Abstract Several targeted therapeutic options exist for non-small cell lung cancer (NSCLC) patients whose tumors harbor mutations of EGFR, including L858R, exon 19 deletions, and the acquired resistance mutation, T790M. However, there is no approved targeted therapy for patients with EGFR exon 20 insertions, highlighting an ongoing unmet medical need. Here, we describe a potent and selective inhibitor of EGFR exon 20 insertion mutants with decreased activity on the wild-type EGFR for an improved therapeutic window. Our compound, BAY-568, exhibits greater than 20-fold selectivity for EGFR exon 20 insertions compared to wild-type EGFR in isogenic Ba/F3 models and in cancer cell lines endogenously expressing EGFR exon 20 insertions. This activity is also observed in xenograft models in vivo, correlating with reductions in phospho-EGFR and phospho-Erk in tumors but not skin samples from treated mice. BAY-568 furthermore has even greater activity towards the “classical” erlotinib-sensitive mutations, L858R and exon 19 deletions. Importantly, our compound is reversible, differentiating it from other investigational compounds currently in clinical trials. Consistent with this, the presence of a C797S mutation, typically found in patients with acquired resistance to osimertinib, has no effect on the activity of BAY-568. Taken together, these results demonstrate the ability of BAY-568 to kill cancer cells harboring exon 20 insertions and other EGFR mutations with decreased activity on wild-type EGFR, irrespective of C797S mutation status. Citation Format: Franziska Siegel, Stephan Siegel, Keith Graham, Bethany Kaplan, Kirstin Petersen, Ulf Boemer, Uwe Eberspaecher, Daniel Korr, Ursula Moenning, Detlev Suelzle, Jens Schroeder, Florian Prinz, Sabine Zitzmann-Kolbe, Gizem Karsli-Uzunbas, Timothy Lewis, Mario Hermsen, Andrew Cherniack, Franz von Nussbaum, Knut Eis, Matthew Meyerson, Heidi Greulich. Preclinical activity of the first reversible, potent and selective inhibitor of EGFR exon 20 insertions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1470.

The aim of this first-in-man study was to demonstrate the feasibility, safety, and tolerability, as well as provide dosimetric data and evaluate the imaging properties, of the bombesin analogue BAY 864367 for PET/CT in a small group of patients with primary and recurrent prostate cancer (PCa). Methods: Ten patients with biopsy-proven PCa (5 with primary PCa and 5 with prostate-specific antigen recurrence after radical prostatectomy) were prospectively selected for this exploratory clinical trial with BAY 864367, a new 18F-labeled bombesin analogue. PET scans were assessed at 6 time points, up to 110 min after intravenous administration of 302 ± 11 MBq of BAY 864367. Imaging results were compared with 18F-fluorocholine PET/CT scans. Dosimetry was calculated using the OLINDA/EXM software. Results: Three of 5 patients with primary disease showed positive tumor delineation in the prostate, and 2 of 5 patients with biochemical relapse showed a lesion suggestive of recurrence on the BAY 864367 scan. Tumor-to-background ratio averaged 12.9 ± 7.0. The ratio of malignant prostate tissue to normal prostate tissue was 4.4 ± 0.6 in 3 patients with tracer uptake in the primary PCa. Mean effective dose was 4.3 ± 0.3 mSv/patient (range, 3.7–4.9 mSv). Conclusion: BAY 864367, a novel 18F-labeled bombesin tracer, was successfully investigated in a first-in-man clinical trial of PCa and showed favorable dosimetric values. Additionally, the application was safe and well tolerated. The tracer delineated tumors in a subset of patients, demonstrating the potential of gastrin-releasing-peptide receptor imaging.

Carbon-11- and fluorine-18-labeled choline derivatives are commonly used in prostate cancer imaging in the clinical setting for staging and re-staging of prostate cancer. Due to a limited detection rate of established positron emission tomography (PET) tracers, there is a clinical need for innovative tumor-specific PET compounds addressing new imaging targets. The aim of this study was to compare the properties of [18F]Bombesin (BAY 86-4367) as an innovative biomarker for prostate cancer imaging targeting the gastrin-releasing peptide receptor and [11C]Choline ([11C]CHO) in a human prostate tumor mouse xenograft model by small animal PET/X-ray computed tomography (CT).

The radiosynthesis of [(18)F]fluoropyruvate was investigated using numerous precursors were synthesized from ethyl 2,2-diethoxy-3-hydroxypropanoate (5) containing different leaving groups: mesylate, tosylate, triflate, and nonaflate. These precursors were evaluated for [(18)F]fluoride incorporation with triflate being superior. The subsequent hydrolysis step was investigated, and an acidic hydrolysis was optimized. After establishing suitable purification and formulation methods, the [(18)F]fluoropyruvate could be isolated in ca. 50% d.c. yield. The [(18)F]fluoropyruvate was evaluated in vitro for its uptake into tumor cells using adenocarcinomic human alveolar basal epithelial cells (A549) and unfortunately showed an uptake of approximately 0.1% of the applied dose per 100,000 cells after 30 min. Initial pharmacokinetic properties were assessed in vivo using nude mice showed a high degree of bone uptake from defluorination, which will limit its potential as an imaging agent for metabolic processes.

(D)-F-18-fluoromethyltyrosine (D-F-18-FMT), or BAY 86-9596, is a novel F-18-labeled tyrosine derivative rapidly transported by the L-amino acid transporter (LAT-1), with a faster blood pool clearance than the corresponding L-isomer. The aim of this study was to demonstrate the feasibility of tumor detection in patients with non-small cell lung cancer (NSCLC) or head and neck squamous cell cancer (HNSCC) compared with inflammatory and physiologic tissues in direct comparison to F-18-FDG. Methods: 18 patients with biopsy-proven NSCLC (n = 10) or HNSCC (n = 8) were included in this Institutional Review Board-approved, prospective multicenter study. All patients underwent F-18-FDG PET/CT scans within 21 d before D-F-18-FMT PET/CT. For all patients, safety and outcome data were assessed. Results: No adverse reactions were observed related to D-F-18-FMT. Fifty-two lesions were F-18-FDG-positive, and 42 of those were malignant (34 histologically proven and 8 with clinical reference). Thirty-two of the 42 malignant lesions were also D-F-18-FMT-positive, and 10 lesions had no tracer uptake above the level of the blood pool. Overall there were 34 true-positive, 8 true-negative, 10 false-negative, and only 2 false-positive lesions for D-F-18-FMT, whereas F-18-FDG was true-positive in 42 lesions, with 10 false-positive and only 2 false-negative, resulting in a lesion-based detection rate for D-F-18-FMT and F-18-FDG of 77% and 95%, respectively, with an accuracy of 78% for both tracers. A high D-F-18-FMT tumor-to-blood pool ratio had a negative correlation with overall survival (P = 0.050), whereas the F-18-FDG tumor-to-blood pool ratio did not correlate with overall survival. Conclusion: D-F-18-FMT imaging in patients with NSCLC and HNSCC is safe and feasible. The presented preliminary results suggest a lower sensitivity but higher specificity for D-F-18-FMT over F-18-FDG, since there is no D-F-18-FMT uptake in inflammation. This increased specificity may be particularly beneficial in areas with endemic granulomatous disease and may improve clinical management. Further clinical investigations are needed to determine its clinical value and relevance for the prediction of survival prognosis.

PURPOSE:Prostate-specific membrane antigen (PSMA) is a transmembrane protein overexpressed in prostate cancer and is therefore being explored as a biomarker for diagnosing and staging of the disease. Here we report preclinical data on BAY 1075553 (a 9:1 mixture of (2S,4S)- and (2R,4S)-2-[(18)F]fluoro-4-phosphonomethyl-pentanedioic acid), a novel (18)F-labelled small molecule inhibitor of PSMA enzymatic activity, which can be efficiently synthesized from a direct radiolabelling precursor.METHODS:The (18)F-radiolabelled stereoisomers of 2-[(18)F]fluoro-4-(phosphonomethyl)-pentanedioic acid were synthesized from their respective isomerically pure precursors dimethyl 2-{[bis(benzyloxy)phosphoryl]methyl}-4-(tosyloxy)pentanedioate. In vivo positron emission tomography (PET) imaging and biodistribution studies were conducted in mice bearing LNCaP, 22Rv1 and PC-3 tumours. Pharmacokinetic parameters and dosimetry estimates were calculated based on biodistribution studies in rodents. For non-clinical safety assessment (safety pharmacology, toxicology) to support a single-dose human microdose study, off-target effects in vitro, effects on vital organ functions (cardiovascular in dogs, nervous system in rats), mutagenicity screens and an extended single-dose study in rats were conducted with the non-radioactive racemic analogue of BAY 1075553.RESULTS:BAY 1075553 showed high tumour accumulation specific to PSMA-positive tumour-bearing mice and was superior to other stereoisomers tested. Fast clearance of BAY 1075553 resulted overall in low background signals in other organs except for high uptake into kidney and bladder which was mainly caused by renal elimination of BAY 1075553. A modest uptake into bone was observed which decreased over time indicating organ-specific uptake as opposed to defluorination of BAY 1075553 in vivo. Biodistribution studies found highest organ doses for kidneys and the urinary bladder wall resulting in a projected effective dose (ED) in humans of 0.0219 mSv/MBq. Non-clinical safety studies did not show off-target activity, effects on vital organs function or dose-dependent adverse effects.CONCLUSION:BAY 1075553 was identified as a promising PET tracer for PSMA-positive prostate tumours in preclinical studies. BAY 1075553 can be produced using a robust, direct radiosynthesis procedure. Pharmacokinetic, toxicology and safety pharmacology studies support the application of BAY 1075553 in a first-in-man microdose study with single i.v. administration.

The [(18)F]fluorocyclobutyl group has the potential to be a metabolically stable prosthetic group for PET tracers. The synthesis of the radiolabeling precursor cis-cyclobutane-1,3-diyl bis(toluene-4-sulfonate) 8 was obtained from epibromohydrin in 7 steps (2% overall yield). The radiolabeling of this precursor 8 and its conjugation to L-tyrosine as a model system was successfully achieved to give the new non-natural amino acid 3-[(18)F]fluorocyclobutyl-L-tyrosine (L-3-[(18)F]FCBT) [(18)F]17 in 8% decay-corrected yield from the non-carrier-added [(18)F]fluoride. L-3-[(18)F]FCBT was investigated in vitro in different cancer cell lines to determine the uptake and stability. The tracer [(18)F]17 showed a time dependent uptake into different tumor cell lines (A549, NCI-H460, DU145) with the best uptake of 5.8% injected dose per 5×10(5) cells after 30min in human lung carcinoma cells A549. The stability of L-3-[(18)F]FCBT in human and rat plasma and the stability of the non-radioactive L-3-FCBT in rat hepatocytes were both found to be excellent. These results show that the non-natural amino acid L-3-[(18)F]FCBT is a promising metabolically stable radiotracer for positron emission tomography.

Silicon-containing prosthetic groups have been conjugated to peptides to allow for a single-step labeling with (18)F radioisotope. The fairly lipophilic di-tert-butylphenylsilane building block contributes unfavorably to the pharmacokinetic profile of bombesin conjugates. In this article, theoretical and experimental studies toward the development of more hydrophilic silicon-based building blocks are presented. Density functional theory calculations were used to predict the hydrolytic stability of di-tert-butylfluorosilanes 2-23 with the aim to improve the in vivo properties of (18)F-labeled silicon-containing biomolecules. As a further step toward improving the pharmacokinetic profile, hydrophilic linkers were introduced between the lipophilic di-tert-butylphenylsilane building block and the bombesin congeners. Increased tumor uptake was shown with two of these peptides in xenograft-bearing mice using positron emission tomography and biodistribution studies. The introduction of a hydrophilic linker is thus a viable approach to improve the tumor uptake of (18)F-labeled silicon-bombesin conjugates.

Fluorine-18 labeled (2S,4S)-2-[F-18]fluoro-4-(phosphonomethyl)pentanedioic acid (BAY 1075553) has been identified as a prostate specific membrane antigen inhibitor ligand and is being investigated in clinical PET studies for its application in the diagnosis and staging of prostate cancer. To facilitate its transfer to the clinics, it was imperative to develop sensitive analytical methods to characterize the final product with regard to chemical identity, specific activity, and ratio of stereoisomers. This Letter reports on a new rapid quantitative derivatization method for converting an aliquot of the final aqueous formulated product (concentration <10(-6) M) into a compound suitable for chiral HPLC analysis to determine the ratio of stereoisomers. This novel analytical method was critical for allowing BAY 1075553 to enter the clinical setting. (C) 2013 Elsevier Ltd. All rights reserved.

Inflammation is a pathophysiological hallmark of many diseases of the brain. Specific imaging of cells and molecules that contribute to cerebral inflammation is therefore highly desirable, both for research and in clinical application. The 18 kDa translocator protein (TSPO) has been established as a suitable target for the detection of activated microglia/macrophages. A number of novel TSPO ligands have been developed recently. Here, we evaluated the high affinity TSPO ligand DPA-714 as a marker of brain inflammation in two independent animal models. For the first time, the specificity of radiolabeled DPA-714 for activated microglia/macrophages was studied in a rat model of epilepsy (induced using Kainic acid) and in a mouse model of stroke (transient middle cerebral artery occlusion, tMCAO) using high-resolution autoradiography and immunohistochemistry. Additionally, cold-compound blocking experiments were performed and changes in blood-brain barrier (BBB) permeability were determined. Target-to-background ratios of 2 and 3 were achieved in lesioned vs. unaffected brain tissue in the epilepsy and tMCAO models, respectively. In both models, ligand uptake into the lesion corresponded well with the extent of Ox42- or Iba1-immunoreactive activated microglia/macrophages. In the epilepsy model, ligand uptake was almost completely blocked by pre-injection of DPA-714 and FEDAA1106, another high-affinity TSPO ligand. Ligand uptake was independent of the degree of BBB opening and lesion size in the stroke model. We provide further strong evidence that DPA-714 is a specific ligand to image activated microglia/macrophages in experimental models of brain inflammation.