[go: up one dir, main page]

WO2025199495A1 - Compositions et méthodes de traitement et de prévention de la maladie d'alzheimer - Google Patents

Compositions et méthodes de traitement et de prévention de la maladie d'alzheimer

Info

Publication number
WO2025199495A1
WO2025199495A1 PCT/US2025/021013 US2025021013W WO2025199495A1 WO 2025199495 A1 WO2025199495 A1 WO 2025199495A1 US 2025021013 W US2025021013 W US 2025021013W WO 2025199495 A1 WO2025199495 A1 WO 2025199495A1
Authority
WO
WIPO (PCT)
Prior art keywords
tau
vldlr
antagonist
antibody
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/021013
Other languages
English (en)
Inventor
Dudley K. Strickland
Joanna Cooper
Mary M. MIGLIORINI
Nicholas WEINRICH
Bradley T. Hyman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
General Hospital Corp
University of Maryland Baltimore
University of Maryland College Park
Original Assignee
General Hospital Corp
University of Maryland Baltimore
University of Maryland College Park
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by General Hospital Corp, University of Maryland Baltimore, University of Maryland College Park filed Critical General Hospital Corp
Publication of WO2025199495A1 publication Critical patent/WO2025199495A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • AD Alzheimer’s Disease
  • a neurodegenerative disorder characterized by the accumulation of extracellular plaques and intracellular neurofibrillary tangles, which are comprised primarily of aggregated amyloid P and tau proteins, respectively.
  • Tau pathology spreads across the brain as AD progresses, and this process is mediated by cell-to-cell transfer of seed competent tau.
  • a potential mechanism responsible for this is the release of seed-competent tau into the extracellular space and subsequent internalization of pathogenic tau via receptor- mediated endocytosis. The internalized pathogenic tau ultimately reaches the cytoplasm to seed aggregation of endogenous tau.
  • AD apolipoprotein E
  • ApoE4 apolipoprotein E
  • SORL1 single nucleotide polymorphisms in the SORL1 gene, which encodes the sortilin-related receptor 1 and shares structural homology with LDL receptor family members.
  • LRP1 was recently identified as major endocytic receptor for tau and regulates tau internalization, degradation, and seeding in a manner that is modified differentially by ApoE isoforms.
  • VLDLR consists of one polypeptide chain that forms the extracellular portion, the transmembrane domain, and the cytoplasmic domain (Takahashi S, et al., J Atheroscler Thromb 2004; 11: 200-208; Lillis AP, et al., Physiol Rev 2008; 88: 887-918).
  • the extracellular' portion which includes 8 complement-type repeats (CR-domains), and EGF-like, P-propeller, and the O-linked sugar domains, has been expressed in the insect expression system (Ruiz J, et al., J Lipid Res 2005; 46: 1721-1731).
  • mice displayed a “reeler” phenotype which led to the discovery that these two receptors are critical for reelin signaling.
  • This signaling pathway plays a critical role in the development of laminated structures of the brain and in synaptic plasticity of the adult brain (3).
  • Reelin signaling has also been discovered to be relevant for Alzheimer’s disease.
  • the phosphorylation of Disabled 1 occurs which has a number of important consequences (for review see (4)) that ultimately leads to phosphorylation of glycogen synthase kinase 3P (GSK3P) at its inhibitory serine- 9 site. This reduces phosphorylation of tau.
  • Hyperphosphorylation of tau results in tau aggregation and tangle formation in neurons, which is thought to drive progression of Alzheimer’s disease.
  • the invention provides a method of reducing tau internalization and/or trafficking in neuronal cells comprising contacting the cells with an effective amount of a VLDL receptor antagonist.
  • the invention provides a method of treating or preventing Alzheimer’s disease in a subject in need thereof, comprising administering to the subject an effective amount of a VLDL receptor antagonist.
  • the VLDL receptor antagonist blocks the interaction of tau and VLDL receptor. In some embodiments, the VLDL receptor antagonist inhibits the expression of VLDL receptor.
  • the VLDL receptor antagonist is an VLDL receptor antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is selected from the group consisting of 1H5, 1H10, 5F3 and combinations thereof.
  • the antibody is a humanized antibody and comprises CDR sequences of an antibody selected from the group consisting of 1H5, 1H10, and 5F3.
  • the VLDL receptor antagonist is a nucleic acid that inhibits the expression of VLDL receptor.
  • the nucleic acid is an RNA, a DNA, or a combination thereof.
  • the nucleic acid is a ribozyme, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA).
  • the nucleic acid is delivered as a viral vector.
  • the VLDLR antagonist is administered to a subject by topical, intravenous, subcutaneous, intramuscular', intracutaneous, transcutaneous, intrathecal, intranasal, intra-arterial, rectal, intragastric, parenteral, or oral administration.
  • the method further comprises administering one or more additional active agents.
  • the one or more additional active agents comprises i) an effective amount of an LRP1 antagonist; ii) an effective amount of a SorLA antagonist; or iii) an effective amount of an LRP1 antagonist and a SorLA antagonist.
  • the cells are neuronal cells. In some embodiments, the cells are non-neuronal cells. In some embodiments, the cells are from mammals, yeast, Drosophila or E. coli. In some embodiments, the cells express VLDLR endogenously. In some embodiments, the cells have been transfected or engineered to express VLDLR.
  • FIG. 2 VLDLr mediates tau internalization.
  • the uptake of 20 nM 125 I-labeled 2N4R tau was quantified in LRP1 -deficient CHO cells (13-5-1) that stably express VLDLr, and compared to non-expressing 13-5- 1 cells.
  • FIG. 3 Monoclonal antibodies block the binding of tau to VLDLr.
  • FIG. 4 Affinities of VLDLr antibodies for sVLDLr.
  • Recombinantly produced VLDLr protein fragment containing the entire extracellular portion of VLDLr (sVLDLr) was immobilized to the surface of a CM5 sensorchip, and then SPR was used to determine the binding affinities of the VLDLr antibodies to sVLDLr. Shown are means +/- SD, n-3.
  • EIG. 5 Anti-VLDLr antibodies inhibit tau uptake.
  • SPR Surface plasmon resonance
  • sVLDLr soluble VLDLr
  • CM5 sensorchip CM5 sensorchip.
  • Analysis was performed using a nonlinear regression fit using Graphpad Prism’s equation for one site - specific binding. Shown are means ⁇ SEM, n - 3
  • FIG. 7 ApoE binds VLDLr.
  • VLDLr 1-8 Recombinantly produced VLDLr protein containing the ligand binding repeats (VLDLr 1-8) was immobilized to the surface of a CM5 sensorchip, and increasing concentrations of ApoE isoforms were flowed over the flow cells in order to assess binding. Analysis was performed using a nonlinear regression fit using Graphpad Prism’s equation for one site - specific binding. This experiment was performed three times. Shown are means ⁇ SEM.
  • Recombinantly produced VLDLr protein fragment containing the entire extracellular portion of VLDLr (sVLDLr) was immobilized to the surface of a CM5 sensorchip, and then the binding of 20 nM tau +/- 100 nM (a) apoE2, (b) apoE3, or (c) apoE4 was assessed via SPR.
  • Each experiment was repeated 3 times, shown are representative images. Experiments were repeated at least 3 times, (d) shown are means ⁇ SEM.
  • FIG. 9. Monomer vs Fibrils experiment.
  • FIG. 10. Binding of pseudo HMW (D20Q3) and pseudo LMW (D8Q2) tau, compared to unmodified 2N4R tau, to LRP1 assessed using the Biacorc 8K surface plasmon resonance (SPR) system.
  • Full-length LRP1 purified from human placenta was immobilized via amine coupling on the surface of a CM5 Biacore sensorchip.
  • concentrations (3.7, 11.1, 33.3 nM) of recombinant 2N4R, D20Q3, or D8Q2 tau were flowed over the sensorchip in a single cycle kinetic titration experiment at pH7.4.
  • A Representative image of single experiment.
  • FIG. 11 Effect of pH on tau binding to LRP1 and SORL1.
  • Tau cannot bind LRP1 at endosomal pH (5.5) (A) and (B), but continues to bind to SORL1 with similar affinity as seen at pH 7.4 (C) and (D).
  • FIG. 12 Reelin blocks the binding of tau to VLDLr.
  • Recombinantly produced VLDLr protein fragment containing the entire extracellular portion of VLDLr (sVLDLr) was immobilized to the surface of a CMS sensorchip, and then the binding of 20 nM tau +/- 100 nM reelin was assessed via SPR. Shown is a representative image.
  • FIG. 13 ApoE3 vs ApoE3 CH binding LRP1 or VLDLr.
  • FIG. 14 LRP1 regulates tau uptake, degradation, and seeding. Uptake of tau in LRP1 -deficient CHO cells confirms the existence of additional receptors for tau uptake.
  • A steady-state levels of 1251-labeled tau (20 nM) internalized in WT or LRP1 -deficient 13- 5-1 CHO cells when incubated in the absence or the presence of 1 uM RAP for 2 h at 37 °C.
  • B and C time course for internalization of 1251-labeled tau (20 nM) in CHO WT (B) and CHO 13-5-1 (C) cells in the presence or the absence of RAP (1 mM) or heparin (20 mg/ml).
  • D WT, 13-5-1 and HSPG-deficient (CHO HSPG) CHO cells were incubated with 20 nM 1251-labeled tau in the absence or the presence of RAP (1 mM) or heparin (20 mg/ml) at 37 °C for 2 h, and internalized tau was measured.
  • E SPR analysis of 10 nM tau binding to LRP1 in the absence or the presence of 20 mg/ml heparin.
  • CHO Chinese hamster ovary
  • HSPG heparan sulfate proteoglycan
  • LRP low-density lipoprotein receptor-related protein 1
  • RAP receptor-associated protein
  • SPR surface plasmon resonance.
  • FIG. 15. SORL1 provides a mechanism of uptake that supports tau proteopathic seeding in the cytoplasm.
  • Tau binds to SORL1 and SORL1 transfection reconstitutes pathogenic internalization and seeding in HEK293T reporter cells, (a) SPR equilibrium analysis of the binding of increasing concentrations of recombinant 2N4R tau to full length SORL1 (blue circles) and the SORL1 VPS 10 Domain (orange triangles), (b) H4 Cells were incubated with 40 nM tau labeled with Alexaflour594 for 2 h, then fixed and immunostained with anti-SORLl antibody to label endogenous SORL1.
  • CHO cells were transfected with SORL1 plasmid or empty vector (Mock), then incubated with 20 nM 1251-labelled tau ⁇ 1 pM RAP for 2 hours, and the amount of tau internalized by the cells was quantified
  • HEK293T FRET reporter cells were transfected with SORL1 plasmid, then incubated with HMW SEC fractions from AD patient brain (AD) or healthy control (Ctrl) and tau seeding quantified
  • siRNA was used to knockdown SORL1 in H4 cells that stably express the FRET reporter system, then cells were incubated with 300ng/well AD brain derived HMW tau seeding material and tau seeding was quantified. (Means ⁇ SEM; 2-way ANOVA).
  • f Immunoblots confirming transfection and knockdown.
  • FIG. 16 Phosphorylated forms of tau bind weakly to LRP1. Binding of tau to LRP1 was assessed by surface plasmon resonance (SPR) experiments. LRP1 was coupled to a CM5 sensor chip and then increasing concentrations of various forms of tau over the chip.
  • SPR surface plasmon resonance
  • A single-cycle kinetic experiment quantifying binding of monomeric tau (3.8, 11.5, 34.4, 103.3, and 310 nM) to LRP1 in the presence of Ca2+ (blue line) or EDTA (black line).
  • C binding of tau isoforms 2N4R, 2N3R, and tau MBD to LRP1 assessed by SPR equilibrium analysis.
  • D about 1 pg of recombinant tau produced in Escherichia coli or SF9 cells was ran on a 4 to 12% gel and stained with colloidal Coomassie. Image was captured using Licor. Quantification of bands reveals a signal of 2270 for E. coli tau and 2280 for SF9 tau.
  • LRP1 low-density lipoprotein receptor-related protein 1
  • MBD microtubule-binding domain
  • RAP receptor-associated protein
  • SPR surface plasmon resonance.
  • FIG. 17 Less impact of phosphorylation on tau binding to SORLl’s VPS10P domain.
  • Recombinant monomeric tau binds to SORL1 and the VPS 10 domain of SORL1.
  • FIG. 18 Impact of pH on tau binding LRP1 and SORL1.
  • LRP1 or SORL1 was immobilized on the surface of a CMS sensorchip and increasing concentrations (11.1, 33.3, 100, 300, 900 nM) of 2N4R tau were flowed over the surface in the presence of either HEPES buffer pH 7.4 or MES buffer pH 5.5.
  • FIG. 19 LRP1 binds tau isolated from AD patient brains.
  • AD Alzheimer’s disease
  • CT age-matched control
  • 2N4R tau 2N4R tau as a positive control.
  • A&B Single injections of 300nM LRP1 demonstrate that LRP1 binds to LMW tau from AD and CT brains, but no binding was detected to HMW tau from AD brains. No tau was captured from HMW CT samples, consistent with the low amount of HMW tau present in these samples.
  • C Plotted is the amount of LRP1 bound divided by the amount of tau captured.
  • the invention is based on the discovery of therapeutic agents that can block tau binding to VLDLR and suppress or inhibit tau internalization in cells.
  • Tau is an intracellular microtubule-associated protein that is hyperphosphorylated and forms “tangles” in neurons of Alzheimer’s disease patients.
  • aggregated tau can spread from cell to cell and from one region of the brain to other regions.
  • Tau is secreted by neurons and is taken up by receptor-mediated endocytosis.
  • the interest in tau receptors arise from the fact that neuronal transfer of pathological forms of tau has been proposed as a mechanism of Alzheimer’s disease (AD) progression.
  • the present inventors have discovered that the very low density lipoprotein receptor (VLDL receptor) can bind tau and mediate its uptake.
  • the VLDL receptor is one of two reelin receptors that participate in reelin signaling.
  • This signaling is required for development of laminated structures of the brain that occur during development. It is shown herein that two monoclonal antibodies (1H10 and 1H5) that are directed against the VLDL receptor block tau binding. Without being bound by theory, it is believed that these antibodies (alone and/or together) may cluster the VLDL receptor and trigger reelin signaling. Thus, these antibodies may not only block tau transmission, but also reduce tau phosphorylation.
  • the interaction of tau with VLDL receptor has important implications for the progression of Alzheimer’s disease, and the further identification of agents capable of blocking the interaction between tau and VLDL receptor can also be beneficial for the treatment or prevention of Alzheimer’s disease.
  • the invention provides a method of reducing tau internalization and/or trafficking in neuronal cells comprising contacting the cells with an effective amount of a Very Low Density Lipoprotein Receptor (VLDLR) antagonist.
  • VLDLR Very Low Density Lipoprotein Receptor
  • the invention provides a method of treating or preventing Alzheimer’s disease in a subject in need thereof, comprising administering to the subject an effective amount of a Very Low Density Lipoprotein Receptor (VLDLR) antagonist.
  • VLDLR Very Low Density Lipoprotein Receptor
  • the present invention provides a method of reducing the cellular uptake or trafficking of tau and thereby preventing and/or treating Alzheimer’s disease, the method comprising administering to the subject an effective amount of an agent that inhibits the activity of VLDLR.
  • the invention provides a method of screening for potential agents that reduce internalization and/or trafficking of tau in cells, comprising i) providing a cell expressing VLDLR or a functional equivalent of VLDLR; ii) providing tau protein or a fragment or derivative thereof to the cell, wherein the tau protein or a fragment or derivative thereof is extracellular; iii) treating the cell with an effective amount of a test agent that may inhibit binding of tau to VLDLR or reduce expression of VLDLR in cells; and iv) assaying the cellular uptake of tau or the fragment or derivative thereof.
  • Alzheimer's disease is a neurodegenerative disease and the most common cause of dementia. This disease appears as a gradual but progressive decline in memory, thinking ability, and behavior that is accelerated compared to normal aging. There are two major types of this disease. Familial Alzheimer's disease is usually caused by a dominant mutation in one of three genes (APP, PSEN1 or PSEN2). This type of disease is a rare but devastating disease that occurs in middle age. The second and much more prevalent form of this disease is sporadic or late-onset Alzheimer's disease. The onset of Alzheimer's disease usually occurs after age 62.
  • Symptoms of Alzheimer's disease are mainly characterized by memory impairment, language dysfunction, and cognitive impairment including visual and spatial abilities, which can extend to occupational and social problems (e.g., activities of daily living); and depression. Behavioral symptoms, including anxiety, aggression and psychosis, may manifest as a progression of disease severity.
  • AD dementia is used to describe dementia due to the pathophysiology of Alzheimer's disease.
  • probable Alzheimer's disease refers to when a subject exhibits the clinical features of Alzheimer's disease and other possible biological causes of dementia (e.g., Parkinson's disease or stroke). Used during life if excluded.
  • AD Alzheimer's disease
  • these methods include determining an individual's ability to perform daily activities and identifying behavioral and personality changes.
  • Dementia of the AD type is usually also characterized by amnestic symptoms (memory impairment) or impairment of language, visual spatial or executive function.
  • Cognitive/dysfunction includes global cognition (e.g., improved minimental state test (3MS-E)), and visual and verbal memory (e.g., simple visual spatial memory test (revised version) (BVMT-), respectively.
  • HVLT-R Hopkins language learning test
  • GVFT utterance fluency test
  • DST executive function and attention
  • the terms “effective amount” or “therapeutically effective amount” are interchangeable and refer to an amount that results in an improvement or remediation of at least one symptom of the disease or condition. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure of the disease and/or condition.
  • the term “effective amount” corresponds to an amount administered that reduces the internalization or trafficking of tau in cells, mediated by VLDLR.
  • the “effective amount” can correspond to an amount administered to subjects or to cells directly.
  • the term “inhibit” refers to the ability of the compound to block, partially block, interfere, decrease, reduce or deactivate a receptor such as VLDLR.
  • inhibit encompasses a complete and/or partial loss of activity of the receptor.
  • Receptor activity may be inhibited by blockage of ligand binding sites on the receptor, by interference with the mechanism of expression of the receptor protein, or by other means.
  • a complete and/or partial loss of activity of the receptor may be indicated by a reduction in the extent of tau internalization or trafficking into neurons or other mammalian cells or a reduction in NFTs or senile plaques in the brain of a subject.
  • treat and all its forms and tenses (including, for example, treat, treating, treated, and treatment) refer to both therapeutic treatment and prophylactic or preventative treatment.
  • a subject in need of treatment includes those already with a pathological condition of the invention as well as those in which a pathological condition of the invention is to be prevented.
  • Alzheimer’s disease is treated by delaying the development or progression of the disease.
  • inhibitor when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of activity compared to normal.
  • the subject to be administered the therapeutic agent is not limiting.
  • the subject is a mammal including for example, a dog, cat, monkey, goat, pig, chimpanzee, cow, horse, sheep, rabbit, guinea pig, rat, hamster, mouse, and human.
  • the subject is a human.
  • the subject has been diagnosed with Alzheimer’s disease or is at risk of developing the disease.
  • the subject has or is at risk of developing late onset disease.
  • the subject has or is at risk of developing early onset disease, or Familial Alzheimer's (which can develop well before the senile period, e.g., between 35 and 60 years of age).
  • the subject is at risk of developing the disease, c.g., due to a family history, genetic predisposition, lifestyle, or due to the presence of one or more early markers or symptoms of the disease.
  • the VLDLR antagonist is not particularly limiting.
  • the VLDLR antagonist can be a protein, a peptide, a lipid, a carbohydrate, an organic molecule, or an inorganic molecule.
  • Exemplary inhibitors of VLDLR function include, without limitation, soluble VLDLR receptor polypeptides.
  • the antagonist inhibits binding of tau to Very Low Density Lipoprotein Receptor (VLDLR). In some embodiments, the antagonist inhibits expression of VLDLR in cells.
  • VLDLR Very Low Density Lipoprotein Receptor
  • the therapeutically effective amount of an agent that inhibits binding of tau to Very Low Density Lipoprotein Receptor is an antibody.
  • antibody refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof.
  • the term “antibody” refers to a homogeneous molecular entity, or a mixture such as a polyclonal serum product made up of a plurality of different molecular entities, and broadly encompasses naturally- occurring forms of antibodies (for example, IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multispecific antibodies.
  • antibody also refers to fragments and derivatives of all of the foregoing, and may further comprise any modified or derivatized variants thereof that retains the ability to specifically bind an epitope.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • a monoclonal antibody is capable of selectively binding to a target antigen or epitope.
  • Antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, camelized antibodies, single chain antibodies (scEvs), Lab fragments, E(ab').sub.2 fragments, disulfide-linked Evs (sdEv) fragments, for example, as produced by a Lab expression library, anti-idiotypic (anti-Id) antibodies, intrabodies, nanobodies, synthetic antibodies, and epitope-binding fragments of any of the above.
  • Monoclonal antibodies as used herein also include sequences corresponding to human antibodies, animal antibodies, and combinations thereof.
  • chimeric antibody includes antibodies that have variable regions derived from an animal antibody, such as a rat or mouse antibody, fused to another molecule, for example, the constant domains derived from a human antibody.
  • One type of chimeric antibodies, “humanized antibodies,” have had the variable regions altered (through mutagenesis or CDR grafting) to match (as much as possible) the known sequence of human variable regions.
  • CDR grafting involves grafting the CDRs from an antibody with desired specificity onto the FRs of a human antibody, thereby replacing much of the nonhuman sequence with human sequence. Humanized antibodies, therefore, more closely match (in amino acid sequence) the sequence of known human antibodies.
  • the antibody can bind at least one complement-type repeat (CR) domain of VLDLR selected from the group consisting of CR-2, CR-3 and CR-4, or any combination thereof. In some embodiments, the antibody can bind to CR domains 3- 6. In some embodiments, the antibody can bind to CR domains 1-2 and 5-6. In some embodiments, the antibody can bind to CR domains 2 and 5-6.
  • CR complement-type repeat
  • the antibody is a monoclonal antibody. In some embodiments, the antibody is a mouse monoclonal antibody. In some embodiments, the antibody is 1H10. In some embodiments, the antibody is 1H5. In some embodiments, the antibody is 5F3. In some embodiments, the antibody comprises one or more complementarity determining regions (CDRs) identical to the CDRs of 1H10, 1H5 or 5F3. Antibodies 1H10, 1H5 and 5F3 are available commercially from Molecular Innovations (Novi, MI). See also Ruiz et al. (2005) J. Lipid Res. 46:1721-1731; and Oganesian et al. (2008) Mol. Biol. Cell 19:563-571.
  • CDRs complementarity determining regions
  • the agent that inhibits binding of fibrin to Very Low Density Lipoprotein Receptor is a humanized antibody.
  • the antibody is a humanized antibody of antibody 1H10, 1H5 or 5F3, that harbors the CDR sequences of antibody 1H10, 1H5, or 5F3.
  • a combination of antibodies are administered.
  • the antibody is a fully human antibody.
  • the VLDLR antagonist comprises a nucleic acid molecule that comprises a nucleotide sequence that binds to at least a portion of a nucleotide sequence of VLDLR.
  • the nucleic acid molecule can be of any length, so long as at least part of the molecule hybridizes sufficiently to VLDLR nucleic acid such as mRNA.
  • the nucleic acid molecule can bind to any region of VLDLR mRNA. In some embodiments, the nucleic acid molecule binds to a particular domain of VLDLR mRNA.
  • a region of the nucleic acid molecule is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary to at least a portion of SEQ ID NO:1.
  • the portion of SEQ ID NO:1 comprises a nucleic acid sequence corresponding to a portion of Homo sapiens VLDLR.
  • the composition can comprise a DNA molecule, such as an antisense DNA molecule.
  • a target sequence on a target mRNA can be selected from a given cDNA sequence corresponding to the VLDLR, in some embodiments, beginning 50 to 100 nt downstream (z.e., in the 3' direction) from the start codon.
  • the target sequence can, however, be located in the 5' or 3' untranslated regions, or in the region nearby the start codon.
  • the antisense DNA is at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1500 nucleotides, at least about 2000 nucleotides, at least about 2500 nucleotides, at least about 3000 nucleotides,
  • the composition comprises an anti-sense RNA.
  • Anti-sense RNA binds with mRNA and prevents translation of the mRNA.
  • the anti-sense RNA can be complementary to a portion of VLDLR mRNA.
  • the anti-sense RNA is complementary to the entire reading frame of VLDLR.
  • the anti-sense RNA is complementary to the entire reading frame of SEQ ID NO:1.
  • the antisense RNA is complementary to a portion of SEQ ID NO: 1.
  • the antisense RNA is at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, at least about 1000 nucleotides, at least about 1200 nucleotides, at least about 1500 nucleotides, at least about 2000 nucleotides, at least about 2500 nucleotides, at least about 3000 nucleotides
  • RNA interference is used to "knock down” or inhibit a particular gene of interest by simply injecting, bathing or feeding to the organism of interest the double- stranded RNA molecule. This technique selectively “knock downs” gene function without requiring transfection or recombinant techniques.
  • siRNA small interfering RNA
  • a siRNA may comprise a double stranded structure or a single stranded structure, the sequence of which is “substantially identical” to at least a portion of the target gene (see WO 04/046320, which is incorporated herein by reference in its entirety).
  • Identity is the relationship between two or more polynucleotide (or polypeptide) sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match of the order of nucleotides between such sequences. Identity can be readily calculated.
  • the siRNA contains a nucleotide sequence that is completely identical to at least a portion of the target gene.
  • an "identical" RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine.
  • dsRNA double- stranded RNA
  • target gene e.g., X'LDLR
  • a siRNA that is essentially identical to a least a portion of the target gene may also be a dsRNA wherein one of the two complementary strands (or, in the case of a self-complementary RNA, one of the two self-complementary portions) is either identical to the sequence of that portion or the target gene or contains one or more insertions, deletions or single point mutations relative to the nucleotide sequence of that portion of the target gene.
  • siRNA technology thus has the property of being able to tolerate sequence variations that might be expected to result from genetic mutation, strain polymorphism, or evolutionary divergence.
  • There are several methods for preparing siRNA such as chemical synthesis, in vitro transcription, siRNA expression vectors, and PCR expression cassettes.
  • the first step in designing an siRNA molecule is to choose the siRNA target site, which can be any site in the target gene.
  • the target selecting region of the gene which may be an ORF (open reading frame) as the target selecting region and may preferably be 50- 100 nucleotides downstream of the "ATG" start codon.
  • siRNA Target Designer by Promega
  • siRNA Target Finder by GenScript Corp.
  • siRNA Retriever Program by Imgenex Corp.
  • EMBOSS siRNA algorithm siRNA program by Qiagen
  • Ambion siRNA predictor Ambion siRNA predictor
  • Whitehead siRNA prediction Sfold.
  • any of the above programs may be utilized to produce siRNA molecules that can be used in the present invention.
  • the composition is an siRNA targeting VLDLR.
  • the VLDLR siRNA contains a nucleotide sequence that is essentially identical to at least a portion of the target gene.
  • the siRNA contains a nucleotide sequence that is completely identical to at least a portion of the VLDLR gene.
  • an "identical" RNA sequence will contain ribonucleotides where the DNA sequence contains deoxyribonucleotides, and further that the RNA sequence will typically contain a uracil at positions where the DNA sequence contains thymidine.
  • a VLDLR siRNA comprises a double stranded structure, the sequence of which is "substantially identical" to at least a portion of the target gene.
  • Identity is the relationship between two or more polynucleotide (or polypeptide) sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match of the order of nucleotides between such sequences. Identity can be readily calculated by standard practices in the art.
  • polynucleotides of different lengths may be compared over the entire length of the longer fragment. Alternatively, small regions may be compared. Normally sequences of the same length are compared for a final estimation of their utility in the practice of the present invention. In some embodiments, there is 100% sequence identity between the dsRNA for use as siRNA and at least 15 contiguous nucleotides of the target gene, although a dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater may also be used in the present invention.
  • a siRNA that is essentially identical to a least a portion of the target gene may also be a dsRNA wherein one of the two complementary strands (or, in the case of a self-complementary RNA, one of the two self-complementary portions) is either identical to the sequence of that portion or the target gene or contains one or more insertions, deletions or single point mutations relative to the nucleotide sequence of that portion of the target gene.
  • siRNA technology thus has the property of being able to tolerate sequence variations that might be expected to result from genetic mutation, strain polymorphism, or evolutionary divergence.
  • the invention provides a VLDLR siRNA that is capable of triggering RNA interference, a process by which a particular RNA sequence is destroyed (also referred to as gene silencing).
  • VLDLR siRNA are dsRNA molecules that are 100 bases or fewer in length (or have 100 base pairs or fewer in its complementarity region).
  • a dsRNA may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotides or more in length.
  • VLDLR siRNA may be approximately 21 to 25 nucleotides in length. In some cases, it has a two nucleotide 3' overhang and a 5' phosphate.
  • the particular VLDLR RNA sequence is targeted as a result of the complementarity between the dsRNA and the particular VLDLR RNA sequence.
  • dsRNA or siRNA of the disclosure can effect at least a 20, 30, 40, 50, 60, 70, 80, 90 percent or more reduction of expression of a targeted VLDLR RNA in a cell.
  • dsRNA of the invention (the term “dsRNA” will be understood to include “siRNA” and/or “candidate siRNA”) is distinct and distinguishable from antisense and ribozyme molecules by virtue of the ability to trigger RNAi.
  • dsRNA molecules for RNAi differ from antisense and ribozyme molecules in that dsRNA has at least one region of complementarity within the RNA molecule.
  • the complementary (also referred to as "complementarity") region comprises at least or at most 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,
  • long dsRNA are employed in which "long” refers to dsRNA that are 1000 bases or longer (or 1000 base pairs or longer in complementarity region).
  • the term "dsRNA” includes "long dsRNA", “intermediate dsRNA” or “small dsRNA” (lengths of 2 to 100 bases or base pairs in complementarity region) unless otherwise indicated.
  • dsRNA can exclude the use of siRNA, long dsRNA, and/or "intermediate” dsRNA (lengths of 100 to 1000 bases or base pairs in complementarity region).
  • a dsRNA may be a molecule comprising two separate RNA strands in which one strand has at least one region complementary to a region on the other strand.
  • a dsRNA includes a molecule that is single stranded yet has at least one complementarity region as described above (such as when a single strand with a hairpin loop is used as a dsRNA for RNAi).
  • lengths of dsRNA may be referred to in terms of bases, which simply refers to the length of a single strand or in terms of base pairs, which refers to the length of the complementarity region.
  • a dsRNA comprised of two strands are contemplated for use with respect to a dsRNA comprising a single strand, and vice versa.
  • the strand that has a sequence that is complementary to the targeted mRNA is referred to as the "antisense strand” and the strand with a sequence identical to the targeted mRNA is referred to as the "sense strand.”
  • the "antisense region” has the sequence complementary to the targeted mRNA
  • the “sense region” has the sequence identical to the targeted mRNA.
  • sense and antisense region like sense and antisense strands, are complementary (i.e., can specifically hybridize) to each other. Strands or regions that are complementary may or may not be 100% complementary (“completely or fully complementary”). It is contemplated that sequences that are "complementary” include sequences that are at least 50% complementary, and may be at least 50%, 60%, 70%, 80%, or 90% complementary.
  • siRNA generated from sequence based on one organism may be used in a different organism to achieve RNAi of the cognate target gene.
  • siRNA generated from a dsRNA that corresponds to a human gene may be used in a mouse cell if there is the requisite complementarity, as described above.
  • RNAi RNA complementary strands or regions.
  • Mismatches may number at most or at least 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 residues or more, depending on the length of the complementarity region.
  • the single RNA strand or each of two complementary double strands of a dsRNA molecule may be of at least or at most the following lengths: 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180
  • the two strands may be the same length or different lengths. If the dsRNA is a single strand, in addition to the complementarity region, the strand may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the strand or strands of dsRNA are 100 bases (or base pairs) or less. In specific embodiments, the strand or strands of the dsRNA arc less than 70 bases in length. With respect to those embodiments, the dsRNA strand or strands may be from 5-70, 10-65, 20-60, 30-55, 40-50 bases or base pairs in length.
  • a dsRNA that has a complementarity region equal to or less than 30 base pairs (such as a single stranded hairpin RNA in which the stem or complementary portion is less than or equal to 30 base pairs) or one in which the strands are 30 bases or fewer in length is specifically contemplated, as such molecules evade a mammalian's cell antiviral response.
  • a hairpin dsRNA (one strand) may be 70 or fewer bases in length with a complementary region of 30 base pairs or fewer.
  • a dsRNA may be processed in the cell into siRNA.
  • the siRNA of the invention can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
  • One or both strands of the siRNA of the disclosure can comprise a 3' overhang.
  • a "3' overhang” refers to at least one unpaired nucleotide extending from the 3'- end of a duplexed RNA strand.
  • the VLDLR siRNA of the invention comprises at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, from 1 to about 5 nucleotides in length, from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length.
  • each strand of the VLDLR siRNA of the invention can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
  • TT dithymidylic acid
  • uu diuridylic acid
  • the 3' overhangs can be also stabilized against degradation.
  • the overhangs are stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues, c.g., substitution of uridine nucleotides in the 3' overhangs with 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi degradation.
  • the absence of a 2' hydroxyl in the 2'-deoxythymidine significantly enhances the nuclease resistance of the 3' overhang in tissue culture medium.
  • the VLDLR siRNA of the invention comprises the sequence AA(N19)TT or NA(N21), where N is any nucleotide.
  • These VLDLR siRNA comprise approximately 30-70% GC, and in some embodiments comprise approximately 50% G/C.
  • the sequence of the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions 3 to 23), respectively. In the latter case, the 3' end of the sense siRNA is converted to TT.
  • the rationale for this sequence conversion is to generate a symmetric duplex with respect to the sequence composition of the sense and antisense strand 3' overhangs.
  • the antisense RNA strand is then synthesized as the complement to positions 1 to 21 of the sense strand.
  • the 3'-most nucleotide residue of the antisense strand can be chosen deliberately.
  • the penultimate nucleotide of the antisense strand (complementary to position 2 of the 23-nt sense strand in either embodiment) is generally complementary to the targeted sequence.
  • the VLDLR siRNA of the invention comprises the sequence NAR(N17)YNN, where R is a purine (e.g., A or G) and Y is a pyrimidine e.g., C or U/T).
  • R is a purine (e.g., A or G)
  • Y is a pyrimidine e.g., C or U/T).
  • the respective 21 -nt sense and antisense RNA strands of this embodiment therefore generally begin with a purine nucleotide.
  • Such siRNA can be expressed from pol III expression vectors without a change in targeting site, as expression of RNAs from pol III promoters is only believed to be efficient when the first transcribed nucleotide is a purine.
  • the VLDLR siRNA of the disclosure can be targeted to any stretch of approximately 19-25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence”).
  • target sequence any of the target mRNA sequences.
  • Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T et al., "The siRNA User Guide,” revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. "The siRNA User Guide” is available on the worldwide web at a website maintained by Dr.
  • the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA.
  • Transcription factors are regulatory proteins that bind to a specific DNA sequence (e.g., promoters and enhancers) and regulate transcription of an encoding DNA region. Thus, transcription factors can be used to modulate the expression of VLDLR.
  • a transcription factor comprises a binding domain that binds to DNA (a DNA-binding domain) and a regulatory domain that controls transcription. Where a regulatory domain activates transcription, that regulatory domain is designated an activation domain. Where that regulatory domain inhibits transcription, that regulatory domain is designated a repression domain.
  • a transcription factor may be targeted by a composition of the invention.
  • the transcription factor may be one that is associated with a pathway in which VLDLR is involved.
  • the transcription factor may be targeted with an antagonist of the invention, including siRNA to downregulate the transcription factor.
  • Such antagonists can be identified by standard methods in the art, and in particular embodiments the antagonist is employed for treatment and or prevention of an individual in need thereof.
  • the antagonist is employed in conjunction with an additional compound, such as a composition that modulates ApoE.
  • the VLDLR antagonist may be used in combination with an inhibitor of ApoE.
  • the antagonist of a transcription factor of a VLDLR-related pathway may be administered prior to, during, and/or subsequent to the additional compound.
  • an antisense molecule that binds to a translational or transcriptional staid site, or splice junctions can be used as an inhibitor.
  • Antisense, ribozyme, and double- stranded RNA molecules target a particular sequence to achieve a reduction or elimination of a particular polypeptide, such as VLDLR.
  • VLDLR polypeptide
  • antisense, ribozyme, and double-stranded RNA, and RNA interference molecules are constructed and can be used to modulate VLDLR expression.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with complementary sequences.
  • polynucleotides arc those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others, in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNAs are employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs are designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs may include regions complementary to intron/exon splice junctions. Thus, in some embodiments, antisense constructs with complementarity to regions within 50-200 bases of an intron-exon splice junction are used. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • genomic DNA it is advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site- specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction. Ribozyme catalysis has primarily been observed as part of sequence specific cleavage/ligation reactions involving nucleic acids. For example, U.S. Pat.
  • Designing and testing ribozymes for efficient cleavage of a target RNA is a process well known to those skilled in the art.
  • the identification of operative and preferred sequences for use VLDLR targeted ribozymes is simply a matter of preparing and testing a given sequence, and is a routinely practiced screening method known to those of skill in the art.
  • the VLDLR antagonists can be administered alone or in combination with effective amounts of one or more active pharmaceutical agents.
  • the one or more active pharmaceutical agents are other drugs that are useful for treating Alzheimer’s disease in the subject, such as cholinesterase inhibitors, N-methyl-D-aspartic acid (NMDA) receptor antagonists or antibodies directed against beta-amyloid.
  • NMDA N-methyl-D-aspartic acid
  • the one or more additional active agents comprises i) an effective amount of an LRP1 antagonist; ii) an effective amount of a SorLA antagonist; or iii) an effective amount of an LRP1 antagonist and a SorLA antagonist.
  • the one or more additional active agents comprises receptor associated protein (RAP).
  • RAP receptor associated protein
  • the one or more additional active pharmaceutical agents comprises aminocaproic acid, acamprosate, amlodipine, argatroban, baclofen, cilostazol, cinacalcet, clopidogrel, dyphylline, fenoldopam, leflunomide, mepacrine, methimazole, phenformin, prilocaine, rifabutin, sulfisoxazole, tadalafil, terbinafine, cinnarizine, ciclopirox, eplerenone, carbenoxolone, sulodexide, carbamazine, amobarbital, cefotetan, erythrityl tetranitrate, methyclothiazide, risedronate, enprofylline, oxtriphylline, paramethadione, cefmenoxime, aprindine, etomidate, mitiglinide, be
  • the subject is administered one or more atypical antipsychotics, beta-amyloid antibodies, cholinesterase inhibitors, or NMDA antagonists.
  • the subject is administered lecanemab, brexpiprazole, rivastigmine, donepezil, galantamine, memantine, or a combination of memantine and donepezil.
  • the VLDLR antagonist or therapeutic agent can be administered in a variety of ways and is not particularly limiting.
  • the administration of the therapeutic agent and/or the therapies of the present invention may include systemic, local and/or regional administrations.
  • the agent is administered directly (topically), intravenously, subcutaneously, transcutaneously, intrathecally, intramuscularly, intracutaneously, intragastrically, intranasally, rectally, intra-arterially, parenterally, orally, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
  • the agent is administered topically (dermally, transdermally), via catheters, implantable pumps, dermal patches, transdermal patches, etc.
  • Other routes of administration arc also contemplated such as, for example, arterial perfusion, intracavitary, intraperitoneal, intrapleural, intraventricular and/or intrathecal. The skilled artisan is aware of determining the appropriate administration route using standard methods and procedures.
  • the therapeutic compound is administered intrathecally. In some embodiments, the compound is administered intrathecally via an implantable pump. In one embodiment, the implantable pump comprises a SynchroMedTM II pump that stores and delivers medication into the intrathecal space (Medtronic).
  • the antagonist can be administered parenterally or alimentarily.
  • Parenteral administrations include, but are not limited to intravenously, intradermally, transdermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally. See, e.g., U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • Alimentary administrations include, but are not limited to orally, buccally, rectally, or sublingually.
  • the VLDLR antagonist or therapeutic agent can be modified to facilitate transport across the blood-brain barrier. See, e.g., Zhao el al., Antib Ther. 2022 Oct; 5(4): 311-331.
  • Treatment methods involve administering to a subject an effective amount of therapeutic agents as described herein.
  • a specific dose level of active compounds such as an antagonist of VLDLR for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.
  • the compound(s) or composition(s) can be administered to the subject once, such as by a single injection or deposition at or near the site of interest. In some embodiments, the compound(s) or composition(s) can be administered to a subject over a period of days, weeks, months or even years. In some embodiments, the compound(s) or composition(s) is administered at least once a day to a subject. Where a dosage regimen comprises multiple administrations, it is understood that the effective amount of the compound(s) or composition(s) administered to the subject can comprise the total amount of the compound(s) or composition(s) administered over the entire dosage regimen.
  • an effective amount of the antagonist of VLDLR that is administered includes a dose of about 0.0001 nM to about 2000 pM.
  • amount administered is from about 0.01 nM to about 2000 pM; about 0.01 pM to about 0.05 pM; about 0.05 pM to about 1.0 pM; about 1.0 pM to about 1.5 pM; about 1.5 pM to about 2.0 pM; about 2.0 pM to about 3.0 pM; about 3.0 pM to about 4.0 pM; about 4.0 pM to about 5.0 pM; about 5.0 pM to about 10 pM; about 10 pM to about 50 pM; about 50 pM to about 100 pM; about 100 pM to about 200 pM; about 200 pM to about 300 pM; about 300 pM to about 500 pM; about 500 pM to about 1000 pM; about 1000 pM to about 1500 pM; and about 1500 p
  • the total daily dose of the antagonist of VLDLR of the present invention administered to a subject in single or in divided doses can be in amounts, for example, from 0.01 to 25 mg/kg body weight or more usually from 0.1 to 15 mg/kg body weight.
  • Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • treatment regimens according to the present invention comprise administration to a human or other mammal in need of such treatment from about 1 mg to about 1000 mg of the active substance(s) of this invention per day in multiple doses or in a single dose of from 1 mg, 5 mg, 10 mg, 100 mg, 500 mg or 1000 mg.
  • the treatments may include various "unit doses.”
  • Unit dose is defined as containing a predetermined quantity of the therapeutic composition (an antagonist of VLDLR) calculated to produce the desired responses in association with its administration, e.g., the appropriate route and treatment regimen.
  • the quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. Also of importance is the subject to be treated, in particular, the state of the subject and the protection desired.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • the VLDLR antagonists are formulated as pharmaceutical compositions comprising a therapeutically effective amount of one or more of the active agents along with a pharmaceutically acceptable carrier.
  • the invention is directed to a method of treating or preventing Alzheimer’ s disease in a subject by administering to the subject a an effective amount of a composition comprising an antagonist of VLDLR and a pharmaceutically acceptable carrier.
  • compositions can comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection.
  • phrases "pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one Alzheimer’s disease drug or related compounds or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).
  • preservatives e.g., antibacterial agents, antifungal agents
  • isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like
  • exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, intrathecal, subcutaneous, intramuscular, intracutancous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.
  • sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
  • antioxidants examples include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, a-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like
  • oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT
  • the antagonists can be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • compositions of the present invention suitable for administration are provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • composition can be combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the ail.
  • the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of pharmaceutical lipid vehicle compositions that include compounds or compositions of the invention such as Alzheimer’s therapeutics, one or more lipids, and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the ail, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester- linked fatty acids and polymerizable lipids, and combinations thereof.
  • neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester- linked fatty acids and polymerizable lipids, and combinations thereof.
  • lipids are also encompassed by the compositions and methods of the present invention.
  • the Alzheimer’s therapeutics may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • the compounds and compositions of the invention are formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. See, e.g., U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each specifically incorporated herein by reference in its entirety.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as, for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar', or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally in a delayed manner.
  • coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water, isotonic solutions, or saline.
  • Such compositions may also comprise adjuvants, such as wetting agents; emulsifying and suspending agents; sweetening, flavoring and perfuming agents.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycol, which are solid at ordinary temperature but liquid at the rectal temperature and will, therefore, melt in the rectum and release the drug.
  • a suitable non-irritating excipient such as cocoa butter and polyethylene glycol
  • sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • the injectable composition can be administered as a nanoparticle formulation.
  • the most common way to accomplish this is to inject a suspension of crystalline or amorphous material with poor water solubility.
  • the rate of absorption of the drug becomes dependent on the rate of dissolution of the drug, which is, in turn, dependent on the physical state of the drug, for example, the crystal size and the crystalline form.
  • Another approach to delaying absorption of a drug is to administer the drug as a solution or suspension in oil.
  • Injectable depot forms can also be made by forming microcapsule matrices of drugs and biodegradable polymers, such as polylactide-polyglycoside.
  • the rate of drug release can be controlled.
  • biodegradable polymers include polyorthoesters and poly anhydrides.
  • the depot injectables can also be made by entrapping the drug in liposomes or microemulsions, which arc compatible with body tissues.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, dimethyl sulfoxide (DMSO), polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • DMSO dimethyl sulfoxide
  • polyol i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • nonaqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria- retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.
  • the active compound is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • a liquid carrier such as, e.g., water or a saline solution
  • the compounds and compositions of the invention may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
  • compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder.
  • Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also comprise the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
  • the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety).
  • the delivery of drugs using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
  • transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
  • aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
  • Dosage forms for topical or transdermal administration of a compound of this invention further include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • Transdermal patches have the added advantage of providing controlled delivery of active compound to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • the invention provides a method of screening for potential agents that reduce internalization and/or trafficking of tau in cells, comprising i) providing a cell expressing VLDLR or a functional equivalent of VLDLR; ii) providing tau protein or a fragment or derivative thereof to the cell, wherein the tau protein or a fragment or derivative thereof is extracellular; iii) treating the cell with an effective amount of a test agent that may inhibit binding of tau to VLDLR or reduce expression of VLDLR in cells; and iv) assaying the cellular uptake of tau or the fragment or derivative thereof.
  • tau and VLDLR or a functional equivalent thereof can be used in screening assays for compounds which bind one or more of the proteins and which inhibit their interaction.
  • the screening methods can be conducted in cells, cell-free preparations, cellular homogenates, animals, or on one or more substrates, for example on surface plasmon resonance sensor chips.
  • any of a tau antibody, tau or a fragment or derivative thereof (including fractions from brain, including from Alzheimer’s patients), VLDLR, and the potential antagonist/agent can be coupled to a solid surface to assay competitive binding.
  • one or more domains of VLDLR are assayed for competition binding of tau or a fragment or derivative thereof using a test compound.
  • a tau antibody is bound to a surface, such as a surface plasmon resonance chip, and a sample comprising tau or a fragment or derivative thereof is added to the surface.
  • the source of tau comprises a sample from brain, e.g., of an Alzheimer’s patient, such as a homogenate, or size exclusion purified fraction, to bind tau to the antibody.
  • Tau binding can be confirmed by a second tau antibody, in some embodiments.
  • VLDLR, or tau binding fragments or derivatives thereof are added to the immobilized tau, and can be added in combination with a test agent to be assayed for competitive binding to displace the bound VLDLR from the surface or to prevent binding.
  • the invention provides a screening assay to test for compounds that inhibit the interaction of tau with VLDLR comprising i) providing a tau antibody bound to a substrate; ii) adding a sample to the substrate comprising tau or a fragment or derivative thereof; iii) adding VLDLR and a test compound to the substrate; iv) detecting binding of VLDLR to the substrate or detecting the absence or reduction of binding of VLDLR to the substrate in the presence of the test compound.
  • the invention provides a screening assay to test for compounds that inhibit the interaction of tau with VLDLR comprising i) providing a VLDLR antibody bound to a substrate; ii) adding a sample to the substrate comprising VLDLR or a fragment or derivative thereof; iii) adding a source of tau or a fragment or derivative thereof and a test compound to the substrate; iv) detecting binding of tau or a fragment or derivative thereof to the substrate or detecting the absence or reduction of binding of tau or a fragment or derivative thereof to the substrate in the presence of the test compound.
  • the substrate is a surface plasmon resonance sensor chip.
  • tau or VLDLR binding can be confirmed by a second tau or VLDLR antibody.
  • the invention provides a method of screening for potential antagonists of VLDLR that reduce internalization and/or trafficking of tau in cells.
  • the method comprises providing a cell expressing VLDLR or a functional equivalent of VLDLR; providing tau or a fragment or derivative thereof protein to the cell, wherein the tau protein or fragment or derivative thereof is extracellular; treating the cell a potential VLDLR antagonist; and assaying the cellular uptake of tau or the fragment or derivative thereof.
  • the method comprises comparing the cellular uptake of tau or the fragment or derivative thereof in the cell with cells that have not been treated with the antagonist.
  • the screening procedures involve producing appropriate cells, which can be neuronal cells which express VLDLR or functional equivalents thereof.
  • Such cells can include neuronal or non-neuronal cells from mammals, yeast, Drosophila or E. coli.
  • the cells express the polypeptide endogenously.
  • the cells have been transfected or engineered to express the polypeptide.
  • cells expressing the protein (or extracts or purified preparations from cells) are contacted with a test compound to observe stimulation or inhibition of a functional response.
  • the levels of VLDLR mRNA or protein can be assayed after contacting the cells with the test compound.
  • the expression level of an endogenous VLDLR target gene is assayed.
  • the cells can comprise a reporter gene located downstream of one or more VLDLR promoter elements and inhibition of the reporter gene is assayed.
  • antagonists can include antibodies, peptides, carbohydrates, lipids, or small molecules which bind to one or more of the proteins so that binding between tau and VLDLR is inhibited. These agents can be selected and screened 1) at random, 2) by a rational selection or 3) by design using for example, protein or ligand modeling techniques (preferably, computer modeling). All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
  • VLDL receptor interacts with tau and mediates its cellular uptake.
  • LRP1 low-density lipoprotein receptor-related protein
  • SORLA sortilin-related receptor
  • VLDLr very low-density lipoprotein receptor
  • SPR Surface plasmon resonance
  • VLDLr as a third receptor capable of binding tau and mediating its internalization. This activity of the VLDLr’ s raises the possibility that the VLDLr may contribute to the spreading of pathogenic forms of tau in the AD brain.
  • Example 2 The VLDL receptor binds and internalizes tau in a process that is inhibited by monoclonal antibodies 1H10 and 1H5.
  • VLDLr Another receptor that mediates tau uptake
  • the interest in tau receptors arise from the fact that neuronal transfer of pathological forms of tau has been proposed as a mechanism of Alzheimer’s disease (AD) progression.
  • AD Alzheimer’s disease
  • the accumulation of misfolded tau aggregates initiates in the entorhinal cortex and spreads across connected neural pathways (7-13).
  • VLDLr is one of two receptors that participate in reelin signaling, which decreases the extent of tau phosphorylation. Phosphorylation generates the pathogenic forms of tau. Since receptor dimerization using antibodies have also been shown to activate the reelin signaling pathway (5), we anticipate that 1H10 and 1H5 will not only block tau uptake, but also activate the reelin signaling pathway decreasing tau phosphorylation.
  • VLDLr Cells that express the VLDLr mediate tau internalization. Having demonstrated a direct interaction between tau and the VLDLr, we next tested if the VLDLr can mediate tau internalization. For this experiment, we selected Chinese hamster ovary (CHO) cells lacking LRP1 (a major endocytic receptor for tau) that were stably transfected with the VLDLr. These cells, along with parental 13-5-1 cells were incubated with 20 nM of 125 I- labclcd tau for 2h, and the amount of tau internalized quantified. The results arc shown in Figure 2 and demonstrate significant increases in tau internalization in cells expressing the VLDLr. The VLDLr-mediated internalization was blocked with the receptor-binding protein (RAP), a molecule that antagonizes binding of ligands to this receptor and other LDL-receptor family members.
  • RAP receptor-binding protein
  • Monoclonal antibodies 1H10 and 1H5 block tau binding to the VLDLr and VLDLr- mediated tau uptake.
  • 100 nM 1H10 was highly effective in blocking 20 nM tau from binding to immobilized VLDLr (Fig 3a).
  • antibody 1H5 was less effective at blocking binding, while 5F3 was not able to block binding. While these antibodies bind to different regions of the ligand binding domain of the VLDLr, the lack of inhibition of 5F3 might result from its low affinity for the VLDLr ( Figure 4).
  • Example 3 Administration of VLDL antibodies in a pathogenic tau animal model.
  • mice which express human 1N4R tau protein with two pathogenic mutations (P301S and G272V) under the control of the neuron- specific Thy 1.2 promotor and/or HtauP301L transgenic mice (which express human 2N4R tau protein with the P301L mutation under the Thy 1.2 promotor) will receive stereotaxic injections of tau isolated from human Alzheimer patients, with or without anti- VLDLr antibodies 1H10, 1H5, 5F3 (30mg/kg) into the CAI layer of the hippocampus. 2 days after injection, mice will be sacrificed and whole brains processed for immunohistochemical (IHC) analysis using human specific anti-tau antibodies. The amount of tau internalized will be quantified based on the integrated tau signal.
  • IHC immunohistochemical

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Neurology (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Neurosurgery (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Psychiatry (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)

Abstract

La présente invention concerne des méthodes et des compositions pour réduire l'internalisation et/ou le trafic de Tau dans des cellules neuronales, comprenant la mise en contact des cellules avec une quantité efficace d'antagoniste du récepteur des VLDL. L'invention concerne en outre une méthode de traitement ou de prévention de la maladie d'Alzheimer chez un sujet en ayant besoin, comprenant l'administration au sujet d'une quantité efficace d'un antagoniste du récepteur des VLDL.
PCT/US2025/021013 2024-03-22 2025-03-21 Compositions et méthodes de traitement et de prévention de la maladie d'alzheimer Pending WO2025199495A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463568862P 2024-03-22 2024-03-22
US63/568,862 2024-03-22

Publications (1)

Publication Number Publication Date
WO2025199495A1 true WO2025199495A1 (fr) 2025-09-25

Family

ID=97140347

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/021013 Pending WO2025199495A1 (fr) 2024-03-22 2025-03-21 Compositions et méthodes de traitement et de prévention de la maladie d'alzheimer

Country Status (1)

Country Link
WO (1) WO2025199495A1 (fr)

Similar Documents

Publication Publication Date Title
AU2016369407B2 (en) Treatment of fibrosis
US20170175125A1 (en) Inhibition of Bruton's Tyrosine Kinase (Btk) in the Lung to Treat Severe Lung Inflammation and Lung Injury
JP7225115B2 (ja) リソソーム蓄積症およびリソソーム蓄積障害を処置するための組成物および方法
Portillo et al. Disruption of retinal inflammation and the development of diabetic retinopathy in mice by a CD40-derived peptide or mutation of CD40 in Müller cells
JP2014088393A (ja) トレフォイル因子およびそれを用いた増殖性疾患の処置方法
JP7612575B2 (ja) 筋萎縮性側索硬化症を治療及び予防するための組成物及び方法
JP4807802B2 (ja) Ｖｅｇｆ媒介性活性をブロックすることによるｉ型糖尿病を処置する方法
US8038992B2 (en) Target for regulating multiple sclerosis
US20100196393A1 (en) Modulation of blood brain barrier protein expression
JP2025127483A (ja) Ｒａｓ変異体癌を治療するための組成物および方法
CN102921007B (zh) 防治胰岛素抵抗和糖尿病的方法和试剂
KR20160130986A (ko) K-ras를 침묵화시키는 비대칭 간섭 rna 조성물 및 이의 사용 방법
US20080249038A1 (en) Bone Morphogenetic Protein (Bmp) 2A and Uses Thereof
US20220332813A1 (en) Compositions and methods for treatment and prevention of alzheimer's disease
WO2025199495A1 (fr) Compositions et méthodes de traitement et de prévention de la maladie d'alzheimer
EP3037817B1 (fr) Procédé de criblage des inhibiteurs de la douleur et composition pharmatheutique pour la prevention ou le traitement de la douleur
CN103957937B (zh) 用于治疗阿尔茨海默病的化合物
US20230391868A1 (en) Compositions for and methods of treating cancer
Li et al. RETRACTED: Long noncoding RNA MEG3 contributes to dysfunction of brain microvascular endothelial cells after intracerebral hemorrhage by regulating the miR-1930-5p/Mllt1 axis
JP7527001B2 (ja) 疼痛の予防及び／又は治療用医薬組成物、並びに、疼痛抑制物質のスクリーニング方法
US20250228918A1 (en) Methods of controlling body weight and/or energy expenditure
WO2010112034A2 (fr) Compositions et méthodes pour le traitement et le diagnostic de synucléinopathies
WO2024173483A2 (fr) Compositions comprenant un inhibiteur de mtor destiné à être utilisé dans le traitement de maladies et de troubles associés à la résistance à la leptine et à l'obésité
EP4665382A2 (fr) Compositions comprenant un inhibiteur de mtor destiné à être utilisé dans le traitement de maladies et de troubles associés à la résistance à la leptine et à l'obésité
EP4253540A1 (fr) Acide nucléique antisens et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25774220

Country of ref document: EP

Kind code of ref document: A1