Preparation and preclinical evaluation of a 68Ga-labelled c(RGDfK) conjugate comprising the bifunctional chelator NODIA-Me

Background We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane with up to three, five-membered azaheterocyclic arms for the development of 68Ga- and 64Cu-based radiopharmaceuticals. Here, a 68Ga-labelled conjugate comprising the bifunctional chelator NODIA-Me in combination with the αvß3-targeting peptide c(RGDfK) has been synthesized and characterized. The primary aim was to evaluate further the potential of our NODIA-Me chelating system for the development of 68Ga-labelled radiotracers. Results The BFC NODIA-Me was conjugated to c(RGDfK) by standard peptide chemistry to obtain the final bioconjugate NODIA-Me-c(RGDfK) 3 in 72% yield. Labelling with [68Ga]GaCl3 was accomplished in a fully automated, cGMP compliant process to give [68Ga]3 in high radiochemical yield (98%) and moderate specific activity (~ 8 MBq nmol− 1). Incorporation of the Ga-NODIA-Me chelate to c(RGDfK) 2 had only minimal influence on the affinity to integrin αvß3 (IC50 values [natGa]3 = 205.1 ± 1.4 nM, c(RGDfK) 2 = 159.5 ± 1.3 nM) as determined in competitive cell binding experiments in U-87 MG cell line. In small-animal PET imaging and ex vivo biodistribution studies, the radiotracer [68Ga]3 showed low uptake in non-target organs and specific tumor uptake in U-87 MG tumors. Conclusion The results suggest that the bifunctional chelator NODIA-Me is an interesting alternative to existing ligands for the development of 68Ga-labelled radiopharmaceuticals.

We recently developed a chelating platform based on the macrocycle 1,4,7-triazacyclononane (TACN) with additional five-membered azaheterocyclic arms for the coordination of the PET radionuclides gallium-68 and copper-64 (Gotzmann et al. 2016;Schmidtke et al. 2017). Initial work revealed that these chelators are characterized by their excellent complexation properties for both radiometals. Labelling with copper-64 was achieved rapidly under very mild conditions (< 1 min incubation time, room temperature) over a pH range of 4.0 to 8.0 to give products with high specific activities (120-180 MBq nmol − 1 ). Stability studies also demonstrated that 64 Cu-labelled complexes have high kinetic stability in vitro (Gotzmann et al. 2016). In subsequent work, we discovered that the imidazole-type ligands can also be labelled with gallium-68 (Schmidtke et al. 2017). Complexation properties were comparable to the ligand NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) (Schmidtke et al. 2017). More recently, we described the bifunctional chelator (BFC) NODIA-Me (2-(4,7-bis((1-methyl-1H-imidazol-2-yl)methyl)-1,4,7-triazonan-1-yl)acetic acid), in which one of the methylimidazole arms was replaced with an acetic acid group. This acetic acid group served as site for the attachment of a prostate-specific membrane antigen targeting vector via peptide bond formation (Schmidtke et al. 2017). In more recent small-animal imaging and ex vivo biodistribution studies, 64 Cu-and 68 Ga-labelled PSMA-targeting conjugates comprising the BFC NODIA-Me specifically delineated PSMA-positive LNCaP tumors (Läppchen et al. 2018). Moreover, no significant decomplexation/transchelation of the radiometal chelate was noted in vivo, underscoring the potential use of our chelating platform for radiopharmaceutical applications.
In the present study, we sought to expand the scope of the BFC NODIA-Me for the development of 68 Ga-based radiopharmaceuticals. Here, we report studies on a 68 Ga-labelled α v ß 3 -targeting probe conjugated to the BFC NODIA-Me. The α v ß 3 -targeting bioconjugate NODIA-Me-c(RGDfK) was evaluated in vitro by a competitive cell binding assay, followed by small-animal PET imaging and ex vivo biodistribution studies.

General
Chemicals and solvents were purchased from Sigma-Aldrich and TCI Europe, and used as received. The bifunctional chelator NODIA-Me 1 was prepared as previously described (Schmidtke et al. 2017). The peptide c(RGDfK) 2 was purchased from ABX (Radeberg, Germany). The radioligand [ 125 I]I-echistatin was obtained from Perkin Elmer (Boston, USA). Low resolution electrospray ionisation mass spectrometry (LR-ESI(+)-MS) was performed on a PerkinElmer Flexar SQ 300 MS Detector. Radiolabelling with [ 68 Ga]GaCl 3 was accomplished using a fully automated synthesis module (Pharmtracer, Eckert & Ziegler, Berlin, Germany) with an IGG100 generator (Eckert & Ziegler, Berlin, Germany). High performance liquid chromatography (HPLC) was performed on an Agilent 1260 Infinity System equipped with an Agilent 1200 DAD UV detector (UV detection at 220 nm) and a Raytest Ramona radiation detector (Raytest GmbH, Straubenhardt, Germany) in series. A Phenomenex Jupiter Proteo (250 × 4.60 mm) column was used for analytical HPLC. The solvent system was A = H 2 O (0.1% TFA) and B = acetonitrile (0.1% TFA). The gradient was 0-1 min 5% B, 1-20 min 40% B at a flow rate of 1 mL min − 1 . Semi-preparative HPLC was performed on a Knauer Smartline 1000 HPLC system in combination with a Macherey Nagel VP 250/21 Nucleosil 120-5 C18 column. Semi-preparative HPLC gradient was 0-40 min 5-60% B at a flow rate of 12 mL min − 1 . Samples were lyophilized using a Christ Alpha 1-2 LD plus lyophilizer. All instruments measuring radioactivity were calibrated and maintained in accordance with previously reported routine quality-control procedures (Zanzonico 2009). Radioactivity was measured using an Activimeter ISOMED 2010 (Nuklear-Medizintechnik, Dresden, Germany). For accurate quantification of radioactivity, experimental samples were counted for 1 min on a calibrated Perkin Elmer (Waltham, MA, USA) 2480 Automatic Wizard Gamma Counter by using a dynamic energy window of 400-600 keV for gallium-68 (511 keV emission).

Lipophilicity (log D oct/PBS ) measurements
For log D oct/PBS measurements, 1-2 MBq of [ 68 Ga]3 in 20 μL labelling buffer were added to a mixture of phosphate buffered saline (PBS) pH 7.4 (Gibco Life Science Technologies, 4 mM phosphate buffer, 0.15 M NaCl) (480 μL) and octanol (500 μL). Samples were shaken for 30 min at room temperature, centrifuged at 13,200 rpm for 5 min and 100 μL of each phase were counted using a Packard Cobra gamma counter. Experiments were performed in triplicate.

Competitive binding assay
The binding affinity of [ nat Ga]3 was determined by a cell-based competitive binding assay in the human glioma cell line U-87 MG with [ 125 I]I-echistatin as the radioligand as previously described (Dumont et al. 2011). Binding assays were performed in 24-well plates precoated with poly-L-lysine. Briefly, each compound at different concentrations (0-10,000 nM) was incubated for 2 h at r.t. with [ 125 I]I-echistatin (30.000 cpm well − 1 ) and 2 × 10 5 U-87 MG cells well − 1 . After incubation, cells were washed three times with ice cold binding buffer and cell-associated activity recovered by addition of 1 M NaOH. Radioactivity was measured by a gamma counter and data fitted using non-linear regression (GraphPad Prism). Experiments were performed two times in triplicate.

Small-animal PET imaging
All animal experiments complied with the current laws of the Federal Republic of Germany and were conducted according to German Animal welfare guidelines. Normal female athymic Balb/c nude mice (17-20 g, 4-6 weeks old) were obtained from Janvier SAS (St. Berthevin Cedex, France). Mice were provided with food and water ad libitum. U-87 MG tumors were inoculated on the right shoulder by sub-cutaneous injection of 5×10 6 cells in a 100 μL cell suspension of a 1:1 v/v mixture of media with reconstituted basement membrane (GFR BD Matrigel™, Corning BV, Amsterdam, Holland).
For PET imaging studies, mice (n = 3) were injected with 100 μL sterile filtered phosphate buffered saline formulations pH 7.4 of [ 68 Ga]3 (7-11 MBq) by intravenous tailvein injection and anesthetized with isoflurane (2-4% in air) 5-10 min prior image acquisition. PET imaging was performed on a Focus 120 microPET scanner at 1 h after administration. Data were acquired 1 h post administration in list mode. Reconstruction was performed using unweighted OSEM2D. Image analysis was performed using AMIDE. Image counts per second per voxel (cps/voxel) were calibrated to activity concentrations (Bq mL − 1 ) by measuring a 3.5 cm cylinder phantom filled with a known concentration of radioactivity. For data analysis, it was explicitly assumed that the density of tissue equals 1.0 g cm − 3 , hence the reported units of %IA g − 1 are identical to %IA cm − 3 . Specificity of [ 68 Ga]3 was confirmed by competitive inhibition (blocking) co-injecting the peptide c(RGDfK) 2 (5 mg kg − 1 =~100 nmol mouse − 1 ; n = 3) in approximately 100fold excess compared to the radiotracer.

Ex vivo biodistribution
For each compound, a total of five animals were injected with [ 68 Ga]3 (7-11 MBq) in 100 μL sterile filtered phosphate buffered saline via a tail vein. At 1 h p.i., animals were sacrificed by isoflurane anesthesia. Organs of interest were dissected, weighed and assayed for radioactivity in a gamma counter. The percent injected activity per gram (%IA g − 1 ) for each tissue was calculated by comparison of the tissue counts to a standard sample prepared from the injectate. Specificity of [ 68 Ga]3 was determined by co-injection of the peptide c(RGDfK) 2 (5 mg kg − 1 =~100 nmol mouse − 1 ).

Bioconjugate synthesis
The bifunctional chelator NODIA-Me 1 bearing an acetic acid residue for the covalent attachment of appropriate targeting vectors was successfully conjugated to the peptide c(RGDfK) 2 in DMF using HATU as coupling reagent (Scheme 1). The final bioconjugate 3 was obtained in 72% yield after purification by semi-preparative RP-HPLC. The identity and purity of compound 3 (> 98%) was determined by mass spectrometry and analytical RP-HPLC.

Radiochemistry
Radiosynthesis of [ 68 Ga]3 was performed using an automated synthesis module by heating 20 μg of compound 3 with [ 68 Ga]GaCl 3 (aq.) at 95°C for 10 min as previously described (Schmidtke et al. 2017). The RCP and the decay corrected RCY for [ 68 Ga]3 were measured to > 98% with mean specific activities of~8 MBq nmol − 1 . The identity of [ 68 Ga]3 was confirmed by analytical HPLC using the non-radioactive reference compound.

Lipophilicity
The radiolabelled conjugate [ 68 Ga]3 is highly hydrophilic with a log D oct/PBS value of − 3.89 ± 0.02. Interestingly, in a series of c(RGDfK) based radiopharmaceuticals with different bifunctional chelators that give gallium complexes of different overall charge, [ 68 Ga]3 with a positively charged metal chelate was more hydrophilic than [ 68 Ga]Ga-NODAGA-c(RGDfK) or [ 68 Ga]Ga-DOTA-c(RGDfK), which have a neutral and negative overall charge on the metal chelate (log D values: − 3.27 ± 0.01 and − 2.86 ± 0.01), respectively (Dumont et al. 2011).

Competitive binding assay
The binding affinity of [ nat Ga]3 was compared to c(RGDfK) 2 in a competitive binding assay on α v ß 3 -positive U-87 MG cells using [ 125 I]I-echistatin as radioligand. Both compounds inhibited the binding of [ 125 I]I-echistatin in a dose dependent manner. The IC 50 values for 2 and [ nat Ga]3 were determined to IC 50 = 159.5 ± 1.3 nM and IC 50 = 205.1 ± 1.4 nM, which are in accordance to previously reported IC 50 values (Dumont et al. 2011). Introduction of the metal chelate had only a minimal effect on receptor binding resulting in a slightly lower affinity of [ nat Ga]3 compared to peptide 2. Corresponding inhibition curves are given in Fig. 1.

In vivo studies
In our efforts to demonstrate the applicability of our novel chelating system for radiopharmaceutical applications, we assessed the stability and pharmacokinetic profile of [ 68 Ga]3 by small-animal experiments.

Ex vivo biodistribution
Biodistribution data for [ 68 Ga]3 are presented in Table 1. For comparison, equivalent data taken from the literature are also given for compounds [ 68 Ga]Ga-NODAGA-c(RGDfK) and [ 68 Ga]Ga-DOTA-c(RGDfK) (Dumont et al. 2011). The primary difference between these three compounds is the change in chelator. The highest activity accumulation of [ 68 Ga]3 among all tissues at 1 h p.i. was seen for the α v ß 3 -positive U-87 MG tumors with 2.10 ± 0.09 %IA g − 1 . Despite differences in the tumor models used, accumulation of [ 68 Ga]3 in U-87 MG xenografts is comparable to that of previously reported 68 Ga-labelled RGD peptides in α v ß 3 -expressing tumors Ferreira et al. 2012;Pohle et al. 2012 significantly reduced by 86% (P value < 0.0001) by co-injection of c(RGDfK), indicating receptor specific binding in tumor tissue. Accumulation in the kidneys and the liver was low considering the positively charged NODIA-Me metal chelate because positively charged compounds might be retained in both organs (Dearling et al. 2013;Dearling et al. 2015;Sprague et al. 2007). In previous animal studies of our 64 Cu-labelled chelators (without a targeting vector), substantial uptake and retention of activity was observed in the kidneys by small-animal PET imaging (Gotzmann et al. 2016). This kidney Table 1 Ex vivo biodistribution of [ 68 Ga]3 in mice bearing α v ß 3 -positive U-87 MG tumors at 1 h p.i. along with blocking studies in comparison to [ 68 Ga]Ga-NODAGA-c(RGDfK) and [ 68 Ga]Ga-DOTAc(RGDfK) (data taken from ref. Dumont et al. 2011). Data are expressed as %IA g − 1 and represent mean ± SD (n = 5)  Table 1). The specificity of [ 68 Ga]3 for integrin α v ß 3 was confirmed in blocking studies by coinjection of an~100fold excess c(RGDfK), which resulted in a significant reduction of tracer uptake in all tissues (P values < 0.05 for all tissues). This is in accordance with the literature where α v ß 3 imaging probes demonstrate low but blockable uptake in normal tissues (Chen et al. 2004;Decristoforo et al. 2008;Dijkgraaf et al. 2011;Li et al. 2007;Wei et al. 2009).

Small-animal PET imaging
In addition to the ex vivo biodistribution studies, the distribution profile of [ 68 Ga]3 was also assessed 1 h after administration by small-animal PET imaging. Corresponding transverse and coronal maximum intensity projections of [ 68 Ga]3 in α v ß 3 xenograft bearing mice along with blocking studies are given Fig. 2. The results of the biodistribution study were confirmed by PET imaging. The α v ß 3 positive U-87 MG tumors as well as the liver and the kidneys were clearly visible on the PET images. The tumor uptake of [ 68 Ga]3 was determined to 2.48 ± 0.14 %IA g − 1 . The differences in blood and tissue activity versus tumor activity between [ 68 Ga]3, [ 68 Ga]Ga-NODAGAc(RGDfK) and [ 68 Ga]Ga-DOTA-c(RGDfK) gave a tumor-to-background ratio of 6.76 for [ 68 Ga]3 that was lower than that of [ 68 Ga]Ga-NODAGA-c(RGDfK) (11.97) but about 2-fold higher than that of [ 68 Ga]Ga-DOTA-c(RGDfK) (3.28) (Dumont et al. 2011). The specificity of [ 68 Ga]3 was confirmed in blockade studies by co-injecting c(RGDfK) shown in Fig. 2b, which resulted in a significant reduction of tumor accumulation (0.67 ± 0.10 %IA g − 1 ) (P value < 0.0001) and reduced uptake in all other organs.

Conclusions
In this work, we successfully developed a 68 Ga-labelled bioconjugate comprising our novel chelator NODIA-Me for imaging the α v ß 3 integrin receptor. The final bioconjugate was readily obtained in a single reaction step by standard peptide chemistry in good yields. The resulting bioconjugate was labelled with [ 68 Ga]GaCl 3 in a cGMP compliant process in high yields and moderate specific activity. Introduction of the novel metal chelate to the peptide c(RGDfK) had only a minimal impact on receptor binding. U-87 MG tumors overexpressing the α v ß 3 integrin receptor were specifically delineated in ex vivo biodistribution and small-animal PET imaging studies by the corresponding 68 Ga-labelled bioconjugate. Uptake of the novel tracer in non-target tissues was low providing acceptable tumor-to-background ratios. Even though tumor uptake and tumor-to-background ratios were lower compared to [ 68 Ga]Ga-NODAGA- c(RGDfK), the low uptake in non-target tissues indicates kinetically stable complexation of gallium-68 in the bifunctional chelator NODIA-Me. Altogether, our results demonstrate that our novel chelating system is an interesting alternative to existing bifunctional chelators such as DOTA and NODAGA for 68 Ga-based radiopharmaceuticals.