700 spectrometer (Bruker UK Limited, Coventry, UK). Spectra were referenced to residual solvent signals. Mass spectra were recorded in the positive ion mode on an Agilent 6510 Q-TOF LC/MS mass spectrometer coupled to an Agilent 1200 LC system (Agilent, Palo Alto, CA). Data were acquired and reference mass-corrected via a dual-spray electrospray ionization source, using the factory-defined calibration procedure. Analytical reverse-phase LCMS and radio-LCMS traces were acquired using an Agilent Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm). Instant thin layer chromatography strips (ITLC-SG) were obtained from Varian Medical Systems UK, Ltd. (Crawley, UK), and ITLC strips were visualized using a Raytest Rita-Star TLC scanner. Semipreparative reverse-phase HPLC was conducted using an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm) coupled to an Agilent 1200 LC system, with a 3 mL min^–1 flow rate and UV spectroscopic detection at 220 nm. Mobile phase A contained water with 0.2% TFA and mobile phase B contained acetonitrile with 0.2% TFA. All methods started with 100% A at 0 min. For method 1, the concentration of B increased at a rate of 2% min^–1; for method 2, the concentration of B increased at a rate of 1% min^–1; and for method 3, the concentration of B increased at a rate of 0.5% min^–1. Analytical reverse-phase HPLC and radio-HPLC traces were acquired using two different instruments: (1) an Agilent 1200 LC system and an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) with a 0.25–1 mL min^–1 flow rate and UV spectroscopic detection at either 214 or 220 nm. The radio-HPLC was coupled to a LabLogic Flow-Count detector with a sodium iodide probe (B-FC-3200). Aliquots (10–200 μL) of each radiolabeled sample were injected onto the column. Mobile phase A contained water with 0.1% TFA and mobile phase B contained acetonitrile with 0.1% TFA. For method 4, flow rate was 1 mL min^–1, the concentration of B increased at a rate of 5% min^–1, with 100% A at 0 min, and 100% B at 20 min. For method 5, flow rate was 0.25 mL min^–1, the concentration of B increased at a rate of 2% min^–1, with 100% A at 0–5 min, and 100% B at 55 min. (2) An Agilent Zorbax Eclipse XDB-C18 column (4.6 × 150 mm, 5 μm) with a 1 mL min^–1 flow rate and UV spectroscopic detection at either 214 or 220 nm coupled to a Shimadzu HPLC consisting of a SCL-10AVP system controller, a SIL-10ADVP autoinjector, a LC-10 ATVP solvent delivery unit, a FCV-10AL control valve, a DGU-14A degasser, and a SPD-10AVP UV detector. This was coupled to a radiation detector consisting of an Ortec model 276 Photomultiplier Base with Preamplifier, Amplifier, BIAS supply, and SCA and a Bicron 1 M 11.2 Photomultiplier Tube. For method 6, the concentration of B increased at a rate of 6.67% min^–1, with 100% A at 0 min, and 80% B at 12 min. For initial radiolabeling and characterization studies that utilized <400 MBq, an Eckert and Ziegler ⁶⁸Ge/⁶⁸Ga generator (Berlin, Germany) was used. For biodistribution studies, and experiments that utilized >600 MBq ⁶⁸Ga, an iThemba Laboratories 1.8 GBq ⁶⁸Ge/⁶⁸Ga generator (IDB Holland BV, Netherlands) was used.

Tricarboxylate 1,¹⁷ hydroxypyridinone 2,⁶⁹ SCN-HP₃, and HP₃-RGD were synthesized as previously described.¹⁸ The cyclic peptide c(RGDfK) was provided by Peptide Synthetics (Fareham, UK).

Compound 4

A solution of tricarboxylate 1 (18 mg, 40 μmol), hydroxypyridinone 2 (170 mg, 160 μmol), HATU (60 mg, 158 μmol), and diisopropylethylamine (30 μL) in DMF (2–5 mL) was heated in a microwave reactor (120 °C, 300 W, 30 min), after which the reaction solution was evaporated to dryness under reduced pressure. The residue was applied to a silica column, and eluted initially with methanol/chloroform (1:4), followed by methanol/chloroform/40% ammonium hydroxide (10:40:1). Fractions containing 3 (identified using mass spectrometry) were dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness. Compound 3: ESI-MS: m/z for C₁₉₅H₂₂₈N₂₆O₃₆ + 3H⁺ calc 1170.90, found 1170.90 (monoisotopic signal). Compound 3 (105 mg, 30 μmol) was added to a solution of ethanol/aqueous 5.5% hydrazine (9:1) (5 mL), and the mixture was heated in a microwave reactor (90–100 °C, 300 W, 30 min). The solution was cooled in an ice bath, and acidified with concentrated hydrochloric acid to pH 1, filtered, and evaporated to dryness. The residue was redissolved in water/acetonitrile (2:1) and applied to a semipreparative HPLC column. Using HPLC method 1, 4 eluted with 44% B (22–25 min). Fractions containing the desired product were lyophilized. Yield = 55 mg, 40% yield (based on tricarboxylate 1). ¹H NMR (CD₃OD, 400 MHz): δ 1.84 (m, 6H), 1.91 (m, 18H), 2.05 (m, 6H), 2.14 (m, 18H), 2.30 (t, J = 7.2, 6H), 2.53 (s, 27H), 2.56 (broad, 2H), 3.10 (t, J = 6.1, 2H), 3.34 (broad, 6H), 3.78 (s, 27H), 4.56 (s, 18H), 5.17 (s, 18H), 6.95 (s, 9H), 7.34 (m, 27H), 7.43 (m, 18H). ¹³C NMR (CD₃OD, 100 MHz): δ 21.2, 31.2, 30.9, 36.5, 37.3, 37.4, 38.9, 59.0, 75.7, 117.5, 129.70, 129.71, 130.1, 137.9, 146.0, 146.4, 152.9, 170.1, 171.5, 173.2, 175.4, 175.5, quaternary carbons of tripodal scaffold obscured by CD₃OD peaks. ESI-MS: m/z for C₁₈₇H₂₂₆N₂₆O₃₄ + 3H⁺ calc 1127.57, found 1127.57 (monoisotopic signal).

SCN-HP₉

An excess of p-phenylene diisothiocyanate (22 mg) and diisopropylethylamine (20 μL) in DMF (0.3 mL) were added to a solution of 4 (15 mg, 4.4 μmol) in DMF (0.5–1 mL). The reaction solution was agitated, and after 5–10 min, applied to a semipreparative HPLC column. Using HPLC method 1, product 5 eluted with 48% B (24–25 min). Fractions containing compound 5 were lyophilized. Compound 5: yield = 7 mg, 45%. ¹H NMR (CD₃OD, 400 MHz): δ 1.86 (m, 6H), 1.90 (m, 18H), 2.07 (m, 6H), 2.14 (m, 18H), 2.28 (t, J = 6.7, 6H), 2.46 (t, J = 6.1, 2H), 2.55 (s, 27H), 3.31 (presumed 6H, under CHD₂OD signal), 3.76 (broad, 2H), 3.80 (s, 27H), 4.58 (s, 18H), 5.18 (s, 18H), 7.00 (s, 9H), 7.14 (d, J = 9.0, 2H) 7.33 (m, 27H), 7.42 (m, 18H), 7.45 (under peak at 7.42, 2H). ESI-MS: m/z for C₁₉₅H₂₃₀N₂₈O₃₄S₂ + 4H⁺ calc 893.92, found 893.92 (monoisotopic signal). A solution of chilled boron trichloride in dichloromethane (5 mL, 1 M) was added to a sealed vial containing compound 5 (7 mg, 2 μmol), and the reaction was stirred at ambient temperature for 1 h. The reaction vial was then cooled in an ice bath, and methanol (5–10 mL) was added dropwise to the flask. The reaction solution was evaporated to near dryness under reduced pressure, and acetone (50 mL) was added to the residue, resulting in a flocculant white precipitate. This suspension was transferred to a 50 mL centrifuge tube, and the mixture centrifuged at 3000 rpm for 10 min. After this, the supernatant was decanted and discarded, acetone added (50 mL), the suspension agitated, and centrifuged again for 10 min. This process of washing with acetone was repeated again. Finally, the product was dissolved in water/acetonitrile (50/50), filtered, frozen, and lyophilized. The residue was applied to a semipreparative HPLC column. Using HPLC method 2, SCN-HP₉ eluted with 35% B (35–36 min). Fractions containing the desired product were lyophilized. Yield (trifluoroacetate salt) = 3 mg, 30–40%. ¹H NMR (CD₃OD, 700 MHz): δ 1.93 (t, J = 7.5, 6H), 1.96 (t, J = 7.5 18H), 2.15 (t, J = 7.5, 6H), 2.22 (t, J = 7.5, 18H), 2.33 (t, J = 6.3, 6H), 2.52 (broad, 2H), 2.57 (s, 27H), 3.35 (broad, 6H), 3.79 (broad, 2H), 3.92 (s, 27H), 4.70 (s, 18H), 6.93 (s, 9H), 7.20 (d, splitting = 8.7, 2H), 7.48 (d, splitting = 8.7, 2H). ¹³C NMR (CD₃OD, 175 MHz): δ 19.7, 29.3, 29.7, 34.9, 35.8, 36.1, 37.9, 57.5, 112.8, 123.9, 125.7, 127.0, 135.1, 137.8, 144.0, 148.9, 161.9, 172.0, 174.3, 174.9, 175.0, 180.6, quaternary carbons of tripodal scaffold obscured by CD₃OD peaks. ESI-MS: m/z for C₁₃₂H₁₇₆N₂₈O₃₄S₂ + 4H⁺ calc 691.32, found 691.32 (monoisotopic signal).

RGD₃

A solution of DMF (5 mL) containing tricarboxylate 1 (100 mg, 220 μmol), diisopropylcarbodiimide (10 equiv), N-hydroxysuccinimide (4 equiv), and diisopropylethylamine (4 equiv) was stirred for 16 h, after which it was filtered, and the filtrate applied to a semipreparative HPLC column. Using HPLC method 2, product 6 eluted with 38% B (38–42 min), and fractions containing the desired compound were lyophilized. Compound 6: isolated yield = 50 mg, 30%. ¹H NMR (CDCl₃, 400 MHz): δ 2.22 (t, J = 7.4, 6H), 2.50 (t, J = 7.0, 2H), 2.77 (s, 12H), 2.59 (t, J = 7.4, 6H), 3.92 (t, J = 7.0, 2H), 7.67 (q, splitting = 3.0, 5.5, 2H), 7.79 (q, splitting = 3.0, 5.5, 2H). ¹³C NMR (CDCl₃, 100 MHz): δ 25.5, 25.8, 28.6, 34.0, 34.6, 50.0, 123.3,132.2, 134.0, 168.3, 168.5, 169.6. ESI-MS: m/z for C₃₃H₃₃N₅O₁₅ + H⁺ calc 740.20, found 740.21. Activated triester 6 (5 mg, 7 μmol) was added to a solution of c(RGDfK) (20 mg, 24 μmol) and diisopropylethylamine (5 μL) in dimethylformamide (0.1–0.5 mL), and the solution was stirred for 10 min reaction at ambient temperature. After this time, the solution was applied to a semipreparative HPLC column. Using HPLC method 2, compound 7 eluted with 32% B (32 min). Fractions containing the desired product were lyophilized. Compound 7: yield of trifluoroacetate salt = 7.5 mg, 42%. ESI-MS: m/z for C₁₀₂H₁₄₁N₂₉O₂₇ + 2H⁺ calc 1103.04, found 1103.04 (monoisotopic signal). Finally, phthalimide-protected compound 7 (15 mg) was added to a solution of ethanol/aqueous 5.5% hydrazine (9:1) (5 mL), and the mixture was heated in a microwave reactor (90–100 °C, 300 W, 30 min). The solution was then cooled in an ice bath, and acidified with concentrated hydrochloric acid to pH 1, filtered, and lyophilized. The solution was redissolved in deionized water and applied to a semipreparative HPLC column. Using HPLC method 2, RGD₃ eluted with 28% B (28 min). Fractions containing RGD₃ were lyophilized. Yield: 6 mg, 40%. ESI-MS: m/z for C₁₀₂H₁₄₁N₂₉O₂₇ + 4H⁺ calc 519.52, found 519.52 (monoisotopic signal); HPLC: 220 nm, RT = 7.90 min, >97% purity (HPLC method 4).

Synthesis of RGD Conjugates

RGD₃ was dissolved in dimethyl sulfoxide (100–300 μL) and added to a solution of either SCN-HP₃ or SCN-HP₉ in dimethyl sulfoxide (100–300 μL), and diisopropylethylamine (5–10 μL) was added. The reaction solutions were heated in a microwave (120 °C, 300 W, 30 min) and then applied to a reverse-phase HPLC column. Fractions containing the desired conjugate in sufficient purity were combined and lyophilized. Using HPLC method 3, HP₃-RGD₃ eluted with 26% solvent B (52 min) and HP₉-RGD₃ eluted with 25% solvent B (51 min). Isolated yields = 30–40%. HP₃-RGD₃: ESI-MS: m/z for C₁₃₉H₁₉₅N₃₉O₃₅S₂ + 5H⁺ calc 607.89, found 607.89 (monoisotopic signal); HPLC: 220 nm, RT = 8.02 min, >99% purity (HPLC method 4). HP₉-RGD₃: ESI-MS: m/z for C₂₂₆H₃₁₅N₅₇O₅₉S₂ + 6H⁺ calc 806.89, found 806.89 (monoisotopic signal); HPLC: 220 nm, RT = 7.77 min, >99% purity (HPLC method 4).

Complexation with ⁶⁸Ga³⁺ and ^natGa³⁺

Initial radiolabeling experiments utilized an Eckert and Ziegler ⁶⁸Ge/⁶⁸Ga generator. Aqueous HCl solution (0.1 M, 5 mL) was passed through the generator and the eluate was fractionated (5 × 1 mL). The second fraction (1 mL, containing 90–100 MBq ⁶⁸Ga) was added directly to an ethanol/water solution (50%/50%, 50–100 μL) of either HP₃-RGD (25 μg, 12 nmol), HP₃-RGD₃ (50 μg, 13 nmol), or HP₉-RGD₃ (25 μg, 4 nmol), immediately followed by a solution of ammonium acetate (1 M, 300 μL). This solution was immediately applied to an analytical reverse-phase C18 HPLC column. [⁶⁸Ga(HP₃-RGD)]: radiochemical yield >99% (HPLC), HPLC: RT = 8.38 min (HPLC method 4). [⁶⁸Ga(HP₃-RGD₃)]: radiochemical yield >99% (HPLC), HPLC: RT = 8.37 min (HPLC method 4). [⁶⁸Ga(HP₉-RGD₃)]: radiochemical yield >99% (HPLC), HPLC: RT = 8.32 min (HPLC method 4). For [⁶⁸Ga(HP₃-RGD)] and [⁶⁸Ga(HP₃-RGD₃)], specific activity at point of synthesis = 8–9 MBq nmol^–1 conjugate and for [⁶⁸Ga(HP₉-RGD₃)], specific activity = 24–27 MBq nmol^–1.

The nonradioactive analogues, [^natGa(HP₃-RGD)], [^natGa(HP₃-RGD₃)], and [^natGa₃(HP₉-RGD₃)], were also prepared. An aqueous solution of Ga(NO₃)₃ (2 mg mL^–1, 5 μL, 30–40 nmol, excess) was added to HP₃-RGD (25 μg, 12 nmol), HP₃-RGD₃ (50 μg, 13 nmol), or HP₉-RGD₃ (25 μg, 4 nmol) dissolved in an ethanol/water solution (50%/50%, 50–100 μL). The solutions were applied to an analytical reverse-phase C18 HPLC column and subjected to LCMS analysis. [^natGa(HP₃-RGD)]: HPLC RT = 8.93 min (HPLC method 4); MS C₇₂H₉₄N₁₉O₁₇S₂Ga + 2H⁺, observed monoisotopic peak = 815.80, calculated = 815.80 (monoisotopic signal); [^natGa(HP₃-RGD₃)]: HPLC RT = 9.05 min (HPLC method 4); MS C₁₃₉H₁₉₂N₃₉O₂₅S₂Ga + 3H⁺, observed monoisotopic peak =1034.45, calculated 1034.45 (monoisotopic signal); [^natGa₃(HP₉-RGD₃)]: HPLC RT = 8.88 min (HPLC method 4); MS C₂₂₆H₃₀₆N₅₇O₅₉S₂Ga₃ + 4H⁺, observed monoisotopic peak = 1259.26, calculated 1259.25 (monoisotopic signal). For more detailed LCMS analysis of HP₉-RGD₃, HP₉-RGD₃ was titrated with 0, 1, 2, and 3 mol equiv of Ga(NO₃)₃, prior to analysis using LCMS (HPLC method 5).

For biodistribution with U87MG tumor bearing mice, ⁶⁸Ga eluate from an iThemba Lab generator was preconditioned as previously described.⁵⁰ Briefly, a cation exchange cartridge containing AG 50Wx4 resin (50 mg) was conditioned by passing through aqueous HCl solution (4 M, 1 mL) and deionized water (1 mL) sequentially. To elute the ⁶⁸Ge/⁶⁸Ga generator, aqueous HCl solution (0.4 M, 5 mL) was passed through the generator and transferred directly onto the cation exchange cartridge. The cartridge was dried with air (1 mL), washed with 0.15 M HCl in water/ethanol (20%/80%), and again dried with air (1 mL). A solution of 0.9 M HCl in water/ethanol (200 μL, 10%/90%) was used to elute ⁶⁸Ga (800–1000 MBq), which was diluted to a volume of 1 mL with deionized water. Lyophilized peptide conjugate (trifluoroacetate salt)—HP₃-RGD (25 μg, 12 nmol), HP₃-RGD₃ (50 μg, 13 nmol), or HP₉-RGD₃ (25 μg, 4 nmol)—dissolved in 20–40 μL of water/ethanol (50%/50%) was added to the solution containing ⁶⁸Ga, immediately followed by a solution of ammonium acetate (2 M, 400 μL) and 0.9% saline (1100 μL). An aliquot for ITLC analysis was immediately applied to an ITLC-SG plate. The ITLC-SG plate was developed using an aqueous citrate buffer (0.1 M, pH 5.5) mobile phase. [⁶⁸Ga(HP₃-RGD)], [⁶⁸Ga(HP₃-RGD₃)], and [⁶⁸Ga(HP₉-RGD₃)]: R_f < 0.1; [⁶⁸Ga(citrate)₂]^3–: R_f > 0.8. [⁶⁸Ga(HP₃-RGD)]: radiochemical yield > 95% (ITLC). HPLC (method 6): RT = 9.78 min; [⁶⁸Ga(HP₃-RGD₃)]: radiochemical yield > 95% (ITLC), HPLC: RT = 9.54 min; [⁶⁸Ga(HP₉-RGD₉)]: radiochemical yield > 95% (ITLC), HPLC: RT = 9.50 min.

Log P_OCT/PBS Determination

A solution containing ⁶⁸Ga-conjugate (50 μL, synthesized using eluate from an Eckert and Ziegler generator as described above) was added to 550 μL of octanol and 500 μL of aqueous phosphate buffered saline solution. The mixture was agitated using a vortex, and the phases separated by centrifugation (1000 rpm, 2 min). Aliquots from each phase were counted for radioactivity in a gamma counter. The experiment was repeated four times.

Determination of IC₅₀

The relative affinity of [^natGa(HP₃-RGD)], [^natGa(HP₃-RGD₃)], [^natGa₃(HP₉-RGD₃)], and c(RGDfK) for integrin α_vβ₃ was determined in a solid-phase competitive binding assay¹⁸ with ¹²⁵I-echistatin (PerkinElmer, Boston). In brief, wells of a 96 well plate were coated with integrin α_vβ₃ (150 ng mL^–1) in coating buffer (100 μL, 25 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, and 1 mM MnCl₂) overnight at 4 °C. Wells were then washed twice in binding buffer (coating buffer containing 0.1% bovine serum albumin (w/v) (BSA)) before being incubated for 2 h at room temperature with blocking buffer (coating buffer containing 1% BSA (w/v)). After a further two washes in binding buffer, ¹²⁵I-echistatin (0.5 kBq) and either [^natGa(HP₃-RGD)], [^natGa(HP₃-RGD₃)], [^natGa₃(HP₉-RGD₃)] or c(RGDfK) were added simultaneously (to a total volume of 100 μL, and a conjugate/c(RGDfK) concentration of 10000 nM to 0.001 nM) for 1 h at room temperature, before being washed twice as before. Finally, the amount of activity bound to the wells via integrin α_vβ₃ was counted using a Wallac 1282 Compugamma Universal Gamma Counter. Measurements at each concentration for each compound were obtained in sextuplicate. IC₅₀ values were calculated using a nonlinear regression model (Binding/Saturation, one site–total) in GraphPad Prism 5.04.

PET Scanning and Biodistribution of U87MG Tumor Bearing Animals

All animal experiments with U87MG tumor bearing animals were performed with approval from the Peter MacCallum animal experimentation ethics committee. Six- to eight-week-old female Balb/c nude mice (Animal Resources Centre, Western Australia) were implanted subcutaneously on the right flank with 4 million U87MG cells. Once the tumors reached a volume >250 mm³ (two to three weeks) the animals were injected intravenously with 13–30 MBq of [⁶⁸Ga(HP₃-RGD)] (1 μg of conjugate), [⁶⁸Ga(HP₃-RGD₃)] (2 μg of conjugate), and [⁶⁸Ga(HP₉-RGD₃)] (1 μg of conjugate) (100 μL of solutions described above). At 1 and 2 h, the animals were anaesthetized and imaged on a Philips MOSAIC small animal PET scanner for 10 min each. For blocking studies, animals were coinjected with c(RGDfK) peptide (400 μg), anaesthetized at 1 h, and imaged for 10 min each. The images were reconstructed using a 3D RAMLA algorithm, and radiotracer uptake was determined by ex vivo tissue counting as described previously.⁷⁰ On completion of the scan animals were euthanized by cervical dislocation and tissues harvested, weighed, and radioactivity counted using a gamma counter (Biomedex). Confidence intervals and p values were calculated using a two-tailed t-test in GraphPad Prism 5.04.

PET/CT Scanning and Biodistribution of Animals with Induced Rheumatoid Arthritis

All animal experiments on mice with induced rheumatoid arthritis complied with the Animals (Scientific Procedures) Act (UK 1986) and Home Office (UK) guidelines. A K/BxN serum transfer model of arthritis was utilized in these experiments, and serum was produced as previously reported.^59,60 Wild type C57Bl/6 male 6–8-week-old mice (Charles River) were injected with 150 μL arthritogenic serum (intraperitoneally) on days 0 and 2. The arthritic serum was either diluted 2-fold with PBS (to induce severe arthritis) or 8-fold with PBS (to induce mild arthritis). PBS alone was also injected into a third group to assess uptake in nonarthritic mice. Disease severity was evaluated in mice on days 0, 2 and 8 post-injection, by measuring weight loss, thickness of swollen paws, using microcalipers (Kroeplin), and visual clinical scoring on a scale of 0–3 per paw added for a total score out of 12 per animal. On day 8, animals from the control, the mild arthritis and severe arthritis groups (n = 3) were injected intravenously with [⁶⁸Ga(HP₃-RGD₃)] (8–14 MBq, 4 μg HP₃-RGD₃). A fourth group of animals (n = 3) with severe arthritis were coadministered [⁶⁸Ga(HP₃-RGD₃)] (8–14 MBq, 4 μg HP₃-RGD₃) and c(RGDfK) peptide (400 μg). [⁶⁸Ga(HP₃-RGD₃)] was prepared using protocols described above for radiolabeling with an Eckert and Ziegler ⁶⁸Ge/⁶⁸Ga generator. Animals were injected under isoflurane anesthesia, after which they were immediately allowed to recover. One hour post-injection, animals were culled by CO₂ asphyxiation and PET scanned using a nanoScanPET/CT (Mediso Medical Imaging Systems, Budapest, Hungary) and organs/tissues harvested, weighed, and radioactivity counted using a gamma counter. PET/CT images were reconstructed using VivoQuant (inviCRO, LLC, Boston, USA). Confidence intervals and p values were calculated using a two-tailed t-test in GraphPad Prism 5.04.

Glossary

Abbreviations

HPLC

high performance liquid chromatography

HATU

1,2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

PET

positron emission tomography

TIC

total ion count

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00621.

• Concentration response curves for solid-phase binding assay, ex vivo biodistribution data (PDF)

Author Contributions

C.I. undertook synthesis and radiolabeling, and coauthored the manuscript; C.C. and R.J.H. conceived of and/or undertook PET imaging and biodistribution studies on U87MG cancer mouse models; S.Y.A.T., F.C., G.H.C., N.K.R., and A.P.C. conceived and/or undertook PET/CT and biodistribution studies on rheumatoid arthritis mouse models; P.R. assisted with radiolabeling; P.J.B. jointly conceived the study, advised on experimental methods and coauthored the manuscript; M.T.M. conceived the study, coauthored the manuscript, undertook synthesis, radiolabeling, LCMS analysis, and competitive binding assays, and conceived of and assisted with biodistribution studies. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.