Psma 617

The radiolabeling of PSMA-617 with ¹⁶¹Tb or ¹⁷⁷Lu and the quality control of both, [¹⁶¹Tb]Tb-PSMA-617 and [¹⁷⁷Lu]Lu-PSMA-617, were accomplished in analogy to the methodology published previously for [¹⁷⁷Lu]Lu-PSMA-617 38 (link). For a typical labeling of [¹⁶¹Tb]Tb-PSMA-617, ¹⁶¹Tb ([¹⁶¹Tb]TbCl₃ in 0.05 M HCl, TERTHERA B.V., Breda, Netherlands) was added to sodium acetate buffer (1.0 M, pH 4.5) containing PSMA-617 (Advanced Biochemical Compounds, ABX GmbH, Radeberg, Germany) to reach a specific activity of ~42 MBq/nmol. The reaction mixture (pH 4.5) was incubated for 25 min at 95 °C. A quality control per reversed-phase high-performance liquid chromatography (Shimadzu LC-20AT high pressure liquid chromatography (HPLC) system) was performed. The radiochemical yields and purities of both radiotracers were ≥ 99%.

Chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Germany) or otherwise as indicated. Used water and acetonitrile for reagents were from Merck. For staining, PSMA-specific antibody (Abcam Cat# ab19071, RRID:AB_444751) and the goat anti mouse DyLight488 antibody (Abcam Cat# ab96871, RRID:AB_10680543) were used. The CD31-specific antibody (Cat# MA3100, RRID:AB_223516) was purchased from ThermoFisher Scientifics. Precursors PSMA-11 and PSMA-617 were purchased from ABX (Germany) and Endocyte (USA). The nuclides ⁶⁸Ga and ¹⁷⁷Lu were supplied by iThemba (South Africa, generator system) and IDB (The Netherlands), respectively.

PSMA-617 (ABX GmbH, Radeberg, Germany) was labeled with ¹⁶⁹Er under standard conditions at a molar activity of 10 MBq/nmol. A stock solution of PSMA-617 (1 mM) was mixed with a solution of ¹⁶⁹Er in 0.05 M HCl and the pH adjusted to 4.0 with the addition of sodium acetate solution (0.5 M, pH 8). The reaction mixture was incubated for 20 min at 95°C. Quality control of ¹⁶⁹Er-PSMA-617 was performed using HPLC, as reported above.
As a control compound, PSMA-617 was labeled with ¹⁷⁷Lu (carrier-free in 0.04 M HCl; ITM, Germany) at a molar activity of 10 MBq/nmol, according to a previously-published procedure (49 (link)). Quality control of ¹⁷⁷Lu-PSMA-617 was also performed using HPLC.

Radiolabeling was conducted as previously described by Kratochwil et al. [34 (link)]. 1 GBq of non-carrier added Lutetium-177 (ITG GmbH, Garching, Germany), 30–40 MBq/µL, was mixed with 100 µL sterile 0.4 M sodium acetate solution, pH 5.5, and 1.25 µL 20% w/w ascorbic acid solution; thereafter, 2 µL of a 10 mM solution of PSMA-617 (ABX, Radeberg, Germany) was added. The solution was heated to and maintained at 95 °C on a shaker for 15 min. Samples, 1 µL, were taken for instant Thin Layer Chromatography (iTLC) at 0 and 15 min. The iTLC strips were placed in 0.2 M sodium citrate solution (mobile phase), which was allowed to migrate up the strip. In this system the free, unbound, ¹⁷⁷Lu migrates with the solvent front of the mobile phase. The iTLC strips were then analyzed on a phosphor imager system (Cyclone Plus Phosphor Imager, PerkinElmer, Inc., Waltham, MA, USA). The reaction was terminated by removing the sample from the shaker and cooling it down to room temperature. A sterile 0.9% sodium chloride solution was added, and the radiotracer diluted (1:3 or 1:7), a sample for iTLC taken, and pH tested, before injections in mice.

Ibu-PSMA-02 (1 mM stock solution) was labeled with lutetium-177 (no-carrier-added lutetium-177 in 0.04 M HCl; ITM Medical Isotopes GmbH, Germany) at molar activities of 5–100 MBq/nmol at pH ~4.5 as previously reported (Supplementary Materials) [16 (link)]. The reaction mixture was incubated for 10 min at 95 °C, followed by quality control using HPLC as previously reported (Supplementary Materials) [11 (link),16 (link)]. Ibu-PSMA-01, which was previously developed in our group [17 (link)], and PSMA-617 (ABX GmbH, Radeberg, Germany) were radiolabeled under the same conditions.
The radiolytic stability of [¹⁷⁷Lu]Lu-Ibu-PSMA-02 was assessed in three independent experiments. [¹⁷⁷Lu]Lu-Ibu-PSMA-02 (50 MBq/nmol) was diluted with saline to an activity concentration of 250 MBq/500 µL and incubated at room temperature with or without the addition of l-ascorbic acid (3 mg) for a total period of 24 h. HPLC was performed after 1, 4 and 24 h to determine the radioligand’s integrity as previously reported (Supplementary Materials) [17 (link)].

PSMA-11 and PSMA-617 precursors were obtained from ABX advanced biochemical compounds (Radeberg, Germany). PSMA-TO-1 precursor was obtained from Dr. H.-J. Wester (Technische Universität München, Germany). Gallium-68 was eluted from an Eckert & Ziegler IGG100 Generator and the peptide precursors (5 nmol for PSMA-11 and PSMA-TO-1; 10 nmol for PSMA-617) were labeled with ⁶⁸Ga to obtain 19–24 mCi of final products according to previously published protocols [13 (link)].
No-carrier-added ¹⁷⁷LuCl₃ was obtained from Spectron MRC and PSMA-617 and PSMA-TO-1 were radiolabeled as previously described with a molar activity of 84 GBq/µmol [14 ]. Actinium-225 was supplied by the Isotope Program within the Office of Nuclear Physics in the Department of Energy’s Office of Science and radiolabeled as previously described (molar activity of 130 MBq/µmol) [13 (link)].

PSMA-617 was obtained from ABX GmbH (Radeberg, Germany), and radiolabeling with ¹⁷⁷LuCl₃ was carried out as described in detail before [10 (link), 16 (link)]. Quality control was overseen by experienced radiochemists and physicians with respective training in the field. ¹⁷⁷Lu-PSMA-617 was administered by slow intravenous injection over 30–60 s. Infusion of 1000 mL of saline was initiated 30 min before application at a continuous rate of 300 mL/h. With the intention to limit the uptake to the parotid and submandibular, icepacks were locally applied 30 min before therapy and continued for 1 h [24 (link)]. All therapies were performed as in-patient procedures at our nuclear medicine therapy ward. As mandated by radiation protection legislation, patients remained hospitalized for a minimum 48 h; median hospitalization was 3 (range 2–5) days per cycle.