Fluorinated Cromolyn Derivatives for Potential Alzheimer’s Disease Treatment

114 Objectives: Cromolyn is an anti-inflammatory mast cell stabilizer with a multi-faceted mechanism of action on amyloid beta (Aβ), currently in Phase III clinical trials for Alzheimer’s disease (AD). We reported that cromolyn acts as an anti-neuroinflammatory modulator that enhances microglial clearance of Aβ and inhibits Aβ aggregation. Here, we report the effects of fluoro-cromolyn derivatives on aggregation and microglial clearance of Aβ. We also report the synthesis of F-18 labeled fluoro-cromolyn analogs and their PET imaging pharmacokinetics in monkey brain. The overall goal of this study was to determine the most effective fluoro-cromolyn candidate for AD treatment and the possibility of monitoring treatment by PET imaging with the same drug. Methods: Aβ aggregation inhibition assays were performed using synthetic Aβ42 at concentrations of 5 µM which were incubated with 0, 10, 100, 1,000 nM of ten fluoro-cromolyn derivatives for 1 hour. Aggregation was initiated by addition of heparin at 0.5 mg/ml in final concentration and were measured by intensity of Thioflavin T binding. Detection was by fluorescent excitation/emission at 450 nm/480 nm in a kinetic mode using a Spectra Max M3 plate reader. Aggregation kinetics were recorded and calculated to express Vmax (units/sec) by the plate reader’s software. Microglial clearance assays were performed with Aβ in BV2 murine microglial cell cultures incubated with increasing doses of compound. Synthesis of ethyl 5,5′-(2-[18F]fluoropropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylate) is representative of the labeling method. A DMSO solution containing 2-[18F]fluoropropane 1,3-ditosylate (produced from the corresponding triflate and K18F/Kryptofix in acetonitrile, 80oC/10 min), K2CO3 (10 mg) and ethyl 5-hydroxy-4-oxo-4H-chromene-2-carboxylate (10 mg) was heated at 130oC for 10 min. The solution was acidified and 18F-cromolyn diethyl ester was purified by HPLC. Synthesis was complete within 90 min; yield was 20% (decay corrected to EOB) and the chemical purity was greater than 95%. Dynamic PET imaging with arterial blood sampling and radiometabolite analysis was performed for three F-18 analogs (diethyl ester, di-tert-butyl ester, diacid) in a rhesus macaque. Results: Fluoro-cromolyn derivatives inhibited >90% Aβ42 aggregation at 5 nM concentration and were comparable or better than that of their parent compound cromolyn in vitro. Both cromolyn and the fluoro-cromolyn diethyl ester derivative enhanced microglial phagocytosis and clearance of Aβ in a dose-dependent manner with no cell lethality. Monkey imaging for three 18F-cromolyn analogs showed the order of brain tracer penetration was diethyl ester > di-tert-butyl ester > diacid. Diethyl ester 18F-cromloyn showed uptake in all regions of the brain with the highest in putamen, grey matter and cerebellum followed by caudate, thalamus and white matter. Uptake was immediate, reaching maximum at 2 min (2.3 SUV) and washout was slow, 2 SUV at 20 min, 1.5 at 60 min. Blood sampling for this tracer study showed 18F-diacid cromolyn as the only metabolite at 20 min. The t-butyl ester also metabolized to the 18F-diacid but more slowly. Hydrolysis to the diacid form appears to take place in the blood as evidenced by ex vivo stability tests. Conclusions: The anti-neuroinflammatory properties, pro-phagocytic activity, and inhibition of Aβ aggregation of fluoro-cromolyn derivatives are comparable or superior to cromolyn, suggesting that these derivatives are potentially more efficacious for AD treatment. The diethyl ester and di-tert-butyl ester [18F]fluoro-cromolyn derivatives degrade to the diacid derivative, but unlike the diacid they have significant brain penetration. The diethyl ester and di-tert-butyl ester fluoro-cromolyn derivatives may have therapeutic potential and the radiofluorinated versions might be used for following the efficacy of AD drug treatment.