Nap peptide for treatment of conditions related to a low level of sex hormone

Abstract

The present invention provides methods for treating conditions, diseases or disorders associated with sex hormone imbalance and specifically with a low sex hormone level or response using NAP peptide, specifically conditions, diseases or disorders mediated via unfolded protein response.

Description

[0001] NAP PEPTIDE FOR TREATMENT OF CONDITIONS RELATED TO A LOW

[0002] LEVEL OF SEX HORMONE

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to methods of treating conditions associated with, or caused by, a low level of sex hormone in an individual using a peptide with the sequence NAPVSIPQ, also known as davunetide (NAP).

[0005] BACKGROUND OF THE INVENTION

[0006] Hormone replacement therapy employing estradiol and molecules with similar properties toward estrogen receptors is used to treat a multitude of conditions, from those related to menopause and ovarian insufficiency to neuropsychiatric conditions, including depression and schizophrenia. Unfortunately, administration of estrogen and certain estrogen analogs has off-target consequences in several other organs, including the uterus and breasts, which can result in cancer and other undesirable effects. In addition, there are many medical conditions in which estrogen replacement therapy is not feasible. Likewise, prescribing hormone replacement therapy using testosterone is limited by many contraindications.

[0007] NAP (NAPVSIPQ single amino acid letter code, also known as davunetide, AL-108, CP201 and sometimes referred to as NAP peptide) is the smallest neuroprotective peptide site of ADNP. NAP and pipeline products protect nerve cells by associating with microtubule end-binding proteins (EB1 / EB3), through the SxIP motif (NAPVSIPQ), thus enhancing microtubule dynamics and Tau-microtubule interaction, protecting the synapse. NAP further enhances ADNP-EB1 / EB3, interactions, protecting against ADNP deficits (Oz, S. et al. Molecular Psychiatry 19, 1115-1124 (2014). https: / / doi.org: 10.1038 / mp.2014.97; Hacohen-Kleiman, et al., The Journal of Clinical Investigation 2018; 128(11): 4956-4969; Gozes, I. & Shazman, S. Eur J Neurosci 58, 2641-2652 (2023). https: / / doi.org: 10.1111 / ejn. l5920). US2018344809 describes compositions comprising NAP.

[0008] There remains an unmet need for effective alternatives to hormone replacement therapy to mitigate the effects of low sex hormone levels or low sensitivity to said hormones in individuals. SUMMARY OF THE INVENTION

[0009] According to the principles of the present invention, NAP (davunetide), a peptide which has heretofore been useful in treating neurodegenerative diseases, is shown for the first time to be an effective treatment for the amelioration of symptoms related to conditions associated with low sex hormones.

[0010] According to the teachings of the present invention, certain sex hormones, specifically estrogen and testosterone, are involved in the regulation of unfolded protein response (UPR), a stress response that is activated upon detection of excess unfolded or misfolded proteins within the lumen of the endoplasmic reticulum (ER). Decreased levels of estrogen or testosterone in females or males lead to an increase in ER stress due to dysregulation of the UPR, thereby contributing to conditions, diseases or disorders associated with low sex hormones.

[0011] The present invention is based on an unexpected finding that an activity-dependent neuroprotective protein (ADNP) is a key regulator of the UPR. The present invention is based on the ability of NAP, a fragment of ADNP, to alleviate symptoms associated with or caused by, inter alia, by an upregulated UPR mediated by low levels of sex hormones.

[0012] According to one aspect, the present invention provides a method of treating a condition, disease or disorder associated with sex hormone imbalance in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject. In some examples, sex hormone imbalance comprises a low sex hormone level. Thus, in some examples, the present invention provides a method of treating a condition, disease or disorder associated with a low sex hormone level or response thereto in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject.

[0013] According to some examples, the sex hormone is estrogen.

[0014] According to some examples, the condition associated with a low level of estrogen is an iatrogenic condition.

[0015] According to some examples, the iatrogenic condition results from a medical intervention selected from a unilateral oophorectomy, bilateral oophorectomy, partial hysterectomy, radical hysterectomy, chemotherapy, or radiation therapy.

[0016] According to some examples, the condition, disease or disorder is selected from aging, perimenopause, menopause, primary ovarian insufficiency, premature ovarian failure, polycystic ovarian syndrome, an autoimmune condition, pelvic inflammatory disease (PID), a genetic condition, a pituitary gland condition, a metabolic disorder, an affective disorder, a skin disorder, a cardiovascular disease, osteopenia, osteoporosis, diabetes, a cancer, stroke, sexual dysfunction, infertility, hirsuteness, a genitourinary condition, a vasomotor symptom, a sleep disturbance, an exposure to tobacco smoke, an exposure to gonadotoxic chemicals, and an exposure to pesticides or herbicides.

[0017] According to some examples, the sex hormone is testosterone.

[0018] According to some examples, the condition associated with a low level of testosterone is an iatrogenic condition.

[0019] According to some examples, the iatrogenic condition results from a medical intervention selected from a unilateral orchidectomy, bilateral orchidectomy, chemotherapy, or radiation therapy.

[0020] According to some examples, the condition, disease or disorder is selected from aging, an infection, an inflammatory disease, varicocele, testicular injury, hemochromatosis, a pituitary gland condition, an autoimmune condition, a genetic condition, obesity, a decreased libido, erectile dysfunction, infertility, decreased energy, osteopenia, osteoporosis, sarcopenia, decreased facial or body hair, undescended testicles, long-term exposure to tobacco smoke, exposure to gonadotoxic chemicals, or exposure to pesticides.

[0021] According to some examples, the condition, disease or disorder is associated with an unfolded protein response.

[0022] According to some examples, the condition, disease or disorder comprises a condition, disease or disorder in which hormone replacement therapy is contraindicated.

[0023] According to some examples, the peptide is applied in an administration mode selected from an intranasal, an intravenous, a subcutaneous, an intramuscular, or a sublingual mode of administration.

[0024] According to any one of the above examples, it is provided that the condition, disease or disorder is not a neurodegenerative condition, disease or disorder.

[0025] According to some examples, the present invention provides a method of hormone replacement therapy in a subject in need thereof, the method comprises administering to said subject a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide).

[0026] According to one aspect, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1 ; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with sex hormone imbalance. According to some examples, the sex hormone imbalance comprises a low sex hormone level. Therefore, according to some examples, the present invention provides for a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with a low sex hormone level. According to some examples, the condition, disease or disorder is associated with an unfolded protein response.

[0027] In some examples, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1 ; NAP peptide) and a pharmaceutically acceptable carrier, for use in hormone replacement therapy.

[0028] According to some examples, the pharmaceutical composition is formulated for intranasal, intravenous, subcutaneous, intramuscular, or sublingual administration.

[0029] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0030] BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figs. 1A-1C: Adnp-specific regulated hippocampal expression in male mice genome edited to carry the most prevalent ADNP autism mutation (mouse orthologue, p.Tyr 719*), namely, Tyr males. (Karmon, G. et al. Biol Psychiatry 92, 81-95 (2022), Fig. 1A) Heatmap of differential expression colored according to log2FoldChange for each gene (row) at each comparison (column). Significant differential expression is (FDR<0.05) and the cell is marked with a star. If differential expression was only significant at the alternatively spliced transcript level, the gene name is marked with a circle suffix. Arrows and ellipses are mentioned in the text. (Fig. IB) A graph of relationships between differentially expressed genes (small nodes) and terms they are associated with. (Fig. 1C) GSEA plot of genes upregulated by the HSP-90 inhibitor Geldanamycin according to their differential expression in the Tyr male comparison. The position of the enriched genes is marked on a ranked list at the bottom, starting from the most downregulated (bottom) to the most upregulated (top).

[0032] Fig. 2: Differential expression across multiple RNA-Seq experiments, differing in Adnp mutations and mouse models. In short, hippocampal differential expression of our heterozygous Tyr model (Karmon, G. et al. IBID), Fig. 1, Tyr or NAP, depending on treatment), heterozygous deletion of exon 5 of Adnp from Cho et al. Molecular Psychiatry 28, 3548-3562 (2023) (prefixed Delx5) and heterozygous deletion of Adnp from Amram et al. (Molecular Psychiatry 21, 1467-1476 (2016), prefixed Adnp+ / '). Heatmap of genes significantly differentially expressed (FDR<0.05) in 5 comparisons or more. Significant results (FDR<0.05) are marked with a star, near-significant results (0.05<FDR<0.1) are marked with a dot.

[0033] Figs. 3A-3C: Comparison of bromodeoxyuridine (BrdU, a marker of neurogenesis)-labeled cell concentrations in hippocampal sub -ventricular zone (SVZ) of two ADNP mouse models, Adnp haplo-insufficient (dr / / / / ? -) mice on ICR background (Amram et al., IBID) or mice CRSIPR / Cas9-edited to present the most prevalent neurodevelopmental ADNP syndrome mutation, p.Tyr718* (Tyr) Adnp+ / ' on C57BL6 / NJ background. (Fig. 3A and 3B) Adnp+ / ' mice, (Fig. 3A) Tyr mice, (Fig. 3B) wild type, ICR and C57BL6 / NJ comparisons (Fig. 3C). Group differences in BrdU positive cells / mm2(mean ± SEM) were compared using a oneway analysis of variance with Tukey’s post hoc test. Technical replicates (6 per animal) were used during statistical analysis. Outliers were excluded using the Grubb’s test.

[0034] (Fig. 3A) For Adnp+ / ' males, a statistically significant difference for BrdU positive cells was discovered (F2,77 = 33.030, p < 0.001), with Tukey’s post hoc test revealing a significant reduction in BrdU positive cells in Adnp+ / ' placebo (termed DD) treated males (N=4, biological repeats) compared to WT (N=5) (***P<0.001), which was significantly corrected with NAP treatment (***P<0.001, N=5). No such effect was found in females (N=5 per group). Significant sex differences were discovered in the WT and the NAP treated Adnp+ / ' group (***P<0.001 for both comparisons).

[0035] (Fig. 3B) For Tyr females, a statistically significant difference for BrdU positive cells was discovered (F2,77 = 11.272, ***P<0.001), with Tukey’s post hoc test revealing a significant reduction in BrdU positive cells in NAP treated Tyr females (N=5) compared to WT (N=4) (***p<0.001), with no difference when compared to DD treated Tyr females (N=5). For Tyr males, a statistically significant difference for BrdU positive cells was discovered (F2,89 = 12.777, ***p< 0.001), with Tukey’s post hoc test revealing a significant reduction in BrdU positive cells in DD and NAP treated Tyr males (N=5 for both groups) compared to WT (N=5) (***P<0.001). Significant sex differences were discovered in the WT and the DD- treated Tyr groups (P<0.05 for both comparisons).

[0036] (Fig. 3C) Significant differences were discovered between males of different tested strains (P<0.01). Fig- 4 shows heterozygous hippocampal Adnp expression in Tyr mice. The figure shows a boxplot of total Adnp gene expression (normalized expression). Note that allelic expression is based solely on reads overlapping the Tyr718 mutation locus, while overall gene expression is based on all reads overlapping the gene, regardless of genotype.

[0037] Figs. 5A-5C shows the effect of NAP vs. estrogen on front limb grip strength of ovariectomized mice. Fig. 5A shows the effect of ovariectomy (OVX) in the control groups, Fig. 5B shows the effect of NAP vs control OVX, and Fig. 5C shows the effect of estrogen vs. control group OVX.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the patent specification, including definitions, will control.

[0040] According to the principles of the present invention, the peptide NAP, which is a fragment of activity-dependent neuroprotective protein (ADNP), is used in ameliorating symptoms of diseases or conditions that are associated with low sex hormones in both males and females. Specifically, NAP is used to successfully treat conditions associated with low hormone levels that involve an unfolded protein response (UPR) pathway. Among the advantages of the invention is that NAP may be used in place of hormonal replacement therapies and provide the same benefits. Thus, NAP may be used as a hormone replacement therapy. This is a particularly important advantage given that many conditions preclude a patient suffering from conditions related to low sex hormones from safely taking a hormone replacement therapy, thereby causing significant physical and emotional distress. Thus, the invention as disclosed herein provides a new and unexpected treatment option for individuals suffering from conditions related to low sex hormones.

[0041] According to one aspect, the present invention provides a method of treating a condition, disease or disorder associated with sex hormone imbalance in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject. In some examples, sex hormone imbalance comprises a low sex hormone level. Thus, according to some aspects, the present invention provides a method of treating a condition, disease or disorder associated with a low level of a sex hormone in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject. According to some embodiments, the present invention provides a method of hormone replacement therapy in a subject in need thereof, the method comprises administering to said subject a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide).

[0042] According to another aspect, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with sex hormone imbalance. According to some examples, the sex hormone imbalance comprises a low sex hormone level. According to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1 ; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with a low sex hormone level.

[0043] In some examples, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1 ; NAP peptide) and a pharmaceutically acceptable carrier, for use in hormone replacement therapy.

[0044] The terms and embodiments described below refer to all aspects of the present invention.

[0045] The ternf'sex hormone imbalance" is used in the application as well known in the art and refers to conditions in which the body produces too much or too little of key hormones that regulate reproductive and sexual functions. These hormones include estrogen, progesterone, and testosterone, as well as hormones that control their release. In some embodiments, sex hormone imbalance refers to a low level of sex hormone.

[0046] As used herein, the term "hormone replacement therapy" refers to the use of compounds such as NAP peptide to make up for the decline or lack of natural hormones produced in the human body. In some embodiments, hormone replacement therapy refers to estrogen replacement therapy (ERT).

[0047] The term “treating” as used herein refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, ameliorating, abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and / or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). According to some embodiments, the term "treating" comprises reducing symptoms caused by a low level of sex hormone.

[0048] The term “low level of sex hormone” in a subject, as used herein is defined as a level of sex hormone that is lower than the average level of the sex hormone that is considered normal in healthy individuals of the same sex and the same age group, and at the same stage of cycle, if relevant, as the subject and causes an individual to experience adverse effects as a result of low sex hormone. The term “hypogonadism” may be used interchangeably with “low level of sex hormone”. The terms “hypoestrogenism” and “estrogen insufficiency” can be used interchangeably with “low level of estrogen”. The terms “hypoandrogenism” and “androgen insufficiency” can be used interchangeably with “low level of testosterone”. The normal levels of sex hormones, typical to healthy subjects in a particular age group, are well known in the art, and a low level of sex hormones is determined by comparing to normal values. In addition, the term “low level of sex hormone” refers to conditions of hyposensitivity to sex hormone, e.g., hyposensitivity to estrogen, such as estrogen resistance syndrome and hyposensitivity to testosterone, such as Androgen insensitivity syndrome.

[0049] The term “associated with” as used herein refers to any condition, disease or disorder that is caused by or its pathology is mediated via a low sex hormone level in an individual.

[0050] The term “effective amount” as used herein refers to a sufficient amount of the peptide for treating a condition, disease or disorder associated with a low level of a sex hormone at a reasonable benefit / risk ratio applicable to any medical or nutritional treatment. The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of the cognitive impairment, and the therapeutics or combination of therapeutics selected for administration, and the mode of administration. The skilled person can readily determine the effective amount for a given situation by routine experimentation.

[0051] Both estrogen and testosterone are present in the body as a bound form, wherein the hormones are bound to sex hormone binding globulin (SHBG) and albumin, and in a free form. Estrogen and testosterone are only capable of binding to their receptors and thereby exerting their function in the unbound, or free, form. The total level of hormone measures both protein-bound hormone and free hormone. It is also possible to measure the level of unbound, or free, hormone. According to some embodiments, the low level of estrogen is measured as total estrogen. According to other embodiments, the low level of estrogen is measured as free estrogen. According to yet other embodiments, the low level of testosterone is measured as total testosterone. According to still other embodiments, the low level of testosterone is measured as free testosterone.

[0052] The terms "NAP" and "NAP protein" are used herein interchangeably and refer to an 8-amino acid peptide consisting of amino acids NAPVSIPQ set forth in SEQ ID NO: 1 and known also as davunetide (AL-108, also known as CP201). In its broadest definition, the term "NAP peptide" refers to peptides comprising the amino acid sequence NAPVSIPQ and to any derivative or analog of the peptide comprising the amino acids NAPVSIPQ or related sequences and having the same biological activity, e.g., as peptides defined in W02008084483, W02006099739, US2012208763 and US20150141345 and incorporated herein by reference in their entirety. According to some embodiments, the formulation of NAP protein is as described in US2012208763 and US2018344809, incorporated herein by reference.

[0053] According to some embodiments, the term “NAP” refers to one of the derivatives of NAP having an amino acid sequence selected from amino acid sequences selected from amino acid sequences SEQ ID NO: 2-49 as disclosed in WO2023112025 and incorporated herein by reference in its entirety or to a NAP alpha-aminoisobutyric acid analog, or SKIP, (Ivashko-Pachima et al., J Mol Neurosci. 2021 Aug;71(8): 1515-1524) or Ac-SKIP (Ivashko- Pachima and Gozes (Front Cell Neurosci. 2019 Oct 1;13:435. doi: 10.3389 / fncel.2019.00435). According to some embodiments, the methods of treatment of the present invention include use of other compounds that have an activity that is similar to that of NAP, i.e., alternatives of NAP, regulating ADNP. Examples of such NAP alternatives, regulating ADNP are vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) described e.g. in Sragovich et al (Translational Psychiatry volume 9, Article number: 235 (2019); SNV described in Eger et al., (Front Pharmacol. 2021 May 5;12:638128. doi: 10.3389 / fphar.2021.638128), gene therapy to increase ADNP expression (e.g. synthetics oligodeoynucleotides) or ketamine (Brown et al., Neuroscience. 2015 Apr 2;290:31-40). In some embodiments, NAP is modified by any known in the art method, such as chemical modification, conjugation or cyclization. In some embodiments, NAP is acylated, acetylated, amidated, lapidated, stearylated, pegylated, biotinylated or modified by any other way.

[0054] According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 50 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 30 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 20 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 15 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 12 amino acids.

[0055] According to any one of the embodiments of the present invention, the subject is human.

[0056] According to some embodiments, the subject is a female. According to other embodiments, the subject is a male. It is understood that the normal level of different sex hormones is different in males and females and subsequently, a low level of sex hormones is defined according to the sex of the subject.

[0057] According to some embodiments, the sex hormone is estrogen.

[0058] According to some embodiments, the subject is a female (human) subject. Therefore, according to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with a low level of estrogen in a female subject. According to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with a low level of estrogen in a male subject. In some embodiments, the present invention provides a method of treating a condition, disease or disorder associated with a low level of estrogen in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject, wherein the subject is a female subject. The term “estrogen” as used herein refers to a group of related steroid sex hormones that are responsible for the development and regulation of the female reproductive system as well as female secondary sex characteristics. There are three major estrogen hormones, namely, estrone (El), estradiol (E2), and estriol (E3). A fourth estrogen, estetrol (E4) is produced only during pregnancy. E2 is largely produced by granulose cells of the ovaries and is the dominant estrogen molecule in females during the period between menarche and menopause. In contrast, estrone (El), is the dominant estrogen after menopause and is produced by the ovaries as well as by the adrenal glands and adipose tissue.

[0059] Estrogen, specifically estradiol, is also present in males, albeit in lower levels than in females, and serves critical roles in, for example, male sexual function, including sperm production, cardioprotective effects, and bone health.

[0060] According to other embodiments, the sex hormone is estrogen and the subject is a male.

[0061] According to some embodiments, the sex hormone is estrogen selected from estrone (El), estradiol (E2), estriol (E3) or a combination thereof. Each possibility represents a separate embodiment. According to yet other embodiments, the sex hormone is estradiol (E2). According to yet other embodiments, the sex hormone is estrone (El).

[0062] According to some embodiments, the condition, disease or disorder associated with a low level of estrogen is an iatrogenic condition, disease or disorder. The term “iatrogenic” as used herein refers to a condition, disease or disorder associated with a low level of sex hormone caused by, induced by, or resulting from a medical intervention. According to some embodiments, the medical intervention is selected from a surgery, treatment or diagnostic procedure.

[0063] According to further embodiments, the iatrogenic condition is caused by a medical intervention selected from a unilateral oophorectomy (also known as an ovariotomy), a bilateral oophorectomy, a partial hysterectomy, a radical hysterectomy, chemotherapy, or radiation therapy. Each possibility represents a separate embodiment.

[0064] The term “chemotherapy” as used herein refers to a treatment with drugs (i.e., chemical compounds) or prodrugs known to, or suspected of being able to treat a cancer (i.e., to kill cancer cells, prohibit proliferation of cancer cells, or treat a symptom related to cancer). More traditional or conventional chemotherapies can be described by mechanism of action or by chemical compound class, and can include, but are not limited to, alkylating agents (e.g., melphalan or cyclophosphamide), anthracyclines (e.g., doxorubicin), cytoskeletal disruptors (e.g., paclitaxel), inhibitors of topoisomerase I or II (e.g., irinotecan or etoposide), kinase inhibitors (e.g., bortezomib), nucleotide analogs or precursors thereof (e.g., methotrexate), platinum-based agents (e.g., cisplatin or oxaliplatin), and vinca alkaloids (e.g., vinblastine). Other examples include aromatase inhibitors, and estrogen receptor modulators such as tamoxifen, elacestrant, and fulvestrant, and kinase inhibitors such as genistein, dasatinib, erlotinib, nilotinib, regorafenib and sorafenib.

[0065] According to some embodiments, the medical intervention is a chemotherapy. According to other embodiments, the chemotherapy is gonadotoxic. According to some embodiments, the chemotherapy is selected from an anthracycline, a cytoskeletal disruptor, an alkylating agent, a vinca alkaloid, a platinum-based agent, a kinase inhibitor, an estrogen receptor modulator (i.e., tamoxifen), and an aromatase inhibitor. Each possibility represents a separate embodiment.

[0066] According to some embodiments, the condition, disease or disorder associated with low estrogen level is selected from aging, perimenopause, menopause, primary ovarian insufficiency, premature ovarian failure, polycystic ovarian syndrome, an autoimmune condition, pelvic inflammatory disease (PID), a genetic condition, a pituitary gland condition, a metabolic disorder, an affective disorder, a skin disorder, a cardiovascular disease, osteopenia, osteoporosis, diabetes, a cancer, stroke, sexual dysfunction, infertility, hirsuteness, a genitourinary condition, a vasomotor symptom, a sleep disturbance, an exposure to tobacco smoke, an exposure to gonadotoxic chemicals, and an exposure to pesticides or herbicides. According to some embodiments, the condition, disease or disorder associated with low estrogen level is selected from aging, perimenopause, and menopause.

[0067] According to some embodiments, the low level of estrogen in a subject is caused by primary hypogonadism, i.e., malfunction or dysfunction of gonads. Causes for primary hypogonadism might be Genetic conditions (e.g., Klinefelter syndrome in males, Turner syndrome in females), testicular or ovarian injury or surgery, Chemotherapy / radiation damage, infections (e.g., mumps orchitis) or Autoimmune diseases.

[0068] According to other embodiments, the low level of estrogen in a subject is caused by secondary hypogonadism, i.e., caused by a failure in hormonal signaling to the gonads.

[0069] According to some embodiments, the low level of estrogen is associated with an autoimmune condition. According to other embodiments, the autoimmune condition associated with a low level of estrogen is selected from autoimmune oophoritis and rheumatoid arthritis. Each possibility represents a separate embodiment. According to some embodiments, autoimmune oophoritis is related to systemic lupus erythematosus.

[0070] According to some embodiments, the low level of estrogen is associated with pelvic inflammatory disease (PID). According to some embodiments, the cause of PID is selected from Chlamydia trachomatis, Neisseria gonorrhoeae, Prevotella spp., Streptococcus pyogenes, Prevotella bivia, Prevotella disiens, Bacteroides spp., Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, Gardnerella vaginalis, Escherichia coli, Group B streptococcus, a-hemolytic streptococcus, Coagulase-negative staphylococcus, Atopobium vaginae, Acinetobacter spp., Dialister spp., Fusobacterium gonidiaf ormans, Gemella spp., Leptotrichia spp., Mogibacterium spp., Porphyromonas spp., Sphingomonas spp., Veillonella spp., Cutibacterium acnes, Mycoplasma genitalium, Mycoplasma hominis, or Ureaplasma spp. Each possibility represents a separate embodiment.

[0071] According to some embodiments, the low level of estrogen is associated with a genetic condition. According to some embodiments, the genetic condition associated with the low level of estrogen is selected from Turner syndrome (monosomy X), Fragile X syndrome, or aromatase deficiency syndrome. Each possibility represents a separate embodiment.

[0072] According to some embodiments, the low level of estrogen is associated with a pituitary gland condition. According to some embodiments, the pituitary gland condition is selected from a hypopituitarism or a pituitary tumor. Each possibility represents a separate embodiment. According to some embodiments, the hypopituitarism is luteinizing hormone (LH) and follicle-stimulating hormone (FSH) deficiency.

[0073] According to some embodiments, the low level of estrogen is associated with a metabolic condition. According to some embodiments, the metabolic condition is selected from metabolic syndrome (MetS), metabolic dysfunction-associated steatohepatitis (MASH), weight gain, and obesity. Each possibility represents a separate embodiment.

[0074] According to some embodiments, the low level of estrogen is associated with an affective disorder. According to some embodiments, the affected disorder associated with the low level of estrogen is selected from major depressive disorder, a mood disorder, a mood episode in bipolar disorder, schizophrenia, and depression. Each possibility represents a separate embodiment.

[0075] Considering that estrogen may have a protective role against the development of Alzheimer's disease, and / or other tauopathies, according to some embodiments, the disease or condition is pre- Alzheimer's disease and the use or method of the present invention provides inhibition of the onset or prevention of the development of Alzheimer's disease.

[0076] According to some embodiments, the low level of estrogen is associated with a skin disorder. According to some embodiments, the skin disorder is selected from atrophic vulvovaginitis, lichen sclerosus, vulvodynia, keratoderma climactericum, skin flushing, and androgenic alopecia. Each possibility represents a separate embodiment. According to some embodiments, the low level of estrogen is associated with a sexual dysfunction. According to some embodiments, the sexual dysfunction is selected from dyspareunia and decreased libido. Each possibility represents a separate embodiment.

[0077] According to some embodiments, the low level of estrogen is associated with exposure to a gonadotoxic chemical. According to some embodiments, the gonadotoxic chemical is a heavy metal selected from Pb, Cd, As, Hg, Cu, Ni, Zn, Mo, and Mn. According to other embodiments, the gonadotoxic chemical is an endocrine-disrupting chemical. According to further embodiments, the endocrine-disrupting chemical is selected from bisphenols, phthalates, parabens, and polychlorinated biphenyls. Each possibility represents a separate embodiment.

[0078] According to some embodiments, the low level of estrogen is associated with exposure to a pesticide or herbicide. According to some embodiments, the pesticide is an organophosphorus insecticide. According to other embodiments, the herbicide is atrazine.

[0079] According to some embodiments, the sex hormone is testosterone.

[0080] According to another embodiment, the subject is a male.

[0081] Thus, according to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with a low level of testosterone in a male subject. Thus, according to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with a low level of testosterone in a female subject. According to some embodiments, the present invention provides a method of treating a condition, disease or disorder associated with a low level of testosterone in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject, wherein the subject is a male or female subject.

[0082] The term “testosterone” as used herein refers to the primary male sex hormone (androgen). Testosterone is largely responsible for the development of primary male sexual characteristics, including descent of testicles, musculoskeletal development, voice deepening, enlargement of penis and testes, spermatogenesis, and increasing libido. As a male enters puberty, the gonadotropic hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), act on receptors on the gonads to effect sexual development. LH acts on Leydig cells in the testes to produce testosterone. In turn, testosterone exerts its effect on the body by binding to androgen receptor (AR), also known as NR3C4.

[0083] In addition to the role testosterone plays in male sexual development and maintenance, testosterone is also an essential hormone in females and is critical for maintaining sexual desire. Furthermore, testosterone is also the starting material for the sex hormones estrogen and dihydrotestosterone. Testosterone is converted to estrogen by the enzyme aromatase. Dihydrotestosterone (DHT), a second androgen, is formed from testosterone by the enzyme 5a-reductase. Relative to testosterone, DHT is considerably more potent as an agonist of the androgen receptor. DHT is involved in the development of secondary sex characteristics such as pubic, axillary, facial and body hair growth.

[0084] According to some embodiments, the sex hormone is testosterone or DHT. According to other embodiments, the sex hormone is testosterone. According to yet other embodiments, the sex hormone is DHT.

[0085] According to some embodiments, the subject is a male. According to other embodiments, the subject is a female.

[0086] According to some embodiments, low testosterone in a subject is caused by primary hypogonadism. According to other embodiments, low testosterone in a subject is caused by secondary hypogonadism.

[0087] According to some embodiments, the condition, disease or disorder associated with low level of testosterone is an iatrogenic condition, disease or disorder. According to other embodiments, the iatrogenic condition results from a medical intervention selected from a unilateral orchidectomy, a bilateral orchidectomy, a chemotherapy, or radiation therapy. Each possibility represents a separate embodiment.

[0088] According to some embodiments, the medical intervention is a chemotherapy. According to other embodiments, the chemotherapy is gonadotoxic. According to some embodiments, the chemotherapeutic is selected from an anthracycline, a cytoskeletal disruptor, an alkylating agent, a vinca alkaloid, a platinum-based agent, and a kinase inhibitor. Each possibility represents a separate embodiment.

[0089] According to some embodiments, the condition, disease or disorder associated with low testosterone is selected from aging, an infection, an inflammatory disease, varicocele, testicular injury, hemochromatosis, a pituitary gland condition, an autoimmune condition, a genetic condition, obesity, a decreased libido, erectile dysfunction, infertility, decreased energy, osteopenia, osteoporosis, sarcopenia, decreased facial or body hair, undescended testicles, long-term exposure to tobacco smoke, exposure to gonadotoxic chemicals, or exposure to pesticides. Each possibility represents a separate embodiment of the invention.

[0090] According to some embodiments, the condition, disease or disorder associated with low testosterone level is an infection. As used herein, the term “infection” refers to an invasion and growth of germs in the body, wherein germs may be bacteria, viruses, yeast, fungi, or other microorganisms. Infections can begin anywhere in the body and may spread to other parts of the body. According to some embodiments, the infection is selected from mumps, HIV / AIDS, and tuberculosis. Each possibility represents a separate embodiment.

[0091] According to some embodiments, the condition, disease or disorder associated with low testosterone level is an inflammatory disease. According to some embodiments, the inflammatory disease is sarcoidosis or histiocytosis. Each possibility represents a separate embodiment.

[0092] According to some embodiments, the condition, disease or disorder associated with low testosterone is a pituitary gland condition.

[0093] According to some embodiments, the condition, disease or disorder associated with low testosterone is a genetic condition. According to some embodiments, the genetic condition is selected from Kleinfelter syndrome, XXYY syndrome, and Kallmann's syndrome. Each possibility represents a separate embodiment.

[0094] According to some embodiments, the condition, disease or disorder associated with low testosterone is an autoimmune disease. According to some embodiments, the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus, and Addison’s disease. Each possibility represents a separate embodiment.

[0095] According to some embodiments, the condition, disease or disorder is associated with exposure to gonadotoxic chemicals. According to some embodiments, the gonadotoxic chemical is a heavy metal selected from Pb, Cd, As, Hg, Cu, Ni, Zn, Mo, and Mn. According to some embodiments, the gonadotoxic chemical is a hormone-disrupting chemical. According to some embodiments, the gonadotoxic chemical is selected from an opioid, a phthalate, excess alcohol, a paraben, a per-fluoroalkyl, a poly-fluoroalkyl. Each possibility represents a separate embodiment.

[0096] According to some embodiments, the condition, disease or disorder is associated with exposure to a pesticide and / or an herbicide. According to some embodiments, the pesticide and / or an herbicide is selected from an organophosphate and an N-methyl carbamate. Each possibility represents a separate embodiment. According to some of the embodiments, the condition, disease or disorder is associated with a low sensitivity to sex hormones, i.e. reduced activity of the estrogen or the testosterone receptor, including, but not limited to Kennedy disease.

[0097] According to any one of the above embodiments, the condition, disease or disorder associated with low estrogen or low testosterone levels is associated with or mediated by an unfolded protein response. According to any one of the above embodiments, the condition, disease or disorder associated with low estrogen or low testosterone is associated with an upregulated, induced unfolded protein response. The unfolded protein response (UPR) is an adaptive intracellular signaling pathway that is activated upon detection of an excess of unfolded / misfolded protein with the endoplasmic reticulum (ER). The ER is the cellular organelle responsible for, amongst other things, the production of secretory proteins that are synthesized by ER-bound ribosomes. ER stress is monitored by 3 major ER-associated proteins: the inositol-requiring transmembrane kinase / endoribonuclease 1 (IRE1); (2) the double-stranded RNA (PKR)-activated protein kinase-like eukaryotic initiation factor 2a kinase (PERK); and (3) the activating transcription factor-6 (ATF6). Each of these monitoring proteins bind to the ER luminal chaperone BiP. Upon accumulation of misfolded proteins in the ER, the unfolded proteins bind to and sequester BiP, thereby activating the UPR. In addition, alternative pathways of activating IRE1 have been reported. In mammals, the homolog IREla cleaves the mRNA of X-box binding protein-1 (XBP1), which is then translated into the potent transcription factor, XBPls, which targets a wide variety of genes, for example, those encoding proteins involved in ER protein folding, ER-associated protein degradation, ER membrane biogenesis, and protein secretion from the cell.

[0098] According to some embodiments, the condition, disease or disorder associated with low estrogen or low testosterone comprises a condition, disease or disorder in which hormone replacement therapy is contraindicated. Therefore, according to some embodiments, the present invention provides method of treating a condition, disease or disorder associated with a low level of a sex hormone and in which hormone replacement therapy is contraindicated in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject.

[0099] The term “hormone replacement therapy” as used herein refers to a treatment in which a synthetic or synthesized hormone or a substitute thereof is administered to a patient with a low level of a sex hormone who experiences symptoms associated with the low level of the sex hormone. According to some embodiments, the hormone replacement therapy comprises administering estrogen and progesterone or a substitute thereof. According to some embodiments, the hormone replacement therapy comprises administering estrogen or a substitute thereof. According to other embodiments, the hormone replacement therapy comprises administering testosterone or a substitute thereof.

[0100] According to some embodiments, the condition, disease or disorder is a condition, disease or disorder in which hormone replacement therapy is contraindicated. As used herein, the term “contraindicated” is defined as anything, including the existence of a symptom or medical condition, that renders a particular line of treatment, including the administration of drugs, undesirable or improper due to the harm that it would cause the subject. This condition may be preexisting, or may develop while the patient is taking the drugs, including conditions which may result directly or indirectly from the treatment.

[0101] According to some embodiments, the condition, disease or disorder comprises a condition, disease or disorder in which hormone replacement therapy is contraindicated and the subject is a female. According to some embodiments, hormone replacement therapy comprises administering estrogen and / or progesterone. According to some embodiments, the hormone replacement therapy is contraindicated in a female subject who uses tobacco. According to some embodiments, the hormone replacement therapy is contraindicated in the female subject due to a hormone-sensitive condition. According to further embodiments, the hormone-sensitive condition is a hormone-sensitive malignancy. According to yet further embodiments, the hormone-sensitive malignancy is selected from uterine cancer, breast cancer, endometrial cancer, or a combination thereof. According to other embodiments, the hormone replacement therapy is contraindicated due to a condition selected from ischemic stroke, hypertension, a uterine leiyomyoma (uterine fibroid), a seizure disorder, coronary arterial disease, thromboembolism, thrombophlebitis, liver disease, endometriosis, a cognitive disorder or dementia, a gallbladder disease, hypertension, migraines, a hypercoagulable disease (i.e., Protein C or Protein S deficiencies or Factor V Leiden syndrome) or a combination thereof. Each possibility represents a separate embodiment.

[0102] According to some embodiments, the condition, disease or disorder comprises a condition, disease or disorder in which hormone replacement therapy is contraindicated and the subject is a male. According to other embodiments, the subject is a female. According to some embodiments, the hormone replacement therapy comprises administering testosterone. According to other embodiments, the hormone replacement therapy is contraindicated due to a condition selected from a history of breast cancer, uncontrolled heart failure, myocardial infarction or cerebrovascular accident within the past six months, men planning fertility, a history of prostate cancer, hematocrit over 50%, a palpable undiagnosed prostate nodule, an elevated prostate-specific antigen (PSA) above 4 ng / mL, and untreated obstructive sleep apnea. Each possibility represents a separate embodiment.

[0103] According to any one of the above embodiments, the condition, disease or disorder is not a neurodegenerative condition, disease or disorder. According to any one of the above embodiments, the condition, disease or disorder is not Parkinson's disease, Alzheimer's disease, autism, progressive supranuclear palsy (PSP), and ADNP syndrome.

[0104] Pharmacology

[0105] According to the principles of the present invention, NAP can be formulated into various pharmaceutical forms for purposes of administration. The pharmaceutical composition of interest may comprise at least one additive selected from a disintegrating agent, binder, flavoring agent, preservative, colorant, and a mixture thereof, as detailed for example, in "Handbook of Pharmaceutical Excipients"; Ed. A. H. Kibbe, 3rd Ed., American Pharmaceutical Association, USA. For example, a compound of the invention, or its salt form or a stereochemically isomeric form, can be combined with a pharmaceutically acceptable carrier.

[0106] A "carrier" as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, excipient, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. For example, in preparing the compositions in oral form, media such as water, glycols, oils, and alcohols can be used in liquid preparations such as suspensions, syrups, elixirs, and solutions. Alternatively, solid carriers such as starches, sugars, kaolin, lubricants, binders, and disintegrating agents can be used, for example, in powders, pills, capsules, or tablets. For parenteral compositions, the carrier can comprise sterile water or aqueous solutions, e.g. in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer, glucose solution, or a mixture thereof. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. In addition, solid preparations that are converted to liquid form shortly before use can be made. Other suitable natural or synthetic carriers are well-known in the art (Pillai et al., 2001, Curr. Opin. Chem. Biol. 5, 447).

[0107] The pharmaceutically acceptable excipient(s) useful in the composition of the present invention are selected from but not limited to a group of excipients generally known to persons skilled in the art, e.g., diluents such as lactose (Pharmatose DCL 21), starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose- based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, and bentonite; disintegrants; binders; fillers; bulking agent; organic acid(s); colorants; stabilizers; preservatives; lubricants; glidants / antiadherants; chelating agents; vehicles; bulking agents; stabilizers; preservatives; hydrophilic polymers; solubility enhancing agents such as glycerin, various grades of polyethylene oxides, transcutol and glycofiirol; tonicity adjusting agents; pH adjusting agents; antioxidants; osmotic agents; chelating agents; viscosifying agents; wetting agents; emulsifying agents; acids; sugar alcohol; reducing sugars; non-reducing sugars and the like, used either alone or in combination thereof.

[0108] The disintegrants useful in the present invention include, but not limited to, starch or its derivatives, partially pregelatinized maize starch (Starch 1500®), croscarmellose sodium, sodium starch glycollate, clays, celluloses, alginates, pregelatinized corn starch, crospovidone, gums and the like used either alone or in combination thereof.

[0109] The lubricants useful in the present invention include but not limited to talc, magnesium stearate, calcium stearate, sodium stearate, stearic acid, hydrogenated vegetable oil, glyceryl behenate, glyceryl behapate, waxes, Stearowet, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate and the like used either alone or in combination thereof.

[0110] The anti -adherents or glidants useful in the present invention are selected from, but not limited to, a group comprising talc, corn starch, DLleucine, sodium lauryl sulfate, and magnesium, calcium, and sodium stearates, and the like or mixtures thereof. In another embodiment of the present invention, the compositions may additionally comprise an antimicrobial preservative such as benzyl alcohol.

[0111] The wetting agents useful in the present invention are selected from, but not limited to, a group comprising oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate and the like, or mixtures thereof. In yet another embodiment, the dosage form of the present invention additionally comprises at least one complexing agent such as cyclodextrin selected from a group comprising but not limited to alpha-cyclodextrin, beta-cyclodextrin, betahydroxycyclodextrin, gammacyclodextrin, and hydroxypropyl beta-cyclodextrin, or the like.

[0112] In an embodiment of the present invention, the composition may additionally comprise a conventionally known antioxidant such as ascorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, and / or tocopherol.

[0113] It can be advantageous to formulate the compositions of the invention in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” refers to physically discrete units suitable as unitary dosages, each unit containing a pre-determined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the chosen carrier.

[0114] In another embodiment, the dosage form of the present invention additionally comprises at least one wetting agent(s) such as a surfactant selected from a group comprising anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, or mixtures thereof. In yet another embodiment, the dosage form of the present invention additionally comprises of lipid(s) selected from, but not limited to, glyceryl behenate such as Compritol® ATO888, Compritol® ATO 5, and the like; hydrogenated vegetable oil such as hydrogenated castor oil e.g. Lubritab®; glyceryl palmitostearate such as Precirol® ATO 5 and the like, or mixtures thereof used either alone or in combination thereof. It will be appreciated that any given excipient may serve more than one function in the compositions according to the present invention.

[0115] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0116] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants for example polyethylene glycol are generally known in the art.

[0117] For intranasal administration, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch. Additional examples of NAP formulations are described in W02017130190 incorporated herein by reference in its entirety. Additional methods for increasing bioavailability can be used. Additional methods to enhance bioavailability and enhance specifically BBB penetration of NAP includes using ultrasound as described e.g. in Shin J, et al., Fluids Barriers CNS. 2025 Aug 25;22(1):87. doi: 10.1186 / sl2987-025-00695- 0 or Li W et al., Proc Natl Acad Sci U S A. 2025 Sep 2;122(35):e2421800122. doi: 10.1073 / pnas.2421800122. Epub 2025 Aug 27.PMID: 40864654.

[0118] For sublingual administration, the compositions may take the form of tablets or lozenges formulated in a conventional manner.

[0119] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active ingredient in water-soluble form. Alternatively, the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0120] According to some embodiments, the use or the method comprises administering the peptide intranasally, intravenously, subcutaneously, intramuscularly, or sublingually. Each possibility represents a separate embodiment. According to some embodiments, the method comprises administering the peptide intranasally. In some embodiments, the pharmaceutical compositions of the invention are formulated for local administration. In some embodiments, the compositions may be formulated for subcutaneous administration.

[0121] According to some embodiments, the formulation of NAP protein is as described in US2018344809 and US2012208763 incorporated herein by reference. Other formulations pertaining to intranasal administration, including nanoparticle formulations, are described (Galushkin and Gozes, Adv Drug Deliv Rev. 2025 May:220: 115573. doi: 10.1016 / j.addr.2025.115573. Epub 2025 Apr 2).

[0122] The composition of the present invention may be administered by any known method. The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, inhalation, orally (by ingestion), intranasally, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including selfadministration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and / or who provides a patient with a prescription for a drug is administering the drug to the patient.

[0123] The pharmaceutical composition may be administered as described hereinabove. According to some embodiments, the pharmaceutical composition is formulated for and administered via intranasal, intravenous, subcutaneous, intramuscular, or sublingual administration.

[0124] The precise dosage and frequency of administration depends on the particular compound of the invention being used, as well as the particular condition being treated, the severity of the condition, the age, weight, and general physical condition of the subject being treated, as well as other medication being taken by the subject, as is well known to those skilled in the art. It is also known that the effective daily amount can be lowered or increased depending on the response of the subject or the evaluation of the prescribing physician. Thus, the ranges mentioned above are only guidelines and are not intended to limit the scope of the use of the invention.

[0125] The combination of NAP with another therapeutic agent can be used. Such a combination can be used simultaneously, sequentially, or separately.

[0126] In some embodiments, NAP is used as a single hormone replacement agent.

[0127] According to some embodiments, the present invention provides a method of reducing an unfolded protein response in a subject in need thereof, the method comprising administering an effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject.

[0128] According to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide), for use in reducing an unfolded protein response in a subject in need thereof. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0129] All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0130] The terms “a,” “an,” and “the” are used herein interchangeably and mean one or more.

[0131] The term “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes (A and B) and (A or B).

[0132] The term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive.

[0133] The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0134] As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / -10%, or + / -5%, + / -1%, or even + / -0.1% from the specified value.

[0135] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

[0136] EXAMPLES

[0137] Example 1: Osteoporosis in female mice and osteoporosis and sarcopenia in male mice Female mice (ADNP intact and ADNP deficient, e.g., Tyr mice) are ovariectomized (OVX) and male mice are orchidectomized (ORX) at the age of between 2 and 5 months, according to the protocols described in Gozes I., etal., Mol Neurosci. 1989; 1(1):55-61. Female mice are randomly separated into four groups (n=6 per group): Sham, OVX + vehicle, OVX + estradiol (2 pg / mouse / day IP injection, or 1 pg / mouse / day IP intranasal administration), and OVX + NAP (0.5 pg / mouse / day intranasally).

[0138] Male mice are likewise randomly separated into four groups (n=6 per group): Sham, ORX + vehicle, ORX + testosterone (20 pg / mouse / day IP injection), and ORX + NAP (0.5 pg / mouse / day intranasally).

[0139] Under tribromoethanol anesthesia, the female groups are separately subjected to sham surgery without ovariectomy or with bilateral ovariectomy and the male groups separately subjected to sham surgery without orchidectomy or with orchidectomy to induce osteoporosis. Either concomitantly or seven days after the operation, the mice in OVX + NAP and ORX + NAP groups are administered 0.5 pg NAP intranasally. The OVX + estradiol group is injected intraperitoneally with 2 pg estradiol and the ORX + testosterone group is injected intraperitoneally with 20 pg testosterone. In parallel, steroid hormones are given intranasally (as above). The sham cohort in both females and males and the OVX + Vehicle and ORX + Vehicle groups are either intraperitoneally injected with placebo (n=3) or intranasally administered (n=3) placebo. Each group is treated once a day for 42 days. Measurement of grip strength

[0140] After the treatment period and prior to sacrifice, male mice are subjected to assessment of grip strength. The measurement of grip strength is carried out for all groups of male mice by using a metal mesh attached to a grip strength meter. The male mice are allowed to grasp the metal mesh attachment by their forelimbs. Once the mice grasp the metal mesh, it is pulled approximately at a rate of 2 cm / s continuously until the mice lose their grip. The maximum force exerted by the grip is recorded. For each mouse, 10 successive measurements are carried out to replicate data, and the maximum values are used for data analysis to derive muscle force.

[0141] The mice are then sacrificed, the femurs are extracted, and excess tissue is removed for microscopic computed tomography (micro-CT) and histological evaluation. The male mice are further evaluated for differences in muscle mass, as low testosterone in males is associated with sarcopenia.

[0142] Gait analysis

[0143] Falls are often associated with osteoporosis, as well as muscle weakening. In mice, gait is easily analyzed by CatWalk XT, a complete gait analysis system for quantitative assessment of footfalls and locomotion in rodents. Micro-CT Data Analysis

[0144] The left femur is scanned with a Micro-CT instrument. The region of interest (ROI) is selected, and 8 mm sections of the ROI of the distal femur are scanned. After the scan is completed, the region of interest (thickness 0.9 mm) is selected for 3D reconstruction and is analyzed for the following bone parameters: cortical bone thickness (Ct. Th); bone volume fraction (BV / TV); trabecular number (Tb.N), connectivity density (Conn.Dn), thickness (Tb.Th) and spacing (Tb.Sp); bone surface area (BS).

[0145] Histological Analysis

[0146] The gastrocnemius muscles of all male mice are likewise stained with HE. For HE staining, the femur tissue sections are stained with hematoxylin dye for about 4 min, washed with running water, dehydrated with 85 and 95% gradient alcohol, and are then stained with eosin dye for 5 min. The images of all stained slides are captured under a microscope with a CCD camera.

[0147] To look specifically at osteoclast activity, tartrate-resistant acid phosphatase staining (TRAcP) is used along with immunohistochemical detection of Cathepsin K+ (Ctsk) and Osteocalcin (OCN). An appropriate amount of TRAcP staining solution is added and incubated at 37°C for 20 min, while for immunohistochemical detection of Ctsk and OCN, femur tissue sections are incubated with corresponding antibodies and then stained with Diaminobenzidine (DAB), and finally re-stained with hematoxylin staining solution for 15 s. After staining, the femur tissue sections are scanned and the images are analyzed at 5* and 20 x magnification.

[0148] Statistical Analysis

[0149] Each experiment and data are repeated three times or more, and all the experimental data are expressed as the mean ± standard deviation (SD). The statistical significance is determined by one-way ANOVA or Student’ s / -test. A / ?-value of less than 0.05 is considered statistically significant.

[0150] Example 2.

[0151] Methods

[0152] Adult mouse neurogenesis: BrdU incorporation

[0153] Two and half month-old Adnp+ / ~ mice on an ICR background and Tyr mice (heterozygous for Adnp p.Tyr718*, also referred as "our Tyr model") on a C57BL6 / NJ background, were handled as described in Hacohen-Kleiman, G. et al. The Journal of clinical investigation 128, 4956-4969 (2018); Karmon, G. et al. Biol Psychiatry 92, 81-95 (2022)). Mice were habituated during the same time under the exact same conditions. At the age of 4 weeks, mice were treated intranasally with 0.5 pg NAP or vehicle (DD, in which each milliliter included 7.5 mg of NaCl, 1.7 mg of citric acid monohydrate, 3 mg of disodium phosphate dihydrate and 0.2 mg of a 50% benzalkonium chloride solution) for 5-6 weeks (Hacohen- Kleiman; Karmon et al). The mice were then injected (i.p.) with BrdU (80mg / kg), 4 injections at 2 h intervals, as previously described in Velazco-Mendoza et al., Neuroscience 396, 166-174 (2019). Mice were then euthanized -one hour from the last injection. Tissues were processed as previously described in Hacohen -Kleiman and in Karmon et al. At least three mice were used per experimental group.

[0154] BrdU immunohistochemistry

[0155] Immunofluorescence for BrdU detection was performed. In short, fixed tissue sections were incubated overnight with a primary antibody against rat BrdU (ab6326, Abeam, Cambridge, UK, 1 : 800), followed by a 2-hour incubation with a secondary antibody (goat anti -rat Alexa Fluor 555, a21434 Invitrogen, Waltham, MA, USA, 1 :500). All cell counting (cells / mm2) was executed by two different investigators, blind to the experiment, on six areas per section (near the lateral ventricles) spaced at least 50 pm apart. Only Dapi+ and BrdU+ nuclei were considered. Images were captured using an Axioplan- 2 optical / widefield fluorescent (Zeiss, Oberkochen, Germany) while confocal images were captured with Nikon Eclipse Ti and were assembled in Imaged (Fiji) software. Statistical analysis was performed using Sigmaplot (Grafiti LLC, CA, USA) as detailed in the figure legend (Fig. 1).

[0156] Hippocampal RNA-seq

[0157] RNA was extracted from 2.5-month-old Tyr mouse hippocampi, treated, handled, sequenced on NextSeq500 (Illumina, CA, USA), and analyzed as previously described in Karmon, G. et al. Originally, six groups of mice were used separated by sex, males and females (wild type, Tyr and Tyr treated with NAP), each including at least three biological replicates. Bioinformatic analysis

[0158] Raw sequencing data was trimmed and filtered using fastp, followed by transcript quantification with Salmon according to the GRCm38 reference genome, length and positional corrections enabled. Batch effect was corrected using surrogate variables derived from the sva package, used as model covariates to DESeq2, or applied to the count data using limma. Gene-level analysis was performed using DESeq2, including normalization of expression levels, differential expression tests and test statistic correction. Differential transcript expression levels were further tested with the Swish package, controlling for uncertainty by generating 20 inferential replicates via Gibbs sampling. Heatmaps were created using the complexHeatmaps package, with colors restricted to a reduced log2- foldchange range, due to dynamic range considerations. To make the log2FoldChange values more representative of the magnitude of differential gene expression, we used a transformed value, produced by the empirical bayes method ashr.

[0159] Enrichment testing was performed using the set-overlap and pre-ranked functions of the GSEApy package, with gene-sets from Enrichr and rummagene. All gene-sets mentioned in this work had significant enrichment results in one or more comparisons. Genotyping was performed according to the GATK best practices workflow for RNAseq short variant discovery. In short, processed reads were aligned to the GRCm38 reference genome using STAR (two pass alignment), followed by post-processing by picard and variant calling with GATK.

[0160] Results

[0161] In males, the Tyr mutation induced 34 DEGs and 23 DETs, while NAP treatment largely maintained expression, with just 10 DEGs and 6 DETs.

[0162] A large set of genes associated ER-driven cellular stress response, commonly referred to as unfolded protein response (UPR) genes, were downregulated in Tyr males exclusively, an effect that was either weakened or made insignificant upon NAP treatment (Fig. 1A, IB). Gene-set enrichment analysis indicates these genes are a part of a broader downregulated HSP-90-inhibition transcriptional signature of 26 genes. Genes induced by the HSP90 inhibitor Geldanamycin were strongly downregulated in male TYR mice (NES=-2.59; FDR<le-5) (Fig. 3C), including HSP90 co-chaperones Cdc3711 and Stipl.

[0163] Hippocampal expression of UPR genes is known to be essential for long-term memory formation and impair learning when disrupted. We further discovered that Hspa5 is downregulated in Tyr males (at the transcript level, Fig. 1A, dashed arrow). It was previously shown that over-expressed Hspa5 reverses the dysregulation of Nr4a, which in turn induces downregulation of UPR genes. While we found no significant expression difference in genes of the Nr4a family, there was a significant enrichment of downregulated glucocorticoid- induced genes (FDR<l x lO'4, Fig. IB) which are known to regulate NR4A. We also found that downregulation of neuroprotective genes such as Manf and Sipal 13 (Fig. 1 A, ellipses), known to facilitate neurological benefit in the hippocampi of old mice, was reversed by NAP treatment. The genes Sunl and Ifnarl, associated with neuronal injury and potential disruption of synaptic plasticity, were upregulated in Tyr male mice and kept unchanged by NAP treatment. NAP induced downregulation of various ER stress genes in males, including the pro- neuroinflammatory Nkd2, Hsphl (Hspl 10 / 105) and Faml07a (DRR1), which were shown to exacerbate cerebral ischemia, and Mertk, the mediator of alpha-synuclein fibril uptake. There were few male Tyr DEGs that were maintained regardless of the treatment, including upregulation of Pla2g4e, which is associated with cognitive resilience in late-onset AD models and downregulation of the glycosylation-related Man2b2.

[0164] Comparative analysis of ADNP mutation models reveal Protocadherin gene expression as a shared mechanism

[0165] We further compared the hippocampal differential expression of our heterozygous Tyr model (prefixed Tyr or NAP, depending on treatment), heterozygous deletion of exon 5 of Adnp from Cho et al. (prefixed Delx5) and heterozygous deletion of Adnp from Amram et al. (prefixed Adnp+ / ') (Fig. 2).

[0166] The relatively few differentially expressed genes that were common to 5 or more comparisons (including at least one from our Tyr models) were enriched with axon guidance genes (Col6a2, Col6al, Slitl, Scn7a), thyroid hormone signaling (Pfkl, Atplal, Myh7), and PI3K-Akt signaling (Chrml, Col6a2, Col6al, Ywhah) (Fig. 2).

[0167] The most common differentially expressed gene by far was protocadherin gamma Al l (Pcdhgal l), which was uniformly upregulated by 50% (FDR<le-10 in all comparisons) in all comparisons of Delx5 and Tyr models, regardless of treatment (Fig. 2). However, in the adult Adnp+ / ' female, a contrasting decrease was observed. Many pcdh genes were further differentially expressed due to the Tyr mutation, for example, Pcdhga9 was decreased by 50%, respectively but not in NAP female. As follows: Higher male / female neurogenesis is regulated by the major neurodevelopmental / intellectual disability / autism-linked ADNP

[0168] Twice-higher BrdU labeling in the hippocampal sub -ventricular zone (SVZ) of Adnp+ / +male mice, compared \oAdnp+ / +female mice (wild type, WT, ICR background) was identified as depicted in Fig. 3A (BrdU immunohistochemistry), densitometry results (4-5 different animals, each with left and right hippocampi / condition). Similarly, twice higher BrdU labeling was also apparent in WT males (ADNP -intact) compared to WT females of C57BL6 / NJ mice (Fig. 3B). Interestingly, the mouse strain affects BrdU labeling as well with ICR males showing 1.53-fold higher incorporation compared to C57BL6 / NJ (**P<0.01, Fig. 3C). Adnp haploinsufficiency (Adnp+ / ', ICR background) or CRSIPR / Cas9 editing to carry the most prevalent neurodevelopmental ADNP syndrome mutation, mouse equivalent p.Tyr718* (heterozygous Tyr mice, C57BL6 / NJ background) showed dramatic reductions in BrdU incorporation, resulting in mutated females presenting higher BrdU labeling than males (*P<0.05, Tyr mice, Fig. 3B). NAP treatment resulted in significantly increased Adnp+ / ~ male mice BrdU incorporation, while in the Tyr mice only a trend was observed, coupled with a significant decrease in NAP -treated Tyr females compared to WT C57BL6 / NJ (Figs. 3A-3C).

[0169] Distinct hippocampal gene and transcript differential expression induced by the ADNP Tyr mutation and NAP treatment

[0170] Analysis of RNA sequencing data from hippocampal samples revealed that about 50% of the Adnp transcripts from Tyr mice were mutated regardless of sex and treatment. Total Adnp gene expression was slightly downregulated in the mutated groups, but the difference was statistically significant only in NAP -treated Tyr females (Fig. 4), paralleling BrdU incorporation levels (Fig. 3B).

[0171] Overall, Adnp-mutation-related differential expression was almost entirely distinct between males and females, with few genes commonly differentially expressed in both sexes and / or treatments (data not shown). In addition to differentially expressed genes (DEGs), we found many differentially expressed transcripts (DETs), suggesting an effect on RNA splicing, most prominent in the Tyr groups (data not shown). The male differential expression of mRNA splicing regulators, such as the X-chromosome-linked cold-response protein RNA Binding Motif 3 (Rbm3) and the cold-inducible RNA binding protein (Cirbp, Fig. 3 A, marked by arrows), might underlie some of the extensive differential splicing. Discussion

[0172] Our results suggest intact ADNP content as a key regulator of increased male neurogenesis through the unfolded protein response (UPR). In other word, male neurogenesis deficits induced by heterozygous Adnp Tyr mutation were mediated by mostly separate, sex-specific pathways, and partial alleviation by NAP was underlined by prevention of ADNP Tyr mutation-induced differential expression, as well as possible compensation by induced neuroprotective factors.

[0173] We further propose the involvement of protocadherin genes that are commonly differentially expressed in the hippocampus of multiple Adnp loss-of-function models regardless of age, sex, or treatment, suggesting a consistent functional link that is seemingly unaffected by NAP treatment.

[0174] Regardless, Pcdhga9 was decreased by 50%, respectively, but not in the NAP female. A recent study connected Pcdhga9 to atherosclerotic cardiovascular disease (ASCVD), the leading cause of mortality worldwide. Laminar shear stress (LSS) from blood flow in straight regions of arteries protects against ASCVD by upregulating the Klf2 / 4 anti-inflammatory program in endothelial cells (ECs). Conversely, disturbed shear stress (DSS) at curves or branches predisposes these regions to plaque formation. A recently discovered suppressor of Klf2 / 4 is Pcdhga9, a member of the clustered protocadherin gene family. Pcdhg deletion increases Klf2 / 4 levels in vitro and in vivo and suppresses inflammatory activation of ECs. Pcdhg suppresses Klf2 / 4 by inhibiting the Notch pathway via physical interaction of cleaved Notchl intracellular domain (NICD Vall744) with nuclear Pcdhg C-terminal constant 47 domain (CCD). Pcdhg inhibition by EC knockout (KO) or blocking antibody protects from atherosclerosis. Pcdhg is elevated in the arteries of human atherosclerosis (https: / / www.biorxiv.org / content / 10.1101 / 2024.01.16.575958v2.full.pdf). Thus, NAP is suggested for the first time for treating inflammatory vascular disease.

[0175] We have discovered here differential expression of mRNA splicing regulators, such as the X-Rbm3 as well as Cirbp (Fig. 1A, IB, males) involving extensive differential splicing, with female Tyr compared to WT showing 43 DETs and 4 additional DEGs, while NAP treatment had 27 DEGs and 9 DETs, in Tyr males, the picture was reversed with 34 DEGs and 23 DETs, while NAP treatment largely maintained expression, with just 10 DEGs and 6 DETs. In this respect, immunoprecipitations suggested Brm-ADNP interaction coupled to ADNP - polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF)-binding, with PSF being a direct regulator of Tau transcript splicing and with Tau deposition (tauopathy) in both Adnp+and Tyr mice as well as ADNP syndrome postmortem tissue.

[0176] Downregulation of unfolded protein response in Tyr males, moderated or prevented by NAP Our results indicated that the Tyr mutation in ADNP caused a significant downregulation of numerous hippocampal UPR genes exclusively in males, which was either moderated or prevented by NAP treatment. This downregulation of male UPR signaling might contribute to the substantial decrease in hippocampal neurogenesis, since it is a pivotal regulator of neurodevelopment, in a sex-dependent manner.

[0177] The synaptogenesis role of the protocadherins is directly related to ADNP critical effect on dendritic spine formation, which exhibits sexual dimorphism accentuated in the Tyr mice (e.g. most significant mutation effect in the male hippocampus corrected by NAP treatment, versus most significant mutation effect in the female motor cortex, implicated in more extensive gait aberrations compared to males, corrected by NAP treatment, Karmon et al., IBID). Furthermore, Pcdhgs differentially expressed in this study are positive regulators of Wnt (Pcdhga8, Pcdhga9, Pcdhgbl, Pcdhgc5), except for the neutral Pcdhgal l. Adnp prevents P-catenin degradation by binding its armadillo domain through the NAP motif, thereby disassociating P-catenin from the AXIN+APC degradation complex, enhancing WNT signaling and promoting neural induction / neurogenesis (Sun X. et al., Nat Commun 11, 2984 (2020)). Hence, the interaction between ADNP / NAP and Pcdhg is multifunctional, acting through expression regulation, as well as protein interaction / WNT signaling pathway.

[0178] Example 3

[0179] The aim of this experiment was to assess the efficacy of NAP peptide as estrogen replacement therapy. In this experiment, the effect of administering estrogen or NAP was compared using grip strength model of ovariectomized mice. Ovariectomy, surgical removal of ovaries, leads to decreased grip strength in animal models, as described e.g. in Lowe et al., Exerc Sport Sci Rev. 2010 April ; 38(2): 61-67. doi: 10.1097 / JES.0b013e3181d496bc. The effect of estrogen's level reduction may be due to muscle weakness and mitochondrial dysfunction in skeletal muscle.

[0180] 8-weeks mice were ovariectomized and assigned to one of four treatment groups:

[0181] Control- intact mice receiving vehicle.

[0182] OVX- ovariectomized mice receiving vehicle. OVX+NAP- ovariectomized mice treated with NAP.

[0183] OVX+Estrogen- ovariectomized mice treated with estrogen.

[0184] The treatments began on the 6thday after ovariectomy. The mice were administered intranasally in a fixed volume of 5 pL per mouse, once daily, five days per week for at least 7 weeks.

[0185] The administered compositions were prepared as follows:

[0186] NAP was first dissolved in water and then diluted in double-distilled water (PDD- H2O) to a final concentration of 0.5 pg in 5 pL.

[0187] Estrogen was first dissolved in ethanol, then in water, and then diluted in I DD-H2O to a final concentration of 1 pg in 5 pL.

[0188] Mice in the Control and OVX groups received only the vehicle solution (1 xDD-EEO), ensuring uniform treatment conditions across all groups with NAP or estrogen being the only variables. (1XDD= 7.5 mg of NaCl, 1.7 mg of citric acid monohydrate, 3 mg of disodium phosphate dihydrate, and 0.2 mg of benzalkonium chloride solution (50 %) per milliliter - (R.N. Alcalay, E. Giladi, C.G. Pick, I. Gozes. Intranasal administration of NAP, a neuroprotective peptide, decreases anxiety-like behavior in aging mice in the elevated plus maze. Neurosci Lett, 361 (1-3) (2004), pp. 128-131)

[0189] At 16 weeks of age (after 7 weeks of treatment), the front limb grip strength was assessed using the Ugo Basile Grip Strength Meter (GSM), which measures the maximum force exerted by rodents during a pull-back movement, leveraging their instinctive resistance to backward motion. For each mouse, five individual measurements were taken, and the average of the top three highest values was calculated to represent the final grip strength score.

[0190] The results are presented in Figs. 5A-5C. As can be seen, both estrogen and NAP provided similar significant effect on grip strength. NAP revised grip strength ssimilarly to estrogen. This provides support that NAP may be used as estrogen replacement.

[0191] Example 4.

[0192] One of the well-known results of estrogen deficiency is reduction in bone density and development of osteoporosis. CT of the thighbone (femur) of mice from Example 3 was performed 9 weeks after initiation of treatment using the VECTor platform, an integrated PET / SPECT system that enables sub-mm imaging of PET and SPECT tracers separately or simultaneously with unprecedented spatial resolutions. The U-PET system is capable of performing quantitative 3D functional imaging of PET tracers at a resolution of less than 0.6 mm. It allows for both focused and whole-body imaging of rodents, both in static and dynamic PET modes. The SPECT technology offers uniform resolution down to 0.13 mm ex vivo and 0.25 mm in vivo. The stationary detectors of the VECTor allow for fast PET and SPECT imaging in both focused and whole-body modes. The U-CT system is high-speed and can scan a mouse’s total body in 5 seconds with ultra-low radiation doses, down to less than 2 mGy whole-body. By default, both mice and rats can be imaged. Optionally, the system can be upgraded to image medium-sized animals, enabling whole-body rabbit imaging. The U-CTHR system provides high-resolution imaging with reconstructed voxel resolution down to 10 pm. It is optimized for in vivo imaging, including dynamic contrast-enhanced (DCE) CT imaging using single- or dual-energy scans, ultra-fast fluoroscopic imaging with up to 66 frames per second, and cardiac and respiratory gating.

[0193] From the results, it can be seen that NAP and estrogen have similar positive trend of improvement in bone density versus the control. Additional results are obtained from micro- CT inspection of the bones of said mice as discussed in Gozes I et 1., Transl Psychiatry. 2017 Feb 21;7(2):el043. doi: 10.1038 / tp.2017.27.PMID: 28221363.

[0194] Example 5

[0195] This experiment aims to assess the efficacy of NAP peptide as a testosterone replacement therapy. In this experiment, the effect of administering testosterone or NAP is compared on grip strength of orchiectomized mice.

[0196] 8-weeks mice were orchiectomized and assigned to one of four treatment groups: Control- intact mice receiving vehicle.

[0197] OVX- orchiectomized mice receiving vehicle.

[0198] OVX+NAP- orchiectomized mice treated with NAP.

[0199] OVX+ (testosterone or DHT)- orchiectomized mice treated with (testosterone or DHT).

[0200] The treatment begins on the 6thday after orchiectomy. The mice are administered intranasally in a fixed volume of 5 pL per mouse, once daily, five days per week.

[0201] The administered compositions are prepared as follows: NAP was first dissolved in water and then diluted in double-distilled water (1 xddEEO) to a final concentration of 0.5 pg in 5 pL.

[0202] Testosterone was first dissolved in ethanol, then in water, and then diluted in 1 xDD-ILO to a final concentration of 1 jug in 5 jiL.

[0203] Mice in the Control and OVX groups receive only the vehicle solution (1 xDD-ILO), ensuring uniform treatment conditions across all groups with NAP or estrogen being the only variables. (1XDD= 7.5 mg of NaCl, 1.7 mg of citric acid monohydrate, 3 mg of disodium phosphate dihydrate, and 0.2 mg of benzalkonium chloride solution (50 %) per milliliter - (R.N. Alcalay, E. Giladi, C.G. Pick, I. Gozes. Intranasal administration of NAP, a neuroprotective peptide, decreases anxiety -like behaviour in aging mice in the elevated plus maze.

[0204] At 16 weeks of age, the front limb grip strength is assessed using the Ugo Basile Grip Strength Meter (GSM), which measures the maximum force exerted by rodents during a pull-back movement, leveraging their instinctive resistance to backward motion. For each mouse, five individual measurements are taken, and the average of the top three highest values is calculated to represent the final grip strength score.

[0205] According to the results, NAP may be used as a testosterone replacement.

[0206] Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

CLAIMS1. A pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a condition, disease or disorder associated with sex hormone imbalance.

2. The pharmaceutical composition for use of claim 1, wherein sex hormone imbalance comprises a low sex hormone level.

3. The pharmaceutical composition for use according to claim 1 or 2, wherein the sex hormone is estrogen.

4. The pharmaceutical composition for use according to claim 3, wherein the low level of estrogen is caused by an iatrogenic condition.

5. The pharmaceutical composition for use according to claim 4, wherein the iatrogenic condition is a result of a medical intervention selected from oophorectomy, bilateral oophorectomy, partial hysterectomy, radical hysterectomy, chemotherapy, or radiation therapy.

6. The pharmaceutical composition for use according to claim 3, wherein the condition, disease or disorder is selected from aging, perimenopause, menopause, primary ovarian insufficiency, premature ovarian failure, polycystic ovarian syndrome, an autoimmune condition, pelvic inflammatory disease (PID), a genetic condition, a pituitary gland condition, a metabolic disorder, an affective disorder, a skin disorder, a cardiovascular disease, osteopenia, osteoporosis, diabetes, a cancer, stroke, sexual dysfunction, infertility, hirsuteness, a genitourinary condition, a vasomotor symptom, a sleep disturbance, an exposure to tobacco smoke, an exposure to gonadotoxic chemicals, and an exposure to pesticides or herbicides.

7. The pharmaceutical composition for use according to claim 1 or 2, wherein the sex hormone is testosterone.

8. The pharmaceutical composition for use according to claim 7, wherein the low level of testosterone is caused by an iatrogenic condition.

9. The pharmaceutical composition for use according to claim 8, wherein the iatrogenic condition is caused by a medical intervention selected from a unilateral orchidectomy, bilateral orchidectomy, chemotherapy, or radiation therapy.

10. The pharmaceutical composition for use according to claim 9, wherein the condition, disease or disorder is selected from aging, an infection, an inflammatory disease, varicocele, testicular injury, hemochromatosis, a pituitary gland condition, an autoimmune condition, a genetic condition, obesity, a decreased libido, erectile dysfunction, infertility, decreased energy, osteopenia, osteoporosis, sarcopenia, decreased facial or body hair, undescended testicles, long-term exposure to tobacco smoke, exposure to gonadotoxic chemicals, or exposure to pesticides.

11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the condition, disease or disorder is associated with an unfolded protein response.

12. The pharmaceutical composition for use according to any one of claims 1 to 11, wherein the condition, disease or disorder comprises a condition, disease or disorder in which a hormone replacement therapy is contraindicated.

13. The pharmaceutical composition for use according to any one of claims 1 to 12, wherein the peptide is administered in an administration mode selected from an intranasal, an intravenous, a subcutaneous, an intramuscular, or a sublingual mode of administration.

14. The pharmaceutical composition for use according to any one of claims 1 to 13, provided that the condition, disease or disorder is not a neurodegenerative condition, disease or disorder.

15. The pharmaceutical composition for use according to any one of claims 1 to 14, wherein the use comprises hormone replacement therapy.

16. A pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in hormone replacement therapy.

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