Background: Peptide radioligands may serve as radionuclide carriers to tumor sites overexpressing their cognate receptor for diagnostic or therapeutic purposes. Treatment of mice with the neprilysin (NEP)-inhibitor phosphoramidon was previously shown to improve the metabolic stability and tumor uptake of biodegradable radiopeptides. Aiming to clinical translation of this methodology, we herein investigated the impact of the approved pill Entresto, releasing the potent NEP-inhibitor LBQ657 in vivo, on the stability and tumor uptake of two radiopeptides. Methods: The metabolic stability of [^99mTc]Tc-DB4 (DB4, N₄-Pro-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH₂) and [¹¹¹In]In-SG4 (SG4, DOTA-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH₂) was tested in LBQ657/Entresto-treated mice vs. untreated controls. The uptake in gastrin-releasing peptide receptor (GRPR)-, or cholecystokinin subtype 2 receptor (CCK₂R)-positive tumors respectively, was compared between LBQ657/Entresto-treated mice and untreated controls. Results: LBQ657/Entresto treatment induced marked stabilization of [^99mTc] Tc-DB4 and [¹¹¹In]In-SG4 in peripheral mice blood, resulting in equally enhanced tumor uptake at 4 h post-injection. Accordingly, the [^99mTc]Tc-DB4 uptake of 7.13 ± 1.76%IA/g in PC-3 tumors increased to 16.17 ± 0.71/17.50 ± 3.70%IA/g (LBQ657/Entresto) and the [¹¹¹In]In-SG4 uptake of 3.07 ± 0.87%IA/g in A431-CCK₂R(+) tumors to 8.11 ± 1.45/9.61 ± 1.70%IA/g. Findings were visualized by SPECT/CT. Conclusions: This study has shown the efficacy of Entresto to notably improve the profile of [^99mTc]Tc-DB4 and [¹¹¹In]In-SG4 in mice, paving the way for clinical translation of this approach.

Radiolabeled gastrin analogues have been proposed for theranostics of cholecystokinin subtype 2 receptor (CCK₂R)-positive cancer. Peptide radioligands based on other receptor antagonists have displayed superior pharmacokinetics and higher biosafety than agonists. Here, we present DGA1, a derivative of the nonpeptidic CCK₂R antagonist Z-360 carrying an acyclic tetraamine, for [^99mTc]Tc labeling. Preclinical comparison of [^99mTc]Tc-DGA1 with [^99mTc]Tc-DG2 (CCK₂R-agonist reference) was conducted in HEK293-CCK₂R/CCK_2i4svR cells and mice models, qualifying [^99mTc]Tc-DGA1 for further study in patients with CCK₂R-positive tumors and single-photon emission computed tomography/CT.

(1) Background: Theranostic approaches in the management of cholecystokinin subtype 2 receptor (CCK₂R)-positive tumors include radiolabeled gastrin and CCK motifs. Moving toward antagonist-based CCK₂R-radioligands instead, we herein present three analogs of the nonpeptidic CCK₂R-antagonist Z360, GAS1/2/3. Each was conjugated to a different chelator (DOTA, NODAGA or DOTAGA) for labeling with medically relevant trivalent radiometals (e.g., Ga-68, In-111, Lu-177) for potential use as anti-CCK₂R cancer agents; (2) Methods: The in vitro properties of the thee analogs were compared in stably transfected HEK293-CCK₂R cells. Biodistribution profiles were compared in SCID mice bearing twin HEK293-CCK₂R and wtHEK293 tumors; (3) Results: The GAS1/2/3 analogs displayed high CCK₂R-affinity (lower nM-range). The radioligands were fairly stable in vivo and selectively targeted the HEK293-CCK₂R, but not the CCK₂R-negative wtHEK293 tumors in mice. Their overall pharmacokinetic profile was found strongly dependent on the radiometal-chelate. Results could be visualized by SPECT/CT for the [¹¹¹In]In-analogs; (4) Conclusions: The present study highlighted the high impact of the radiometal-chelate on the end-pharmacokinetics of a new series of Z360-based radioligands, revealing candidates with promising properties for clinical translation. It also provided the impetus for the development of a new class of nonpeptidic radioligands for CCK₂R-targeted theranostics of human cancer.

Aiming to expand the application of the SST₂R-antagonist LM4 (DPhe-c[DCys-4Pal-DAph(Cbm)-Lys-Thr-Cys]-DTyr-NH₂) beyond [⁶⁸Ga]Ga-DATA^5m-LM4 PET/CT (DATA^5m, (6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate), we now introduce AAZTA⁵-LM4 (AAZTA⁵, 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-[pentanoic-acid]perhydro-1,4-diazepine), allowing for the convenient coordination of trivalent radiometals of clinical interest, such as In-111 (for SPECT/CT) or Lu-177 (for radionuclide therapy). After labeling, the preclinical profiles of [¹¹¹In]In-AAZTA⁵-LM4 and [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 were compared in HEK293-SST₂R cells and double HEK293-SST₂R/wtHEK293 tumor-bearing mice using [¹¹¹In]In-DOTA-LM3 and [¹⁷⁷Lu]Lu-DOTA-LM3 as references. The biodistribution of [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 was additionally studied for the first time in a NET patient. Both [¹¹¹In]In-AAZTA⁵-LM4 and [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 displayed high and selective targeting of the HEK293-SST₂R tumors in mice and fast background clearance via the kidneys and the urinary system. This pattern was reproduced for [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 in the patient according to SPECT/CT results in a monitoring time span of 4–72 h pi. In view of the above, we may conclude that [¹⁷⁷Lu]Lu-AAZTA⁵-LM4 shows promise as a therapeutic radiopharmaceutical candidate for SST₂R-expressing human NETs, based on previous [⁶⁸Ga]Ga-DATA^5m-LM4 PET/CT, but further studies are needed to fully assess its clinical value. Furthermore, [¹¹¹In]In-AAZTA⁵-LM4 SPECT/CT may represent a legitimate alternative diagnostic option in cases where PET/CT is not available.

Sentinel lymph node detection (SLND) is rapidly entering common practice in the management of patients with tumors. The introduction of mannose molecules to ^99mTc-labeled dextrans, so far, showed that the sentinel node could trap these agents due to their recognition by the mannose receptors of lymph node macrophages. The current study aimed to synthesize, characterize, and biologically evaluate a series of mannosylated dextran derivatives labeled with ^99mTc for potential use in SLND. The compounds were designed to have a dextran with a molecular weight of 10–500 kDa as a backbone, S-derivatized cysteines, efficient SNO chelators, and mannose moieties for binding to mannose receptors. They were successfully synthesized, thoroughly characterized using NMR techniques, and labeled with the fac-[^99mTc(CO)₃]⁺ synthon. Labeling with high yields and radiochemical purities was achieved with all derivatives. In vivo biodistribution and imaging studies demonstrated high uptake in the first lymph node and low uptakes in the following node and confirmed the ability to visualize the SLN. Among the compounds studied, ^99mTc-D75CM demonstrated the most attractive biological features, and in combination with the high radiochemical yield and stability of the compound, its further evaluation as a new radiopharmaceutical for sentinel lymph node detection was justified.

In the current work, a pix2pix conditional generative adversarial network has been evaluated as a potential solution for generating adequately accurate synthesized morphological X-ray images by translating standard photographic images of mice. Such an approach will benefit 2D functional molecular imaging techniques, such as planar radioisotope and/or fluorescence/bioluminescence imaging, by providing high-resolution information for anatomical mapping, but not for diagnosis, using conventional photographic sensors. Planar functional imaging offers an efficient alternative to biodistribution ex vivo studies and/or 3D high-end molecular imaging systems since it can be effectively used to track new tracers and study the accumulation from zero point in time post-injection. The superimposition of functional information with an artificially produced X-ray image may enhance overall image information in such systems without added complexity and cost. The network has been trained in 700 input (photography)/ground truth (X-ray) paired mouse images and evaluated using a test dataset composed of 80 photographic images and 80 ground truth X-ray images. Performance metrics such as peak signal-to-noise ratio (PSNR), structural similarity index measure (SSIM) and Fréchet inception distance (FID) were used to quantitatively evaluate the proposed approach in the acquired dataset.