Stealth Liposomes Encapsulating a Potent ACAT1/SOAT1 Inhibitor F12511: Pharmacokinetic, Biodistribution, and Toxicity Studies in Wild-Type Mice and Efficacy Studies in Triple Transgenic Alzheimer's Disease Mice

Abstract

Cholesterol is essential for cellular function and is stored as cholesteryl esters (CEs). CEs biosynthesis is catalyzed by the enzymes acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2), with ACAT1 being the primary isoenzyme in most cells in humans. In Alzheimer's Disease, CEs accumulate in vulnerable brain regions. Therefore, ACATs may be promising targets for treating AD. F12511 is a high-affinity ACAT1 inhibitor that has passed phase 1 safety tests for antiatherosclerosis. Previously, we developed a nanoparticle system to encapsulate a large concentration of F12511 into a stealth liposome (DSPE-PEG₂₀₀₀ with phosphatidylcholine). Here, we injected the nanoparticle encapsulated F12511 (nanoparticle F) intravenously (IV) in wild-type mice and performed an HPLC/MS/MS analysis and ACAT enzyme activity measurement. The results demonstrated that F12511 was present within the mouse brain after a single IV but did not overaccumulate in the brain or other tissues after repeated IVs. A histological examination showed that F12511 did not cause overt neurological or systemic toxicity. We then showed that a 2-week IV delivery of nanoparticle F to aging 3xTg AD mice ameliorated amyloidopathy, reduced hyperphosphorylated tau and nonphosphorylated tau, and reduced neuroinflammation. This work lays the foundation for nanoparticle F to be used as a possible therapy for AD and other neurodegenerative diseases.

Keywords: ACAT1/SOAT1; Alzheimer’s disease; DSPE-PEG; F12511; amyloid; cholesterol; cholesteryl ester; nanoparticles; phosphatidylcholine; tau.

Figure 1

IV injection of nanoparticle F reduces ACAT activities in both adrenal glands and brain, while nanoparticle K604 only reduces ACAT activity in adrenal glands but not in brain. WT mice were IV injected with either nanoparticle F with F12511 at low concentration (30 mM DSPE-PEG2000 with 5.8 mg F per kg mouse bodyweight) or with empty nanoparticle at zero time and sacrificed after (A) 1 h, (B) 4 h, or (C) 8 h. (D) WT mice were IV injected with nanoparticle K604, with K604 at high concentration (30 mM DSPE-PEG2000 with 24 mg K604 per kg) or with empty nanoparticle at zero time and sacrificed after 4 h. A1^−/− refers to Acat1^−/− mouse as a negative control. Relative ACAT activity was determined by using the ACAT enzyme assay as described in the methods. WT mice per group, at age 4–5 months with gender matched. N = 1 for A1^−/− mouse. Two-way ANOVA was conducted for statistics. p < 0.001 ***; p < 0.01 **; p < 0.05 *; n.s., not significant.

Figure 2

HPLC/MS/MS analyses of F12511. HPLC/MS/MS results (in (top) panel) shown F12511 content in the WT mouse plasma, liver, adrenal glands, and brain after 4, 12, and 24 h post IV delivery of nanoparticle F (at 46 mg F per kg mouse body weight. Most of the mice were perfused with PBS for 15 min to avoid blood contamination of tissues before sample collections. For each mouse tissue measured, each point represents the average of 3 replicates, with 3 HPLC column injections per replicate. DSPE-PEG₂₀₀₀/PC-treated mice were also measured by HPLC/MS/MS. As expected, values for F12511 were under the detectable limit. Results from the table on (top) were replotted in graphs shown at (bottom) by using results from perfused tissue.

Figure 3

The ACAT activity in forebrain, cerebellum, liver, and adrenals gradually return to normal 24 to 48 h after a single IV of nanoparticle F. Adult WT mice were given single IV of nanoparticle F at high dose (~46 mg/kg). At various time points indicated, mice were perfused with PBS. The forebrain, cerebellum/brain stem, adrenal gland, and liver were isolated and homogenized to measure ACAT enzyme activity. One-way ANOVA was conducted to determine statistics. p < 0.001 ***, p < 0.01 **.

Figure 4

Treating normal mice with nanoparticle F or nanoparticles alone produces no overt morphological alterations in central nervous and peripheral tissues. Representative images from WT mice (n = 3–4/group) either untreated or treated with IV injections once per day with DSPE-PEG₂₀₀₀/PC or with nanoparticle F for 7 days. Tissues were collected 48 h after the last injection. (A) Images show hippocampus and dentate gyrus regions (left panels). Images on the right panels show cerebellar folia region. (B) Images show adrenal glands (left panels), and livers (right panels). For adrenal gland images, symbols used: m = medulla; ZR = zona reticularis; ZF = zona fasciculata; ZG = zona glomerulosa. Scale bars: 200 pixels: ~109 µm for 10× enlargement and ~54 µm for 20× enlargement. Scale bar: 200 pixels.

Figure 4

Treating normal mice with nanoparticle F or nanoparticles alone produces no overt morphological alterations in central nervous and peripheral tissues. Representative images from WT mice (n = 3–4/group) either untreated or treated with IV injections once per day with DSPE-PEG₂₀₀₀/PC or with nanoparticle F for 7 days. Tissues were collected 48 h after the last injection. (A) Images show hippocampus and dentate gyrus regions (left panels). Images on the right panels show cerebellar folia region. (B) Images show adrenal glands (left panels), and livers (right panels). For adrenal gland images, symbols used: m = medulla; ZR = zona reticularis; ZF = zona fasciculata; ZG = zona glomerulosa. Scale bars: 200 pixels: ~109 µm for 10× enlargement and ~54 µm for 20× enlargement. Scale bar: 200 pixels.

Figure 5

Biodistribution of nanoparticles. 3-month-old sex-matched WT mice were injected with 8.3 mM of 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Iodide (DIR) encapsulated in DSPE-PEG/PC nanoparticles (NP). Mice were sacrificed after 4 h and perfused with 20 mL of cold 1xPBS. Brains were collected and separated into 2 hemispheres. The right hemisphere was homogenized in lysis buffer and loaded into capillary tubes for imaging capture on the Pearl 500. (A) Representative of mouse brain homogenates in capillary tubes. (B) Quantitative analysis of DiR in mouse brain homogenate. Raw fluorescence read was applied to the generated standard curve to calculate the amount of DỉR in the brain homogenate of injected mice. All images were analyzed in Fiji. N = 3–4 mice/treatment group. Scale bar = 2500 μm. n.d.: not detectable.

Figure 6

IV injections of nanoparticle F reduce mutant hAPP and reduce total acid extractable Aβ1-42 in advance-aged 3xTg AD mice. Male (M) and female (F) 3xTg AD mice at 16–20 months of age were either untreated or treated with DSPE-PEG₂₀₀₀/PC (vehicle) or with nanoparticle F (F12511 at 46 mg/kg), as indicated with daily IV injections once daily for 2 weeks. Mouse brain homogenates were prepared according to procedure described in PMID: 15980612 and PMID: 20133765. (A) Western blots were used to monitor the immature and mature forms of hAPP (two adjacent bands at 105-kDa and 115-kDa) by using mouse monoclonal antibody anti-6E10 (recognizing amyloid Aβ; 1:5000 from Covance). Results are shown as light and dark exposures. Two-way ANOVA was conducted to analyze the signals for mature and immature bands individually. One-way ANOVA was used to analyze total signals comprising both mature and immature bands. Mouse anti-beta-actin antibodies were used as loading controls. (B) ELISA assay used on mouse monoclonal antibody anti-6E10 to monitor total formic acid extractable Aβ1-42 levels in brain homogenates. Procedure for Aβ1-42 extraction was described in PMID: 15980612 and PMID: 20133765. ELISA plates were from Invitrogen. Each sample was measured in quadruplicate. Results are presented as Aβ1-42 signal intensity from mice treated with DSPE-PEG₂₀₀₀/PC (nanoparticle/vehicle) or with nanoparticle F relative to that of untreated control using one-way ANOVA analysis. N.S. not significant; p < 0.01 **, p < 0.05 *.

Figure 7

Nanoparticle F reduces total unphosphorylated mutant human tau (hTau) and hyperphosphorylated human tau (HPTau) while nanoparticles alone reduce HPTau but do not reduce htau in advance-aged 3xTg AD mice. The same brain homogenate samples described in Figure 6 were used for Western blot analyses to monitor hTau (A) and HPTau (B), according to procedure described in PMID: 25930235. Mouse anti-HT7 used for total unphosphorylated human tau (~50 kDa) and mouse anti-AT8 used for hyperphosphorylated human tau (~55–60 kDa) were from Thermo Fisher Scientific, and mouse anti-beta-actin (42 kDa) antibodies served as loading control. Dark and light exposure of each blot is shown. One-way ANOVA was conducted to determine statistics. N.S. not significant; p < 0.01 **, p < 0.05 *.

Figure 8

DSPE-PEG/PC and nanoparticle F alter proinflammatory cytokines profile in 3xTg AD mice (16–20 month after 2 weeks of daily IV/RO injections). Mice were perfused with 20 mL of cold 1xPBS and their brains were collected and homogenized. Forebrain homogenates were analyzed by using MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (32 plex), and data were normalized by using total protein content determined by Lowry assay. (A) Heatmap visualizing average cytokines readings from several biological replicates of each treatment group with a Z-score transformation. (B) Alzheimer’s-related cytokines were plotted individually with 3xTg AD mice forebrain cytokines content. For B, values obtained from each individual cytokine from PBS injected animal is normalized to 1. Unpaired student t-test (two tailed) were performed; p < 0.01 **, p < 0.05 *. N = 4 mice/group.