Synthesis, Radiosynthesis, and Preliminary in vitro and in vivo Evaluation of the Fluorinated Ceramide Trafficking Inhibitor (HPA-12) for Brain Applications

Abstract

Ceramide levels are increased in blood and brain tissue of Alzheimer's disease (AD) patients. Since the ceramide transporter protein (CERT) is the only known protein able to mediate non-vesicular transfer of ceramide between organelle membranes, the modulation of CERT function may impact on ceramide accumulation. The competitive CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide (HPA-12) interferes with ceramide trafficking. To understand the role of ceramide/CERT in AD, HPA-12 can be a useful tool to modulate ceramide trafficking. Here we first report the synthesis and in vitro properties of HPA-12 radiolabeled with fluorine-18 and present preliminary in vitro and in vivo positron emission tomography (PET) imaging and biodistribution data. In vitro results demonstrated that the fluorination did not alter the biological properties of HPA-12 since the [fluorine-19]HPA-12, interferes with 5-DMB-ceramide trafficking in HeLa cells. Radiolabeled HPA-12, [fluorine-18]HPA-12, was obtained with a radiochemical yield of 90% and a specific activity of 73 MBq/μmol. PET imaging on wild-type mice showed hepatobiliary clearance and a brain uptake on the order of 0.3 standard uptake value (SUV) one hour post injection. Furthermore, the biodistribution data showed that after removal of the blood by intracardial perfusion, radioactivity was still measurable in the brain demonstrating that the [fluorine-18]HPA-12 crosses the blood brain barrier and is retained in the brain.

Natural ligand-nonmimetic inhibitors of the lipid-transfer protein CERT

“…9b), suggesting gradual degradation of the inhibitors in the cell culture. This is in agreement with a recent study showing in vivo degradation of fluorinated HPA-12 in mice 26 . The cell-associated levels of these compounds reached peaks during the initial 4 h, partially decreased, and then plateaued up to 72 h (Supplementary Fig.…”

“…The availability of LTP inhibitors remains very limited except for a few LTP types, including sterol or oxysterol transfer proteins [27][28][29][30] and CERT 13,14,17,19 . (1R, 3S)-HPA-12 and its closely related compounds have so far been the only potent inhibitors of CERT effective in living cells 13,14,17,26,31 . Iminosugar-based structures were recently shown to be another type of ceramidemimicking CERT inhibitor, although their effectiveness toward living cells remains unclear 19 .…”

“…CERT inhibitors may serve as pharmaceutical seed compounds to prevent or ameliorate human diseases. A recent study suggested that a fluorinated derivative of HPA-12 can serve as an in vivo probe of CERT in positron emission tomography (PET) imaging 26 . A fluorinated HPCB-5 may also serve as a PET probe of CERT.…”

CERTL Reduces C16 Ceramide, Amyloid-β Levels and Inflammation in a Model of Alzheimer's Disease

“…Recently, the pharmacokinetics of HPA-12was described,and it was proven that the compound reaches the brain intact (24). Here, we found that after 4 weeks,the treatment of HPA-12 increases Cer andAβ levels.…”

“…CERTs are found in at least two isoforms,which differ for the presence of a 26-amino acid serine-rich domain (23). CERTs are expressed in the central nervous system, and they are crucial in embryogenesis and brain development (23)(24)(25). When CERTs' activity is blocked pharmacologically or genetically, by compromising the START domain of the protein, SM production decreases signi cantly (26,27).…”