A novel ¹⁸F-labeled MAG lipase biomarker for differentiating brown and white adipose tissue in the lipid network

Objectives: Brown adipose tissue (BAT) consumes stored lipids and provides non-shivering thermogenesis. Recent studies proposed that enhancing/activating BAT could be a novel therapeutic approach to counteract metabolic diseases, including obesity and type II diabetes. Herein we describe the development of a reversible and peripheral-specific ¹⁸F-ligand targeting monoacylglycerol lipase (MAGL), and its PET application for differentiating brown and white adipose tissue in the lipid network, followed by molecular mechanism investigation. Methods: A novel MAGL inhibitor, (4-(1-(4'-chloro-2-(2-fluoroethoxy)-3'-(trifluoromethyl)-[1,1'-biphenyl]-4-carbonyl)azetidin-3-yl)piperazin-1-yl)(thiazol-2-yl)methanone (FEPAD) was synthesized and evaluated in cell-based and human recombinant MAGL assays for potency in vitro. Target selectivity was verified in fatty acid amide hydrolase (FAAH), cannabinoid receptors (CB₁/CB₂), and activity-based protein profiling (ABPP) assays. ¹⁸F-Isotopologue of FEPAD was synthesized by reacting its phenolic precursor and 1-bromo-2-[¹⁸F]fluoroethane. PET studies and whole body biodistribution were carried out in rodents with focus on adipose tissues, followed by ex vivo validation using immunohistochemistry (IHC), immunofluorescence (IF) and H&E stains. The expression level of MAGL in BAT and WAT was also quantified by western blots. Results: FEPAD was synthesized from methyl 4-bromo-3-hydroxybenzoate in ~10% yield over 4 steps. FEPAD showed strong binding affinity in vitro to hydrolysis assay and human recombinant MAGL inhibition assay with IC₅₀ values of 77.6 and 23.8 nM, respectively, and exhibited excellent target selectivity (> 100 fold) among other major serine hydrolases, including FAAH, and CB₁/CB₂ receptors. The reversible binding profile was confirmed by time-dependent ABPP assay. [¹⁸F]FEPAD was synthesized in 13% RCY with >99% radiochemical purity and >2 Ci/µmol molar activity. The whole body distribution of [¹⁸F]FEPAD was peripheral-specific and consistent with MAGL distribution, and in vivo specificity was confirmed by KML29 pretreatment (3 mg/kg, 70% blockade). Furthermore, [¹⁸F]FEPAD showed characteristic high uptake in BAT (peak value 21.4% %ID/g,) but not in white adipose tissue (WAT, 3.5 %ID/g). Static PET imaging studies showed ~300% and ~200% higher uptake in BAT than that of WAT at 30 and 60 min post injection. Using [¹⁸F]FDG as positive control, FEPAD is not sensitive to cold-stimulation at 4°C. Ex vivo validation was also performed to quantify MAGL in adipose tissues, providing the underlying mechanism for differentiating BAT. Histological observations showed that adipocytes from WAT were much larger than that from BAT. The quantitative measurement also indicated the number of adipocytes from BAT were significantly higher (4.5 fold) than that from WAT in the same size area. In addition, MAGL protein staining was substantially higher in BAT both in IHC and IF staining, indicating BAT contained considerably higher level (4-6 fold) of MAGL-positive cells than WAT. It is worthy of note that although MAGL expression was similar between BAT and WAT based on same amount of protein (60 μgs protein loading per western blot), BAT exhibited significantly higher MAGL density (4.2 fold) compared to WAT. Because WAT contains large mass of lipid within the adipose tissue, resulting a rather lower content of MAGL in terms of micrograms per gram of wet tissue of protein. Conclusion: We have developed the first-in-kind PET probe, FEPAD, for imaging MAGL based on a unique piperidinyl azetidine amide scaffold. This PET tracer not only represents the most potent and specific MAGL PET probe that operates on a reversible binding, but also demonstrated promise for differentiating BAT in adipose tissues. We expect this work will extend our understanding of the roles of MAGL in vivo and provide a new imaging tool for BAT.