Liver X Receptor Activation with an Intranasal Polymer Therapeutic Prevents Cognitive Decline without Altering Lipid Levels

The progressive accumulation of amyloid-beta (Aβ) in specific areas of the brain is a common prelude to late-onset of Alzheimer’s disease (AD). Although activation of liver X receptors (LXR) with agonists decreases Aβ levels and ameliorates contextual memory deficit, concomitant hypercholesterolemia/hypertriglyceridemia limits their clinical application. DMHCA (N,N-dimethyl-3β-hydroxycholenamide) is an LXR partial agonist that, despite inducing the expression of apolipoprotein E (main responsible of Aβ drainage from the brain) without increasing cholesterol/triglyceride levels, shows nil activity in vivo because of a low solubility and inability to cross the blood brain barrier. Herein, we describe a polymer therapeutic for the delivery of DMHCA. The covalent incorporation of DMHCA into a PEG-dendritic scaffold via carboxylate esters produces an amphiphilic copolymer that efficiently self-assembles into nanometric micelles that exert a biological effect in primary cultures of the central nervous system (CNS) and experimental animals using the intranasal route. After CNS biodistribution and effective doses of DMHCA micelles were determined in nontransgenic mice, a transgenic AD-like mouse model of cerebral amyloidosis was treated with the micelles for 21 days. The benefits of the treatment included prevention of memory deterioration and a significant reduction of hippocampal Aβ oligomers without affecting plasma lipid levels. These results represent a proof of principle for further clinical developments of DMHCA delivery systems.


Materials
All chemicals were purchased from Sigma-Aldrich or Fluka unless otherwise noted. All solvents were HPLC grade, purchased from Scharlab, Sigma-Aldrich or Acros Organics.
Et3N was dried under 4Å molecular sieves. DMF and CH2Cl2 were dried using a SPS800 solvent purification system from MBRAUN. H2O of Milli-Q grade was obtained from a Millipore water purification system. PEG-[G1]-N3, 1,2 BocHN-PEG-[G1]-N3, 3  MegaView G2. A drop of a solution of micelles (0.1 mg/mL) was settled on a PELCO ® TEM carbon type-B film copper grid (Ted Pella, Inc.) and allowed to dry at room temperature for 12 h. Negative staining was performed by using a droplet of 2% uranyl acetate following standard procedures. The size of the micelles was determined with ImageJ software (version 1.51j8) measuring the line intensity profile across the assemble.
An average diameter of 17±2 nm was obtained by measuring the size of 25 micelles. S6

Integrity of the Ester Bond in PEG-[G1]-DMHCA Micelles by 1 H NMR
The NMR chemical shift of 1 H in alpha to oxygen atoms is very sensitive to the chemical environment. This has been exploited to study the integrity of the ester bond linking DMHCA to the dendritic block inside the micelles during their preparation, storage and lyophilization.
The 1 H in alpha to the hydroxyl group in DMHCA appears as a multiplet at 3.57-3.42 ppm in CDCl3 (highlighted in green in Figure S6). However, after ester formation, this

Immunohistochemistry
Free floating immunohistochemical staining was performed as previously described. 7 Mouse monoclonal primary antibodies used were:

In Vivo Cytotoxicity
The

Data Analysis
All data were analyzed using the Graph-Pad Prism 6 software. Comparison was done by unpaired Student's T-test. Significance was set at p<0.05. Data are presented as mean ± S.E.M.