5-Hydroxypyrroloindoline Affords Tryptathionine and 2,2'-bis-Indole Peptide Staples: Application to Melanotan-II.

With peptides increasingly favored as drugs, natural product motifs, namely the tryptathionine staple, found in amatoxins and phallotoxins, and the 2,2'-bis-indole found in staurosporine represent unexplored staples for unnatural peptide macrocycles. We disclose the efficient condensation of a 5-hydroxypyrroloindoline with either a cysteine-thiol or a tryptophan-indole to form a tryptathionine or 2-2'-bis-indole staple. Judicious use of protecting groups provides for chemoselective stapling using α-MSH, which provides a basis for investigating both chemoselectivity and affinity. Both classes of stapled peptides show nanomolar Ki's, with one showing a sub-nanomolar Ki value.

68Ga-Labeled [Thz14]Bombesin(7-14) Analogs: Promising GRPR-Targeting Agonist PET Tracers with Low Pancreas Uptake.

With overexpression in various cancers, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer imaging and therapy. However, the high pancreas uptake of reported GRPR-targeting radioligands limits their clinical application. Our goal was to develop 68Ga-labeled agonist tracers for detecting GRPR-expressing tumors with positron emission tomography (PET), and compare them with the clinically validated agonist PET tracer, [68Ga]Ga-AMBA. Ga-TacBOMB2, TacBOMB3, and TacBOMB4, derived from [Thz14]Bombesin(7-14), were confirmed to be GRPR agonists by a calcium mobilization study, and their binding affinities (Ki(GRPR)) were determined to be 7.62 ± 0.19, 6.02 ± 0.59, and 590 ± 36.5 nM, respectively, via in vitro competition binding assays. [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA clearly visualized PC-3 tumor xenografts in a PET imaging study. [68Ga]Ga-TacBOMB2 showed comparable tumor uptake but superior tumor-to-background contrast ratios when compared to [68Ga]Ga-AMBA. Moreover, [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 showed a much lower rate of uptake in the pancreas (1.30 ± 0.14 and 2.41 ± 0.72%ID/g, respectively) than [68Ga]Ga-AMBA (62.4 ± 4.26%ID/g). In conclusion, replacing Met14 in the GRPR-targeting sequence with Thz14 retains high GRPR-binding affinity and agonist properties. With good tumor uptake and tumor-to-background uptake ratios, [68Ga]Ga-TacBOMB2 is promising for detecting GRPR-expressing tumors. The much lower pancreas uptake of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 suggests that [Thz14]Bombesin(7-14) is a promising targeting vector for the design of GRPR-targeting radiopharmaceuticals, especially for radioligand therapy application.

Synthesis and Evaluation of 68Ga-Labeled (2S,4S)-4-Fluoropyrrolidine-2-Carbonitrile and (4R)-Thiazolidine-4-Carbonitrile Derivatives as Novel Fibroblast Activation Protein-Targeted PET Tracers for Cancer Imaging.

Fibroblast activation protein α (FAP-α) is a cell-surface protein overexpressed on cancer-associated fibroblasts that constitute a substantial component of tumor stroma and drive tumorigenesis. FAP is minimally expressed by most healthy tissues, including normal fibroblasts. This makes it a promising pan-cancer diagnostic and therapeutic target. In the present study, we synthesized two novel tracers, [68Ga]Ga-SB03045 and [68Ga]Ga-SB03058, bearing a (2S,4S)-4-fluoropyrrolidine-2-carbonitrile or a (4R)-thiazolidine-4-carbonitrile pharmacophore, respectively. [68Ga]Ga-SB03045 and [68Ga]Ga-SB03058 were evaluated for their FAP-targeting capabilities using substrate-based in vitro binding assays, and in PET/CT imaging and ex vivo biodistribution studies in an HEK293T:hFAP tumor xenograft mouse model. The IC50 values of natGa-SB03045 (1.59 ± 0.45 nM) and natGa-SB03058 (0.68 ± 0.09 nM) were found to be lower than those of the clinically validated natGa-FAPI-04 (4.11 ± 1.42 nM). Contrary to the results obtained in the FAP-binding assay, [68Ga]Ga-SB03058 demonstrated a ~1.5 fold lower tumor uptake than that of [68Ga]Ga-FAPI-04 (7.93 ± 1.33 vs. 11.90 ± 2.17 %ID/g), whereas [68Ga]Ga-SB03045 (11.8 ± 2.35 %ID/g) exhibited a tumor uptake comparable to that of [68Ga]Ga-FAPI-04. Thus, our data suggest that the (2S,4S)-4-fluoropyrrolidine-2-carbonitrile scaffold holds potential as a promising pharmacophore for the design of FAP-targeted radioligands for cancer diagnosis and therapy.

Synthesis and Preclinical Evaluation of Three Novel 68Ga-Labeled Bispecific PSMA/FAP-Targeting Tracers for Prostate Cancer Imaging.

Tumor heterogeneity limits the efficacy and reliability of monospecific radiopharmaceuticals in prostate cancer diagnosis and therapy. To overcome this limitation and improve lesion detection sensitivity, we developed and evaluated three bispecific radiotracers that can target both prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are the two key proteins overexpressed in prostate cancer. Three FAP-targeting ligands with various linker lengths were synthesized through multistep organic synthesis, and then connected to the PSMA-targeting motif. IC50(PSMA) and IC50(FAP) values of Ga-complexed bispecific ligands, Ga-AV01017, Ga-AV01030, and Ga-AV01038 were 25.2-71.6 and 1.25-2.74 nM, respectively. The uptake values in PSMA-expressing LNCaP tumor xenografts were 4.38 ± 0.55, 5.17 ± 0.51, and 4.25 ± 0.86 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were lower than the monospecific PSMA-targeting tracer [68Ga]Ga-HTK03041 (23.1 ± 6.11 %ID/g). The uptake values in FAP-expressing HEK293T:hFAP tumor xenografts were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were also lower than the monospecific FAP-targeting tracer, [68Ga]Ga-FAPI-04 (12.5 ± 2.00 %ID/g). We observed that the bispecific tracers had prolonged blood retention, in which tracers with a longer linker tend to have a higher blood uptake and lower tumor uptake. Further investigations are needed to optimize the linker selection to generate promising bispecific PSMA/FAP-targeting tracers for prostate cancer imaging.

[213Bi]Bi3+/[111In]In3+-neunpa-cycMSH: Theranostic Radiopharmaceutical Targeting Melanoma─Structural, Radiochemical, and Biological Evaluation.

With the emergence of [225Ac]Ac3+ as a therapeutic radionuclide for targeted α therapy (TAT), access to clinical quantities of the potent, short-lived α-emitter [213Bi]Bi3+ (t1/2 = 45.6 min) will increase over the next decade. With this in mind, the nonadentate chelator, H4neunpa-NH2, has been investigated as a ligand for chelation of [213Bi]Bi3+ in combination with [111In]In3+ as a suitable radionuclidic pair for TAT and single photon emission computed tomography (SPECT) diagnostics. Nuclear magnetic resonance (NMR) spectroscopy was utilized to assess the coordination characteristics of H4neunpa-NH2 on complexation of [natBi]Bi3+, while the solid-state structure of [natBi][Bi(neunpa-NH3)] was characterized via X-ray diffraction (XRD) studies, and density functional theory (DFT) calculations were performed to elucidate the conformational geometries of the metal complex in solution. H4neunpa-NH2 exhibited fast complexation kinetics with [213Bi]Bi3+ at RT achieving quantitative radiolabeling within 5 min at 10-8 M ligand concentration, which was accompanied by the formation of a kinetically inert complex. Two bioconjugates incorporating the melanocortin 1 receptor (MC1R) targeting peptide Nle-CycMSHhex were synthesized featuring two different covalent linkers for in vivo evaluation with [213Bi]Bi3+ and [111In]In3+. High molar activities of 7.47 and 21.0 GBq/µmol were achieved for each of the bioconjugates with [213Bi]Bi3+. SPECT/CT scans of the [111In]In3+-labeled tracer showed accumulation in the tumor over time, which was accompanied by high liver uptake and clearance via the hepatic pathway due to the high lipophilicity of the covalent linker. In vivo biodistribution studies in C57Bl/6J mice bearing B16-F10 tumor xenografts showed good tumor uptake (5.91% ID/g) at 1 h post-administration with [213Bi][Bi(neunpa-Ph-Pip-Nle-CycMSHhex)]. This study demonstrates H4neunpa-NH2 to be an effective chelating ligand for [213Bi]Bi3+ and [111In]In3+, with promising characteristics for further development toward theranostic applications.

H3TPAN-Triazole-Bn-NH2: Tripicolinate Clicked-Bifunctional Chelate for [225Ac]/[111In] Theranostics.

A new, high-denticity, bifunctional ligand─H3TPAN-triazole-Bn-NH2─has been synthesized and studied in complexation with [225Ac]Ac3+ and [111In]In3+ for radiopharmaceutical applications. The bifunctional chelator is readily synthesized, using a high-yielding four-step prep, which is highly adaptable and allows for straightforward incorporation of different covalent linkers using CuI-catalyzed alkyne-azide cycloaddition (click) chemistry. Nuclear magnetic resonance (NMR) studies of H3TPAN-triazole-Bn-NH2 with La3+ and In3+ metal ions show the formation of a single, asymmetric complex with each ion in solution, corroborated by density functional theory (DFT) calculations. Radiolabeling studies with [225Ac]Ac3+ and [111In]In3+ showed highly effective complexation, achieving quantitative radiochemical conversions at low ligand concentrations (<10-6 M) under mild conditions (RT, 10 min), which is further accompanied by high stability in human serum. The bioconjugate─H3TPAN-triazole-Bn-Aoc-Pip-Nle-CycMSHhex─was prepared for targeting of MC1R-positive tumors, and the corresponding 111In-radiolabeled tracer was studied in vivo. SPECT/CT and biodistribution studies in C57BL/6J mice bearing B16-F10 tumors were performed, with the radiotracer showing good in vivo stability; tumor uptake was achieved. This work highlights a new promising and versatile bifunctional chelator, easily prepared and encouraging for 225Ac/111In theranostics.

H4picoopa─Robust Chelate for 225Ac/111In Theranostics.

The nuclear decay characteristics of 225Ac (Eα = 5-8 MeV, linear energy transfer (LET) = ∼100 keV/µm, t1/2 = 9.92 days) are well recognized as advantageous for the treatment of primary and metastatic tumors; however, suitable chelation systems are required, which can accommodate this radiometal. Since 225Ac does not possess any suitable low-energy, high abundance γ-ray emissions for nuclear imaging, there is a clear need for the development of other companion radionuclides with similar coordination characteristics and comparable half-lives, which can be applied in diagnostics. H4picoopa was designed and executed as a high-denticity ligand for chelation of [225Ac]Ac3+, and the complexation characteristics have been explored through nuclear magnetic resonance (NMR) spectroscopy, solution thermodynamic stability studies, and radiolabeling. The ligand shows highly favorable complexation with La3+ (pM = 17.6), Lu3+ (pM = 21.3), and In3+ (pM = 31.2) and demonstrates effective radiolabeling of both [225Ac]Ac3+ and [111In]In3+ ions achieving quantitative radiochemical conversions (RCCs) under mild conditions (RT, 10 min), accompanied by high serum stability (>97% radiochemical purity (RCP) over 6 days). A bifunctional analogue of H4picoopa was synthesized and conjugated to the Pip-Nle-CycMSHhex peptide for targeting of MC1R positive melanoma tumors. In vivo single-photon emission computed tomography (SPECT) and biodistribution studies of the 111In-radiolabeled bioconjugate in mice bearing B16-F10 tumors showed good radiotracer stability, although improved tumor targeting could not be achieved for imaging purposes. This work highlights H4picoopa as a very promising platform for application of [225Ac]Ac3+ and [111In]In3+ as a theranostic pair and allows great versatility for the incorporation of other directing vectors. The logical synthetic approach reported here for bifunctional H4picoopa, involving an azide-functionalized covalent linker and CuI-catalyzed alkyne-azide cycloaddition, allows for ease of optimization of bioconjugate pharmacokinetics and will be valuable for further radiopharmaceutical applications moving forward.

68Ga-Labeled [Leu13ψThz14]Bombesin(7-14) Derivatives: Promising GRPR-Targeting PET Tracers with Low Pancreas Uptake.

The gastrin-releasing peptide receptor (GRPR) is a G-protein-coupled receptor that is overexpressed in many solid cancers and is a promising target for cancer imaging and therapy. However, high pancreas uptake is a major concern in the application of reported GRPR-targeting radiopharmaceuticals, particularly for targeted radioligand therapy. To lower pancreas uptake, we explored Ga-complexed TacsBOMB2, TacsBOMB3, TacsBOMB4, TacsBOMB5, and TacsBOMB6 derived from a potent GRPR antagonist sequence, [Leu13ψThz14]Bombesin(7-14), and compared their potential for cancer imaging with [68Ga]Ga-RM2. The Ki(GRPR) values of Ga-TacsBOMB2, Ga-TacsBOMB3, Ga-TacsBOMB4, Ga-TacsBOMB5, Ga-TacsBOMB6, and Ga-RM2 were 7.08 ± 0.65, 4.29 ± 0.46, 458 ± 38.6, 6.09 ± 0.95, 5.12 ± 0.57, and 1.51 ± 0.24 nM, respectively. [68Ga]Ga-TacsBOMB2, [68Ga]Ga-TacsBOMB3, [68Ga]Ga-TacsBOMB5, [68Ga]Ga-TacsBOMB6, and [68Ga]Ga-RM2 clearly show PC-3 tumor xenografts in positron emission tomography (PET) images, while [68Ga]Ga-TacsBOMB5 shows the highest tumor uptake (15.7 ± 2.17 %ID/g) among them. Most importantly, the pancreas uptake values of [68Ga]Ga-TacsBOMB2 (2.81 ± 0.78 %ID/g), [68Ga]Ga-TacsBOMB3 (7.26 ± 1.00 %ID/g), [68Ga]Ga-TacsBOMB5 (1.98 ± 0.10 %ID/g), and [68Ga]Ga-TacsBOMB6 (6.50 ± 0.36 %ID/g) were much lower than the value of [68Ga]Ga-RM2 (41.9 ± 10.1 %ID/g). Among the tested [Leu13ψThz14]Bombesin(7-14) derivatives, [68Ga]Ga-TacsBOMB5 has the highest tumor uptake and tumor-to-background contrast ratios, which is promising for clinical translation to detect GRPR-expressing tumors. Due to the low pancreas uptake of its derivatives, [Leu13ψThz14]Bombesin(7-14) represents a promising pharmacophore for the design of GRPR-targeting radiopharmaceuticals, especially for targeted radioligand therapy application.

High-Contrast CXCR4-Targeted 18F-PET Imaging Using a Potent and Selective Antagonist.

C-X-C chemokine receptor 4 (CXCR4) is highly expressed in cancers, contributing to proliferation, metastasis, and a poor prognosis. The noninvasive imaging of CXCR4 can enable the detection and characterization of aggressive cancers with poor outcomes. Currently, no 18F-labeled CXCR4 positron emission tomography (PET) radiotracer has demonstrated imaging contrast comparable to [68Ga]Ga-Pentixafor, a CXCR4-targeting radioligand. We, therefore, aimed to develop a high-contrast CXCR4-targeting radiotracer by incorporating a hydrophilic linker and trifluoroborate radioprosthesis to LY2510924, a known CXCR4 antagonist. A carboxy-ammoniomethyl-trifluoroborate (PepBF3) moiety was conjugated to the LY2510924-derived peptide possessing a triglutamate linker via amide bond formation to obtain BL08, whereas an alkyne ammoniomethyl-trifluoroborate (AMBF3) moiety was conjugated using the copper-catalyzed [3+2] cycloaddition click reaction to obtain BL09. BL08 and BL09 were radiolabeled with [18F]fluoride ion using 18F-19F isotope exchange. Pentixafor was radiolabeled with [68Ga]GaCl3. Side-by-side PET imaging and biodistribution studies were performed on immunocompromised mice bearing Daudi Burkitt lymphoma xenografts. The biodistribution of [18F]BL08 and [18F]BL09 showed tumor uptake at 2 h postinjection (p.i.) (5.67 ± 1.25%ID/g and 5.83 ± 0.92%ID/g, respectively), which were concordant with the results of PET imaging. [18F]BL08 had low background activity, providing tumor-to-blood, -muscle, and -liver ratios of 72 ± 20, 339 ± 81, and 14 ± 3 (2 h p.i.), respectively. [18F]BL09 behaved similarly, with ratios of 64 ± 20, 239 ± 72, and 17 ± 3 (2 h p.i.), respectively. This resulted in high-contrast visualization of tumors on PET imaging for both radiotracers. [18F]BL08 exhibited lower kidney uptake (2.2 ± 0.5%ID/g) compared to [18F]BL09 (7.6 ± 1.0%ID/g) at 2 h p.i. [18F]BL08 and [18F]BL09 demonstrated higher tumor-to-blood, -muscle, and -liver ratios compared to [68Ga]Ga-Pentixafor (18.9 ± 2.7, 95.4 ± 36.7, and 5.9 ± 0.7 at 2 h p.i., respectively). In conclusion, [18F]BL08 and [18F]BL09 enable high-contrast visualization of CXCR4 expression in Daudi xenografts. Based on high tumor-to-organ ratios, [18F]BL08 may prove a valuable new tool for CXCR4-targeted PET imaging with potential for translation. The use of a PepBF3 moiety is a new approach for the orthogonal conjugation of organotrifluoroborates for 18F-labeling of peptides.

Comparison of biological properties of [177 Lu]Lu-ProBOMB1 and [177 Lu]Lu-NeoBOMB1 for GRPR targeting.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate cancer and other solid malignancies. Following up on our work on [68 Ga]Ga-ProBOMB1 that had better imaging characteristics than [68 Ga]Ga-NeoBOMB1, we investigated the effects of substituting 68 Ga for 177 Lu to determine if the resulting radiopharmaceuticals could be used with a therapeutic aim. We radiolabeled the bombesin antagonist ProBOMB1 (DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH2 ) with lutetium-177 and compared it with [177 Lu]Lu-NeoBOMB1 (obtained in 54.2 ± 16.5% isolated radiochemical yield with >96% radiochemical purity and 440.8 ± 165.1 GBq/µmol molar activity) for GRPR targeting. Lu-NeoBOMB1 had better binding affinity for GRPR than Lu-ProBOMB1 (Ki values: 2.26 ± 0.24 and 30.2 ± 3.23nM). [177 Lu]Lu-ProBOMB1 was obtained in 53.7 ± 5.4% decay-corrected radiochemical yield with 444.2 ± 193.2 GBq/µmol molar activity and >95% radiochemical purity. In PC-3 prostate cancer xenograft mice, tumor uptake of [177 Lu]Lu-ProBOMB1 was 3.38 ± 1.00, 1.32 ± 0.24, and 0.31 ± 0.04%ID/g at 1, 4, and 24 hours pi. However, the uptake in tumor was lower than [177 Lu]Lu-NeoBOMB1 at all time points. [177 Lu]Lu-ProBOMB1 was inferior to [177 Lu]Lu-NeoBOMB1, which had better therapeutic index for the organs receiving the highest doses.

[68Ga]Ga/[177Lu]Lu-BL01, a Novel Theranostic Pair for Targeting C-X-C Chemokine Receptor 4.

C-X-C chemokine receptor type 4 (CXCR4) is overexpressed in hematological and solid malignancies. LY2510924 is a potent peptide antagonist of CXCR4. A derivative of LY2510924, BL01, was evaluated for theranostic applications targeting CXCR4. Methods: BL01 was synthesized by solid phase approach. A Lys(ivDde) residue was added at the C-terminus of LY2510924 (cyclo[Phe-Tyr-Lys(iPr)-d-Arg-2-Nal-Gly-d-Glu]-Lys(iPr)-NH2). A DOTA chelator was conjugated to the side chain of the deprotected exogenous Lys residue. The binding affinity of Ga/Lu-BL01 was determined by competitive radioligand binding assays. BL01 was radiolabeled with 68GaCl3 or 177LuCl3. Biodistribution studies were performed in mice bearing Daudi Burkitt's lymphoma tumor xenografts at selected time points. PET imaging studies were performed with [68Ga]Ga-BL01, with blocking experiments performed with preinjection of LY2510924. The stability of [68Ga]Ga/[177Lu]Lu-BL01 was assessed in mouse plasma. Results: Ga-BL01 and Lu-BL01 have nanomolar affinity for CXCR4. [68Ga]Ga-BL01 was obtained in 58 ± 5% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 40 ± 11 GBq/µmol, while [177Lu]Lu-BL01 was obtained in 65 ± 6% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 120 ± 21 GBq/µmol. [68Ga]Ga-BL01 and [177Lu]Lu-BL01 were excreted primarily through the renal pathway. Daudi xenografts were clearly delineated in PET images with good contrast. On the basis of biodistribution data, tumor uptake of [68Ga]Ga-BL01 was 10.2 ± 2.56% injected dose per gram (%ID/g) at 1 h postinjection (p.i.). Spleen (12.6 ± 2.36 %ID/g) and lungs (13.2 ± 2.98 %ID/g), organs that express CXCR4, had high uptake as well. Preinjection of LY2510924 reduced average uptake of [68Ga]Ga-BL01 in tumors by 88%, demonstrating target specificity. The uptake of [68Ga]Ga-BL01 in tumor increased to 15.3 ± 1.86 %ID/g at 2 h p.i., with improved contrast. [177Lu]Lu-BL01 has similar pharmacokinetics as [68Ga]Ga-BL01 at 1 h p.i. The highest uptake was observed in tumor (14.0 ± 1.11 %ID/g), followed by the lungs (13.0 ± 1.27 %ID/g) and spleen (11.6 ± 1.78 %ID/g). The tumor uptake increased to 16.2 ± 2.69 %ID/g at 4 h p.i., before declining slightly to 10.1 ± 1.41 %ID/g at 24 h p.i. Both compounds were stable in vivo, as no metabolites were observed at 5 min p.i. Conclusions: [68Ga]Ga-BL01 and [177Lu]Lu-BL01 are a promising theranostic pair for imaging and endoradiotherapy of CXCR4-expressing malignancies.

Fluorescent Isoindole Crosslink (FlICk) Chemistry: A Rapid, User-friendly Stapling Reaction.

The stabilization of peptide secondary structure via stapling is a ubiquitous goal for creating new probes, imaging agents, and drugs. Inspired by indole-derived crosslinks found in natural peptide toxins, we employed ortho-phthalaldehydes to create isoindole staples, thus transforming inactive linear and monocyclic precursors into bioactive monocyclic and bicyclic products. Mild, metal-free conditions give an array of macrocyclic α-melanocyte-stimulating hormone (α-MSH) derivatives, of which several isoindole-stapled α-MSH analogues (Ki ≈1 nm) are found to be as potent as α-MSH. Analogously, late-stage intra-annular isoindole stapling furnished a bicyclic peptide mimic of α-amanitin that is cytotoxic to CHO cells (IC50 =70 µm). Given its user-friendliness, we have termed this approach FlICk (fluorescent isoindole crosslink) chemistry.

Melanoma Imaging Using 18F-Labeled α-Melanocyte-Stimulating Hormone Derivatives with Positron Emission Tomography.

Melanocortin 1 receptor (MC1R) is specifically expressed in the majority of melanomas, a leading cause of death related to skin cancers. Accurate staging and early detection is crucial in managing melanoma. Based on the α-melanocyte-stimulating hormone (αMSH) sequence, MC1R-targeted peptides have been studied for melanoma imaging, predominately for use with single-photon emission computed tomography, with few attempts made for positron emission tomography (PET). 18F is a commonly used PET isotope due to readily available cyclotron production, pure positron emission, and a favorable half-life (109.8 min). In this study, we aim to design and evaluate αMSH derivatives that enable radiolabeling with 18F for PET imaging of melanoma. We synthesized three imaging probes based on the structure of Nle4-cyclo[Asp5-His-d-Phe7-Arg-Trp-Lys10]-NH2 (Nle-CycMSHhex), with a Pip linker (CCZ01064), an Acp linker (CCZ01070), or an Aoc linker (CCZ01071). 18F labeling was enabled by an ammoniomethyl-trifluoroborate (AmBF3) moiety. In vitro competition binding assays showed subnanomolar inhibition constant ( Ki) values for all three peptides. The 18F radiolabeling was performed via a one-step 18F-19F isotope exchange reaction that resulted in high radiochemical purity (>95%) and good molar activity (specific activity) ranging from 40.7 to 66.6 MBq/nmol. All three 18F-labeled peptides produced excellent tumor visualization with PET imaging in C57BL/6J mice bearing B16-F10 tumors. The tumor uptake was 7.80 ± 1.77, 5.27 ± 2.38, and 5.46 ± 2.64% injected dose per gram of tissue (%ID/g) for [18F]CCZ01064, [18F]CCZ01070, and [18F]CCZ01071 at 1 h post-injection (p.i.), respectively. Minimal background activity was observed except for kidneys at 4.99 ± 0.20, 4.42 ± 0.54, and 13.55 ± 2.84%ID/g, respectively. The best candidate [18F]CCZ01064 was further evaluated at 2 h p.i., which showed increased tumor uptake at 11.96 ± 2.31%ID/g and further reduced normal tissue uptake. Moreover, a blocking study was performed for CCZ01064 at 1 h p.i., where tumor uptake was significantly reduced to 1.97 ± 0.60%ID/g, suggesting the tumor uptake was receptor mediated. In conclusion, [18F]CCZ01064 showed high tumor uptake, low normal tissue uptake, and fast clearance and is therefore a suitable and promising candidate for PET imaging of melanoma.

Radiolabeled R954 Derivatives for Imaging Bradykinin B1 Receptor Expression with Positron Emission Tomography.

Peptide receptors have emerged as promising targets for diagnosis and therapy. The aberrant overexpression of these receptors in different cancer subtypes allows for the adoption of new treatment strategies that complement conventional chemotherapies. Bradykinin B1 receptor (B1R) is a G protein-coupled receptor that is overexpressed in many cancers, with limited expression in healthy tissues. Previously, we developed 68Ga- and 18F-labeled derivatives of B1R antagonist peptides B9858 and B9958, and successfully targeted B1R-expressing tumor xenografts in vivo. R954 (Ac-Orn-Arg-Oic-Pro-Gly-αMePhe-Ser-d-2-Nal-Ile), a potent B1R antagonist, is reportedly more stable than B9858 against peptidase degradation. We evaluated two radiolabeled derivatives of R954 (68Ga-HTK01083 and 18F-HTK01146) for B1R PET imaging. Peptides were synthesized via solid phase strategy. Nonradioactive standards were obtain by reacting GaCl3 with DOTA-dPEG2-R954 and by clicking N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate with azidoacetyl-dPEG2-R954. Binding affinity for B1R was determined by an in vitro competition binding assay. 68Ga-HTK01083 was obtained by incubating DOTA-dPEG2-R954 with 68GaCl3 under acidic conditions, while 18F-HTK01146 was prepared via an 18F-19F isotope exchange reaction. Biodistribution and imaging studies were conducted at 1 h postinjection (p.i.) in mice inoculated with B1R-expressing (B1R+) and B1R-nonexpressing (B1R-) cells. HTK01083 and HTK01146 bound B1R with good affinity (Ki = 30.5 and 24.8 nM, respectively). 68Ga/18F-labeled R954 were obtained on average in ≥10% decay-corrected radiochemical yield with >99% radiochemical purity and ≥52 GBq/µmol specific activity. For both tracers, clearance was predominantly renal with minimal involvement of the hepatobiliary system. For PET images, B1R+ tumors, kidneys, and bladder were visible. At 1 h p.i., uptake in B1R+ tumor was comparable between 68Ga-HTK01083 (8.46 ± 1.44%ID/g) and 18F-HTK01146 (9.25 ± 0.69%ID/g). B1R+ tumor-to-blood and B1R+ tumor-to-muscle ratios were 6.32 ± 1.44 and 20.7 ± 3.58 for 68Ga-HTK01083, and 7.24 ± 2.56 and 19.5 ± 4.29 for 18F-HTK01146. Our results indicate R954 is a good lead sequence for optimization of B1R tracers for cancer imaging.

Synthesis and evaluation of a 68Ga-labeled bradykinin B1 receptor agonist for imaging with positron emission tomography.

A novel 68Ga-labeled bradykinin B1 receptor (B1R) agonist, 68Ga-Z01115, was synthesized and evaluated for imaging with positron emission tomography (PET). Z01115 exhibited good binding affinity (Ki=25.4±5.1nM) to hB1R. 68Ga-Z01115 was prepared in 74±5 decay-corrected radiochemical yield with >99% radiochemical purity and 155±89GBq/µmol (4.2±2.4Ci/µmol) specific activity. 68Ga-Z01115 was stable in vitro in mouse plasma (93% remaining intact after 60min incubation), and relatively stable in vivo (51±5% remaining intact at 5min post-injection). PET imaging and biodistribution studies in mice showed that 68Ga-Z01115 cleared rapidly from nontarget tissues/organs, and generated high target-to-nontarget contrast images. The uptake of 68Ga-Z01115 in B1R-positive (B1R+) tumor was 5.65±0.59%ID/g at 1h post-injection. Average contrast ratios of B1R+ tumor-to-B1R- tumor, -to-blood and -to-muscle were 24.3, 24.4 and 82.9, respectively. Uptake of 68Ga-Z01115 in B1R+ tumors was reduced by ∼90% with co-injection of cold standard, confirming it was mediated by B1R. Our data suggest that 68Ga-Z01115 is a promising tracer for imaging the expression of B1R that is overexpressed in a variety of cancers.

Targeting the Neuropeptide Y1 Receptor for Cancer Imaging by Positron Emission Tomography Using Novel Truncated Peptides.

The neuropeptide Y1 receptor (Y1R) is overexpressed in many human cancers, particularly breast cancer. Due to stability issues, limited success has been achieved for Y1R imaging agents, including full length and truncated neuropeptide Y (NPY) analogues. The goal of this study was to evaluate the possibility of using radiolabeled truncated NPY analogues to visualize Y1R expression in a preclinical model of Y1R-positive tumor. Four truncated NPY analogues were synthesized based on the sequence of [Pro30, Tyr32, Leu34]NPY(28-36), also known as BVD15. We substituted Tyr5 and Arg6 with unnatural amino acids aiming to enhance plasma stability while maintaining good receptor binding affinity to Y1R. In addition, we substituted Leu4 to Lys4 in order to conjugate via an optional linker the DOTA chelator for 68Ga labeling. Receptor binding affinity and plasma stability of these compounds were evaluated. Positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies were performed using immune-compromised mice bearing HEK293T::WT and HEK293T::hY1R tumors. [Lys(Ga-DOTA)4, Bip5]BVD15 (CCZ01035), [Lys(Ahx-Ga-DOTA)4, Bip5]BVD15 (CCZ01053), and [Lys(Pip-Ga-DOTA)4, Bip5]BVD15 (CCZ01055) demonstrated good binding affinity to Y1R (Ki = 23.4-32.3 nM), while [Lys(Ga-DOTA)4, Har6]BVD15 (P05067) showed poor binding affinity (Ki > 1000 nM). In addition, CCZ01055 exhibited low binding affinity (Ki > 1000 nM) to Y2R and Y4R, demonstrating its selectivity to Y1R. The former three peptides showed improved in vitro plasma stability of 7-16% remaining intact after 1 h incubation. PET/CT imaging and biodistribution studies for 68Ga-labeled CCZ01053, CCZ01035, and CCZ01055 showed that radioactivity was mainly cleared by the renal pathway, and HEK293T::hY1R tumors were clearly visualized with minimal background activity with the latter two. Of these two tracers, [68Ga]CCZ01055 provided lower kidney accumulation and higher contrast, i.e., average uptake ratios of Y1R tumor to wild type tumor, blood, and muscle are 3.87 ± 0.83, 4.12 ± 1.14, and 17.6 ± 4.64, respectively. Furthermore, Y1R tumor uptake with [68Ga]CCZ01055 was significantly reduced with coinjection of 100 µg of peptide YY, confirming the specificity of tumor accumulation was receptor mediated. We successfully developed the first Y1R-targeting truncated NPY analogues for PET imaging in a preclinical model, and [68Ga]CCZ01055 is a critical template for designing improved imaging agents to detect Y1R expressing cancers.

Radiolabeled B9958 Derivatives for Imaging Bradykinin B1 Receptor Expression with Positron Emission Tomography: Effect of the Radiolabel-Chelator Complex on Biodistribution and Tumor Uptake.

Bradykinin B1 receptor (B1R), which is upregulated in a variety of malignancies, is an attractive cancer imaging biomarker. In this study we optimized the selection of radiolabel-chelator complex to improve tumor uptake and tumor-to-background contrast of radiolabeled analogues of B9958 (Lys-Lys-Arg-Pro-Hyp-Gly-Cpg-Ser-d-Tic-Cpg), a potent B1R antagonist. Peptide sequences were assembled on solid phase. Cold standards were prepared by incubating DOTA-/NODA-conjugated peptides with GaCl3, and by incubating AlOH-NODA-conjugated peptide with NaF. Binding affinities were measured via in vitro competition binding assays. (68)Ga and (18)F labeling experiments were performed in acidic buffer and purified by HPLC. Imaging/biodistribution studies were performed in mice bearing both B1R-positive (B1R+) HEK293T::hB1R and B1R-negative (B1R-) HEK293T tumors. Z02176 (Ga-DOTA-Pip-B9958; Pip: 4-amino-(1-carboxymethyl)piperidine), Z02137 (Ga-NODA-Mpaa-Pip-B9958; Mpaa: 4-methylphenylacetic acid), and Z04139 (AlF-NODA-Mpaa-Pip-B9958) bound hB1R with high affinity (Ki = 1.4-2.5 nM). (68)Ga-/(18)F-labeled peptides were obtained on average in ≥32% decay-corrected radiochemical yield with >99% radiochemical purity and 100-261 GBq/µmol specific activity. Biodistribution/imaging studies at 1 h postinjection showed that all tracers cleared rapidly from background tissues (except kidneys) and were excreted predominantly via the renal pathway. Only kidneys, bladders, and B1R+ tumors were clearly visualized in PET images. Uptake in B1R+ tumor was higher by using (68)Ga-Z02176 (28.9 ± 6.21 %ID/g) and (18)F-Z04139 (22.6 ± 3.41 %ID/g) than (68)Ga-Z02137 (14.0 ± 4.86 %ID/g). The B1R+ tumor-to-blood and B1R+ tumor-to-muscle contrast ratios were also higher for (68)Ga-Z02176 (56.1 ± 17.3 and 167 ± 57.6) and (18)F-Z04139 (58.0 ± 20.9 and 173 ± 42.9) than (68)Ga-Z02137 (34.3 ± 15.2 and 103 ± 30.2). With improved target-to-background contrast (68)Ga-Z02176 and (18)F-Z04139 are promising for imaging B1R expression in cancers with PET.

Design, synthesis and evaluation of (18)F-labeled bradykinin B1 receptor-targeting small molecules for PET imaging.

Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers.

Preclinical evaluation of MC1R targeting theranostic pair [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH.

BACKGROUND: Targeted radionuclide therapy is established as a highly effective strategy for the treatment of metastatic tumors; however, the co-development of suitable imaging companions to therapy remains significant challenge. Theranostic isotopes of terbium (149Tb, 152Tb, 155Tb, 161Tb) have the potential to provide chemically identical radionuclidic pairs, which collectively encompass all modes of nuclear decay relevant to nuclear medicine. Herein, we report the first radiochemistry and preclinical studies involving 155Tb- and 161Tb-labeled crown-αMSH, a small peptide-based bioconjugate suitable for targeting melanoma. METHODS: 155Tb was produced via proton induced spallation of Ta targets using the isotope separation and acceleration facility at TRIUMF with isotope separation on-line (ISAC/ISOL). The radiolabeling characteristics of crown-αMSH with 155Tb and/or 161Tb were evaluated by concentration-dependence radiolabeling studies, and radio-HPLC stability studies. LogD7.4 measurements were obtained for [161Tb]Tb-crown-αMSH. Competitive binding assays were undertaken to determine the inhibition constant for [natTb]Tb-crown-αMSH in B16-F10 cells. Pre-clinical biodistribution and SPECT/CT imaging studies of 155Tb and 161Tb labeled crown-αMSH were undertaken in male C57Bl/6 J mice bearing B16-F10 melanoma tumors to evaluate tumor specific uptake and imaging potential for each radionuclide. RESULTS: Quantitative radiolabeling of crown-αMSH with [155Tb]Tb3+ and [161Tb]Tb3+ was demonstrated under mild conditions (RT, 10 min) and low chelator concentrations; achieving high molar activities (23-29 MBq/nmol). Radio-HPLC studies showed [161Tb]Tb-crown-αMSH maintains excellent radiochemical purity in human serum, while gradual metabolic degradation is observed in mouse serum. Competitive binding assays showed the high affinity of [natTb]Tb-crown-αMSH toward MC1R. Two different methods for preparation of the [155Tb]Tb-crown-αMSH radiotracer were investigated and the impacts on the biodistribution profile in tumor bearing mice is compared. Preclinical in vivo studies of 155Tb- and 161Tb- labeled crown-αMSH were performed in parallel, in mice bearing B16-F10 tumors; where the biodistribution results showed similar tumor specific uptake (6.06-7.44 %IA/g at 2 h pi) and very low uptake in nontarget organs. These results were further corroborated through a series of single-photon emission computed tomography (SPECT) studies, with [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH showing comparable uptake profiles and excellent image contrast. CONCLUSIONS: Collectively, our studies highlight the promising characteristics of [155Tb]Tb-crown-αMSH and [161Tb]Tb-crown-αMSH as theranostic pair for nuclear imaging (155Tb) and radionuclide therapy (161Tb).

Mutant frizzled-4 disrupts retinal angiogenesis in familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Loci associated with FEVR map to 11q13-q23 (EVR1; OMIM 133780, ref. 1), Xp11.4 (EVR2; OMIM 305390, ref. 2) and 11p13-12 (EVR3; OMIM 605750, ref. 3). Here we have confirmed linkage to the 11q13-23 locus for autosomal dominant FEVR in one large multigenerational family and refined the disease locus to a genomic region spanning 1.55 Mb. Mutations in FZD4, encoding the putative Wnt receptor frizzled-4, segregated completely with affected individuals in the family and were detected in affected individuals from an additional unrelated family, but not in normal controls. FZD genes encode Wnt receptors, which are implicated in development and carcinogenesis. Injection of wildtype and mutated FZD4 into Xenopus laevis embryos revealed that wildtype, but not mutant, frizzled-4 activated calcium/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC), components of the Wnt/Ca(2+) signaling pathway. In one of the mutants, altered subcellular trafficking led to defective signaling. These findings support a function for frizzled-4 in retinal angiogenesis and establish the first association between a Wnt receptor and human disease.