⁴⁴Sc-PSMA-617 for radiotheragnostics in tandem with ¹⁷⁷Lu-PSMA-617—preclinical investigations in comparison with ⁶⁸Ga-PSMA-11 and ⁶⁸Ga-PSMA-617

Abstract

Background

The targeting of the prostate-specific membrane antigen (PSMA) is of particular interest for radiotheragnostic purposes of prostate cancer. Radiolabeled PSMA-617, a 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)-functionalized PSMA ligand, revealed favorable kinetics with high tumor uptake, enabling its successful application for PET imaging (⁶⁸Ga) and radionuclide therapy (¹⁷⁷Lu) in the clinics. In this study, PSMA-617 was labeled with cyclotron-produced ⁴⁴Sc (T _1/2 = 4.04 h) and investigated preclinically for its use as a diagnostic match to ¹⁷⁷Lu-PSMA-617.

Results

⁴⁴Sc was produced at the research cyclotron at PSI by irradiation of enriched ⁴⁴Ca targets, followed by chromatographic separation. ⁴⁴Sc-PSMA-617 was prepared under standard labeling conditions at elevated temperature resulting in a radiochemical purity of >97% at a specific activity of up to 10 MBq/nmol. ⁴⁴Sc-PSMA-617 was evaluated in vitro and compared to the ¹⁷⁷Lu- and ⁶⁸Ga-labeled match, as well as ⁶⁸Ga-PSMA-11 using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu prostate cancer cells. In these experiments it revealed similar in vitro properties to that of ¹⁷⁷Lu- and ⁶⁸Ga-labeled PSMA-617. Moreover, ⁴⁴Sc-PSMA-617 bound specifically to PSMA-expressing PC-3 PIP tumor cells, while unspecific binding to PC-3 flu cells was not observed. The radioligands were investigated with regard to their in vivo properties in PC-3 PIP/flu tumor-bearing mice. ⁴⁴Sc-PSMA-617 showed high tumor uptake and a fast renal excretion. The overall tissue distribution of ⁴⁴Sc-PSMA-617 resembled that of ¹⁷⁷Lu-PSMA-617 most closely, while the ⁶⁸Ga-labeled ligands, in particular ⁶⁸Ga-PSMA-11, showed different distribution kinetics. ⁴⁴Sc-PSMA-617 enabled distinct visualization of PC-3 PIP tumor xenografts shortly after injection, with increasing tumor-to-background contrast over time while unspecific uptake in the PC-3 flu tumors was not observed.

Conclusions

The in vitro characteristics and in vivo kinetics of ⁴⁴Sc-PSMA-617 were more similar to ¹⁷⁷Lu-PSMA-617 than to ⁶⁸Ga-PSMA-617 and 68Ga-PSMA-11. Due to the almost four-fold longer half-life of ⁴⁴Sc as compared to ⁶⁸Ga, a centralized production of ⁴⁴Sc-PSMA-617 and transport to satellite PET centers would be feasible. These features make ⁴⁴Sc-PSMA-617 particularly appealing for clinical application.

Electronic supplementary material

The online version of this article (doi:10.1186/s13550-017-0257-4) contains supplementary material, which is available to authorized users.

Background

Prostate cancer is the second most frequently diagnosed cancer type in men in the US [1]. Patients with localized disease have a good prognosis for survival; however, those diagnosed with metastatic castration-resistant prostate cancer, have much less chance of successful treatment [1, 2]. Sensitive and specific imaging tools are needed in order to enable tumor localization and staging of the disease, as well as monitoring therapy [3]. Currently, nuclear imaging of prostate cancer and related metastases is performed using ¹¹C- and ¹⁸F-choline for positron emission tomography (PET) and ^99mTc-methanediphosphonic acid for single photon emission computed tomography (SPECT), respectively [4, 5]. However, the clinical value of these radiotracers has been controversially discussed due to the low specificity and sensitivity [6, 7]. More promising may be a class of radioligands for targeting prostate-specific membrane antigen (PSMA) that has been widely investigated over the last two decades [8–10]. PSMA is a transmembrane protein, upregulated in poorly differentiated, metastatic, and hormone-refractory prostate carcinomas, while physiological expression is restricted to only a few sites, including the kidneys [11]. In recent years, a number of PSMA-targeted nuclear imaging agents were developed [12–14], among those PSMA-11, which comprises an acyclic N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid (HBED-CC)-chelator suitable for coordination of ⁶⁸Ga (T _1/2 = 68 min, Eβ⁺ _av = 830 keV; Fig. 1, Table 1) [15, 16]. This PSMA radioligand has been used successfully in clinics for PET imaging of prostate cancer [17–19]. More recently, a structurally modified PSMA ligand, referred to as PSMA-617, has been designed with a 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) chelator (Fig.1, Table 1). This derivative allows coordination of diagnostic and therapeutic radionuclides and, hence, it paved the way towards a theragnostic approach [20, 21]. Clinical studies performed so far demonstrated the promising potential of ⁶⁸Ga- and ¹⁷⁷Lu-labeled PSMA-617 to be used for the management of prostate cancer (Table 1) [21, 22]. ⁶⁸Ga-PSMA-11 and ⁶⁸Ga-PSMA-617 showed similar accumulation in most organs, albeit the renal clearance of ⁶⁸Ga-PSMA-617 was significantly faster than for ⁶⁸Ga-PSMA-11 [20, 21]. In patients, application of ⁶⁸Ga-PSMA-11 resulted in an increased contrast on delayed images; however, all lesions were already visible on the 1 h post injection (p.i.) scans [21, 23]. On the other hand, it was suggested to scan patients 2–3 h after injection of ⁶⁸Ga-PSMA-617 due to the improving image quality over time [21]. For the same reason, the scanning at even later time points (>3 h p.i.) was supposed to enable discovering additional lesions [21]; however, longer-lived radionuclides would be required for this purpose. Several ¹⁸F-based PSMA-ligands are currently under development, and the first clinical applications with ¹⁸F-DCFPyl and ¹⁸F-PSMA-1007 revealed promising results [24, 25]. The concept of radiotheragnostics, as proposed herein, is based on the use of a diagnostic and a therapeutic radionuclide with the same PSMA-targeting ligand.

Fig. 1.

Chemical structures of a PSMA-617 and b PSMA-11 suitable for labeling with the corresponding radionuclides for theragnostic or diagnostic intervention

Table 1.

Overview of radionuclides and corresponding ligands used in this study

Nuclide	Half-life	Radiation energy	Ligand	Chelator	Application	Refs.
44Sc	4.04 h	Eβ+ av = 632 keV Eγ = 1157 keV*	PSMA-617	DOTA	PET (β+-radiation)	Evaluated in this study
177Lu	6.65 days	Eβ− av = 134 keV Eγ = 113 keV, 208 keV	PSMA-617	DOTA	Therapy/SPECT (β−/γ-radiation)	[22, 37–39]
68Ga	68 min	Eβ+ av = 830 keV Eγ = 1077 keV**	PSMA-617 PSMA-11	DOTA HBED-CC	PET (β+-radiation)	[21] [17, 18, 40]

*I = 99.9%; **I = 3.2%

In this regard, we proposed ⁴⁴Sc (T _1/2 = 4.04 h, Table 1 [26]) as an alternative radionuclide to ⁶⁸Ga for PET imaging allowing to use ⁴⁴Sc-PSMA-617 as a diagnostic match to ¹⁷⁷Lu-PSMA-617. The emitted positrons have a lower energy (Eβ⁺ _av = 632 keV) as compared to ⁶⁸Ga (Eβ⁺ _av = 830 keV), enabling PET imaging with a potentially favorable spatial resolution [27, 28]. Recently, the production of ⁴⁴Sc via the ⁴⁴Ca(p,n)⁴⁴Sc nuclear reaction has been implemented at the research cyclotron at Paul Scherrer Institut, providing this radionuclide with high radionuclidic purity (>99%) and at high activities (>2 GBq) [29]. There is great potential of using ⁴⁴Sc clinically, as it can be produced at medical cyclotrons typically installed in PET centers worldwide. Another means of producing ⁴⁴Sc is via the ⁴⁴Ti/⁴⁴Sc generator [30]; however, the quantity of eluted ⁴⁴Sc activity is very limited (<200 MBq) in this case. In contrast, the proposed production route of ⁴⁴Sc at a cyclotron would address the clinical needs and, thus, be a favorable option for application of this radionuclide in patients.

The aim of this study was to investigate PSMA-617, labeled with cyclotron-produced ⁴⁴Sc, in preclinical experiments. ⁴⁴Sc-PSMA-617 was prepared and evaluated for direct comparison with ¹⁷⁷Lu- and ⁶⁸Ga-PSMA-617, as well as with ⁶⁸Ga-PSMA-11. PSMA-positive (PC-3 PIP) and PSMA-negative (PC-3 flu) prostate cancer cells were used for the performance of in vitro studies and, as xenografts in mice, for preclinical in vivo experiments including PET imaging.

Methods

PSMA-ligands and radionuclides

The PSMA ligands PSMA-617 and PSMA-11 were obtained from Advanced Biochemical Compounds (ABX GmbH, Radeberg, Germany). ⁴⁴Sc was prepared by proton irradiation of enriched ⁴⁴Ca targets at the Injector 2 cyclotron at PSI, as previously reported [29]. The irradiation of targets with ~11 MeV protons at a beam current of 50 μA lasted for 90 min. The separation of the produced ⁴⁴Sc from the target material was carried out by chromatographic methods using DGA resin [29]. ⁴⁴Sc was provided in an acidic solution (~0.1 M HCl, pH ~1 in ~700 μL) and was used directly for labeling reactions. No-carrier added ¹⁷⁷Lu in HCl 0.5 M was provided by Isotope Technologies Garching (ITG GmbH, Germany). ⁶⁸Ga was obtained from a ⁶⁸Ge/⁶⁸Ga generator (Eckert & Ziegler, Berlin, Germany) using 0.1 M HCl as an eluent (radionuclidic purity >99%).

Radiolabeling of PSMA-617 and PSMA-11

PSMA-617 was labeled with ⁴⁴Sc, ⁶⁸Ga, and ¹⁷⁷Lu in a mixture of sodium acetate (0.5 M, pH 8) and HCl (0.05–0.1 M, pH ~1) at a pH of 3.5–4.5. The reaction mixture was incubated for 10 min at 95 °C. PSMA-11 was labeled with ⁶⁸Ga under the same reaction conditions. Quality control of the radiolabeled PSMA ligands was performed using high-performance liquid chromatography (HPLC) with a C-18 reversed-phase column (Xterra^TM MS, C18, 5 μm, 150 × 4.6 mm; waters) (Additional file 1).

Determination of n-octanol/PBS distribution coefficients

The distribution coefficients (logD values) of the radioligands were determined by a shake-flask method using liquid-liquid extraction followed by phase separation, as previously reported (Additional file 1) [31]. In brief, the PSMA ligands were labeled at a specific activity of 5 MBq/nmol. Samples containing 1.25 MBq (250 pmol) of the radioligand in a volume of 25 μL were added to each vial containing 1475 μL of PBS (pH 7.4) and 1500 μL of n-octanol. The vials were vortexed vigorously for 1 min and then centrifuged for 6 min for phase separation. The concentration of radioactivity in a defined volume of each layer was measured in a γ-counter (Perkin Elmer, Wallac Wizard 1480). The distribution coefficients were expressed as the logarithm of the ratio of counts per minute (cpm) measured in the n-octanol phase to the cpm measure in the PBS phase. The values are reported as the average of at least three independent measurements (± standard deviation, SD), each performed with five replicates. Data were analyzed for significance using a one-way ANOVA test (GraphPad Prism software, version 7). A p value of <0.05 was considered as statistically significant.

Cell culture

The PC-3 PIP (PSMA^pos) and PC-3 flu (PSMA^neg) tumor cells were kindly provided by Prof. Dr. Martin Pomper (John Hopkins Institutions, Baltimore, USA) [32]. The cells were grown in RPMI cell culture medium supplemented with 10% fetal calf serum, L-glutamine, antibiotics, and puromycin (2 μg/mL) to maintain PSMA expression (Additional file 1) [33].

Cell experiments

Determination of the PSMA affinity was performed by saturation binding assays using PC-3 PIP cells and different concentrations of ^nat/44Sc-, ^nat/177Lu-, ^nat/68Ga-PSMA-617, or ^nat/68Ga-PSMA-11, respectively (Additional file 1). The relative affinities were defined as the average of at least three independent experiments and expressed as inverse molar ratio of compound needed for half-maximal binding to PSMA and the relative affinity of ¹⁷⁷Lu-PSMA-617 was set to 1.

Cell uptake and internalization experiments were performed with ⁴⁴Sc-, ¹⁷⁷Lu-, and ⁶⁸Ga-PSMA-617 as well as ⁶⁸Ga-PSMA-11 using PSMA^pos PC-3 PIP and PSMA^neg PC-3 flu cells in order to investigate whether they behaved equally and whether the uptake was PSMA-specific. For this purpose, cells were seeded in 12-well plates (~5 × 10⁵ cells in 2 mL RPMI medium/well) allowing adhesion and growth overnight at 37 °C. After removal of the supernatant, cells were washed once with PBS prior to the addition of RPMI medium without supplements (975 μL/well), followed by the addition of the radiolabeled PSMA ligands (MBq/nmol) to each well (25 μL, 7.5 pmol). Some of the cell samples were co-incubated with excess 2-(phosphonomethyl)pentane-1,5-dioic acid (2-PMPA; 100 μM) to block PSMA on the surface of PC-3 PIP cells. The well plates were incubated at 37 °C for 2 and 4 h, respectively. The cells were washed three times with ice-cold PBS to determine the total uptake of the radioligands (PSMA-bound fraction on the surface and internalized fraction). The internalized fraction of the radioligands was determined in cells which were washed with ice-cold PBS, then incubated for 10 min with acidic stripping buffer (0.05 M glycine stripping buffer in 100 mM NaCl, pH 2.8) followed by an additional washing step with ice-cold PBS. Cell samples were lysed by addition of NaOH (1 M, 1 mL) to each well. The samples of the cell suspensions were measured in a γ-counter (Perkin Elmer, Wallac Wizard 1480). After homogenization of the cell suspensions, the protein concentration was determined for each sample using a Micro BCA Protein Assay kit (Pierce, Therma Scientific). The results were expressed as percentage of total added radioactivity per 300 μg/mL protein.

Tumor mouse model

In vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection. All mice were obtained from Charles River Laboratories (Sulzfeld, Germany), at the age of 5–6 weeks. Female, athymic nude Balb/c mice were subcutaneously inoculated with PC-3 PIP cells (6 × 10⁶ cells in 100 μL Hank’s balanced salt solution (HBSS) with Ca²⁺/Mg²⁺) on the right shoulder and with PC-3 flu cells (5 × 10⁶ cells in 100 μL HBSS with Ca²⁺/Mg²⁺) on the left shoulder 2 weeks before the performance of the experiments.

Biodistribution studies

⁴⁴Sc-, ¹⁷⁷Lu-, and ⁶⁸Ga-PSMA-617 as well as ⁶⁸Ga-PSMA-11 were intravenously injected (5 MBq, 1 nmol, 100–200 μL). Mice were sacrificed at different time points post injection (p.i.) of the radioligands. Selected tissues and organs were collected, weighed and measured using a γ-counter. The results were decay-corrected and listed as a percentage of the injected activity per gram of tissue mass (% IA/g).

Imaging studies

PET/CT scans were performed using a small-animal bench-top PET/CT scanner (G8, Sofie Biosciences, Culver City, California, USA, and Perkin Elmer, Massachusetts, USA) and a small-animal SPECT/CT scanner (NanoSPECT/CT^TM, Mediso Medical Imaging Systems, Budapest, Hungary), respectively (Additional file 1). During the scans, the mice were anesthetized with a mixture of isoflurane and oxygen.

Static whole-body PET scans of 20 min duration were performed at 30 min, 2 and 4 h after injection of ⁴⁴Sc-PSMA-617 (~5 MBq, 1 nmol) and of 10 min duration at 30 min and 2 h after injection of ⁶⁸Ga-PSMA-617 and ⁶⁸Ga-PSMA-11 (~5 MBq, 1 nmol), respectively. The PET scans were followed by a CT of 1.5 min. The SPECT scan of 45 min duration was performed 2 h after injection of ¹⁷⁷Lu-PSMA-617 (~50 MBq, 1 nmol) followed by a CT of 7.5 min. Reconstruction of the acquired data was performed by using the software of the respective scanner.

All images were prepared using VivoQuant post-processing software (version 2.10, inviCRO Imaging Services and Software, Boston USA). A Gauss post-reconstruction filter (full width at half maximum = 1 mm) was applied to the images, which were presented with the scale adjusted to allow visualization of the most important organs and tissues, usually by cutting 0.5–1% of the lower scale.

Results

Radiolabeling and in vitro evaluation of PSMA-targeted radioligands

The radiochemical yield was always >97% for all radiolabeled compounds at a specific activity of up to 10 MBq/nmol (Additional file 1: Figure S1). The n-octanol/PBS distribution coefficients (logD values) were in the same range for ⁴⁴Sc-, ¹⁷⁷Lu-, and ⁶⁸Ga-PSMA-617, but somewhat reduced for ⁶⁸Ga-PSMA-11 (Table 2). The K_D values obtained for ⁴⁴Sc-PSMA-617 and ¹⁷⁷Lu-PSMA-617 were in the same range, but somewhat higher values were determined for the ⁶⁸Ga-labeled PSMA ligands (Additional file 1: Figure S2). The results were converted into relative PSMA-binding affinities, which were similar for ⁴⁴Sc-PSMA-617 and ¹⁷⁷Lu-PSMA-617, but slightly reduced for the ⁶⁸Ga-labeled PSMA ligands (Table 2).

Table 2.

In vitro characteristics of the radiolabeled PSMA-ligands

Radioligand	LogD valuesa	Relative PSMA-binding affinityb
44Sc-PSMA-617	−4.21 ± 0.04	1.2
177Lu-PSMA-617	−4.18 ± 0.06	1.0
68Ga-PSMA-617	−4.30 ± 0.10	0.5
68Ga-PSMA-11	−4.82 ± 0.07	0.4

^aLogD values represent the average (±SD) of 3–5 independent experiments performed in triplicate

^bBinding affinity is the inverse molar ratio of the average K_D values determined in four independent experiments performed in triplicates

Cell internalization studies of PSMA radioligands

Uptake and internalization of all four radioligands was investigated using PC-3 PIP/flu cells (Fig. 2). The uptake of all radioligands into PC-3 PIP cells (PSMA^pos) was comparable and in the range of 55–70%, whereas the internalized fraction was about 10–15% of total added activity (Fig. 2a). The uptake of all radioligands dropped to <0.5% when PC-3 flu cells (PSMA^neg) were used, which proves PSMA-specific uptake/internalization of all four radioligands (Fig. 2b).

Fig. 2.

Uptake and internalization of all radioligands using a PSMA^pos PC-3 PIP and b PSMA^neg PC-3 flu tumor cells

Biodistribution studies in tumor-bearing mice

The tissue distribution profiles of ⁴⁴Sc-PSMA-617 and ¹⁷⁷Lu-PSMA-617 were investigated in PC-3 PIP/flu tumor-bearing mice over a period of 6 h (Fig. 3, Additional file 1: Tables S1/S2). Relatively high radioactivity levels were detected in the blood shortly after injection (~7% IA/g, 15 min p.i.); however, blood activity decreased quickly over time to less than 0.5% IA/g at 2 h p.i. The uptake of both radioligands in PC-3 PIP tumors was already high 15 min after injection (36.5 ± 7.44 and 32.3 ± 3.54% IA/g) and increased further to reach a maximum uptake (51.9 ± 4.05 and 56.0 ± 8.0% IA/g) after 4 h (Fig. 3a). In PC-3 flu tumor xenografts, however, the accumulated activity was clearly below the blood level, indicating that unspecific accumulation of the radioligands did not occur (Fig. 3b). The uptake of the radioligands in the kidneys (37.7 ± 0.82 and 30.8 ± 4.52% IA/g, 15 min p.i.) was cleared quickly, resulting in renal retention of ~6% IA/g after 2 h and ~3% IA/g after 6 h (Fig. 3c). Most importantly, the tissue distribution kinetics and excretion pattern of ⁴⁴Sc-PSMA-617 was comparable to that of ¹⁷⁷Lu-PSMA-617 (Fig. 3).

Fig. 3.

Biodistribution data of ⁴⁴Sc-PSMA-617 and ¹⁷⁷Lu-PSMA-617, respectively, in PC-3 PIP/flu tumor-bearing mice. a Uptake of the radioligands in PSMA^pos PC-3 PIP tumors, b in PSMA^neg PC-3 flu tumors, and c clearance through the kidneys

All four radioligands were investigated under the same in vivo conditions over a period of 2 h (Fig. 4, Table 3, Additional file 1: Tables S1–S5). Clearance of radioactivity from the blood pool was fast (<0.5% IA/g, 2 h p.i) resulting in very high tumor-to-blood ratios (>200) at 2 h p.i. The accumulation of activity in PC-3 PIP tumors was comparable for all radioligands (45–49% IA/g, 2 h p.i.). The tumor-to-background ratios of ⁴⁴Sc-PSMA-617 were virtually the same as those of ¹⁷⁷Lu-PSMA-617. Renal retention of ⁴⁴Sc-PSMA-617 was comparable to the ⁶⁸Ga- and ¹⁷⁷Lu-labeled versions; however, ⁶⁸Ga-PSMA-11 showed a much higher uptake in the kidneys (~60% IA/g, 2 h p.i., Fig. 4). This resulted in reduced tumor-to-kidney ratios of ⁶⁸Ga-PSMA-11 as compared to the other radioligands. Moreover, uptake of ⁶⁸Ga-PSMA-11 in the spleen was also slightly increased. In comparison to ⁴⁴Sc-PSMA-617 and ¹⁷⁷Lu-PSMA-617, ⁶⁸Ga-PSMA-617 showed reduced tumor-to-liver ratios which is due to the increased liver uptake of this radioligand.

Fig. 4.

Time-dependent uptake and retention of radioactivity in different organs and tissues over the first 2 h after injection of ⁴⁴Sc-PSMA-617 (green), ¹⁷⁷Lu-PSMA-617 (yellow-brown), ⁶⁸Ga-PSMA-617 (red) and ⁶⁸Ga-PSMA-11 (blue)

Table 3.

Tumor-to-background ratios obtained in PC-3 PIP/flu tumor-bearing mice

	15 min after injection
PSMA-ligand	44Sc-PSMA-617	177Lu-PSMA-617	68Ga-PSMA-617	68Ga-PSMA-11
Tumor-to-blood	5.38 ± 0.90	4.45 ± 0.66	4.15 ± 0.24	6.10 ± 1.84
Tumor-to-liver	18.8 ± 5.02	20.4 ± 5.48	6.02 ± 0.59	15.3 ± 3.96
Tumor-to-kidney	0.97 ± 0.21	1.06 ± 0.14	1.26 ± 0.25	0.41 ± 0.14
	30 min after injection
	44Sc-PSMA-617	177Lu-PSMA-617	68Ga-PSMA-617	68Ga-PSMA-11
Tumor-to-blood	17.6 ± 1.23	16.0 ± 3.52	16.9 ± 4.22	20.5 ± 6.40
Tumor-to-liver	55.6 ± 7.36	53.1 ± 6.68	19.0 ± 2.32	31.7 ± 2.48
Tumor-to-kidney	1.60 ± 0.22	2.09 ± 0.24	3.62 ± 0.81	0.58 ± 0.10
	2 h after injection
	44Sc-PSMA-617	177Lu-PSMA-617	68Ga-PSMA-617	68Ga-PSMA-11
Tumor-to-blood	>200	>200	>200	>200
Tumor-to-liver	>200	>200	35.2 ± 9.94	71.3 ± 7.48
Tumor-to-kidney	7.98 ± 1.71	11.6 ± 0.87	15.9 ± 3.26	0.69 ± 0.12

Imaging studies in PC-3 PIP/flu tumor-bearing mice

Nuclear imaging studies were performed with PC-3 PIP/flu tumor-bearing mice 2 h after injection of ⁴⁴Sc-, ¹⁷⁷Lu-, and ⁶⁸Ga-PSMA-617 as well as ⁶⁸Ga-PSMA-11 (Fig. 5, Additional file 1: Figure S3). PC-3 PIP tumor xenografts, located on the right shoulder, showed high uptake of activity in all four cases. No activity was detected, however, in PC-3 flu tumors on the left shoulder, demonstrating the PSMA-specific tumor accumulation of the radioligands. The PET images obtained with ⁴⁴Sc- and ⁶⁸Ga-PSMA-617 as well as the SPECT image obtained with ¹⁷⁷Lu-PSMA-617 showed a similar distribution pattern and confirmed the post mortem data. The tissue distribution of ⁶⁸Ga-PSMA-11 was, however, different in that it accumulated to a significantly higher extent in the kidneys.

Fig. 5.

Images (PET/CT and SPECT/CT, respectively) as maximum intensity projections (MIPs) of PC-3 PIP/flu tumor-bearing mice 2 h after injection of a ⁴⁴Sc-PSMA-617, b ¹⁷⁷Lu-PSMA-617, c ⁶⁸Ga-PSMA-617, and d ⁶⁸Ga-PSMA-11. PSMA- PC-3 flu tumor, PSMA+ PC-3 PIP tumor, ki kidney, bl urinar bladder

PET/CT scans were performed at 30 min, 2 and 4 h after injection of ⁴⁴Sc-PSMA-617 to visualize the tissue distribution in the same animal over time (Fig. 6). The PC-3 PIP tumor (PSMA^pos) was visible already 30 min p.i.; however, at this time point, retention of radioactivity was also seen in the kidneys. At delayed time points, when renal activity was cleared, the tumor became better visible and was finally the only site showing accumulated activity. These results demonstrated increasing tumor-to-background contrast over time and confirmed the advantage of late imaging.

Fig. 6.

PET/CT images as maximum intensity projections (MIPs) of PC-3 PIP/flu tumor-bearing mice a 0.5, b 2, and c 4 h after injection of ⁴⁴Sc-PSMA-617. PSMA- PC-3 flu tumor, PSMA+ PC-3 PIP tumor, ki kidney, bl urinary bladder

Discussion

In this study, ⁴⁴Sc-PSMA-617 was compared to ⁶⁸Ga-PSMA-617 and ⁶⁸Ga-PSMA-11 in vitro and in tumor-bearing mice as a potential diagnostic match to the therapeutically employed ¹⁷⁷Lu-PSMA-617 (Fig. 1). The PC-3 PIP/flu tumor cells enabled the investigation of PSMA-specific and -unspecific uptake of the radioligands in vitro and in vivo by using a PC-3 PIP and PC-3 flu tumor xenograft in the same animal.

A comparable in vitro behavior of ⁴⁴Sc-PSMA-617 and ¹⁷⁷Lu-PSMA-617, as was determined in this study, was expected due to the chemical similarities of ⁴⁴Sc and ¹⁷⁷Lu with regard to their complexation using DOTA [34]. Our data revealed almost identical characteristics of ⁴⁴Sc-PSMA-617 and ¹⁷⁷Lu-PSMA-617 with regard to their hydrophilic properties and PSMA-binding affinities (Table 2). ⁴⁴Sc-PSMA-617 showed PSMA-specific cell uptake and internalization, as was the case for all three other radioligands (Fig. 2).

The evaluation of the radioligands in mice revealed a favorable tumor accumulation of ⁴⁴Sc-PSMA-617 already shortly after injection and a relatively fast clearance of background activity through the kidneys (Fig. 4). These circumstances enabled PET/CT imaging with increasing tumor-to-background contrast over time (Fig. 6). The tissue distribution profile of ⁴⁴Sc-PSMA-617 was largely identical to that obtained with ¹⁷⁷Lu-PSMA-617 over the investigated period of 6 h (Fig. 3). Comparable pharmacokinetic properties of ⁴⁴Sc- and ¹⁷⁷Lu-labeled compounds were expected as previously shown in preclinical experiments with a DOTA-functionalized folate conjugate [35]. The chemical similarities of these nuclides and, as a consequence, comparable pharmacokinetics of ligands labeled with ⁴⁴Sc and ¹⁷⁷Lu, respectively, would thus, allow predicting the exact tissue distribution of ¹⁷⁷Lu-PSMA-617 based on the PET imaging results obtained with ⁴⁴Sc-PSMA-617.

The chemical structure of ⁶⁸Ga-PSMA-11 is fundamentally different (Fig. 1). Not surprisingly, the tissue distribution profile of ⁶⁸Ga-PSMA-11 in mice varied significantly from those of the other investigated radioligands, as was observed previously in preclinical experiments [15, 20]. High uptake of activity was found in the kidneys promptly after injection of ⁶⁸Ga-PSMA-11 (Fig. 4). This observation was in agreement with the fact that in patients ⁶⁸Ga-PSMA-11 showed an increased retention of activity in the kidneys as compared to ⁶⁸Ga-PSMA-617 [21, 23].

Even when using the same chelator, the chemical properties of ⁶⁸Ga- and ¹⁷⁷Lu-labeled compounds are not identical due to the different coordination chemistry of these radiometals [34] which may potentially result in different in vivo kinetics [36]. In the case of ⁶⁸Ga-PSMA-617, the pharmacokinetics were similar to those of the ⁴⁴Sc- and ¹⁷⁷Lu-labeled versions, with the exception of an increased uptake of ⁶⁸Ga-PSMA-617 in the liver (Fig. 4). Exactly the same phenomenon has been previously observed in mice, when they were injected with ⁶⁸Ga-labeled DOTA peptides [28]. In patients, increased hepatic uptake of radioactivity was not detected after injection of ⁶⁸Ga-PSMA-617 [21, 23].

Although ⁶⁸Ga-labeled PSMA ligands—in particular ⁶⁸Ga-PSMA-11—are successfully employed in the clinics, it is indisputable that the high cost for a ⁶⁸Ge/⁶⁸Ga generator is a disadvantage when considering the limited activity that can be eluted daily. The short half-life of ⁶⁸Ga is also a limiting factor, making transportation of activity over long distances unattractive. The almost fourfold longer half-life of ⁴⁴Sc as compared to ⁶⁸Ga would enable the shipment of ⁴⁴Sc-based radiopharmaceuticals to PET centers without cyclotron and radiopharmaceutical facilities. Concerns of radiation safety with regard to the high-energy γ-radiation of ⁴⁴Sc (E_γ = 1157 keV) may be addressed by using tungsten-based containers as are employed for other commercial PET nuclides such as ⁸⁹Zr, which emits high-energy γ-radiation (E_γ = 909 keV) as well.

The use of ⁴⁴Sc-PSMA-617 as a diagnostic radioligand would be novel and favorable, since the longer half-life of ⁴⁴Sc permits a greater flexibility of patient scheduling to accommodate urgent cases in between, thus, allowing optimization of patient management in nuclear medicine departments. Moreover, ⁴⁴Sc-PSMA-617 would allow PET/CT imaging of patients several hours after injection, potentially allowing dosimetry estimations and enabling the detection of small pathological lesions due to increased tumor-to-background ratios and, consequently, improved image contrast.

A clinical translation of the proposed concept, as is planned for the near future, will allow addressing the diverse aspects of this promising approach. Appropriate acquisition parameters for PET imaging of patients with prostate cancer remain to be determined after collecting practical experiences with ⁴⁴Sc-PSMA-617 in a clinical setting.

Conclusions

In this study, the potential of ⁴⁴Sc-PSMA-617 for PET imaging was demonstrated in a preclinical setting. The results indicate more similar characteristics of ⁴⁴Sc-PSMA-617 to ¹⁷⁷Lu-PSMA-617 than is the case for ⁶⁸Ga-PSMA-11. An important advantage of using ⁴⁴Sc over ⁶⁸Ga is the feasibility of transporting ⁴⁴Sc-based PSMA-ligands to PET centers without cyclotron and radiopharmaceutical facilities due to its longer half-life. The fact that ⁴⁴Sc-PSMA-617 can enable delayed PET imaging makes it particularly appealing for clinical application as it may allow pretherapeutic dosimetry and an improved image quality at later time points after injection.

Abbreviations

2-PMPA

2-(Phosphonomethyl)pentane-1,5-dioic acid

PSMA

prostate-specific membrane antigen

DOTA

1,4,7,10-Tetraazacyclododecane- N,N′,N′′,N′′′-tetraacetic acid

HBED-CC

N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid

HBSS

Hank’s balanced salt solution

HPLC

High performance liquid chromatography

IA

Injected activity

p.i.

Post injection

PBS

Phosphate-buffered saline pH 7.4

PET

Positron emission tomography

PSMA

Prostate-specific membrane antigen

SPECT

Single photon emission computed tomography

Additional file

Additional file 1: ^{(680.1KB, docx)}

Supporting information. (DOCX 671 kb)