Identification of a PET Radiotracer for Imaging of the Folate Receptor-α: A Potential Tool to Select Patients for Targeted Tumor Therapy.

The aim of this study was to identify a folate receptor-α (FRα)-selective PET agent potentially suitable for the selection of patients who might profit from FRα-targeted therapies. The 6R and 6S isomers of 18F-aza-5-methyltetrahydrofolate (MTHF) were assessed regarding their binding to FRα and FRß, expressed on cancer and inflammatory cells, respectively, and compared with 18F-AzaFol, the folic acid-based analog. Methods: FR selectivity was investigated using FRα-transfected (RT16) and FRß-transfected (D4) CHO cells. The cell uptake of 18F-folate tracers was investigated, and receptor-binding affinities were determined with the nonradioactive analogs. In vitro autoradiography of the 18F-folate tracers was performed using RT16 and D4 tissue sections. Biodistribution studies and PET/CT imaging of the radiotracers were performed on mice bearing RT16 and D4 xenografts. Results: The uptake of 18F-6R-aza-5-MTHF was high when using RT16 cells (62% ± 10% of added activity) but much lower when using D4 cells (5% ± 2%). The FRα selectivity of 18F-6R-aza-5-MTHF was further demonstrated by its approximately 43-fold higher binding affinity to FRα (half-maximal inhibitory concentration [IC50], 1.8 ± 0.1 nM) than to FRß (IC50, 77 ± 27 nM). The uptake of 18F-6S-aza-5-MTHF and 18F-AzaFol was equal in both cell lines (52%-70%), with similar affinities to FRα (IC50, 2.1 ± 0.4 nM and 0.6 ± 0.3 nM, respectively) and FRß (0.8 ± 0.2 nM and 0.3 ± 0.1 nM, respectively). The autoradiography signal obtained with 18F-6R-aza-5-MTHF was 11-fold more intense for RT16 than for D4 tissue sections. Biodistribution data showed high uptake of 18F-6R-aza-5-MTHF in RT16 xenografts (81% ± 20% injected activity per gram [IA]/g 1 h after injection) but significantly lower accumulation in D4 xenografts (7.3% ± 2.1% IA/g 1 h after injection), which was also visualized using PET. The uptake of 18F-6S-aza-5-MTHF and 18F-AzaFol was similar in RT16 (53% ± 10% IA/g and 45% ± 2% IA/g, respectively) and D4 xenografts (77% ± 10% IA/g and 52% ± 7% IA/g, respectively). Conclusion: This study demonstrated FRα selectivity for 18F-6R-aza-5-MTHF but not for 18F-6S-aza-5-MTHF or 18F-AzaFol. This characteristic, together with its favorable tissue distribution, makes 18F-6R-aza-5-MTHF attractive for clinical translation to enable detection of FRα-positive cancer while preventing undesired accumulation in FRß-expressing inflammatory cells.

Development of Folate Receptor-Targeted PET Radiopharmaceuticals for Tumor Imaging-A Bench-to-Bedside Journey.

The folate receptor-α (FR-α) is overexpressed in many epithelial cancers, including ovary, uterus, kidneys, breast, lung, colon and prostate carcinomas, but shows limited expression in normal tissues such as kidneys, salivary glands, choroid plexus and placenta. FR-α has therefore emerged as a promising target for the delivery of therapeutic and imaging agents to FR-positive tumors. A series of folate-based PET (positron emission tomography) radiopharmaceuticals have been developed for the selective targeting of FR-positive malignancies. This review provides an overview on the research progress made so far regarding the design, radiosynthesis and the utility of the folate-derived PET radioconjugates for targeting FR-positive tumors. For the most part, results from folate radioconjugates labeled with fluorine-18 (t1/2 = 109.8 min) and gallium-68 (t1/2 = 67.7 min) have been presented but folates labeled with "exotic" and new PET radionuclides such as copper-64 (t1/2 = 12.7 h), terbium-152 (t1/2 = 17.5 h), scandium-44 (t1/2 = 3.97 h), cobalt-55 (t1/2 = 17.5 h) and zirconium-89 (t1/2 = 78.4 h) are also discussed. For tumor imaging, none of the reported PET radiolabeled folates reported to date has made the complete bench-to-bedside journey except [18F]AzaFol, which made it to patients with metastatic ovarian and lung cancers in a multicenter first-in-human trial. In the near future, however, we expect more clinical trials with folate-based PET radiopharmaceuticals given the increasing clinical interest in imaging and the treatment of FR-related malignancies.

Diastereomerically Pure 6R- and 6S-3'-Aza-2'-18F-Fluoro-5-Methyltetrahydrofolates Show Unprecedentedly High Uptake in Folate Receptor-Positive KB Tumors.

The aim of this study was to develop the radiosyntheses of diastereomerically pure 6R- and 6S-3'-aza-2'-18F-fluoro-5-methyltetrahydrofolate (MTHF) (6R-18F-1 and 6S-18F-1) using the integrated approach and to compare the in vitro and in vivo performance characteristics of both radioligands with the previously reported 3'-aza-2'-18F-fluorofolic acid tracer (18F-2), the oxidized form. Methods: 6R-18F-1, 6S-18F-1, and 18F-2 were radiolabeled with 18F using aromatic nucleophilic substitution reaction. In vitro cell uptake studies and binding affinity assays were performed using folate receptor (FR)-α-expressing KB cells. PET/CT imaging and biodistribution experiments were performed with KB tumor-bearing mice. Results: Reference compounds 6R-1 and 6S-1 were obtained after acidic hydrolysis of the corresponding protected intermediates 6R-3 and 6S-3 in high chemical yields (81%-87%) and chemical purities of more than 95%. 6R-18F-1, 6S-18F-1, and 18F-2 were obtained after a 2-step radiosynthetic procedure in a decay-corrected radiochemical yield of up to 5% and molar radioactivities ranging from 20 to 250 GBq/µmol. In vitro binding affinity studies using FR-α-positive KB cells gave half-maximal inhibitory concentrations of 27.1 ± 3.7 and 23.8 ± 4.0 nM for 6R-1 and 6S-1, respectively, which were higher than for the previously reported 3'-aza-2'-fluorofolic acid 2 (1.4 ± 0.5 nM). Comparably high cell uptake values in FR-α-expressing KB cells were found for all 3 radiofolates. In biodistribution studies, exceptionally high KB tumor uptake value of over 32% injected activity per gram of tissue for both 6R-18F-1 and 6S-18F-1 was observed at 180 min after injection, whereas for 18F-2 only 15% injected activity per gram was found in the KB tumors. Radioactivity uptake in the kidneys, liver, salivary glands, and spleen was substantially different for the 6R- and 6S-diastereoisomers and 18F-2 Excellent KB tumor visualization was found in PET/CT images with 6R-18F-1 and 6S-18F-1, both of which outperformed the corresponding oxidized 18F-2. Conclusion: We have successfully radiolabeled 6R- and 6S-3'-aza-2'-18F-fluoro-5-MTHF with 18F using the integrated approach. Our results suggest that both 6R- and 6S-3'-aza-2'-18F-fluoro-5-MTHF are promising reduced radiofolates for imaging FR-α-expressing cancers.

Ketamine and Ceftriaxone-Induced Alterations in Glutamate Levels Do Not Impact the Specific Binding of Metabotropic Glutamate Receptor Subtype 5 Radioligand [18F]PSS232 in the Rat Brain.

Several studies showed that [11C]ABP688 binding is altered following drug-induced perturbation of glutamate levels in brains of humans, non-human primates and rats. We evaluated whether the fluorinated derivative [18F]PSS232 can be used to assess metabotropic glutamate receptor 5 (mGluR5) availability in rats after pharmacological challenge with ketamine, known to increase glutamate, or ceftriaxone, known to decrease glutamate. In vitro autoradiography was performed on rat brain slices with [18F]PSS232 to prove direct competition of the drugs for mGluR5. One group of rats were challenged with a bolus injection of either vehicle, racemic ketamine, S-ketamine or ceftriaxone followed by positron emission tomography PET imaging with [18F]PSS232. The other group received an infusion of the drugs during the PET scan. Distribution volume ratios (DVRs) were calculated using a reference tissue model. In vitro autoradiography showed no direct competition of the drugs with [18F]PSS232 for the allosteric binding site of mGluR5. DVRs of [18F]PSS232 binding in vivo did not change in any brain region neither after bolus injection nor after infusion. We conclude that [18F]PSS232 has utility for measuring mGluR5 density or occupancy of the allosteric site in vivo, but it cannot be used to measure in vivo fluctuations of glutamate levels in the rat brain.

Reduced 18F-Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging.

5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on 18F-labeled 6 S- and 6 R-5-MTHF radiotracers for imaging folate receptor (FR)-α-positive tissues. Therefore, the goal of this study was to synthesize four 18F-labeled 5-MTHF derivatives conjugated at either the α- or γ-carboxylic functionality of glutamate and to assess their suitability for FR-targeting. Organic syntheses of the precursors and the four reference compounds, namely, 6 S-α, 6 S-γ, 6 R-α, and 6 R-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated 18F-labeled folate derivatives were accomplished in approximately 100 min, low radiochemical yields (1-7% d.c.) and high molar activities (139-245 GBq/µmol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and l-cysteine. In vitro, all four 5-MTHF conjugates showed similar binding affinities to FR-α (IC50 = 17.7-24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-α. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high in vivo stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6 S- and 6 R-5-MTHF conjugates exhibited a different accumulation pattern in various organs including the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the α- and γ-conjugated diastereoisomers. Despite the considerably lower binding affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8-11% IA/g). Based on these results, we conclude that 18F-labeled 5-MTHF conjugates are a promising new class of radiotracers for targeting FR-positive tumor tissues.

Discovery of a Selective Aurora A Kinase Inhibitor by Virtual Screening.

Here we report the discovery of a selective inhibitor of Aurora A, a key regulator of cell division and potential anticancer target. We used the atom category extended ligand overlap score (xLOS), a 3D ligand-based virtual screening method recently developed in our group, to select 437 shape and pharmacophore analogs of reference kinase inhibitors. Biochemical screening uncovered two inhibitor series with scaffolds unprecedented among kinase inhibitors. One of them was successfully optimized by structure-based design to a potent Aurora A inhibitor (IC50 = 2 nM) with very high kinome selectivity for Aurora kinases. This inhibitor locks Aurora A in an inactive conformation and disrupts binding to its activator protein TPX2, which impairs Aurora A localization at the mitotic spindle and induces cell division defects. This phenotype can be rescued by inhibitor-resistant Aurora A mutants. The inhibitor furthermore does not induce Aurora B specific effects in cells.

Comparative Studies of Three Pairs of α- and γ-Conjugated Folic Acid Derivatives Labeled with Fluorine-18.

The folate receptor (FR) is upregulated in various epithelial cancer types (FR α-isoform), while healthy tissues show only restricted expression. FR-targeted imaging using folate radiopharmaceuticals is therefore a promising approach for the detection of FR-positive cancer tissue. Almost all folate-based radiopharmaceuticals have been prepared by conjugation at the γ-carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of fluorinated α- and γ-conjugated folate derivatives were synthesized and their in vitro and in vivo properties compared. The syntheses of all six regioisomers were obtained in good chemical yields using a multistep synthetic approach including the highly selective Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radiosyntheses of the α- and γ-conjugated (18)F-labeled folate derivatives were accomplished in moderate to good radiochemical yields, high radiochemical purities (>95%), and specific activities ranging from 25 to 196 GBq/µmol. In vitro, all folate derivatives showed high binding affinity to the FR-α (IC50 = 1.4-2.2 nM). In vivo PET imaging and biodistribution studies in FR-positive KB tumor-bearing mice demonstrated similar FR-specific tumor uptake for both regioisomers of each pair of compounds. However, FR-unspecific liver uptake was significantly lower for the α-regioisomers compared to the corresponding γ-regioisomers. In contrast, kidney uptake was up to 50% lower for the γ-regioisomers than for the α-regioisomers. These results show that the site of conjugation in the glutamyl moiety of folic acid has a significant impact on the in vivo behavior of (18)F-based radiofolates, but not on their in vitro FR-binding affinity. These findings may potentially stimulate new directions for the design of novel (18)F-labeled folate-based radiotracers.

Discovery of potent positive allosteric modulators of the α3ß2 nicotinic acetylcholine receptor by a chemical space walk in ChEMBL.

While a plethora of ligands are known for the well studied α7 and α4ß2 nicotinic acetylcholine receptor (nAChR), only very few ligands address the related α3ß2 nAChR expressed in the central nervous system and at the neuromuscular junction. Starting with the public database ChEMBL organized in the chemical space of Molecular Quantum Numbers (MQN, a series of 42 integer value descriptors of molecular structure), a visual survey of nearest neighbors of the α7 nAChR partial agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU-282,987) pointed to N-(2-halobenzyl)-3-aminoquinuclidines as possible nAChR modulators. This simple "chemical space walk" was performed using a web-browser available at www.gdb.unibe.ch . Electrophysiological recordings revealed that these ligands represent a new and to date most potent class of positive allosteric modulators (PAMs) of the α3ß2 nAChR, which also exert significant effects in vivo. The present discovery highlights the value of surveying chemical space neighbors of known drugs within public databases to uncover new pharmacology.