Synthesis, Radiolabelling and In Vitro Characterization of the Gallium-68-, Yttrium-90- and Lutetium-177-Labelled PSMA Ligand, CHX-A''-DTPA-DUPA-Pep

Since prostate-specific membrane antigen (PSMA) has been identified as a diagnostic target for prostate cancer, many urea-based small PSMA-targeting molecules were developed. First, the clinical application of these Ga-68 labelled compounds in positron emission tomography (PET) showed their diagnostic potential. Besides, the therapy of prostate cancer is a demanding field, and the use of radiometals with PSMA bearing ligands is a valid approach. In this work, we describe the synthesis of a new PSMA ligand, CHX-A''-DTPA-DUPA-Pep, the subsequent labelling with Ga-68, Lu-177 and Y-90 and the first in vitro characterization. In cell investigations with PSMA-positive LNCaP C4-2 cells, KD values of ≤14.67 ± 1.95 nM were determined, indicating high biological activities towards PSMA. Radiosyntheses with Ga-68, Lu-177 and Y-90 were developed under mild reaction conditions (room temperature, moderate pH of 5.5 and 7.4, respectively) and resulted in nearly quantitative radiochemical yields within 5 min.


Introduction
Prostate cancer (PCa) is still one of the leading causes of cancer deaths among men. Despite using novel therapeutic approaches, mortality from metastasizing prostate cancer is still high [1]. Therefore, the early diagnosis of prostate cancer to prevent tumor dissemination is highly desirable. Furthermore, effective treatment strategies for disseminated prostate cancer are urgently needed. Comprehensibly, targeting of PCa or its metastases is a demanding task in the field of molecular imaging with positron emission tomography (PET) and for targeted internal radiation therapy.
Prostate-specific membrane antigen (PSMA) is a peptidase that catalyzes the hydrolysis of N-acetyl-L-aspartyl-L-glutamate (NAAG) into the corresponding N-acetyl-L-aspartate (NAA) and L-glutamate [2]. The use of PSMA as a target for diagnostic and therapeutic agents is a highly valid approach. Compared to healthy human prostate tissue, in almost all PCa tumors, the expression of PSMA is 10-80 fold higher.
Besides, the PSMA levels are increased in the neovasculature of other solid tumors, as well [3][4][5][6]. Therefore, selective addressing of PSMA with small molecules labelled with a positron emitting radionuclide is a considerable approach for the diagnosis of prostate cancer with PET.
Based on the chemical structure of NAAG, several glutamate-urea-glutamate-based peptides bearing a 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) moiety were developed in the last few years [7]. These molecules showed high affinity and specific binding to PSMA, as demonstrated in binding studies using PSMA expressing LNCaP cell lines. Beside the slightly modified chemical structure, these molecules differ mainly in the selection of the chelator for the complexation of the desired radionuclide [7][8][9].
Due to their proven excellent affinity to PSMA, it is desirable to develop radionuclide-based therapeutic strategies using adapted PSMA targeting. In radiometal therapy approaches, the application of Y-90 and Lu-177 is favored. The use of Y-90 (E βmax : 2.3 MeV, t ½ : 64 h) is more appropriate in the treatment of larger tumor lesions, while Lu-177 (E βmax : 0.5 MeV, t ½ : 6.7 d) is more suitable for the treatment of smaller lesions and metastases, accompanied by a minimization of kidney dose in comparison to the application of Y-90 labelled peptides [10]. Moreover, due to the contemporary beta-and gamma-emission, Lu-177 is a useful diagnostic tool for scintigraphy of tumoral uptake [11].
For both diagnostic and therapeutic application, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) is the mostly used chelator for the complexation of radiometals, like Ga-68, Lu-177 and Y-90, to small molecules [12,13]. However, DOTA can show some undesirable characteristics for therapy. Possible immunogenicity in humans has been published, as well as unfavorable kinetics in the complexation of radiometals [14,15]. Furthermore, labelling reactions using DOTA as the chelating agent are usually carried out at high temperatures under acidic conditions and long reaction times [16]. The preferred labelling procedure for peptides should consist of a simple, fast and quantitative labelling step at room temperature and neutral pH to avoid decomposition. Therefore, the use of alternative chelators is a demanding approach. In addition, the chelator must provide sufficient stability in vivo.

Production of [ 68 Ga]Ga-Chloride
Ga-68 (t ½ = 67.7 min, β + : 89%, E β + max , 1.9 MeV; EC: 11%, E γ max : 4.0 MeV) was prepared as described by Meyer et al. [21]. Briefly, Ga-68 was eluted from a TiO 2 -based 68 Ge/ 68 Ga generator with ca. 2 mL of 1 M HCl and collected into a vial containing 1 mL of 9.5 M HCl. The resulting solution was loaded onto a strong anion exchange resin (100 mg Dowex 1 × 8), and the activity was retained as the [GaCl 4 ] − complex. After elution with 200 µL of water, the pH of the solution was neutralized by adding the appropriate volume of 2 M Na 2 CO 3 .

Radio-TLC Analysis
RCYs of the labelling reactions described above were determined in dependency on reaction time and peptide concentration. One microliter was withdrawn from every reaction mixture at different definite time points for radio-TLC analysis. RP-18 Silica gel plates were used as the stationary phase and 0.1% TFA/MeOH 30/70 v/v as the mobile phase, in which the free radiometal remains at the baseline (R f : 0.01). The R f value of CHX-A''-DTPA-DUPA-Pep was found to be 0.73 (λ: 254 nm), while the R f value of radio labelled compound was found to be ca. 0.78 (phosphorimager). Quantitative assays of radioactive spots were carried out by phosphorimager to determine the amount of radio labelled chelate and free radiometal.

Stability of Labelled CHX-A''-DTPA-DUPA-Pep
The stability of the labelled peptide in human serum and phosphate buffered saline (PBS) was verified via radio-HPLC after 30 min, 2, 4 and 8 h for
To determine the binding coefficient (K D ) of the radio-labelled peptide, 5 × 10 5 cells/well were grown in coated 12-well plates in 1 mL of medium for 48 h. The cells were washed twice with PBS, and 900 µL fresh media were added. Radiolabelled peptide was added, resulting in final concentrations of 480, 240, 120, 60, 30, 15 and 7.5 nM. In parallel, the PSMA-inhibitor (2-PMPA) was applied in a final concentration of 30 µM to determine unspecific binding. All samples were prepared in triplicate. Following 60 min of incubation at 37 °C , cells were washed twice to remove unbound activity and afterwards lysed in 1 mL of 0.5 M NaOH. Activity was measured in a gamma counter (COBRA TM II, Packard Instrument). Aliquots of the solution added to the cells were also measured for the calculation of the cellular uptake as %ID. Data were analyzed using GraphPad Prism 5.02 (one site, total and non-specific binding evaluation).

Statistical Aspects
All experiments were conducted with n ≥ 3. Data are expressed as the mean ± SD.

Organic Synthesis of CHX-A''-DTPA-DUPA-Pep
CHX-A''-DTPA-DUPA-Pep synthesis was successfully achieved by coupling DUPA-Pep and p-SCN-Bn-CHX-A''-DTPA in the presence of DIPEA as the base. After preparative HPLC purification and lyophilization of the purified fraction, the peptide was obtained as a white powder in yields of 72% and a purity of ≥98% (HPLC).

Radiochemistry
Radiolabelling with Ga-68 was performed in HEPES buffer (pH = 7.4) at room temperature by adding the prepared solution of [ 68 Ga]GaCl 3 to the peptide reaction mixture. The radiochemical yields were evaluated in dependence on the reaction time and the amount of peptide via radio-TLC (Table 1, Figure 2). Starting from 18 nM (25 µg) of CHX-A''-DTPA-DUPA-Pep, the RCY lay over 95% after 30 min of reaction time. Increasing the amount of peptide, to 72 nM (100 µg), a RCY >95% was obtained after 1 min of reaction time. Due to nearly quantitative labelling, the product solution was applied without further purification.   Regarding radiometals for radiotherapy (Lu-177 and Y-90), the radiolabelling was performed at pH 5.5 in 0.5 M NH 4 OAc buffer and showed RCYs >95% for low amounts of peptide (25 µg (18 nM) in the case of 90 Y-labelling and 10 µg (7.2 nM) in the case of 177 Lu-labelling) after 5 min of reaction time. A practically quantitative RCY of >99% was obtained in both radiosyntheses after 30 min of reaction time when the highest amount of peptide (100 µg; 72 nM) was used (Tables 2 and 3, Figures 3 and 4).

Stability Investigations
A high incorporation level and high complex stability is strongly desired in therapeutic applications, due to the severe myelotoxicity associated with Y-90 and the long half-life of Lu-177 (6.74 d). Thus, in order to verify the stability of the Y-90 and Lu-177 complexes, stability studies were performed in human serum and PBS at 37 °C. Additionally, the stability of the Ga-68 labelled compound was investigated.   (4). The first peak shows the total activity injected (the detector bypass for the recovery rate calculation).

Conclusions
The synthesis of the DTPA-derivative bearing the PSMA targeting ligand, CHX-A''-DTPA-DUPA-Pep, was straightforward and efficient.

Time (min)
The radiolabelling of the new PSMA ligand with diverse radiometals, for both diagnosis and therapy, was investigated, and optimized conditions were developed.
Labelling with Ga-68 was performed at room temperature under neutral conditions. Significant differences in RCY were observed. Radio labelling with Ga-68 succeeds in a short reaction time with high radiochemical yields; ≥95% when 50 µg (36 nM) of the peptide was used. Additionally, the labelling of CHX-A''-DTPA-DUPA-Pep with Y-90 and Lu-177 was successfully developed. In both cases, the RCY was >95% after 5 min at room temperature using 25 µg (18 nM) peptide Y-90 and 10 µg (7.2 nM) Lu-177, respectively. In conclusion, RCYs over 95% up to quantitative yields were obtained for each radionuclide at room temperature at a moderate pH value. In