Regulation of neuroinflammation

Abstract

Compositions and methods for treating neuroinflammation and neurodegeneration and / or reducing the risk of onset or progression of neuroinflammation and neurodegeneration, comprising, for example, HSP60 for nasal administration and IGFBPL1 for, for example, nasal, systemic, or ocular, e.g., intravitreal, administration.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 813,556, filed March 4, 2019, the entire contents of which are hereby incorporated by reference.

[0002] This invention was made with government support under Grant No. EY025259 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Described herein are compositions and methods for treating neuroinflammation and neurodegeneration and / or reducing the risk of onset or progression of neuroinflammation and neurodegeneration, comprising, for example, HSP60 and / or HSP27 for nasal administration, and IGFBPL1 for, for example, nasal, systemic, or ocular, e.g., intravitreal, administration. [Background technology]

[0004] Glaucoma is a leading cause of blindness and remains a global unmet medical challenge. Summary of the Invention

[0005] Provided herein is a method for treating neuroinflammation and neurodegeneration and / or reducing the risk of developing or progressing neuroinflammation and neurodegeneration in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of one or more of (i) HSP60 and / or HSP27, or an active fragment thereof, and / or (ii) IGFBPL, or an active fragment thereof.

[0006] In some embodiments, the therapeutically effective amount is sufficient to reduce inflammation and neuronal cell death in the subject.

[0007] In some embodiments, the method comprises nasal administration of HSP60 and / or HSP27 and systemic (eg, nasal or oral) or ocular administration of an IGFBPL.

[0008] In some embodiments, ocular administration of the IGFBPL comprises intravitreal injection.

[0009] In some embodiments, the subject has glaucoma, autism, multiple sclerosis, Alzheimer's disease, Parkinson's disease, ischemic retinopathy, age-related macular degeneration, stroke, ischemic and traumatic optic neuropathy, or diabetic retinopathy.

[0010] In some embodiments, the method reduces inflammation and neuronal cell death in the eye of the subject.

[0011] In some embodiments, the method reduces inflammation and neuronal cell death in the brain or spinal cord of the subject.

[0012] Also provided herein are kits comprising compositions comprising HSP60 and / or HSP27, and compositions comprising IGFBPL1, for use in the methods described herein.

[0013] Further provided herein are compositions comprising HSP60 and / or HSP27, and / or compositions comprising IGFBPL1 for use in methods for treating neuroinflammation and neurodegeneration and / or reducing the risk of onset or progression of neuroinflammation and neurodegeneration.

[0014] In some embodiments, HSP60 and / or HSP27 are formulated for nasal administration, and IGFBPL1 is formulated for ocular, for example, intravitreal, administration. In some embodiments, HSP60 and / or HSP27 are formulated for nasal administration, and IGFBPL1 is formulated for systemic, for example, ocular or nasal administration. In some embodiments, HSP60 and / or HSP27 are formulated for nasal administration, and IGFBPL1 is formulated for nasal administration. In some embodiments, one, two, or all three of HSP60 and / or HSP27 and IGFBPL1 are formulated together in a single composition for nasal administration.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art can also be used.The materials, methods, and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In the event of any conflict, the present specification, including definitions, will control.

[0016] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

[0017] The patent or application file contains at least one screen executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0018] [Figure 1]Figure 1 shows that microbead (MB)-induced elevation of intraocular pressure (IOP) was not affected by treatment with heat shock protein (HSP) 60 via nasal spray. Left panel: Anterior chambers of mouse eyes filled with saline and MB. Right panel: IOP levels over time in untreated saline-injected control mice (light gray), saline-treated MB-injected mice (gray), and HSP60-treated MB-injected mice (black). [Figure 2] Figure 1 shows the induction of regulatory T cells in mice receiving HSP60 nasal spray. Top panel: Live gating of CD4+ cells from eye-draining lymph nodes and superior cervical lymph nodes. Bottom panel: Treg+ count (CD4+CD25+FOXP3+) histograms 2 weeks after MB injection comparing HSP60-treated and saline-treated mice. [Figure 3A] Figure 1 shows rescue of vision by HSP60 nasal spray in MB-induced glaucoma mice. Visual contrast sensitivity (CS) assessed at 2, 4 and 6 weeks after IOP elevation in all groups, ***P<0.001, **P<0.01, *P<0.05. [Figure 3B] Figure 1 shows visual acuity rescue by HSP60 nasal spray in MB-induced glaucoma mice. Visual acuity (VA) assessed at 2, 4, and 6 weeks after IOP elevation in all groups, ***P<0.001, **P<0.01, *P<0.05. [Figure 3C] Figure 1 shows visual acuity rescue by HSP60 nasal spray in MB-induced glaucoma mice. Positive scotopic threshold potential (pSTR) amplitude assessed at 2, 4, and 6 weeks after IOP elevation in all groups, ***P<0.001, **P<0.01, *P<0.05. [Figure 4A] Figure 1: Rescue of retinal ganglion cells (RGCs) by HSP60 nasal spray in MB-induced glaucoma mice. Quantification of retinal ganglion cells (RGCs; immunohistochemistry with Brn3a) at 2, 4, and 6 weeks after IOP elevation in all groups; ***P<0.001, **P<0.01, *P<0.05. [Figure 4B]Figure 1 shows axonal rescue by HSP60 nasal spray in MB-induced glaucoma mice. Axon quantification at 2, 4 and 6 weeks after IOP elevation in all groups, ***P<0.001, **P<0.01, *P<0.05. [Figure 5] FIG. 1 is a schematic diagram showing the hypothesized mechanism of HSP60 nasal spray. [Figure 6A] Figure 1 shows that IGFBPL1 protects RGCs from elevated IOP-induced damage and prevents loss of RGC function and vision in a glaucoma model. IOP levels in sham-injected (triangles) or microbead-injected mice receiving intravitreal injections of saline (squares) or IGFBPL1 (circles). [Figure 6B] Figure 1 shows that IGFBPL1 protects RGCs from elevated IOP-induced damage and prevents loss of RGC function and vision in a glaucoma model: RGC counts in untreated and sham-injected mice and MB-injected mice receiving saline or IGFBPL1 treatment. [Figure 6C] Figure 1 shows that IGFBPL1 protects RGCs from elevated IOP-induced damage and prevents loss of RGC function and vision in a glaucoma model. Quantification of ERG positive scotopic threshold potential (pSTR) in saline- and IGFBPL1-treated mice shows that RGC function was significantly improved in IGFBPL1-treated mice compared to saline-treated mice. [Figure 6D] Figure 1 shows that IGFBPL1 protects RGCs from elevated IOP-induced damage and prevents loss of RGC function and vision in a glaucoma model. OKR tests show that visual acuity (VA) and visual contrast sensitivity (CS) were significantly improved in IGFBPL1-treated groups compared with saline-treated groups, as determined 2-8 weeks after MB injection. [Figure 7](FIG. 7A) Microglial expression of IGFBPL1, IGF-1 receptor (IGF-1R), and IGF-1. Double immunolabeling of microglia for the microglial marker Iba-1 (green) and IGFBPL1 (red) in culture and in retinal whole mounts counterstained with the nuclear marker DAPI (blue). (FIG. 7B) Microglial expression of IGFBPL1, IGF-1 receptor (IGF-1R), and IGF-1. Double immunolabeling of the microglial marker Iba-1 (green) and IGF-1R (red) or IGF-1 (red) in flat mounts of adult mouse retina. [Figure 8A] Figure 1 shows IGFBPL1 inhibition of microglial activation in glaucomatous mouse retinas. Activated microglia counts in retinal whole mounts from untreated (normal), microbead and saline-injected (MB+saline), and microbead and IGFBPL1-injected (MB+IGFBPL1) mice 5-14 days after IOP elevation. [Figure 8B] 1 shows IGFBPL1 suppression of microglial activation in glaucomatous mouse retina. qPCR results show induction of activated microglial markers following elevated IOP and suppression of activated microglial markers by IGFBPL1 administration. [Figure 8B-2] (as above) [Figure 9] Figure 1 shows that IGFBPL1 inhibits pro-inflammatory cytokine production in the glaucomatous retina. qPCR results show the induction of activated pro-inflammatory cytokines following elevated IOP and its suppression by IGFBPL1 administration. [Figure 9-2] (as above) [Figure 10A] Figure 1 shows that IGFBPL1 deficiency leads to microglial activation in the adult retina. Quantification of activated microglia in retinal whole mounts. [Figure 10B] Figure 1 shows that IGFBPL1 deficiency leads to microglial activation in the adult retina. qPCR results show increased levels of activated microglial markers in IBKO retinas compared to WT retinas. [Figure 11] 1 shows progressive RGC loss in IGFBPL1-deficient mice. Quantification of RGC density shows the gradual loss of RGCs in IBKO mice from 4 weeks to 7 months of age. [Figure 12] Figure 1 shows IGFBPL1 suppression of LPS-induced inflammation. Quantification of pro-inflammatory cytokine levels in isolated microglial cultures treated with control, IGFBPL1, LPS and LPS+IGFBPL1. [Figure 13] Figure 1 shows the protection of RGCs against ischemia-reperfusion-induced death and functional damage. Data are presented as mean ± SD (n = 5 mice per group). *p < 0.05 by Student's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention describes a method for treating neuroinflammation and neurodegeneration and reducing the risk of the onset or progression of neuroinflammation and neurodegeneration.The method comprises one or more of (i) administering HSP60 or HSP27 or its active fragment, and / or (ii) administering IGFBPL or its active fragment.The method can be used to reduce inflammation and neuronal cell death in other places, including the eye and CNS and PNS.The method can comprise, for example, the nasal administration of HSP60 or HSP27, and the nasal, systemic or ocular administration of IGFBPL1, for example, intravitreal injection or local administration in the eye, for the treatment of glaucoma.

[0020] HSP60 (HSPD1, heat shock protein family D (Hsp60) member 1) A growing body of new evidence implicates an autoimmune mechanism in glaucoma, although its relative importance in pathogenesis remains to be proven. As shown herein, neuronal and visual loss in glaucoma and other immune-related conditions is associated with pre-existing memory T cells that are presensitized by exposure to bacterial HSP60 early in life. Uncovering the immune mechanism in glaucoma and its relationship to commensal microorganisms in progressive neurodegeneration will provide the basis for new diagnostic procedures.

[0021] The amino acid sequence of human hsp60 is provided under GenBank Accession No. NP_002147.2, and the amino acid sequence of bacterial hsp60 is provided under GenBank Accession No. WP_000729117.1, both of which are incorporated herein by reference. HSP60 is preferably formulated for nasal administration to induce tolerance to HSP60. Alternatively, oral or subcutaneous administration can be used. See also WO2012118863.

[0022] HSP27 (heat shock protein family B (small) member 1 (HSPB1)) HSP27, also known as HSPB1, is shown herein to directly induce the pro-inflammatory response of HMC3 cells. The amino acid sequence of human HSP27 is provided under GenBank accession number NP_001531.1, and this amino acid sequence is incorporated herein by reference. HSP27 is preferably formulated for nasal administration to induce tolerance to HSP27. Alternatively, oral or subcutaneous administration can be used. See also WO2012118863.

[0023] IGFBPL (insulin-like growth factor binding protein-like 1) Insulin growth factor binding protein-like 1 (IGFBPL1) promotes the survival and neurite outgrowth of neonatal mouse retinal ganglion cells (RGCs), which are regulated via the insulin-like growth factor 1-mediated signaling pathway (Guo et al., Sci Rep. 2018 Feb 1;8(1):2054). As shown herein, IGFBPL1 actively suppresses neuroinflammatory microglia in adult / post-neonatal animals. The amino acid sequence of human IGFBPL1 is provided in GenBank Accession No. NP_001007564, which is incorporated herein by reference. See also WO2012118796.

[0024] Treatment and prevention methods The compositions described herein can be administered to a subject to treat or prevent disorders associated with abnormal or unwanted immune responses, such as neuroinflammatory or neurodegenerative disorders associated with excessive or abnormal activation of microglia. Examples of such disorders include, but are not limited to, non-arteritic ischemic optic neuropathy (NAION), autism, multiple sclerosis, Alzheimer's disease, Parkinson's disease, ischemic retinopathy, glaucoma, age-related macular degeneration, stroke, ischemic and traumatic optic neuropathy, and diabetic retinopathy; in some embodiments, the disease is associated with vision loss and / or increased intraocular pressure. The method can be used to treat subjects with these diseases, for example, to reduce the risk of or treat vision loss and neuronal cell loss associated with these diseases. See also US8 / 198,284; WO / 2017 / 213504; WO2012118863; and WO2012118796. All of the above patent documents are incorporated herein by reference.

[0025] The methods of treatment or prevention described herein can include, for example, administering to a subject a nasal or subcutaneous HSP60 or HSP27 composition sufficient to stimulate the mucosal immune system. In some embodiments, the method includes administering a nasal HSP60 or HSP27 composition sufficient to increase regulatory T cell levels by, for example, about 50%, 75%, 100%, 200%, 300% or more above baseline.

[0026] In some embodiments, the method involves administering an intranasal or subcutaneous HSP60 or HSP27 composition and / or IGFBPL1 composition in an amount sufficient to result in improvement in one or more clinical markers of vision loss (e.g., decreased vision) or disability; for example, in multiple sclerosis, such markers could include gadolinium-enhancing lesions visualized by MRI, or by the Patty, Fazekas, or Barkhof MRI criteria, or the McDonald diagnostic criteria. The IGFBPL1 composition can be administered intranasally, systemically, or ocularly, for example, by eye drops or intravitreal administration.

[0027] In some embodiments, the treatment is administered to a subject diagnosed with a disorder associated with microglial activation; such a diagnosis can be made by a skilled practitioner using known methods and routine techniques. In some embodiments, the method includes diagnosing, identifying, or selecting a subject with a disorder associated with microglial activation, or identifying or selecting a subject based on the presence or diagnosis of a disorder associated with microglial activation. In some embodiments, the subject is an adult, for example, a human who is at least 18 years old, or a post-neonatal human, for example, at least 6 months or 1 year old.

[0028] Pharmaceutical Compositions and Methods of Administration The methods described herein involve the use of pharmaceutical compositions that include one or more of HSP60, HSP27, or IGFBPL1 as an active ingredient.

[0029] Pharmaceutical compositions typically contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0030] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration.

[0031] Examples of administration routes include systemic parenteral administration, such as intravenous, intraperitoneal, intradermal, or subcutaneous; local administration to the eye, such as topical, intravitreal, intraocular, intraorbital, periorbital, subconjunctival, subretinal, subtenon, or transscleral; and systemic oral administration. In some embodiments, intraocular administration or, in particular, administration by eye drops, ointments, creams, gels, or lotions may be used. In some embodiments, the composition is administered systemically, for example, orally; in preferred embodiments, the composition is administered to the eye, for example, via topical (eye drops, lotions, or ointments) administration or by local injection, for example, periocular or intravitreal injection; see, for example, Gaudana et al., AAPS J. 12(3):348-360 (2010); Fischer et al., Eur J Ophthalmol. 21 Suppl 6:S20-6 (2011). Administration may be provided as a periodic bolus (e.g., intravitreally or intravenously) or as a continuous infusion from an internal reservoir (e.g., from an implant placed in an intraocular or extraocular location (see U.S. Pat. Nos. 5,443,505 and 5,766,242)) or from an external reservoir (e.g., from an intravenous bag or contact lens sustained release system). The composition may be administered locally, for example, by continuous release from a sustained-release drug delivery device fixed to the inner wall of the eye or via targeted transscleral controlled release into the choroid (see, for example, PCT / US00 / 00207, PCT / US02 / 14279, PCT / US2004 / 004625, Ambati et al. (2000) Invest. Ophthalmol. Vis. Sci. 41:1181-1185, and Ambati et al. (2000) Invest. Ophthalmol. Vis. Sci. 41:1186-1191). A variety of devices suitable for administering drugs locally inside the eye are known in the art. See, e.g., U.S. Patent Nos. 6,251,090, 6,299,895, 6,416,777, 6,413,540, and 6,375,972, and PCT / US00 / 28187.

[0032] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include systemic (e.g., parenteral, nasal, subcutaneous, and oral) and topical (ocular, e.g., intravitreal or topical) administration. Thus, compositions comprising the compositions described herein in formulations for ocular administration, such as eye drops, lotions, and creams, including, for example, microcapsules, microemulsions, or nanoparticles, are also within the scope of the present disclosure. Methods for formulating pharmaceutical compositions suitable for ocular delivery are known in the art; see, e.g., Losa et al., Pharmaceutical Research 10:1(80-87 (1993); Gasco et al., J. Pharma Biomed Anal., 7(4):433-439 (1989); Fischer et al., Eur J Ophthalmol. 21 Suppl 6:S20-6 (2011); and Tangri and Khurana, Intl J Res Pharma Biomed Sci., 2(4):1541-1442 (2011).

[0033] General methods for formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid, or phosphate, and agents for adjusting osmotic pressure such as sodium chloride or glucose. pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0034] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to exist for easy syringability. The composition should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0035] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in a suitable solvent, optionally with one or a combination of the above-listed ingredients, followed by filtration sterilization.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients from the above-listed ingredients.For sterile powder preparation of sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which can obtain a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0036] Oral compositions generally contain an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or cornstarch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0037] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0038] The systemic administration of the therapeutic compound described herein can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be passed is used in the formulation.Such penetrants are generally known in the art, and for example, for transmucosal administration, include surfactants, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams that are generally known in the art.

[0039] Pharmaceutical compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0040] In one embodiment, the therapeutic compound is prepared with a carrier that protects the therapeutic compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques or purchased commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These compounds can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0041] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0042] kit Also provided herein is a kit for use in the methods described herein.For example, the kit can include, for example, a composition comprising HSP60 or HSP27 for nasal administration, and a composition comprising IGFBPL1 for nasal, systemic (for example, oral) or ocular (for example, topical or intravitreal) administration.Instructions for use can also be included in the kit. [Example]

[0043] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0044] [Example 1] Immune tolerance to HSP60 attenuates neurodegeneration in a mouse model of glaucoma Primary open-angle glaucoma (POAG), a leading cause of blindness worldwide, is a disease that damages the optic nerve. POAG has been primarily associated with high intraocular pressure (IOP), but treatments that affect IOP do not completely prevent vision loss and blindness. 1,2 .

[0045] Elevated intraocular pressure (IOP) induces a T cell-mediated autoimmune response against HSP60, and in mice maintained without bacterial flora, HSP-specific T cell responses and neuronal loss are abolished after elevated IOP 3~9 In this example, we tested whether induction of tolerance to HSP60 attenuates glaucomatous damage.

[0046] Immune tolerance to HSP60 was induced in 6- to 8-week-old male and female G57BK / 6J mice by intranasal administration of a low dose of HSP60 (2 μM HSP60 daily for 7 days). Control mice were treated with saline. Glaucoma was induced by two injections of microbeads (MB) into the anterior chamber and elevated IOP maintained for 8 weeks. IOP was monitored weekly. Visual function was assessed by optokinetic motor response (OMR) and electroretinogram scotopic threshold potential (pSTR). Mice were sacrificed at 2, 4, and 8 weeks. Immune responses and T cell tolerance to HSP60 were analyzed by fluorescence-activated cell sorting (FACS). Glaucomatous nerve damage was quantified by counting retinal ganglion cells (RGCs) and axons.

[0047] By 4 weeks, MB-injected eyes showed an IOP of 20.1 ± 0.56 mmHg or higher compared to 11.6 ± 0.21 mmHg in the contralateral uninjected eyes (Figure 1). As shown by FACS analysis, intranasal administration of low-dose HSP60 induced immune tolerance and increased Treg levels (Figure 2). No significant differences in IOP levels were observed between HSP60- or saline-treated mice. Treatment with HSP60 did not alter visual acuity (VA), contrast sensitivity (CS), or pSTR prior to MB injection when compared with saline-treated mice. However, when assessed by OMR, HSP60-treated mice exhibited significantly higher VA and CS than saline-treated mice at all time points after MB injection (Figure 3A-C). Consistently, RGC function, assessed by pSTR, was also significantly improved in HSP60-treated mice compared with saline-treated non-immune tolerant mice at all time points after MB injection (Figure 4A-B).

[0048] As shown in Figure 5, these results indicate that immune tolerance to HSP60 attenuates glaucomatous RGC loss and functional loss in mice.

[0049] [Example 2] A role for insulin-like growth factor binding protein-like protein 1 in microglia in adult mice Insulin-like growth factor binding protein-like protein 1 (IGFBPL1) plays a crucial role in promoting axon outgrowth and survival of retinal ganglion cells (RGCs) during development (Guo et al., Sci Rep. 2018 Feb 1;8(1):2054). This requires the presence of insulin-like growth factor 1 (IGF1) and is mediated through the IGF1 receptor (IGF1R). Because adult RGCs are known to express low or undetectable levels of IGF1R, this example investigated whether IGFBPL1 supports RGC survival after injury to the adult retina.

[0050] The expression of IGF1, IGFBPL1, and IGF1R in the adult retina was examined using immunohistochemistry in retinal whole mounts. Retinal whole mounts were double-immunolabeled with primary antibodies against the RGC marker Brn-3 or the microglial marker Iba-1 to identify cell-type-specific expression of IGF1, IGF1R, and IGFBPL1.

[0051] Coculture of purified microglia isolated from postnatal day 5 Cx3CR1 / GFP mice with RGCs was performed in the presence or absence of LPS and / or IGFBPL1 and IGF1. Neuronal survival was determined using a LIVE / DEAD cell viability kit, and the percentage of surviving RGCs was quantified using Image J software.

[0052] The results showed that IGF1, IGF1R, and IGFBPL1 were expressed by microglia but not by RGCs in the adult mouse retina (Figure 6A-B). Addition of IGF1 and / or IGFBPL1 to purified RGC cultures did not promote neuronal survival, whereas LPS stimulated microglial activation and caused significant RGC death compared with control cultures (P<0.05). IGF1 and / or IGFBPL1 significantly attenuated neuronal death in LPS-treated microglia-induced RGC death in microglia-RGC cocultures (see Figure 12).

[0053] In a glaucoma model, intravitreal injection of IGFBPL1 at 3 and 10 days after elevated IOP protected RGCs against elevated IOP-induced damage and prevented loss of RGC function and vision (Figure 7A-D). IGFBPL1 suppressed microglial activation in the glaucomatous retina (Figure 8A-B) and also inhibited microglial activation, reactive gliosis, and pro-inflammatory cytokine production (Figure 9). As shown in Figure 10A-B, deficiency of IGFBPL1 leads to microglial activation and elevated levels of pro-inflammatory cytokines in the adult retina. Figure 11 shows that intravitreal injection of IGFBPL1 rescued RGC loss and functional deficits in IGFBPL1-deficient mice.

[0054] This study demonstrates that IGFBPL1 is expressed by microglia rather than RGCs in adult mice. IGFBPL1 exerts its neuroprotective effects by acting on microglia. These results suggest that IGFBPL1 protects against neuronal death by regulating neuroinflammation.

[0055] [Example 3] IGFBPL1 with ocular hypertension - / - IGFBPL1 protected against neuronal and visual loss in mice Insulin growth factor binding protein-like 1 (IGFBPL1) promotes survival and neurite outgrowth of neonatal mouse retinal ganglion cells (RGCs), which is regulated via the insulin-like growth factor 1-mediated signaling pathway (Guo et al., Sci Rep. 2018 Feb 1;8(1):2054). Neonatal IGFBPL1 deficiency (IGFBPL1 - / - ) mice had approximately 20% fewer RGCs than wild-type control mice. This study compared adult IGFBP1 mice with or without elevated intraocular pressure (IOP). - / - We investigated the role of IGFBPL1 protein on neuronal survival and visual performance in mice.

[0056] IGFBPL1 at 1, 2, and 7 months of age - / - The RGC density of mice was determined by Brn3a immunolabeling in retinal flat mounts. To induce elevated IOP, 2 microliters of polystyrene microbeads (MB; 5 × 10 6 / ml) in adult male and female IGFBP1 - / -The mice were injected intravitreally with IGFBPL1 recombinant protein or sterile saline (control) on days 3, 7, and 17 after MB injection. Four weeks after MB injection, two researchers recorded the contrast sensitivity and visual acuity of the mice in a masked manner. The mice were sacrificed, and the retinas were flat-mounted and processed for Brn3a immunolabeling to reveal surviving RGCs. Statistical analysis was performed using a Student's t-test.

[0057] According to the data, IGFBP1 - / - In mice, the absence of IGFBPL1 led to the progressive loss of RGCs. - / - In mice, transient elevation of IOP resulted in significant impairment of visual performance and RGC loss. IGFBPL1 treatment improved the visual performance of wild-type and IGFBPL1 mice with ocular hypertension. - / - Visual performance (P<0.05) and RGC survival (P<0.05) were significantly improved in mice.

[0058] Furthermore, retinal ischemia-reperfusion injury was induced unilaterally in mice, and saline (isotype) or IGFBPL1 (BPL1) was intravitreally injected on day 1 (early) or day 10 (late) after injury. Mice were sacrificed 4 weeks after injury and quantified for RGC density. RGC function was assessed by pSTR amplitude 2 and 4 weeks after injury (before sacrifice), and visual contrast sensitivity (CS) and visual acuity (VA) were measured using the optokinetic reflex (OKR) assay. As shown in Figure 13, after ischemic injury, IGFBPL1-treated mice showed significantly increased RGC density and improved pSTR amplitude, as well as CS and VA values, compared with the saline-treated group.

[0059] IGFBPL1 is known to be strongly expressed in the embryonic retina but barely detectable in the adult retina. - / -In mice, the absence of IGFBPL1 during embryonic development was shown to induce progressive degeneration of RGCs. Administration of IGFBPL1 protected mice with ocular hypertension from RGC loss and vision loss. Overall, IGFBPL1 is an important neuroprotective agent in the retina undergoing progressive degeneration, such as in glaucoma.

[0060] [Example 4] Heat shock proteins 27 and 60 directly activate human microglia Glaucoma has an autoimmune component driven by CD4+ T cells primed by commensal bacteria, which then enter the retina and cross-react with heat shock protein (HSP)-expressing neurons through a mechanism of molecular mimicry. As shown herein, microglial activation contributes to the immune response and retinal degeneration in glaucoma. Given the limited availability of primary human microglia, the immortalized human microglial clone 3 cell line (HMC3) is useful for examining microglial behavior under pathological conditions. To test whether HSP27 and HSP60 can induce microglial activation, cytokine expression and morphological changes were examined in HMC3. Other known pro-inflammatory agents were used as positive controls.

[0061] Methods: HMC3 cell line (ATCC) was cultured in EMEM medium and challenged with 10 μg / ml HSP27, 10 μg / ml HSP60, 200 ng / ml LPS, or 100 ng / ml LPS with or without 5 mM ATP for an additional 30 minutes. Cells receiving medium alone served as a control. After 24 hours, RNA from HMC3 cells was collected using the ZYMO Research Quick-RNA Microprep Kit, and RNA reverse transcription was performed using PrimeScript™ RT Master Mix. Sybr green RT-PCR mixtures containing different primers and cDNA samples were subjected to PCR using an EP realplex real-time PCR instrument. Relative fold changes in mRNA transcripts were presented and compared with the control group. Additionally, low-density HMC3 cell cultures were prepared, and images of cell morphology were recorded 24 hours after LPS, HSP27, or HSP60 treatment, and morphological changes were quantified.

[0062] Results: The data showed that LPS with or without ATP induced increased expression of pro-inflammatory cytokines such as IFNγ in HMC3, while HSP27 and HSP60 could also activate HMC3 to express higher levels of IFNγ and TNFα. Quantification of cell morphology showed that dendrites were shortened and round cell body size was enlarged in the groups stimulated with LPS, HSP27, and HSP60 compared with the vehicle control group (P<0.05).

[0063] Conclusions: This study demonstrated that HMC3 cells respond to known pro-inflammatory agents in a manner similar to primary microglia. HSP27 and HSP60 can directly induce pro-inflammatory responses in HMC3 cells, supporting the idea that HSPs may induce microglial activation as an early driver of glaucoma-associated immune responses.

[0064] [Example 5] Investigating the spatiotemporal dynamics of microglia / macrophage polarization after ischemia / reperfusion in the retina Background: Microglia / macrophages exhibit diverse functional phenotypes under various microenvironmental stimuli and disease processes. The phenotypic dynamics of microglia during ischemia / reperfusion (I / R) have remained unclear. Identifying the spatiotemporal patterns of microglia / macrophage polarization after I / R may improve our understanding of post-I / R injury and recovery.

[0065] Methods: I / R was induced in rats by cannulating with a 30-gauge needle connected to a saline reservoir to maintain an intraocular pressure of 110 mmHg for 60 minutes. Rat retinas were harvested on postoperative days 1, 2, 7, and 14. Flow cytometry, reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemical staining for M1 and M2 markers were performed to characterize the phenotypic changes of retinal cells, including microglia and infiltrating macrophages.

[0066] Results: Flow cytometry results showed that CD11b + CD45 high , possibly a significant increase in macrophages and / or activated microglia, followed by CD11b - CD45 high Both showed an increase in lymphocytes, peaking on day 7. + Iba1 + (M1 marker) cells and Ym - 1 + Iba1 + A rapid increase in both M2 and M3 cells was found in the retina on days 1 and 2 after I / R. These cells displayed round bodies with rare short dendrites and were distributed from the inner nuclear layer to the ganglion cell layer on days 1–7.

[0067] Conclusion: I / R induced an initial response of variably activated microglia / macrophages toward both M1 and M2 responses, leading to the recruitment of lymphocytes. Therefore, microglia / macrophages may play a leading role in the recruitment of infiltrated lymphocytes following I / R.

[0068] [Example 6] Targeting HSPs: Immune tolerance to HSP60 attenuates neurodegeneration in glaucoma Objective: Previous studies have suggested that bacterially primed T cell-mediated autoimmune mechanisms underlie the pathogenesis of glaucoma, with heat shock proteins (HSPs) acting as pathogenic autoantigens. We hypothesized that in glaucoma, induction of immune tolerance to bacterial HSP60 may block such pathogenic immune responses and attenuate neuronal loss.

[0069] Methods: Adult C57BL / 6J mice received low doses of recombinant bacterial HSP60, ovalbumin (OVA), or saline (both as controls) daily in the nostrils for 7 days. Elevated IOP was induced unilaterally by anterior chamber injection of polystyrene microbeads (MB). Visual and retinal function were assessed by optokinetic response (OMR) and electroretinogram positive scotopic threshold potential (pSTR). Mice were sacrificed 2, 4, and 8 weeks after MB injection. T cell responses to bacterial HSP60 were analyzed by ear DTH (delayed-type sensitive response) testing and flow cytometry. Glaucomatous nerve damage was quantified by counting retinal ganglion cells (RGCs) and axons.

[0070] Results: Intranasal administration of a low dose of HSP60 induced immune tolerance, as indicated by a reduced DTH response, and increased levels of regulatory T cells, as seen by flow cytometry analysis. MB-injected eyes maintained an IOP level of 25 ± 3 mmHg compared with 12 ± 2 mmHg in the contralateral uninjected eyes. No significant differences in IOP levels were observed between mice treated with HSP60, OVA, or saline. Treatment with HSP60 or OVA did not alter basal levels of visual acuity (VA), contrast sensitivity (CS), or pSTR prior to MB injection compared with untreated or saline-treated mice. However, after MB injection, VA and CS, assessed by OMR, were significantly better in HSP60-treated mice compared with saline- or OVA-treated mice at all time points. Consistently, both RGC function, assessed by pSTR amplitude, and RGC counts were significantly higher in HSP60-treated mice compared with saline- or OVA-treated mice after MB injection.

[0071] Conclusions: Intranasal administration of low doses of multiple bacterial HSP60s induced immune tolerance and attenuated RGC loss and functional deterioration in a MB-induced mouse model of glaucoma. These results suggest an attractive antigen-specific therapeutic strategy for preventing vision loss in glaucoma. Such studies may aid our understanding of the pathogenesis of brain neurodegenerative disorders and may also provide innovative therapeutic interventions for the treatment of neurodegeneration affecting other parts of the central nervous system. References

[0072] [Table 1]

[0073] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate, but not limit, the scope of the invention, which is limited by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] A method for treating neuroinflammation and neurodegeneration and / or reducing the risk of onset or progression of neuroinflammation and neurodegeneration, comprising administering to a subject in need thereof a therapeutically effective amount of one or more of: (i) HSP60 or HSP27, or an active fragment thereof, and / or (ii) IGFBPL or an active fragment thereof. [Invention 2] The method of claim 1, wherein the therapeutically effective amount is sufficient to reduce inflammation and neuronal cell death in the subject. [Invention 3] 3. The method according to claim 1 or 2, comprising intranasal or subcutaneous administration of HSP60 and systemic or ocular administration of IGFBPL. [Invention 4] The method of invention 3, wherein ocular administration of the IGFBPL comprises intravitreal injection. [Invention 5] 5. The method according to any one of inventions 1 to 4, wherein the subject has non-arteritic ischemic optic neuropathy (NAION), glaucoma, autism, multiple sclerosis, Alzheimer's disease, Parkinson's disease, ischemic retinopathy, age-related macular degeneration, stroke, ischemic and traumatic optic neuropathy, or diabetic retinopathy. [Invention 6] 6. The method according to any one of claims 1 to 5, wherein inflammation and neuronal death are reduced in the eye of the subject. [Invention 7] A kit comprising a composition comprising HSP60 and / or HSP27, and a composition comprising IGFBPL1 for use in the methods described herein. [Invention 8] A composition comprising HSP60 and / or HSP27, and / or a composition comprising IGFBPL1, for use in a method for treating neuroinflammation and neurodegeneration and / or reducing the risk of onset or progression of neuroinflammation and neurodegeneration. [Invention 9] The kit according to Invention 7 or the composition for use according to Invention 8, wherein the HSP60 and / or HSP27 is formulated for intranasal administration and the IGFBPL1 is formulated for ocular, e.g., intravitreal, administration. [Invention 10] The kit according to Invention 7 or the composition for use according to Invention 8, wherein the HSP60 and / or HSP27 is formulated for nasal or subcutaneous administration, and the IGFBPL1 is formulated for systemic administration. [Invention 11] The kit according to Invention 7 or the composition for use according to Invention 8, wherein the HSP60 and / or HSP27 is formulated for nasal or subcutaneous administration, and the IGFBPL1 is formulated for nasal administration. [Invention 12] 12. A kit or composition for use according to invention 11, wherein one, two or all three of said HSP60 and / or HSP27 and said IGFBPL1 are formulated together in a single composition for nasal or subcutaneous administration.

Claims

1. A pharmaceutical composition for use in a method for treating a neuroinflammatory or neurodegenerative disorder associated with excessive or abnormal activation of microglia in a subject, wherein the disorder is selected from non-arteritic ischemic optic neuropathy (NAION), ischemic retinopathy, and ischemic and traumatic optic neuropathy, the pharmaceutical composition comprising a therapeutically effective amount of IGFBPL1 or an active fragment thereof.

2. The composition of claim 1, which is used for systemic or ocular administration of IGFBPL1 or an active fragment thereof.

3. The composition of claim 2 , wherein the ocular administration comprises an intravitreal injection.

4. The composition according to any one of claims 1 to 3, wherein the neuroinflammatory or neurodegenerative disorder associated with excessive or abnormal activation of microglia is ischemic retinopathy or ischemic and traumatic optic neuropathy.

5. The composition according to any one of claims 1 to 4, wherein the neuroinflammatory or neurodegenerative disorder associated with excessive or abnormal activation of microglia is NAION.

6. The composition of any one of claims 1 to 5, which reduces inflammation and neuronal death in the eye of the subject.

7. 3. The pharmaceutical composition of claim 1 or 2, which reduces inflammation and neuronal death in the brain or spinal cord of the subject.

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