CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No. 63/292,191, filed on December 21, 2021, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Exploratory/Developmental Research Grant 1R21NS084328-01A1, National Institute on Aging Grant No. 1K01AG047954-01/05, National Institute on Aging Grant No. 1R56AG062271-01A1, Clinical and Translational Science Awards Grant No. 5TL1TR001875-05, and National Center for Advancing Translational Sciences TRx Program Pilot Grant awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates to compositions and method for regulation of lipid dyshomeostasis and compositions of lecithin cholesterol acyltransferase (LCAT) activators for prophylaxis or amelioration of neurodegeneration, such as Alzheimer’s disease (AD) and Niemann-Pick type C (NPC).

BACKGROUND

Lipid effectors have been identified in a genome wide association study GWAS for AD (Cuyvers and Sleegers, Lancet Neurol, 2016). Furthermore, carriers of the Apolipoprotein E 4 (APOE4) genetic variant associated with AD can exhibit greater brain uptake of Docosahexaenoic acid (DHA), which is an omega-3 fatty acid that is commonly found in the brain (Yassine et al., Alz Res & Therapy, 2017).

Lipid changes, such as enlarged endosomes, have been observed before the accumulation of amyloid 0 (A0) in AD patients (Nixon et al., Neurobio of Aging, 2005 and Cataldo et al., American J. Pathology, 2000). Membrane trafficking and membrane constituents alter cleavage of amyloid precursor protein (APP) can also affect accumulation of A0 (Di Paolo and Kim, Neuroscience Reviews, 2011). Altered lipid metabolism can also be associated with tau accumulation and abnormal clearance (Dall’Armi et al., Nature Comm, 2010 and Dyment et al., Neurobiol Aging, 2015).

Furthermore, ApoE has been reported as an activator of lecithi cholesterol acyltransferase (LCAT), which is an enzyme responsible for the maturation of high density lipoprotein particles in the brain, including ApoE containing particles. Genetic ablation of ApoE and LCAT can lead to complete loss of long chain polyunsaturated cholesteryl ester (Zhao et al., Biochem, 2005; Furbee et al., J Lipid Res, 2002). LCAT enzymatic activity can be reduced by 50% in CSF from patients with a probable AD diagnosis (Demeester et al., J Lipid Res, 2000).

Accordingly, lipid composition and metabolism in the brain can play a role in AD. There is a need for compositions for regulation of lipid dyshomeostasis and LCAT activators for prophylaxis or amelioration of neurodegeneration.

SUMMARY

The present disclosure provides a method for treating and/or preventing a neurodegenerative disorder in a subject in need thereof. In certain embodiments, the disorder is selected from the group consisting of Alzheimer’s Disease, Niemann-Pick type C (NPC), familial LCAT deficiency (FLD) and fish eye disease (FED), and cholesteryl ester storage disease (CESD).

In certain embodiments, the method comprising administering a therapeutically effective amount of an agent that regulates lipid dyshomeostasis. In certain embodiments, the agent is administered via intracerbroventricular injection.

In certain embodiments, the agent prevents the accumulation of amyloid 0 (A0) and/or tau in subject. In certain embodiments, the agent disrupts the expression of a phospholipid modifying enzyme.

In certain embodiments, the agent comprises a functional nucleic acid molecule that targets a transcript encoding a phospholipid modifying enzyme. In certain embodiments, the agent comprises a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule. In certain embodiments, the functional nucleic acid molecule comprises an inhibitory RNA molecule.

In certain embodiments, the phospholipid modifying enzyme is selected from the group consisting of synaptojaninl (Synj l), phospholipase A2 (PLA2), phospholipase DI (PLD1), phospholipase D2 (PLD2), phospholipase C (PLC), phosphoinositide 3 kinase-C2a (P12K), and Myc box-dependent-interacting protein 1 (BINI) and combinations thereof.

In certain embodiments, the agent comprises a small molecule inhibitor. In certain embodiments, the small molecule inhibitor comprises an inhibitor of phospholipase D 1 and phosphlipase D2. In certain embodiments, the small molecule inhibitor comprises MI. 299.

In certain embodiments, the method comprising administering a therapeutically effective amount of a lecithin cholesterol acyltransferase (LCAT) activator. In certain embodiment the LCAT activator comprises the general formula as set forth in Figure 13.

In certain embodiments, the method further comprising administering a polyunsaturated fatty acid (PUFA). In certain embodiments, the PUFA is a phosphatidylcholine (PC) or lysoPC (LPC) containing 22:6 at the sn-2 position.

The present disclosure also provides a composition comprising a therapeutically effective amount of an agent that regulates lipid dyshomeostasis for use in treating and/or preventing a neurodegenerative disorder in a subject in need thereof. In certain embodiments, the agent comprises a functional nucleic acid molecule that targets a transcript encoding a phospholipid modifying enzyme.

In certain embodiments, the disorder is selected from the group consisting of Alzheimer’s Disease, Niemann-Pick type C (NPC), familial LCAT deficiency (FLD) and fish eye disease (FED), and cholesteryl ester storage disease (CESD).

In certain embodiments, the agent comprises a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule. In certain embodiments, the functional nucleic acid molecule targets a transcript encoding a phospholipid modifying enzyme that contributes to lipid dyshomeostasis. In certain embodiments, the functional nucleic acid molecule comprises an inhibitory RNA molecule.

In certain embodiments, the phospholipid modifying enzyme is selected from the group consisting of synaptojaninl (Synj l), phospholipase A2 (PLA2), phospholipase DI (PLD1), phospholipase D2 (PLD2), phospholipase C (PLC), phosphoinositide 3 kinase-C2a (P12K), and Myc box-dependent-interacting protein 1 (BINI) and combinations thereof.

In certain embodiments, the agent comprises a small molecule inhibitor. In certain embodiments, the small molecule inhibitor comprises an inhibitor of phospholipase D 1 and phosphlipase D2. In certain embodiments, the small molecule inhibitor comprises ML 299.

In certain embodiments, the composition comprising a therapeutically effective amount of a lecithin cholesterol acyltransferase (LCAT) activator. In certain embodiments, the LCAT activator comprises the general formula as set forth in Figure 13. In certain embodiments, the method further comprising administering a polyunsaturated fatty acid (PUFA). In certain embodiments, the PUFA is a phosphatidylcholine (PC) or lysoPC (LPC) containing 22:6 at the sn-2 position.

The present disclosure also provides a kit for use in treating and/or preventing a neurodegenerative disorder in a subject in need thereof. In certain embodiments, the kit comprises an agent that regulates lipid dyshomeostasis. In a further embodiment, the kit comprises a therapeutically effective amount of a lecithin cholesterol acyltransferase (LCAT) activator.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the disclosed subject matter can be acquired by referring to the following description taken in conjunction with the accompanying drawings, which relate to embodiments of the present disclosure. Certain abbreviations used in these figures and the descriptions thereof are explained in further detail in the remainder of this specification.

Figure 1 is a schematic diagram of the genetic disruption-based screening method used to identify enzymes active in brain lipid metabolism in a mouse AD model.

Figure 2, which includes (A)-(F), presents results from the optimization of 384-well plate platform for detection of synapses in mESN.

Figure 3, which includes (A) and (B), presents results of a pilot screen of multiple phospholipid modifying enzymes.

Figure 4 is a diagram of a novel metabolic pathway identified in the cell model of AD.

Figure 5, which includes (A)-(D), presents results of the investigation of effects on pathologically enriched lipids in AD.

Figure 6 is a diagram showing experimental setup for single intracerebro ventricular injection (ICV) injection for proof of concept for LCAT activation with Compound 2 ( Manthei et al., eLife, 2018) and subsequent behavioral testing.

Figures 7A and 7B demonstrate the LCAT activator Compound 2 ameliorates memory in contextual fear conditioning (FC), fear conditioning (FC). The cued FC, a hippocampusindependent task, was used as a control. Briefly, mice were exposed for 2 minutes to the context before the onset of a tone (a 30 second, 85 dV sound at 2800 Hz, serving as a condition stimulus (CS). In the last 2 seconds of the CS, mice received a 2 second, 0.80 mA foot shock unconditional stimulus (US) though the bars of the floor. Freezing, which is defined as a species-specific defensive reaction characterized by the lack of movement, associated with crouching posture was measured right after the end of the CS/US for 30 seconds. The contextual memory test was performed 24 h later, by reexposure of the mice to the same context and by measuring the proportion of freezing time during 5 minutes. To evaluate cued fear learning, 24 hours after contextual testing, mice were placed into a novel context for 2 minutes (pre-CS test), followed by an exposure to the S for 3 minutes (S test, during which freezing was measured.

Figure 7A shows plot of average percent of time spent freezing (+/- SEM) during initial exposure to the training context (baseline) and 24 hours after footshock for the indicated groups after 3 days of the single intracerebro ventricular injection (ICV) injection of the Compound 2 (50 pg/Kg) ( Manthei et al., eLife, 2018). An ANOVA analysis for freezing at 24 hours showed a significant difference among groups (F (1,9) = 6.696, P = 0.029). Tukey’s multiple comparison show that only the Tg/Vehicle group was significantly different compared to WT/Vehicle (p = 0.023). No differences in baseline freezing day 1 were observed among groups (ANOVA: F(1 ,9) = 0.339), p = 0.574). Figure 7B shows a plot of average percent of time spent freezing (+/- SEM) during pre-cue and post-cue F for the indicated groups. No difference in pre- and post-cue on day 3 were observed among groups (ANOVA: pre-cue f( 1 ,9) = 0.195, p = 0.669; post-cue (F(l,9) = 1.164, p = 0.309). N= 3 WT/Vehicle, N = 4 WT/Compound 2, 2 Tg/Vehicle, and 4 Tg/Compound 2.

Figure 8 demonstrates the LCAT activator Compound 2 ( Manthei et al., eLife, 2018) ameliorates memory in the Novel Object Recognition test. The plot shows the average object preference ratio for the indicated groups after 3 days of single ICV injection of the Compound 2 (50 ug/Kg). Statistical analysis was done with Kruskal -Wallis Test resulting in [X ² (3)=15.819; p=0.001; Tg/Vehicle Tg/Compound 2: p= 0.020; Tg/Vehicle WT/Compound 2: p= 0.001.

Figure 9 demonstrates ICV injection of the LCAT activator Compound 2 (Manthei et al., eLife, 2018) did not affect motor behavior in the Open Field Test as indicated by no difference found in (A) Distance traveled (B) Center Crossings (C) Time spent in the center of the open field (Time center), and (D) Speed.

Figure 10 demonstrates ICV injection of the LCAT activator Compound 2 ( Manthei et al., eLife, 2018) did not affect amyloid 0-peptide 42 or amyloid 0-peptide 40 levels in the hippocampus (A) or cortex (B). Figure 11 demonstrates ICV injection of the LCAT activator Compound 2 (Manthei et al., eLife, 2018) resulted in a significant decrease in interleukin 4 (IL -4) levels from mouse brain.

Figures 12A-12D demonstrate the results of assay optimization and LCAT activator synthesis and activity. Figure 12A shows the chemical structured for synthesized LCAT Activators Compound A (Freeman et al., J. Pharmacol and Exp. Therap. 2017) and Compound 2 ( Manthei et al., eLife, 2018). Figure 12B shows rLCAT incubated with increasing amount of substrate MUP and fluorescence read over 45 minutes. The miniaturization MUP assay was performed in a 1536-well platform. Figure 12C shows the detection of LCAT activity using a MUP assay 384-well platform. Figure 12D shows the effect of Compound A on LCAT activity. Recombinant LCAT was incubated in presence or absence of synthesized LCAT activator Compound A. Fluorescence increased over time in reactions containing LCAT and increasing concentrations of Compound A. The increased fluorescence was due to LCAT activity compared to the no enzyme control (no ENZ).

Figure 13 provides a generic structure of LCAT activators.

Figure 14 provides specific examples of LCAT activators disclosed herein (a) and a blood-brain barrier permeable prodrug.

Figure 15 provides a general scheme for synthesis of LCAT activators according to the present disclosure.

DETAILED DESCRIPTION

The present disclosure provides compositions and methods for regulation of lipid dyshomeostasis and compositions of lecithin cholesterol acyltransferase (LCAT) activators for prophylaxis or amelioration of neurodegeneration.

Non-limiting embodiments of the present disclosure are described by the present specification and Examples.

For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

1. Definitions;

2. Regulation of Lipid Dyshomeostasis;

3. LCAT Activators;

4. Compositions and Methods of Treatment; and

5. Kits. 1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosed subject matter belongs. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g. , within 5 -fold, or within 2-fold, of a value.

An “individual” or “subject" herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

An “effective amount” or “therapeutically effective amount” is an amount effective, at dosages and for periods of time necessary, that produces a desired effect, e.g., the desired therapeutic or prophylactic result. In certain embodiments, an effective amount can be formulated and/or administered in a single dose. In certain embodiments, an effective amount can be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen. As used herein, the term “treating” or “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing cancer, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By “preventing” progression of a disease or disorder, a treatment can prevent deterioration due to a disorder (e.g., a cancer) in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment can prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder. The decrease can be a 10- 99% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%) decrease in severity of complications, impairments, or symptoms. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

By “increase” is meant to alter positively by at least about 5%. A positive alteration can be an increase of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

By “reduce” is meant to alter negatively by at least about 5%. A negative alteration can be a decrease of about 5%, about 10%, about 25%, about 30%, about 50%, about 75% or more, even by about 100%.

The term “dosage” is intended to encompass a formulation expressed in terms of total amounts for a given timeframe, for example, as pg/kg/hr, pg/kg/day, mg/kg/day, or mg/kg/hr. The dosage is the amount of an ingredient administered in accordance with a particular dosage regimen. A “dose” is an amount of an agent administered to a mammal in a unit volume or mass, e.g., an absolute unit dose expressed in mg of the agent. The dose depends on the concentration of the agent in the formulation, e.g., in moles per liter (M), mass per volume (m/v), or mass per mass (m/m). The two terms are closely related, as a particular dosage results from the regimen of administration of a dose or doses of the formulation. The particular meaning, in any case, will be apparent from the context.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Ranges disclosed herein, for example, “between about X and about Y” are, unless specified otherwise, inclusive of range limits about X and about Y as well as X and Y. With respect to sub-ranges, “nested sub-ranges” that extend from either endpoint of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 can include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, test tubes and cell cultures.

As used herein, the term “behavioral impairment” or “behavioral deficit” refers to an acquired deficit in one or more of memory function, problem solving, orientation, attention, visual conceptualization, spatial conceptualization, executive and/or abstraction that impinges on an individual's ability to function independently.

The term “nucleic acid” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. A “nucleic acid fragment” is a portion of a given nucleic acid molecule.

A “nucleotide sequence” is a polymer of DNA or RNA that can be single- or doublestranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” are used interchangeably and can also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

The term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, “gene” refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. “Genes” also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. “Genes” can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and can include sequences designed to have desired parameters. An “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome.

“Naturally occurring,” “native,” or “wild-type” is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and that has not been intentionally modified by a person in the laboratory, is naturally occurring.

The term “inhibitory RNA” is meant to include a functional nucleic acid molecule that contains a sequence that is complementary to a target nucleic acid that mediates a decrease in the level or activity of the target nucleic acid. Non-limiting examples of inhibitory RNAs include interfering RNA, small hairpin RNA (shRNA), small interfering RNA (siRNA), ribozymes, antagomirs, and antisense oligonucleotides. Methods of making inhibitory RNAs are known in the art. An “interfering RNA” can refer to any double stranded or single stranded RNA sequence, capable of either directly or indirectly (i.e., upon conversion) of inhibiting or down-regulating gene expression by mediating RNA interference. “RNA interference” refers to the selective degradation of a sequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. As used herein, the phrase “post-transcriptional processing” refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzymes Dicer and/or Drosha. A “short hairpin RNA” or “short interfering RNA” or “shRNA” or “small interfering RNA” or “siRNA” or “antisense nucleic acid” is a RNA duplex of nucleotides that is targeted to a nucleic acid sequence of interest, for example, synaptojaninl (Synj 1), phospholipase A2 (PLA2), and phospholipase D2 (PLD2). As used herein, the term “shRNA” is a generic term encompassed by RNA interference. A “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule. shRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the shRNA is complementary to a nucleotide sequence of the targeted gene.

2. Regulation of Lipid Dyshomeostasis

The present disclosure provides for a prophylactic or ameliorative effect of compositions for regulation of lipid dyshomeostasis corresponds to the observations contained herein regarding the role of lipid dyshomeostatis in AD. Prevention, reduction, or reversal of lipid dyshomeostasis can provide prophylactic or ameliorative effects for AD or other similar neurodegenerative diseases. Compositions and methods according to the present disclosure can disrupt the function of synaptojaninl (Synjl), phospholipase A2 (PLA2), phospholipase D2 (PLD2), or any combinations thereof. Compositions and methods according to the present disclosure can disrupt acyl chain remodeling in the brain of patients with AD or at risk of developing AD.

Lipidomic data from autopsy brain, human plasma and animal models highlights severe lipid dyshomeostasis in AD. The importance of lipid metabolism in AD is supported by GWAS studies, which have identified multiple lipid modifying enzymes and interacting proteins. Further, certain mouse genetic studies have shown benefits of genetic disruption of Synjl, PLA2, and PLD2. Haploinsufficiency of synaptojaninl (Synjl), a phosphoinositide phosphatase, and concurrent maintenance of phosphoinositide levels, ameliorates behavioral deficits in a mouse model of AD even when pathological amyloid accumulation is present. Similarly, disruption of phospholipid modifying enzymes PLD2 and PLA2 resulted in rescue of behavioral deficits despite the pathological accumulation of amyloid. These observations indicate that certain specific pathways in lipid metabolism can underlie AD disease mechanisms leading to behavioral impairment.

Specifically, the loss of polyunsaturated fatty acids among multiple phospholipid classes is common in AD affected human brain and mouse models. Lipid species can be altered in mouse brain in a regionally specific manner determined by imaging mass spectrometry.

Synthesis of these findings and the fact that disruption of phospholipid modifying enzymes Synjl, PLA2 and PLD2 rescue behavioral deficits in spite of pathological accumulation of amyloid, implicate acyl chain remodeling as a therapeutic target in AD.

The present disclosure provides for high content screening for Ap-induced synapse loss in mature embryonic stem cell derived neurons (ESN) identified components of lipid metabolism responsible for resistance to A -triggered synapse loss using knock-down in mice. It also provides that acyl chain remodeling/Land’s cycle in lipid metabolism is an important component of AD disease pathology and a target for the development of biomarkers and therapeutics. In certain embodiments, the present disclosure provides for additional proteins for preventive and therapeutic targeting, including but not limited to lecithin-cholesterol acyltransferase (LCAT; discussed below), cholesterol ester transfer protein (CETP), ATP- binding cassette sub-family A member 1 (ABCA1), ATP-binding cassette sub-family A member 7 (ABCA7), clusterin (apolipoprotein J) (CLU (ApoJ)), apolipoprotein E (ApoE), acyl-CoA synthetase long chain family member 6 (ACSL6), major facilitator superfamily domain containing 2A (MFSD2a), and combinations thereof.

3. LCAT Activators

The present disclosure provides compositions including a combination of LCAT activator moieties and phosphatidylcholine (PC) or lysoPC (LPC) containing 22:6 at the sn-2 position, as well as compositions comprising PC or LPC 22:6 as a carrier of polyunsaturated fatty acids (PUFA) for incorporation into phospholipid and cholesterol metabolism in the brain.

Alzheimer’s disease and Niemann-Pick type C (NPC) are terminal, neurodegenerative diseases. Certain research on AD therapeutics has been focused on decreasing biogenesis or clearance of amyloid plaques and tau, two hallmarks of the disease based on protein aggregates, and have failed to prevent cognitive decline in multiple Phase III clinical trials. Certain therapeutics target AD symptoms but have limited long term efficacy often losing effectiveness after several years. Further, many late stage clinical trials for new AD therapeutic strategies targeting clearance or biogenesis of amyloid have failed efficacy trials. Therefore, there is an unmet need for therapeutic intervention in Alzheimer’s disease and NPC. Apolipoprotein E (ApoE) variants are a strong risk factor for developing Alzheimer’s disease, after age. However, cholesterol dyshomeostasis in the brain associated with AD is not yet fully understood. Certain studies showed depletion of specific cholesteryl esters in ventricular fluid including docosahexaenoic cholesteryl ester (CE 22:6) (Montine et al., 1997. Am J. Path). Other studies have corroborated the dysregulation of lipid classes defined by lipid head group as well as the disregulation of docosahexaenoic acid across multiple lipid classes including phosphatidylcholine, phosphatidylethanoloamine and phosphatidylserine (Whiley et al., 2014, Neurobiol Aging; Soderberg et al., 1991. Lipids; Cunnane et al., 2012, JAD). It has also been shown that there is a global depletion of polyunsaturated fatty acids (PUFA) including docosahexaenoic acid in AD patient brain and mouse models suggesting that dysregulation of PUFA and DHA can be critical factors in AD pathogenesis. The transfer of docosahexaenoic acid from the sn-2 position of phospholipids resulting in the esterification of cholesterol is controlled by Lecithin: Cholesterol Acyltransferase (LCAT). LCAT is responsible for the maturation of high density lipoprotein particles in the brain including ApoE containing particles.

Multiple cell types are involved in the cholesterol transport in the brain. Nascent ApoE containing particles are made in astrocytes, subsequently lipidated though unknown mechanisms and responsible for distribution of cholesterol and lipids to neurons (Vitali et al., 2014, Cardiovasc. Res.). ApoE has been reported as the major activator of LCAT activity and genetic ablation of ApoE and LCAT leads to complete loss of long chain polyunsaturated CE (Zhao et al., 2005, Biochem; Furbee et al., 2002, J. Lipid Res.). Further, it has been reported that LCAT enzymatic activity is reduced by 50% in CSF from patients with a probable AD diagnosis (Demeester et al, 2000, J. of Lipid Research). LCAT has been shown to be inhibited by certain phosphatidylcholine hydroperoxides which can accumulate during aging (Davit- Spraul et al., 1999, FEBS Letters) and physiologically relevant aldehydes (acetaldehyde, acrolein, hexanal, 4-hydroxynonenal, and malondialdehyde) (McCall et al, 1995, Arterioscler Thromb Vase Biol.). Certain sulfhydryl-reactive beta-lactams were developed as class of LCAT activators for use with familial LCAT deficiency (FLD) and cardiovascular disease (Freeman, et al., 2017, J. Pharmacology and Exp. Therapeutics).

Certain data has indicated that aggressive intranasal supplementation with phosphatidylcholine containing docosahexaenoic acid at the sn-2 position (PC 18:0,22:6) was able to partially rescue behavioral deficits in Tg2576, AD mouse model (results not significant due to a small n). Intranasal PC 18:0,22:6 can deliver 22:6 to critical neuronal site of action perhaps through formation of CE 22:6 and interaction with ApoE. Alternatively, lipid head group modification of PC 22:6 while preserving the LCAT catalytic activity (described in Freeman, et al., 2017, J. Pharmacology and Exp. Therapeutics and Davit-Spraul) can allow use of PC or LPC 22:6 as a carrier of PUFA for incorporation into phospholipid and cholesterol metabolism in the brain. The present disclosure furthermore relates to compositions including small molecules that are capable of activating LCAT activity. The small molecule compositions can lead to cholesteryl ester formation and can be capable of mobilizing cholesterol for amelioration of NPC and AD associated behavioral phenotypes in mouse models.

Aberrant cholesterol metabolism in the brain has been associated with AD risk in patient populations with a protective valine variant in Cholesteryl Ester Transfer Protein (CETP), which slows memory decline and incidence of dementia and interacts with ApoE genotype (Sanders et al., 2010, JAMA; Sundermann et al., 2016, Neurobiol Aging). Further, an acyl-coenzyme A:cholesterol acyltranserase (ACAT) inhibitor has been shown to reduce amyloid pathology in a mouse model of AD (Hutter-Pai er et al., 2004). The targeting HDL/cholesterol metabolism for AD is reviewed in Osherovich, Cholesterol Metabolism Adds up, SciBX, 2010 and for cardiovascular disease it is reviewed in Verdier et al., Arch Cardiovasc Disease, 2013. LCAT can be more effective due to direct regulation, functional and genetic interaction with ApoE. This can corroborate the ApoE associated HDL pathological changes found in AD patient CSF (Yang et al., 2015, J Neuropath Exp. Neurol).

The present disclosure provides that a small molecule can be capable of activating activity of LCAT. The treatment strategy can be both a prophylactic supplement which can delay or prevent onset of Alzheimer’s disease as well as an interventional therapeutic strategy in AD and NPC. Therefore, the therapeutic strategy can impact on Alzheimer’s disease prevention, the standard of care for disease management, and modify disease progression in AD and NPC.

Cholesterol metabolism has been established as a critical pathogenic mechanism in Neimann Pick-type C disease (NPC) and Alzheimer’s disease (AD). In NPC, mutations in NPC gene lead to aberrant cholesterol accumulation in the endosomal compartment of neurons. The gene product of NPC Niemann Pick type-C proteins type-1 and -2 (NPC1/NPC2) are responsible for transport of free cholesterol to intracellular membranes within neurons. In AD, variants of ApoE4, an apolipoprotein involved in cholesterol transport between astrocytes and neurons, is an important risk factor for late onset sporadic AD (Chen et al., 2014). Interestingly, these two diseases converge on pathological mechanisms based on cholesterol dyshomeostasis which have potential to uncover a common link to neurodegeneration (Nixon, 2004, Malnar, 2014). In AD, cholesterol has been shown to affect the processing of Amyloid Precursor Protein (APP) and to accumulate with pathological amyloid -peptide (A ) containing plaques (Malnar et al., 2014; Panchai et al., 2010). In NPC, pathological accumulation of histological hallmarks of AD, plaques (A ) and neurofibrillary tangles (tau) and have been described (Malnar et al., 2014). Further, increased cerebrospinal fluid (CSF) levels of A and tau in patients and aberrant APP processing in mouse models and has been shown described (Mattsson et al, 2012a; Mattsson et al., 2012b, Mattsson et al., 2011, Kodam et al., 2010).

In certain embodiments, a LCAT activator comprises a piperidinylprazolopyridinge or piperidinylimidazopyridine LCAT activator, or derivatives thereof. In certain embodiments, the LCAT activator binds exclusively to the membrane-binding domain of LCAT. In a further embodiments, a LCAT activator comprises a compound having the general formula as set forth in Figure 13 (Compound 2), or a structurally related compound. In a further embodiments, a LCAT activator comprises, but is not limited to, the compound 3-[5-(ethylthio)-l ,3,4- thiadiazol-2-ylthio]pyrazine-2 -carbonitrile (Compound A; Fig. 12A), and other sulfhydrylreactive compounds based on monocyclic -lactams.

4. Compositions and Methods of Treatment

The present disclosure provides for compositions for use in the methods disclosed herein. In certain embodiments, a composition can include a molecule, e.g., therapeutic agent, that modulates or regulates lipid metabolism or lipid dyshomeostasis. In certain embodiments, a composition disclosed herein can be used for prophylaxis or amelioration of neurodegeneration.

In certain embodiments, the therapeutic agent is a functional nucleic acid molecule. In certain embodiments, the therapeutic agent is an inhibitory RNA. In certain embodiments, the shRNAs are targeted to the sequence encoding Synjl, PLA2, and PLD2.

In some embodiments, the length of the duplex of shRNAs is less than 30 base pairs. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length. In some embodiments, the length of the duplex is 19 to 25 base pairs in length. In certain embodiments, the length of the duplex is 19 or 21 base pairs in length. The RNA duplex portion of the shRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. In certain embodiments, the loop is 9 nucleotides in length. The hairpin structure can also contain 3' or 5' overhang portions. In some embodiments, the overhang is a 3' or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

In certain embodiments, the therapeutic agent targets a phospholipid modifying enzyme, including but not limited to synaptojaninl (Synjl), phospholipase A2 (PLA2), phospholipase DI (PLD1), phospholipase D2 (PLD2), phospholipase C (PLC), phosphoinositide 3 kinase-C2a (P12K), and Myc box-dependent-interacting protein 1 (BINI), and combinations thereof.

In certain embodiments, the antisense nucleic acid, a shRNA, or a siRNA is homologous to at least a portion of an Synj 1, PLA2, and PLD2 nucleic acid sequence, wherein the homology of the portion relative to the Synj 1, PLA2, and PLD2 sequence is at least about 75 or at least about 80 or at least about 85 or at least about 90 or at least about 95 or at least about 98 percent, where percent homology can be determined by, for example, BLAST or FASTA software.

In certain embodiments, the antisense nucleic acid, shRNA, or siRNA homologous portion constitutes at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides. In certain embodiments, the antisense nucleic acid, shRNA, or siRNA molecules have up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 75, or up to 100 nucleotides in length. In certain embodiments, the antisense nucleic acid, shRNA, or siRNA molecules include DNA or atypical or non- naturally occurring residues, for example, but not limited to, phosphorothioate residues.

In certain embodiments, the antisense nucleic acid, shRNA, or siRNA molecules disclosed herein can be expressed from a vector or produced chemically or synthetically. Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art.

In certain embodiments, the therapeutic agent is a small molecule activator. In certain embodiments, the therapeutic agent is a lecithin cholesterol acyltransferase (LCAT) activator. In certain embodiments, the LCAT activator has a chemical structure set forth in Figures 13- 15.

In certain embodiments, the compositions disclosed herein can comprising a combination of LCAT activator moieties and phosphatidylcholine (PC) or lysoPC (LPC) containing 22:6 at the sn-2 position, as well as compositions comprising PC or LPC 22:6 as a carrier of polyunsaturated fatty acids (PUFA) for incorporation into phospholipid and cholesterol metabolism in the brain.

The present disclosure provides methods for the treatment of a subject having a neurodegenerative disease or disorder. In certain embodiments, the neurodegenerative disease or disorder is associated with altered lipid metabolism or lipid dyshomeostasis. In certain embodiments, the neurodegenerative disease or disorder is associated with increased protein aggregates, for example, A or tau accumulation and abnormal clearance. In certain embodiments, the neurodegenerative disease or disorder is associated with lysosomal storage defects.

In certain embodiments the neurodegenerative disorder or disease that can be treated according to the disclosed subject matter includes, but is not limited to, alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), ataxiatelangiectasia, zeroidea neuronal lipofuscinosis, Batten's disease, bovine spongiform encephalopathy ( BSE), Canavan disease, childhood cerebral palsy, cholesteryl ester storage disease (CESD), Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, Familial LCAT deficiency (FLD), fish eye disease (FED), frontotemporal lobular degeneration, Huntington's disease, HIV-associated dementia, Kennedy disease, Lewy body dementia, neuroborreliosis, disease of Machado-Joseph, multisystemic atrophy, multiple sclerosis, multiple sulfatase deficiency, mucolipidosis, narcolepsy, Niemann Pick disease, Parkinson's disease, Pick's disease, Pompe's disease, primary lateral sclerosis, prion diseases, progressive supranuclear paralysis, Refsum, Schilder's disease, degener Subacute combined action of spinal cord secondary to pernicious anemia, Spielmeyer-Vogt-Sjogren-Batten disease, spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease or dorsal tabes. In a further embodiment, lipid storage disorder (LSD) is Niemann-Pick type C (defect NPC1 and / or NPC2), Smith-Lemli-Opitz syndrome (SLOS), a congenital error of cholesterol synthesis, Tangier disease, Pelizaeus-Merzbacher disease, a zeroide neuronal lipofuscinosis, a primary glycosphingolipidosis, Farber's disease or multiple sulfatase deficiency. For example, primary glycosphingolipidosis is Gaucher disease, Fabry disease, GM1 gangliosidosis, GM2 gangliosidosis, Krabbe disease or metachromatic leukodystrophy (LDM). In addition, LSD is NPC, Tay-Sachs disease, Sandhoff disease, GM1 gangliosidosis, disease of Fabry, a neurodegenerative mucopolysaccharidosis, MPS I, MPS IH, MPS IS, MPS II, MPS III, MPS IIIA, MPS IIIB, MPS IIIC, MPS HID, MPS, IV, MPS IV A, MPS IV B, MPS VI, MPS VII, MPS IX, a disease with secondary lysosomal involvement, SLOS or Tangier disease. In a further embodiment, the neurodegenerative disease is cerebellar ataxia, Niemann Pick's disease, parkinsonism, Gaucher's neuropathic disease, Sandhoffs disease, Louis-Barr syndrome, Alzheimer's disease, Parkinson's disease, atrophy multisystemic, frontotemporal dementia or Parkinson's syndrome of the lower limbs. In still a further embodiment of the kits or uses, the neurodegenerative disease is Niemann Pick disease, Niemann Pick type C, Niemann Pick type A, Tay-Sachs disease, Sandhoff disease, amyotrophic lateral sclerosis (ALS), cerebellar-type multisystemic atrophy (AMS-C), frontotemporal dementia with parkinsonism, corticobasal degeneration syndrome, progressive supranuclear paralysis or cerebellar downward nystagmus. In certain embodiments, LSD is Niemann Pick disease, Niemann Pick type C, Niemann Pick type A, Tay-Sachs disease, Sandhoff disease or type II mucolipidosis.

As a further non-limiting example, where the disorder is Alzheimer’s Disease or Niemann Pick type C, signs or symptoms that can be reduced or otherwise ameliorated according to the disclosed subject matter include impairment of short-term memory, impairment of abstract thinking, impairment of judgment, impairment of language skills, and mood changes.

In certain embodiments, the therapeutic agent or LCAT activator disclosed herein can be administered to the subject by any suitable route known in the art, including, but not limited to, oral, parenteral, topical, intravenous, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional, intra-arterial, intrathecal, or intracerbroventricular. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

5. Kits

The present disclosure provides kits for use in the disclosed methods. In certain embodiments, a kit can include a container that includes a therapeutic agent or a pharmaceutical formulation thereof. In certain embodiments, the container can include a single dose of the therapeutic agent or a pharmaceutical formulation thereof or multiple doses of the therapeutic agent or a pharmaceutical formulation thereof. A container can be any receptacle and closure suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.

In certain embodiments, the kit can further include a second container that includes a solvent, carrier, and/or solution for diluting and/or resuspending the therapeutic agent or a pharmaceutical formulation thereof. For example, but not by way of limitation, the second container can include sterile water.

In certain embodiments, the kits include a sterile container that contains the therapeutic agent or a pharmaceutical formulation thereof; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

In certain embodiments, the kit can further include instructions for administering the therapeutic agent or a pharmaceutical formulation thereof. The instructions can include information about the use of the therapeutic agent or a pharmaceutical formulation thereof for treating a subject having neurodegenerative disorder. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treating a neurodegenerative disorder; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. For example, but not by way of limitation, the instructions can describe the method for administration and the dosage amount.

In certain embodiments, the kit can further include a device for administering the therapeutic agent or a pharmaceutical formulation thereof. For example, but not by way of limitation, the device can include a syringe, catheter, e.g., implantable catheter, and/or pump.

EXAMPLES

Example 1: Candidate based screen for resistance to A f- triggered synapse loss

As illustrated in Figure 1, various candidate enzymes were screened using genetic disruption in a mouse AD model to determine which enzymes, if disrupted, rendered the mice resistance to Ap-triggered synapse loss. Synj 1 , PLA2 and PLD2 were found to rescue behavior deficits.

Example 2: Optimization of 384-well plate platform for detection of synapses in mESN

In Figure 2A cells were plated at increasing density, matured for 21 days and fixed. Nuclei were detected with Hoechst Stain and detected using In Cell Analysis Software.

In Figure 2B postsynaptic density protein 95 (PSD-95) and synaptophysin (S-physin) were detected by immunostaining (antibodies from Millipore and Epitomics, respectively) and granules were detected using In Cell Analysis Software.

In Figure 2C the ratio of PSD-95 granules to S-physin granules was calculated for each cell density and found to be approximately 1: 1 for densities greater than IxlO ⁵ cells/cm ² .

Figure 2D shows representative images for nuclei (detected by Hoechst stain, Sigma); PSD-95 (detected with Alexa 488, Molecular Probes) and S-physin (detected with Alexa 568, Molecular Probes).

In Figure 2E duplicate wells at a density of 1.5xl0 ⁵ cells/cm ² were treated with Abeta, fixed and stained for PSD-95 and S-physin and In Cell Analyzer/CCD camera was used to scan the plate.

In Figure 2F the In Cell Investigator software was used to detect the granule (based on size and intensity) at low and high sensitivity comparing 1 or 6 fields/well andPSD-95/S-physin ratio was calculated as above from which Z-factor was calculated. Example 3: Pilot screen identifies multiple phospholipid modifying enzymes

Results of a pilot screen of candidate phosphoinositide metabolizing enzymes and effectors using short hairpin RNA (shRNA) Lentiviral particles (Sigma Mission Particles) are presented in Figure 3.

In Figure 3A (ESN) were differentiated and matured for functional synapse formation (div 21) in 384-well glass bottom plates. Neurons were infected with shRNA viral particles for 3 days and challenged with A 42 or DMSO (0.2%, control) containing media. PSD-95 and S- physin positive puncta were identified by immunoreactivity to specific antibodies (Millipore and Epitomics respectively) and detected using an In Cell 2000 Analyzer equipped with a large CCD camera with 20x objective using deconvolution. Puncta with defined intensity, particle area, particle shape and size were identified using Granule mode in the In Cell Investigator. Non-silencing, green fluorescent protein (GFP)-silencing and GFP expressing lenti-viral particles were used as negative controls (-) and Synj 1 shRNA was used as a positive control (+)•

Figure 3B shows resulting hits from the pilot screen of candidate phosphoinositide metabolizing enzymes and effectors using shRNA Lenti-viral particles (Sigma Mission Particles). ESN were challenged with A [342 containing media or control media (DMSO) for 24 hours. The ratio of PSD-95/S-physin was determined in 3 fields of duplicate well shRNA. Synj 1 was able to prevent synapse loss in presence of A 42 (McIntire et al., 2012) and was the positive control (+). Hits were defined by maintenance of PSD-95/S-physin ratio 4 standard deviations (shown by dashed blue line) greater than the mean of the negative control (A [342 treated neurons). Lipid phosphatases including Synj 1 (positive control) were identified as well as phospholipase C, phospholipase D, phosphoinositide 3 kinase-C2a, and a novel interacting protein Synjl, BINI. Candidates able to completely prevent A(342 induced synapse loss (redline in A) are indicated in bold red.

Example 4: Novel metabolic pathway identified in cell model of AD

Figure 4 presents the results of lipidomic analysis of metabolic pathways affected by phospholipase D (PLD) inhibition. Figure 4 includes a diagram of a method for PLD activity detection. Human embryonic kidney cells (HEK293T) overexpressing APP with the Swedish mutation were treated with the PLD inhibitor ML-299 (Caymen Chemicals and O’Reily et al., J. Med Chem, 2013) for 24 hours. Phosphatidylethanol (PEtOH), a unique lipid formed by PLD in the presence of alcohol, was detected. Lipidomic analysis indicated only cholesteryl esters (CE) were affected and further analysis showed that CE species are up-regulated after PLD inhibition.

Example 5: Lipidomics identified pathological lipid dyshomeostasis in AD cell and mouse models

Figure 5 A shows a scheme for immunoisolation of Ap-containing particles.

Figure 5B shows that immunoisolation of A particles IP/Westem was detected by the 6el0 antibody.

Figure 5C shows the specific mass/charge ratio determined by liquid chromatography mass spectrometry (LC-MS/MS) lipidomics and independently by Desorption Electrospray Ionization (DESI) with SYNAPT G2-Si Mass Spectrometry. DESI MS-imaging using a Prosolia source of AD mouse model (Tg2576) or wild type mouse brain, coronal section.

Figure 5D shows fold enrichment of CE in immuno-isolated Ap-containing particles from HEK293 APP cell line (using 6el0) compared to control media. Lipidomic analyses were done with an Agilent 6490 Triple Quadrupole LC/MS system.

Example 6: Small molecule therapeutic for amelioration of behavioral impairment in mouse model

A prominent strategy for amelioration of NPC phenotype has been to accelerate cholesterol clearance using cholesterol lowering drugs including cyclodextrin or derivatives (Aqul et al., 2011, Malnar et al., 2012; Yao et a., 2012; Maulik et al, 2012). However, these strategies can be limited by ability to effectively alter brain cholesterol which is metabolically distinct from cholesterol metabolism in the periphery (Quan et a., 2003; Madra et al., 2010). Further, effectiveness of this strategy can be impaired by low brain penetrance of cyclodextrin when given systemically (Aqul et al., 2011). The metabolically active cholesterol pool in systemic tissues such as liver, spleen has been well documented by increased levels of cholesterol esters (CE) and reduced sterol synthesis and suppression of SREBP target genes (Liu et a., 2009, 2010). The sequestration of cholesterol in NPC mouse models can be overcome by expansion of metabolically active cholesterol pool shown by increased level of cholesterol esters (CE) when cyclodextrin derivative is administered directly to the brain (Aqul et al., 2011).

The administration directly to the brain in humans can be clinically complicated and can be detrimental to normal brain function. The formation of cholesterol esters (metabolically active cholesterol pool) has been shown to be deficient in NPC patients (Pentchev et al., 1985). Cholesterol esters have also found to be selectively depleted in ventricular fluid from AD brain (Montine et al., 1997). Therefore, activating the metabolically active pool of cholesterol through cholesterol ester formation in brain can overcome the block in cholesterol trafficking in both NPC and AD allowing normal trafficking through the endo/lysosomal compartment and plasma membrane. Moreover, mobilization of cholesterol through cholesterol ester formation can be stimulated by fatty acids as has been shown in liver (Daumerie et al., 1992). In the periphery, unsaturated dietary fatty acids were greatly enriched in cholesterol esters and decreased low density lipoprotein cholesterol (LDL) (Daumerie et al., 1992; Wang et al., 1993; Woollett et al., 1992; Rampresath et al., 2013). This mobilization of the metabolically active pool of cholesterol in neurons has promise to be ameliorative in NPC and AD.

In order to test the ability of LCAT activation to rescue behavioral deficits in a mouse model of Alzheimer’s disease, the disclosed subject matter bypassed the blood brain barrier (BBB) with intracerbroventricular injection of a known LCAT activator Compound 2 (Manthei et al., eLIFE 2018). Injection was accomplished with a Hamilton syringe, 1 ul 3.4mM Compound 2 in ACSF diluted from a 50mM stock, 50ug/kg final. Vehicle alone was used as a control (6.4% DMSO in ACSF). Single intracerebroventricual injection was administered to the ventrical 0.5mm posterior and 1mm lateral to the Bregma at a depth of 2.4mm. Location of injection was confirmed with 3% methylene blue injection at the site and sectioning the brain (Fig- 6).

Compound 2 treatment was accomplished by intracerebroventricular injection (ICV). Aliquots of Compound 2 (50 pg/Kg) or vehicle (0.64% DMSO) were diluted in aCSF (124 mM NaCl, 4.4 mM KC1, 1 mM Na ₂ HPO ₄ , 25 mM NaHCO ₃ , 2 mM CaCh, 2 mM MgCh, and 10 mM glucose). For experiments involving bilateral single intracerebroventricular (ICV) injection, animals received infusion into ICV part (coordinates: posterior = 0.5 mm, lateral=1.0 mm to a depth of 2.40 mm) (Paxinos and Franklin, 2013) under anesthesia with 20 mg/kg Avertin. Volume of 1 pL of Compound 2 was infused ICV at a concentration of 50 pg/Kg over a period of 1 min through cannulas connected to a microsyringe by a polyethylene tubing (Hamilton Company, 10 pL, 26-gauge, #84853). After infusion, the needle was kept in place for an additional minute to allow diffusion of Compound 2 or vehicle into the tissue. Mice were allowed to recover for 2 days and on the third day, behavioral assessment using contextual fear conditioning commenced (Puzzo et al., 2009).

Contextual fear conditioning (FC) is a hippocampus amygdala-dependent task in which Tg2576 mice show deficits (Barnes and Good, 2005) (Fig. 7). The test was performed on 8-to- 11-month-old mice, and cued FC, a hippocampus-independent task, was used as a control (Oliveira et al., 2010). Animals were placed into atransparent Plexiglas conditioning chamber (33 cm; 20 cm, 22 cm) (Noldus PhenoTyper). Animal movement was recorded using an overhead video camera connected to a personal computer, and freezing behavior was scored and analyzed using Ethovision XT software (Noldus). Footshocks were administered through a removable metal grid floor, and the entire apparatus was cleaned and deodorized between animals with distilled water and 70% ethanol. Animals were placed in the conditioning chamber once on each of 3 consecutive days. On the first day of exposure, mice were placed in the conditioning chamber for 2 min before the onset of a discrete 30 s, 2800Hz, 85dB tone, the last 2 s of which coincided with a 0.8 mA. After the tone and shock exposure, the mice were left in the conditioning chamber for another 30 s before returning to their home cages. At 24 h after their first exposure, animals were returned to the conditioning chamber for 5min without footshock or tone presentation.

Figure 8 demonstrated the LCAT activator Compound 2 ameliorated memory in the Novel Object Recognition test. The plot shows the average object preference ratio for the indicated groups after 3 days of single ICV injection of the Compound 2 (50 ug/Kg).

Figure 9 demonstrated ICV injection of the LCAT activator Compound 2 did not affect motor behavior in the Open Field Test as indicated by no difference found in (A) Distance traveled (B) Center Crossings (C) Time spent in the center of the open field (Time center), and (D) Speed.

Figure 10 demonstrated ICV injection of the LCAT activator Compound 2 did not affect amyloid P-peptide 42 or amyloid P-peptide 40 levels in the hippocampus (A) or cortex (B).

Figure 11 demonstrated ICV injection of the LCAT activator Compound 2 resulted in a significant decrease in interleukin 4 (IL -4) levels from mouse brain.

Example 7: Results

The present disclosure demonstrated the activation of LCAT by a small molecule activator result in behavioral rescue in a mouse model of Alzheimer’s disease in both Contextual Fear Conditioning (Fig. 7) and Novel Object Recognition (Fig. 8) without significant changes in motor behavior (Fig. 9). This effect is independent of amyloid accumulation in the mouse model brain since amyloid-P peptide 40 (Ap40) and amyloid-P peptide 42 (Ap42) did not change after treatment with Compound 2 (Fig. 10). Moreover, Compound 2 activation of LCAT results in anti-inflammatory effects based on the significant reduction in Interleukin 4 (IL-4) (Fig.l 1). Thus, the activation of LCAT can be ameliorative in Alzheimer’s disease through mechanisms independent of amyloid accumulation. The present disclosure demonstrates LCAT stimulation can result in anti-inflammatory effects leading to behavioral rescue in spite of accumulation of amyloid. LCAT activation also has potential to target orphan indications including Niemann-pick Type C, Familial LCAT deficiency (FLD) and fish eye disease (FED) and Cholesteryl ester storage disease (CESD).

The disclosed subject matter can provide a brain permeable small molecule activator of LCAT to engage physiologically relevant pathways such as HDL maturation in the case of AD or cholesterol trafficking and efflux in the case of NPC. A brain penetrant small molecule activator of LCAT can lead to increase in CE formation in the brain to facilitate clearance of pathologically accumulated cholesterol from intracellular stores in NPC. Further, facilitating HDL maturation and cholesterol trafficking in the brain can lead to prophylactic and therapeutic benefit in AD as well. The disclosed subject matter can serve as both a prophylactic target which can safely delay onset of AD as well as therapeutic intervention for NPC and AD where none currently exist. An improved in vitro assay for LCAT activity can be used together with certain published LCAT activators (Manthei et al., 2018; Freeman et al., 2017, assay Fig. 12).

The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Publications are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties.