68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Abstract

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment.

Theranostics of Primary Prostate Cancer: Beyond PSMA and GRP-R

“…The radioligands used in this study, their respective targets and their affinities towards the target are summarized as follows: [ 111 In]In-PSMA-617 targets PSMA (Ki = 2.34 ± 2.94 nM [ 14 ]), [ 111 In]In-RM2 targets GRP-R (Kd = 2.9 ± 0.4 nM [ 15 ], [ 111 In]In-JMV 6659 is a radioligand of NTS 1 (Kd = 6.29 ± 1.37 nM [ 16 ]), [ 111 In]In-JMV 7488 is a radioligand of NTS 2 (Kd = 36.39 ± 4.02 nM) [ 17 ], [ 177 Lu]Lu-DOTATATE targets SST 2 (Kd = 2.0 ± 0.8 nM [ 18 ]) and [ 67 Ga]Ga-pentixafor is a radioligand of CXCR4 (Kd = 24.6 ± 2.5 nM [ 19 ]). The production and control of the radiopharmaceuticals used are described in the Supplementary Materials .…”

Cold Kit Labeling: The Future of 68Ga Radiopharmaceuticals?

“…This has to be optimized finely to prevent saturation of targeted receptors with unlabeled biomolecule. On the other hand, this, plus shorter production time and higher radiolabeling yield with the kit than with automated module synthesis, might allow for multiple patient preparations, although kits are usually recommended as single-dose preparations ( 118 , 124 , 125 ). Regarding outcomes of both processes, in terms of product characteristics, quality control gave comparable results ( Figure 6A ), and both methods are reliable and comply with Good Manufacturing Practices and European Pharmacopeia specifications ( 113 , 124 , 126 , 127 ).…”

“…It should be noted that a commercial kit would be more expensive. In a study on [ 68 Ga]Ga-RM2 synthesis, consumable costs for a synthesis was reported to be 282.1 vs. 65.7 € for a module and a kit, respectively, whereas the shorter production time and higher radiolabeling yield with the kit would allow for the injection of at least two additional patients ( 125 ). This gain in production time may also improve patient workflow, improving, again, cost efficiency.…”

“…In vitro studies demonstrated that [ 68 Ga]Ga-RM2 prepared this way was suitable for GRPR imaging. Soon after, Chastel et al developed a similar kit formulation (50 μg of peptide precursor, same reaction conditions) usable with a GalliAd® generator ( 125 ). Besides demonstrating high uptake in GRP-R-expressing PC-3 cells, they established the importance of excipients (scavenger, bulking agent) in the formulation of freeze-dried kits, since composition without trehalose did not meet criteria for lyophilization.…”

Modular One-Pot Strategy for the Synthesis of Heterobivalent Tracers

“…A K D value of 1.9 ± 1.2 nM toward the GRPR was obtained on PC3 cells, and a value of 17.7 ± 4.9 nM toward the PSMA was obtained on 22Rv1 cells (22Rv1 expresses a small amount of GRPR; in this case, the GRPR interaction was blocked by coincubation with 10 μM of bombesin). The affinity of [ 68 Ga]Ga-18 toward the GRPR and the PSMA was lower than GRPR-and PSMAtargeting standards such as [ 68 Ga]Ga-RM2 (K D = 0.3 ± 0.2 nM on PC3 cells) 42 and [ 68 Ga]Ga-PSMA-617 (K i = 2.3 ± 2.9 nM on LnCaP cells) 43 but still in the nanomolar range. 43 To assess [ 68 Ga]Ga-18 in vivo, a model of athymic nude mice doubly xenografted with PC3 cells on the left shoulder and 22Rv1 cells on the right shoulder was used.…”