1 Introduction
Prostate cancer (PC), as the most common men cancers in Europe, is still one of the outstanding reasons among cancer deaths [1]. However, the 5-year survival rate for an early detection of localized gland prostate cancers is almost 100%, the cancer spread beyond the prostate lead to significant falling of the survival rates [2] and unfortunately mortality from metastasizing prostate cancer is still high [1]. Nowadays, an early detection of the metastatic lesions has significant impact on the clinical staging and therapy management of the patients [3,4].
The prostate-specific membrane antigen (PSMA) also called glutamate carboxypeptidase II (GCP II), is expressed by almost all prostate cancers and gained the highest clinical impact in the past years. Due to the high quality PET imaging of prostate cancer, PSMA has been known as an ideal target for this purpose [5-7] and some PSMA inhibitors in labeling with 68Ga [8-11] and 123I [12] have shown promising results in first human studies.
Studies on the recently prepared Glu-NH-CO-NH-Lys-(Ahx)-[68Ga(HBED-CC)] (68Ga-PSMA-11) as a 68Ga-labelled PSMA ligand showed the ability of tracer for high contrast detection of PC relapses and metastases [8,13]. Also, the comparison of this PET imaging agent with 18F-fluoromethylcholine PET/CT as the choline based PET/CT, demonstrated the detection of lesions with improved contrast, especially at low PSA levels [9].
Beside the slightly modified chemical structure of the molecules synthesized for binding to PSMA, these molecules differ mainly in the selection of the chelator for the complexation of the desired radionuclide [14-16]. 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) is the mostly used chelator for the complexation of radiometals such as 68Ga, 177Lu and 166Ho, especially to small molecules [17-19] either for diagnostic or for therapeutic applications. 2-[3-(1-Carboxy-5-{3-naphthalen-2-yl-2-[(4-{[2-(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetylamino]-methyl}-cyclohexanecarbonyl)-amino]-propionylamino}-pentyl)-ureido]-pentanedioic acid (DKFZ-PSMA-617) as the recently DOTA-based synthesized PSMA (Fig. 1) labelled with 177Lu has indicated a high potential to improve the clinical management of advanced PC in its first human study [20].
68Ga is an excellent positron emitting radioisotope suitable for clinical PET imaging. Having physical characteristics of positron emission (89%), low abundance of 1077 keV photon emission (3.22%) and relatively short half-life (t½=67.71 min)[22], it permits PET applications of the 68Ga-radiopharmacuticals, with an acceptable radiation dose to the patient [23]. With the extension of PET and the construction of 68Ge/68Ga generators with suitable eluates for labeling, the use of 68Ga has arisen recently [19].
The clinical impact of the PSMA PET tracers is growing very fast. Recently, 68Ga-DKFZ-PSMA-617 was designed and its first individual clinical experience showed comparable results with 68Ga-PSMA-11 [21], but it is better than 68Ga-PSMA-11 in the uptake ratio of tumor to blood and muscle after 1 h [21]. However, more preclinical data on its biodistribution in different organs and at specified times after injection are required for performance evaluation of this new compound before clinical usage.
In this paper, we present the additional preclinical data of 68Ga-DKFZ-PSMA-617 and relevant aspects of its production. The 68Ga-DKFZ-PSMA-617 was prepared at the optimized conditions (pH, temperature, ligand concentration and reaction time) and the appropriate systems for HPLC and RTLC analysis were introduced. The biodistribution data of the radiolabelled compound was investigated in male Syrian rats at given intervals from 15 min to 2 h after injection by sacrifice and PET imaging. 68GaCl3 was injected to the same type rats for biodistribution comparison of the organ uptake of the radiolabelled compound.
2 Experimental section
A prototype 40-mCi 68Ge/68Ga generator, developed at Pars Isotope Co. (Tehran, Iran), was used in this study. 68Ge/68Ga generator was eluted with Supra-Pure HCl (0.6 mM, 5 mL) in 0.5 mL fractions. Three fractions with the highest 68GaCl3 activity were used for labeling purposes. DKFZ-PSMA-617 was provided from ABX (Radeberg, Germany). The other chemical reagents were from Sigma-Aldrich Chemical Co. (UK). Whatman No. 2 paper was from Whatman (UK). Radio-chromatography was performed by Whatman paper using a thin layer chromatography scanner, Bioscan AR2000 (Paris, France). Activities of the samples were measured by a p-type coaxial high-purity germanium detector (HPGe, EGPC 80-200R) coupled with a multichannel analyzer. Calculations were based on the 511 keV peak for 68Ga. All values were expressed as mean ± SD and the data were compared using Student's T-test. Statistical significance was defined as P<0.05. Animal studies were performed in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations, second edition.
2.1 Elution of 68Ge/68Ga generator
For selecting appropriate eluent, the generator were eluted by 5 mL HCl with different concentrations from 0.1 to 1.0 M and the activity of the eluted 68Ga was measured utilizing HPGe detector at each time. Also, in order to optimize the minimum required volume for the elution of 68Ga with the maximum yield and radioactive concentration, the generator was eluted with the equal volume of HCl and the activity of each fraction containing 0.5 mL of the eluate was measured.
2.2 Quality control of the eluted 68Ga
The radionuclidic purity of the product was investigated by gamma spectrometry. This step was carried out utilizing an HPGe detector coupled to a Canberra™ multichannel analyzer for 1000 s. Breakthrough was measured by counting the same sample at 48 h after the first test for the detection of a small amount of 68Ge in the sample.
The chemical purity control of the sample was carried out by the ICP-OES method, to ensure that concentrations of tin (from generator material), iron (from the sealing parts and acid impurities), zinc (as the decay product) and gallium (as the target material) were acceptable regarding the internationally accepted limits.
Radiochemical purity of the eluted 68Ga was studied using ITLC. ITLC chromatograms of 68GaCl3 solution were performed in 10 % ammonium acetate:methanol on silicagel sheets and in 10 mM DTPA solution (pH=4) on Whatman No. 2 paper.
2.3 Radiolabeling of DKFZ-PSMA-617 with 68GaCl3
A stock solution of DKFZ-PSMA-617 in concentration of 1 µg/µL in distilled water was prepared. The first fraction of the eluted 68Ga was put away and the next three fractions including 1.5 mL of 68GaCl3 (approximately 925 MBq) were used for radiolabeling. Certain amount of DKFZ-PSMA-617 was added to the vial containing 68GaCl3 and the pH of the reaction mixture was adjusted utilizing HEPES (1 M in H2O). In order to obtain the optimized conditions, several experiments were performed by changing the ligand concentration, pH, temperature and incubation time.
Then, 8 mL of water was added to the final solution and the mixture was passed through a C18 Sep-Pak column which preconditioned with 5 mL ethanol, 10 mL water and 10 mL air, respectively. The column was then washed with 0.5 mL ethanol and 1 mL of 0.9% NaCl. The volume of the final solution was adjusted to 5 mL by 0.9% NaCl before injection.
2.4 Quality control of the radiolabelled complex
Radiochemical purity of the radiolabelled complex was checked using both HPLC and ITLC methods. Paper chromatography was carried out using Whatman No. 2 paper and 0.9 % NaCl and 0.1 M sodium citrate as the mobile phases.
HPLC was performed on the final preparation utilizing a C18ODS column with the dimensions of 100 mm × 4.6 mm and 5 µm particle size. Gradient elution was applied with the following parameters: A= water + 1% TFA, B= Acetonitrile, flow rate: 2.6 mL/min, 100 % A: 0 % B for 3 min, 50 % A: 50 % B for 7 min, 0 % A: 100 % B for 5 min.
2.5 Stability Tests
In order to check the stability in the final product, a sample of 68Ga-DKFZ-PSMA-617 (37 MBq) was kept at room temperature for up to 2 h while being checked by ITLC at the specified intervals.
Also, for the stability assessment of 68Ga-DKFZ-PSMA-617 in human serum, 11.1 MBq of the final solution (50 μL) was added to the 300 μL of the freshly prepared serum and kept at 37°C for 2 h. Every 30 min, trichloroacetic acid (10 %, 100 μL) was added to a portion of the mixture (50 μL), and the mixture was centrifuged at 3,000 rpm for 5 min followed by decanting the supernatant from the debris. The stability was determined by performing frequent ITLC analysis of the supernatant using the above-mentioned ITLC system.
2.6 Biodistribution of 68GaCl3 and the radiolabeled complex in Syrian rats
The 100 μL of final 68Ga-DKFZ-PSMA-617 solution with approximately 5.55 MBq radioactivity was injected intravenously into male Syrian rats through their tail vein. Also, for better comparison, biodistribution of 68GaCl3 in 0.9% normal saline (pH=7) was investigated followed by intravenous administration of 100 μL of the solution (5.55 MBq). The total amount of radioactivity injected into each animal was measured by counting the 1-mL syringe before and after injection in a dose calibrator with fixed geometry. The biodistribution of the solutions among tissues was determined by sacrificing of four rats for each selected intervals (15, 30, 60 and 120 min) after injection using the animal care protocols.
Blood samples were taken immediately after sacrifice. The tissues were weighed and rinsed with normal saline and their activities were determined with a p-type coaxial HPGe detector coupled with a multichannel analyzer.
The percentage of injected dose per gram (%ID/g) for different organs was calculated by dividing the activity amount of each tissue (A) to the decay-corrected injected activity and the mass of each organ. All values were expressed as mean ± SD and the data were compared using Student's T-test.
2.7 Imaging studies
PET/CT imaging was performed with a PET/CT scanner (Biograph 6 TrueX; Siemens Medical Solutions). Static PET images were acquired for 5 min with three sets of emission images in two hour after 68Ga-DKFZ-PSMA-617 injection in the rats. In addition, PET emission scans were preceded by CT scans performed for anatomical reference and attenuation correction (spatial resolution 1.25 mm, 80 kV, 150 mA) with a total CT scanning time of 20 s. Reconstruction was performed using the iterative algorithm with attenuation correction. The reconstruction settings were 4 iterations and 21 subsets to a 256×256 matrix, with a post filtering of 2 mm. Transmission data were reconstructed into a matrix of equal size by means of filtered back-projection, yielding a co-registered image set.
3 Results and Discussion
3.1 Elution of 68Ge/68Ga generator
While the generator was eluted by 5 mL HCl with different concentrations of 0.1–1.0 M, activity of the eluted 68Ga increased with the increment of HCl concentration. However, this data indicates higher elution yield for 1.0 M HCl, 0.6 M HCl was determined as the more suitable solvent for radiolabeling purposes.
The generator was eluted with the equal volumes of 0.6 M HCl. The second, third and fourth fractions showed the maximum activities (>1110 MBq) and therefore, these fractions were used for radiolabeling purpose.
3.2 Quality control of the eluted 68Ga
Radionuclidic control showed the presence of 511 and 1077 keV peaks, all originating from 68Ga. The radionuclidic purity was higher than 99.9 %. Also, the calculations for Ge breakthrough demonstrated the 68Ge/68Ga activity ratio of about 9×10−6 at the time of elution. The HPGe spectrum of the solution is presented in Fig. 2.
The concentrations (in part per million) of tin (from generator material), iron (from the sealing parts and acid impurities), zinc (as the decay product) and gallium (as the target material) were 0.220, 0.230, 0.135 and <0.1, respectively. This chemical purity is crucial and usually suffices for the procedure.
The radiochemical purity of the 68GaCl3 solution was checked in two solvent systems. In 10 mM DTPA solution, free 68Ga3+ is coordinated to a more lipophilic moiety as 68Ga(DTPA)2− and migrates to a higher Rf. On the other hand, in a 10% ammonium acetate:methanol mixture (1:1), 68Ga3+ would remain at the origin, while any other ionic cation of 68Ga3+ would migrate to higher Rf which was not observed here (Fig. 3).
3.3 Radiolabeling of DKFZ-PSMA-617 with 68GaCl3
In order to obtain the maximum complexation yield, experiments were performed by varying the reaction parameters of ligand concentration, pH, temperature and reaction time. The effect of pH on complexation yield was studied at pH3–5 of the reaction mixture using HEPES. As shown in Fig.4 (a), the results indicate that the optimum pH for radiolabeling is 3.5–4.
The effect of the ligand amount on the radiochemical yield is shown in Fig 4(b). Even at very low concentration of the ligand (2.5 µg, 2.4 nmol), the high radiochemical purity can be achievable.
The effects of temperature and reaction time on radiochemical yield are presented in Table 1. While the temperature of 90–95°C is required, the experiments show that 10 min is sufficient for radiolabeling.
| Temperature /°C (2.5 µg ligand, pH.3.5, 10 min reaction) | Reaction time /min (2.5 µg ligand, pH.3.5, 95°C) | ||||||
|---|---|---|---|---|---|---|---|
| 50 | 70 | 85 | 95 | 5 | 10 | 20 | 30 |
| 32.4±1.0 | 65.3±0.9 | 88.6±0.4 | 96.1±0.7 | 92.6±0.3 | 96.1±0.6 | 96.0±0.5 | 96.3±0.8 |
Totally, 68Ga-DKFZ-PSMA-617 was prepared with the complexation yield of higher than 96% and specific activity of 308.3 MBq/nmol at the optimized conditions which is about 2 times greater than the previous reported literature (140 MBq/nmol) [23].
3.4 Quality control of the radiolabelled complex
Radiochemical purity of the radiolabelled complex was checked using HPLC and ITLC. HPLC analysis demonstrated that the fast eluting compound was hydrophilic 68GaCl3 cation (1.3 min), while 68Ga- DKFZ-PSMA-617 with high molecular weight was eluted after 4.5 min (Fig. 5a). HPLC chromatogram showed the radiochemical purity of more than 96%.
ITLC was applied for detecting the radiolabelled compound from the free gallium cation. In both 0.1 M sodium citrate and 0.9% NaCl as the mobile phases with Whatman No.2 as the stationary phase, the radiolabelled compound remains at the origin, while free gallium cation migrates to higher Rf (Figs. 5b and 5c). ITLC chromatogram also proved the radiochemical purity of over 96%.
3.5 Stability studies
The stability of 68Ga-DKFZ-PSMA-617 was investigated at room temperature and in human serum at 37°C. The radiochemical purity of the complex remained >96% at room temperature and in freshly prepared human serum at 37°C even after 120 min of preparation.
3.6 Biodistribution of 68GaCl3 and the radiolabelled complex in Syrian rats
The tissue uptakes of 68GaCl3 and the radiolabelled complex were calculated as the area percentage under the curve of the related photo peak per gram of tissue (% ID/g) (Tables 2 and 3). 68Ga is mainly excreted from the gastrointestinal tract (GIT) with high blood contents due to the transferrin binding at early intervals. Also, the colon, bone and stomach activity contents are significant.
| Organs | 15 min | 30 min | 60 min | 120 min |
|---|---|---|---|---|
| Blood | 3.18±0.17 | 2.98±0.12 | 2.53±0.11 | 2.44±0.14 |
| Kidney | 0.70±0.09 | 0.41±0.10 | 1.42±0.12 | 0.91±0.08 |
| Spleen | 0.51±0.09 | 0.70±0.06 | 1.03±0.10 | 1.43±0.12 |
| Stomach | 0.41±0.11 | 0.56±0.07 | 1.21±0.09 | 1.54±0.14 |
| Intestine | 0.71±0.06 | 0.82±0.08 | 0.89±0.05 | 0.61±0.07 |
| Liver | 0.91±0.02 | 1.50±0.12 | 0.78±0.08 | 0.60±0.05 |
| Bone | 0.56±0.06 | 0.91±0.10 | 1.20±0.11 | 1.05±0.09 |
| Organs | 15 min | 30 min | 60 min | 120 min | Organs | 15 min | 30 min | 60 min | 120 min |
|---|---|---|---|---|---|---|---|---|---|
| Blood | 0.81±0.05 | 0.36±0.03 | 0.26±0.01 | 0.18±0.01 | Skin | 0.25±0.03 | 0.15±0.01 | 0.18±0.01 | 0.06±0.00 |
| Herat | 0.38±0.02 | 0.14±0.01 | 0.12±0.01 | 0.00±0.00 | Bone | 0.08±0.00 | 0.03±0.00 | 0.04±0.00 | 0.07±0.00 |
| Kidney | 23.74±0.85 | 9.05±0.54 | 3.63±0.18 | 1.76±0.10 | Muscle | 0.08±0.00 | 0.04±0.00 | 0.01±0.00 | 0.04±0.00 |
| Spleen | 0.13±0.01 | 0.14±0.02 | 0.10±0.00 | 0.10±0.01 | Thyroid | 0.31±0.02 | 0.23±0.01 | 0.45±0.03 | 0.21±0.04 |
| Stomach | 0.11±0.02 | 0.09±0.02 | 0.12±0.01 | 0.13±0.00 | Adrenal | 0.18±0.02 | 0.46±0.03 | 0.29±0.03 | 0.47±0.04 |
| Intestine | 0.14±0.02 | 0.09±0.01 | 0.15±0.01 | 0.57±0.04 | Salivary gland | 0.24±0.03 | 0.20±0.02 | 0.18±0.01 | 0.24±0.05 |
| Lung | 0.50±0.06 | 0.30±0.02 | 0.15±0.01 | 0.10±0.00 | Pancreases | 0.18±0.01 | 0.14±0.02 | 0.20±0.01 | 0.10±0.00 |
| Liver | 0.22±0.04 | 0.14±0.01 | 0.12±0.02 | 0.08±0.00 | Prostate | 0.86±0.06 | 0.76±0.05 | 0.99±0.07 | 0.90±0.04 |
68Ga-DKFZ-PSMA-617 demonstrated significant uptake in the kidney as the major route of excretion. The maximum uptake in the kidney occurred at 15 min post injection while decreased with time. As expected, small accumulation was perceived in the prostate. The results indicated rapid clearance form blood. Approximately no activity was found after 30 min in blood samples. No significant accumulation was observed in the other organs.
The biodistribution pattern of 68Ga-DKFZ-PSMA-617 is completely different from 68GaCl3 (Fig. 6). Whereas the activity of 68GaCl3 in blood decreases slightly with the time, the radiolabelled complex is cleared from blood rapidly (0.36% after 30 min). The kidney is the major route of the excretion for the compound, while no considerable activity was accumulated in the kidney after 68GaCl3 administration. Generally, accumulation of the complex in the most organs such as the intestine, liver, spleen and the bone is so much smaller than 68GaCl3, which is a major advantage for this radiopharmaceutical.
Our study on 68Ga-DKFZ-PSMA-617 indicates the benefit of its application over 68Ga-PSMA-11 as the routinely used PSMA radiopharmaceutical, but the injected dose per gram of tissue is only presented at 1 h post injection. With regard to the importance of activity distribution in non-target organs, to avoid undesirable absorbed dose, biodistribution of this new radiopharmaceutical in different organs was studied from 15 min to 2 h post injection. Significant accumulation was observed in the kidney, which is in accordance with the literatures [23]. Activity aggregation in the other organs is insignificant.
3.7 PET/CT Imaging studies
Figure 7 shows typical PET/CT images acquired immediately and 30/60 min after 68Ga-DKFZ-PSMA-617 injection in the normal Syrian rats. It can be seen that the only visible organs were the kidney and bladder. This confirms the imaging results in Ref. [23].
4. Conclusion
In this work, 68Ga was obtained from the SnO2 based 68Ge/68Ga generator. The results of quality control analysis including radionuclidic, chemical and radiochemical purities, indicated high purity of the eluted 68Ga. The conditions for preparation of 68Ga-DKFZ-PSMA-617 were optimized, with radiochemical purity of over 96% and specific activity of 308.3 MBq/nmol in less than 10 min at 2.5 µg PSMA, pH 3.5–4 and 90–95°C. We presented biodistribution of the 68Ga-DKFZ-PSMA-617 complex in different intervals (15–120 min) in the organs of male Syrian rats after intravenous injection. Most of the injected activity was accumulated in the kidney which can be considered as the major route of the excretion. With regard to the insignificant amount of activity in other organs, this compound can be considered as a good agent for PET imaging applications.
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