Preclinical evaluation of new GRPR-antagonists with improved metabolic stability for radiotheranostic use in oncology

Abstract

Background: The gastrin-releasing peptide receptor (GRPR) has been extensively studied as a biomolecular target for peptide-based radiotheranostics. However, the lack of metabolic stability and the rapid clearance of peptide radioligands, including radiolabeled GRPR-antagonists, often impede clinical application. Aiming at circumventing these drawbacks, we have designed three new GRPR-antagonist radioligands using [^99mTc]Tc-DB15 ([^99mTc]Tc-N₄-AMA-DIG-_DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt; AMA: p-aminomethylaniline; DIG: diglycolate) as a motif, due to its high GRPR-affinity and stability to neprilysin (NEP). The new analogues carry the DOTAGA-chelator (1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid) through different linkers at the N-terminus to allow for labeling with the theranostic radionuclide pair In-111/Lu-177. After labeling with In-111 the following radioligands were evaluated: (i) [¹¹¹In]In-AU-SAR-M1 ([¹¹¹In]In-DOTAGA-AMA-DIG-_DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt), (ii) [¹¹¹In]In-AU-SAR-M2 ([¹¹¹In]In-[DOTAGA-Arg]AU-SAR-M1) and (iii) [¹¹¹In]In-AU-SAR-M3 ([¹¹¹In]In-[DOTAGA-_DArg]AU-SAR-M1).

Results: These radioligands were compared in a series of in vitro assays using prostate adenocarcinoma PC-3 cells and in murine models. They all displayed high and GRPR-specific uptake in PC-3 cells. Analysis of mice blood collected 5 min post-injection (pi) revealed similar or even higher metabolic stability of the new radioligands compared with [^99mTc]Tc-DB15. The stability could be further increased when the mice were treated with Entresto® to in situ induce NEP-inhibition. In PC-3 xenograft-bearing mice, [¹¹¹In]In-AU-SAR-M1 displayed the most favourable biodistribution profile, combining a good tumor retention with the highest tumor-to-organ ratios, with the kidneys as the dose-limiting organ.

Conclusions: These findings strongly point at AU-SAR-M1 as a promising radiotherapeutic candidate when labeled with Lu-177, or other medically appealing therapeutic radiometals, especially when combined with in situ NEP-inhibition. To this goal further investigations are currently pursued.

Keywords: GRPR-antagonist; Metabolic stability; NEP-inhibition; PC-3 tumors; Prostate cancer; Radiotheranostics.

Fig. 1

Chemical structures of DB15, AU-SAR-M1, AU-SAR-M2 and AU-SAR-M3

Fig. 2

Representative HPLC radiochromatograms for a [¹¹¹In]In-AU-SAR-M1—radiochemical purity: 91%, b [¹¹¹In]In-AU-SAR-M2—radiochemical purity: 96%, (c) [¹¹¹In]In-AU-SAR-M3—radiochemical purity: 94%

Fig. 3

Percentage of activity linked to PC-3 cells in the absence (control—left blue bars) or in the presence of excess NOTA-PEG2-RM26 (blocked—right green, bars too low to be visible) for a [¹¹¹In]In-AU-SAR-M1, b [¹¹¹In]In-AU-SAR-M2 and c [¹¹¹In]In-AU-SAR-M3 (**** = p < 0.0001)

Fig. 4

Ligandtracer sensograms for a [¹¹¹In]In-AU-SAR-M1, b [¹¹¹In]In-AU-SAR-M2 and c [¹¹¹In]In-AU-SAR-M3; association was measured at 1 nM and 3 nM radioligand

Fig. 5

Normalized in vitro cellular processing showing % of total cell associated activity (solid line) and fraction of activity internalized (dotted line) for a [¹¹¹In]In-AU-SAR-M1, b [¹¹¹In]In-AU-SAR-M2 and c [¹¹¹In]In-AU-SAR-M3

Fig. 6

a Comparison of biodistribution results at 4 h pi across [¹¹¹In]In-AU-SAR-M1 (blue bars), [¹¹¹In]In-AU-SAR-M2 (red bars) and [¹¹¹In]In-AU-SAR-M3 (green bars) (from left to right), presented as average %IA/g ± sd (error bars), n = 4. Results for gastrointestinal tract (GI) and carcass are presented as average %IA ± sd.** = p < 0.01; *** = p < 0.001; and **** = p < 0.0001; b T/O ratios for across [¹¹¹In]In-AU-SAR-M1 (blue bars), [¹¹¹In]In-AU-SAR-M2 (red bars) and [¹¹¹In]In-AU-SAR-M3 (green bars) at 4 h pi

Fig. 7

a Biodistribution results of (from left to right): GRPR-specificity (black bars), [¹¹¹In]In-Au-SAR-M1 at 4 h (blue bars) and 24 h (red bars). Results are given as average %IA/g ± sd (error bars), ** = p < 0.01; **** = p < 0.0001. b T/O ratios for [¹¹¹In]In-AU-SAR-M1 at 4 h pi (blue bars) and 24 h pi (red bars), given as mean values ± sd (as error bars), n = 4 for both groups

Fig. 8

SPECT/CT images of a PC-3 tumor-bearing mouse, at a 4 h and b 24 h pi of [¹¹¹In]In-AU-SAR-M1, red arrows indicate tumor-sites