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Previously, we designed the EuK-based PSMA ligand BQ0413 with an maE3 chelator for labeling with technetium-99m. It showed efficient tumor targeting, but our preclinical data and preliminary clinical results indicated that the renal excretion levels need to be decreased. We hypothesized that this could be achieved by a decrease in the ligand's total negative charge, achieved by substituting negatively charged glutamate residues in the chelator with glycine. The purpose of this study was to evaluate the tumor targeting and biodistribution of two new PSMA inhibitors, BQ0411 and BQ0412, compared to BQ0413. Conjugates were radiolabeled with Tc-99m and characterized in vitro, using PC3-pip cells, and in vivo, using NMRI and PC3-pip tumor-bearing mice. [99mTc]Tc-BQ0411 and [99mTc]Tc-BQ0412 demonstrated PSMA-specific binding to PC3-pip cells with picomolar affinity. The biodistribution pattern for the new conjugates was characterized by rapid excretion. The tumor uptake for [99mTc]Tc-BQ0411 was 1.6-fold higher compared to [99mTc]Tc-BQ0412 and [99mTc]Tc-BQ0413. [99mTc]Tc-BQ0413 has demonstrated predominantly renal excretion, while the new conjugates underwent both renal and hepatobiliary excretion. In this study, we have demonstrated that in such small targeting ligands as PSMA-binding EuK-based pseudopeptides, the structural blocks that do not participate in binding could have a crucial role in tumor targeting and biodistribution. The presence of a glycine-based coupling linker in BQ0411 and BQ0413 seems to optimize biodistribution. In conclusion, the substitution of amino acids in the chelating sequence is a promising method to alter the biodistribution of [99mTc]Tc-labeled small-molecule PSMA inhibitors. Further improvement of the biodistribution properties of BQ0413 is needed. [ABSTRACT FROM AUTHOR]

Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC50 value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound's metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn2+ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding. [ABSTRACT FROM AUTHOR]

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]

Introduction: Prostate specific membrane antigen (PSMA), highly expressed in metastatic castration-resistant prostate cancer (mCRPC), is an established therapeutic target. Theranostic PSMA-targeting agents are widely used in patient management and has shown improved outcomes for mCRPC patients. Earlier, we optimized a urea-based probe for radionuclide visualization of PSMAexpression in vivo using computer modeling. With the purpose to develop a targeting agent equally suitable for radionuclide imaging and therapy, the agent containing DOTA chelator was designed (BQ7876). The aim of the study was to test the hypothesis that 177Lu-labeled BQ7876 possesses target binding and biodistribution properties potentially enabling its use for radiotherapy. Methods: BQ7876 was synthesized and labeled with Lu-177. Specificity and affinity of [177Lu]Lu-BQ7876 to PSMA-expressing PC3-pip cells was evaluated and its processing after binding to cells was studied. Animal studies in mice were performed to assess its biodistribution in vivo, target specificity and dosimetry. [177Lu]Lu-PSMA-617 was simultaneously evaluated for comparison. Results: BQ7876 was labeled with Lu-177 with radiochemical yield >99%. Its binding to PSMA was specific in vitro and in vivo when tested in antigen saturation conditions as well as in PSMA-negative PC-3 tumors. The binding of [177Lu]Lu-BQ7876 to living cells was characterized by rapid association, while the dissociation included a rapid and a slow phase with affinities KD1 = 3.8 nM and KD2 = 25 nM. The half-maximal inhibitory concentration for natLu-BQ7876 was 59 nM that is equal to 61 nM for natLu-PSMA-617. Cellular processing of [177Lu]Lu-BQ7876 was accompanied by slow internalization. [177Lu]Lu-BQ7876 was cleared from blood and normal tissues rapidly. Initial elevated uptake in kidneys decreased rapidly, and by 3 h post injection, the renal uptake (13 ± 3% Frontiers in ID/g) did not differ significantly from tumor uptake (9 ± 3%ID/g). Tumor uptake was stable between 1 and 3 h followed by a slow decline. The highest absorbed dose was in kidneys, followed by organs and tissues in abdomen. Discussion: Biodistribution studies in mice demonstrated that targeting properties of [177Lu]Lu-BQ7876 are not inferior to properties of [177Lu]Lu-PSMA-617, but do not offer any decisive advantages. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer continues to be the most frequently diagnosed form of cancer and the leading cause of cancer-related deaths among men. For a successful treatment plan and outcome, an early diagnosis, correct staging, and monitoring of treatment response are crucial. To improve this, a radiotracer could be used to target the two most abundant proteins overexpressed in prostate cancer: prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR). To date, no such heterodimeric radiotracer is used in the clinic. In this study, we have preclinically characterized and evaluated a galium-68 labelled PSMA/GRPR-targeting radiotracer for PET imaging of prostate cancer. We hope that the findings from this study will be able to contribute to designing better heterodimeric ligands, promote clinical translation of a heterodimer, and serve as a step towards a first-in-human study. The development of radioligands targeting prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) has shown promising results for the imaging and therapy of prostate cancer. However, studies have shown that tumors and metastases can express such targets heterogeneously. To overcome this issue and to improve protein binding, radioligands with the ability to bind both PSMA and GRPR have been developed. Herein, we present the preclinical characterization of [68Ga]Ga-BQ7812; a PSMA/GRPR-targeting radioligand for the diagnostic PET imaging of prostate cancer. This study aimed to evaluate [68Ga]Ga-BQ7812 to promote the translation of such imaging probes into the clinic. [68Ga]Ga-BQ7812 demonstrated rapid and specific binding to both targets in a PSMA/GRPR-expressing PC3-pip cell line. Results from the biodistribution study in PC3-pip xenografted mice showed specific binding to both targets, with the highest activity uptake at 1 h pi in tumor (PSMA+/GRPR+, 10.4 ± 1.0% IA/g), kidneys (PSMA+, 45 ± 16% IA/g), and pancreas (GRPR+, 5.6 ± 0.7% IA/g). At 3h pi, increased tumour-to-organ ratios could be seen due to higher retention in the tumor compared with other PSMA or GRPR-expressing organs. These results, together with low toxicity and an acceptable estimated dosimetry profile (total effective dose = 0.0083 mSv/MBq), support the clinical translation of [68Ga]Ga-BQ7812 and represent a step towards its first clinical trial. [ABSTRACT FROM AUTHOR]

Prostate-specific membrane antigen (PSMA) is overexpressed in the majority of prostate cancer cells and is considered to be an important target for the molecular imaging and therapy of prostate cancer. Herein, we present the design, synthesis, and evaluation of 11 PSMA-binding radioligands with modified linker structures, focusing on the relationship between molecular structure and targeting properties. The linker design was based on 2-naphthyl-L-alanine-tranexamic acid, the linker structure of PSMA-617. X-ray crystal-structure analysis of PSMA and structure-based design were used to generate the linker modifications, suggesting that substitution of tranexamic acid could lead to interactions with Phe546, Trp541, and Arg43 within the binding cavity. After synthesis through SPPS, analogues were labelled with indium-111 and evaluated in vitro for their specific binding, affinity, and cellular retention. Selected compounds were further evaluated in vivo in PSMA-expressing tumour-bearing mice. Based on the results, 2-naphthyl-L-alanine appears to be crucial for good targeting properties, whereas tranexamic acid could be replaced by other substituents. [111In]In-BQ7859, consisting of a 2-naphthyl-L-alanine-L-tyrosine linker, demonstrated favourable targeting properties. The substitution of tranexamic acid for L-tyrosine in the linker led to an improved tumour-to-blood ratio, highlighting [111In]In-BQ7859 as a promising PSMA-targeting radioligand. [ABSTRACT FROM AUTHOR]

Insulin‐regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non‐peptide IRAP inhibitors derived from a spiro‐oxindole dihydroquinazolinone screening hit (pIC50 5.8). The compounds were synthesized either by a simple microwave (MW)‐promoted three‐component reaction, or by a two‐step one‐pot procedure. For decoration of the oxindole ring system, rapid MW‐assisted Suzuki‐Miyaura cross‐couplings (1 min) were performed. A small improvement of potency (pIC50 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S‐configuration of the spiro‐oxindole dihydroquinazolinones accounts for the inhibition of IRAP. [ABSTRACT FROM AUTHOR]

Gastrin‐releasing peptide receptors (GRPRs) are overexpressed in prostate cancer and are suitable for targeted radionuclide therapy (TRT). We optimized the bombesin‐derived GRPR‐antagonist PEG2‐RM26 for labeling with 177Lu and further determined the effect of treatment with 177Lu‐labeled peptide alone or in combination with the anti‐HER2 antibody trastuzumab in a murine model. The PEG2‐RM26 analog was coupled to NOTA, NODAGA, DOTA and DOTAGA chelators. The peptide‐chelator conjugates were labeled with 177Lu and characterized in vitro and in vivo. A preclinical therapeutic study was performed in PC‐3 xenografted mice. Mice were treated with intravenous injections (6 cycles) of (A) PBS, (B) DOTAGA‐PEG2‐RM26, (C) 177Lu‐DOTAGA‐PEG2‐RM26, (D) trastuzumab or (E) 177Lu‐DOTAGA‐PEG2‐RM26 in combination with trastuzumab. 177Lu‐DOTAGA‐PEG2‐RM26 demonstrated quantitative labeling yield at high molar activity (450 GBq/μmol), high in vivo stability (5 min pi >98% of radioligand remained when coinjected with phosphoramidon), high affinity to GRPR (KD = 0.4 ± 0.2 nM), and favorable biodistribution (1 hr pi tumor uptake was higher than in healthy tissues, including the kidneys). Therapy with 177Lu‐DOTAGA‐PEG2‐RM26 induced a significant inhibition of tumor growth. The median survival for control groups was significantly shorter than for treated groups (Group C 66 days, Group E 74 days). Trastuzumab together with radionuclide therapy significantly improved survival. No treatment‐related toxicity was observed. In conclusion, based on in vitro and in vivo characterization of the four 177Lu‐labeled PEG2‐RM26 analogs, we concluded that 177Lu‐DOTAGA‐PEG2‐RM26 was the most promising analog for TRT. Radiotherapy using 177Lu‐DOTAGA‐PEG2‐RM26 effectively inhibited tumor growth in vivo in a murine prostate cancer model. Anti‐HER2 therapy additionally improved survival. What's new? Targeted radionuclide therapy (TRT) using radiolabeled peptides seeking gastrin‐releasing peptide receptors (GRPRs) in tumors is a promising approach to treat disseminated prostate cancer. The possibility to improve the therapeutic index via combination therapies also warrants further investigation. Here, the authors developed and characterized a promising GRPR‐targeting radioligand and demonstrated its therapeutic efficacy in prostate cancer xenografts. Moreover, this study using the anti‐HER2 antibody trastuzumab presents the first in vivo proof‐of‐principle that the effects of anti‐GRPR radiotherapy can be amplified by co‐administration of anti‐HER2 treatment leading to prolonged survival. [ABSTRACT FROM AUTHOR]

The feasibility of targeted imaging and therapy using radiolabeled albumin-binding domain-derived affinity proteins (ADAPTs) has been demonstrated. However, high renal uptake of radioactivity limits the maximum tolerated dose. Successful reduction of renal retention of radiolabeled Fab fragments has been demonstrated by incorporating a cleavable linker between the targeting agent and the radiometal chelator. The present study investigated if the introduction of a glycine-leucine-glycine-lysine (GLGK)-linker would reduce the kidney uptake of radiolabeled ADAPT6 and also compared it with the non-residualizing [125I]I-[(4-hydroxyphenyl)ethyl]maleimide ([125I]I-HPEM) labeling strategy. GLGK was site-specifically coupled to human epidermal growth factor receptor 2 (HER2)-targeting ADAPT6. Conjugates without the cleavable linker were used as controls and all constructs were labeled with lutetium-177 (177Lu). [125I]I-HPEM was coupled to ADAPT6 at the C-terminus. Biodistribution of all constructs was evaluated in NMRI mice 4 h after injection. Specific binding to HER2-expressing cells in vitro was demonstrated for all constructs. No significant difference in kidney uptake was observed between the [177Lu]Lu-2,2′,2",2"'-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid-GLGK-conjugates and the controls. The renal activity of [125I]I-HPEM-ADAPT6 was significantly lower compared with all other constructs. In conclusion, the incorporation of the cleavable GLGK-linker did not result in lower renal retention. Therefore, the present study emphasized that, in order to achieve a reduction of renal retention, alternative molecular design strategies may be required for different targeting agents. [ABSTRACT FROM AUTHOR]

Aims: Radiolabeled Exendin-4 has been proposed as suitable imaging marker for pancreatic beta cell mass quantification mediated by Glucagon-like peptide-1 receptor (GLP-1R). However, noticeable species variations in basal pancreatic uptake as well as uptake reduction degree due to selective beta cell ablation were observed. Methods: In vitro and ex vivo autoradiography studies of pancreas were performed using [Lu]Lu-DO3A-VS-Cys-Exendin4, in order to investigate the mechanism of uptake as well as the islet uptake contrast in mouse, rat, pig, and non-human primate. The autoradiography results were compared to the in vivo pancreatic uptake as assessed by [Ga]Ga-DO3A-VS-Cys-Exendin4 Positron Emission Tomography (PET) in the same species. In vitro, ex vivo, and in vivo data formed the basis for calculating the theoretical in vivo contribution of each pancreatic compartment. Results: [Lu]Lu-DO3A-VS-Cys-Exendin4 displayed the highest islet-to-exocrine pancreas ratio (IPR) in rat (IPR 45) followed by non-human primate and mouse at similar levels (IPR approximately 5) while pigs exhibited negligible IPR (1.1). In vivo pancreas uptake was mainly GLP-1R mediated in all species, but the magnitude of uptake under basal physiology varied significantly in decreasing order: non-human primate, mouse, pig, and rat. The theoretical calculation of islet contribution to the total pancreatic PET signal predicted the in vivo observation of differences in pancreatic uptake of [Ga]Ga-DO3A-VS-Cys-Exendin4. Conclusions: IPR as well as the exocrine GLP-1R density is the main determinants of the species variability in pancreatic uptake. Thus, the IPR in human is an important factor for assessing the potential of GLP-1R as an imaging biomarker for pancreatic beta cells. [ABSTRACT FROM AUTHOR]

High gastrin releasing peptide receptor (GRPR) expression is associated with numerous cancers including prostate and breast cancer. The aim of the current study was to develop a 55Co-labeled PET agent based on GRPR antagonist RM26 for visualization of GRPR-expressing tumors. Labeling with 57Co and 55Co, stability, binding specificity, and in vitro and in vivo characteristics of 57Co-NOTA-PEG2-RM26 were studied.NOTA-PEG2-RM26 was successfully radiolabeled with 57Co and 55Co with high yields and demonstrated high stability. The radiopeptide showed retained binding specificity to GRPR in vitro and in vivo. 57Co-NOTA-PEG2- RM26 biodistribution in mice was characterized by rapid clearance of radioactivity from blood and normal non-GRPR-expressing organs and low hepatic uptake. The clearance was predominantly renal with a low degree of radioactivity reabsorption. Tumor-toblood ratios were approximately 200 (3 h pi) and 1000 (24 h pi).The favorable biodistribution of cobalt-labeled NOTA-PEG2-RM26 translated into high contrast preclinical PET/CT (using 55Co) and SPECT/CT (using 57Co) images of PC-3 xenografts. The initial biological results suggest that 55Co-NOTA-PEG2-RM26 is a promising tracer for PET visualization of GRPR-expressing tumors. [ABSTRACT FROM AUTHOR]

Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes. The 384-well microplate-based absorbance assay was used to screen a diverse set of 10,500 compounds for their inhibitory capacity of IRAP. The assay performance was robust with Z′-values ranging from 0.81 to 0.91, and the screen resulted in 23 compounds that displayed greater than 60% inhibition at a compound concentration of 10 μM. After hit confirmation experiments, purity analysis, and promiscuity investigations, three structurally different compounds were considered particularly interesting as starting points for the development of small-molecule-based IRAP inhibitors. After resynthesis, all three compounds confirmed low μM activity and were shown to be rapidly reversible. Additional characterization included activity in a fluorescence-based orthogonal assay and in the presence of a nonionic detergent and a reducing agent, respectively. Importantly, the characterized compounds also showed inhibition of the human ortholog, prompting our further interest in these novel IRAP inhibitors. [ABSTRACT FROM AUTHOR]

The inhibition of insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) by angiotenesin IV is known to improve memory and learning in rats. Screening 10 500 low-molecular-weight compounds in an enzyme inhibition assay with IRAP from Chinese Hamster Ovary (CHO) cells provided an arylsulfonamide ( N-(3-(1 H-tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfonamide), comprising a tetrazole in the meta position of the aromatic ring, as a hit. Analogues of this hit were synthesized, and their inhibitory capacities were determined. A small structure-activity relationship study revealed that the sulfonamide function and the tetrazole ring are crucial for IRAP inhibition. The inhibitors exhibited a moderate inhibitory potency with an IC50=1.1±0.5 μ m for the best inhibitor in the series. Further optimization of this new class of IRAP inhibitors is required to make them attractive as research tools and as potential cognitive enhancers. [ABSTRACT FROM AUTHOR]

The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG2) spacer (NOTA-PEG2-RM26) and labeled with 68Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC50 values of natGa-NOTA-PEGn-RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68Ga-NOTA-PEG3-RM26 showed lower liver uptake. Biodistribution of 68Ga-NOTA-PEG3-RM26 was evaluated in nude mice bearing PC-3 (prostate cancer) and BT-474 (breast cancer) xenografts. High uptake in tumors (4.6 ± 0.6%ID/g and 2.8 ± 0.4%ID/g for PC-3 and BT-474 xenografts, respectively) and high tumor-to-background ratios (tumor/blood of 44 ± 12 and 42 ± 5 for PC-3 and BT-474 xenografts, respectively) were found already at 2 h p.i. of 68Ga-NOTA-PEG3-RM26. Results of this study suggest that variation in the length of the PEG spacer can be used for optimization of targeting properties of peptide-chelator conjugates. However, the influence of the mini-PEG length on biodistribution is minor when di-, tri-, tetra- and hexaethylene glycol are compared. [ABSTRACT FROM AUTHOR]

There is an unmet medical need for non-invasive, sensitive, and quantitative methods for the assessment of fibrosis. Herein, an improved collagelin analogue labelled with gallium-68 for use with positron emission tomography (PET) is presented. A cyclic peptide, c[CPGRVNleHGLHLGDDEGPC], was synthesized by solid-phase peptide synthesis, conjugated to 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid, and labelled with gallium-68. High performance liquid chromatography (HPLC) was used for the quality and stability assessment of the collagelin analogue. Non-specific organ distribution, blood clearance, and excretion rates were investigated in healthy mice and rats using ex vivo organ distribution analysis and dynamic in vivo PET/CT. Mice with carbon tetrachloride (CCl4) induced liver fibrosis were used for the investigation of specific binding via in vitro frozen section autoradiography, ex vivo organ distribution, and in vivo PET/CT. A non-decay corrected radiochemical yield (48 ± 6%) of [68Ga]Ga-NOTA-PEG2-c[CPGRVNleHGLHLGDDEGPC] ([68Ga]Ga-NO2A-[Nle13]-Col) with a radiochemical purity of 98 ± 2% was achieved without radical scavengers. The 68Ga-labelling was regioselective and stable at ambient temperature for at least 3 h. The autoradiography of the cryosections of fibrotic mouse liver tissue demonstrated a distinct heterogeneous radioactivity uptake that correlated with the fibrosis scores estimated after Sirius Red staining. The blood clearance and tissue washout from the [68Ga]Ga-NO2A-[Nle13]-Col was fast in both normal and diseased mice. Dosimetry investigation in rats indicated the possibility for 4–5 PET/CT examinations per year. Radiolytic stability of the collagelin analogue was achieved by the substitution of methionine with norleucine amino acid residue without a deterioration of its binding capability. [68Ga]Ga-NO2A-[Nle13]-Col demonstrated a safe dosimetry profile suitable for repeated scanning. [ABSTRACT FROM AUTHOR]

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are promising targets for molecular imaging of prostate cancer (PCa) lesions. Due to the heterogenic overexpression of PSMA and GRPR in PCa, a heterodimeric radiotracer with the ability to bind to both targets could be beneficial. Recently, our group reported the novel heterodimer BQ7800 consisting of a urea-based PSMA inhibitor, the peptide-based GRPR antagonist RM26 and NOTA chelator. The study reported herein, aimed to improve the affinity of BQ7800 towards PSMA by changing the composition of the two linkers connecting the PSMA- and GRPR-targeting motifs. Three novel heterodimeric analogues were synthesized by incorporation of phenylalanine in the functional linker of the PSMA-binding motif and/or shortening the PEG-linker coupled to RM26. The heterodimers were labeled with indium-111 and evaluated in vitro. In the competitive binding assay, BQ7812, featuring phenylalanine and shorter PEG-linker, demonstrated a nine-fold improved affinity towards PSMA. In the in vivo biodistribution study of [111In]In-BQ7812 in PC3-pip tumor-bearing mice (PSMA and GRPR positive), the activity uptake was two-fold higher in the tumor and three-fold higher in kidneys than for [111In]In-BQ7800. Herein, we showed that the affinity of a bispecific PSMA/GRPR heterodimer towards PSMA could be improved by linker modification. [ABSTRACT FROM AUTHOR]

Gastrin-releasing peptide receptors (GRPRs) are promising targets in oligometastatic prostate cancer. We have recently used 55Co (T1/2 = 17.5 h) as a label for next day PET imaging of GRPR expression obtaining high imaging contrast. The radionuclide-chelator combination can significantly influence the biodistribution of radiopeptides. Therefore, in this study, we hypothesized that the properties of 55Co-labeled PEG2-RM26 can be improved by identifying the optimal macrocyclic chelator. All analogues (X-PEG2-RM26, X = NOTA,NODAGA,DOTA,DOTAGA) were successfully labeled with radiocobalt with high yields and demonstrated high stability. The radiopeptides bound specifically and with picomolar affinity to GRPR and their cellular processing was characterized by low internalization. The best binding capacity was found for DOTA-PEG2-RM26. Ex vivo biodistribution in PC-3 xenografted mice was characterized by rapid blood clearance via renal excretion. Tumor uptake was similar for all conjugates at 3 h pi, exceeding the uptake in all other organs. Higher kidney uptake and longer retention were associated with N-terminal negative charge (DOTAGA-containing conjugate). Tumor-to-organ ratios increased over time for all constructs, although significant chelator-dependent differences were observed. Concordant with affinity measurements, DOTA-analog had the best retention of activity in tumors, resulting in the highest tumor-to-blood ratio 24 h pi, which translated into high contrast PET/CT imaging (using 55Co). [ABSTRACT FROM AUTHOR]

Simultaneous targeting of the prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) could improve the diagnostic accuracy in prostate cancer (PCa). The aim of this study was to develop a PSMA/GRPR-targeting bispecific heterodimer for SPECT and positron emission tomography (PET) diagnostic imaging of PCa. The heterodimer NOTA-DUPA-RM26 was produced by manual solid-phase peptide synthesis. NOTA-DUPA-RM26 was labeled with 111In and 68Ga, with yields >98%, and demonstrated a high stability and binding specificity to PSMA and GRPR. IC50 values for natIn-NOTA-DUPA-RM26 were 4 ± 1 nM towards GRPR and 824 ± 230 nM towards PSMA. An in vivo binding specificity 1 h pi of 111In-NOTA-DUPA-RM26 in PC3-PIP-xenografted mice demonstrated partially blockable tumor uptake when co-injected with an excess of PSMA- or GRPR-targeting agents. Simultaneous co-injection of both agents induced pronounced blocking. The biodistribution of 111In-NOTA-DUPA-RM26 and 68Ga-NOTA-DUPA-RM26 revealed fast activity clearance from the blood and normal organs via the kidneys. Tumor uptake exceeded normal organ uptake for both analogs 1 h pi. 68Ga-NOTA-DUPA-RM26 had a significantly lower tumor uptake (8 ± 2%ID/g) compared to 111In-NOTA-DUPA-RM26 (12 ± 2%ID/g) 1 h pi. Tumor-to-organ ratios increased 3 h pi, but decreased 24 h pi, for 111In-NOTA-DUPA-RM26. MicroPET/CT and microSPECT/CT scans confirmed biodistribution data, suggesting that 68Ga-NOTA-DUPA-RM26 and 111In-NOTA-DUPA-RM26 are suitable candidates for the imaging of GRPR and PSMA expression in PCa shortly after administration. [ABSTRACT FROM AUTHOR]

Radiolabelled antagonistic bombesin analogues are successfully used for targeting of gastrin-releasing peptide receptors (GRPR) that are overexpressed in prostate cancer. Internalization of antagonistic bombesin analogues is slow. We hypothesized that the use of a non-residualizing radioiodine label might not affect the tumour uptake but would reduce the retention in normal organs, where radiopharmaceutical would be internalized. To test this hypothesis, tyrosine was conjugated via diethylene glycol linker to N-terminus of an antagonistic bombesin analogue RM26 to form Tyr-PEG2-RM26. [111In]In-DOTA-PEG2-RM26 was used as a control with a residualizing label. Tyr-PEG2-RM26 was labelled with 125I with 95% radiochemical purity and retained binding specificity to GRPR. The IC50 values for Tyr-PEG2-RM26 and DOTA-PEG2-RM26 were 1.7 ± 0.3 nM and 3.3 ± 0.5 nM, respectively. The cellular processing of [125I]I-Tyr-PEG2-RM26 by PC-3 cells showed unusually fast internalization. Biodistribution showed that uptake in pancreas and tumour was GRPR-specific for both radioconjugates. Blood clearance of [125I]I-Tyr-PEG2-RM26 was appreciably slower and activity accumulation in all organs was significantly higher than for [111In]In-DOTA-PEG2-RM26. Tumor uptake of [111In]In-DOTA-PEG2-RM26 was significantly higher than for [125I]I-Tyr-PEG2-RM26, resulting in higher tumour-to-organ ratio for [111In]In-DOTA-PEG2-RM26 at studied time points. Incorporation of amino acids with hydrophilic side-chains next to tyrosine might overcome the problems associated with the use of tyrosine as a prosthetic group for radioiodination. [ABSTRACT FROM AUTHOR]

Gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) are overexpressed in most prostate cancers. GRPR expression is higher in early stages while PSMA expression increases with progression. The possibility of targeting both markers with a single theranostics radiotracer could improve patient management. Three GRPR/PSMA-targeting bispecific heterodimers (urea derivative PSMA-617 and bombesin-based antagonist RM26 linked via X-triazolyl-Tyr-PEG2, X = PEG2 (BO530), (CH2)8 (BO535), none (BO536)) were synthesized by solid-phase peptide synthesis. Peptides were radio-iodinated and evaluated in vitro for binding specificity, cellular retention, and affinity. In vivo specificity for all heterodimers was studied in PC-3 (GRPR-positive) and LNCaP (PSMA-positive) xenografts. [125I]I-BO530 was evaluated in PC-3pip (GRPR/PSMA-positive) xenografts. Micro single-photon emission computed tomography/computed tomography (microSPECT/CT) scans were acquired. The heterodimers were radiolabeled with high radiochemical yields, bound specifically to both targets, and demonstrated high degree of activity retention in PC-3pip cells. Only [125I]I-BO530 demonstrated in vivo specificity to both targets. A biodistribution study of [125I]I-BO530 in PC-3pip xenografted mice showed high tumor activity uptake (30%–35%ID/g at 3 h post injection (pi)). Activity uptake in tumors was stable and exceeded all other organs 24 h pi. Activity uptake decreased only two-fold 72 h pi. The GRPR/PSMA-targeting heterodimer [125I]I-BO530 is a promising agent for theranostics application in prostate cancer. [ABSTRACT FROM AUTHOR]