Cognitive function improving agent containing CX3CL1

Abstract

To provide a novel use of CX3CL1, which is one of chemokine.SOLUTION: There is provided a cognitive function improver containing CX3CL1: more specifically, a pharmaceutical composition containing a polypeptide containing a chemokine domain of CX3CL1 based on the discoveries that the polypeptide containing the chemokine domain of CX3CL1 has the actions of improving the cognitive functions decreased with aging and recovering neurogenesis in an adult hippocampus. The composition is prepared into a medicine for administration excluding intrathecal administration.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the use of CX3CL1 for improving cognitive function. The present invention also provides the use of CX3CL1 for restoring neurogenesis. [Background technology]

[0002] CX3CL1 is a membrane-bound chemokine discovered in 1997 by searching an EST database. Human CX3CL1 is known as fractalkine, and mouse CX3CL1 is known as neurotactin. Many other chemokines are secreted proteins, whereas CX3CL1 is expressed on the cell surface as a membrane-bound protein having a chemokine domain, a mucin domain (mucin-like shaft), a cell membrane-spanning domain, and an intracytoplasmic domain from the N-terminus (Non-Patent Document 1), and the mucin domain functions to present the chemokine domain on the cell membrane surface. When CX3CL1 is expressed as a membrane-bound protein, the extracellular domain containing the chemokine domain and the mucin domain is cleaved by a protease to become free CX3CL1 (Non-Patent Document 1). While the membrane-bound protein CX3CL1 is involved in cell-cell adhesion, free CX3CL1 promotes the migration of immune cells, similar to other chemokines (Non-Patent Document 2).

[0003] CX3CL1 is known to be induced by inflammatory stimuli in vascular endothelial cells, renal mesangial cells, airways, renal tubules, and intestinal epithelial cells, and is also expressed in dendritic cells, neurons, and activated astrocytes. CX3CL1 is also expressed in pancreatic β cells and is involved in the control of insulin secretion (Non-Patent Document 3). Furthermore, CX3CL1 is frequently expressed in the hippocampus in the brain and suppresses excessive activation of microglia (Non-Patent Document 4). It is known that the concentration of CX3CL1 in the hippocampus and pancreas decreases with aging (Non-Patent Document 5).

[0004] Patent Document 1 describes an invention relating to an inhibitor of the decline in neurogenesis that contains as an active ingredient a substance that inhibits the activity or expression of stress-inducible factors in the brain, and in this invention, CX3CL1 is described as one of the stress-inducible factors.

[0005] In addition, maintaining and improving cognitive function is required for a wide range of generations, from young to old, and maintaining and improving memory, thinking ability, judgment, etc. is particularly important for daily life, and in the elderly, it can prevent cognitive function decline associated with aging, extend healthy lifespan, and suppress decline in quality of life. For this reason, the development of drugs that maintain and improve cognitive function is an urgent task.

[0006] Hippocampal neurogenesis is necessary for memory formation (Non-Patent Document 6), and is reduced in dementia patients compared to healthy individuals (Non-Patent Document 7). It has also been shown that cognitive ability can be improved by enhancing hippocampal neurogenesis (Non-Patent Document 8).

[0007] It has also been reported that CX3CL1 upregulates memory-related synaptic plasticity in the rat hippocampus (Non-Patent Document 9), and that treatment of aged rats with fractalkine attenuates age-related microglial activation (Non-Patent Document 10).

[0008] However, all of the reports in the prior art that relate CX3XL1 to brain function involve intraventricular injection of CX3CL1 or treatment of extracted brain tissue with CX3CL1, and cells in the brain must be directly treated with CX3CL1. Considering the fact that brain tissue is highly protected by the blood-brain barrier, the disclosures in the above prior art documents do not suggest that CX3CL1 can be a practical cognitive function improving agent.

[0009] In addition, substances that can pass through the blood-brain barrier are basically lipophilic substances with small molecular weights, and it is known that hydrophilic substances have difficulty passing through the lipid membrane of the blood-brain barrier. The chemokine domain of CX3CL1 is a hydrophilic polypeptide, and according to common technical knowledge, it is not thought to be able to pass through the blood-brain barrier. In addition, there have been no reports that CX3CL1 from peripheral tissues passes through the blood-brain barrier and enters the brain. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] JP 2020-63229 A [Non-patent literature]

[0011] [Non-Patent Document 1] Bazan JF, Bacon KB, Hardiman G et al (1997) A new class of membrane-bound chemokine with a CX3C motif. Nature 385:640-644 [Non-Patent Document 2] Imai T, Hieshima K, Haskell C et al (1997) Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91:521-530 [Non-Patent Document 3] Lee YS, Morinaga H, Kim JJ et al (2013) The fractalkine / CX3CR1 system regulates β cell function and insulin secretion. Cell 153:413-425 [Non-Patent Document 4] Harrison JK, Jiang Y, Chen S et al (1998) Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia. Proc Natl Acad Sci U S A 95:10896-10901 [Non-Patent Document 5] Lyons A, Lynch AM, Downer EJ et al (2009) Fractalkine-induced activation of the phosphatidylinositol-3 kinase pathway attentuates microglial activation in vivo and in vitro. J Neurochem 110:1547-1556 [Non-Patent Document 6] Goncalves JT, Schafer ST, Gage FH (2016) Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior. Cell 167:897-914 [Non-Patent Document 7] Moreno-Jimenez EP, Flor-Garcia M, Terreros-Roncal J et al (2019) Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat Med 25:554-560 [Non-Patent Document 8] Berdugo-Vega G, Arias-Gil G, Lopez-Fernandez A et al (2020) Increasing neurogenesis refines hippocampal activity rejuvenating navigational learning strategies and contextual memory throughout life. Nat Commun 11:135

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

[0012] The present invention provides a novel use of CX3CL1. [Means for solving the problem]

[0013] The novel use of CX3CL1 provided by the present inventors is as a cognitive function improving agent, particularly as an agent for improving material cognitive ability that has declined due to aging. The present inventors also provide a use of CX3CL1 as an agent for restoring neurogenesis in the hippocampus.

[0014] It is important that CX3CL1 administered in the present invention contains a chemokine domain. The present invention also provides a pharmaceutical composition containing CX3CL1, particularly a pharmaceutical composition formulated for administration other than intrathecal administration (intraventricular administration or lumbar puncture), for example, oral administration, intravenous administration, intraperitoneal administration or subcutaneous administration. Specifically, the present invention relates to a pharmaceutical composition containing a cognitive function improving agent containing a polypeptide containing a chemokine domain of CX3CL1, and formulated for oral administration, intravenous administration, intraperitoneal administration or subcutaneous administration, particularly preferably a pharmaceutical composition formulated in a form suitable for intraperitoneal administration.

[0015] It is an important feature of the present invention that the pharmaceutical compositions of the present invention can be administered by routes other than intrathecal administration.

[0016] The present inventors found that administering a peptide corresponding to the chemokine domain of CX3CL1 into the peritoneal cavity of aged mice improved the material cognition ability that had declined with aging (Figure 1). In addition, when measuring hippocampal neurogenesis, which decreases with aging, it was shown that neurogenesis was increased in aged mice that had received an intraperitoneal administration of a peptide corresponding to the chemokine domain of CX3CL1 (Figure 2), and further found that aged macrophages in the peritoneal cavity were reduced (Figure 3). On the other hand, there was no change in the proportion of macrophages in peritoneal cells.

[0017] Considering that it is known that removing senescent cells restores various functions of aged organs (Non-Patent Documents 11, 12), it is possible that administration of CX3CL1 reduces aged macrophages in the peritoneal cavity, thereby improving the decline in immunity caused by aging, and as a result, improving cognitive function.

[0018] To demonstrate this, the inventors collected peritoneal cells from mice that had been intraperitoneally administered CX3CL1 and transplanted them into mice, and conducted a novel substance discovery test similar to that in Example 1, confirming the same effect of improving cognitive function as in the case of administration of CX3CL1 (Figure 4).

[0019] Such an action of CX3CL1 has not been reported so far, and the present invention provides an industrially useful novel invention.

[0020] Specific embodiments of the present invention are exemplified below. (1) A cognitive function improving agent comprising a polypeptide including the chemokine domain of CX3CL1. (2) The cognitive function improving agent according to (1), wherein the cognitive function improvement improves cognitive function that has declined due to aging. (3) The cognitive function improving agent according to (1), wherein the polypeptide is a polypeptide corresponding to a chemokine domain of CX3CL1. (4) The cognitive function improving agent according to (1), wherein the polypeptide is a polypeptide comprising a chemokine domain of CX3CL1 and at least a portion of a mucin domain of CX3CL1. (5) The cognitive function improving agent according to any one of (1) to (4), wherein the polypeptide may form a complex with another substance. (6) The cognitive function improving agent according to any one of (1) to (5), wherein the CX3CL1 is human CX3CL1. (7) A pharmaceutical composition comprising the cognitive function improver according to any one of (1) to (6). (8) The pharmaceutical composition according to (7), which is formulated for administration by a method other than intrathecal administration. (9) The pharmaceutical composition according to (8), which is formulated for oral, intravenous, intraperitoneal or subcutaneous administration. (10) The pharmaceutical composition according to item (9), which is formulated into a form suitable for intraperitoneal administration. (11) The pharmaceutical composition according to any one of (7) to (10), in the form of a sustained release formulation. (12) A neurogenesis recovery agent comprising a polypeptide including the chemokine domain of CX3CL1. (13) The neurogenesis recovery agent according to (12), wherein the polypeptide is a polypeptide corresponding to a chemokine domain of CX3CL1. (14) The neurogenesis recovery agent according to (12), wherein the polypeptide is a polypeptide comprising a chemokine domain of CX3CL1 and at least a portion of a mucin domain of CX3CL1. (15) The neurogenesis recovering agent according to any one of (12) to (14), wherein the polypeptide may form a complex with another substance. (16) The neurogenesis recovering agent according to any one of (12) to (15), wherein the CX3CL1 is human CX3CL1. Effect of the Invention

[0021] The medicine containing CX3CL1 of the present invention as an active ingredient shows excellent effect of improving material cognition ability, and is useful as a medicine for improving cognitive function deteriorated by aging, for example. Moreover, the medicine is preferably for mammals, and more preferably for humans.

[0022] The pharmaceutical agent of the present invention improves cognitive function or restores neurogenesis in the hippocampus without being administered directly to the brain. Considering that there have been no reports that CX3CL1 from peripheral tissues enters the brain by passing through the blood-brain barrier, and furthermore, that CX3CL1 is unlikely to pass through the blood-brain barrier due to its nature, it is surprising that the pharmaceutical agent exerts its effect on the brain by oral, intravenous, intraperitoneal, subcutaneous administration, or the like, without being administered directly to the brain.

[0023] As described above, the present invention provides a practical cognitive function improving agent, and is of great social significance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention provides a pharmaceutical composition for improving cognitive function, which contains CX3CL1 as an active ingredient.

[0025] CX3CL1 It is important that the polypeptide containing the chemokine domain of CX3CL1, which is an active ingredient in the pharmaceutical composition of the present invention, substantially maintains the structure corresponding to the chemokine domain. However, as long as the structure corresponding to the chemokine domain is substantially maintained, it may contain the mucin domain of CX3CL1, or may form a complex with other polypeptides, proteins, polymers, etc., such as PEG, albumin, Fc, etc. For example, known strategies for extending the blood half-life of protein therapeutics include conjugation, binding, or fusion to FcRn-binding proteins or domains (Fc, albumin), as well as multimerization, chemical binding to polymers or carbohydrates (such as PEG, colominic acid, or hydroxyethyl starch), and incorporation of N-glycosylation sites.

[0026] In addition, a polypeptide containing the chemokine domain of CX3CL1 may be CX3CL1 expressed as a membrane-bound protein, and the extracellular region containing the chemokine domain and mucin domain may be cleaved by a protease to become free CX3CL1, or it may be synthesized by genetic engineering techniques or chemically.

[0027] As CX3CL1, for example, human CX3CL1 or mouse CX3CL1 is used.

[0028] Pharmaceutical Compositions When administering a polypeptide comprising the chemokine domain of CX3CL1 of the present invention, it is generally beneficial to prepare a pharmaceutical composition suitable for the intended application. The pharmaceutical composition may include a pharma- ceutical acceptable carrier, excipient or additive.

[0029] The present invention also provides a pharmaceutical composition comprising a cognitive function improving agent comprising a polypeptide comprising a chemokine domain of CX3CL1 as described herein, together with at least one pharma- ceutically acceptable carrier. Such a composition can be used to improve cognitive function in a patient (preferably a human or a pet animal) in need of cognitive function improvement. Such patients include patients with cognitive function decline due to aging. If desired, other active ingredients that contribute to the improvement of cognitive function can be included in the pharmaceutical composition. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with CX3CL1. A physiologically acceptable carrier can be a sterile liquid such as water or oil, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, or sesame oil). When the pharmaceutical composition is administered intravenously or intraperitoneally, water is a preferred carrier. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, and ethanol. The composition, if desired, can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents.

[0030] The pharmaceutical composition may be formulated for any suitable administration method, for example, oral administration, or parenteral administration, such as intravenous administration, intraperitoneal administration, or subcutaneous administration, for improving cognitive function. These compositions may take any of a variety of well-known forms, such as solutions, suspensions, tablets, pills, capsules, powders, aerosols, and sustained release formulations, that are compatible with the administration method. The composition may be formulated as a suppository with traditional binders and carriers, such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, and the like. For parenteral administration, the bispecific fusion protein may be suspended or dissolved in a carrier. Generally, sterile aqueous carriers, such as water, buffer, saline, or phosphate-buffered saline, are preferred. Sterile fixed oils may also be used as a solvent or suspending agent. For this purpose, any sterile fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, are also used in the preparation of injectable compositions. Medically acceptable auxiliary substances such as pH adjusting and buffering agents, isotonicity agents, dispersing agents, suspending agents, wetting agents, detergents, preservatives, local anesthetics and buffers may also be included to approximate physiological conditions.

[0031] In one preferred embodiment, the pharmaceutical composition is formulated for intraperitoneal administration to a patient (e.g., a human). Typically, the composition for intraperitoneal administration is a sterile, isotonic, aqueous buffer solution. Where necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the site of injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, e.g., a lyophilized powder or anhydrous concentrate in a sealed (e.g., hermetically sealed) container, such as an ampoule or sachet indicating the amount of active agent. Where the composition is to be administered by infusion, it is dispensed into an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is to be administered by injection, an ampoule of water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0032] Compositions intended for oral use may be present, for example, as tablets, troches, drops, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Such compositions may contain one or more ingredients such as sweeteners, flavorings, colorants and preservatives. Tablets contain the active ingredient mixed with physiologically acceptable excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents, granulating and disintegrating agents, binding agents and lubricating agents. Formulations for oral use may also be present as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium. Aqueous suspensions contain the active ingredient mixed with one or more excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents and dispersing or wetting agents. Dispersible powders and granules suitable for preparation of aqueous suspensions by the addition of water provide the active ingredient in a mixture with a dispersing or wetting agent, a suspending agent and one or more preservatives.

[0033] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil) or a mineral oil such as liquid paraffin. The pharmaceutical composition can also be an oil-in-water emulsion. The oil phase can be a vegetable oil, a mineral oil, or a mixture thereof. Suitable emulsifying agents include, for example, naturally occurring gums, naturally occurring phospholipids and anhydrides.

[0034] The pharmaceutical composition can be sterilized by conventional sterilization or can be sterile filtered. The sterile aqueous solution can be packaged for use as is or can be lyophilized as a lyophilized product that is combined with a sterile aqueous carrier before administration. The pH of the aqueous pharmaceutical composition will typically be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, most preferably between 7 and 8, such as between 7 and 7.5.

[0035] CX3CL1 provided herein, as a polypeptide comprising the chemokine domain of CX3CL1, is generally present in a pharmaceutical composition at a concentration such that a therapeutically effective amount is delivered in a single dose to a patient.

[0036] The therapeutically effective amount can be approximated from the amount that was observed to improve cognitive function in the animal model exemplified in Example 1. Nevertheless, it will be clear that various factors will affect the therapeutically effective amount, including the activity of the CX3CL1 used; body weight, general health, sex, and diet; time and route of administration; excretion rate; any concurrent treatments, such as combination drugs; and the severity of the patient being treated. The optimal dose can be established using routine tests and procedures well known in the art. In general, the dose ranges from about 0.5 mg to about 400 mg of CX3CL1 per dose (e.g., 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, or 400 mg per dose). In general, compositions that provide a dosage level ranging from about 0.1 mg to about 100 mg per kilogram of body weight per day are preferred. In one embodiment, the unit dose form contains between about 10 mg and 100 mg of CX3CL1.

[0037] Treatment method The pharmaceutical composition can be administered to a patient (preferably a human or a pet animal) to improve the patient's cognitive function, particularly to improve cognitive function that has declined due to aging. The pharmaceutical composition is administered once or multiple times. For example, it is administered 3 to 30 times at intervals of 2 to 6 days. The number of administrations can be reduced by using a sustained release formulation. EXAMPLES

[0038] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0039] Novel substance discovery test in CX3CL1-administered mice 1. Method In the following experiments, a synthetic peptide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) corresponding to the extracellular N-terminal chemokine domain was used as CX3CL1. CX3CL1 was dissolved in sterile saline at a concentration of 10 μg / ml (hereinafter referred to as "CX3CL1 solution"). Saline or CX3CL1 solution was injected intraperitoneally at 50 μg / kg five times at 3-day intervals. Six weeks after the last injection, the mice were subjected to the exploratory test described below.

[0040] Male C57BL / 6J mice (purchased from Charles River Japan) were used.

[0041] 2. Test methods for discovering novel substances The novel substance exploration test was performed with slight modifications to previously reported methods (references 1-3). The test consisted of three phases: "habituation," "training," and "testing." In the "habituation" phase, mice were allowed to freely explore an empty transparent box (60 cm wide x 30 cm long x 30 cm high) or a Y-maze (each arm 30 cm wide x 15 cm long x 6 cm high) made of transparent acrylic boards (5 min / day for 3 days). On the fourth day, two objects (known substances) were placed in the arena, and the mice explored freely for 10 min (training phase). After 6 h, one of the two objects was changed to another object (novel substance), and the mice explored again for 10 min (test phase). The time spent on each substance was recorded.

[0042] This behavioral analysis is based on the tendency of mice to spend more time exploring novel objects than familiar ones. Figure 1(A) shows a schematic diagram of the exploration test using a transparent box, and Figure 1(C) shows a schematic diagram of the exploration test using a Y-maze.

[0043] 3.Results As shown in the leftmost graph in Figure 1(B), young (4-month-old) mice spent more time exploring the novel substance than the familiar substance. This indicates that the young mice still had memory of the exploration even after 6 hours. In contrast, the time spent exploring the novel substance was the same as the time spent exploring the familiar substance in the 15-16-month-old mice that had not been injected (second graph from the left) and the 15-16-month-old mice that had been administered saline (third graph from the left). This indicates that the aged mice had no memory of the exploration after 6 hours. However, the 15-16-month-old mice that had been administered CX3CL1 had significantly increased time exploring the novel substance compared to the 15-16-month-old mice that had been administered saline and the 15-16-month-old mice that had not been injected. The proportion of time spent exploring the novel substance was equivalent to that of the 4-month-old mice, and no age-dependent decline in recognition memory was observed in the aged mice that had been administered CX3CL1.

[0044] These results indicate that administration of CX3CL1 improved material cognitive function that had deteriorated due to physiological aging. EXAMPLES

[0045] Effect of CX3CL1 on neurogenesis in the adult hippocampus 15-16 month old mice were intraperitoneally administered CX3CLl (50μg / kg) or saline six times with 3-day intervals. Brain sections were prepared 7 weeks after the last administration. Sox2 positive, Ngn2 negative, and Ngn2 positive cells in the hippocampus were immunofluorescently stained and then quantified.

[0046] Sox (SRY-box2) is a transcription factor that is highly expressed in type 1 NSCs in the adult hippocampus (Ref. 4), and Ngn2 (Neurogenin 2) is a basic helix-loop-helix transcription factor that is highly expressed in type 2 NSCs in the adult hippocampus (Refs. 5, 6).

[0047] The results are shown in Figure 2. As shown in Figure 2(B), the number of Ngn2-positive cells increased by CX3CL1 administration, but no significant change in the number of Sox2-positive Ngn2-negative cells was observed. This shows that intraperitoneal injection of CX3CL1 increased type 2 NSCs in the hippocampus, while leaving type 1 NSCs unchanged. This indicates that neural production was activated, and is consistent with the significant improvement in material cognitive function in aged mice administered CX3CL1, as shown in Example 1.

[0048] Next, because oxidative stress is thought to be a major cause of aging (Reference 7), we examined the effect of CX3CL1 administration on oxidative stress.

[0049] Reactive oxygen species induce DNA double-strand breaks (DSBs), one of the most severe forms of DNA damage, but cells containing DSBs are eliminated from tissues of young mice, whereas they accumulate in tissues of old mice (References 8, 9). Therefore, we quantified γ-H2AX (phosphorylated H2AX), a sensitive marker of DNA damage that has recently been used as an indicator of cell senescence.

[0050] As shown in FIG. 2C, the results showed that aged mice that received intraperitoneal injection tended to have a decreased number of γ-H2AX-positive cells in the hippocampus. EXAMPLES

[0051] Effect of CX3CL1 on the phenotype of peritoneal macrophages CX3CLl (50 μg / kg) or saline was administered intraperitoneally to 15-month-old mice six times at 3-day intervals. Peritoneal cells were prepared the day after the last administration. Peritoneal cells were prepared from 2-month-old and 15-month-old mice as controls.

[0052] With age, the cyclin-dependent kinase inhibitor p16 INK4a Macrophages expressing β-lactam IgG accumulate in the peritoneal cavity (Reference 10), and phagocytosis of peritoneal cells is reduced (Reference 11).

[0053] p16 in peritoneal cells Ink4a p16+ macrophages accumulate with age, and intraperitoneal injection of CX3CL1 suppressed p16+ macrophages in the peritoneal cavity of aged mice. Ink4a , and reduced the proportion of macrophages expressing IL-1 (Figure 3(A)).

[0054] FIG. 3(B) shows the number of FITC-positive cells after intraperitoneal injection of latex beads-rabbit Ig-FITC, demonstrating that administration of CX3CL1 increases the in vivo phagocytic activity of peritoneal cells in old mice.

[0055] These results suggest that CX3CL1 mediates two age-related phenotypes of macrophages, namely, phagocytic activity and p16 Ink4a It has been shown to induce changes in the expression of

[0056] It is known that removing senescent cells restores various functions of aged organs (Non-Patent Documents 11, 12), so administration of CX3CL1 is expected to improve various functional declines caused by aging. EXAMPLES

[0057] Transplantation of peritoneal cells As in Example 3, mouse peritoneal cells were prepared on the day following the sixth injection of CX3CL1 or saline and washed once with PBS. Peritoneal cells from control mice, mice treated with saline, and mice treated with CX3CL1 were transplanted into 56-week-old male C57BL / 6 mice (2.5 × 10 6 Six weeks later, the mice were subjected to the novel substance exploration test described in Example 1. The results are shown in FIG. 4. The mice transplanted with peritoneal cells derived from the mice treated with CX3CL1 showed a significant increase in the exploration time for the novel substance, and as in Example 1, improvement in cognitive function was confirmed.

[0058] Considering these results together with the experimental results of Example 3, it is speculated that the improvement in cognitive function by administration of CX3CL1 is not due to CX3CL1 migrating into the brain, but rather that administration of CX3CL1 reduces aged macrophages in the peritoneal cavity, thereby improving the decline in immunity caused by aging, and ultimately resulting in improvement in cognitive function.

[0059] 1. Akkerman, S. et al. Object recognition testing: methodological considerations on exploration and discrimination measures. Behav Brain Res 232, 335-347 (2012). 2. Antunes, M. & Biala, G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cogn Process 13, 93-110 (2012). 3. Leger, M. et al. Object recognition test in mice. Nat Protoc 8, 2531-2537 (2013). 4. Brazel, CY et al. Sox2 expression defines a heterogeneous population of neurosphere-forming cells in the adult murine brain. Aging Cell 4, 197-207 (2005). 5. Yu, DX, Marchetto, MC & Gage, FH How to make a hippocampal dentate gyrus granule neuron. Development 141, 2366-2375 (2014). 6. Uda, Y., Xu, S., Matsumura, T. & Takei, Y. P2Y4 Nucleotide Receptor in Neuronal Precursors Induces Glutamatergic Subtype Markers in Their Descendant Neurons. Stem Cell Reports 6, 474-482 (2016). 7. Gorbunova, V. & Seluanov, A. Making ends meet in old age: DSB repair and aging. Mech Ageing Dev 126, 621-628 (2005). 8. Mah, L. J., El-Osta, A. & Karagiannis, T. C. GammaH2AX as a molecular marker of aging and disease. Epigenetics 5, 129-136 (2010). 9. Maynard, S., Fang, E. F., Scheibye-Knudsen, M., Croteau, D. L. & Bohr, V. A. DNA Damage, DNA Repair, Aging, and Neurodegeneration. Cold Spring Harb Perspect Med 5, (2015). 10. Hall, B. M. et al. Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells. Aging (Albany NY) 8, 1294-1315 (2016). 11. Linehan, E. et al. Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis. Aging Cell 13, 699-708 (2014). [Brief description of the drawings]

[0060] [Figure 1] Figure 1 shows a schematic diagram of a novel substance discovery test and the test results. Figure 1(A) is a schematic diagram of a novel substance discovery test in which a transparent box was used. Figure 1(B) shows the results of the novel substance discovery test in which a transparent box was used. Figure 1(C) is a schematic diagram of a novel substance discovery test in which a Y-maze was used. Figure 1(D) shows the results of the novel substance discovery test in which a Y-maze was used. [Diagram 2] Figure 2 shows the effect of CX3CL1 on neurogenesis in the adult hippocampus. Figure 2(A) shows the number of Sox2+Ngn2-cells in the hippocampus of 15-16 month old mice that were intraperitoneally administered CX3CL1 at 50 μg / kg six times with 3-day intervals, Figure 2(B) shows the number of Ngn2-positive cells, and Figure 2(C) shows the number of γ-H2AX-positive cells. [Diagram 3] Figure 3 shows the effect of CX3CL1 on the phenotype of peritoneal macrophages, Figure 3(A) shows the percentage of p16Ink4a positive cells in peritoneal macrophages, and Figure 3(B) shows the percentage of FITC positive cells. [Figure 4] FIG. 4 shows the results of a novel substance discovery test in mice transplanted with peritoneal cells derived from mice treated with CX3CL1 cells. [Industrial Applicability]

[0061] According to the present invention, a novel pharmaceutical composition for improving cognitive function is provided. The pharmaceutical composition of the present invention has a great practical advantage in that it does not need to be administered directly to the brain.

Claims

1. A pharmaceutical composition for improving cognitive function, comprising a polypeptide including a chemokine domain of CX3CL1, the pharmaceutical composition being formulated for oral, intravenous, intraperitoneal or subcutaneous administration.

2. The pharmaceutical composition according to claim 1 , wherein the improvement of cognitive function is improvement of cognitive function that has declined due to aging.

3. A pharmaceutical composition for restoring neurogenesis, comprising a polypeptide including the chemokine domain of CX3CL1, the pharmaceutical composition being formulated for oral, intravenous, intraperitoneal or subcutaneous administration.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the polypeptide is a polypeptide corresponding to the chemokine domain of CX3CL1.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the polypeptide is a polypeptide comprising a chemokine domain of CX3CL1 and at least a portion of a mucin domain of CX3CL1.

6. The pharmaceutical composition according to claim 1 , wherein the polypeptide is optionally complexed with another substance.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the CX3CL1 is human CX3CL1.

8. A pharmaceutical composition according to any one of claims 1 to 7, formulated in a form suitable for intraperitoneal administration.

9. 9. A pharmaceutical composition according to any one of claims 1 to 8 in the form of a sustained release formulation.

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