Hip / PAP protein or derivatives thereof for the treatment of cognitive disorders related

Abstract

The present invention relates to the use of HIP / PAP proteins or derivatives thereof in therapy and prophylaxis, in particular to the use of said HIP / PAP proteins or derivatives thereof in the treatment of cognitive disorders associated with one or more anxiety disorders in an individual in need thereof, and for improving cognitive function in an individual affected by a nervous system condition associated with one or more anxiety disorders, or for alleviating cognitive deficits in a subject suffering from a condition selected from the group consisting of obsessive-compulsive disorder, attention deficit disorder, Lewy body dementia, early onset dementia, epilepsy-related cognitive dysfunction, frontotemporal dementia, posterior cortical atrophy, Huntington's disease (HD), Parkinson's disease, bipolar affective disorder, substance abuse, attention deficit disorder, etc. A psychiatric disorder and an SARS-CoV-2 infection; and for the prevention and / or treatment of diet-induced cognitive and anxiety deficits in an individual in need thereof, in particular for the prevention and / or treatment of cognitive and anxiety deficits caused by high fat diet.

Description

Technical Field

[0001] This invention relates to the use of HIP / PAP protein or derivatives thereof in treatment and prevention, and more particularly to the use of said HIP / PAP protein or derivatives thereof in treating cognitive impairment associated with one or more anxiety disorders in individuals with such need, and for improving cognitive function in individuals affected by neurological disorders associated with one or more anxiety disorders, or for alleviating cognitive deficits in individuals affected by specific cognitive impairments associated with one or more anxiety disorders as described below. Background Technology

[0002] Occasional anxiety is a normal part of life, but anxiety disorders are far more than just temporary worry or fear. For people with anxiety disorders, anxiety doesn't go away on its own; instead, it tends to worsen over time, and symptoms can interfere with daily activities such as work performance, studies, and interpersonal relationships. These symptoms need to be distinguished from normal anxiety and fear, which are healthy emotional responses to everyday stressors such as interpersonal relationships, social interactions, education, and career demands. Anxiety disorders can manifest as elevated anxiety levels (feeling tense, irritable, or anxious, with a sense of impending danger, panic, or doom, difficulty controlling worry, and difficulty taking risks or making decisions) or decreased anxiety levels, the latter also known as behavioral inhibition disorders and externalization disorders (preferring to exhibit strong approach behaviors, lack of self-control, inappropriate laughter, loss of verbal and behavioral control in unfamiliar situations, high novelty-seeking, low harm avoidance; having a thrill-seeking personality, poor impulse control, and a liking for risky behavior—NR Marmorstein, J Anxiety Disord. 2007;21(3):420-32; JT Nigg, PsycholBull. March 2000;126(2):220-46; Dina R Hirshfeld-Becker et al., Biol Psychiatry. June 1, 2003;53(11):985-99) or both (NR Marmorstein, J Anxiety Disord. 2007;21(3):420-32; JT Nigg, PsycholBull. March 2000;126(2):220-46; Dina R Hirshfeld-Becker et al., Biol Psychiatry. June 1, 2003;53(11):985-99) or both (NR Marmorstein, J Anxiety Disord. 2007;21(3):420-32; JT Nigg, PsycholBull. March 2000;126(2):220-46; Dina R Hirshfeld-Becker et al., Biol Psychiatry. June 2003;53(11):985-99). Disord. 2007;21(3):420-32; Audra K Langley et al., Eur Child Adolesc Psychiatry. Aug. 2010;19(8):637-45; B Wanner et al., Psychol Med. Nov. 2012;42(11):2373-82; Joan PYoo et al., Am J Orthopsychiatry. Oct. 2009;79(4):532-40), especially in young people. In some cases, behavioral inhibition may be a precursor to anxiety disorder, suggesting an intricate relationship between the two types of disorders (Dina R Hirshfeld-Becker et al., Biol Psychiatry. Jun. 1, 2003;53(11):985-99).

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

[0004] It is estimated that in 2015, more than 58 million people in the Americas suffered from anxiety disorders, accounting for 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, accounting for the majority of lifetime mental health disorders worldwide (Kessler et al., Epidemiol Psichiatr Soc. Jan–Mar 2009;18(1):23–33).

[0005] Anxiety disorders (including externalization disorders) are often associated with cognitive impairment and may be primary or secondary symptoms.

[0006] Obsessive-compulsive disorder (OCD) is an example of a cognitive disorder with anxiety disorder as the primary symptom. OCD is characterized by the recurring occurrence of unwanted thoughts and fears (obsessive ideas), which prompt patients to repeat certain behaviors or mental activities (compulsive behaviors) to alleviate the anxiety associated with these obsessive ideas (Anu E Castaneda et al., J Affect Disord. Feb. 2008; 106(1-2): 1-27; Ashwini Vishwanathan, Indian J Psychol Med. Nov. 2022; 44(6): 558-566). Cognitive impairment (decreased learning and working memory) has also been demonstrated in externalization disorders and behavioral disinhibition disorders of anxiety disorders, such as bipolar disorder, as well as substance and alcohol abuse (Michael J Endres et al., J Abnorm Psychol. May 2011; 120(2): 336-51).

[0007] Anxiety symptoms can also be secondary to other conditions, especially certain cognitive impairments. In these impairments, changes in cognitive function, specifically when accompanied by mild to severe memory loss, can alter normal responses to anxious situations, thus triggering anxiety disorders.

[0008] Not all cognitive impairments are associated with anxiety disorders. For example, certain degrees of mild cognitive impairment, normal pressure hydrocephalus, or neurological conditions associated with cognitive dysfunction can all be linked to anxiety disorders.

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

[0010] Both share a common mechanism, which includes an imbalance in neurotransmitter transmission in areas that control emotions and anxiety.

[0011] Therefore, there is an increasing need for novel active medicines that can prevent and / or treat cognitive impairments associated with one or more anxiety disorders for individuals in need.

[0012] Furthermore, there is an increasing need to prevent individuals from altering their emotional responses to stressors, particularly for individuals with cognitive impairments, as these impairments are expected to trigger one or more anxiety disorders.

[0013] There is indeed a need for new active pharmaceutical ingredients to restore healthy emotional responses to stress, specifically for individuals affected by cognitive impairments associated with anxiety disorders.

[0014] In addition, there is a need for novel active drugs that can restore an individual's physiological anxiety levels, specifically for individuals affected by cognitive impairments associated with anxiety disorders.

[0015] In addition, there is a need to develop novel active pharmaceutical ingredients to improve memory in individuals affected by cognitive impairments associated with one or more anxiety disorders.

[0016] Therefore, there is an increasing need for novel active pharmaceutical ingredients that can improve cognitive function in individuals affected by one or more neurological disorders associated with anxiety disorders, selected from the group consisting of autism spectrum disorders, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0017] In addition, there is a need to develop novel active medicines to alleviate cognitive deficits in individuals with conditions selected from the following groups: 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 SARS-CoV-2 infection.

[0018] This invention provides solutions to these problems. Summary of the Invention

[0019] The applicant has demonstrated, unexpectedly, that HIP / PAP proteins or their derivatives can improve memory in a mouse model of anxiety-related cognitive impairment. Furthermore, HIP / PAP proteins or their derivatives can rescue cognitive deficits, particularly in this model and in a high-fat diet (HFD) model, restoring normal anxiety-like behavior under anxiety stimuli.

[0020] Without being bound by any particular theory, the inventors hypothesize that HIP / PAP can restore neurotransmitter homeostasis through direct or indirect action (acting on the GABAergic and glutamatergic systems). Indeed, the accompanying examples demonstrate that the anxiety physiology in mice treated with HIP / PAP was restored (to control levels).

[0021] Furthermore, the inventors unexpectedly demonstrated that administration of HIP / PAP protein or its derivatives could both prevent cognitive alteration and improve cognitive function in individuals exhibiting cognitive alteration.

[0022] Therefore, the HIP / PAP protein can be advantageously used for:

[0023] - Treatment and / or prevention, specifically treatment of cognitive impairments associated with one or more anxiety disorders in individuals who require this treatment;

[0024] Specifically, it is the treatment and / or prevention of cognitive impairments related to externalization disorders, which are specifically selected from the following groups: bipolar disorder, substance abuse, attention deficit disorder, and psychotic disorders;

[0025] - To improve cognitive function in individuals affected by one or more neurological disorders associated with anxiety disorders, more specifically, said one or more neurological disorders associated with anxiety disorders being selected from the group consisting of: autism spectrum disorder, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders, such as chronic meningitis, autoimmune encephalitis, or sarcoidosis; and

[0026] - To alleviate cognitive deficits in individuals with conditions selected from the following groups: 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 SARS-CoV-2 infection.

[0027] Therefore, the present invention relates to the following items:

[0028] Project 1: HIP / PAP protein or derivatives thereof, for the treatment and / or prevention of cognitive impairment associated with one or more anxiety disorders in individuals with such needs.

[0029] Project 2: The HIP / PAP protein or its derivatives used according to Project 1, wherein one or more anxiety disorders related to cognitive impairment are:

[0030] (i) Primary symptoms, such as obsessive-compulsive disorder, and / or

[0031] (ii) Secondary symptoms, such as neurodegenerative diseases selected from the following groups: 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 SARS-CoV-2 infection.

[0032] Project 3: A HIP / PAP protein or a derivative thereof, used to improve cognitive function in individuals affected by one or more neurological disorders associated with anxiety disorders, said one or more neurological disorders associated with anxiety disorders being selected from the group consisting of: autism spectrum disorder, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders, such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0033] Project 4: A HIP / PAP protein or a derivative thereof, for the purpose of alleviating cognitive deficits in individuals with conditions selected from the following groups: 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 SARS-CoV-2 infection.

[0034] Project 5: HIP / PAP protein or its derivatives used according to any one of Projects 1 to 4, wherein the anxiety disorder is an externalizing disorder and the cognitive disorder is specifically selected from the group consisting of: bipolar disorder, substance abuse, attention deficit disorder and psychotic disorders.

[0035] Project 6: A HIP / PAP protein or a derivative thereof, used for the prevention and / or treatment of diet-induced cognitive and anxiety deficits in individuals with such needs, specifically cognitive and anxiety deficits induced by a high-fat diet.

[0036] Item 7: The HIP / PAP protein or a derivative thereof used according to any one of Items 1 to 6, wherein the individual is a mammal, specifically a human.

[0037] Item 8: A HIP / PAP protein or a derivative thereof for any of the applications described in any of Items 1 to 7, wherein the HIP / PAP protein comprises an amino acid sequence selected from the group consisting of sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, specifically the sequence shown in SEQ ID NO: 4.

[0038] Project 9: A HIP / PAP protein or a derivative thereof according to any one of Projects 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 sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and having the same biological activity as 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, more specifically, the amino acid sequence having at least 80% sequence identity with an amino acid sequence shown as SEQ ID NO: 4, and having the same biological activity as an amino acid sequence shown as SEQ ID NO: 4.

[0039] Item 10: The HIP / PAP protein or a derivative thereof for any of the applications described in any of Items 1 to 9, wherein the HIP / PAP protein or a derivative thereof is in a composition comprising a physiologically acceptable medium.

[0040] Item 11: HIP / PAP protein or its derivatives as described in Item 10, wherein the composition is for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, tracheal, nasal, or rectal administration.

[0041] Item 12: HIP / PAP protein or a derivative thereof used according to Item 10 or 11, wherein the composition is for oral, subcutaneous, intravenous, topical or local administration.

[0042] Item 13: HIP / PAP protein or a derivative thereof used according to any one of Items 10 to 12, wherein the composition further comprises at least one agent known for the prevention and / or treatment of cognitive impairment, such as agents selected from the group consisting of: cholinesterase inhibitors, such as donepezil, rivastigmine, or galantamine; glutamate modulators; such as memantine; cholinesterase inhibitors, specifically cholinesterase inhibitors in combination with glutamate modulators, such as a combination of donepezil and memantine; methylphenidate; amphetamine; atormoxetine; AMPA-R agonists; "alpha7 nicotinic agonist"; guanifaxine; bupropion; vortioxetine; and D-cyclic serine.

[0043] Item 14: The HIP / PAP protein or a derivative thereof used according to any one of Items 10 to 13, wherein the composition further comprises at least one pharmaceutical agent known for the prevention and / or treatment of anxiety disorders, specifically an agent selected from the group consisting of: selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium channel modulators, azaspiron, monoamine oxidase A reversible inhibitors, agomelatine, quetiapine, and vortioxetine, and more specifically selected from citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine, and vortioxetine.

[0044] Project 15: The HIP / PAP protein or its derivatives used according to Project 14, wherein the cognitive impairment associated with one or more anxiety disorders 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 SARS-CoV-2 infection.

[0045] Item 16: The HIP / PAP protein or a derivative thereof used according to any one of Items 10 to 15, wherein the composition further comprises at least one agent known to be used to relieve symptoms of conditions selected from the group consisting of: autism spectrum disorder, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, wherein the agent is specifically selected from the group consisting of: antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids such as prednisone; and immunosuppressants such as methotrexate or azathioprine. Attached Figure Description

[0046] Figure 1A shows the spontaneous activities of different groups of mice, specifically their movement distances in the open field experiment: the horizontal axis from left to right is as follows: normal control mice (Ntg–n=10); APP / PS1 mice treated with rAAV9 empty capsids (TgVeh–control group–n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP–n=8).

[0047] Vertical axis: Total distance traveled (meters)

[0048] Figure 1 B shows the resting time of different groups of mice in the open field experiment: the horizontal axis from left to right is as follows: normal control mice (Ntg–n=10); APP / PS1 mice treated with rAAV9 empty capsids (Tg Veh–control group–n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP–n=8).

[0049] Vertical axis: Total stationary time (seconds)

[0050] Figure 2 The results of the novel object recognition test were shown for different groups of mice: the horizontal axis from left to right is as follows: normal control mice (Ntg–n=10); APP / PS1 mice treated with rAAV9 empty capsids (Tg Veh–control group–n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP–n=8).

[0051] The results for each group are shown in cases where a familiar object (left) or a new object (right) is present.

[0052] Vertical axis: Time spent with the object (seconds)

[0053] Two-way ANOVA was used, followed by Tukey multiple comparisons post-hoc tests, with a significance level set at p < 0.05. Data analysis was performed using GraphPad Prism version 9 (GraphPad software, San Diego, California, www.Graphpad.com).

[0054] Figure 3 A and 3B show the results of the radial arm water maze test on different groups of mice: normal control mice (Ntg–n=10); APP / PS1 mice treated with rAAV9 empty capsids (Tg Veh–control group–n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP–n=8).

[0055] Figure 3A shows the average number of errors for each group of mice, calculated in blocks of 3 consecutive trials. There were 15 trials on day 1 (groups 1 to 5) and 15 trials on day 2 (groups 6 to 10). From left to right, they are: Ntg, Tg Veh, and Tg HIP / PAP.

[0056] y-axis: number of errors

[0057] Figure 3 B represents the sum of the total number of mistakes made by mice in each group on day 2 (groups 5 to 10) (Ntg ;Tg Veh Tg HIP / PAP ).

[0058] y-axis: number of errors

[0059] For error groups within 2 days ( Figure 3 A) A two-way repeated measures ANOVA was used, followed by Tukey's multiple comparisons post-hoc test, with the significance level set at p < 0.05. For the total number of errors on day 2 ( Figure 3 B) One-way ANOVA was used, followed by Tukey multiple comparisons post-hoc tests, with a significance level set at p < 0.05. Data analysis was performed using GraphPad Prism version 9 (GraphPad software, San Diego, California, www.Graphpad.com).

[0060] Figure 4 A and 4B show the results of the radial arm water maze reversal test on different groups of mice: normal control mice (Ntg–n=10); APP / PS1 mice treated with rAAV9 empty capsids (Tg Veh–control group–n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP–n=8).

[0061] Figure 4 A shows the groups of mice (Ntg) ;Tg Veh Tg HIP / PAP The average number of errors at the end of the day was calculated by averaging every 3 consecutive trials as a group, with a total of 15 trials per group.

[0062] Vertical axis: Groups 1 to 5 (from left to right).

[0063] Figure 4 B shows the total number of errors made by each group of mice in all trials (from left to right: Ntg, Tg Veh, Tg HIP / PAP).

[0064] For error group ( Figure 4 A) A two-way repeated measures ANOVA was used, followed by Tukey's multiple comparisons post-hoc test, with the significance level set at p < 0.05. For the total number of errors ( Figure 4 B) One-way ANOVA was used, followed by Tukey multiple comparisons post-hoc tests, with a significance level set at p < 0.05. Data analysis was performed using GraphPad Prism version 9 (GraphPad software, San Diego, California, www.Graphpad.com).

[0065] Figure 5 The anxiety-like behaviors of the mice in each group were assessed based on the time spent in the open and closed arms of the elevated cross maze test (EPM). Figure 5 A), and a comparison of the residence time in the closed arm between different groups ( Figure 5 B).

[0066] The horizontal axis from left to right represents: normal control mice (Ntg–n=10); APP / PS1 mice treated with rAAV9 empty capsids (Tg Veh–control group–n=8); or APP / PS1 mice treated with rAAV9-hHIP / PAP (Tg HIP / PAP–n=8).

[0067] The results for each group are shown in the cases of open arm (left) and closed arm (right).

[0068] Vertical axis: Number of entries

[0069] Figure 6 A demonstrates a construct of recombinant adeno-associated virus serotype 9 (rAAV9) for overexpression of hemagglutinin (HA)-tagged human HIP / PAP.

[0070] Figure 6 B shows the results of immunohistochemical staining of the hippocampus with anti-HA antibody in APP / PS1 mice injected with empty rAAV9 capsids 6 months after expression.

[0071] Figure 6 C shows the results of immunohistochemical staining of the hippocampus with anti-HA antibody in APP / PS1 mice injected with rAAV9-HIP / PAP capsid 6 months after expression.

[0072] Figure 6D shows the level of human HIP / PAP protein in the cerebral cortex of APP / PS1 mice measured by ELISA 6 months after injection of rAAV9-HIP / PAP. HA refers to human influenza hemagglutinin. Scale bar: 100 µm, inset scale bar: 30 µm.

[0073] Horizontal axis: Cortex; Vertical axis: HIP / PAP concentration (pg / mg).

[0074] Figure 7 A and Figure 7 B shows the rAAV9-empty capsid in the APP / PS1 mouse groups respectively. Figure 8 A) Processed or via rAAV9-HIP / PAP ( Figure 8 B) Microscopic images of Congo red staining 6 months after treatment. Scale bar: 100 µm.

[0075] Figure 7 C shows the quantitative results of the percentage of Congo red positive staining area in the anterior cortex (ACX), hippocampus (HPC), and posterior cortex (PCX) of the two groups of mice (APP / PS1 mouse groups, treated with rAAV9-empty capsid or rAAV9-HIP / PAP for 6 months, respectively) using Nearcyte software.

[0076] Vertical axis: Area of ​​positive staining (ratio).

[0077] The horizontal axis, from left to right, represents ACX, HPC, and PCX. Within each region, from left to right, are: mouse groups treated with rAAV9-empty capsid (APP / PS1 empty) and mouse groups treated with rAAV9-HIP / PAP (APP / PS1 HIP / PAP).

[0078] Data are represented as mean ± SEM.

[0079] Figure 7 D shows the results of ELISA quantification of β-amyloid protein levels in the ACX and HPC regions of the two groups of mice (APP / PS1 mice, treated with rAAV9-empty capsid or rAAV9-HIP / PAP for 6 months, respectively).

[0080] Vertical axis: Concentration (ng / mL).

[0081] Horizontal axis: from left to right: ACX and HPC. Each region is divided from left to right into: mouse group treated with rAAV9-empty capsid (APP / PS1 empty) and mouse group treated with rAAV9-HIP / PAP (APP / PS1 HIP / PAP).

[0082] Data are represented as mean ± SEM.

[0083] Figure 8 The results of Western blot analysis include micrographs of the blots. Figure 8 A) and catalase in the hippocampus of APP / PS1 mice treated with rAAV9 empty capsid (APPPS1 empty) or rAAV9-HIP / PAP (APPPS1 HIP / PAP). Figure 8 B) Heme oxygenase 1 (Ho-1) Figure 8 C) and superoxide dismutase (SOD-1) Figure 8 D) Results of quantitative analysis of band density, compared with the non-transgenic control group (Ntg). N=6-10. Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01. One-way ANOVA was used, followed by Tukey multiple comparison post-hoc tests. Outliers were removed using Prism software according to the Grubb test.

[0084] Figure 8 The vertical axis from B to 8D represents the ratio relative to total protein.

[0085] Figure 8 The horizontal coordinates from B to 8D are: Ntg, APP / PS1 empty, and APP / PS1 HIP / PAP, respectively.

[0086] Figure 9 A shows the concentrations of HIP / PAP in plasma and brain tissue samples 28 days after subcutaneous administration of rHIP / PAP (or ALF5755) to mice (n=7) via an Alzet pump.

[0087] Left vertical axis: Concentration (mg / mL)

[0088] Right vertical axis: Concentration (pg / mL)

[0089] Figure 9 B indicates an IVIS (in vivo imaging system) image of non-transgenic mice 30 minutes after intravenous injection of 250 µg bovine serum albumin (BSA) (top image) or rHIP / PAP labeled with vivotag 680XL (Revvity, Hopkinton, Massachusetts, USA) (bottom image).

[0090] Figure 9 C and 9D represent the above-mentioned intravenous injections (heart, Figure 9 C, n=3) or intramuscular injection (hind limb, Figure 9 D, n=2)rAAV9-HIP / PAP 2x10 13One month after VG, plasma samples ( Figure 9 C) and brain samples ( Figure 9 The concentration of HIP / PAP in D).

[0091] Figure 10 A represents the 10-week weight follow-up of non-transgenic mice on a control diet (Ntg CD, n=6), a high-fat diet (Ntg HFD, n=5), or a high-fat diet combined with rAAV9-HIP / PAP treatment (Ntg HFD HIP / PAP, n=4).

[0092] Vertical axis: Weight (% of baseline weight)

[0093] Horizontal axis: Time (weeks, from 0 to 10)

[0094] Figure 10 B shows the time mice spent in the open arm "o" and the closed arm "c" in the elevated cross maze (EPM) test. Data are presented as mean ± SEM. * p<0.05, ** p<0.01, *** p < 0.001. Depending on the circumstances, one-way ANOVA, two-way ANOVA, or repeated measures ANOVA should be used, followed by Tukey's multiple comparisons post-hoc test.

[0095] Vertical axis: Dwell time of each arm (s)

[0096] The horizontal axis, from left to right, represents: the control diet non-transgenic mouse group (Ntg CD), the high-fat diet non-transgenic mouse group (Ntg HF), and the high-fat diet non-transgenic mouse group treated with rAAV9-HIP / PAP (Ntg HFD HIP / PAP). For each group, the left side represents the closed arm results, and the right side represents the open arm results. Detailed Implementation

[0097] definition -

[0098] In the context of this invention, the terms "prevention," "avoidance," and "avoidance" all refer to reducing the risk or probability of a specific phenomenon occurring. Specifically, in this invention, it refers to preventing anxiety-related cognitive impairment, particularly preventing complications selected from the group consisting of diabetic foot ulcers, foot infections, and amputations. The terms "prevention," "avoidance," and "avoidance" also include preventing the worsening of the condition, slowing its progression, or preventing its recurrence.

[0099] As used herein, the terms “treatment,” “management,” or “therapy” include relieving and / or eliminating symptoms associated with a particular symptom or condition; that is, in this invention, it refers to treating cognitive impairments related to anxiety.

[0100] Cognitive impairment refers to any condition that severely impairs an individual's cognitive function to the point that, without treatment, they cannot integrate normally into society. Cognitive impairment affects a person's thinking, learning, memory, judgment, and decision-making abilities. The signs and symptoms of cognitive impairment vary depending on the specific disease, but most conditions share some common signs and symptoms. Some of the most common signs and symptoms of cognitive impairment include: confusion, poor motor coordination, identity impairment, impaired judgment, memory loss (short-term or long-term), inattention, difficulty completing tasks, comprehension difficulties, memory problems, difficulty following instructions, and decreased problem-solving abilities. Other common signs may include mood or behavioral changes, lack of motivation, and a lack of awareness of the surrounding environment. The severity of cognitive impairment can range from mild to severe. Some cognitive impairments develop in stages, with symptoms worsening as the disease progresses. Cognitive instability has both short-term and long-term effects. Some common short-term effects include decreased thinking and reasoning abilities, memory loss, confusion, and decreased coordination. Long-term effects include progressive loss of declarative memory, such as forgetting names and important faces, a general lack of emotional stability and control over one's own behavior, and dementia (altered mental state and level of consciousness, attention deficit, mood swings, violence or abnormal behavior, and hallucinations). According to the present invention, the cognitive impairment of interest is cognitive impairment associated with one or more anxiety disorders. "Cognitive impairment associated with one or more anxiety disorders" refers to an individual studied who is simultaneously affected by at least one cognitive impairment and at least one anxiety disorder, wherein the anxiety disorder is the primary or secondary symptom of the cognitive impairment. The term "primary symptom" refers to a symptom not caused by other diseases. "Secondary symptom" refers to a symptom caused by other diseases. Therefore, according to the present invention, the term "cognitive impairment associated with one or more anxiety disorders" includes both cognitive impairment caused by one or more anxiety disorders and cognitive impairment that leads to the development of anxiety disorders.

[0101] Different methods can be used to diagnose cognitive impairment, including the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), Mini-Cog and Cognitive Assessment Method (CAM), Glasgow Coma Scale (GCS), and Richmond Agitation and Sedation Scale (RASS) (Kelvin KF Tsoi et al., JAMA Intern Med. Sep 2015; 175(9):1450-8).

[0102] Anxiety disorders are defined in detail. While occasional anxiety is a common experience, anxiety disorders differ from typical or temporary feelings of worry or fear. Anxiety disorders can manifest in various forms, including elevated anxiety levels, which can lead to tension, restlessness, and a sense of impending danger, as well as difficulty controlling worry and making decisions. On the other hand, anxiety disorders can also manifest as decreased anxiety levels, which can lead to behavioral instability and externalization disorders. These disorders are characterized by a strong tendency to approach new environments, a lack of self-control, inappropriate laughter, and verbal and behavioral instability. They may also include a high degree of novelty-seeking, low risk aversion to harm, poor impulse control, and a tendency to engage in risky behaviors. In some cases, behavioral instability can even be a precursor to anxiety disorders. Both mood disorders and anxiety disorders are complex illnesses involving disturbances in the neuroendocrine, neurotransmitter, and neuroanatomical systems. Differentiating between these disorders can be difficult due to the complex interactions between different neural circuits in the brain. However, high and low anxiety levels may stem from similar underlying causes, namely, an imbalance in neurotransmitter signaling.

[0103] Various questionnaires can be used to diagnose anxiety disorders, such as the State-Trait Anxiety Scale (STAI), Generalized Anxiety Disorder 7 (GAD-7), Beck Anxiety Scale (BAI), Zung Self-Rating Anxiety Scale, and Tyler Overt Anxiety Scale (Matthias Rose and Janine Devine, Dialogues Clin Neurosci. 2014 June;16(2):197-211). Diagnosis of anxiety disorders is based on symptoms, triggering factors, and the patient's personal and family history. Currently, there are no objective biomarkers or laboratory tests that can diagnose anxiety disorders.

[0104] Substance abuse refers to the excessive and persistent use of a substance, such as alcohol or drugs, despite the negative impacts on an individual's physical, mental, or social health. Substance abuse can lead to addiction, a chronic and relapsing condition characterized by compulsive seeking of the substance and continued use despite knowing the harmful consequences. The diagnosis of substance abuse is typically based on multiple criteria, such as tolerance (requiring increased dosage to achieve the desired effect), withdrawal symptoms, continued use despite knowing the harmful consequences, and failed attempts to quit or reduce dosage. It is important to note that the definition of substance abuse can vary depending on the substance involved and the context of its use.

[0105] Attention deficit disorder (ADD), including attention deficit hyperactivity disorder (ADHD), is a disorder characterized by inattention, hyperactivity, and impulsivity. Symptoms of inattention may include difficulty concentrating, forgetfulness, disorganization, and easy distractibility. Hyperactivity symptoms may include restlessness, agitation, difficulty sitting still, and excessive talking. Impulsivity symptoms may include acting without thinking, interrupting others, and difficulty waiting for one's turn. The diagnosis of ADD / ADHD is usually based on the combination of symptoms, duration, and extent of functional impairment, while ruling out other medical or psychological conditions that may cause similar symptoms.

[0106] Psychotic disorders are a group of mental illnesses characterized by a loss of connection with reality, and can include a variety of symptoms such as delusions, hallucinations, thought disorders, and abnormal behavior. Delusions are fixed beliefs not based on reality, often manifesting as bizarre or paranoid. Hallucinations are sensory experiences not based on external stimuli, such as hearing sounds that are not present or seeing things that are not present. Thought disorders may include difficulty with logical thinking, incomprehensible speech, and jumping between unrelated topics. Abnormal behavior may include bizarre gestures or movements, lack of emotional expression, and social withdrawal.

[0107] Psychotic disorders can significantly impact a patient's ability to live daily lives, ranging in severity from mild to severe. The most common psychotic disorders are schizophrenia, schizoaffective disorder, and delusional disorder.

[0108] Cholinesterase inhibitors are chemical substances that prevent the breakdown of the neurotransmitter acetylcholine. Examples of cholinesterase inhibitors include donepezil, rivastigmine, and galantamine. Glutamate modulators are chemical substances that regulate the activity of the chemical messenger glutamate, which helps the brain process information. These modulators are known for improving memory, attention, reasoning, language skills, and the ability to perform simple tasks. An example of a glutamate modulator is memantine. NMDA receptor antagonists are chemical substances that non-selectively reduce the abnormal activity of NMDA receptors.

[0109] "α7 nicotinic acetylcholine receptor agonists" are chemical substances used to treat attention disorders. Examples of such agonists include, for example, clozapine or 3-2,4-dimethoxybenzyl anabasin (DMXBA). Other examples of α7 nicotinic acetylcholine receptor agonists are also known, for example, Laura F Martin et al., Psychopharmacology (Berlin), June 2004; 174(1):54-64, which are incorporated herein by reference.

[0110] Selective serotonin reuptake inhibitors (SSRIs) are currently the most commonly used antidepressants. Examples of such inhibitors include citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, or sertraline.

[0111] "Serotonin-norepinephrine reuptake inhibitors" are a class of antidepressants that inhibit the reuptake of serotonin and norepinephrine. Examples of such inhibitors are duloxetine and venlafaxine.

[0112] "Calcium channel modulators," also known as calcium channel blockers, include gabapentin, which is currently the most widely prescribed medication. They work by inhibiting pain signals transmitted through voltage-gated calcium channels located at the nerve endings in the central nervous system. Pregabalin is an example of such a modulator.

[0113] "Reversible monoamine oxidase A inhibitors (RIMAs)" can inhibit the breakdown of three major neurotransmitters—serotonin, norepinephrine, and dopamine—thus providing a multi-neurotransmitter strategy for treating depression. Moclobemide is an example of such a chemical.

[0114] Antiepileptic drugs are medications that prevent or treat epileptic seizures or convulsions by controlling abnormal electrical activity in the brain. They are used to treat epilepsy and other epileptic disorders. They are also used to treat a variety of medical conditions, such as bipolar disorder, neuralgia, migraines, fibromyalgia, and restless legs syndrome.

[0115] "Antifungal drugs" are medications that can kill or prevent the growth of various fungi that cause infections. They are also known as antifungal agents.

[0116] "Corticosteroids," commonly known as steroids, are anti-inflammatory drugs. For example, prednisone is a corticosteroid.

[0117] Immunosuppressants, also known as immunosuppressive drugs, are key treatments that suppress or prevent the activity of the immune system. For example, methotrexate and azathioprine are both immunosuppressants.

[0118] "Normal response to anxiety situations" refers to physiological and psychological responses that are proportionate to the perceived level of threat or stress in the anxiety situation.

[0119] The term "anxiety situation" refers to any situation or circumstance that may trigger or exacerbate an individual's anxiety. These situations may include, but are not limited to, public speaking, conflict, social occasions, uncertainty about the future, or situations involving potential threats to personal safety or well-being. In short, anxiety situations are those that are likely to trigger anxiety or fear in an individual.

[0120] The term "physiologically acceptable medium" refers to a medium compatible with the constitution of the individual to whom the composition must be administered. For example, it can be a non-toxic solvent, such as water. Specifically, the medium is suitable for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, tracheal, nasal, or rectal administration, and is particularly suitable for oral, subcutaneous, intravenous, topical, or local administration.

[0121] The terms “sequence homology,” “sequence identity,” “homology,” or “identity” are used interchangeably herein. For the purposes of this invention, it is defined herein that, in order to determine the percentage of sequence homology or sequence identity between two amino acid sequences or two nucleic acid sequences, it is necessary to align the sequences for optimal comparison. To optimize the alignment between two sequences, a gap may be introduced in either of the two sequences to be compared. Such alignments can be performed over the full length of the sequences to be compared. Alternatively, alignments can be performed over a shorter length, such as about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Sequence identity refers to the percentage of identical matches between two sequences in the reported alignment region. The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. Those skilled in the art will recognize that several different computer programs are available for comparing two sequences and determining their identity (Kruskal, JB (1983) An overview of sequence comparison, in D. Sankoff and JB Kruskal, (eds.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44, Addison Wesley).

[0122] The Needleman-Wunsch algorithm, used to align two sequences, can determine the percentage of sequence identity between two amino acid sequences or two nucleotide sequences (Needleman, SB and Wunsch, CD (1970) J.Mol. Biol. 48, 443-453). This algorithm can be used for both amino acid and nucleotide sequence alignment. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.

[0123] This invention uses the NEEDLE program (version 2.8.0 or later) from the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, the substitution matrix is ​​EBLOSUM62. For nucleotide sequences, EDNAFULL is used. Optional parameters are: 10 for nick opening penalty and 0.5 for nick extension penalty. No end nick penalty is added. In the output section, the "Brief Identity and Similarity" option is set to "Yes", and the output alignment format is set to "SRS Paired".

[0124] As described above, after alignment using the NEEDLE program, the percentage of sequence similarity between the query sequence and the sequence of this invention is calculated as follows: the number of corresponding positions with the same amino acid or nucleotide in the two sequences in the alignment result, divided by the total length of the alignment result (excluding the total number of gaps in the alignment result). The identity defined herein can be obtained by using the NOBRIEF option of NEEDLE and marked as "longest-identity" in the program output.

[0125] Nucleotide and amino acid sequence similarity, i.e., the percentage of sequence identity, can be determined by sequence alignment using a variety of other commonly used algorithms. Preferred algorithms include the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), hmmalign (HMMER software package, http: / / hmmer.wustl.edu / ), CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80, available for example from https: / / www.ebi.ac.uk / Tools / msa / clustalo / ), GAP program (mathematical algorithm from the University of Iowa), the mathematical algorithm of Myers and Miller (1989–Cabios 4: 11-17), or Clone Manager 9. The preferred parameters are the default parameters set at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0126] The level of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are also integrated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. The BLASTN program is used to perform a BLAST polynucleotide sequence search (score=100, word length=12) to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins.

[0127] BLAST protein search (score=50, word length=3) can be performed using the BLASTP program to obtain amino acid sequences homologous to the SHC peptide. For cleavage alignment results for comparative purposes, cleavage BLAST can be used as described by Altschul et al. (1997) NucleicAcids Res. 25, 3389-3402. When using BLAST and cleavage BLAST programs, the default parameters for the respective programs can be used. Sequence matching analysis can be supplemented by established homology mapping techniques, such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Supplement 1: 154-162) or Markov random fields. Unless otherwise specified, the percentage of sequence identity mentioned in this application is calculated based on the full length of the longer sequence.

[0128] In a specific implementation, CLUSTAL O (version 1.2.4) is used to determine the identity between two sequences.

[0129] As used herein, the term "peptide" refers to a molecule containing more than five amino acid residues linked by peptide bonds. Amino acids can be represented by a single letter or a three-letter symbol. As used herein, the terms "protein" and "peptide" are synonymous and can also refer to two or more polypeptides. Therefore, the terms "protein," "peptide," and "peptide" are used interchangeably. Polypeptides may optionally be modified (e.g., glycosylation, phosphorylation, acylation, farnesylation, isopreneation, sulfonation, etc.) to add functional groups.

[0130] HIP / PAP protein and its derivatives according to the present invention

[0131] HIP / PAP protein is known for its anti-apoptotic and mitotic activities against hepatocytes (US 13 / 032,521, WO 2004 / 112824, Simon et al., FASEB J. Aug 2003; 17(11): 1441-50).

[0132] The study also showed that a 15-amino acid peptide derived from the Reg IIIa family (HIP / PAP), namely HIP (human islet propeptide), has regenerative activity on the islets, thereby stimulating insulin production (US2010 / 0093605).

[0133] The HIP / PAP protein according to the present invention may comprise an amino acid sequence selected from the group consisting of sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, specifically the sequence shown in SEQ ID NO: 4.

[0134] The amino acid sequence SEQ ID NO: 1 corresponds to the HIP / PAP protein with the sequence SEQ ID NO: 4, which lacks the N-terminal 26 amino acid signal peptide.

[0135] In a specific embodiment, the HIP / PAP protein according to the present invention comprises or is composed of the following amino acid sequence as shown in SEQ ID NO: 4.

[0136] In a specific embodiment, the HIP / PAP protein according to the present invention comprises or is composed of the following amino acid sequence as shown in SEQ ID NO: 1.

[0137] The amino acid sequence SEQ ID NO:2 corresponds to the short form of the HIP / PAP protein, which, compared to the amino acid sequence SEQ ID NO:1, lacks 11 amino acid propeptides at the N-terminal position.

[0138] In a specific embodiment, the HIP / PAP protein according to the present invention comprises or is composed of the following amino acid sequence as shown in SEQ ID NO: 2.

[0139] The sequence SEQ ID NO: 3 corresponds to the sequence SEQ ID NO: 1, with a methionine added to its N-terminus. The HIP / PAP derivative of sequence SEQ ID NO: 3 is also known as rcHIP / PAP or ALF5755. This derivative can be specifically produced by recombinant synthesis in E. coli. The 12-amino acid N-terminal propeptide (an 11-amino acid propeptide plus the added methionine) can be cleaved to obtain the short form of the HIP / PAP protein (SEQ ID NO: 2).

[0140] In a specific embodiment, the HIP / PAP protein according to the present invention comprises or is composed of the following amino acid sequence as shown in SEQ ID NO: 3.

[0141] According to the present invention, short or long forms of HIP / PAP protein or its derivatives may be used without distinction.

[0142] The HIP / PAP protein derivatives according to the present invention refer to the bioactive derivatives of HIP / PAP proteins of any of the sequences SEQ ID No. 1 to 4. The term "bioactive" means that the HIP / PAP protein derivatives have the same bioactivity as HIP / PAP proteins of any of the sequences SEQ ID No. 1 to 4.

[0143] The HIP / PAP protein derivative according to the present invention comprises or consists of an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, specifically SEQ ID NO: 4; and having the same biological activity as an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, specifically SEQ ID NO: 4.

[0144] As previously stated, the bioactivity of the HIP / PAP protein according to the present invention, having the same properties, can treat and / or prevent peripheral neuropathy in an individual, specifically diabetic peripheral neuropathy, and correspondingly prevent complications of peripheral neuropathy in an individual, specifically the complications described in detail elsewhere herein.

[0145] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses 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 having the same biological activity as the reference amino acid sequence.

[0146] In some embodiments, HIP / PAP proteins or derivatives thereof can be linked or bound to non-HIP / PAP portions via non-covalent bonds. For example, HIP / PAP proteins or derivatives thereof can be linked to liposome particles. Depending on the type of liposome or the manufacturing process, HIP / PAP proteins or derivatives thereof can be attached to the surface of the liposome or encapsulated within the liposome.

[0147] HIP / PAP proteins or their derivatives can also be covalently linked to non-HIP / PAP moieties. These non-HIP / PAP moieties can be selected from protein or non-protein compounds, such as polyethylene glycol, to form PEGylated HIP / PAP derivatives.

[0148] The HIP / PAP protein derivatives according to the present invention also include derivatives that are only biologically active when administered to patients.

[0149] Finally, derivatives of HIP / PAP proteins also include chimeric or fusion proteins. These proteins are fused with non-HIP / PAP peptides. The latter can be fused with the N-terminal or C-terminal portion. Typically, to facilitate the purification of recombinant proteins, HIP / PAP proteins or their derivatives can be fused with a GST sequence at their C-terminal portion.

[0150] In some embodiments of the present invention, the HIP / PAP protein or its derivatives according to the present invention are recombinantly produced in bacterial or animal cells (including insect and mammalian cells) using techniques known to those skilled in the art.

[0151] In other embodiments, the HIP / PAP protein or its derivatives according to the present invention can be isolated from cells or tissues using known purification techniques.

[0152] HIP / PAP proteins and their derivatives can also be produced by chemical synthesis.

[0153] In this article, the term "HIP / PAP protein" encompasses the HIP / PAP protein itself, as well as its derivatives as described above.

[0154] Composition according to the present invention

[0155] The present invention also relates to the application of a previously defined HIP / PAP protein or a derivative thereof in a composition comprising a physiologically acceptable medium.

[0156] Physiologically acceptable media, as previously defined herein, may be selected from commonly used excipients known to those skilled in the art, depending on the desired form of medicine and route of administration (see Remington's Pharmaceutical Sciences, 16th edition, Osol, ed. A, 1980).

[0157] For example, the composition according to the present invention may comprise, according to the therapeutic indication and HIP / PAP protein or a derivative thereof:

[0158] a) HIP / PAP protein or its derivatives; and

[0159] b) A buffer solution capable of maintaining pH within its maximum stable range, preferably pH 1 to 9, more specifically pH 4 to 8, and more specifically pH 6 to 7.5; and / or

[0160] c) Detergents or surfactants that stabilize proteins or peptides to prevent aggregation caused by agitation; and / or

[0161] d) Isotonic agents; and / or

[0162] e) Preservatives, such as those selected from the group consisting of phenols, benzyl alcohol, benzothiazolium halides, and chlorides; and / or

[0163] f) Water.

[0164] If the detergent or surfactant used is nonionic, it can be selected from polysorbate, PLURONIC™, polyethylene glycol (PEG), or poloxamer.

[0165] Isotonic agents maintain the isotonicity of a composition and typically contain polyols such as glycerol, erythritol, arabinitol, xylitol, sorbitol, or mannitol, which can be used alone or in combination. Alternatively, sodium chloride and / or any other inorganic salt can be used as an isotonic agent.

[0166] Depending on the required pH, the buffer can be, for example, acetate, citrate, succinate, phosphate buffer, or any other inorganic buffer.

[0167] Phenol, benzyl alcohol, benzothiazolium halides, and chloride preservatives are known antimicrobial agents. Typical preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkyl esters of p-hydroxybenzoate (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0168] Other excipients may include antioxidants (such as ascorbic acid and methionine), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), etc.

[0169] The HIP / PAP protein or its derivatives according to the present invention can exist in pharmaceutically acceptable salt forms. This refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases, including organic salts and acids, and inorganic salts and acids. For example, alkali metal salts (sodium and potassium salts), alkaline earth metal salts (calcium and magnesium salts), ammonium salts, organic base salts (pyridinium or triethylamine salts), inorganic acid salts (hydrochloride, sulfate, nitrate), and organic acid salts (acetate, oxalate, p-toluenesulfonate) may be mentioned.

[0170] The compositions according to the present invention can be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, topically, locally, intratracheally, intranasally, or rectally, and are particularly suitable for oral, subcutaneous, intravenous, topical, or local administration.

[0171] According to a preferred embodiment, the HIP / PAP protein is administered in an effective amount, i.e., the amount required to achieve the intended effect of the invention. The amount of HIP / PAP is typically determined empirically based on the patient being treated and their pathological condition. The effective amount also depends on the intended administration method. Adjustments to determine the effective amount required to obtain the maximum therapeutic effect are routine techniques commonly used by clinicians.

[0172] For example, the effective dose of HIP / PAP protein or its derivatives can be from 0.1 µg / day / kg body weight to 100 mg / day / kg body weight (for individuals who must receive the administration). Although in some embodiments, the effective dose of HIP / PAP protein or its derivatives can be more than 10 mg / kg, according to the present invention, the effective dose of HIP / PAP protein or its derivatives is generally less than 5 mg / kg body weight, including less than 4.5 mg / kg, 4 mg / kg, 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, or 2000 μg / kg. More specifically, the effective amount of the HIP / PAP protein or its derivatives described in this invention comprises 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, 60 μg / kg, 70 μg / kg, 80 μg / kg, 90 μg / kg, 100 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg, 400 μg / kg, 450 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, relative to the individual body weight to which the protein is administered or must be administered. μg / kg, 800 μg / kg, 900 μg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg or higher.

[0173] According to a specific implementation, the HIP / PAP protein or its derivatives are administered at a dose of 10 to 5000 µg / kg relative to body weight, preferably 100 to 2000 µg / kg.

[0174] In the compositions according to the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, external, topical, intratracheal, intranasal, or rectal administration, the active ingredient (HIP / PAP protein or its derivative) may be mixed with a pharmaceutical excipient and administered in a single-dose manner.

[0175] When the composition is intended for oral administration, the composition may be selected from the group consisting of: food, beverage, pharmaceutical, nutritional supplement, food additive, food supplement and dairy products.

[0176] The preferred routes of administration are oral, subcutaneous, intravenous, topical, or local, and more specifically, subcutaneous, intravenous, topical, or local.

[0177] The compounds or compositions of the present invention may be administered, for example, by means of a sheath, patch, pad, dressing, bandage, tape, gauze dressing, woven or non-woven sponge, or syringe.

[0178] HIP / PAP proteins or their derivatives may be sterilized before in vivo administration. Sterilization can be achieved through filtration using a sterile filter membrane and can be performed before or after lyophilization or reconstitution. HIP / PAP proteins or their derivatives administered systemically are preferably lyophilized or stored in solution form. Lyophilized forms of HIP / PAP proteins or their derivatives are typically formulated in combination with excipients for reconstitution with appropriate diluents at the time of use.

[0179] HIP / PAP protein or its derivatives can be taken once daily or in divided doses (e.g., 2 to 3 times daily) until the desired therapeutic effect is achieved. Long-term use is also possible.

[0180] HIP / PAP protein or its derivatives may also be administered in courses of treatment, such as 15 days to 3 months, optionally repeated 1 to 6 times according to a determined dose and time interval.

[0181] According to the present invention, HIP / PAP proteins or derivatives thereof may also be used in combination with agents known for the treatment and / or prevention of cognitive impairment in cases of multidrug therapy. Other agents known for the prevention and / or treatment of cognitive impairment are well known to those skilled in the art and may specifically be selected from the group consisting of: cholinesterase inhibitors, such as donepezil, rivastigmine, or galantamine; glutamate modulators, such as memantine; cholinesterase inhibitors, specifically cholinesterase inhibitors used in combination with glutamate modulators, such as a combination of donepezil and memantine; methylphenidate; amphetamine; atomoxetine; AMPA receptor agonists; "α7 nicotinic acetylcholine receptor agonists"; guanfacine; bupropion; vortioxetine; and D-cyclic serine.

[0182] According to the present invention, HIP / PAP protein or its derivatives may also be used in combination with pharmaceuticals known for the treatment and / or prevention of anxiety disorders in cases of polypharmacy. Other pharmaceutical agents known to be used for the prevention and / or treatment of anxiety disorders are well known to those skilled in the art and may be specifically selected from the group consisting of: selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium channel modulators, azaspiron, monoamine oxidase A reversible inhibitors, agomelatine, quetiapine, and vortioxetine; more specifically, may be selected from the group consisting of: citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine, and vortioxetine.

[0183] The compositions according to the present invention may, in addition to the HIP / PAP protein or its derivatives of the present invention, contain at least one agent known for the prevention and / or treatment of cognitive deficits and at least one agent known for the prevention and / or treatment of anxiety disorders, including cases where one agent exerts both effects simultaneously, i.e., known for the prevention and / or treatment of cognitive deficits and anxiety disorders, such as vortioxetine.

[0184] The term "combined administration" refers to the simultaneous or sequential administration of the HIP / PAP protein, its derivatives, or compositions containing the protein according to the present invention with other agents or compounds. When they are present in separate compositions, compositions containing the HIP / PAP protein, its derivatives, and compositions containing at least one other agent according to the present invention can be administered via the same or different routes.

[0185] According to the present invention, HIP / PAP protein or its derivatives and at least one adjuvant may be administered in the same composition or in separate compositions.

[0186] "At the same time" means that these medications can be taken at the same time, on the same day, or within several days.

[0187] The term "in sequence" means that these compositions can be taken at least a few days apart, for example, at least two days apart.

[0188] Implementation of HIP / PAP proteins and / or their derivatives

[0189] As previously stated, HIP / PAP proteins, their derivatives, and compositions containing such proteins can be used for:

[0190] - Treatment and / or prevention, specifically treating cognitive impairments associated with one or more anxiety disorders in individuals with this need; and / or

[0191] Specifically, this involves the treatment and / or prevention of cognitive impairments related to externalizing disorders, specifically selected from the group consisting of bipolar disorder, substance abuse, attention deficit disorder, and psychotic disorders; and / or

[0192] - To restore the physiological anxiety level of an individual, specifically an individual affected by cognitive impairment associated with anxiety disorders; and / or

[0193] - To improve cognitive function in individuals affected by one or more neurological disorders associated with anxiety disorders, which are selected from the group consisting of: autism spectrum disorder, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0194] - To alleviate cognitive deficits in individuals with conditions selected from the following groups: 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 SARS-CoV-2 infection; and / or

[0195] - To treat and / or prevent dietary-related cognitive and anxiety deficits in individuals who require this treatment, specifically cognitive and anxiety deficits caused by a high-fat diet.

[0196] Environmental factors, such as diet, have indeed been shown to influence neurobiology and cognitive function and affect healthy brain aging (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 diets are associated with obesity, insulin resistance, diabetes, age-related cognitive impairment, and neurodegenerative diseases (Buckman et al., 2014, Brain BehavImmun. 35, 33–42; Sanchez et al., 2018, Int J Mol Sci. 19(2): 533). The mechanisms by which a high-fat diet affects brain function have not been fully established, although neuroimmune signaling may be a contributing factor (Butler, 2021, Brain Behav Immun Health, 16, 100298).

[0197] Studies have shown that even exposure to a high-fat diet (HFD) for just three days can induce neuroinflammation and impair memory consolidation in aged rats (Spencer et al., 2019, Neurobiol Aging. 74, 121–134). Furthermore, HFD has been shown to increase anxiety-related behaviors in young rats and impair their learning and memory abilities (Gainey et al., 2016, FrontBehav). Long-term high-fat diet (HFD)-induced obesity leads to 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).

[0198] As mentioned earlier, anxiety disorders related to cognitive impairment can specifically include:

[0199] (i) Primary symptoms, such as obsessive-compulsive disorder, and / or

[0200] (ii) Secondary symptoms, such as neurodegenerative diseases selected from the following groups: 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 SARS-CoV-2 infection.

[0201] The HIP / PAP protein, its derivatives, or a combination thereof, for use according to the present invention, may be given to an individual, which may be a mammal, specifically a human.

[0202] The HIP / PAP protein, derivatives, or compositions thereof, as described in this invention, can be administered in conjunction with standard medical care for cognitive impairment and / or anxiety disorders.

[0203] Therefore, the HIP / PAP protein, derivatives, or compositions thereof, according to the present invention, may be administered to individuals in need together with at least one agent known to be used for the prevention and / or treatment of cognitive impairment, wherein the at least one agent is selected from the group consisting of: cholinesterase inhibitors, such as donepezil, rivastigmine, or galantamine; glutamate modulators; such as memantine; cholinesterase inhibitors, specifically cholinesterase inhibitors in combination with glutamate modulators, such as a combination of donepezil and memantine; methylphenidate; amphetamine; atormoxetine; AMPA-R agonists; "α7 nicotinic acetylcholine receptor agonists"; guanifaxine; bupropion; vortioxetine, and D-cyclic serine.

[0204] Alternatively or additionally, the HIP / PAP protein, derivatives, or compositions thereof, used according to the present invention, may be administered to individuals in need together with at least one agent known for the prevention and / or treatment of cognitive impairment, wherein said at least one agent is selected from the group consisting of: selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium channel blockers, azaspiron, monoamine oxidase A reversible inhibitors, agomelatine, quetiapine, and vortioxetine, and more specifically from the group consisting of citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine, and vortioxetine.

[0205] When such agents, known to be used for the prevention and / or treatment of anxiety disorders, are administered together with the HIP / PAP protein, its derivatives, or compositions according to the present invention to patients in need, the cognitive impairment associated with one or more anxiety disorders may specifically 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 (PD), bipolar disorder, substance abuse, attention deficit disorder, psychotic disorders, and SARS-CoV-2 infection.

[0206] According to the HIP / PAP protein of the present invention, its derivatives or compositions may be administered more specifically with at least one agent known to be used to relieve symptoms associated with conditions selected from the group consisting of: autism spectrum disorder, Angleman's syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders, such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, said agents specifically selected from the group consisting of: antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids, such as prednisone; and immunosuppressants, such as methotrexate or azathioprine. Specifically, the compositions of the present invention may also comprise at least one agent known to be used to relieve symptoms associated with conditions selected from the group consisting of: autism spectrum disorder, Angleman's syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders, such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, said agents specifically selected from the group consisting of: antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids, such as prednisone; and immunosuppressants, such as methotrexate or azathioprine.

[0207] The present invention also relates to a treatment and / or prevention, specifically a method for treating cognitive impairment associated with one or more anxiety disorders in an individual in need, the method comprising administering to the individual a HIP / PAP protein, a derivative thereof, or a composition comprising a HIP / PAP protein, a derivative thereof.

[0208] The present invention also relates to a method for improving cognitive function in an individual affected by one or more neurological disorders associated with anxiety disorders, said neurological disorders being selected from the group consisting of autism spectrum disorder, Angleman syndrome, Down syndrome, and other neurological disorders associated with cognitive impairment, such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, said method comprising administering to the individual HIP / PAP protein, derivatives thereof, or a composition comprising HIP / PAP protein, derivatives thereof.

[0209] The present invention also relates to a method for alleviating cognitive deficits in individuals suffering from conditions 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 SARS-CoV-2 infection, the method comprising administering to the individual a HIP / PAP protein, a derivative thereof, or a composition comprising a HIP / PAP protein, a derivative thereof.

[0210] The present invention also relates to a method for treating and / or preventing diet-induced cognitive and anxiety deficits in individuals in need, the method comprising administering to the individual the HIP / PAP protein, a derivative thereof, or a composition comprising the HIP / PAP protein, a derivative thereof, or a composition comprising the HIP / PAP protein, a derivative thereof, according to the present invention.

[0211] Furthermore, the present invention relates to the use of HIP / PAP protein, its derivatives, or compositions containing HIP / PAP protein or its derivatives in the treatment and / or prevention, specifically in the treatment of cognitive impairment associated with one or more anxiety disorders in individuals in need of such treatment.

[0212] Furthermore, the present invention relates to the use of HIP / PAP proteins, derivatives thereof, or compositions containing HIP / PAP proteins and derivatives thereof in improving cognitive function in individuals suffering from one or more neurological disorders associated with anxiety disorders selected from the group consisting of autism spectrum disorder, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

[0213] The present invention also relates to the use of HIP / PAP proteins, derivatives thereof, or compositions containing HIP / PAP proteins and derivatives thereof in alleviating cognitive deficits in individuals suffering from conditions 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 SARS-CoV-2 infection.

[0214] Furthermore, the present invention also relates to the use of HIP / PAP protein, its derivatives, or compositions containing HIP / PAP protein, its derivatives, in the treatment and / or prevention of dietary-induced cognitive and anxiety deficits in individuals in need, said use including administering to said individual the HIP / PAP protein, its derivatives, or compositions containing HIP / PAP protein, its derivatives, according to the present invention.

[0215] The invention will be described in more detail below through the following examples, which are for illustrative purposes only.

[0216] Unless otherwise stated, all percentages are by weight.

[0217] Example

[0218] Example 1

[0219] Animal injection

[0220] Animals were anesthetized with isoflurane and fixed to a World Precision Instruments stereotactic surgical instrument. Incisions were made in the sagittal plane of the skull. In 6-month-old APP / PS1 mice (referred to as transgenic or Tg), incisions were made in the right and left hippocampus (HPC) (X=+ / -2.7, Y=-2.7, Z=-3, with bregma as a reference point) and the right and left cortex (X=+ / -2, Y=-2, Z=-3, with bregma as a reference point) at 5 x 10 mm incisions. 11 Inject 2 μL of rAAV9-empty capsid (transgenic carrier, Tg Veh, n=8) at µg / mL or rAAV9-hHIP / PAP (rAAV9-HIP / PAP (see...) Figure 6 A) Tg HIP / PAP, n=8). Non-transgenic littermates (Ntg n=10) were used as behavioral baseline controls. Six months post-surgery, animals underwent a two-week behavioral test. After the behavioral test, the animals were euthanized and tissues were collected.

[0221] Behavioral Analysis

[0222] Prior to euthanasia, the mice underwent a series of behavioral tasks, including the open field test, novel object recognition test, elevated cross maze test, and radial arm water maze test. Age-matched wild-type mice were used as controls. The tasks were conducted from least (open field test) to most (RAWM) stress levels, as described below.

[0223] One-way or two-way ANOVA was used for measurement analysis. Repeated measures ANOVA was used for tasks requiring multiple training / trials, such as the water maze. Tukey's HSD post-hoc mean comparisons were performed on significant ANOVA measurements using GraphPad Prism software. Outliers were identified using the Grubb test.

[0224] Open field experiment

[0225] Open field tests were used as a standard test of overall activity. Animals were monitored for 15 minutes in a 40 cm square open field under moderate lighting conditions using video tracking software (ANY-Maze, Stoelting, Illinois).

[0226] Overall activity levels are assessed by measuring horizontal and vertical activity.

[0227] New Object Recognition Test (NOR)

[0228] Mice were placed in a 40x40 cm experimental area and monitored and quantified using a video tracking system (ANY-Maze, Stoelting, Illinois). Two objects of similar size to the mice were placed approximately 3-5 cm from the outer wall along the centerline of the experimental area.

[0229] Each animal underwent three adaptation tests, each lasting 5 minutes, with a 5-minute interval between tests.

[0230] After each experiment, the experimental area and object cues were cleaned with 70% ethanol to minimize olfactory cues. After the adaptation test, one of the adapted objects was replaced with a new object. Subsequently, the animals underwent a 5-minute exploratory test, during which their object exploration behavior was monitored via video recording.

[0231] Assess working memory capacity by measuring the time spent exploring new objects and becoming familiar with them.

[0232] Radial arm water maze and its reversal experiment

[0233] The radial arm water maze comprises six swimming paths (arms) radiating outwards from a central open area, with an escape platform hidden at the end of one arm. Multiple external cues are placed around the pool to assist the mice in spatial navigation. In each trial, the mice were allowed a maximum of 60 seconds to locate the escape platform. In all trials, the platform was located within the same arm.

[0234] On Day 1, mice underwent 15 trials, during which visible platforms (above the water surface) and hidden platforms (below the water surface) alternated. On Day 2, mice underwent 15 additional trials, all using hidden platforms. The starting arm positions varied in each trial to encourage mice to rely on spatial cues rather than learning movement rules to complete the tasks (e.g., the second arm was on the right side).

[0235] To avoid olfactory cues revealing the target arm, the target arm was different for each mouse. Entering the wrong arm (all four limbs entering the arm) was counted as an error. Failure to enter the target arm within 15 seconds was also counted as an error. In data analysis, the average error was calculated for each of three consecutive trials. Mice with an average of 1 or less error by the end of the second day were considered to have met the learning criteria.

[0236] On the third day, a reversal test was conducted, placing the target platform in the arm 180° away from its original position. The mice underwent a total of 15 trials, all conducted with the platform hidden. Animal activity was monitored using video tracking software (Ethovision, Noldus).

[0237] elevated cross maze

[0238] The elevated cross maze test is one of the most widely used tests for measuring anxiety-like behaviors.

[0239] This test was based on mice’s natural aversion to open, high ground and their spontaneous exploration behavior in new environments. Mice were placed at the intersection of a four-armed maze, which consisted of two open arms without walls and two enclosed arms with walls 15.25 cm high, 30 cm long, and 5 cm wide.

[0240] Under moderate lighting conditions, animals were monitored for 5 minutes using video tracking software (ANY-Maze, Stoelting, Illinois). Anxiety-like behaviors were assessed by measuring the number of times the animals entered the open and closed arms and the duration of their stay.

[0241] The longer the mice stayed in the closed arm, the higher their anxiety levels became, a normal behavior observed in non-transgenic control mice.

[0242] result

[0243] (i) open field

[0244] Regardless of treatment, there was no difference in overall activity between non-transgenic control mice and transgenic mice. Overexpression of HIP / PAP in the brains of APP / PS1 mice did not cause any visible changes in spontaneous activity (see [link to relevant documentation]). Figure 1 A (open field experiment - distance of movement), Figure 1 B (open field experiment - stationary time)).

[0245] (ii) New Object Recognition Test (NOR)

[0246] Compared to familiar objects, non-transgenic control mice spent significantly longer time with new objects upon exposure, indicating short-term memory of previous trials. Conversely, transgenic mice spent the same amount of time with both types of objects regardless of treatment, indicating a deficit in their short-term memory. Overexpression of HIP / PAP in the brains of APP / PS1 mice did not salvage the genotype-related effects on short-term memory observed during new object recognition (see [link to relevant documentation]). Figure 2 ).

[0247] (iii) Radial Arm Water Maze

[0248] Compared to non-transgenic control mice, APP / PS1 mice treated with the loading agent made significantly more errors when attempting to reach the platform, as reflected in the number of errors per group ( Figure 3 A) and the total number of errors on day 2 ( Figure 3 B) Both were significantly increased. These results indicate cognitive impairment in mice, particularly in learning and memory. HIP / PAP-treated APP / PS1 mice made significantly fewer errors than APP / PS1 mice treated with the loading agent, suggesting that cognitive function was restored in this test (see [link]). Figure 3 (A and 3B).

[0249] (iv) Reversal test

[0250] As expected, the error rate in APP / PS1 control mice was significantly higher than that in non-transgenic littermates (see [link to article]). Figure 4 B). No significant reduction in error rate was observed in APP / PS1 mice treated with HIP / PAP (see [link]). Figure 4 A).

[0251] (v) Elevated Cross Maze

[0252] like Figure 5 As shown in Figure A, non-transgenic control mice spent significantly longer in the closed arm than in the open arm, exhibiting normal anxiety and fear of open spaces (a physiological defense mechanism). In contrast, transgenic mice treated with the loading agent spent the same amount of time in both arms, indicating a defect in their anxiety-like behavior. Figure 5A). Furthermore, when comparing the time spent in the closed arm, transgenic mice treated with the loading agent spent significantly less time in the closed arm compared to non-transgenic control mice, indicating the release of behavioral inhibition and an increase in risk-taking behavior (Figure 5B). After HIP / PAP treatment, the normal anxiety-like behavior of transgenic mice was restored, manifested by a significantly longer time spent in the closed arm than in the open arm. Moreover, the increase in time spent in the closed arm in HIP / PAP-treated mice was significantly different from that in transgenic mice treated with the loading agent, but not significantly different from that in non-transgenic mice, suggesting that HIP / PAP treatment is a phenotypic recovery rather than an anxiety-inducing effect. Figure 5 A).

[0253] in conclusion

[0254] Overexpression of HIP / PAP in transgenic mouse models significantly improved memory in the radial arm water maze (RAWM) test, rescuing cognitive deficits in this test. Compared to transgenic mice treated with the transgenic agent, HIP / PAP treatment also rescued anxiety-like behavioral deficits observed in the elevated cross maze test. Therefore, HIP / PAP treatment can restore normal responses to anxious situations in mice. HIP / PAP treatment indeed restored the anxiety-like behaviors lost in transgenic control mice.

[0255] In fact, non-transgenic control mice spent significantly longer in the closed arm than in the open arm, exhibiting normal anxiety and fear of open spaces (a physiological defense mechanism). In contrast, control APP / PS1 mice spent the same amount of time in both arms, indicating a defect in their 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 control mice, indicating deinhibition of behavioral inhibition and increased risk-taking behavior.

[0256] After HIP / PAP treatment, the transgenic mice regained their normal anxiety-like behavior, which was manifested by spending significantly longer time in the closed arm than in the open arm.

[0257] The difference was not significant compared to non-transgenic mice, indicating that the transgenic mice experienced phenotypic recovery rather than an anxiety effect.

[0258] Improved cognitive function was not associated with a reduction in amyloid pathology in the hippocampus, as evidenced by the similar levels of Aβ and Congo red-stained amyloid plaques. Figure 7However, Western blot analysis of the hippocampus showed that, compared with non-transgenic mice and APP / PS1 control mice, the levels of the antioxidant enzymes sodium superoxide dismutase-1 (SOD-1) and heme oxygenase-1 (HO-1) were significantly increased. Figure 8 (A, C, D), and the level of catalase also showed an increasing trend. Figure 8 A, B, p=0.06).

[0259] These data suggest that the antioxidant effects of HIP / PAP are unexpectedly exerted in the brain and contribute to the unexpected beneficial effects of HIP / PAP on cognitive function.

[0260] HIP / PAP expression

[0261] After a series of behavioral tests on the mice, their tissues were collected at 12 months of age (6 months after expression).

[0262] HIP / PAP expression in the brain was measured by hemagglutinin (HA) staining. Figure 6 C), and measured the concentration of HIP / PAP in the cortex using a human HIP / PAP ELISA (R&D Systems, Minneapolis, USA). Figure 6 D).

[0263] No HA-positive staining was detected in the control group. Figure 6 B) No HIP / PAP was detected in the plasma (not shown).

[0264] Example 2

[0265] HIP / PAP can cross the blood-brain barrier (BBB) ​​via peripheral injection of rHIP / PAP or rAAV. Pharmacokinetic studies of [3H]-rHIP / PAP in mice showed that, 24 hours after intravenous administration of 1 mg / kg [3H]-rHIP / PAP, significant levels of radioactivity were detected not only in plasma (18.20 + / - 15.82 ng / g) and liver (64.22 + / - 11.73 ng / g), but also in brain tissue (85.57 + / - 11.55 ng / g).

[0266] In addition, brain tissue from non-transgenic mice was tested. These mice were subcutaneously administered rHIP / PAP via an ALZET pump at a dose of 43 µg / day for one month.

[0267] Brain and plasma samples were collected, and rHIP / PAP levels were assessed using ELISA (Abcam, R&D systems).

[0268] One month later, significant levels of rHIP / PAP were found in the brain and plasma. Figure 9 A).

[0269] Furthermore, the brain permeability of fluorescently labeled rHIP / PAP was determined using an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA) and a vivotag 680 (Revity, Waltham, MA). Thirty minutes after cardiac injection of rHIP / PAP-680 or control bovine serum albumin (BSA)-680, a strong signal was detected in the brains of animals injected with rHIP / PAP-680, while no strong signal was detected in the brains of animals injected with BSA-680, indicating that rHIP / PAP can cross the blood-brain barrier. Figure 9 B).

[0270] Therefore, whether HIP / PAP is administered subcutaneously or intravenously, the protein can significantly cross the blood-brain barrier.

[0271] Peripheral rAAV9-HIP / PAP

[0272] rAAV9-HIP / PAP was administered peripherally via intravenous or intramuscular injection (hind limb). One month after expression, plasma from injected mice was used for further analysis. Figure 9 C) and brain ( Figure 9 Significant levels of rHIP / PAP were detected in both D)

[0273] One month after peripheral injection, the intracranial rHIP / PAP level was similar to that observed after central injection. Figure 9 D) indicates that peripheral injection can be used for this purpose. No rHIP / PAP signal was detected in the control group animals (not shown).

[0274] Example 3 – Testing of a high-fat diet model in non-transgenic wild-type mice

[0275] Six-month-old non-transgenic mice were fed either a control diet (n=6), a high-fat diet (60% kcal / fat, HFD, n=5), or an intracerebral injection of 2 μL of rAAV9-hHIP / PAP (5×10¹¹ µg / mL) followed by HFD for two weeks (n=4). The injection sites were the right and left hippocampus (HPC) (X=+ / -2.7Y=-2.7, Z=-3, with bregma as the reference point) and the right and left cortex (X=+ / -2Y=-2, Z=-3, with bregma as the reference point).

[0276] Weight and food intake were monitored weekly during the study.

[0277] Ten weeks after the diet, behavioral analysis was conducted to assess the effects of the diet on motor activity, cognition, and anxiety.

[0278] result

[0279] After 10 weeks of diet, mice fed a high-fat diet showed a significant increase in body weight compared to mice fed a control diet. Figure 10 A) Regardless of whether or not the matter is processed.

[0280] As mentioned above, in the elevated cross maze experiment, mice on the control diet spent significantly longer in the closed arm than in the open arm, exhibiting normal physiological responses to anxiety. Figure 10 B).

[0281] However, mice fed a high-fat diet for 10 weeks spent the same amount of time in both arms and exhibited deficits in anxious behavior.

[0282] The difference was that mice fed a high-fat diet and treated with HIP / PAP exhibited similar behavior to control mice fed a control diet, with a significantly increased time spent in the closed arm compared to the open arm.

[0283] In summary, these results indicate that:

[0284] A high-fat diet successfully induced obesity, while administering HIP / PAP to the brain did not prevent obesity from occurring; and

[0285] - The obesity phenotype induces anxiety-related behavioral deficits, which are prevented in mice treated with HIP / PAP.

[0286] These results confirm the beneficial effects of HIP / PAP on cognitive and anxiety-related behaviors, and therefore have high research value in diet-induced cognitive and anxiety deficits.

[0287] sequence list

[0288] SEQ ID NO: 1 is the amino acid sequence of the HIP / PAP protein, in which the signal peptide of the N-terminus 26 amino acids is missing.

[0289] EEPQRELPSARIRCPKGSKAYGSHCYALFLSPKSWTDADLACQKRPSGNLVSVLSGAEGSFVSSLVKSIGNSYSYVWIGLHDPTQGTEPNGEGWEWSSSDVMNYFAWERNPSTISSPGHCASLSRSTAFLRWKDYNCNVRLPYVCKFTD

[0290] SEQ ID NO: 2 is the amino acid sequence of the HIP / PAP protein, in which the N-terminal 26-amino acid signal peptide and the N-terminal 11-amino acid propeptide are missing.

[0291] IRCPKGSKAYGSHCYALFLSPKSWTDADLACQKRPSGNLVSVLSGAEGSFVSSLVKSIGNSYSYVWIGLHDPTQGTEPNGEGWEWSSSDVMNYFAWERNPSTISSPGHCASLSRSTAFLRWKDYNCNVRLPYVCKFTD

[0292] SEQ ID NO: 3 is the amino acid sequence of the HIP / PAP protein, in which the N-terminal 26-amino acid signal peptide is deleted and a methionine is added to the N-terminus.

[0293] MEEPQRELPSARIRCPKGSKAYGSHCYALFLSPKSWTDADLACQKRPSGNLVSVLSGAEGSFVSSLVKSIGNSYSYVWIGLHDPTQGTEPNGEGWEWSSSDVMNYFAWERNPSTISSPGHCASLSRSTAFLRWKDYNCNVRLPYVCKFTD

[0294] SEQ ID NO: 4 is the amino acid sequence of the full-length HIP / PAP protein.

[0295] MLPPMALPSVSWMLLSCLMLLSQVQGEEPQRELPSARIRCPKGSKAYGSHCYALFLSPKSWTDADLACQKRPSGNLVSVLSGAEGSFVSSLVKSIGNSYSYVWIGLHDPTQGTEPNGEGWEWSSSDVMNYFAWERNPSTISSPGHCASLSRSTAFLRWKDYNCNVRLPYVCKFTD

[0296] The amino acid sequences disclosed in this specification are for reference only. These sequences are also listed in a sequence listing format conforming to patent standard requirements. In the event of any discrepancy with the standard sequence listing, the sequences described in this specification shall prevail.

Claims

1. HIP / PAP protein or derivatives thereof, used for the treatment and / or prevention, specifically for the treatment of cognitive impairment associated with one or more anxiety disorders in individuals with such needs.

2. The HIP / PAP protein or its derivatives as described in claim 1, wherein the one or more anxiety disorders associated with cognitive impairment are: (i) Primary symptoms, such as obsessive-compulsive disorder, and / or (ii) Secondary symptoms, such as neurodegenerative diseases selected from the following groups: 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 SARS-CoV-2 infection.

3. A HIP / PAP protein or a derivative thereof, for use in improving cognitive function in an individual affected by one or more neurological disorders associated with anxiety disorders, said one or more neurological disorders associated with anxiety disorders being selected from the group consisting of: autism spectrum disorder, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders, such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis.

4. A HIP / PAP protein or a derivative thereof, for use in alleviating cognitive deficits in individuals affected by conditions selected from the following groups: 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 SARS-CoV-2 infection.

5. The HIP / PAP protein or a derivative thereof for any one of claims 1 to 4, wherein the anxiety disorder is an externalizing disorder, and the cognitive disorder is specifically selected from the group consisting of bipolar disorder, substance abuse, attention deficit disorder, and psychotic disorders.

6. A HIP / PAP protein or a derivative thereof, used for the prevention and / or treatment of dietary-induced cognitive and anxiety deficits in individuals with such needs, specifically cognitive and anxiety deficits induced by a high-fat diet.

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

8. The HIP / PAP protein or a derivative thereof for 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 in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, specifically the sequence shown in SEQ ID NO:

4.

9. The HIP / PAP protein or a derivative thereof according to any one of claims 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 sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and having the same biological activity as 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, more specifically, the amino acid sequence having at least 80% sequence identity with an amino acid sequence shown as SEQ ID NO: 4, and having the same biological activity as an amino acid sequence shown as SEQ ID NO:

4.

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

11. The HIP / PAP protein or its derivatives according to claim 10, wherein the composition is for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, tracheal, nasal, or rectal administration.

12. The HIP / PAP protein or a derivative thereof as described in claim 10 or 11, wherein the composition is for oral, subcutaneous, intravenous, topical or local administration.

13. The HIP / PAP protein or a derivative thereof for any one of claims 10 to 12, wherein the composition further comprises at least one agent known for the prevention and / or treatment of cognitive impairment, such as agents selected from the group consisting of: cholinesterase inhibitors, such as donepezil, rivastigmine, or galantamine; glutamate modulators; such as memantine; cholinesterase inhibitors, specifically cholinesterase inhibitors in combination with glutamate modulators, such as a combination of donepezil and memantine; methylphenidate; amphetamine; atormoxetine; AMPA-R agonists; α7 nicotinic acetylcholine receptor agonists; guanifaxine; bupropion; vortioxetine; and D-cyclic serine.

14. The HIP / PAP protein or a derivative thereof for any one of claims 10 to 13, wherein the composition further comprises at least one agent known for the prevention and / or treatment of anxiety disorders, specifically an agent selected from the group consisting of: selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tricyclic antidepressants, calcium channel blockers, azaspiron, monoamine oxidase A reversible inhibitors, agomelatine, quetiapine, and vortioxetine, and more specifically selected from citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, duloxetine, venlafaxine, clomipramine, pregabalin, buspirone, moclobemide, agomelatine, quetiapine, and vortioxetine.

15. The HIP / PAP protein or a derivative thereof for the application of claim 14, wherein the cognitive impairment associated with one or more anxiety disorders 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 SARS-CoV-2 infection.

16. The HIP / PAP protein or a derivative thereof for any one of claims 10 to 15, wherein the composition further comprises at least one agent known to be effective in alleviating symptoms associated with conditions selected from the group consisting of: autism spectrum disorder, Angleman syndrome, Down syndrome, and other cognitive impairments associated with neurological disorders such as chronic meningitis, autoimmune encephalitis, or neurosarcoidosis, wherein the agent is specifically selected from the group consisting of: antiepileptic drugs; amino acid supplements; antifungal drugs; corticosteroids such as prednisone; and immunosuppressants such as methotrexate or azathioprine.

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