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Radionuclide imaging using radiolabeled inhibitors of prostate-specific membrane antigen (PSMA) can be used for the staging of prostate cancer. Previously, we optimized the Glu-urea-Lys binding moiety using a linker structure containing 2-napththyl-L-alanine and L-tyrosine. We have now designed a molecule that contains mercaptoacetyl–triglutamate chelator for labeling with Tc-99m (designated as BQ0413). The purpose of this study was to evaluate the imaging properties of [99mTc]Tc-BQ0413. PSMA-transfected PC3-pip cells were used to evaluate the specificity and affinity of [99mTc]Tc-BQ0413 binding in vitro. PC3-pip tumor-bearing BALB/C nu/nu mice were used as an in vivo model. [99mTc]Tc-BQ0413 bound specifically to PC3-pip cells with an affinity of 33 ± 15 pM. In tumor-bearing mice, the tumor uptake of [99mTc]Tc-BQ0413 (38 ± 6 %IA/g in PC3-pip 3 h after the injection of 40 pmol) was dependent on PSMA expression (3 ± 2 %IA/g and 0.9 ± 0.3 %IA/g in PSMA-negative PC-3 and SKOV-3 tumors, respectively). We show that both unlabeled BQ0413 and the commonly used binder PSMA-11 enable the blocking of [99mTc]Tc-BQ0413 uptake in normal PSMA-expressing tissues without blocking the uptake in tumors. This resulted in an appreciable increase in tumor-to-organ ratios. At the same injected mass (5 nmol), the use of BQ0413 was more efficient in suppressing renal uptake than the use of PSMA-11. In conclusion, [99mTc]Tc-BQ0413 is a promising probe for the visualization of PSMA-positive lesions using single-photon emission computed tomography (SPECT). [ABSTRACT FROM AUTHOR]

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [99mTc]Tc-(HE)3-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)3), provided clear imaging of HER2 expressing breast cancer 2–4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G3C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)3-linker (designated as G3-(G3S)3C) would further improve the contrast of imaging using 99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9–5 nM. Biodistribution of [99mTc]Tc-G3-G3C, [99mTc]Tc-G3-(G3S)3C, and [99mTc]Tc-G3-E3C in mice was compared with the biodistribution of [99mTc]Tc-(HE)3-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-(HE)3-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications. [ABSTRACT FROM AUTHOR]

177 Lu-labeled small-molecule prostate-specific membrane antigen (PSMA) targeted tracers are therapeutic agents for metastatic castration-resistant prostate cancer. Optimizing molecular design holds the potential to further enhance the pharmacokinetic properties of PSMA-targeted agents while preserving their potent therapeutic effects. In this study, six novel N -[ N -[( S )-1,3-dicarboxypropyl]carbamoyl]-( S )-l-lysine (DCL) urea-based PSMA ligand 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid conjugates were synthesized. These conjugates feature polypeptide linkers containing the Phe-Phe peptide sequence and an aromatic fragment at the ε-NH-Lys group of the DCL fragment. The synthesis yielded products with satisfactory yields ranging from 60% to 72%, paving the way for their preclinical evaluation. The labeling of the new variants of urea-based PSMA inhibitors provided a radiochemical yield of over 95%. The 177 Lu-labeled conjugates demonstrated specific and moderate affinity binding to PSMA-expressing human cancer cells PC3-pip in vitro and specific accumulation in PSMA-expressing xenografts in vivo. Based on the results, both the lipophilicity and the type of substituent in the linker significantly influence the binding properties of the PSMA inhibitor and its biodistribution profile. Specifically, the studied variants containing a bromine substituent or a hydroxyl group introduced into the aromatic fragment of the phenylalanyl residue in DCL exhibit higher affinities to PSMA compared to variants with only a chlorine-substituted aromatic fragment or variants without any substituents. The [ 177 Lu]Lu- 13C with the bromine substituent was characterized by the highest activity accumulation in blood, salivary glands, muscle, bone, and gastrointestinal tract and had inasmuch as an unfavorable pharmacokinetic profile. The negative charge of the carboxyl group in the phenyl moiety of the [ 177 Lu]Lu- 13A variant has demonstrated a positive effect on reducing the retention of activity in the liver and the kidneys (the ratio of tumor to kidneys was 1.3-fold). Low accumulation in normal tissues in vivo indicates that this novel PSMA-targeting inhibitor is a promising radioligand., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Gastrin releasing peptide receptor (GRPR) is overexpressed in prostate cancer (PC-3) and can be used for diagnostic purposes. We herein present the design and preclinical evaluation of two novel NOTA/NODAGA-containing peptides suitable for labeling with the positron emission tomography (PET) radionuclide Ga-68. These analogs are based on the previously reported GRPR-antagonist DOTAGA-PEG2-[Sar 11 ]RM26, developed for targeted radiotheraostic applications. Both NOTA-PEG2-[Sar 11 ]RM26 and NODAGA-PEG2-[Sar 11 ]RM26 were successfully labeled with Ga-68 and evaluated in vitro and in vivo using PC-3 cell models. Both, [ 68 Ga]Ga-NOTA-PEG2-[Sar 11 ]RM26 and [ 68 Ga]Ga-NODAGA-PEG2-[Sar 11 ]RM26 displayed high metal-chelate stability in phosphate buffered saline and against the EDTA-challenge. The two [ 68 Ga]Ga-labeled conjugates demonstrated highly GRPR-mediated uptake in vitro and in vivo and exhibited a slow internalization over time, typical for radioantagonistis. The [ nat Ga]Ga-loaded peptides displayed affinity in the low nanomole range for GRPR in competition binding experiments. The new radiotracers demonstrated biodistribution profiles suitable for diagnostic imaging shortly after administration with fast background clearance. Their high tumor uptake (13 ± 1 and 15 ± 3% IA/g for NOTA and NODAGA conjugates, respectively) and high tumor-to-blood ratios (60 ± 10 and 220 ± 70, respectively) 3 h pi renders them promising PET tracers for use in patients. Tumor-to-normal organ ratios were higher for [ 68 Ga]Ga-NODAGA-PEG2-[Sar 11 ]RM26 than for the NOTA-containing counterpart. The performance of the two radiopeptides was further supported with the PET/CT images. In conclusion, [ 68 Ga]Ga-NODAGA-PEG2-[Sar 11 ]RM26 is a promising PET imaging tracer for visualization of GRPR-expressing lesions with high imaging contrast shortly after administration., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three 99m Tc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G 3 C), (Gly-Gly-Gly-Ser) 3 -Cys ((G 3 S) 3 C), or Glu-Glu-Glu-Cys (E 3 C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with 99m Tc(CO) 3 using the (HE) 3 -tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of 99m Tc-labeled DARPin G3 variants to HER2 in vitro; however, [ 99m Tc]Tc-G3-(G 3 S) 3 C-HYNIC had the highest affinity. Comparative biodistribution of [ 99m Tc]Tc-G3-G 3 C-HYNIC, [ 99m Tc]Tc-G3-(G 3 S) 3 C-HYNIC, [ 99m Tc]Tc-G3-E 3 C-HYNIC, and [ 99m Tc]Tc-(HE) 3 -G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [ 99m Tc]Tc-G3-E 3 C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [ 99m Tc]Tc-G3-(G 3 S) 3 C-HYNIC and [ 99m Tc]Tc-(HE) 3 -G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [ 99m Tc]Tc-G3-(G 3 S) 3 C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [ 99m Tc]Tc-(HE) 3 -G3. Radiolabeling of DARPin G3-HYNIC conjugates with 99m Tc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the 99m Tc-tricarbonyl-labeled DARPin G3. At this stage, [ 99m Tc]Tc-(HE) 3 -G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.