HIP / PAP proteins or their derivatives for treating cognitive impairment associated with anxiety disorders

Abstract

The present invention relates to the use of HIP / PAP proteins or derivatives thereof in the treatment and prevention, particularly in the treatment of anxiety-related cognitive impairment in individuals requiring treatment, and in improving cognition in individuals suffering from anxiety-related neurological disorders, or in reducing cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection, and in the prevention and / or treatment of diet-induced cognitive impairment and anxiety-related dysfunction, particularly high-fat diet-induced cognitive impairment and anxiety-related dysfunction, in individuals requiring prevention and / or treatment.

Description

Technical Field

[0001] The present invention relates to the treatment and prevention, particularly the treatment and prevention of cognitive impairment associated with anxiety disorders in individuals requiring treatment, and particularly in treatment, and for improving cognition in individuals suffering from a neurological disorder associated with anxiety disorders, or for reducing cognitive dysfunction in individuals suffering from a specific cognitive impairment associated with anxiety disorders as described below, to the use of HIP / PAP protein or a derivative thereof.

Background Art

[0002] While occasional anxiety is normal and a part of life, anxiety disorders go beyond temporary worry or fear. For people with anxiety disorders, anxiety doesn't go away, worsens over time, and can be accompanied by symptoms that interfere with daily activities such as work performance, schoolwork, or relationships. These symptoms need to be distinguished from ordinary anxiety and fear, which are healthy emotional responses to daily stressors related to interpersonal, social, educational, and occupational requirements. Anxiety disorders, particularly in young adults, are characterized by increased anxiety levels (having a sense of nervousness, restlessness, or tension, imminent danger, panic, or impending doom, difficulty controlling worry, difficulty with risk or decision-making), decreased anxiety levels (high approach, lack of inhibition, inappropriate laughter, disinhibition of speech and behavior in novel situations, high novelty-seeking tendencies and low harm-avoidance tendencies, high novelty-stimuli personality, lack of impulse control, and a tendency toward dangerous behaviors) (NR. Marmorstein, J Anxiety Disord., 2007; 21(3):420~32; J.T. Nigg, Psychol Bull., March 2000; 126(2):220~46; Dina R. Hirshfeld-Becker et al., Biol Psychiatry., June 1, 2003; 53(11):985~99), or a combination of both (NR. Marmorstein, J It can manifest as Anxiety Disord., 2007;21(3):420~32;Audra K Langley et al., Eur Child Adolesc Psychiatry., August 2010;19(8):637~45;B Wanner et al., Psychol Med., November 2012;42(11):2373~82;Joan P Yoo et al., Am J Orthopsychiatry. October 2009;79(4):532~40). In certain cases, disinhibition of behavior may be a precursor to anxiety disorder, indicating a complex relationship between both types of disorders (Dina R Hirshfeld-Becker et al., Biol Psychiatry. June 1, 2003;53(11):985~99).

[0003] Mood disorders and anxiety disorders are characterized by a variety of neuroendocrine, neurotransmitter, and neuroanatomical imbalances. Identifying the most functionally relevant differences is complex due to the high degree of interconnectivity between neurotransmitter-containing and neuropeptide-containing circuits in the limbic system, brainstem, and higher cortical regions. Therefore, both high and low anxiety levels may share the same mechanism, namely, an imbalance in neurotransmitter signaling (Elizabeth I Martin et al., Psychiatr Clin North Am. 2009 September;32(3):549~75).

[0004] In 2015, it was estimated that more than 58 million people in the United States suffered from anxiety disorders, representing 7.7% of the female population and 3.6% of the male population. In fact, anxiety disorders are more prevalent than any other mental health disorder and account for the majority of lifelong mental health disorders worldwide (Kessler et al., Epidemiol Psichia Soc. 2009 January-March; 18(1):23-33).

[0005] Anxiety disorders (including externalization disorders) are often associated with cognitive impairment, either as a primary or secondary symptom.

[0006] An example of cognitive impairment with anxiety disorder as a primary symptom is obsessive-compulsive disorder, which is characterized by patterns of repetitive behaviors or mental activities, or compulsions, that arise from unwanted thoughts or fears, i.e., obsessions, and are intended to alleviate the anxiety associated with the obsessions (Anu E Castaneda et al., J Affect Disord., February 2008; 106(1-2):1~27; Ashwini Vishwanathan, Indian J Psychol Med., November 2022; 44(6):558~566). Cognitive impairment (impaired learning and working memory) has also been demonstrated in externalization disorders of anxiety and disinhibition disorders of behavior, as seen in bipolar disorder, as well as in substance and alcohol abuse (Michael J Endres et al., J Abnorm Psychol., May 2011; 120(2):336~51).

[0007] Anxiety symptoms may occur as a result of or in connection with other disorders, particularly certain cognitive impairments. In these cognitive impairments, especially when associated with mild to severe memory loss, changes in cognitive function can lead to altered normal responses to anxiety-inducing situations, resulting in anxiety disorder.

[0008] Not all cognitive impairments are associated with anxiety disorders. For example, some levels of mild cognitive decline, normal pressure hydrocephalus, or neurological disorders associated with cognitive impairment may be linked to anxiety.

[0009] Depending on the cognitive impairment considered, or the stage of the cognitive impairment considered, changes in normal responses to anxiety-provoking situations may manifest as either an increase or decrease in anxiety-like behaviors.

[0010] Both systems share common mechanisms, including imbalances in neurotransmission in areas that control mood and anxiety.

[0011] Therefore, there is a growing need for novel active substances that can prevent and / or treat cognitive impairments associated with anxiety disorders in individuals who require prevention and / or treatment.

[0012] Furthermore, there is a growing need to prevent changes in an individual's emotional response to stressors, particularly in individuals suffering from cognitive impairments that are expected to trigger the onset of anxiety disorders.

[0013] In fact, there is a need for novel active substances that can restore healthy emotional responses to stressors, particularly in individuals suffering from cognitive impairments associated with anxiety disorders.

[0014] There is also a need for novel active substances that can restore anxiety at a physiological level in individuals, particularly those suffering from cognitive impairments associated with anxiety disorders.

[0015] There is also a need for novel active substances that can improve the memory of individuals suffering from cognitive impairments associated with anxiety disorders.

[0016] Therefore, there is a growing need for novel active substances that can improve cognition in individuals suffering from anxiety-related neurological disorders, selected from a group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and cognitive impairment associated with other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0017] Novel active substances are needed that can alleviate cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease (PD), bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

[0018] This invention provides solutions to these problems. [Prior art documents] [Patent Documents]

[0019]

Patent Document 1

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Non-licensed literature

[0020] [Non-licensed document 1] NR Marmorstein, J Anxiety Disord., 2007;21(3):420~32 [Non-licensed document 2] JT Nigg, Psychol Bull, March 2000; 126(2):220-46 [Non-licensed document 3] Dina R Hirshfeld-Beckerら, Biol Psychiatry., June 1, 2003; 53(11):985~99

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[0021] The applicant has surprisingly demonstrated that HIP / PAP proteins or their derivatives can improve memory in a mouse model of anxiety-related cognitive impairment. HIP / PAP proteins or their derivatives can also restore cognitive impairment, and more specifically, can restore normal anxiety-like behavior in response to anxiety-inducing situations in this model as well as in a high-fat diet (HFD) model.

[0022] While we do not wish to be bound by any theory, we hypothesize that HIP / PAP can restore neurotransmitter homeostasis through direct or indirect effects (on GABAergic and glutamatergic systems). Indeed, the results of the accompanying examples show the restoration of physiological levels of anxiety (to control levels) in mice treated with HIP / PAP.

[0023] Furthermore, the inventors have surprisingly demonstrated both the ability to prevent cognitive changes and the ability to improve cognition in individuals who exhibit cognitive changes after administration of HIP / PAP protein or its derivatives.

[0024] Therefore, the HIP / PAP proteins are as follows: - Treatment and / or prevention, especially treatment, of cognitive impairments associated with anxiety disorders in individuals requiring treatment; In particular, treatment and / or prevention, especially treatment, of cognitive impairments associated with externalization disorders, wherein the cognitive impairment is selected in particular from the group consisting of bipolar disorder, substance abuse, attention deficit disorder and psychotic disorder; - To improve cognition in individuals suffering from neurological disorders associated with anxiety disorders, more specifically, neurological disorders selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and cognitive impairment associated with other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis; and - It can be advantageously used to mitigate cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease (PD), bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

[0025] Therefore, the present invention relates to the following items.

[0026] Item 1: Treatment and / or prevention, in particular, of cognitive impairment associated with anxiety disorder in individuals requiring treatment, in particular, of HIP / PAP proteins or derivatives thereof for use in treatment.

[0027] Item 2: Anxiety disorders associated with cognitive impairment, (i) Major symptoms of obsessive-compulsive disorder, and / or (ii) Secondary symptoms of neurodegenerative diseases selected from the group consisting of attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection. The HIP / PAP protein or its derivative for use as described in item 1.

[0028] Item 3: HIP / PAP proteins or derivatives thereof for use in improving cognition in individuals with anxiety-related neurological disorders selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and cognitive impairment associated with other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0029] Item 4: HIP / PAP proteins or derivatives thereof for use in reducing cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

[0030] Item 5: Anxiety disorder is an externalization disorder, and in particular, cognitive impairment is selected from the group consisting of bipolar disorder, substance abuse, attention deficit disorder and psychotic disorder, and is a HIP / PAP protein or derivative thereof for use as described in any one of items 1 to 4.

[0031] Item 6: HIP / PAP proteins or derivatives thereof for use in the prevention and / or treatment of diet-induced cognitive impairment and anxiety-related dysfunction, particularly high-fat diet-induced cognitive impairment and anxiety-related dysfunction, in individuals requiring treatment and / or prevention.

[0032] Item 7: HIP / PAP protein or derivative thereof for use as described in any one of items 1 through 6, wherein the individual is a mammal, in particular a human.

[0033] Item 8: A HIP / PAP protein or derivative thereof for use according to any one of items 1 to 7, comprising an amino acid sequence selected from the group consisting of sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and in particular the sequence shown as SEQ ID NO: 4.

[0034] Item 9: A HIP / PAP protein or derivative thereof for use as described in any one of Items 1 to 8, wherein the derivative comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and having the same properties of biological activity as the amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, more specifically, an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown as SEQ ID NO: 4, and having the same properties of biological activity as the amino acid sequence shown as SEQ ID NO: 4.

[0035] Item 10: A HIP / PAP protein or derivative thereof for use as described in any one of items 1 to 9, wherein the HIP / PAP protein or derivative thereof is contained in a composition comprising a physiologically acceptable medium.

[0036] Item 11: HIP / PAP protein or derivative thereof for use as described in Item 10, wherein the composition is for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, tracheal, nasal, or rectal administration.

[0037] Item 12: A composition for use as described in Item 10 or 11, which is for oral, subcutaneous, intravenous, topical, or local administration.

[0038] Item 13: A composition further comprising at least one agent known to be useful for the prevention and / or treatment of cognitive impairment, for example, a cholinesterase inhibitor such as donepezil, rivastigmine or galantamine; a glutamate modulator such as memantine; a cholinesterase inhibitor in combination with a glutamate modulator, particularly a combination of donepezil and memantine; methylphenidate; amphetamine; atomoxetine; AMPA-R agonists; α7 nicotinic agonists; guanfacine; bupropion; vortioxetine and D-cycloserine, for use according to any one of items 10 to 12.

[0039] Item 14: A composition comprising at least one agent known to be useful for the prevention and / or treatment of anxiety disorders, in particular an agent selected from the group consisting of selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium modulators, azapirone, reversible inhibitors of monoamine oxidase A, agomelatine, quetiapine and vortioxetine, more specifically an agent selected from the group consisting of citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine and vortioxetine, for use as described in any one of items 10 to 13.

[0040] Item 15: HIP / PAP protein or derivative thereof for use as described in Item 14, wherein the cognitive impairment associated with anxiety disorder is selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

[0041] Item 16: A composition for use according to any one of items 10 to 15, comprising at least one agent known to be useful in alleviating symptoms associated with disorders selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, further comprising an agent selected from the group consisting of antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids such as prednisone; and immunosuppressants such as methotrexate or azathioprine. [Brief explanation of the drawing]

[0042] [Figure 1] Figure 1A shows the spontaneous motility activity of various mouse groups, particularly the distance traveled in open fields: horizontal axis from left to right: normal control mice (Ntg-n=10); APP / PS1 mice treated with rAAV9-empty capsid (Tg veh-control group-n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP-n=8). vertical axis: total distance traveled (m). Figure 1B shows the resting time in open fields for various mouse groups: horizontal axis from left to right: normal control mice (Ntg-n=10); APP / PS1 mice treated with rAAV9-empty capsid (Tg veh-control group-n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP-n=8). vertical axis: total resting time (seconds). [Figure 2]Figure 2 shows the results of a novel object recognition test conducted on various groups of mice: From left to right on the horizontal axis: normal control mice (Ntg-n=10); APP / PS1 mice treated with rAAV9-empty capsid (Tg veh-control group-n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP-n=8). Each group shows results in the presence of either a familiar object (left) or a novel object (right). Vertical axis: Contact time with the object (seconds). Two-way ANOVA was performed, followed by Tukey's multiple comparison post-hoc test with a significance level of p<0.05. Data were analyzed using GraphPad Prism version 9 (GraphPad Software, San Diego, California, USA, www.GraphPad.com). [Figure 3] Figures 3A and 3B show the results of radial arm water maze experiments conducted on various groups of mice: normal control mice (Ntg-n=10); APP / PS1 mice treated with rAAV9-empty capsid (Tg veh-control group-n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP-n=8). Figure 3A shows the average number of errors per block of 3 consecutive trials for each group of mice at the end of the experiment, for a total of 15 trials on day 1 (blocks 1-5) and 15 trials on day 2 (blocks 6-10), from left to right: Ntg; Tg Veh; Tg HIP / PAP. Vertical axis: number of errors. Figure 3B shows the number of errors for each group (Ntg [ka] ;Tg Veh [ka] ;Tg HIP / PAP [ka] This represents the total number of errors committed by the mouse on day 2 (blocks 5-10). Vertical axis: Number of errors For the error blocks over two days (Figure 3A), a two-way ANOVA with repeated measures was performed, followed by Tukey's multiple comparison post-hoc test with a significance level of p<0.05. For the total number of errors on day 2 (Figure 3B), a one-way ANOVA was performed, followed by Tukey's multiple comparison post-hoc test with a significance level of p<0.05. Data were analyzed using GraphPad Prism version 9 (GraphPad Software, San Diego, California, USA, www.GraphPad.com). [Figure 4] Figure 4 shows the results of inversion trials of the radial arm water maze experiment performed on various groups of mice: normal control mice (Ntg-n=10); APP / PS1 mice treated with rAAV9-empty capsid (Tg veh-control group-n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP-n=8). Figure 4A shows each group (Ntg [ka] ;Tg Veh [ka] ;Tg HIP / PAP [ka] The number of errors committed by each mouse group is shown as the average value at the end of each day, with three consecutive trials forming one block, for a total of 15 trials. Horizontal axis: Blocks 1-5 (left to right). Figure 4B shows the total number of errors committed by each group of mice in all trials (from left to right: Ntg; Tg Veh; Tg HIP / PAP). For the error blocks (Figure 4A), a two-way ANOVA with repeated measures was performed, followed by Tukey's multiple comparison post-hoc test with a significance level of p<0.05. For the total number of errors (Figure 4B), a one-way ANOVA was performed, followed by Tukey's multiple comparison post-hoc test with a significance level of p<0.05. The data were analyzed using GraphPad Prism version 9 (GraphPad Software, San Diego, California, USA, www.GraphPad.com). [Figure 5] Figure 5 shows the anxiety-like behavior of each mouse group detailed above, evaluated by measuring the duration of activity in the open arm versus closed arm of an elevated cross maze (EPM) (Figure 5A), and by comparing the duration of activity in the closed arm only between different groups (Figure 5B). From left to right on the horizontal axis: normal control mice (Ntg-n=10); APP / PS1 mice treated with rAAV9-empty capsid (Tg Veh-control group-n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP-n=8). Each group shows results for either the open arm (left) or the closed arm (right). Vertical axis: number of entries. [Figure 6] Figure 6A shows a recombinant adeno-associated virus serotype 9 (rAAV9) construct for overexpression of human HIP / PAP tagged with hemagglutinin (HA). Figure 6B shows the results of anti-HA immunohistochemistry in the hippocampus of APP / PS1 mice injected with rAAV9-empty capsid 6 months after expression. Figure 6C shows the results of anti-HA immunohistochemistry in the hippocampus of APP / PS1 mice injected with rAAV9-HIP / PAP capsid 6 months after expression. Figure 6D shows the levels of human HIP / PAP in the cortex of APP / PS1 mice injected with rAAV9-HIP / PAP, measured by ELISA 6 months after injection. HA is human influenza hemagglutinin. Scale: 100 μm, insert scale: 30 μm. Horizontal axis: cortex; Vertical axis: HIP / PAP concentration (pg / mg). [Figure 7]Figures 7A and 7B show micrographs of Congo red stained mice treated with rAAV9-empty capsid (Figure 7A) or rAAV9-HIP / PAP (Figure 7B) for 6 months in the APP / PS1 mouse group. Scale: 100 μm. Figure 7C shows the quantification of the percentage of Congo red stained area in the anterior cortex (ACX), hippocampus (HPC), and posterior cortex (PCX) for the two aforementioned mouse groups, using nearcyte software analysis. The APP / PS1 mouse group was treated with rAAV9-empty capsid or rAAV9-HIP / PAP for 6 months. Vertical axis: Positive staining area (percentage). Horizontal axis: From left to right: ACX, HPC, and PCX. For each group, from left to right, are the mouse group treated with rAAV9-empty capsid (APP / PS1 empty) and the mouse group treated with rAAV9-HIP / PAP (APP / PS1 HIP / PAP). Data are shown as mean ± SEM. Figure 7D shows the quantification of amyloid beta levels in ACX and HPC quantified by ELISA for the two mouse groups described above. The APP / PS1 mouse group was treated with either rAAV9-empty capsid or rAAV9-HIP / PAP for 6 months. Vertical axis: concentration (ng / mL). Horizontal axis: from left to right: ACX and HPC. For each group, from left to right, are the mouse group treated with rAAV9-empty capsid (APP / PS1 empty) and the mouse group treated with rAAV9-HIP / PAP (APP / PS1 HIP / PAP). Data are shown as mean ± SEM. [Figure 8]Figure 8 shows the analysis of Western blot analysis using blot micrographs (Figure 8A) and band densitometry quantification of catalase (Figure 8B), heme oxygenase 1 (Ho-1) (Figure 8C), and superoxide dismutase (SOD-1) (Figure 8D) in the hippocampus of APP / PS1 mice treated with rAAV9-empty capsid (APPPS1 empty) or rAAV9-HIP / PAP (APPPS1 HIP / PAP) and non-transgenic controls (Ntg) in Figures 8B and 8D. N=6-10. Data are shown as mean ± SEM. *p<0.05, **p<0.01. One-way ANOVA was used, followed by Tukey's multiple comparison post-hoc test. Outliers were removed according to Grubbs' test with prisms. Vertical axis of 8B-8D: Ratio to total protein. Horizontal axis of 8B-8D: Ntg, APPPS1 empty, and APPPS1 HIP / PAP. [Figure 9] Figure 9A shows the concentrations of HIP / PAP in plasma and brain samples after subcutaneous administration of rHIP / PAP (or ALF5755) to mice (n=7) via an Alzet pump for 28 days. Left vertical axis: concentration (mg / mL) Right vertical axis: concentration (pg / mL) Figure 9B shows IVIS (in vivo imaging system) imaging of non-transgenic mice 30 minutes after intravenous injection of 250 μg of bovine serum albumin (BSA) (top) or rHIP / PAP (bottom) labeled with vivotag 680 XL (Revvity, Hopkinton, Massachusetts, USA). Figures 9C and 9D show the concentrations of HIP / PAP in plasma samples (Figure 9C) and brain samples (Figure 9D) one month after the above mice were injected with 2 × 10¹³ vg of rAAV9-HIP / PAP intravenously (heart, Figure 9C, n=3) or intramuscularly (hind limb, Figure 9D, n=2). [Figure 10]Figure 10A shows the weight course of non-transgenic mice over 10 weeks on a control diet (Ntg CD, n=6), a high-fat diet (Ntg HFD, n=5), or a high-fat diet treated with rAAV9-HIP / PAP (Ntg HFD HIP / PAP, n=4). Vertical axis: Weight (% basal) Horizontal axis: Time (0-10 weeks) Figure 10B shows the time spent in open arm "o" versus closed arm "c" during the elevated cross maze (EPM) test. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. One-way, two-way, or repeated measures ANOVA were used as appropriate, followed by Tukey's multiple comparison post-hoc test. Vertical axis: Time spent in the arm (s). Horizontal coordinates: From left to right: Non-transgenic mice on a control diet (Ntg CD), non-transgenic mice on HFD (Ntg HF), and non-transgenic mice treated with rAAV9-HIP / PAP on HFD (Ntg HFD HIP / PAP). For each group, the left portion represents the results in the closed arm, and the right portion represents the results in the open arm. [Modes for carrying out the invention]

[0043] definition In the context of the present invention, the terms “prevent,” “prevent,” and “prevent” mean reducing the risk or probability of a given phenomenon occurring, i.e., in the present invention, the prevention of anxiety-related cognitive impairment, particularly complications selected from the group consisting of diabetic foot ulcers, foot infections, and amputations. The terms “prevent,” “prevent,” and “prevent” also include preventing the worsening of the given phenomenon, slowing its progression, or preventing its recurrence.

[0044] As used herein, the terms “to treat,” “to treat,” or “to heal” mean the reduction and / or elimination of symptoms associated with a particular disorder or condition, i.e., in the present invention, the treatment of cognitive impairments associated with anxiety.

[0045] "Cognitive impairment" is any disorder that significantly impairs an individual's cognitive function to the extent that normal social functioning is impossible without treatment. It involves problems with a person's ability to think, learn, remember, judge, and make decisions. The signs of cognitive impairment vary depending on the specific disorder, but some common signs and symptoms overlap across most disorders. Some of the most common signs of cognitive impairment include confusion, impaired motor coordination, confused self-awareness, impaired judgment, memory loss (loss of short-term or long-term memory), difficulty concentrating, difficulty completing tasks, difficulty understanding, difficulty remembering, difficulty following instructions, and difficulty solving problems. Other common signs may include mood and behavioral changes, decreased motivation, and unawareness of one's surroundings. Cognitive impairment can be mild or severe. Some cognitive impairments develop gradually, and symptoms become more severe as the disorder progresses. Cognitive instability can have both short-term and long-term effects. Some common short-term effects include impaired thinking and reasoning abilities, memory loss, confused states, and lack of coordination. Long-term effects include progressive loss of declarative memory, such as forgetting names or faces of important people, as well as a general lack of emotional stability and difficulty controlling behavior, and dementia (alterations in mental state and level of consciousness, attention shifts, mood swings, violent or unusual behavior, and hallucinations). According to the present invention, the cognitive impairment of interest is cognitive impairment associated with anxiety disorder. "Cognitive impairment associated with anxiety disorder" means that the individual in question suffers from both at least one cognitive impairment and at least one anxiety disorder, the anxiety disorder being the primary or secondary symptom of the cognitive impairment. The term "primary symptom" is used to describe a condition not caused by another medical condition. The term "secondary symptom" means that it is a result of another condition. Therefore, according to the present invention, the term "cognitive impairment associated with anxiety disorder" includes both cognitive impairment that is a result of anxiety disorder and cognitive impairment that led to the onset of anxiety disorder.

[0046] The diagnosis of cognitive impairment can be made using different methods, including the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), MiniCog, and the Cognitive Assessment Method (CAM), Glasgow Coma Scale (GCS), and Richmond Agitation-Sedation Scale (RASS) (Kelvin KF Tsoi et al., JAMA Intern Med. 2015 September;175(9):1450~8).

[0047] Anxiety disorders are well defined above. While feeling anxious from time to time is a common experience, anxiety disorders differ from typical or transient feelings of worry or fear. Anxiety disorders can take various forms and may include increased anxiety levels that can cause nervousness, restlessness, and a sense of imminent danger, as well as difficulty controlling worry and making decisions. Alternatively, anxiety disorders may manifest as decreased anxiety levels, which may result in disinhibition of behavior and externalization disorders. These disorders are characterized by a tendency to exhibit high approachability, lack of inhibition, inappropriate laughter, and disinhibition of speech and behavior in new situations. They may also include a high tendency to seek novelty, a low tendency to avoid harm, lack of impulse control, and a tendency toward risky behaviors. In some cases, disinhibition of behavior may even be a precursor to anxiety disorders. Both mood disorders and anxiety disorders are complex conditions involving disturbances in the neuroendocrine, neurotransmitter, and neuroanatomical systems. Identifying the differences between these disorders can be challenging due to the complex interactions between different neural circuits in the brain. Nevertheless, both high and low levels of anxiety may stem from the same underlying cause: an imbalance in neurotransmitter signaling.

[0048] Anxiety disorders can be diagnosed using questionnaires such as the State-Trait Anxiety Inventory (STAI), the Generalized Anxiety Disorder 7 (GAD-7), the Beck Depression Inventory (BAI), the Zung Self-Rating Anxiety Scale, and the Taylor Manifest Anxiety Scale (Matthias Rose and Janine Devine, Dialogues Clin Neurosci. June 2014; 16(2): 197-211). The diagnosis of anxiety disorders is based on symptoms, triggers, and the individual's and family's medical history. There are no objective biomarkers or clinical trials that can diagnose anxiety.

[0049] The term "substance abuse" refers to the excessive and persistent use of a substance, such as alcohol or drugs, despite its potential to have adverse effects on a person's physical, psychological, or social health. Substance abuse can lead to addiction, a chronic and relapsing condition characterized by compulsive drug-seeking behavior and use despite harmful consequences. Diagnosis of substance abuse is usually based on criteria such as tolerance (the need to increase the amount of the substance to achieve the desired effect), withdrawal symptoms, continued use despite adverse effects, and unsuccessful attempts to stop or discontinue use. It should be noted that the definition of substance abuse can vary depending on the substance in question and the context in which it is used.

[0050] Attention-deficit disorder (ADD), including attention-deficit hyperactivity disorder (ADHD), is a medical condition characterized by symptoms of inattention, hyperactivity, and impulsivity. Symptoms of inattention may include lack of attention, forgetfulness, lack of planning ability, and distractibility. Symptoms of hyperactivity may include restlessness, fidgeting, inability to sit still, and excessive talking. Symptoms of impulsivity may include acting without thinking, interrupting others, and difficulty waiting one's turn. Diagnosis of ADD / ADHD is usually based on the combination, duration, and impairment of symptoms, as well as the exclusion of other possible medical or psychological conditions that may cause similar symptoms.

[0051] Psychotic disorders are a group of mental illnesses characterized by a loss of contact with reality, and may include symptoms such as delusions, hallucinations, confusion of thought, and abnormal behavior. Delusions are fixed beliefs not based on reality, and are often bizarre or paranoid in nature. Hallucinations are sensory experiences not based on external stimuli, such as hearing voices or seeing things that are not there. Confusion of thought may include difficulty with logical thinking, illogical speech, and leaps between unrelated topics. Abnormal behavior may include strange habits or actions, lack of emotional expression, and social withdrawal.

[0052] Psychotic disorders can have a significant impact on an individual's functioning in daily life and can range in severity from mild to severe. The most common psychotic disorders are schizophrenia, schizoaffective disorder, and delusional disorder.

[0053] Cholinesterase inhibitors are chemicals that prevent the breakdown of the neurotransmitter acetylcholine. Examples of cholinesterase inhibitors include donepezil, rivastigmine, and galantamine. Glutamate modulators are chemicals that modulate the activity of glutamate, a chemical neurotransmitter involved in assisting brain information processing. Such modulators are known to improve memory, attention, reasoning, language ability, and the ability to perform simple tasks. An example of a glutamate modulator is memantine. NMDA-R antagonists are chemicals that can non-selectively reduce the abnormal activity of NMDA-R.

[0054] "α7 nicotinic agonists" are chemical substances implemented to normalize attentional dysfunction. Examples of such agonists may be clozapine or 3-2,4-dimethoxybenzylidene anabaceine (DMXBA). Other examples of α7 nicotinic agonists are publicly known, for example, as exemplified by Laura F Martin et al., Psychopharmacology (Berl). January 2004; 174(1): pp. 54-64, incorporated by reference.

[0055] Selective serotonin reuptake inhibitors are particularly well-known as the most commonly prescribed antidepressants. Examples of such inhibitors may include citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, or sertraline.

[0056] Serotonin-norepinephrine reuptake inhibitors (Serotonin-Norepinephrine Reuptake Inhibitors) are a family of antidepressants that inhibit the reuptake of both serotonin and norepinephrine. Examples of such inhibitors include duloxetine and venlafaxine.

[0057] Calcium modulators, also known as calcium channel modulators, inhibit pain signals transmitted via voltage-gated calcium channels located in the central nervous system terminals. Of these, gabapentinoids are by far the most commonly prescribed. Pregabalin is one example of such a modulator.

[0058] Reversible inhibitors of monoamine oxidase A (RIMAs) inhibit the breakdown of three major neurotransmitters—serotonin, norepinephrine, and dopamine—and offer a multi-neurotransmitter strategy, particularly in the treatment of depression. An example of such a chemical is moclobemide.

[0059] "Antiepileptic drugs" are a type of medication used to prevent or treat seizures or convulsions by controlling abnormal electrical activity in the brain. Antiepileptic drugs are used to treat epilepsy and other seizure disorders. Antiepileptic drugs are also used to treat medical conditions such as bipolar disorder, neuralgia, migraines, fibromyalgia, and restless legs syndrome.

[0060] Antifungal drugs are medications that kill or stop the growth of fungi (or multiple fungi) that cause infections. Antifungal drugs are also called antifungal agents.

[0061] Corticosteroids, often known simply as steroids, are anti-inflammatory drugs. An example of a corticosteroid is prednisone.

[0062] Immunosuppressants, or immunosuppressant drugs, are important therapeutic tools that inhibit or prevent the activity of the immune system. Examples of immunosuppressants include methotrexate and azathioprine.

[0063] "A normal response to an anxiety-inducing situation" means a physiological and psychological response that is appropriate and proportional to the level of threat or stress perceived in the anxiety-inducing situation.

[0064] The term “anxiety-inducing situation” refers to any situation or environment that is likely to induce or increase anxiety in an individual. These situations may include, but are not limited to, public speaking, confrontation, social situations, uncertainty about the future, or situations involving a potential threat to one's safety or well-being. Overall, anxiety-inducing situations are those that tend to induce anxiety or fear in an individual.

[0065] The term "physiologically acceptable medium" is intended to mean a medium that is compatible with the body of the individual to whom the composition must be administered. For example, a non-toxic solvent such as water. In particular, the medium is suitable for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, or rectal administration, more specifically oral, subcutaneous, intravenous, topical, or local administration.

[0066] The terms “sequence homology,” “sequence identity,” “homology,” and “identity” are used interchangeably herein. For the purposes of the present invention, it is defined herein that sequences are aligned for the purpose of optimal comparison in order to determine the percentage of sequence homology or sequence identity between two amino acid sequences or two nucleic acid sequences. To optimize the alignment between two sequences, gaps can be introduced in either of the two sequences being compared. Such alignment can be performed over the entire length of the sequences being compared. Alternatively, the alignment can be performed over a shorter length, for example, about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of the reported aligned region that matches identically between the two sequences. The comparison of sequences and the determination of the percentage of sequence identity between two sequences can be achieved using mathematical algorithms. Those skilled in the art are aware that several different computer programs are available for aligning two sequences and determining the identity between them (D. Sankoff and J.B. Kruskal (eds.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Kruskal, J.B. (1983), An overview of sequence comparison, pp. 1-44, Addison Wesley).

[0067] The percentage of sequence identity between two amino acid sequences or two nucleotide sequences can be determined using the Needleman-Wunsch algorithm for aligning two sequences. (Needleman, S.B. and Wunsch, C.D. (1970), J.Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned using this algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE.

[0068] For the purposes of this invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or later, EMBOSS: The European Molecular Biology Open Software Suite (2000), Rice, P., Longden, I., and Bleasby, A., Trends in Genetics 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 was used as the substitution matrix. EDNAFULL was used for the nucleotide sequence. The optional parameters used were a gap start penalty of 10 and a gap extension penalty of 0.5. No end-gap penalty was applied. In the output section, "Yes" was indicated for the question "Brief identity and similarity," and "SRS pairwise" was specified as the output alignment format.

[0069] Following alignment using the NEEDLE program as described above, the percentage of sequence identity between the query sequence and the sequence of the present invention is calculated as follows: the number of corresponding positions in the alignment that indicate the same amino acid or the same nucleotide in both sequences, divided by the total length of the alignment, after subtracting the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled as "longest identity" in the program's output.

[0070] The percentage of nucleotide and amino acid sequence similarity, i.e., sequence identity, is calculated using several other algorithms known in the art, preferably such as the Karlin and Altschul mathematical algorithm (Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA 90:5873~5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm available at https: / / www.ebi.ac.uk / Tools / msa / clustalo / (Thompson, JD, Higgins, DG and Gibson, TJ (1994), Nucleic Acids Res. 22, 4673~80), or the GAP program (mathematical algorithm from the University of Iowa), or the Myers and Miller mathematical algorithm (1989 - Cabios 4:11~17), or Clone Manager. This can be determined by sequence alignment using parameter 9. The preferred parameters used are the default parameters set at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0071] The degree of sequence identity (sequence matching) can be calculated, for example, using BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs in Altschul et al. (1990), J.Mol.Biol.215, 403-410. BLAST polynucleotide searching is performed using the BLASTN program, score=100, word length=12, to obtain polynucleotide sequences homologous to the nucleic acid encoding the relevant protein.

[0072] BLAST protein search is performed using the BLASTP program, score=50, word length=3 to obtain amino acid sequences homologous to the SHC polypeptide. To obtain gapped alignment for comparison purposes, Gapped BLAST is used as described by Altschul et al. (1997), Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Sequence matching analysis can be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. Where sequence identity percentages are referred to in this application, these percentages are calculated with respect to the full length of longer sequences unless otherwise specified.

[0073] In certain embodiments, the identity % between two sequences is determined using CLUSTAL O (version 1.2.4).

[0074] As used herein, the term “polypeptide” refers to a molecule containing more than five amino acid residues linked by peptide bonds. Amino acids are identified by either single-letter or three-letter abbreviations. As used herein, the term “protein” is synonymous with the term “polypeptide” and may refer to two or more polypeptides. Therefore, the terms “protein,” “peptide,” and “polypeptide” can be used interchangeably. Polypeptides may be modified (e.g., glycosylation, phosphorylation, acylation, farnesylation, prenylation, sulfonation, etc.) to add functionality.

[0075] HIP / PAP proteins and their derivatives implemented according to the present invention HIP / PAP proteins are known for their anti-apoptotic and pro-mitotic activity against hepatocytes (U.S. Patent No. 13 / 032,521, International Publication No. 2004 / 112824, Simon et al., FASEB J. August 2003; 17(11):1441-50).

[0076] The 15-amino acid peptide HIP (human islet precursor peptide) peptide, derived from the Reg IIIa family (HIP / PAP), has been shown to have regenerative activity on pancreatic islets and therefore stimulate insulin production (U.S. Patent No. 2010 / 0093605).

[0077] The HIP / PAP protein of the present invention may include an amino acid sequence selected from the group consisting of sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, particularly the sequence shown as SEQ ID NO: 4.

[0078] The amino acid sequence of SEQ ID NO: 1 corresponds to the HIP / PAP protein sequence of SEQ ID NO: 4, but the signal peptide at the N-terminus of that protein is deleted.

[0079] In certain embodiments, the HIP / PAP protein according to the present invention comprises or consists of the amino acid sequence shown as SEQ ID NO: 4.

[0080] In certain embodiments, the HIP / PAP protein according to the present invention comprises or consists of the amino acid sequence shown as SEQ ID NO: 1.

[0081] The amino acid sequence of SEQ ID NO: 2 corresponds to the short-chain form of the HIP / PAP protein, and compared to the amino acid sequence of SEQ ID NO: 1, it lacks 11 amino acid propeptides at the N-terminus.

[0082] In certain embodiments, the HIP / PAP protein according to the present invention comprises or consists of the amino acid sequence shown as SEQ ID NO: 2.

[0083] The sequence of Sequence ID No. 3 corresponds to the sequence of Sequence ID No. 1, with methionine added to the N-terminus. The HIP / PAP derivative of Sequence ID No. 3 is also called rcHIP / PAP or ALF5755. This derivative can be produced by recombination, particularly in E. coli (E. coli) cells. The 12-amino acid N-terminal propeptide (11 amino acids + additional methionine propeptide) may be cleaved to obtain the short-chain form of the HIP / PAP protein (Sequence ID No. 2).

[0084] In certain embodiments, the HIP / PAP protein according to the present invention comprises or consists of the amino acid sequence shown as SEQ ID NO: 3.

[0085] According to the present invention, short-chain or long-chain forms of HIP / PAP proteins or their derivatives can be used without distinction.

[0086] The HIP / PAP protein derivatives according to the present invention exhibit a biologically active derived form of any one of the sequences of SEQ ID NOs: 1 to 4 of the HIP / PAP protein. The term "biologically active" means that the HIP / PAP protein derivative has the same biological activity as any one of the sequences of SEQ ID NOs: 1 to 4 of the HIP / PAP protein.

[0087] The HIP / PAP protein derivative according to the present invention has at least 80% sequence identity with an amino acid sequence selected from the group consisting of Sequence ID No. 1, Sequence ID No. 2, Sequence ID No. 3, and Sequence ID No. 4, particularly Sequence ID No. 4, and comprises or consists of an amino acid sequence having the same properties of biological activity as the amino acid sequence selected from the group consisting of Sequence ID No. 1, Sequence ID No. 2, Sequence ID No. 3, and Sequence ID No. 4, particularly Sequence ID No. 4.

[0088] The biological activity of the HIP / PAP protein according to the present invention is, as stated above, the ability to treat and / or prevent peripheral neuropathy in an individual, particularly diabetic peripheral neuropathy, and accordingly, the ability to prevent complications of peripheral neuropathy in an individual, particularly the complications detailed elsewhere in this specification.

[0089] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% amino acid identity with the reference amino acid sequence, and also includes biological activity of the same nature as the reference amino acid sequence.

[0090] In certain embodiments, HIP / PAP proteins or their derivatives can associate or bind to non-HIP / PAP moieties via non-covalent bonding. For example, HIP / PAP proteins or their derivatives can associate with liposome particles. Depending on the type of liposome or the manufacturing method, HIP / PAP proteins or their derivatives may be bound to the surface of the liposome or encapsulated within the liposome.

[0091] HIP / PAP proteins or their derivatives can also be covalently bonded to non-HIP / PAP moieties. Such non-HIP / PAP moieties are selected from proteins or non-protein compounds, such as polyethylene glycol, and thus form pegylated HIP / PAP derivatives.

[0092] The HIP / PAP protein derivatives of the present invention also include derivatives that become biologically active only when administered to a patient.

[0093] Finally, derivatives of HIP / PAP proteins also include chimeric or fusion proteins. Such proteins are fused with non-HIP / PAP polypeptides, the latter of which may be fused to the N-terminal or C-terminal portion. Typically, HIP / PAP proteins or their derivatives may be fused with a GST sequence at the C-terminal portion to facilitate the purification of recombinant proteins.

[0094] In certain embodiments of the present invention, the HIP / PAP protein or derivative thereof according to the present invention is recombinantly produced in bacterial cells or animal cells, including insect cells and mammalian cells, according to techniques known to those skilled in the art.

[0095] In other embodiments, the HIP / PAP protein or derivative thereof according to the present invention can be isolated from cells or tissues by known purification techniques.

[0096] HIP / PAP proteins and their derivatives can also be produced by chemical synthesis.

[0097] In this text, the term "HIP / PAP protein" includes both the HIP / PAP protein itself and its derivatives as described above.

[0098] Composition implemented according to the present invention The present invention also relates to the implementation of the previously defined HIP / PAP protein or its derivative in a composition comprising a physiologically acceptable medium.

[0099] The physiologically acceptable media as defined herein may be selected from common excipients known to those skilled in the art, according to the desired pharmaceutical form and mode of administration (see Remington's Pharmaceutical Sciences, 16th edition, Osol, A., 1980).

[0100] For example, compositions implemented according to the present invention may be used according to the therapeutic indication and the HIP / PAP protein or its derivatives. a) HIP / PAP protein or its derivatives; and b) A buffer capable of maintaining the pH within the maximum stable range, preferably 1–9, more specifically 4–8, and even more specifically 6–7.5; and / or c) Detergents or surfactants that stabilize proteins or polypeptides against aggregation induced by stirring; and / or d) Isotonic agents; and / or e) Preservatives selected from the group consisting of, for example, phenol, benzyl alcohol, benzosyl halogenates, and chlorides; and / or f) water It can include...

[0101] If the detergent or surfactant used is nonionic, it can be selected from polysorbate, Pluronic®, polyethylene glycol (PEG), or poloxamer.

[0102] Isotonic agents, which enable the composition to maintain its isotonicity, typically include polyhydric alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol, and are used alone or in combination. Alternatively, sodium chloride and / or any other inorganic salt can be used as the isotonic agent.

[0103] The buffer may be, for example, an acetate, citrate, succinate, phosphate buffer or any other inorganic buffer, depending on the desired pH.

[0104] Phenol, benzyl alcohol, halogenated benzosyl, and chloride-type preservatives are known antimicrobial agents. Typical preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0105] Additional excipients may also include antioxidants such as ascorbic acid and methionine, chelating agents such as EDTA, and sugars such as sucrose, mannitol, trehalose, or sorbitol.

[0106] The HIP / PAP protein or derivative thereof according to the present invention may be in the form of a pharmaceutically acceptable salt. This is intended to mean a salt prepared from a pharmaceutically acceptable non-toxic acid or a pharmaceutically acceptable non-toxic base, including organic and inorganic salts and acids. Examples include alkali metal salts (sodium and potassium salts), alkaline earth metal salts (calcium and magnesium salts), ammonium salts, salts of organic bases (pyridine or triethylamine salts), salts of inorganic acids (hydrochloride, sulfate, nitrate), and salts of organic acids (acetate, oxalate, p-toluenesulfonate).

[0107] The compositions implemented in accordance with the present invention may be for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, or rectal administration, and may be particularly for oral, subcutaneous, intravenous, topical, or local administration.

[0108] According to one preferred embodiment, the HIP / PAP protein is administered in an effective dose, i.e., the amount necessary to obtain the expected effect of the present invention. Such a dose of HIP / PAP protein is generally determined empirically according to the subject being treated and its pathological condition. The effective dose also depends on the intended mode of administration. The adjustments necessary to determine the effective dose for obtaining the maximum therapeutic effect are a matter of routine practice for clinicians.

[0109] The effective dose of HIP / PAP protein or its derivative may be, for example, 0.1 μg / day per kg of body weight of the individual to be administered the drug to, or 100 mg / day per kg of body weight. In certain embodiments, the effective dose of HIP / PAP protein or its derivative may reach more than 10 mg / kg, but the effective dose of HIP / PAP protein or its derivative according to the present invention is generally less than 5 mg per kg of body weight, including amounts of 4.5 mg / kg, 4 mg / kg, 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, or less than 2000 μg / kg. More specifically, the effective amount of the HIP / PAP protein or derivative thereof according to the present invention is at least 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 40 μg / kg, 50 μg / kg, relative to the body weight of the individual to whom it is administered or must be administered. 60μg / kg, 70μg / kg, 80μg / kg, 90μg / kg, 100μg / kg, 150μg / kg, 200μg / kg, 250μg / kg, 300μg / kg, 350μg / kg, 400μg / kg, 45 Contains amounts of 0μg / kg, 500μg / kg, 600μg / kg, 700μg / kg, 800μg / kg, 900μg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg or more.

[0110] According to certain embodiments, HIP / PAP protein or its derivatives are administered in doses of 10 to 5000 μg / kg relative to body weight, preferably 100 to 2000 μg / kg.

[0111] In compositions implemented according to the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, or rectal administration, the active ingredient (HIP / PAP protein or its derivative) can be administered in unit dose form as a mixture with pharmaceutical excipients.

[0112] If the composition is for oral administration, it may be selected from the group consisting of foods, beverages, pharmaceuticals, functional foods, food additives, nutritional supplements, and dairy products.

[0113] The preferred mode of administration is orally, subcutaneously, intravenously, topically, or locally, more specifically, subcutaneously, intravenously, topically, or locally.

[0114] The compound or composition of the present invention can be administered, for example, by using a sheath, patch, pad, compress, bandage, tape, gauze-based bandage, woven or non-woven sponge, or syringe.

[0115] HIP / PAP proteins or their derivatives may be sterilized before in vivo administration. Sterilization can be achieved by filtration through a sterile filter membrane before or after lyophilization or reconstitution. Systemically administered HIP / PAP proteins or their derivatives can be advantageously lyophilized or stored in solution. In lyophilized form, HIP / PAP proteins or their derivatives can generally be formulated in combination with excipients that can be reconstituted with an appropriate diluent at the time of use.

[0116] HIP / PAP proteins or their derivatives can be administered once daily or in divided doses (e.g., two to three times daily) until the desired therapeutic effect is achieved. They may also be administered chronically.

[0117] HIP / PAP proteins or their derivatives can also be administered in the form of a course lasting, for example, 15 days to 3 months, and may be repeated 1 to 6 times at predetermined doses and time intervals.

[0118] The HIP / PAP protein or derivative thereof according to the present invention may also be combined with agents known to treat and / or prevent cognitive impairment in the context of polypharmacy. Such additional agents known to be useful in preventing and / or treating cognitive impairment are known to those skilled in the art and can be selected from the group consisting of agents selected from the group consisting of cholinesterase inhibitors such as donepezil, rivastigmine or galantamine; glutamate modulators such as memantine; cholinesterase inhibitors in combination with glutamate modulators such as the combination of donepezil and memantine; methylphenidate; amphetamine; atomoxetine; AMPA-R agonists; α7 nicotinic agonists; guanfacine; bupropion; vortioxetine and D-cycloserine.

[0119] The HIP / PAP protein or derivative thereof according to the present invention may also be combined with agents known to treat and / or prevent anxiety disorders in the context of polypharmacy. Such additional agents known to be useful in preventing and / or treating anxiety disorders are known to those skilled in the art and, in particular, can be selected from the group consisting of selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium modulators, azapirone, reversible inhibitors of monoamine oxidase A, agomelatine, quetiapine, and vortioxetine, more specifically, from the group consisting of citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine, and vortioxetine.

[0120] Accordingly, compositions implemented in accordance with the present invention may include, in addition to the HIP / PAP protein or derivative thereof according to the present invention, at least one agent known to be useful for the prevention and / or treatment of cognitive impairment and at least one agent known to be useful for the prevention and / or treatment of anxiety disorders, including, for example, a single agent that plays both roles, i.e., an agent known to be useful for the prevention and / or treatment of cognitive impairment and anxiety disorders, such as botioxetine.

[0121] Combined administration means that the HIP / PAP protein, its derivatives, or compositions containing the same according to the present invention can be administered simultaneously or sequentially with other drugs or compounds. When they are in separate compositions, the compositions containing the HIP / PAP protein, its derivatives, and compositions containing at least one additional drug according to the present invention can be administered via the same route or via different routes.

[0122] The HIP / PAP protein or its derivative according to the present invention and at least one additional agent can be administered in the same composition or in separate compositions.

[0123] "Simultaneously" is understood to mean that the compositions can be administered at the same time, or on the same day or within a few days.

[0124] "Continuously" is understood to mean that the composition can be administered with intervals of at least several days, for example, at least two days.

[0125] Use of HIP / PAP proteins and / or derivatives thereof As mentioned above, HIP / PAP proteins, their derivatives, and compositions containing them are - Treatment and / or prevention, especially treatment, of cognitive impairments associated with anxiety disorder in individuals requiring treatment; and / or In particular, treatment and / or prevention of cognitive impairment associated with externalization disorder, especially treatment, wherein the cognitive impairment is selected in particular from the group consisting of bipolar disorder, substance abuse, attention deficit disorder and psychotic disorder; and / or - To restore physiological anxiety levels in individuals, particularly those suffering from cognitive impairments associated with anxiety disorders; and / or - To improve cognition in individuals with anxiety-related neurological disorders, selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and cognitive impairment associated with other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis; - To reduce cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease (PD), bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection; and / or - Treatment and / or prevention of diet-induced cognitive impairment and anxiety-related dysfunction in individuals requiring treatment and / or prevention. It is used.

[0126] Environmental factors such as diet have been shown to actually influence the aging of healthy brains and affect neurobiology and cognitive function (Evans et al., 2022, Front Pharmacol. 13, 1030609; Gonzalez Olmo et al., 2021, Nutrients. 13; Spencer et al., 2019, Neurobiol Aging. 74, 121-134; Wieckowska-Gacek et al., 2021, Ageing Res Rev. 70, 101397). High-fat diet intake has been associated with obesity, insulin resistance, diabetes, age-related cognitive decline, and neurodegenerative disorders (Buckman et al., 2014, Brain Behav Immun. 35, 33-42; Sanchez et al., 2018, Int J Mol Sci. 19(2):533). The mechanisms by which HFD affects brain function are not yet fully established, but neuroimmunological signaling is likely a contributing factor (Butler, 2021, Brain Behav Immun Health.16, 100298).

[0127] Acute exposure to HFD, even for just three days, has been shown to induce neuroinflammation and impair memory consolidation in aged rats (Spencer et al., 2019, Neurobiol Aging. 74, 121-134). HFD has also been shown to increase anxiety-related behaviors and impair learning and memory in young mice (Gainey et al., 2016, Front Behav Neurosci. 10, 156). Diet-induced obesity after chronic HFD administration contributes to the formation of a systemic inflammatory environment, which may affect neuroinflammation, neurobiology, and cognitive function (Buckman et al., 2014, Brain Behav Immun. 35, 33-42; Butler, 2021, Brain Behav Immun Health. 16, 100298).

[0128] As previously shown, anxiety disorders associated with cognitive impairment are particularly, (i) Major symptoms of obsessive-compulsive disorder, and / or (ii) Secondary symptoms of neurodegenerative diseases selected from the group consisting of attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease (PD), bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection (SARS-CoV-2). That's fine.

[0129] The individuals to whom the HIP / PAP protein, its derivatives, or compositions for use in accordance with the present invention are administered may be mammals, and in particular may be humans.

[0130] HIP / PAP proteins, derivatives thereof, or compositions for use according to the present invention can be administered in combination with standard medical treatment for cognitive impairment and / or anxiety disorders.

[0131] Accordingly, HIP / PAP proteins, derivatives thereof, or compositions for use in accordance with the present invention may be administered to individuals in need of prevention and / or treatment of cognitive impairment together with at least one agent known to be useful for the prevention and / or treatment of cognitive impairment, for example, a cholinesterase inhibitor such as donepezil, rivastigmine, or galantamine; a glutamate modulator such as memantine; a combination of a cholinesterase inhibitor and a glutamate modulator, particularly a combination of donepezil and memantine; methylphenidate; amphetamine; atomoxetine; AMPA-R agonists; α7 nicotinic agonists; guanfacine; bupropion; vortioxetine, and D-cycloserine.

[0132] Alternatively, or furthermore, HIP / PAP proteins, derivatives thereof, or compositions for use according to the present invention may be administered to individuals in need of prevention and / or treatment of anxiety disorders together with at least one agent known to be useful for the prevention and / or treatment of anxiety disorders, in particular an agent selected from the group consisting of selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium modulators, azapirone, reversible inhibitors of monoamine oxidase A, agomelatine, quetiapine, and vortioxetine, more specifically an agent selected from the group consisting of citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine, and vortioxetine.

[0133] When such agents known to be useful for the prevention and / or treatment of anxiety disorders are implemented together with HIP / PAP proteins, derivatives thereof, or compositions for use in accordance with the present invention in patients requiring the prevention and / or treatment of anxiety disorders, the cognitive impairment associated with anxiety disorders may be selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

[0134] HIP / PAP proteins, derivatives thereof, or compositions for use in accordance with the present invention can be administered together with at least one agent known to be useful in alleviating symptoms associated with disorders selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, particularly agents selected from the group consisting of antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids such as prednisone; and immunosuppressants such as methotrexate or azathioprine. In particular, compositions for use according to the present invention may further include at least one agent known to be useful in alleviating symptoms associated with disorders selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, in particular an agent selected from the group consisting of antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids such as prednisone; and immunosuppressants such as methotrexate or azathioprine.

[0135] The present invention also relates to a method for treating and / or preventing, particularly therapeutic, anxiety-related cognitive impairment in an individual requiring treatment and / or prevention of anxiety-related cognitive impairment, comprising administering to the individual a HIP / PAP protein, a derivative thereof, or a composition comprising the individual an anxiety-related cognitive impairment.

[0136] The present invention also relates to a method for improving cognition in an individual suffering from an anxiety-related neurological disorder, comprising administering to the individual a HIP / PAP protein, a derivative thereof, or a composition containing the same, wherein the anxiety-related neurological disorder is selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and cognitive impairment associated with other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0137] The present invention also relates to a method for mitigating cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

[0138] The present invention also relates to a method for treating and / or preventing diet-induced cognitive impairment and anxiety-related dysfunction in an individual requiring treatment and / or prevention of diet-induced cognitive impairment and anxiety-related dysfunction, comprising administering to the individual a HIP / PAP protein according to the present invention, a derivative thereof, or a composition containing the same.

[0139] The present invention further relates to the use of HIP / PAP proteins, derivatives thereof, or compositions containing them, particularly for the treatment and / or prevention, of cognitive impairment associated with anxiety disorder in individuals requiring treatment.

[0140] The present invention further relates to the use of HIP / PAP proteins, derivatives thereof, or compositions comprising them for improving cognition in individuals suffering from anxiety-related neurological disorders, wherein the anxiety-related neurological disorders are selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and cognitive impairment associated with other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0141] The present invention further relates to the use of HIP / PAP proteins, derivatives thereof, or compositions containing them, for mitigating cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease (PD), bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

[0142] The present invention further relates to the use of HIP / PAP proteins, derivatives thereof, or compositions thereof for treating and / or preventing diet-induced cognitive impairment and anxiety-related dysfunction in individuals requiring treatment and / or prevention of diet-induced cognitive impairment and anxiety-related dysfunction, comprising administering the HIP / PAP proteins, derivatives thereof, or compositions thereof according to the present invention to individuals.

[0143] The present invention will be described in more detail by the following examples, which are provided for illustrative purposes only.

[0144] All references to percentages refer to weight percentages unless otherwise specified. [Examples]

[0145] (Example 1) Injections into animals The animals were anesthetized with isoflurane and placed in a stereotactic surgical device from World Precision Instruments. An incision was made in the sagittal plane of the skull. Two microliters of either rAAV9-empty capsid (transgenic vehicle, Tg Veh, n=8) or rAAV9-hHIP / PAP (rAAV9-HIP / PAP (see Figure 6A), Tg HIP / PAP, n=8) were injected into the left and right hippocampi (HPC) (from bregma: X=+ / -2.7, Y=-2.7, Z=-3) and left and right cortices (from bregma: X=+ / -2, Y=-2, Z=-3) of 6-month-old APP / PS1 mice (named transgenic or Tg) in 5 × 10⁻¹⁶ doses.11 The drug was administered by injection at a dose of μg / mL. Non-transgenic littermates (Ntg n=10) were used as behavioral baseline controls. Six months after surgery, the animals underwent a two-week behavioral study. At the end of the behavioral study, the animals were euthanized and tissue samples were collected.

[0146] behavior analysis Before euthanasia, mice were tested in a series of behavioral tasks, including open field, novel object recognition, elevated cruciform maze, and radial arm water maze. A group of age-matched wild-type mice was used for comparison. The tasks were performed from least stressful (open field) to most stressful (RAWM), as described below.

[0147] Measurements were analyzed using one-way or two-way ANOVA. Repeated measures were performed on tasks such as water mazes requiring multiple training / trials. Mean comparisons were performed using Tukey's HSD post-hoc test with GraphPad Prism for significant ANOVA measurements. Outliers were identified using Grubbs' test.

[0148] Open field An open field was used as a standard test for general activities. Animals were monitored for 15 minutes in a 40cm square open field under moderate lighting conditions using video tracking software (ANY-Maze, Stoelting, Illinois).

[0149] General activity levels were assessed by measuring horizontal and vertical activity levels.

[0150] Novel Object Recognition Test (NOR) Mice were placed in a 40 x 40 cm experimental enclosure and monitored and quantified using video tracking (ANY-Maze, Stoelting, Illinois). Two objects of similar size to the mice were placed along the centerline of the enclosure, approximately 3-5 cm from the outer wall.

[0151] Each animal underwent three 5-minute acclimatization tests, with a 5-minute interval between each test.

[0152] After each test, the laboratory and object cues were cleaned with 70% ethanol to minimize olfactory cues. After the acclimatization test, one of the acclimatized objects was replaced with a new object. The animals were given a 5-minute search test, during which their object search was monitored by video recording.

[0153] Working memory was assessed by the time spent searching for both novel and familiar objects.

[0154] Radial arm water maze and its inversion The radial arm water maze featured six swimming paths (arms) radiating from an open central area, with a hidden escape platform at the end of one of the arms. The pool was surrounded by several maze-external cues to enable spatial navigation. In each test, mice were allowed to swim for up to 60 seconds to find the escape platform. The platform was located on the same arm in each test.

[0155] On day 1, the mice underwent 15 tests, alternating between a visible platform (above the water surface) and a hidden platform (below the water surface). On day 2, the mice underwent 15 additional tests, all using the hidden platform. The starting arm was changed for each test, so the mice relied on spatial cues to solve the task (i.e., the second arm was on the right) rather than learning motor rules.

[0156] Each mouse was assigned a different target arm to avoid revealing the target arm through olfactory cues. Entering the wrong arm (any limb within the arm) was scored as an error. Failure to enter an arm within 15 seconds was also scored as an error. Errors across three consecutive test blocks were averaged for data analysis. Mice showing an average of one or fewer errors by the end of day two were considered to have reached the learning criterion.

[0157] On the third day, an inversion test was conducted in which the target platform was positioned on an arm 180° from its original position. The mice underwent 15 tests, all using a hidden platform. The animals were monitored using video tracking software (Ethovision, Noldus).

[0158] elevated cross maze The elevated cusp maze test is one of the most widely used tests for measuring anxiety-like behaviors.

[0159] The experiment was based on the mouse's natural aversion to open, elevated positions, as well as its natural, spontaneous exploratory behavior in a new environment. Mice were placed at the branching point of a four-arm maze consisting of two open arms without walls and two arms enclosed by walls measuring 15.25 cm high, 30 cm long, and 5 cm wide.

[0160] Animals were monitored for 5 minutes under moderate lighting conditions using video tracking software (ANY-Maze, Stoelting, Illinois). Anxiety-like behavior was assessed by measuring the number of times animals entered and stayed in the open and closed arms.

[0161] The longer a mouse spends with its arms closed, the greater its anxiety, which is normal behavior observed in non-transgenic control mice.

[0162] result (i) Open field Regardless of treatment, no difference in general activity levels was observed between non-transgenic control mice and transgenic mice. Overexpression of HIP / PAP in the brains of APP / PS1 mice did not induce any visible changes in spontaneous motor activity (Figure 1A (open field - distance traveled), 1B (open field - resting time)).

[0163] (ii) Novel Object Recognition Test (NOR) Upon encountering a novel object, non-transgenic control mice spent significantly longer in contact with the novel object compared to familiar objects, suggesting short-term memory impairment as seen in previous studies. In contrast, transgenic mice spent similar amounts of time with both objects, regardless of treatment, suggesting short-term memory deficits. Overexpression of HIP / PAP in the brains of APP / PS1 mice did not restore the genotype effect on short-term memory observed during novel object recognition (see Figure 2).

[0164] (iii) Radial arm water maze Vehicle-treated APP / PS1 mice produced significantly more errors in platform arrival trials than non-transgenic control mice, as evidenced by a higher number of errors per block (Figure 3A) and a higher total number of errors on day 2 (Figure 3B). These results indicate cognitive impairment, particularly in learning and memory. HIP / PAP-treated APP / PS1 mice produced significantly fewer errors than vehicle-treated APP / PS1 mice, demonstrating a recovery in cognitive function for this study (see Figures 3A and 3B).

[0165] (iv) Invert As expected, APP / PS1 control mice had significantly more errors than non-transgenic littermates (see Figure 4B). No visible recovery was observed in APP / PS1 mice treated with HIP / PAP (see Figure 4A).

[0166] (v) Elevated plus maze As shown in Figure 5A, non-transgenic control mice spent significantly more time in the closed arm than in the open arm and exhibited normal levels of anxiety and fear towards open spaces, which are physiological defense mechanisms. In contrast, vehicle-treated transgenic mice spent the same amount of time in both arms and showed a deficit in anxiety-like behavior (Figure 5A). Furthermore, comparing the time spent in the closed arm, vehicle-treated transgenic mice spent significantly less time in the closed arm compared to non-transgenic controls, showing a release of behavioral inhibition and an increase in risk-taking behavior (Figure 5B). Treatment with HIP / PAP in transgenic mice restored normal anxiety-like behavior, as indicated by a significant increase in the time spent in the closed arm compared to the open arm. Moreover, the increase in time spent in the closed arm observed during HIP / PAP treatment was significant in vehicle-treated transgenic mice but not in non-transgenic mice, suggesting that it supports phenotypic recovery rather than anxiety induction (Figure 5A).

[0167] conclusion Overexpression of HIP / PAP in transgenic mouse models significantly improved memory in a radial-arm water maze (RAWM), restoring cognitive impairment during the study. Deficiencies in anxiety-like behavior were also restored during HIP / PAP treatment during an elevated cross maze, compared to transgenic vehicle-treated mice. Therefore, HIP / PAP treatment enables the restoration of normal responses to anxiety-inducing situations in mice. HIP / PAP treatment effectively normalized anxiety-like behaviors that were absent in transgenic control mice.

[0168] In fact, non-transgenic control mice spent significantly more time in the closed arm than in the open arm and exhibited normal levels of anxiety and fear towards open spaces, which are physiological defense mechanisms. In contrast, control APP / PS1 mice spent the same amount of time in both arms and showed a lack of anxiety-like behavior. Furthermore, when comparing the time spent in the closed arm, control APP / PS1 mice spent significantly less time in the closed arm compared to non-transgenic controls, indicating a release of behavioral inhibition and an increase in risk-taking behavior.

[0169] Treatment with HIP / PAP in transgenic mice restored normal anxiety-like behavior, as indicated by a significant increase in time spent with closed arms compared to open arms.

[0170] This difference was not significant compared to non-transgenic mice and supported the recovered phenotype rather than an anxiety-inducing effect.

[0171] This improvement in cognition was not associated with a reduction in amyloid pathology in the hippocampus, as indicated by similar levels of amyloid plaques stained with Aβ and Congo Red (Figure 7). However, Western blot analysis of the hippocampus revealed significant increases in the antioxidant enzymes sodium dismutase-1 (SOD-1) and heme oxygenase-1 (HO-1) (Figures 8A, C, D), as well as a trend toward increased catalase levels (Figures 8A, B, p=0.06), compared to both non-transgenic mice and APP / PS1 control mice.

[0172] These data indicate that the antioxidant effects of HIP / PAP are remarkably effective in the brain, contributing to the unexpected beneficial effects of HIP / PAP on cognition.

[0173] HIP / PAP expression After a series of behavioral tests, tissue samples were taken from the mice at 12 months of age (6 months after the onset of symptoms).

[0174] HIP / PAP expression was measured via hemagglutinin (HA) staining in the brain (Figure 6C), and HIP / PAP concentrations in the cortex were measured using human HIP / PAP ELISA (R&D Systems, Minneapolis, USA) (Figure 6D).

[0175] Positive HA staining was not detected in the control (Figure 6B), and HIP / PAP was not detected in the plasma (not shown).

[0176] (Example 2) HIP / PAP crosses the blood-brain barrier (BBB) ​​via peripheral injection of rHIP / PAP or rAAV injection. In pharmacokinetic studies conducted in mice using [3H]-rHIP / PAP, significant radioactivity levels were detected in the brain (85.57+ / -11.55ng / g) as well as in the plasma (18.20+ / -15.82ng / g) and liver (64.22+ / -11.73ng / g) 24 hours after intravenous administration of 1 mg / kg of [3H]-rHIP / PAP.

[0177] Furthermore, brain tissue from non-transgenic mice that were subcutaneously administered rHIP / PAP at a dose of 43 μg / day for one month via an ALZET pump was examined.

[0178] Brain and plasma samples were collected, and rHIP / PAP levels were evaluated by ELISA (Abcam, R&D Systems).

[0179] Significant levels of rHIP / PAP were found in the brain and plasma after one month (Figure 9A).

[0180] Furthermore, the brain permeability of fluorescently labeled rHIP / PAP was established using an in vivo imaging system (IVIS, PerkinElmer, Waltham, Massachusetts, USA) and vivotag 680 (Revvity, Waltham, Massachusetts, USA). Thirty minutes after cardiac injection of rHIP / PAP-680 or control bovine serum albumin (BSA)-680, a strong signal was found in the brains of animals injected with rHIP / PAP-680, but not in the brains of animals injected with BSA-680, indicating that rHIP / PAP crossed the blood-brain barrier (BBB) ​​(Figure 9B).

[0181] Therefore, significant blood-brain barrier crossing of HIP / PAP was established after either subcutaneous or intravenous administration of the protein.

[0182] Peripheral rAAV9-HIP / PAP rAAV9-HIP / PAP was injected peripherally either intravenously or intramuscularly (hind limb). One month after expression, significant levels of rHIP / PAP were found in the plasma (Figure 9C) and brain (Figure 9D) of the injected mice.

[0183] One month after peripheral injection, rHIP / PAP levels in the brain were comparable to those observed after central injection (Figure 9D), demonstrating the appropriateness of peripheral injection for this purpose. rHIP / PAP signals were not detected (not shown) in control animals.

[0184] (Example 3) Tests in a high-fat diet model using non-transgenic wild-type mice Six-month-old non-transgenic mice were given either a control diet (n=6), a high-fat diet of 60% kcal / fat (HFD, n=5), or 2 microliters of rAAV9-hHIP / PAP (n=4) for 5 × 10⁻¹⁰ days. 11Two weeks after intracranial injection at μg / mL into the left and right hippocampi (HPC) (X=+ / -2.7, Y=-2.7, Z=-3 from the bregma) and the left and right cortices (X=+ / -2, Y=-2, Z=-3 from the bregma), the subjects were allowed to freely consume either HFD.

[0185] Body weight and food intake were monitored once a week during the study period.

[0186] After 10 weeks of dietary changes, behavioral analyses were conducted to evaluate the effects of the diet on spontaneous motor activity, cognition, and anxiety.

[0187] result After 10 weeks of dietary therapy, mice that received HFD showed a significant increase in body weight compared to mice that received a control diet, regardless of treatment (Figure 10A).

[0188] During the elevated cruciform maze described in detail above, mice on a control diet spent significantly more time in the closed arm than in the open arm and showed a normal physiological response to anxiety behavior (Figure 10B).

[0189] However, mice given HFD for 10 weeks spent the same amount of time on both arms and showed a lack of anxiety behavior.

[0190] In contrast, mice given HFD and treated with HIP / PAP exhibited similar behavior to control mice given a control diet, and spent significantly more time with their arms closed compared to their arms open.

[0191] These results are as follows: - Diet is effective in inducing obesity, which was not prevented by HIP / PAP cerebral administration; and - The obesity phenotype induced anxiety deficits, which were prevented in mice treated with HIP / PAP.

[0192] These results support the beneficial role that HIP / PAP plays in cognitive and anxiety-related behaviors, and are therefore of great interest in diet-induced cognitive impairment and anxiety-related dysfunction.

[0193] Sequence List Sequence ID 1 is the amino acid sequence of the HIP / PAP protein lacking the N-terminal 26-amino acid signal peptide.

[0194] [ka]

[0195] Sequence ID 2 is the amino acid sequence of a HIP / PAP protein lacking either the N-terminal 26-amino acid signal peptide or the N-terminal 11-amino acid propeptide.

[0196] [ka]

[0197] Sequence ID 3 is the amino acid sequence of the HIP / PAP protein, which has a deletion of the N-terminal 26-amino acid signal peptide and a methionine molecule added to the N-terminal position.

[0198] [ka]

[0199] Sequence ID 4 is the complete amino acid sequence of the HIP / PAP protein.

[0200] [ka]

[0201] This specification discloses amino acid sequences for reference. The same sequences are also presented in sequence listings formatted according to standard requirements for patent purposes. In the event of any sequence discrepancy with standard sequence listings, the sequences described herein shall be referred to.

Claims

1. HIP / PAP proteins or derivatives thereof for use in the treatment and / or prevention of cognitive impairment associated with anxiety disorder, particularly in individuals requiring treatment.

2. The anxiety disorder associated with the aforementioned cognitive impairment, (i) Major symptoms of obsessive-compulsive disorder, and / or (ii) Secondary symptoms of neurodegenerative diseases selected from the group consisting of attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection. A HIP / PAP protein or derivative thereof for use according to claim 1.

3. HIP / PAP proteins or derivatives thereof for use in improving cognition in individuals suffering from anxiety-related neurological disorders selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and cognitive impairment associated with other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

4. HIP / PAP proteins or derivatives thereof for use in reducing cognitive impairment in individuals suffering from disorders selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and COVID-19 infection.

5. HIP / PAP protein or derivative thereof for use according to any one of claims 1 to 4, wherein the anxiety disorder is an externalization disorder, and in particular the cognitive impairment is selected from the group consisting of bipolar disorder, substance abuse, attention deficit disorder and psychotic disorder.

6. HIP / PAP proteins or derivatives thereof for use in the prevention and / or treatment of diet-induced cognitive impairment and anxiety-related dysfunction, particularly high-fat diet-induced cognitive impairment and anxiety-related dysfunction, in individuals requiring prevention and / or treatment.

7. The HIP / PAP protein or derivative thereof for use according to any one of claims 1 to 6, wherein the individual is a mammal, particularly a human.

8. The HIP / PAP protein for use according to any one of claims 1 to 7, wherein the HIP / PAP protein comprises an amino acid sequence selected from the group consisting of sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and in particular the sequence shown as SEQ ID NO:

4.

9. The derivative comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and having the same biological activity as the amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, more specifically, an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown as SEQ ID NO: 4, and having the same biological activity as the amino acid sequence shown as SEQ ID NO: 4, according to any one of claims 1 to 8.

10. The HIP / PAP protein or derivative thereof for use according to any one of claims 1 to 9, wherein the HIP / PAP protein or derivative thereof is contained in a composition comprising a physiologically acceptable medium.

11. The composition is for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, or rectal administration, wherein the HIP / PAP protein or derivative thereof is for use according to claim 10.

12. The composition is for oral, subcutaneous, intravenous, topical, or local administration, wherein the HIP / PAP protein or derivative thereof is for use according to claim 10 or 11.

13. The composition further comprises at least one agent known to be useful for the prevention and / or treatment of cognitive impairment, the agent being selected from the group consisting of, for example, a cholinesterase inhibitor such as donepezil, rivastigmine, or galantamine; a glutamate modulator such as memantine; a cholinesterase inhibitor in combination with a glutamate modulator, particularly a combination of donepezil and memantine; methylphenidate; amphetamine; atomoxetine; AMPA-R agonist; α7 nicotinic agonist; guanfacine; bupropion; vortioxetine, and D-cycloserine, for use according to any one of claims 10 to 12.

14. The composition further comprises at least one agent known to be useful for the prevention and / or treatment of anxiety disorders, in particular an agent selected from the group consisting of selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium modulators, azapirone, reversible inhibitors of monoamine oxidase A, agomelatine, quetiapine and vortioxetine, more specifically an agent selected from the group consisting of citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine and vortioxetine, for use according to any one of claims 10 to 13.

15. HIP / PAP protein or derivative thereof for use according to claim 14, wherein the cognitive impairment associated with the anxiety disorder is selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early-onset dementia, epilepsy-related cognitive impairment, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar disorder, substance abuse, attention deficit disorder, psychotic disorder, and novel coronavirus (SARS-CoV-2) infection.

16. The composition further comprises at least one agent known to be useful in alleviating symptoms associated with disorders selected from the group consisting of autism spectrum disorder, Angelman syndrome, Down syndrome, and other neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, particularly an agent selected from the group consisting of antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids such as prednisone; and immunosuppressants such as methotrexate or azathioprine, for use according to any one of claims 10 to 15.

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