Development, preclinical evaluation and preliminary dosimetry profiling of SB03178, a first-of-its-kind benzo[h]quinoline-based fibroblast activation protein-α-targeted radiotheranostic for cancer imaging and therapy.

Fibroblast activation protein-α (FAP) is a marker of cancer-associated fibroblasts (CAFs) that constitute a significant portion of most carcinomas. Since it plays a critical role in tumor growth and metastasis, its timely detection to identify tumor lesions in early developmental stages using targeted radiopharmaceuticals has gained significant impetus. In the present work, two novel FAP-targeted precursors SB03178 and SB04033 comprising of an atypical benzo[h]quinoline construct were synthesized and either chelated to diagnostic radionuclide gallium-68 or therapeutic radionuclide lutetium-177, with ≥90% radiochemical purities and 22-76% decay-corrected radiochemical yields. natGa-labeled complexes displayed dose-dependent FAP inhibition, with binding potency of natGa-SB03178 being ∼17 times higher than natGa-SB04033. To evaluate their pharmacokinetic profiles, PET imaging and ex vivo biodistribution analyses were executed in FAP-overexpressing HEK293T:hFAP tumor-bearing mice. While both tracers displayed clear tumor visualization that was primarily FAP-arbitrated, with negligible uptake in most peripheral tissues, [68Ga]Ga-SB03178 demonstrated higher tumor uptake and superior tumor-to-background contrast ratios than [68Ga]Ga-SB04033. 177Lu-labeled SB03178 was subjected to tumor retention studies, mouse dosimetry profiling and mouse-to-human dose extrapolations also using the HEK293T:hFAP tumor model. [177Lu]Lu-SB03178 exhibited a combination of high and sustained tumor uptake, with excellent tumor-to-critical organ uptake ratios resulting in a high radiation absorbed dose to the tumor and a low estimated whole-body dose to humans. Our preliminary findings are considerably encouraging to support clinical development of [68Ga]Ga-/[177Lu]Lu-SB03178 theranostic pair for use in a vast majority of FAP-overexpressing neoplasms, particularly carcinomas.

Synthesis and Preclinical Evaluation of Two Novel 68Ga-Labeled Bispecific PSMA/FAP-Targeted Tracers with 2-Nal-Containing PSMA-Targeted Pharmacophore and Pyridine-Based FAP-Targeted Pharmacophore.

Some bispecific radiotracers have been developed to overcome the limitations of monospecific tracers and improve detection sensitivity for heterogeneous tumor lesions. Here, we aim to synthesize two bispecific tracers targeting prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are key markers expressed in prostate cancer. A pyridine-based FAP-targeted ligand was synthesized through multi-step organic synthesis and then connected to the 2-Nal-containing PSMA-targeted motif. The Ki(PSMA) values of Ga-complexed bispecific ligands, Ga-AV01084 and Ga-AV01088, were 11.6 ± 3.25 and 28.7 ± 6.05 nM, respectively, and the IC50(FAP) values of Ga-AV01084 and Ga-AV01088 were 10.9 ± 0.67 and 16.7 ± 1.53 nM, respectively. Both [68Ga]Ga-AV01084 and [68Ga]Ga-AV01088 enabled the visualization of PSMA-expressing LNCaP tumor xenografts and FAP-expressing HEK293T:hFAP tumor xenografts in PET images acquired at 1 h post-injection. However, the tumor uptake values from the bispecific tracers were still lower than those obtained from the monospecific tracers, PSMA-targeted [68Ga]Ga-PSMA-617 and FAP-targeted [68Ga]Ga-AV02070. Further investigations are needed to optimize the selection of linkers and targeted pharmacophores to improve the tumor uptake of bispecific PSMA/FAP tracers for prostate cancer imaging.

Synthesis and Preclinical Evaluation of Novel 68Ga-Labeled (R)-Pyrrolidin-2-yl-boronic Acid-Based PET Tracers for Fibroblast Activation Protein-Targeted Cancer Imaging.

Fibroblast activation protein (FAP) is a membrane-tethered serine protease overexpressed in the reactive stromal fibroblasts of >90% human carcinomas, which makes it a promising target for developing radiopharmaceuticals for the imaging and therapy of carcinomas. Here, we synthesized two novel (R)-pyrrolidin-2-yl-boronic acid-based FAP-targeted ligands: SB02055 (DOTA-conjugated (R)-(1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)glycyl)pyrrolidin-2-yl)boronic acid) and SB04028 (DOTA-conjugated ((R)-1-((6-(3-(piperazin-1-yl)propoxy)quinoline-4-carbonyl)-D-alanyl)pyrrolidin-2-yl)boronic acid). natGa- and 68Ga-complexes of both ligands were evaluated in preclinical studies and compared to previously reported natGa/68Ga-complexed PNT6555. Enzymatic assays showed that FAP binding affinities (IC50) of natGa-SB02055, natGa-SB04028 and natGa-PNT6555 were 0.41 ± 0.06, 13.9 ± 1.29 and 78.1 ± 4.59 nM, respectively. PET imaging and biodistribution studies in HEK293T:hFAP tumor-bearing mice showed that while [68Ga]Ga-SB02055 presented with a nominal tumor uptake (1.08 ± 0.37 %ID/g), [68Ga]Ga-SB04028 demonstrated clear tumor visualization with ~1.5-fold higher tumor uptake (10.1 ± 0.42 %ID/g) compared to [68Ga]Ga-PNT6555 (6.38 ± 0.45 %ID/g). High accumulation in the bladder indicated renal excretion of all three tracers. [68Ga]Ga-SB04028 displayed a low background level uptake in most normal organs, and comparable to [68Ga]Ga-PNT6555. However, since its tumor uptake was considerably higher than [68Ga]Ga-PNT6555, the corresponding tumor-to-organ uptake ratios for [68Ga]Ga-SB04028 were also significantly greater than [68Ga]Ga-PNT6555. Our data demonstrate that (R)-(((quinoline-4-carbonyl)-d-alanyl)pyrrolidin-2-yl)boronic acid is a promising pharmacophore for the design of FAP-targeted radiopharmaceuticals for cancer imaging and radioligand therapy.

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.

Novel 68Ga-Labeled Pyridine-Based Fibroblast Activation Protein-Targeted Tracers with High Tumor-to-Background Contrast.

Compared to quinoline-based fibroblast activation protein (FAP)-targeted radiotracers, pyridine-based FAP-targeted tracers are expected to have faster pharmacokinetics due to their smaller molecular size and higher hydrophilicity, which we hypothesize would improve the tumor-to-background image contrast. We aim to develop 68Ga-labeled pyridine-based FAP-targeted tracers for cancer imaging with positron emission tomography (PET), and compare their imaging potential with the clinically validated [68Ga]Ga-FAPI-04. Two DOTA-conjugated pyridine-based AV02053 and AV02070 were synthesized through multi-step organic synthesis. IC50(FAP) values of Ga-AV02053 and Ga-AV02070 were determined by an enzymatic assay to be 187 ± 52.0 and 17.1 ± 4.60 nM, respectively. PET imaging and biodistribution studies were conducted in HEK293T:hFAP tumor-bearing mice at 1 h post-injection. The HEK293T:hFAP tumor xenografts were clearly visualized with good contrast on PET images by [68Ga]Ga-AV02053 and [68Ga]Ga-AV02070, and both tracers were excreted mainly through the renal pathway. The tumor uptake values of [68Ga]Ga-AV02070 (7.93 ± 1.88%ID/g) and [68Ga]Ga-AV02053 (5.6 ± 1.12%ID/g) were lower than that of previously reported [68Ga]Ga-FAPI-04 (12.5 ± 2.00%ID/g). However, both [68Ga]Ga-AV02070 and [68Ga]Ga-AV02053 showed higher tumor-to-background (blood, muscle, and bone) uptake ratios than [68Ga]Ga-FAPI-04. Our data suggests that pyridine-based pharmacophores are promising for the design of FAP-targeted tracers. Future optimization on the selection of a linker will be explored to increase tumor uptake while maintaining or even further improving the high tumor-to-background contrast.

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.

Development and biological evaluation of[18F]FMN3PA & [18F]FMN3PU for leucine-rich repeat kinase 2 (LRRK2) in vivo PET imaging.

PURPOSE: Among all genetic mutations of LRRK2, the G2019S mutation is the most commonly associated with the late-onset of Parkinson's disease (PD). Hence, one potential therapeutic approach is to block the hyperactivity of mutated LRRK2 induced by kinase inhibition. To date, only a few LRRK2 kinase inhibitors have been tested for in vivo quantification of target engagement by positron emission tomography (PET). In this study, we performed biological evaluations of two radiolabeled kinase inhibitors i.e. [18F]FMN3PA (14) and [18F]FMN3PU for LRRK2 (15). PROCEDURES: Radiosyntheses of [18F]FMN3PA (14) and [18F]FMN3PU (15) were performed using K[18F]-F-K222 complex in a TRACERlab FXN module and purification was carried out via C18 plus (Sep-Pak) cartridges. In vitro specific binding assays were performed in rat brain striatum and kidney tissues using GNE-0877 as a blocking agent (Ki = 0.7 nM). For in vivo blocking, 3 mg/kg of GNE-0877 was injected 30 min before radiotracer injection via tail vein in wild-type (WT) mice (n = 4). Dynamic scans by PET/CT (Siemens Inveon) were performed in WT mice (n = 3). RESULTS: Radiofluorinations resulted in radiochemical yields (RCYs) of 25 ± 1.3% (n = 6) ([18F]FMN3PU, 15) and 37 ± 1.6% (n = 6) ([18F]FMN3PA, 14) with ≥96% radiochemical purity (RCP) and a molar activity (MA) of 3.55 ± 1.6 Ci/µmol (131 ± 56 GBq/µmol) for [18F]FMN3PU (15) and 4.57 ± 1.7 Ci/µmol (169 ± 63 GBq/µmol) for [18F]FMN3PA (14), respectively. Saturation assays showed high specific binding for rat brain striatum with Kd 20 ± 1.3 nM ([18F]FMN3PA, 14) and 23.6 ± 4.0 nM ([18F]FMN3PU, 15). In vivo blocking data for [18F]FMN3PA (14) was significant for brain (p < 0.0001, 77% blocking) and kidney (p = 0.0041, 65% blocking). PET images showed uptake in mouse brain striatum. CONCLUSION: In the presence of GNE-0877 as a blocking agent, the specific binding of [18F]FMN3PA (14) and [18F]FMN3PU (15) was significant in vitro. [18F]FMN3PA (14) showed good brain uptake in vivo, though fast clearance from brain was observed (within 10-15 min).

Lewis Acid-Facilitated Radiofluorination of MN3PU: A LRRK2 Radiotracer.

BACKGROUND: Temperature-sensitive radiopharmaceutical precursors require lower reaction temperatures (<100 °C) during nucleophilic radiofluorination in order to avoid compound thermolysis, often resulting in sub-optimal radiochemical yields (RCYs). To facilitate nucleophilic aromatic substitution (SNAr) of nucleofuges commonly used in radiofluorination (e.g., nitro group), we explored the use of Lewis acids as nucleophilic activators to accelerate [18F]fluoride incorporation at lower temperatures, and thereby increasing RCYs for thermolabile activated precursors. Lewis acid-assisted radiofluorination was exemplified on the temperature-sensitive compound 1-(4-(4-morpholino-7-neopentyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)phenyl)-3-(6-nitropyridin-3-yl)urea (MN3PU, compound 3) targeting leucine-rich repeat kinase 2 (LRRK2), an important target in the study of Parkinson's disease and various cancers. METHODS: To a vessel containing dried K[18F]F-K222 complex, a solution of precursor MN3PU ((3), 1 mg; 1.8 µmol) and Lewis acid (6 µL of 0.2 µmol: chromium II chloride (A), ferric nitrite (B) or titanocene dichloride (C)) in 500 µL of N,N-dimethylformamide (DMF) (with 10% t-BuOH for B) were added. Reactions were stirred for 25 min at 90 °C. In parallel, reactions were conducted without the addition of Lewis acids for baseline comparison. After purification via preconditioned Sep-Pak C18 plus cartridges, aliquots were analyzed by analytical radio-HPLC. RESULTS: Non-decay corrected radiochemical yields (ndc RCYs) for [18F]FMN3PU (7) were improved from 1.7 ± 0.7% (no addition of Lewis acids) to 41 ± 1% using Cr(II) and 37 ± 0.7% using Ti(II)-based Lewis acids, with radiochemical purities of ≥96% and molar activities (Am) of up to 3.23 ± 1.7 Ci/µmol (120 ± 1.7 GBq/µmol). CONCLUSION: RCYs of [18F]FMN3PU (7) improved from ~5% using conventional nucleophilic radiofluorination, up to 41 ± 1% using Lewis-acid supported SNAr.

Evaluation of Met-Val-Lys as a Renal Brush Border Enzyme-Cleavable Linker to Reduce Kidney Uptake of 68Ga-Labeled DOTA-Conjugated Peptides and Peptidomimetics.

High kidney uptake is a common feature of peptide-based radiopharmaceuticals, leading to reduced detection sensitivity for lesions adjacent to kidneys and lower maximum tolerated therapeutic dose. In this study, we evaluated if the Met-Val-Lys (MVK) linker could be used to lower kidney uptake of 68Ga-labeled DOTA-conjugated peptides and peptidomimetics. A model compound, [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (AmBz: aminomethylbenzoyl), and its derivative, [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH, coupled with the PSMA (prostate-specific membrane antigen)-targeting motif of the previously reported HTK01166 were synthesized and evaluated to determine if they could be recognized and cleaved by the renal brush border enzymes. Additionally, positron emission tomography (PET) imaging, ex vivo biodistribution and in vivo stability studies were conducted in mice to evaluate their pharmacokinetics. [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was effectively cleaved specifically by neutral endopeptidase (NEP) of renal brush border enzymes at the Met-Val amide bond, and the radio-metabolite [68Ga]Ga-DOTA-AmBz-Met-OH was rapidly excreted via the renal pathway with minimal kidney retention. [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH retained its PSMA-targeting capability and was also cleaved by NEP, although less effectively when compared to [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH. The kidney uptake of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was 30% less compared to that of [68Ga]Ga-HTK01166. Our data demonstrated that derivatives of [68Ga]Ga-DOTA-AmBz-MVK-OH can be cleaved specifically by NEP, and therefore, MVK can be a promising cleavable linker for use to reduce kidney uptake of radiolabeled DOTA-conjugated peptides and peptidomimetics.