WO2013006076A1 - The use of intranasally administered hsp70 protein to treat neurodegenerative diseases - Google Patents

Abstract

The invention provides a method of treatment of Alzheimer's disease and other neurodegenerative diseases comprising intranasally administering Hsp70 protein and/or active fragments and/or derivatives thereof.

Description

THE USE OF INTRANASALLY ADMINISTERED IISP70 PROTEIN TO TREAT NEURODEGENERATIVE DISEASES

FIELD OF THE INVENTION

[0002] The invention provides a method of treatment of Alzheimer^'s d isease and other neurodegenerative diseases associated with abnormal protein biogenesis and cogn itive disturbances comprising intranasally administering Hsp70 protein and/or active fragments and/or derivatives thereof

BACKGROUND OF THE INVENTION

[0003] Neurodegenerative diseases are a large group of pathologies caused by metabol ic changes in brain cells, loss of synapses and other of neurons, and final ly neuronal death, for review see Neurodegenerative diseases: From Molecular Concepts to Therapeutic Targets. Kditors: R. von Bernhardi, N .C. Inestrosa, Nova Publ ishers, 2008. This group of diseases includes A lzheimer's disease (AD), Lewy Body dementia, Parkinson ^'s d isease ( PI)). Huntington's d isease (HD), frontotemporal dementia (FTD), vascular dementia, multiple sclerosis (MS), amyotroph ic lateral sclerosis (A LS), prion d iseases, d ifferent atax ias, and others. Due to the increased l ifespan, neurodegenerative diseases become very common in developed countries.

|0004J Alzheimer's d isease (A D) is the most prevalent neurodegenerative d isease in the growing population of elderly humans and leads eventually to severe brain damage and death ( 1 ). There are about 6 mi llion people living with AD in the U S only, 70-80 mi l l ion people are in the risk group and $ 148 bi llion are spent in the US for AD patient treatment and care. Despite tremendous effort, no effective treatments for A D are currently avai lable. The sevcrilv of cognitive impairment in patients with AD usual ly correlates with the ex tent of observed pathomorphological abnormal ities, including the decrease of synaptic density, the enhanced concentration of soluble Λβ oligomers, and. most importantly, the amount of neurons that die by apoptosis in speci fic brain areas ( 1 . 2). [0005] Heat shock proteins (HSPs) have emerged as critical regulators of neurodegenerative processes associated with protein misfolding. I n brains A D and Parkinson ^' s disease (PD) patients, HSPs were found to be associated with sen ile plaques and Lewy bodies, respectively (3-8). Live cell imaging experiments showed that Hsp70 associates transiently with huntingtin aggregates impl icated in Huntington's disease (HD) (40). Various data suggest that Hsp70 and other molecular chaperones function as a complex neuroprotective sy stem, wh ich, however, fails in the brains of individuals with neurodegenerative diseases that involve aberrant protein folding and protein damage and aging individuals (6, 9- 12). Indeed, the expression and functional ity of the inducible form of Hsp70 and other major HSPs becomes compromised in aging brain tissue (7).

[0006] Currently, no effective pharmacological therapy has been developed for the treatment of AD or other neurodegenerative diseases. Such therapy is urgently needed .

SUMMARY OF THE INVENTION

[0007] As specified in the Background Section, there is a great need in the art to develop novel therapeutics for the treatment of Alzheimer's disease (AD) and other neurodegenerative diseases associated with abnormal protein biogenesis (e.g., aberrant protein folding and protein damage) and cognitive d isturbances (e.g., Parkinson ^' s d isease ( PD), Huntington ^'s disease (HD), Lewy Body dementia, frontotemporal dementia ( 1 ^"I ^'D), vascular dementia, mi ld cognitive impairment (MCI), mixed dementia, Creutzfeldt-Jakob Disease (CJ D), normal pressure hydrocephalus, Wernicke-Korsakoff syndrome, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), prion diseases, and different ataxias, etc.) or aging.

[0008] The present invention satisfies this and other needs by provid ing a method of treatment of a neurodegenerative disease in a subject (e.g., human) in need thereof comprising intranasal ly administering to the subject a therapeutically effective amount of I lsp70 protein and/or active fragment and/or derivative thereof.

[0009] Neurodegenerative diseases treatable by the method o f the present invention include neurodegenerative diseases associated with abnormal protein biogenesis. Non- l imiting examples of neurodegenerative diseases treatable by the method of the invention include Alzheimer' s disease (AD), Parkinson's disease (PD), Huntington' s disease (H D), l .ewy Body dementia, frontotemporal dementia (FTD), vascular dementia, mi ld cognitive impairment (MC I ), mixed dementia, Creutzfeldt-Jakob Disease (CJD), normal pressure hydrocephal us. Wern icke- Korsakoff syndrome, multiple sclerosis (MS), amyotroph ic lateral sc lerosis (A I .S). prion diseases, and ataxia. In one embodiment, the method of the invention is used to treat neurodegenerative diseases associated with aging.

[0010] In one embodiment, the Hsp70 protein used in the method of the invention is a full-length human Hsp70 protein (e.g., Hsp70 protein which has the amino acid sequence comprising SF,Q ID NO: 1 ). In another embodiment, one or two or more active fragments of Hsp70 protein or derivatives thereof are used.

[0011] In one embodiment, the Hsp70 protein and/or active fragment and/or derivative thereof is formulated in a composition further comprising one or more nasal del ivery- enhancing agents.

[0012] In one embodiment, the Hsp70 protein and/or active fragment and/or derivative thereof is formulated in a composition further comprising one or more agents which faci litate brain delivery.

[0013] In one embodiment, the amount of Hsp70 protein and/or active fragment and/or derivative thereof administered according to the method of the invention is within the range from about 0.2 μg to about 1 mg per kg body weight per day. In another embod iment, the amount of Hsp70 protein and/or active fragment and/or derivative thereof administered according to the method of the invention is within the range from about 0.2 μ to about 1 00 μg per kg body weight per day. In yet another embodiment, the amount of I Isp70 protein and/or active fragment and/or derivative thereof administered according to the method of the invention is within the range from about 1 0 μg to about 1 00 μg per kg body weight per day. I n a further embodiment, the amount of Hsp70 protein and/or active fragment and/or derivative thereof administered according to the method of the invention is within the range from about 100 μ to about 1 mg per kg body weight per day.

[0014] In one embodiment, the Hsp70 protein and/or active fragment and/or derivative thereof is administered as a single daily dose. [0015] I n one embodiment, the Hsp70 protein and/or active fragment and/or derivative thereof is administered for 3 weeks to 5 months.

[0016] In one embodiment, the Hsp70 protein and/or active fragment and/or derivative thereof is administered in combination with at least one add itional therapeutic agent. Non-limiting examples of such additional therapeutic agents include memory enhancement agents, antidepressants, anxiolytics, antipsychotic agents, sleep disorder agenst, antiinflammatory agents, anti-oxidant agents, cholesterol modulating agents, and anti-hypertensive agents.

BRIEF DESCRIP ION OF THE DRAWINGS

[0017] Figures 1Λ-Β. IIsp70 improves memory in OBF mice. The level of spatial memory correlates strongly with the hippocampal concentration of endogenous I Isp70 in OBE mice (Λ and B). Panel (Λ) shows the time spent by OBE mice in a target sector of a Morris water maze during probe period . The values (mean SEM) % represent the ratio between OBE and control SO animals of the same age group expressed as a function of time (months post- operation). After completion of the behavioral experiments, animals from each age group (n 6- 7) were euthanized and their brains were used for Hsp70 measurements. Panel (B) shows the changes of Hsp70 concentration in the hippocampus of OBE mice as a function of time after bu lbectomy. The values (mean I SEM) % represent the ratio of I lsp70 concentrations between OBI⁷, and SO animals of the same age group. The absolute values (ng/g) of l lsp7() in SO animals after 0.5, 3, 6, and 12 months were 6.2±2.1 , 7.8± 1 .4, 5.2^0.9, and 1 2.95 I 3.5, respectively.

[0018] Figures 1C-D. The protective effects of sub-chronic intranasal administration of exogenous Hsp70 on spatial memory of OBE mice. Bars indicate time ( in seconds) spent by m ice in each of the water maze quadrants during probe trial (mean i SEM ). Gray or hatched bars represent time spent in the target sector. Train ing and probe trials with independent groups of animals were performed immediately after Hsp70 treatment one month after bulbectomy (C) or eight months after bulbectomy (D). Asterisks indicate signi ficant differences (*- p<0.05; * * - pO.01 ; * **- pO.001 ) in comparison with the target quadrant (hatched bar). [0019] Figures 2Λ-Ι. Localization of exogenous IIsp70 in different brain regions of mice after intranasal administration. The images show grains of the fluoresccntly- labeled Hsp70 in the brain sections of NMRI mice 3 h after intranasal injection. I Isp70 is distributed non-randomly and concentrated in a few brain structures. Photos show its local ization in the ol factory bulbs (Λ, B), fields of the hippocampus (C, D), in the n. raphe dorsal is (E, F), and in cells of the cerebellum (G, H). In most cases Hsp70 has an intracel lular local ization (B, D, F, H) and is restricted to the perinuclear zone (indicated by arrow in 1) and II). Control mice treated with unlabeled Hsp70 do not exhibit any brain fluorescence (I).

[0020] Figures 3Λ-Β. Neuronal protection by exogenous Hsp70. (Λ)

Representative photomicrographs of frontal sections through the hippocampus and temporal cortex of untreated control SO mice ( 1 , 2, 3); SO mice after Hsp70 treatment (4, 5, 6); untreated OBE mice (7, 8, 9) and OBE mice treated with Hsp70 ( 10, 1 1 , 12). C T-temporal cortex. C I and C3 areas of hippocampus. Different pathological states of neurons such as pyknosis (P), karyolysis (K), cytolysis (C), or vacuolization (V) arc ind icated by arrows. (B) Effect of intranasal Hsp70 administration on neuronal density in CA 1 and CA3 areas of h ippocampus. SO+HSP70 and OBE I HSP70 - sham-operated and bulbectomizcd mice treated intranasal ly for 21 days with Hsp70. Asterisks indicate significant d i fferences * *- p<0.0 1 , * * * - p<().00 l re lative to the SO group. ⁰⁰⁰ - p< 0.001 relative to OBE group.

[0021 ] Figures 4A-B. Exogenous IIsp70 prevents increase of Αβ peptide levels in brains of OBE mice. Bars indicate the level of Αβ peptides (mean i SE ) in the indicated groups of experimental mice (n 6- 1 1 per group). Αβ was determined by DOT analysis (A) or ELI SA (B) (see Example 1 , Materials and Methods). The level of Αβ peptides in di fferent groups (mean I SEM) is shown relative to the content in SO group (B). The absolute value of Αβ in SO animals was 225.5±78.25 pM/g tissue of brain (cortex -t hippocampus). SO U 1 SP70 and OBE+H SP70 - sham-operated and bulbectomized mice treated intranasal ly for 2 1 days with Hsp70, respectively. Differences between the levels of Αβ in the groups were determ ined by using the two-tailed Student's t-test.

[0022] Figures 5Λ-Β. Latency to find invisible platform in seconds (mean±SEM). Training trials were performed one month (A) or eight months (B) after the bulbectomy followed by three weeks of Hsp70 treatment. [0023] Figure 6. Λ representative DOT analysis of Λβ in OBE and SO mice treated by IIsp70. The scale at the bottom shows standards of different dilution of Λβ 1 -40 amyloid. Four repeats of each sample have been routinely measured for each mouse in the investigation and gave similar results. It is evident that the maximal level of Λβ is observed in OBE mice, whi le intranasal administration of Hsp70 significantly reduced the level of Λβ. SO control mice as well as SO animals treated with Hsp70 do not differ in the levels οΓ Λβ. wh ich, however, was lower in comparison with OBE and OBE t Hsp70 mice.

[0024] Figure 7. Localization of IIsp70 labeled with Alexa647 (1-4) and Alexa554 (5, 6) in various brain areas of OBE mice. The images show grains of (luoresccntly- labeled Hsp70 in the brain sections of OBE mice 3 h after intranasal injection. Hsp70 is distributed more randomly in cytoplasm of cells than in control N M R1 mice ( f ig. 2) and concentrated in the same brain structures. Photos show its localization in the frontal and temporal cortex ( 1 and 2). in an area CA3 of the hippocampus (3), in the n. magnocel lu laris and n. raphe dorsalis (4 and 6), and in the cells of the locus coeruleus (5). It is evident that in most cases Hsp70 has an intracellular localization.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention is based on an unexpected observation that intranasal administration for three weeks of recombinant human Hsp70 in the bi lateral ol factory bulbectomy (OBE) mouse model of Alzheimer^'s disease (AD) leads to a dramatic long-lasting (for at least 7 months) neuroprotective effect.

[0026] Olfactory bulbectomised (OBE) mice (i.e., mice with removed ol factory bu lbs) exhibit functional, biochemical and behavioral characteristics, inc lud ing a depression-l ike state and severe memory loss ( 13- 1 8). similar to those described for A D patients ( 1 , 19). OBE mice also d isplay a decreased use of cerebral glucose (20) and an increased level of brain amy loid precursor protein (21 ) and Αβ (22). In OBE guinea pigs, which have an Λβ amino acid sequence identical to that of the human ortholog, intracellular accumulation of Λβ in neurons of the temporal cortex and the formation of extracellular amyloid results in the formation of plaques ( 16). Simi lar to AD patients, OBE mice display a deficit of serotonin-, acety lchol ine-, and glutamatergic brain systems (23-25). Most importantly, in contrast to widely used transgenic mice models of AD (26, 27), OBE animals are characterized by massive death of neurons in the hippocampus and temporal cortex, brain structures most affected in AD individuals (28-30).

[0027] As discussed in detail in the Examples section, below, the present inventors have surprisingly found that intranasally administered Hsp70 (which is a rather large protein of 70kDa) rapidly enters the afflicted brain regions and mitigates multiple AD-like morphological and cognitive abnormalities observed in OBE animals. In particular, intranasal administration of recombinant human Hsp70 normalizes the density of neurons in the hippocampus (which correlates with the diminished accumulation of amyloid β (Αβ) peptide) and fully protects spatial memory (at the level of control animals) for at least seven months following treatment, which is a very long-lasting effect.

[0028] Based on this observation, the present invention provides a method of treatment of neurodegenerative diseases associated with abnormal protein biogenesis and cognitive disturbances (such as, e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Lewy Body dementia, frontotemporal dementia (FTD), vascular dementia, mild cognitive impairment (MCI), mixed dementia, Creutzfeldt-Jakob Disease (CJD), normal pressure hydrocephalus, Wernicke-Korsakoff syndrome, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), prion diseases, and different ataxias, etc.) or aging comprising intranasally administering Hsp70 protein and/or functional fragments and/or derivatives thereof.

Definitions

[0029] As used herein, the term "isolated" means that the material (e.g., Hsp70 protein) being referred to has been removed from the environment in which it is naturally found, and is characterized to a sufficient degree to establish that it is present in a particular sample. Such characterization can be achieved by any standard technique, such as, e.g. , hybridization, immunoassay, functional assay, expression, size determination, or the like. A protein that is associated with other proteins and/or nucleic acids with which it is associated in an intact cell, or with cellular membranes if it is a membrane-associated protein, is considered isolated if it has otherwise been removed from the environment in which it is naturally found and is characterized to a sufficient degree to establish that it is present in a particular sample. A protein expressed from a recombinant vector in a host cell, particularly in a cel l in which the protein is not naturally expressed, is also regarded as isolated .

[0030] An isolated material may or may not be "purified". The term "purified^" as used herein refers to a material (e.g., Hsp70 protein) that has been isolated under cond itions that detectably reduce or eliminate the presence of other contaminating materials. Contaminants may or may not include native materials from which the purified material has been obtained. Λ purified material preferably contains less than about 90%, less than about 75%, less than about 50%, less than about 25%, less than about 10%, less than about 5%, or less than about 2% by weight of other components with which it was original ly associated.

[0031] Methods for purification are wel l-known in the art. For example, polypeptides can be purified by various methods including, without l imitation, preparative d isc- gel electrophoresis, isoelectric focusing, HPLC, reverse-phase I I PLC, gel fi ltration, affinity chromatography, ion exchange and partition chromatography, precipitation and salting-out chromatography, extraction, and counter-current distribution. The term "substantially pure" indicates the highest degree of purity that can be achieved using conventional purification techniques currently known in the art. In the context of analytical testing of the material, "substantially free" means that contaminants, if present, are below the l imits of detection using current techniques, or are detected at levels that are low enough to be acceptable for use in the relevant art, for example, no more than about 2-5% (w/w). Accord ingly, with respect to the purified material, the term "substantial ly pure" or "substantial ly free" means that the puri fied material being referred to is present in a composition where it represents 95% (w/w) or more of the weight of that composition. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, or any other appropriate method known in the art.

[0032] The term "active fragment" refers to smaller portions of the I lsp70 polypeptide that possess therapeutic activity with respect to neurodegenerative diseases. Such therapeutic activity can be tested, e.g., using any of the methods described in the Kxamp les section, below.

[0033] The terms "active derivative", "active variant" or "active mutant^" arc used interchangeably to refer to polypeptides that contain, as compared to the human I Isp7() protein having the amino acid sequence corresponding to SwissProt P08107 (SEQ I D NO: 1 ) and EM BE BC009322, one or more amino acid deletions and/or insertions and/or substitutions, provided that the resulting polypeptides possess therapeutic activity with respect to neurodegenerative diseases. Such therapeutic activity can be tested, e.g., using any of the methods described in the Examples section, below. These terms also encompass Hsp70 orthologs from non-human species and other proteins belonging to Hsp70 fami ly, including inducible and constitutively expressed members.

[0034] The term "about" means within an acceptable error range for the particular value as determined by one of ord inary skil l in the art, which will depend in part on how the value is measured or determined, i.e. , the limitations of the measurement system, l or example, "about" can mean within an acceptable standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to rl 20%, preferably up to I 10%, more preferably up to ±5%, and more preferably still up to ± 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of a value. Where particular values arc described in the appl ication and claims, unless otherwise stated, the term "about" is impl icit and in this context means with in an acceptable error range for the particular value.

[0035] In the context of the present invention insofar as it relates to any of the disease conditions recited herein, the terms "treat", "treatment", and the l ike mean to rel ieve or alleviate at least one symptom associated with such cond ition, or to slow or reverse the progression of such condition. Within the meaning of the present invention, the term "treat" also denotes to arrest, delay the onset (i. e. , the period prior to cl inical man i festation o f a disease) and/or reduce the risk of developing or worsening a disease. For example, in relation to neurodegenerative diseases, the term "treat" may mean to delay mani festation, arrest the progression, relieve or alleviate at least one aspect of cognitive impairment (e.g., as assessed by Mini-Mental State Examination [MMSE], Orientation-Memory-Concentration [ Short-OMC | test, Mattis Dementia Rating Scale, AD Assessment Scale [ADAS], Ncurobehavioral Cogn itive Status Exam, Dementia Assessment Battery, Cambridge Cogn itive Assessment Scale, etc . | scc, e.g., Herndon, Handbook of neurologic rating scales, Demos Medical Publ ish ing, 20061). Within the meaning of the present invention, disease cond itions include neurodegenerative disorders associated with abnormal protein biogenesis (e.g., aberrant protein fold ing and protein damage) and cognitive disturbances (e.g., Alzheimer's disease (AD), Parkinson^' s disease ( PD), Huntington' s disease (HD), Lewy Body dementia, frontotemporal dementia (1 I D), vascular dementia, mild cognitive impairment (MCI), mixed dementia, Creutzfcldt-Jakob Disease (CJ D), normal pressure hydrocephalus, Wernicke-Korsakoff syndrome, multiple sclerosis (M S), amyotrophic lateral sclerosis (ALS), prion diseases, and different ataxias, etc.) or aging.

[0036] As used herein the term "therapeutically effective" applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. W ith in the context of the present invention, the term "therapeutically effective" refers to that quantity of a compound (e.g., Hsp70 protein or active fragment or derivative thereof) or pharmaceutical composition containing such compound that is sufficient to delay manifestation, arrest the progression, relieve or alleviate at least one aspect of cognitive impairment associated with a neurodegenerative disease or aging. Note that when a combination of active ingred ients is administered the effective amount of the combination may or may not include amounts of each ingred ient that would have been effective if administered individually.

[0037] The phrase "pharmaceutically acceptable", as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g. , a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other general ly recognized pharmacopeia for use in mammals, and more particularly in humans.

[0038] As used herein, "nasal delivery-enhancing agents" include agents which enhance the release or solubi lity (e.g., from a formulation delivery vehicle), d i ffusion rate, penetration capacity and timing, uptake, residence time, stabil ity, effective half-l i fe, peak or sustained concentration levels, clearance and other desired nasal del ivery characteristics (e.g., as measured at the site of delivery, or at a selected target site of activity such as the brain) of the Hsp70 protein or active fragment or derivative thereof. Enhancement of mucosal del ivery can thus occur by any of a variety of mechanisms, for example by increasing the d iffusion, transport, persistence or stabi lity of the Hsp70 protein or active fragment or derivative thereof , enzyme inhibition, increasing membrane fluidity, modulating the availabi lity or action of calcium and other ions that regulate intracellular or paracellular permeation, solubil i/.ing mucosal membrane components (e.g., l ipids), changing non-protein and protein sulfhydryl levels in mucosal tissues, increasing water flux across the mucosal surface, modulating epithelial j unctional physiology, reducing the viscosity of mucus overlying the mucosal epithelium, reducing mucoci l iary clearance rates, increasing nasal blood flow and other mechanisms. Suitable mucosal del ivery enhancing agents will be clear to a person ski lled in the art of pharmacology and arc further described hereafter.

[0039] As used herein, the term "subject" refers to any mammal. I n a preferred embodiment, the subject is human.

[0040] I n accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techn iques within the ski l l of the art. See, e.g. , Sambrook, Fritsch and Maniatis, Molecular C loning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1 989 (herein "Sambrook et al. , 1989"); DNA Cloning: A Practical Approach, Volumes I and I I (G lover cd. 1985); Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hamcs and Higgins eds. 1985); Transcription And Translation (Hames and H iggins eds. 1 84); Animal Cel l Culture (Freshney ed. 1986); Immobilized Cells And Enzymes (1RL Press, 1 986); B. Pcrbal, A Practical Guide To Molecular Cloning ( 1984); Ausubel et al. eds.. Current Protocols in Molecular Biology, John Wi ley and Sons, Inc. 1994; among others.

IIsp70 Proteins, Fragments and Derivatives Useful in the Methods of the Invention

[0041] Hsp70 proteins useful in the methods of the present invention include a full-length human Hsp70 protein (both isolated from nature and recombinantly produced) which protein has the amino acid sequence corresponding to SwissProt P081 07 (SLQ I D NO: 1 ) and EM BL BC009322 as wel l as active derivatives thereof (including I lsp70 orthologs from non- human species and other proteins belonging to Hsp70 fami ly, includ ing inducible and constitutively expressed members), that contain, as compared to the human Hsp70 protein, one or more amino acid deletions and/or insertions and/or substitutions, provided that the resulting polypeptides possess therapeutic activity with respect to neurodegenerative diseases (such therapeutic activity can be tested, e.g., using any of the methods described in the Examp les section, below).

[0042] Useful Hsp70 proteins also include active fragments of the human Hsp70 protein or derivatives thereof, which fragments or fragment derivatives possess therapeutic activity with respect to neurodegenerative diseases (such therapeutic activity can be tested, e.g., using any of the methods described in the Examples section, below).

[0043] As specified herein, the present invention contemplates the use of a single active fragment and/or derivative thereof as well as a combination of two or more active fragments and/or derivatives. Such two or more active fragments and/or derivatives can be administered simultaneously (in a single composition or different compositions) or sequential ly.

[0044] To avoid undesired immune response, human Hsp70 or active fragments or derivatives thereof are preferably used for administration to human subjects.

[0045] Hsp70 proteins, active fragments and derivatives thereof useful in the methods of the present invention can be purified from natural sources (in case of ful l-length human Hsp70 protein or its orthologs from other species or other proteins belonging to I lsp7() family, including inducible and constitutively expressed members) or can be rccombinantiv produced. Non-l imiting examples of systems useful for recombinant production include phage T5 promoter B.coli expression system (e.g., pQF30 vector; Qiagen). baculovirus expression system in insect cells (e.g., Bac-to Bac system; Invitrogen), yeast expression systems, and mammalian expression systems. Other suitable expression systems are wel l known to those skilled in the art.

[0046] Recombinant Hsp70 proteins of the invention can be produced as fusion proteins to enhance stability and/or facilitate purification.

[0047] Methods for purification which can be used to puri fy Hsp7() proteins of the invention are well known in the art. Non-l imiting examples include use of affin ity tags (e.g., H is₆) or affinity purification on immobilized A^'fP or ADP (for ful l-length molecu les, fragments and derivatives containing Hsp70 ATP-binding domain).

Compositions and Administration [0048] The Hsp70 protein (fragment or derivative)-containing pharmaceutical compositions of the present invention are administered intranasally. Such compositions can be administered intranasal ly as a powdered or l iquid spray, nose drops, a gel or ointment, through a tube or catheter, by syringe, packtail, pledget or by submucosal infusion.

[0049] As provided in the Examples section, below, the Hsp7() protein-containing compositions of the invention can be simple aqueous (e.g., saline) solutions. A lternatively, they can contain various additional ingredients which enhance stabil ity and/or nasal del ivery of Hsp70 protein. Such additional ingredients are well known in the art. Non-limiting examples of useful additional ingredients for enhancing nasal delivery include, e.g., (a) aggregation inhibitory agents (e.g., polyethylene glycol, dextran, diethylaminoethyl dextran, and carboxy methyl cellulose), (b) charge modifying agents, (c) pH control agents, (d) degradative enzyme inh ibitors (e.g., amastatin and bestatin [see, e.g., O'Hagan et a!., Pharm. Res. 1990, 7: 772-776 and WO 05/12055 1 ]; Hsp70 pegylation with PEG molecules, preferably low molecular weight PEG molecules [e.g. 2 kDa; Lee et al., Calcif Tissue Int. 2003, 73 : 545-549J); (c) mucolytic or mucus clearing agents (e.g., n-acetyl-cysteine, propyl gallate and cysteine methionine dimmers, chaotropes [see, e.g., WO 04/09391 7]), (f) ciliostatic agents; (g) membrane penetration- enhancing agents, (h) modulatory agents of epithelial junction physiology, such as nitric oxide (NO) stimulators, chitosan, and chitosan derivatives; (i) vasodilator agents, j) selective transport-enhancing agents, and (k) stabilizing delivery vehicles, carriers, supports or complex- forming agents. See, e.g., EP 037943, EP 0941 57, EP 1 73990, EP 2 14898, EP 2 1 5697, EP 327756, EP 490806, U .S. Pat. No. 4,476, 1 16, U.S. Pat. No. 5,759,565, WO 04/0939 1 7 and WO 05/ 120551 .

[0050] The activity or physical stability of Hsp70 protein in aqueous solutions or lyophilized preparations can be enhanced by various additives such as, e.g., polyols (inc lud ing sugars [e.g., sucrose and Ficoll 70]), amino acids, and various salts. For example, I lsp7() polypeptide microparticles can be prepared by simply lyophil izing or spray dryi ng a solution containing various stabi lizing additives described above. A wide non-limiting range of suitable methods and anti-aggregation agents are available for incorporation within the compositions of the invention such as disclosed in WO 05/120551 , Breslow et al. (J. Am. Chcm. Soc. 1 996; 1 1 8: 1 1678- 1 1 68 1 ), Breslow et al .(PNAS USA 1997; 94: 1 1 1 56- 1 1 1 58), Breslow et al. ( Tetrahedron Lett. 1 998; 2887-2890), Zutsh i et al. (Curr. Opin. Chem. Biol. 1998; 2: 62-66). Daugherty et al . (J. Am. Chem. Soc. 1999; 121 : 4325-4333), Zutshi et al. (J. Am. Chem. Soc. 1 997; 1 19: 4841 - 4845), Ghosh et al. (Chem. Biol. 1997; 5: 439-445), Hamuro et al . (Angew. Chem. Int. Fd. Fngl . 1997; 36: 2680-2683), Alberg et al., Science 1993 ; 262: 248-250), Tauton et al . (J . Am. Chem. Soc. 1996; 1 1 8: 10412- 10422), Park et al . (J. Am. Chem. Soc. 1999; 1 2 1 : 8- 1 3), Prasanna ct al. (Biochemistry 1998; 37:6883-6893), Tiley et al. (J. Am. Chem. Soc. 1 997; 1 19: 7589-7590), Judice et al . (PNAS U SA 1997; 94: 13426- 1 3430), Fan et al . (J. Am. Chem. Soc. 1998; 120: 8893-8894), Gamboni et al. (Biochemistry 1998; 37: 121 89- 12194).

[0051] Non-limiting examples of membrane penetration-enhancing agents useful in the Hsp70 protein-containing compositions of the invention include, e.g., (i) a surfactant (e.g., Tween 80, Poloxamer 1 88, polysorbates; see also EP 490806, U.S. Pat. No. 5,759,565, and WO 04/09391 7), (ii) a bi le salt or bi le salt derivative (e.g., unsaturated cycl ic ureas and Transcutol). (iii) a phospholipid or fatty acid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) a nitric oxide donor compound (e.g., S-nitroso-N-acetyl- F-pcnicil lamine, NOR 1 , NOR4, which are preferably co-administered with an NO scavenger such as carboxy- PITO or doclofenac sodium), (vii) a long-chain amphipath ic molecu le (e.g., dcacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid) (viii) a smal l hydrophobic penetration enhancer, (ix) sodium salicylate or a salicylic acid derivative (e.g., acetyl salicylate, chol ine salicylate, salicylamide, etc.), (x) a glycerol ester of acetoacetic acid, (xi) a cyclodcxtrin or bcta- cyclodextrin derivative, (xii) a medium-chain fatty acid including mono- and d iglycerides (e.g., sodium caprate - extracts of coconut oil, Capmul), (xi ii) a chelating agent (e.g., citric acid, salicylates), (xiv) an amino acid or salt thereof (e.g. monoaminocarboxl ic acids such as glycine, alanine, phenylalanine, prol ine, hydroxyproline, etc.; hydroxyamino acids such as serine; acid ic amino acids such as aspartic acid, glutamic acid, etc; and basic amino acids such as lysine etc., inclusive of their alkali metal or alkaline earth metal salts), (xv) an N-acctylamino acid or salt (.hereof, (xv i) an enzyme degradative to a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis, (xix) cationic polymers, or any combination thereof. The membrane penetration-enhancing agent can be also selected from smal l hydrophilic molecules, including but not l imited to, dimethyl sulfox ide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrol idoncs. Additional membrane penetration enhancers include emulsifiers (e.g. sodium oleyl phosphate, sod ium lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ethers, polyoxycthy lenc alky 1 esters, etc.). caproic acid, lactic acid, malic acid and citric ac id and alkal i metal salts thereof, pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxyl ic acid esters, N- alkylpyrrolidones, proline acyl esters, and the like; mixed micel les; glycerol esters of acctoacetic acid (e.g., glyceryl- 1 ,3-diacetoacetate or l ,2-isopropylideneglycerine-3-acetoacetate). and triglycerides (e.g., amylodextrin, Estaram 299, iglyol 810); cyclodcxtrins and (3-cyclodcxtrin derivatives (e.g., 2-hydroxypropyl-p-cyclodextrin and heptakis (2,6-di-0-methyl-[3-cyclodextrin) which can be optionally conjugated with Hsp70 and further optional ly formu lated in an oleaginous base; and N-acetylamino acids (N-acetylalaninc, N-acetylphcnylalan inc, N- acetylserine, N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N -acetyl pro l ine, N- acetylhydroxyproline, etc.) and their salts (alkali metal salts and alkaline earth metal salts), as well as other penetration-promoting agents that are physiologically compatible for intranasal del ivery. See, e.g., WO 04/09391 7, WO 05/12055 1 and Davis and I l ium (C l in . Pharmacokinct 2003, 42: 1 1 07- 1 1 28).

[0052] Non-limiting examples of useful absorption enhancers include, e.g., surfactants, glycosides, cyclodextrin and glycols. Non-limiting examples of useful bioadhesive agents include, e.g., carbopol, cellulose agents, starch, dextran, and chitosan.

[0053] In various embodiments of the invention, the I lsp70 protein, fragment or derivative of the invention (or several such proteins and/or fragments and/or derivati ves) is combined with one or more of the nasal delivery-enhancing agents rec ited above. These nasal delivery-enhancing agents may be admixed, alone or together, with the nasal carrier and with the Hsp70 protein, fragment or derivative, or otherwise combined therewith in a pharmaceutical ly acceptable formulation or delivery vehicle. For nasal delivery-enhancing agents to be of value within the invention, it is generally desired that any significant changes in permeabi lity of the mucosa be reversible within a time frame appropriate to the desired duration of drug del ivery. Furthermore, there should be no substantial, cumulative toxicity, nor any permanent deleterious changes induced in the barrier properties of the nasal mucosa with long term use.

[0054] In addition to the Hsp70 protein, fragment or derivative (or several such proteins and/or fragments and/or derivatives), the nasal carrier and, optional ly, one or more further additives and/or agents, the composition of the invention may further comprise one or more add itional therapeutic ingredients (or active substances). These therapeutic ingred ients can be any compound that elicits a desired activity or therapeutic or biological response in the subject. Non-l imiting examples of useful additional therapeutic ingredients is provided in, the Combination Treatments section, below.

[0055] The compositions of the invention may further comprise agents which facilitate brain delivery. Non-limiting examples of such useful agents include, e.g., functionalized nanocarriers (e.g., nanoparticles coated with transferrin or transferrin receptor [TR] antibodies) and liposomes (e.g., liposomes coated with targeting molecu les such as antibodies, Trojan Horses Liposomes [THL]), antibodies (e.g., antibodies against transferrin receptor [TR] and insulin receptor [HIR], BBB transmigrating Llama single domain antibod ies (sdAb)), chimeric peptides (e.g., Angiopeps derived from proteins expressing the Kun it/ domain), low-density lipoprotein receptor related proteins 1 and 2 (LRP- 1 and 2), diphtheria toxin receptor (DTR), mesenchyme stem cells, etc.

[0056] As indicated above, the pharmaceutical compositions of the invention should comprise a therapeutically effective amount of the Hsp70 protein, fragment or derivative of the invention (or several such proteins and/or fragments and/or derivatives). The optimal therapeutic concentration of Hsp70 protein, fragment or derivative in the pharmaceutical compositions of the present invention will necessarily depend upon the activity of the specific Hsp70 protein, fragment, or derivative being used, characteristics of the patient and the nature of the neurodegenerative disease for which the agent is being used. In addition, the concentration of the Hsp70 protein, fragment or derivative will depend upon whether it is being employed in a preventive or treatment capacity. Further, the stage of a particular d isease or disorder, e.g., early vs. late AD, may dictate the optimal concentration of the Hsp7() protein, fragment or derivative.

[0057] Dosage regimens should be adjusted to provide an optimum activity for a specific disease and patient. Dosages should also be adjusted based on the release rate of the administered formulation (e.g., a nasal spray versus drops).

[0058] The amount of active compound wi l l general ly be chosen to provide effective treatment upon as few administrations as possible (th is is particu larly important for achieving compliance in a patient population with memory defects). Preferably, the administration should be once daily. [0059] As disclosed herein, a short-term intranasal administration of recombinant human Hsp70 leads to a dramatic long-lasting neuroprotective effect. Thus, an admin istration regimen of the invention preferably includes a short-term (e.g., weeks to months, e.g., 3 weeks to 5 months for humans), daily treatment, but could also include long-term (e.g., at least 6 months), daily treatment. The cl inician will generally be able to determine a suitable dai ly dose, depend ing on the factors mentioned herein.

[0060] As a non-l imiting example, the Hsp70 protein is suitably present in the composition of the invention in an amount such as to provide a free Hsp70 protein concentration from about 0.2 μg to about 1 mg per kg body weight per day either as a single dai ly dose or as multiple divided doses during the day.

[0061 J The proportion of each further component in the nasal composition of the invention may vary depending on the components used. For example, but without being l imiting, the amount of nasal carrier may be in the range of from 0. 1 to 99.9% by weight of the total weight or volume of the composition. When present, the amount surfactant may be in the range from about 0.01 to about 10% or higher and preferably about 0.05 to about 1 .0% by weight of the total volume or weight of the composition, the amount depending on the speci fic surfactant used. The amount is generally kept as low as possible since above a certain level no further enhancement of absorption can be achieved and also too high of a surf actant level may cause irritation of the nasal mucosa. The amount of delivery enhancing agents may be at least 0. 1 %, suitably in the range from about 0.5 to 10% of the total weight of the composition. Where the composition is liquid, the enhancing agent may suitably be present in an amount of from 0. 1 to 5% w/v of the total composition. Preserving agents may be present in an amount of from about 0.002 to 0.02% by weight of the total weight or volume of the composition.

[0062] The useful delivery volume of the pharmaceutical compositions of the invention is limited by the size of the nasal cavity. Suitable del ivery volumes wi l l be c lear to a person skilled in the art of pharmacology. Preferably, the total composition quantity administered at each nasal appl ication comprises from about 0.02 to 0.5 ml, preferably about 0.07 to 0.3 ml, typically about 0.09-0. 1 ml. A solid composition may comprise from 1 to 30 mg carrier per dosage, more particularly 4 to 20 mg. [0063] The liquid compositions of the invention may be prepared by bringing into intimate admixture the Hsp70 protein, fragment or derivative of the invention in the l iquid carrier optionally together with the further ingredients, add itives and/or agents. Preferably the resulting mixture is then lyophilized and dissolved in water or aqueous sal ine for use in a liquid form according to the invention. The solid nasal composition of the invention may be prepared in conventional manner. The Hsp70 protein, fragment or derivative of the invention may be admixed with the carrier particles, e.g. a polymer base or cel lulose product in conventional manner, optionally with further ingredients, additives and/or agents as ind icated above e.g. a mucosal del ivery enhancing agent or surfactant such as disclosed. The Hsp70 protein, fragment or derivative of the invention may be in solution e.g. an aqueous or alcohol ic solution when being mixed with the carrier particles and the solvent evaporated, e.g. under frceze-drying or spray drying. Such drying may be effected under the conventional conditions. Alternatively the mixture may be compacted or granulated and then be pulverized and/or sieved . I f desired the particles may be coated. According to a preferred embodiment of the invention, the nasal composition is prepared by lyophil isation. A homogeneous solution, preferably aqueous, containing the Hsp70 protein, fragment or derivative of the invention and optional ly containing further ingredients, additives and/or agents as discussed above, is prepared and then submitted to lyophilisation in analogy with known lyophilisation procedures, and to subsequent drying. The resulting powder may then be dissolved in a liquid excipient or nasal carrier before administration, e.g. to reconstitute nasal drops, gel or spray. A lternatively it may be admin istered as such in the form of lyophil ized powder or it may be mixed with further ingred ients, additives and/or agents as discussed above. For example, a lyophil ized powder comprising the I lsp70 protein, fragment or derivative of the invention but free of any nasal carrier may be prepared and then admixed with the desired nasal carrier or mixture of nasal carriers.

[0064] The present invention encompasses any del ivery device that is suitable for nasal administration of the compositions of the invention. Preferably, such means administers a metered dosage of the composition. The composition of the present invention may be packed in any appropriate form or container as long as a means is provided to del iver the composition to the nasal mucosa. Non-limiting examples of useful intranasal del ivery devices include, e.g., instillation catheters, droppers, unit-dose containers, squeeze bottles pump sprays, airless and preservative-fee sprays, compressed air nebulizers, metered-dosc inhalers, insufflators and pressurized metered dose inhalers

[0065] For administration of a liquid in drop form, compositions of the invention can be placed in a container provided with a conventional dropper/closure device, e.g. comprising a pipette or the like, preferably del ivering a substantial ly fixed volume of composition/drop.

[0066] For administration of an aqueous solution as a nasal spray, the aqueous solution may be dispensed in spray form by a variety of methods known to those skil led in the art. For example, such compositions will be put up in an appropriate atomising device, e.g. in a pump-atomiser, or the like. The atomising device will be provided with appropriate means, such as a spray adaptor for delivery of the aqueous spray to the naris. Preferably it wil l be provided with means ensuring delivery of a substantially fixed volume of composition/actuation (i.e. per spray-unit). Examples of nasal sprays include nasal actuators produced by I ng. Krich Pfciffer GmbH, Radolfzel l, Germany (see U .S. Pat. No. 4,5 1 1 ,069, U .S. Pat. No. 4,778.8 1 0, U .S. Pat. No. 5,203,840, U .S. Pat. No. 5,860,567, U.S. Pat. No. 5,893,484, U.S. Pat. No. 6.227,4 1 5. and U.S. Pat. No. 6,364, 166. Additional aerosol delivery forms may include, e.g.. compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers.

[0067] Alternatively the spray may be bottled under pressure in an aerosol device. The propel lant may be a gas or a l iquid (e.g. a fluorinated and/or chlorinated hydrocarbon). The spray composition may be suspended or dissolved in a liquid propellant. Stabi l izing and/or suspending agents and/or co-solvents may be present.

[0068] A dry powder may be readi ly dispersed in an inhalation device as described in U.S. Pat. No. 6,5 14,496 and Garcia-Arieta et al., Biol. Pharm. Bul l . 200 1 ; 24: 14 1 1 - 1416.

[0069] If desired a powder or liquid may be fi l led into a soft or hard capsule or in a single dose device adapted for nasal administration. The powder may be sieved before fi l led into the capsules such as gelatine capsules. The del ivery device may have means to break open the capsule. The powdery nasal composition can be directly used as a powder for a unit dosage form. The contents of the capsule or single dose device may be administered using e.g. an insufflator. Preferably it will be provided with means ensuring dosing of a substantial ly fixed amount of composition.

[0070] Delivery devices are important not only for del ivering the I Isp70 protein of the invention, but also for providing an appropriate environment for storage. This would include protection from microbial contamination and chemical degradation. The device and formulation should be compatible so as to avoid potential leach ing or adsorption.

[0071] The delivery device (or its packaging) can be optional ly provided with a label and/or with instructions for use indicating that the composition should be used intranasal lv.

[0072] In another embodiment, the composition of the invention can be provided as a nasal insert having the Hsp70 protein, fragment or derivative of the invention dispersed therein. The insert may be retained in the naris, but flushed by the nasal mucus, and may be designed to release the Hsp70 protein, fragment or derivative of the invention at the same place in the naris. Suitable nasal insert types include nasal plugs, tampons and the l ike, f urther examples of nasal inserts, their characteristics and preparation are described in HP 490806.

Combination Treatments of the Invention

[0073] In the treatment methods of the invention, intranasal I lsp70 protein, fragment or derivative can be administered in combination with various other treatments which can be useful for neurodegenerative diseases. For example, I Isp70 protein, fragment or derivative can be administered in combination with a memory enhancement agent, e.g.. Aricept™ and/or Namenda™, antidepressant, e.g., Zoloft™, anxiolytic, antipsychotic agent, e.g., Geodon™, sleep disorder agent, anti-inflammatory agent, e.g., Celebrex™, Bcxlra™, etc., antioxidant agent, cholesterol modulating agent (for example, an agent that lowers LDL or increases H DL), e.g., Lipitor™, Caduet™, etc., H istamine (H2) antagonist, e.g., Cimctadinc™, and antihypertensive agent, e.g., Norvasc™, Caduet™, etc.

EXAMPLES

[0074] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not l imited to any particu lar preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the ful l scope of equivalents to which those claims are entitled.

EXAMPLE 1 : Animal Studies Using Intranasal Hsp70 Administration

Materials and Methods

[0075] Mice maintenance and diet. Adult NMRI mice (males) were used in all experiments (Charles River). The animals were maintained in their home cages in a cl imate- controlled room at 21 -23 °C with a 12: 12 hour light-dark cycle and had free access to water and food. Means and SEM were determined for each group. All animal experiments were performed in accordance with the guidance of the National Institutes of Health for care and use of laboratory animals, NI H Publ ications No. 8023, revised 1978.

[0076] Olfactory bulbectomy procedure. Four groups of mice aged 1 0 weeks were employed in experiments: two groups were composed of olfactory bulbcctom ized (OBI !) mice while the other two groups were sham operated (SO). Mice were anaesthetized with Nembutal (40 mg/kg, i.p.) and 0.5% Novocaine for local anesthesia of the scalp and the skin over the skull was surgically uncovered. A single burr hole (2 mm diameter) was dri l led over the olfactory bulbs (2 mm anterior to the bregma, 0 mm laterally from the midl ine). Both ol factory bulbs were carefully aspirated through a blunt needle attached to a water pump. The extent of the lesion was assessed both visually and histologically at the end of the experimental study (29). SO mice were treated similarly, except that the olfactory bulbs were not removed.

[0077] Tissue procedure. On the day after the spatial memory probe trial, test mice were anaesthetized by an overdose of Nembutal (60 mg/kg, i/p). Their brains were perfused through the left cardiac ventricle with 20 ml of 0. 1 M PBS (pi 17.4). Brains were rapid ly removed, verified on the extent of the lesion, and divided into hemispheres. Ind ividuals show ing a partial lesion and/or any damage of the frontal neocortex were excluded from histological and beta- amyloid analyses as well as from behavioral stud ies. Twenty one samples of the brain (SO (n 5), SO-t Hsp70 (n- 5), OBE (n-6) and OBX +Hsp70 (n-6) were extracted to evaluate the beta- amyloid level by DOT and EL1 SA analysis, as well as cellular morphology. One hemisphere was frozen on dry ice and the other was fixed in 4% phosphate-buffered paraformaldehyde at 4°C for 48h before storage in 30% sucrose. Twenty μιη sections of the hcmibrains were cut in the coronal plane on a cryostat and stored in a glycol-based cryoprotectant at -20°C until histological analysis. Sections were washed again, and soaked in distilled water for 1 0 min .

[0078] Histology and morphology. Cresyl-violet (N issl) acetate (Sigma) was used for observations of the morphological state of neurons in the temporal cortex and areas of hippocampus: stratum lucidum of area CA3 and stratum radiatum of area CA l . The sections were stained for the Nissl substance with the Cresyl Violet as previously described (29) and examined with an optical microscope Nikon Eclipse E200. The shape and size of the cel ls, as well as the intensity of staining, represent major morphological detai ls taken into considerations in this analysis. Sections were viewed at a magnification of 2()x or 40x and digitized images were captured using a DXM 1 200 camera mounted on a light microscope. Normal ly every fi fth section was analyzed. Only neurons with well defined cel lular contour, nucleus and nucleoli were taken into account. In order to evaluate morphophysiological state of neurons in the area studied, 1000 cel ls were analyzed in each animal. This was usual ly ach ieved after exam in ing of ten microscopic fields (objective X40, eyepiece 1 0).

[0079] Comparative studies of the cellular composition of the temporal cortex, and the CA l and CA3 areas of the hippocampus ( 1 000 cells for the each structure in the each mouse were counted) were performed using a digitizer computer system PDP- 1 2 (Germany). Functional neurons and neurons with distinct pathologic changes were counted . The fol lowing categories of neuronal abnormalities were distinguished: cytolysis, karyolysis, pyknosis, vacuol ization. Their frequencies were estimated in relation to the quantity of the normal neurons. Ten to twelve view fields were analyzed for each structure. A 40x objective and a compensative ocular with a net magnification of 1 0 were used. A l OOx immersion objective (oi l coeffic ient of the refraction of 1 ,565) was used for a more detai led observation.

[0080] The cell density was determined in 1 mm², To measure neuron density in d ifferent brain structures of OBE and SO mice an eyepiece with a spec ial inbui lt net, with square side estimated by standard object-micrometer was used. The cell number was determined with in ten squares of the net, in the area equal to 0.036 mm² when X40 objective was used . Density measurements were performed in ten microscopic views. Therefore, neuron density (Dn) has been estimated by formula Dn^k x X, where X-average cell number in one microscopic v iew, and k-constant coefficient (27.7), estimated taking into account the square of microscopic view. The data were statistically treated using "Statistica 06" program. Comparisons were performed using a two-tailed Student's t test. The differences were considered to be signi ficant at p<0.05. Therefore, to evaluate morphophysiological state of neurons two independent criteria were explored, i.e., the percentage of various pathologies observed in neuronal population and alterations in the neuron density, monitored as described above.

[0081] Confocal Microscopy. Sections of brains of mice that received intranasal injection of human HSP70 labeled by Alexa Fluor 647 (Invitrogen) were inspected under a scanning confocal microscope "Axioplan 2". Sections were observed using a l Ox/0.3 ΝΛ or 20x/0.5 NA Plan-Neofluar or 63x/1 .4 NA Plan-Apochromat objective on the microscope Axioplan 2 equipped with a Zeiss LSM-5 1 0 ΜΕΤΛ scan head. A I IcNe laser was used to generate the 633 nm excitation wavelength, and pinholes were typical ly set to 1 - 1 .5 airy units. For Alexa Fluor 647, 3-9 images of non-overlapping fields of cells were col lected at low power (total of 56 photomicrographs for analysis). Additionally, 3 images of non-overlapping fields of labeled cells at high power were collected (total of 36 photomicrographs for analysis). I l igh- power images were taken of the olfactory bulb, the temporal cortex, CA I and CA3 fields of the hippocampus, the locus coeruleus, the nucleus raphe dorsalis, and the cerebel lum .

[0082] Hsp70 administration. Two weeks fol lowing the OBK or SO procedures, mice were randomly divided into 6 groups. Animals in two groups, denoted (SO t I lsp70) (n- 1 3) and (OBE * Hsp70) (n - 13), were treated with Hsp70 in 0.9 % saline (2ng/mousc in 4μ1 intranasal injections) daily for 21 days. The control animals were injected intranasal ly with the same doses of heat-inactivated Hsp70 (OBE i inactive Flsp70, n 5) or solution of human scrum albumin (OBE ⁽ albumin, n^:^5). Animals in the remaining two groups, denoted as (SO) (n 14) and (OBE) (iv 3), were administered the same volume of 0.9 % sal ine (intranasal injection) during the same period. [0083] Morris water maze test. Four weeks or eight months after the olfactory bulb removal, learning ability and spatial memory were tested in ten independent groups of animals as described (38). Experiments were performed in a test room with extra-maze cues to facilitate spatial learning.

[0084] A circular swimming tank 80 cm in d iameter with a wal l height of 40 cm and a hidden platform 5 cm in diameter (State Institute of B iological I nstrumentation, RAS, Russia) was fil led to a depth of 30 cm with water at 23°C and rendered opaque by the add ition of powdered milk. The tank was operationally (mentally) d ivided into four sectors: platform target sector (third), opposite sector (first), adjacent clockwise sector (fourth) and ad jacent counterclockwise (second) quadrant. The hidden platform was located in the middle of the third target sector during training trials. It was submerged at a depth of 0.5cm so as to be invisible to a swimming animal. A video monitoring system (TSI, the Germany) was used to record the major behavioral parameters in the water-maze paradigm.

[0085] Initially, all groups of OBE and SO an imals were assessed in the water maze to identify any inherent sector preference. None of them showed any preference. To veri fy that bulbectomy did not induce motor or visual impairments that can affect the results of memory tests, the latencies required for al l sham operated (SO) and bulbcctomiz.cd (OBE) animals to reach the visible platform were determined (in three trials) in the Morris water maze before the main spatial training trials were initiated in each testing period. Since two-way ANOVA revealed no significant d ifference among the all groups in the test with the visible platform, the d i fference in performance at any test point could not be attributed to swimming velocity or impairment of the vision of the OBX mice. Mice were then exposed to a total of 20 training trials for 5 days (i.e. four trials per day; no more than 60s each trial). During each trial, the latency period to locate the hidden platform was evaluated, up to a maximum of 60s. Each an imal was placed in the water facing the wall of the tank in one of three random ly selected sectors other than that containing the hidden platform. I f an animal failed to locate the platform within 60s. it was placed on the platform for 1 0s. Spatial memory was tested on the fol lowing day after completion of training with a single probe trial (60s) in the absence of the hidden platform, beginning from a random position. During the probe trial, occupancy time spent in each sector was recorded . [0086] Differences in behavioral parameters were evaluated with one or two-way ANOVA for repeated measures. Mean latencies and evolution curves for SO and OBE m ice to reach the invisible platform were approximately equal in case of train ing immed iately after Hsp70 treatment as well as eight months after the olfactory bulb removal. The comparison of SO (n- 12), SO+Hsp70 (n=5), OBE (n- 10), and OBE i Hsp70 (n 6), OB i heat-inactivated Hsp70 (n-5), and OBE+ human albumin (n--=5) groups did not show any sign ificant group effect in latent periods to reach the invisible platform, neither in the course of the first scries (F₅ 209^" 0.95, p >0.05) nor in the second series, when training tests were performed using other groups of animals eight months after bulbectomy (SO (n 9); SOU Isp7() (n 8); OBX (n 9); OBX + Hsp70 (n 7)] (F3 161 1 · 14, p>0,05). The comparison of these groups of an imals ind icated high significant effect of days of training in the course of the first series (F4_, 836 38.27. p <().()0 1 ) as wel l as in the second series eight months after bulbectomy, (F_{4 64}4 41 . 14, p<0,()01 ). One-way ANOVA and post hoc analysis revealed significant d ifferences on day 1 and 2.

[0087] On these days, OBE mice found the save platform significantly slower than SO (p<0.05). As illustrated by the learning curves in Figure 5, all groups improved their performance (i .e. time spent to reach invisible platform) with increased training. Post hoc examination revealed no statistical difference in the learning abilities between any groups over the 5-day training of animals tested four weeks as well as eight months after removal of ol factory bulbs. It is important that the performance of all groups reached the same control levels on the last day (day 5) of the train ing phase in both experimental series (Fig. 5 left and right d iagrams).

[0088] At 24 hours after the last learning trial, mice were examined in a probe trial, in which search patterns were monitored in the absence of a platform, fwo-way ANOVA was then performed in order to expose any possible link between groups and sectors as the main factors in the spatial memory test.

[0089] This analysis of sector occupancy time 1 .5 and 8 months after bulbectomy revealed significant effects of sector ( F3. 148.1 i 6 ⁷ 12.5 p<0.05 and 1 7.3 pO.001 ), but not of group (F5. 148 0.22 and F3 _, n ₆ ^™1 .02 for each p >0.05) and there was a sign i ficant interaction between group and sector in both periods (F| _{5 f} ,_l8 6.05 and F₉ n ₆ - 5.3 p<0.0 l for each). Consequently, a substantial l ink between sector and group in the water maze test was establ ished using parameters of spatial memory (i .e., occupancy time) in al l groups of an imals. A group analysis of variance was performed to evaluate the differences in occupancy time in each sector for each group (see Table 1 , below).

[0090] Measurement of Hsp70 concentration. To measure the level of endogenous inducible Hsp70 in the brain tissue of mice, their brains were perfused with saline and the olfactory bulbs, temporal and enthorinal cortex, as well as the hippocampus were excised and l x l x l mm size pieces were homogenized in a solution contain ing 20 mM NaCI; 20 mM Tris-HCl, pH7.5; 0. 1 mM EDTA; 1 % Protease inhibitor cocktail. The extracts were ccntrifuged at 13000 g for 5 min and the protein concentration was measured in the supernatant fraction with the aid of the Bradford reagent using bovine serum albumin as a standard. Then the extracts, having a concentration of total protein between 1 0 and 1 00 μg/ml were applied in wel ls of a 96- well ELI SA microplate covered with ATP-ovalbumin reagent and measured as described elsewhere (39).

[0091] Isolation and labeling of human recombinant IIsp7fA Ful l-length human recombinant Hsp70 expressed in army worm {Spodoptera) cel ls was used for the experiments. The original clone containing human Hsp70 cDNA GcnBank Accession No. M l 1 71 7 corresponding to SwissProt P08107 (SEQ ID NO: 1 ) (pBlucScriptSK( I )-hsp70) (obtained from Dr. R. Morimoto, Northwestern Univ., Evanston, U SA) was subc loned in the donor plasmid pFastBacHTb-hsp70 under regulation of the polyhedrin promoter. The plasm id was subsequently used in the Bac-to Bac system (Invitrogen) to express the human I lsp7() protein. Recombinant protein contained six His residues at its N-tcrminal end enabl ing its isolation from the cell extracts by chromatography on N i-NTA resin columns according to the manufacturer's instructions (QUIAGEN, N i-NTA Superflow BioRobot Hand Book). The purity of Hsp70 preparations was confirmed by PAGE-electrophoresis fol lowed by staining with Coomassie Blue and immunoblotting using monoclonal 3 B5 anti-Hsp70 and N69 anti-Hsc7() antibodies (39). According to the E-Toxate assay (LALA test), the level of EPS in the final Hsp70 preparation was less than 0. 1 MU/ml, i.e., less than the last control standard, f or "heat- inactivated" Hsp7() control, Hsp70 was heat inactivated at 1 00 °C for 5 min and cooled before intranasal injection. [0092] To monitor brain localization of injected human recombinant I Isp70 the protein was conjugated with Alexa Fluor 647 or Alexa Fluor 554 dye (I nvitrogcn, Carlsbad, USA) according to the instructions of manufacturer.

[0093] Beta-amyloid measurements. Αβ in the brains was detected by a modified, higher resolution version of the DOT-analysis as previously described (22) and HI , I SA analysis as previously described (Toyn JH et al. (2010) BC Neurosciencc 1 1 : 143- 1 5 1 ). The levels of Αβ species were presented as ng or pM of Λβ per g of wet brain, taking into account di lution factors introduced by multiple steps throughout the assay (brain homogen ization and extraction procedures). Means and SEM were determined for each group.

[0094] DOT analysis. Specifically, for immunological DOT analysis, the brain specimens from the OBE and SO animals were prepared as follows: 1 50-200 mg of the brain tissue (the cortex and hippocampus together) was homogenized in 0.5 ml of 70% formic acid, maintained for 1 h, centrifuged at 100,000 g for 40 min, then the supernatant fluid was evaporated on a rotor evaporator to the minimum volume, supplemented with 1 ml of twice distilled water, and the solution was neutralized to pH 7.4 with NaOI l and lyophil izcd. During the work, the fol lowing control reactions were performed to determine the endogenous peroxidase activity, nonspecific sorption of secondary antibod ies, and also the activity of immune reagents and the antigen presence under conditions of the experiment. For the calibration curve and subsequent quantitative determination of Λβ in the brain extracts, the intensity of staining of different concentrations of Αβ was used. The staining intensity of spots on the membrane was determined using the ONEDSCAN program, version 1 .3, copyright 1 994- 1997. The following antibodies were used: monoclonal antibodies 4G8 reacting with ' 1 7-24 ' amino acid fragment of Αβ, which has the same structure as that of human and mouse Λβ (dilution 1 : 1000); biotinylated horse antibodies to mouse IgG (di lution 1 : 3500); monoc lonal antibodies to biotin (the clone BN34) conjugated with peroxidase (di lution 1 : 4000).

[0095] Specificity of the antibody binding to Αβ was confirmed by the control results. Absence of staining of the membrane with the samples placed during interaction with 3,3 '-diaminobenzidine (DAB) suggested the absence of endogenous perox idase activity . The absence in the samples of immunoglobul ins cross-reacting with secondary antibodies was shown by testing nonspecific sorption of secondary antibod ies: the reaction was negati ve when the membrane with the samples on it was treated with secondary antibodies and stained with DA B. The activity of immune reagents and antigen validity under the experiment cond itions were also monitored. The membrane with the Αβ marker was treated with primary antibodies, then with secondary antibodies, and stained with DAB. Monoclonal antibod ies 4G8 and biotiny lated horse antibodies against mouse IgG were purchased from Vector Laboratories (Burlingamc, CA, USA), monoclonal antibodies against biotin (the clone BN-34) conjugated with peroxidase were purchased from Sigma (USA).

[0096] ELISA analysis. Frozen samples of the cortex and hippocampus were thawed and homogenized at a concentration of 4 ml/g in ice cold 2% CFIA PS, 20 mM I ris pFI 7.7, in the presence of protease inhibitors ( 1 0 μg/ml leupeptin, 1 0 μg/ml aprotinin and 1 0 μg/ml AEBSF). The homogenates were centrifuged at 21 ,000 x g at 4°C for 30 min. The supernalants from the centrifugation were stored frozen at -80°C, and thawed immediately before use in the ELI SA. Αβ levels were determined using an ELI SA mouse Αβ ( 1 -40) kit, wh ich uses a sol id phase sandwich ELISA (Invitrogen, Camarillo, CA). Briefly, monoclonal antibody speci fic for the N-terminus of mouse Αβ was pre-coated onto wells of the provided microtiter strips. Samples and standard diluted in the standard diluent buffer were incubated for 2 hrs at room temperature allowing the Αβ to bind the capture antibody, followed by extensive wash ing. Samples were incubated with detection rabbit antibody specific for the C-terminus of the Λβ ( 1 -40) for I hr at room temperature. After washing, samples were incubated with FIRP anti-rabbit antibody for 30 min at room temperature. After removal of excess anti-rabbit antibodies, a substrate solution is added, which is acted upon by the bound enzyme to produce color. After incubation for 30 min at room temperature the color reaction was stopped. The absorbance was read at 450 nm. 1 he standards provided a l inear curve were used to calculate the concentration of Λβ in the samples.

[0097] Statistics. All results are reported as means I SEM. Statistical significance for DOT and ELISA analysis data was determined using a two-tai l Student' s t test. Significant differences between mean scores during training trials in the Morris water ma/.e were assessed with two-way ANOVA ("Statistica 06") with Tukey's post-hoc analysis for multiple comparisons using group and trial day block number during training. Statistical analysis of the spatial memory (probe trials) was carried out with two and one-way ANOVA using the program "Statistica 06" and the ANOVA statistical package (CSS). The preference for platform target quadrant in comparison with other indifferent quadrants was assessed by post hoc analysis using a multiple-range LSD test. Comparisons of quantitative characteristics of occurrence of d i fferent neural pathologies and levels of brain Αβ between experimental groups were performed using a two-tailed Student's t test. Significance was defined as *-p <0.05 ; * * - p<0.01 ; * * * p<0.001 .

Results

The level of endogenous Hsp70 correlates with the memory status in OBE mice

[0098] In the first series of experiments the changes in the spatial memory of OBE and sham-operated (SO) mice were correlated with the levels of endogenously expressed, inducible Hsp70 at different times after surgery (Figures 1 A-B). The behavioral studies were carried out 0.5; 1 ; 1 .5; 3; 6; 8; 12 and 16 months after bulbectomy (8 groups of OB E and 8 groups of SO animals, n- 7-8 animals per group). Figure 1 A (top panel) compares the average time spent by OBE mice, represented as percentage relative to SO animals, in a target sector of a Morris water maze (see Methods and Table 1 ), in different time periods after bulbectomy.

(0099] Results demonstrate a distinctive biphasic dynamic of spatial memory in OBE animals. By the end of the first month, the OBE group exhibited sign ificant memory loss, which was almost completely restored by the sixth month post-operation. However, starting from the eighth month and extending to the end time point ( 16 months) OBE an imals displayed a second, more profound wave of memory deterioration (Figure 1 A). A control set of experiments conducted prior to the training trials demonstrated that bulbectomy per se did not induce motor or visual impairments that could affect the results of the memory test in Morris water maze. At 0.5, 3, 6, and 12 month intervals after bulbectomy the concentration of Hsp70 was measured in the brains of corresponding groups of OBE and SO mice by EEI SA. Strikingly, the decrease in the endogenous level of Hsp70 in the hippocampus of OBE animals F igure I correlated well with the decline of memory. The highest level of Hsp70 was observed at six months after bulbectomy and coincided precisely with the almost complete, but transient, memory recovery in OBE mice (Figure I B). The congruency of the dynamic changes in the animals^' cognitive abilities and the levels of Hsp70 in the hippocampus suggest a pivotal compensatory cytoprotective mechanism directed toward restoring learning and memory.

Exogenous Hsp70 protects spatial memory in OBE mice [00100] The results described above provided a rational for the use of exogenous Hsp70 as a potential therapeutic agent to ameliorate the memory impairment in OBE mice. H ighly pure recombinant human Hsp70 was administered intranasal ly for 21 days beginning two weeks after the bulbectomy (n^~ 13) or sham operation (n 1 3) (see Materials and Methods). The training trials in Morris water maze were started four weeks or eight months after olfactory bulb ablation.

[00101] A control set of experiments conducted prior to the training trials demonstrated that bulbectomy per se as well as Hsp70 did not induce motor or visual impairments that could affect the results of the memory test in Morris water maze. Means of latent periods to search the visible platform in water maze did not show the statistic sign i ficant differences either 1 ,5 months (SO 10.62 ± 0.84 sec; OBE 1 3.83 i 2.3 sec; SO I Hsp70 15.37 I . 2.5 sec; OBE -f- Hsp70 1 6. 1 ± 4.2 sec) or 8 months after bulbectomy (SO 1 3.8 1 1 1 .82 sec; OBE - 14.92^■± 2.1 sec; SO + Hsp70 13. 17 ± 1 .5 sec; OBE I Hsp70 1 .2 1 I 2.2 sec).

[00102] The data presented in Table 1 demonstrate that during spatial memory testing (probe trial), OBE animals, as well as control groups of OBI t heat- inactivated HSP70 (n^) and OBE-t human albumin (n ^5), did not show a preference for any of the sectors and. hence, did not remember the position of the target sector, that contained the saving platform during the learning trials. Thus, in agreement with the results described above, spatial memory in d ifferent groups of OBE mice was severely impaired one month as wel l as eight months after bulbectomy . In contrast, the OBE i Hsp70 mice displayed a significant preference for the target sector as determined by ANOVA and post-hoc analysis of their occupancy time ( fable 1 and Figures 1 C-D) where the results of post hoc analysis using LSD (see Materials and Methods) criteria for the occupancy time are presented). It is evident that al l experimental groups, but not the OBE mice and control groups (OBE+inactivated HS 70 and OBE i albumin), spent significantly more time in the target sector than in the other indifferent sectors (f igures 1 C-D and Table 1 ). Hsp70 administration per se or bulbectomy did not influence the visual or motor functions, because there was no significant difference in latencies to locate the visible platform in pretraining sessions between any of the groups one month or eight months after bulbectomy or sham operation. It can be therefore concluded that intranasal injection of Hsp70 protected spatial memory in OBE mice. [00103] Remarkably, the Hsp70-induced memory recovery was maintained throughout the entire study, as revealed by the results of the Morris water maze test in the experiments performed in OBE mice eight months after bulbcctomy followed by IIsp7() treatment (Figure ID; Table 1).

Table 1. Factor analysis of sector occupancy time in the water maze for memory tests in different groups of OBE and SO NMRI mice treated with IIsp7() (1.5 and 8 months after bulbectomy).

Animal groups Means of Factor

occupancy time

F

series (1.5 months after bulbectomy)

SO (n-12) E -55.66 0,00001***

SO ⁽ Hsp70 (n--5) F3 -24.84 0.00001***

OBE (n- 10) I' -1.32 >0.05 ns

OBE+Hsp70 (n 6) f -29.76 O.00001***

OBE+human albumin ( F3J6 -0.147 >0.5 ns

OBE heat-inactivated F3 -1.783 >0.5 ns

Hsp70(n 5)

series (8 months after bulbectomy)

SO (n-9) F ^~13,92 _<0001***

SO- Hsp70 (n -8) F ^"23,29 <0.001***

OBE (n-9) F,.;;' 3.5 >0.05 ns

OBEiHsp70 (11 7) F = 17,82 O.001***

[00104] The data demonstrate that in contrast to SO, SO Hsp70 and OBE H Lsp70 groups, the OBE, OBE human albumin, and OBE ^ inactivated Hsp70 animals do not exhibit preference for any of the four sectors in 1,5 m and 8 m periods after bulbectomy. * - pO.()5; **- pO.01; ***-p<0.001

IIsp70 rapidly enters specific brain regions after intranasal administration both in control and

OBE mice [00105] To trace exogenous Hsp70 and determine whether it could indeed reach the brain to exert its therapeutic effect, the distribution of fluorescently-labclcd I lsp70 in specific areas of the brain usually afflicted in AD patients in control mice (n 6) and in OBE mice (n 3) was monitored by confocal microscopy. Three hours after intranasal admin istration, in control mice Hsp70 was readily detected in the olfactory bulbs, neocortex, hippocampus, n. raphe dorsal is, locus coeruleus, and cerebellum (Figure 2). I n most cases, labeled Hsp70 was localized intracellularly and restricted to the perinuclear zone (e.g., Figure 2H). As expected in simi larly treated OBE mice labeled Hsp70 was observed in the same brain areas as in the control animals (Figure 7). However, in OBE mice the labeled protein was not concentrated in the perinuclear zone but formed aggregates randomly spaced in the cytoplasm (Figure 7). No fluorescent material was detected in the brain slices in mice following intranasal administration of un labeled Hsp70 (n-4) and control not treated animals (n-=2). These resu lts demonstrate that exogenous Hsp70 is able to enter the brain of control and ΟΒΙΐ mice rapid ly and be transported within neurons of brain regions, including specific areas where neurodegeneration predominantly develops in AD patients.

Hsp70 treatment improves neuronal survival and morphology

[00106] To investigate whether the dramatic protective effect of Hsp /O on memory occurred in paral lel with the mitigation of other AD-l ike symptoms in OBE mice, the brai n level of Αβ peptides was monitored and morphological state of neurons in the temporal cortex and areas of the hippocampus (CA 1 and CA3 areas) was analyzed (Figure 3 A; Table 2). because these regions play the pivotal role in the mechanism of learning and memory and arc the most damaged in AD patients.

Table 2. Effect of Hsp70 intranasal administration on morphology of neurons in the temporal cortex and areas of the hippocampus.

±0.66 ±0.26 ±0.51 ±0.28 t 1 .0

9 4 o o o

OBE+Hsp70 3.6 ° ° 1 2 2.3 65.7 ***

±0.68 ±0.43 ±0.82 ±0.28 L I .5

Hippocampus SO 6.3 2.9 5.9 ^"2.2 82.6 area CA 1 ±0.66 ±0.26 ±0.46 1 0.26 1 1 .08

SO±Hsp70 7.4 4 2*** 10.3*** 2.4 75.7***

±0.55 ±0.33 ±0.59 ±0.29 1:0.95

1 5 9 *** 5 4 *** _] 7 J ***

OBE 2.7 59.0 **"

±0.69 ±0.3 1 ±0.61 L0.24 1 0.87

OBE+Hsp70 6.5 ^{0 0 0} 4 2 *** " 19 i *** o J ^ o o o 68.9 **"

±0.33 ±0.38 ±0.89 ±0.21 ± 1 .19

Hippocampus SO 4.6 2.5 5.9 Ί.2 8578 area CA3 ±0.43 ±0.23 ±0.4 ±0.16 1 0.89

SO±Hsp70 8.6** 3 ] *** 16.9*** 1 .8* 69.6***

±0.53 ±0.21 ±0.44 ±0.32 ±0.89

OBE 16.7 *** 2 *** 21 .1 *** 2.6*** 54.0 **-

±0.63 ±0.33 ±0.58 ±0.2 ⁽ 0.74

# * o o o

OBE+Hsp70 5 2 *** 1 8 5 *** ° ° 1 .7 ^{0 0} 67.4 **"

±0.4 ±0.37 ±0.65 ±0.23 ± 1 .02

Data are given as mean±SEM. Comparison between the groups were performed separately for the temporal cortex and both areas of the hippocampus with two-tail Student's t test to compare repl icate means with special emphasis on bulbectomy-induced changes (P values provided as * *p<0.01 ,** * p O.001 ) and effects of Hsp70 treatment in OBE mice (P values provided as pO.001 ).

[00107] No significant changes were identified in the form and si/.e of neural cel ls in SO mice five weeks after the operation. Majority of the neurons in th is group had normal phenotypic characteristics: evenly distributed tigroid and light colored, central ly positioned nuclei with easi ly distinguishable nucleoli (Figure 3A). In contrast, most neurons of the OBE animals displayed pathologic changes, included pyknosis, karyolysis, cytolysis, or vacuol ization (Figure 3A; Table 2). The pyknomorphic neurons were unusually smal l with chromatosis, fragmentation, corkscrew circling of the terminals, and conglutination of tigroid blocks. Erosion of the nuclear and nucleolar membranes and hypertrophy of the nucleol i were characteristic of karyolysis. Cytolysis became apparent in the octopi of the nucleus, with disappearance of the cel lular contour. Lysis of the tigroid was also observed. Vacuol ization was evident in alveolar structures and accompanied by enlargement of the nucleus and nucleolus and the appearance of confluent lacunae in the cytoplasm. [00108] Hsp70 treatment resulted in a striking protection of the neurons in al l brain regions examined in OBE animals (Figure 3A; Table 2). The proportion of pathologic neurons decreased drastically. Representative photomicrographs of the cortical and hippocampal sections (Figure 3A) demonstrate that the Hsp70 treatment prevented the development of neuronal pathology in OBE animals, reducing the number of neurons with pathology. Although cytolysis was occasional ly noticed in the cortex and hippocampal regions of Hsp70-trcatcd OBI - mice, it was different in quality as compared to untreated OBE animals. Consistent with the protection of cells from development of pathology, Hsp70 improved significantly neuronal survival in the OBE mice. Quantitative analysis (Table 2) revealed that OBE mice treated with Hsp70 had significantly more intact neurons in the areas of the hippocampus than untreated OBE animals.

[00109] A representative analysis depicted in Figure 3B demonstrates the preservation of neuronal density in the OBE tHSP70 group in the observed areas of the hippocampus. It can be therefore concluded that Hsp70 treatment radical ly protects neurons from OBE-induced deterioration and death.

Hsp70 treatment reduces the accumulation of Αβ peptides in the brain of OBE mice

[00110] An abnormal ly high accumulation of Αβ peptides in affl icted regions of the brain is a biochemical hallmark of AD and also a prominent characteristic of OB E mice (2, 22). To examine the effect of FIsp70 treatment on this marker of AD. the level of Λβ was measured in the pooled extracts of the cortex and hippocampus by DOT analysis using the 4G8 monoclonal antibodies against mouse and human Αβ peptides and using EEI SA technique as described in the Materials and Methods section, above (Figure 6). Statistical analysis of the data demonstrated that the total amount of Αβ (pool of soluble and insoluble fractions of Αβ40 and Λβ42 peptides) in the untreated OBE group was sign i ficantly higher than that in either the control SO or OBE t Hsp70 group (p<0.001 ) (Figures 4A-B). Importantly, I lsp7() treatment d id not affect the basal level of Αβ in the SO group (Figures 4A-B). Thus, I lsp70 treatment effectively prevented from the accumulation of potentially toxic Λβ peptides in the brain of OBE mice.

[00111 ] Notably, despite the accumulation of Αβ and degenerative changes in morphology of neurons, OB E mice do not develop plaques. On the other hand, OBE guinea pigs, which express Λβ that is identical to the human counterpart, do generate Λβ positive deposits similar to amyloid plaques in AD patients ( 16), which further val idates the use of OBI; an imals as a model for AD-l ike pathology. These observations are consistent with the notion that soluble rather than deposited Αβ is associated with the memory loss and neurotoxicity ( reusch S. Cyr DM, Lindquist S (2009) Cell Cycle 8: 1668- 1674). Plaque formation per sc is not the major cytotoxic factor, but rather a phenotypic manifestation of Αβ misfolding, which varies depending on its precise sequence. Apparently, the extent of memory impairment and neuronal loss in OBE mice represent the consequence of neurotoxicity of high level of soluble Λβ and its smal l oligomers leading to neuropathology ( 14). It is likely that the level of sol uble Λβ and its oligomeric forms are mainly responsible for the cytotoxicity in AD (3 1 ).

Discussion

[00112] The present results demonstrate that intranasal administration of Hsp70 protein can be used as a therapeutic for neurodegenerative diseases. Speci fical ly, as demonstrated herein, the intranasal administration of full-length recombinant human I Isp70 protein to OBE mice, which demonstrate main signs of AD-l ike neurodegeneration, drastical ly protects from development of several degenerative symptoms simi lar to AD pathology, includ ing memory impairment, loss of neurons in specific areas, cellular pathology, and accumu lation of the Αβ peptide. Remarkably, the therapeutic effect of Hsp70 was induced by a three-week single course of treatment with a small dose of protein ^g/mouse/day, see Materials and Methods) and lasted for at least six months (the extent of the experiment).

[00113] The observed Hsp70-mediated neuroprotection required at least some domains of the chaperone to be in a functional conformation rul ing out any indirect effects associated with potential immunoreactive contaminants, such as LPS or other endotoxins (Tsan MF, Gao B (2004) Am J Physiol 286: 739- 744). Indeed, heat-inactivated Hsp70 as wel l as human serum albumin used in the control experiments exhibited no therapeutic activity (figures 3 and 5; Table 1 ).

[00114] Another surprising observation is that exogenous Hsp7() protein promptly enters the mammalian brain and its neuronal cells upon intranasal administration. The intranasal delivery of Alexa-labeled Hsp70 results in distribution of the protein with in specific brain regions in both control and OBE animals (Figures 2 and 7). Importantly, A lexa-labeled I lsp7() injected directly into the brain of mice is localized essentially in the same regions (Ekimova et al. (2010) JNeurochem 115(4): 1035-1044).

[00115] As demonstrated herein using two independent techniques (KUSA and DOT), there is a significant decrease in the level of Αβ peptides in OBEHlsp70 group (figures 4A-B). This should inhibit the formation of toxic soluble forms of Λβ, facilitate its normal utilization, and prevent accumulation in the brain. These effects provide the rationale for intranasal application of exogenous Hsp70 in treatment of AD-like neurodegencration.

[00116] The experimental observations disclosed herein are believed to be of direct relevance to treatment of AD based on the following AD-like characteristics of the OBE mouse model:

(i) Neuronal death in OBE mice is restricted to the same specific brain regions affected in AD patients: temporal cortex, areas of hippocampus, n. Raphe dorsalis and n. agnocellularis (e.g., Nesterova et al.(2008) Neurosc Behav Physiol 38: 349-353; Holland D et al. (2009) Proc Natl Acad Sci U S A 106: 20954-20959).

(ii) Similar to AD patients, OBE mice display a deficit of serotonin-, acetylcholine-, and glutamatergic brain systems (e.g. Bobkova et al.(2001) Bull Exp Biol Med 1 1: 427- 1; llo/umi et al. (2003) Behav Brain Res 138: 9-15; Moriguchi et al.(2006)..1 Neurochem 97.22 29). In this regard, the OBE mouse is a unique model that mimics most of the brain disturbances characteristic of AD patients.

(iii) Similar to AD patients, the OBE mouse displays an increased level of brain amyloid precursor protein (Strube et al. (1998) Brain Res 780:129-137) and Αβ as illustrated in I^'igure 4 (see also Bobkova et al. (2008) Brain Research 1232: 185-194). Due to differences in the Λβ amino acid sequence between humans and mice, the OBE mouse docs not develop amyloid plaques. However, the OBE guinea pigs, in which the Αβ sequence is identical to that of its human ortholog, do develop extracellular amyloid deposits that are very similar to plaques in AD patients (e.g. Bobkova et al. (2005) in New Trends in Alzheimer and Parkinson disorder: ADPD 2005, eds Fisher A, et al. (Medimond, onduzzi) pp 91-95.). [00117] Also of clinical relevance is the fact that the therapeutic effect of intranasal Hsp70 protein observed herein for mice model of AD was not accompanied by any significant anti-Hsp70 immune reaction in the brain. This is not surprising considering the very small amounts of Hsp70 protein needed for the neuroprotective effect. The cl inical safety of intranasal Hsp70 protein is also suggested by the fact that only a background autoantibody IgD response was observed in mice immunized with large amounts of native I lsp70 and G P96 chaperones (Menoret at al., Immunity, 101 : 364-370, 2000).

[00118] Taken together, the present findings reveal neuroprotective functions of Hsp70 and establish exogenous Hsp70 as a promising pharmacological agent for the treatment of various neurodegenerative diseases accompanied by severe neuron loss and cognitive disturbances.

EXAMPLE 2: Human Studies Using Intranasal Hsp70

[00119] 10-30 subjects diagnosed with early stage AD by Min i Mental State Examination (MMSE), Cognistat in combination with Rivermead Behavioural Memory Test and showing worsening in spatial orientation and memory, developing depression, decreasing sense of smell and asymmetrical increase in electrophysiological activity of hippocampus arc enrol led in the study. The subjects are administered single daily doses of 0.9% NaC I solution of the ful l- length human Hsp70 protein via intranasal drops. The Hsp70 protein doses tested arc 0 μ» (control), 2 ^, 20 μg, 100 μg, 1 mg, and 20 mg. The administration is continued dai ly for 5 months and cognitive parameters are assessed by Mini Mental State Examination (M M SE) every 1 month during the administration and every 6 months for 5 years after the adm inistration is completed.

EXAMPLE 3: Investigation of the Neuroprotective Function of IIsp70 Fragments

[00120] Using the methods described in Example 1 , supra, the anti-A D acti vity of several intranasal ly administered recombinantly produced human Hsp70 fragments is tested in OBE mice and compared to the full-length recombinant human Hsp70 protein and buffer control. Effects on memory impairment, loss of neurons in specific areas, cel lular pathology, and accumulation of the Αβ peptide are tested. Hsp70 fragments are tested and compared to the ful l- length Hsp70 protein at various concentrations (0. 1 , 0.5, 1 , 2, 5, 1 0 μ»/ιτιοι^εΛ^). Various combinations of two different fragments are also tested and compared to the full-length Hsp7() protein.

[00121] The following HSP 70 fragments are tested:

1. amino terminal ATP binding domain

MA AAAIGIDLGTTYSCVGVFQHGKVEHANDQGNRr fPSYVAF I D I HRI.IGDAA NQVALNPQNTVFDAKRLIGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSY GET AFYPEEISSMVLT M EIAEAYLGYPVTNAVI^'fVPAYFNDSQRQA I DAG VIAGLNVLRIINEPTAAAIAYGLDRTGKGERNVLIFDEGGG FDVSII.IIDIXjIFEV KATAGDTHLGGEDFDNRLVNHFVEEFKR HKKDISQNKRAVRRLR I ACERAKR TLSSSTQASLElDSLFEGIDFYTSITRARFEELCSDLFRSI^'LliPVE ALRDA LD A QIHDLVLVGGSTRIPKVQKLLQDFFNGRDLNKSINPDEAVAYGAAVQAAIL GD

S (SEQIDNO: 2);

2. substrate binding domain

MKSENVQDLLLLDVAPLSLGLETAGGVMTALIKRNSTIP i Q^'I OU TYSDNQPG VLIQVYEGERAMT DNNLLGRFELSGIPPAPRGVPQIHV l l'Dl ANGll.NV 1 A I DK STG ANKrnTNDKGRLSKEEIERMVQEAE YKAEDi:VQRi;RVSAKNAFi;SYAl NM SAVEDEGL GKISEAD KVLD CQEVISWLDA ILAEKDIiFUI R l-Ei; QVCNPIISGLYQGAGGPGPGGFGAQGP GGSGSGP TIEEVD (SEQ ID NO: 3);

3. C-teminal domain

MG SAVEDEGLKGKISEAD KKVLD COEVISWLDANTLAI:KDI-;I I-:H R F;I.)-: OVCNPIISGLYQGAGGPGPGGFGAQGP GGSGSGP TIEEVD (SEQ ID NO: 4).

4. N-terminal halfof Hsp70 (amino acids 1-430 of SEQIDNO: 1).

5. C-terminal half of Hsp70 (amino acids 420-641 of SEQ ID NO: 1).

Rel^'crenceses

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Cummings CJ et al. (2001 ) Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA 1 mice. Hum Mol Crenel 10: 1 5 1 1 - 1 5 1 8.

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[00122J The present invention is not to be limited in scope by the speci fic embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skil led in the art from the foregoing description. Such modifications are intended to fall within the scope oi the appended claims.

[00123] All patents, applications, publications, test methods, l iterature, and other materials cited herein are hereby incorporated by reference in their entirety as i f physical ly present in this specification.

Claims

1 . A method of treatment of a neurodegenerative disease in a subject in need thereof comprising intranasally administering to the subject a therapeutically effective amount of Hsp7() protein and/or active fragment and/or derivative thereof.

2. The method of claim 1 , wherein the neurodegenerative disease is associated with abnormal protein biogenesis.

3. The method of claim 1 , wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PI)), Huntington 's disease (HD), Lewy Body dementia, frontotemporal dementia (F I D), vascular dementia, mi ld cognitive impairment (MCI), mixed dementia, Creutzfeldt-Jakob Disease (CJD), normal pressure hydrocephalus, Wernicke-Korsakoff syndrome, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), prion d iseases, and ataxia.

4. The method of claim 1 , wherein the neurodegenerative disease is associated with aging.

5. The method of claim 1 , wherein the Hsp70 protein is a full-length human 1 lsp7() protein.

6. The method of claim 5, wherein the Hsp70 protein has the amino acid sec]iiencc comprising SEQ ID NO: 1 .

7. The method of claim 5, wherein the Hsp70 protein has the am ino acid sequence consisting of SEQ ID NO: 1 .

8. The method of claim 1 , wherein the Hsp70 protein and/or active fragment and/or derivative thereof is formulated in a composition further comprising one or more nasal del ivery- enhancing agents.

9. The method of claim 1 , wherein the Hsp70 protein and/or active fragment and/or derivative thereof is formulated in a composition further comprising one or more agents which faci litate brain del ivery.

10. The method of claim 1 , wherein the subject is human.

1 1 . The method of claim 1 , wherein the amount of Hsp70 protein and/or active fragment and/or derivative thereof administered is within the range from about 0.2 μg to about 1 mg per kg body weight per day.

12. The method of claim 1 , wherein the amount of Hsp70 protein and/or active fragment and/or derivative thereof administered is within the range from about 0.2 μg to about 1 00 μ° per kg body weight per day.

13. The method of claim 1 , wherein the amount of Hsp70 protein and/or active fragment and/or derivative thereof administered is within the range from about 1 0 μg to about 100 μg per kg body weight per day.

14. The method of claim 1 , wherein the amount of Hsp70 protein and/or active fragment and/or derivative thereof administered is within the range from about 100 μg to about 1 mg per kg body weight per day.

15. The method of claim 1 , wherein the Hsp70 protein and/or active fragment and/or derivative thereof is administered as a single daily dose.

16. The method of claim 1 , wherein the Hsp70 protein and/or active fragment and/or derivative thereof is administered for 3 weeks to 5 months.

17. The method of claim 1 , wherein the Hsp70 protein and/or active fragment and/or derivative thereof is administered in combination with at least one additional therapeutic agent selected from the group consisting of a memory enhancement agent, antidepressant, anx iolytic, antipsychotic agent, sleep disorder agent, anti-inflammatory agent, anti-oxidant agent, cholesterol modulating agent, and anti-hypertensive agent.

1 8. A method of treatment of a neurodegenerative disease in a subject in need thereof comprising intranasally administering to the subject a therapeutical ly effective amount of two or more active fragments of Hsp70 protein or derivatives thereof.