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

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

ACS Omega

Neurotensin receptor 2 (NTS) is a well-known mediator of central opioid-independent analgesia. Seminal studies have highlighted NTS overexpression in a variety of tumors including prostate cancer, pancreas adenocarcinoma, and breast cancer. Herein, we describe the first radiometalated neurotensin analogue targeting NTS. JMV 7488 (DOTA-(βAla)-Lys-Lys-Pro-(D)Trp-Ile-TMSAla-OH) was prepared using solid-phase peptide synthesis, then purified, radiolabeled with Ga and In, and investigated on HT-29 cells and MCF-7 cells, respectively, and on HT-29 xenografts. [Ga]Ga-JMV 7488 and [In]In-JMV 7488 were quite hydrophilic (logD = -3.1 ± 0.2 and -2.7 ± 0.2, respectively, < 0.0001). Saturation binding studies showed good affinity toward NTS ( = 38 ± 17 nM for [Ga]Ga-JMV 7488 on HT-29 and 36 ± 10 nM on MCF-7 cells; = 36 ± 4 nM for [In]In-JMV 7488 on HT-29 and 46 ± 1 nM on MCF-7 cells) and good selectivity (no NTS binding up to 500 nM). On cell-based evaluation, [Ga]Ga-JMV 7488 and [In]In-JMV 7488 showed high and fast NTS-mediated internalization of 24 ± 5 and 25 ± 11% at 1 h for [In]In-JMV 7488, respectively, along with low NTS-membrane binding (<8%). Efflux was as high as 66 ± 9% at 45 min for [Ga]Ga-JMV 7488 on HT-29 and increased for [In]In-JMV 7488 up to 73 ± 16% on HT-29 and 78 ± 9% on MCF-7 cells at 2 h. Maximum intracellular calcium mobilization of JMV 7488 was 91 ± 11% to that of levocabastine, a known NTS agonist on HT-29 cells demonstrating the agonist behavior of JMV 7488. In nude mice bearing HT-29 xenograft, [Ga]Ga-JMV 7488 showed a moderate but promising significant tumor uptake in biodistribution studies that competes well with other nonmetalated radiotracers targeting NTS. Significant uptake was also depicted in lungs. Interestingly, mice prostate also demonstrated [Ga]Ga-JMV 7488 uptake although the mechanism was not NTS-mediated

Angiogenesis

APJ has been extensively described in the pathophysiology of angiogenesis and cell proliferation. The prognostic value of APJ overexpression in many diseases is now established. This study aimed to design a PET radiotracer that specifically binds to APJ. Apelin-F13A-NODAGA (AP747) was synthesized and radiolabeled with gallium-68 ([Ga]Ga-AP747). Radiolabeling purity was excellent (> 95%) and stable up to 2 h. Affinity constant of [Ga]Ga-AP747 was measured on APJ-overexpressing colon adenocarcinoma cells and was in nanomolar range. Specificity of [Ga]Ga-AP747 for APJ was evaluated in vitro by autoradiography and in vivo by small animal PET/CT in both colon adenocarcinoma mouse model and Matrigel plug mouse model. Dynamic of [Ga]Ga-AP747 PET/CT biodistributions was realized on healthy mice and pigs for two hours, and quantification of signal in organs showed a suitable pharmacokinetic profile for PET imaging, largely excreted by urinary route. Matrigel mice and hindlimb ischemic mice were submitted to a 21-day longitudinal follow-up with [Ga]Ga-AP747 and [Ga]Ga-RGD small animal PET/CT. [Ga]Ga-AP747 PET signal in Matrigel was significantly more intense than that of [Ga]Ga-RGD. Revascularization of the ischemic hind limb was followed by LASER Doppler. In the hindlimb, [Ga]Ga-AP747 PET signal was more than twice higher than that of [Ga]Ga-RGD on day 7, and significantly superior over the 21-day follow-up. A significant, positive correlation was found between the [Ga]Ga-AP747 PET signal on day 7 and late hindlimb perfusion on day 21. We developed a new PET radiotracer that specifically binds to APJ, [Ga]Ga-AP747 that showed more efficient imaging properties than the most clinically advanced tracer of angiogenesis, [Ga]Ga-RGD.France Life Imagin

Breast Cancer Res Treat

Neurotensin receptor-1 (NTS) is increasingly recognized as a potential target in diverse tumors including breast cancer, but factors associated with NTS expression have not been fully clarified. We studied NTS expression using the Tissue MicroArray (TMA) of primary breast tumors from Institut Bergonié. We also studied association between NTS expression and clinical, pathological, and biological parameters, as well as patient outcomes. Out of 1419 primary breast tumors, moderate to strong positivity for NTS (≥ 10% of tumoral cells stained) was seen in 459 samples (32.4%). NTS staining was cytoplasmic in 304 tumors and nuclear in 155 tumors, a distribution which appeared mutually exclusive. Cytoplasmic overexpression of NTS was present in 21.5% of all breast tumors. In multivariate analysis, factors associated with cytoplasmic overexpression of NTS in breast cancer samples were higher tumor grade, Ki67 ≥ 20%, and higher pT stage. Cytoplasmic NTS was more frequent in tumors other than luminal A (30% versus 17.3%; p < 0.0001). Contrastingly, the main "correlates" of a nuclear location of NTS were estrogen receptor (ER) positivity, low E&E (Elston and Ellis) grade, Ki67 < 20%, and lower pT stage. In NTS-positive samples, cytoplasmic expression of NTS was associated with shorter 10-year metastasis-free interval (p = 0.033) compared to NTS nuclear staining. Ancillary analysis showed NTS expression in 73% of invaded lymph nodes from NTS-positive primaries. NTS overexpression was found in about one-third of breast tumors from patients undergoing primary surgery with two distinct patterns of distribution, cytoplasmic distribution being more frequent in aggressive subtypes. These findings encourage the development of NTS-targeting strategy, including radiopharmaceuticals for imaging and therapy.Translational Research and Advanced Imaging Laborator

Membrane and Nuclear Absorbed Doses from 177Lu and 161Tb in Tumor Clusters: Effect of Cellular Heterogeneity and Potential Benefit of Dual Targeting—A Monte Carlo Study

Early use of targeted radionuclide therapy to eradicate tumor cell clusters and micrometastases might offer cure. However, there is a need to select appropriate radionuclides and assess the potential impact of heterogeneous targeting. The Monte Carlo code CELLDOSE was used to assess membrane and nuclear absorbed doses from Lu and Tb (β-emitter with additional conversion and Auger electrons) in a cluster of 19 cells (14-μm diameter, 10-μm nucleus). The radionuclide distributions considered were cell surface, intracytoplasmic, or intranuclear, with 1,436 MeV released per labeled cell. To model heterogeneous targeting, 4 of the 19 cells were unlabeled, their position being stochastically determined. We simulated situations of single targeting, as well as dual targeting, with the 2 radiopharmaceuticals aiming at different targets. Tb delivered 2- to 6-fold higher absorbed doses to cell membranes and 2- to 3-fold higher nuclear doses than Lu. When all 19 cells were targeted, membrane and nuclear absorbed doses were dependent mainly on radionuclide location. With cell surface location, membrane absorbed doses were substantially higher than nuclear absorbed doses, both with Lu (38-41 vs. 4.7-7.2 Gy) and with Tb (237-244 vs. 9.8-15.1 Gy). However, when 4 cells were not targeted by the cell surface radiopharmaceutical, the membranes of these cells received on average only 9.6% of the Lu absorbed dose and 2.9% of the Tb dose, compared with a cluster with uniform cell targeting, whereas the impact on nuclear absorbed doses was moderate. With an intranuclear radionuclide location, the nuclei of unlabeled cells received only 17% of the Lu absorbed dose and 10.8% of the Tb dose, compared with situations with uniform targeting. With an intracytoplasmic location, nuclear and membrane absorbed doses to unlabeled cells were one half to one quarter those obtained with uniform targeting, both for Lu and for Tb. Dual targeting was beneficial in minimizing absorbed dose heterogeneities. To eradicate tumor cell clusters, Tb may be a better candidate than Lu. Heterogeneous cell targeting can lead to substantial heterogeneities in absorbed doses. Dual targeting was helpful in reducing dose heterogeneity and should be explored in preclinical and clinical studies

ACS Med Chem Lett

Bivalent ligands, i.e., molecules having two ligands covalently connected by a linker, have been gathering attention since the first description of their pharmacological potential in the early 80s. However, their synthesis, particularly of labeled heterobivalent ligands, can still be cumbersome and time-consuming. We herein report a straightforward procedure for the modular synthesis of labeled heterobivalent ligands (HBLs) using dual reactive 3,6-dichloro-1,2,4,5-tetrazine as a starting material and suitable partners for sequential SAr and inverse electron-demand Diels-Alder (IEDDA) reactions. This assembly method conducted in a stepwise or in a sequential one-pot manner provides quick access to multiple HBLs. A conjugate combining ligands toward the prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPR) was radiolabeled, and its biological activity was assessed and (receptor binding affinity, biodistribution, imaging) as an illustration that the assembly methodology preserves the tumor targeting properties of the ligands.France Life Imagin

A proof of principle study using radiopharmaceuticals to quantify and localize container-content interactions in medical syringes

The sorption of drugs onto their contents is a known phenomenon that is difficult to analyse precisely. The purpose of this study was to present a non-invasive method for locating and quantifying sorption phenomena using radiopharmaceuticals. Radiopharmaceutical are medicines armed with a radionuclide enabling quantification and imaging using dedicated scanners. The sorption of nine different radiopharmaceuticals on 2- and 3-part syringes was investigated. These syringes were filled with the studied radiopharmaceutical solutions and stored immobile for 3 h. At different times ranging from 0 to 180 min, 10 µL were taken from the syringes and the radioactivity of these samples was determined by a gamma counter. 5 radiopharmaceuticals exhibited no significant sorption at any time point in both 2 and 3-parts syringes, but 4 radiopharmaceuticals exhibited sorption losses varying from 20 to 33% after 3 h contact with 3-part-syringes, but no sorption on 2-part syringes at any time point. [Tc]Tc-tetrofosmine Single Photon Emission Computed Tomography/Computed Tomography imaging indicated clearly that the interactions were located on the rubber plunger of the 3-part-syringes. The specific nature of radiopharmaceuticals allowed their use as an innovative method to quantify and localize drug sorption phenomena

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

International audienceBackground: [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

Comparison of (68)Ga-PSMA-617 PET/CT and (68)Ga-RM2 PET/CT in patients with localized prostate cancer candidate for radical prostatectomy: a prospective, single arm, single center, phase II study

Considering the wide range of therapeutic options for localized prostate cancer (active surveillance, radiation beam therapy, focal therapy, radical prostatectomy, etc), accurate assessment of the aggressiveness and localization of primary prostate cancer lesion are essential for treatment decision making. National Comprehensive Cancer Network guidelines recognize Prostate-Specific Membrane Antigen (PSMA) Positron Emission Tomography/Computed Tomography (PET/CT) for the initial staging of high risk primary prostate cancer. The Gastrin-Releasing Peptide Receptor (GRP-R) is a neuropeptide receptor over-expressed by low-risk prostate cancer cells. We aim to perform the first prospective head-to-head comparison of PSMA and GRP-R targeted imaging at the initial staging to understand how PSMA-PET and GRP-R-PET could be used or combined in clinical practice Methods: This was a prospective, single-center, diagnostic cross-sectional imaging study using anonymized, masked and independent interpretations of PET/CT paired studies in 22 patients with (68)Ga-PSMA-617 (a radiolabelled PSMA-inhibitor) and (68)Ga-RM2 (a radiolabelled GRP-R-antagonist). We enrolled patients with newly diagnosed, biopsy-proven, prostate cancer. No patient had received neoadjuvant hormone therapy or chemotherapy. All patients underwent extended pelvic lymph node dissection. Histology served as reference. Results: On a lesion-based analysis (including lesions <0.1cc), (68)Ga-PSMA-617 PET/CT detected 74.3% (26/35) of all tumor lesions and (68)Ga-RM2 PET/CT detected 78.1% (25/32; one patient could not be offered (68)Ga-RM2 PET/CT). Paired examinations showed positive uptake with the two tracers in 21/32 lesions (65.6%), negative uptake in 5/32 lesions (15.6%), and discordant uptake in 6/32 lesions (18.8%). Uptake of (68)Ga-PSMA-617 was higher in ISUP ≥ 4 vs ≥ 1 (P < 0.0001); and ISUP ≥ 4 vs 2 (P = 0.002). There were no significant differences in uptake between ISUP scores for (68)Ga-RM2. Median (68)Ga-RM2 SUV(max) was significantly higher than median (68)Ga-PSMA-617 SUV(max) in the ISUP 2 subgroup (P = 0.01). Conclusion: (68)Ga-PSMA-617 PET/CT is useful to depict higher, more clinically significant, ISUP score lesions and (68)Ga-RM2 PET/CT has higher detection rate for low-ISUP tumors. Combining PSMA-PET and GRP-R PET allows to better classify intraprostatic lesions