Therapeutic drug compositions for the treatment of mild cognitive impairment, depression, and mental disorders and methods of using the same

Abstract

The present invention generally relates to the use of cyclic prolylglycine ("cyclic PG" or "cPG") and its analogs and mimetics as neuroprotective agents for the treatment or prevention of cognitive and neurological disorders, including, but not limited to, cerebral ischemia or cerebral infarction. The present invention provides therapeutic agents for the treatment and prevention of chronic neurodegenerative diseases such as epilepsy, perinatal asphyxia, anoxia, brain trauma, Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as anticonvulsants. The present invention also generally provides manufacturing methods for preparing dosage forms. The present invention further generally relates to the use of cyclic prolylglycine and its analogs and mimetics as neuroprotective and neuroregenerative agents for the treatment or prevention of depression and other psychiatric disorders.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of priority of U.S. Patent Application No. 62 / 674,855, filed May 22, 2018; U.S. Patent Application No. 62 / 671,485, filed May 15, 2018; and U.S. Patent Application No. 62 / 671,466, filed May 15, 2018, each of which is hereby incorporated by reference in its entirety. Inventor Lloyd Hoon Roy Tran reflects a change of legal name from Roy H.

Technical Field

[0002] The present invention generally relates to novel cyclic dipeptide compounds structurally related to diketopiperazine and methods for their therapeutic use. In particular, the present invention relates to the neuroprotective and neuroregenerative activities of such compounds. In particular, the present invention relates to the use of cyclic prolylglycine (“cyclic GP” or “cPG”) and cPG analogs, and cPG compounds, pharmaceutically effective analogs thereof, and pharmaceutical compositions thereof, in the treatment and prevention of cognitive impairment and related neurodegenerative and mental disorders. The present invention also generally relates to materials and methods for regenerating nerve cells and glial cells, or methods for repairing damaged nerve cells and glial cells.

Background Art

[0003] Mild cognitive impairment (MCI) refers to a state in which cognitive function is reduced more than expected according to age and educational background. Mild cognitive impairment refers to a condition in which there are disorders in cognitive functions such as memory, which is beyond the norm for age but does not reach the level of dementia, which is a characteristic of dementia. The prevalence of MCI varies by age. The age-specific prevalence of MCI is as follows: 6.7% for those aged 60 - 64, 8.4% for those aged 65 - 69, 10.1% for those aged 70 - 74, 14.8% for those aged 75 - 79, and 25.2% for those aged 80 - 84. More than half of people with MCI progress to dementia within 5 years (Non-Patent Document: 10.1212 / WNL.0000000000004826.:https: / / en.wikipedia.org / wiki / PubMed_Identifier “\o”PubMed Identifier 29282327.).

[0004] Dementia is a general term that describes a group of symptoms related to a decline in memory and other thinking abilities, and is severe enough to reduce a person's ability to perform daily activities. Alzheimer's disease accounts for 60 - 80% of dementia cases. Vascular dementia, which develops after a stroke, is the second most common type of dementia. However, there are many other diseases that cause dementia symptoms, including reversible ones such as thyroid problems and vitamin deficiencies. The symptoms of dementia are diverse, but a person cannot be said to have dementia unless at least two of the following core mental functions are significantly impaired: memory, communication, language, concentration and attention, reasoning and judgment, and visual perception.

[0005] In 2018, more than 5.5 million people in the United States and over 50 million people worldwide were suffering from Alzheimer's disease. Reference: Alzheimer's Disease International's World Alzheimer Report 2018) The increasing prevalence of Alzheimer's disease over the next few decades is expected to put significant pressure on social and healthcare systems in both developed and developing countries. There has long been an unmet need for treatments that stop, substantially slow down, or otherwise improve the progression and symptoms of diseases associated with this illness, and provide comfort and palliative care.

[0006] The pioneer researcher Ramón y Cajal at the beginning of the 20th century wrote as follows: "The functional specialization of the brain imposed two major fissures on neurons. This is why, when development is over, the sources of axon and dendrite growth and regeneration become irreversibly depleted" (Non-Patent Document 2 (New Ideas on the Fine Anatomy of the Nervous Center)). This hypothesis, which used to be a basic principle of neuroscience dating back from the second half of the 19th century to the mid-20th century, has now been proven invalid.

[0007] In 1966, Altman and Gopal showed that evidence of adult mammalian neurogenesis was discovered in other regions of the brain including the hippocampus of rodents. They reported autoradiography and histological studies of postnatal neurogenesis in which cell proliferation and migration were seen in the anterior forebrain, referring in particular to the persistence of neurogenesis in the olfactory bulb (Non-Patent Document 3 (https: / / doi.org / 10.1002 / cne.901240303)).

[0008] In Altman's experiment, in the intact adult mammalian brain, nerve regeneration maintains the function and structure of the central nervous system (CNS). Thymidine-H3 was intraperitoneally injected into 6-day-old and 13-day-old rats, and then the animals were allowed to survive for 1 hour to 60 days. Autoradiography data obtained from animals that survived for a short period were used to estimate the local cell proliferation rate. Animals with a long survival period were used to infer the migration of new cells from the reproductive site to the target site via the migration channel and to determine their differentiation pattern. The formation and differentiation of microneurons continue during infancy, but the rate is decreasing in most structures. In the external granular layer of the cerebellar cortex, cell proliferation continues at a very high rate, and cells migrate from there to the molecular layer and the internal granular layer.

[0009] It has been revealed that adult regenerated neurons are integrated into existing brain circuits and contribute to the improvement of nerve disorders (Non-Patent Document 4).

[0010] Interestingly, neurogenesis has been observed to occur not only at the level of the olfactory bulb and hippocampus, but also at the level of neurons. In this regard, this process has been suggested to occur even in the substantia nigra of adult mice, opening up a new field of research for the treatment of neurodegenerative diseases (Non-Patent Document 5).

[0011] According to a review paper by Guo-li Ming, since neural precursors were discovered in the postnatal rat hippocampus, researchers have now firmly established that active neural precursors from neural precursors continue throughout life in discrete regions of the central nervous system (CNS) of all mammals, including humans (Non-Patent Document 6).

[0012] Regarding the generation process and control of adult neurogenesis, significant progress has been made in understanding processes such as proliferation, fate specification, neuronal maturation, targeting, and synaptic integration of newborn neurons.

[0013] Although the exact mechanism for maintaining functional neural stem cells (NSCs) in these regions has not been elucidated, NSCs have shown the ability to restore neurons and glia in response to specific pathological conditions.

[0014] Depression is a mental health disorder characterized by a persistently low mood and a lack of interest in activities, which significantly impairs daily life. Many factors, such as genes, stress, and the chemical nature of the brain, can potentially cause depression. According to John Geddes, a professor of epidemiological psychiatry at the University of Oxford, "Depression is the single largest contributor to the global disorder we have, and a major problem for humanity," affecting approximately 350 million people worldwide, with the number of instances increasing by nearly 20% from 2005 to 2015 (World Health Organization (WHO) and Centers for Disease Control).

[0015] In recent years, a great deal of research effort has been expended on the study of mental depression and its treatment methods. Although many commercially available drugs can improve depression, they all have undesirable side effects. Recently, suicide has been reported as one of the serious side effects of depression, and this has virtually become the sole cause of death due to mental illness.

[0016] The present invention provides a method for treating depression without serious side effects. According to this method, cyclic prolylglycine and its pharmaceutically effective analogs have been demonstrated as potential treatment methods for patients suffering from depression.

Prior Art Documents

Non-Patent Documents

[0017]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Means for Solving the Problems

[0018] One aspect of the present invention provides cyclic prolylglycine compounds suitable for the treatment and prevention of diseases and injuries in animals and humans. Cyclic PGs are selected from the group including cPG, cPG analogs, cPG peptidomimetics, and related compounds that promote or cause the formation of cPG or cPG analogs in vivo.

[0019] An example of a cPG analog is cyclic (glycyl-L-prolyl-L-prolyl), or is abbreviated as cyclic or herein referred to as c(PG)3.

[0020] Another example of a CPG analog is cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, referred to herein as "cGAL".

[0021] Collectively, cPG, c(PG)3, cGAL, and cyclic glycyl-2-methyl-proline, and their pharmaceutically acceptable salts are referred to herein as "cPG compounds".

[0022] Also, cyclic glycyl-2-methyl-proline is a compound belonging to the group of cyclic glycyl-2-alkylprolines of the compound.

[0023] Furthermore, any of the cPG compounds, their derivatives, their analogs, and the like disclosed herein or otherwise known in the art can be provided in the form of pharmaceutically acceptable salts.

[0024] Preferably, the cPG compound is administered in a pharmaceutically acceptable composition such as a pharmaceutically acceptable carrier.

[0025] More preferably, the composition further comprises a therapeutic amount of the cPG compound in combination with a compound selected from growth factors and related derivatives (insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), GPE, transforming growth factor-II). Activin, growth hormone, nerve growth factor, growth hormone binding protein, JQF binding protein (especially JGFBP-3), basic fibroblast growth factor, acidic fibroblast growth factor, hst / Kfgk gene product, FGF-3, FGF-4, FGF-6, keratinocyte growth factor, androgen-induced growth factor. Additional members of the FGF family include, for example, int-2, fibroblast growth factor homologous factor-1 (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growth factor 2, glia activating factor, FGF-10 and FGF-16, ciliary neurotrophic factor, brain-derived growth factor. Neurotrophin 3, neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial cell line-derived neurotrophic factor, activity-dependent neurotrophic factor, cytokine leukemia inhibitory factor, oncostatin M, interleukin), β, α, χ or consensus interferon, TNF-α. Cromethiazole; kynurenic acid, max, FK506 [tacrolimus], L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, adrenocorticotropin-(4-9_analog [ORG2766] and dizolcipine [MK-801], selegiline; glutamic acid ali, for example, NPSl 5O6, GV1505 260, MK-801, GV150526. 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070 and LY300164-like AMPA ali; anti-inflammatory agents directed against addressin MAdCAM-1 and / or integrin α4 receptors (α4β1 and α4β7) such as anti-MAdCAM-1lmAb MECA-367 (ATCC accession number HB-9478), interferons including interferon βLb and interferon alpha con-1.

[0026] Preferably, the cPG compound can be used for the treatment or prevention of cell damage or cell death resulting from septic shock, ischemia, cytokine administration, cytokine overexpression, ulcers, gastritis, ulcerative colitis, Crohn's disease, and diseases and injuries caused thereby. Diabetes, rheumatoid arthritis, asthma, Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, cirrhosis, transplant rejection, encephalomyelitis, meningitis, pancreatitis, peritonitis, vasculitis, lymphocytic choriomeningitis glomerulonephritis uveitis, glaucoma, blepharitis, carradion, allergic eye diseases, corneal ulcers, keratitis, cataracts, retinal disorders, age-related macular degeneration, optic neuritis ileus, inflammation due to overproduction of inflammatory cytokines, hemorrhagic shock anaphylactic shock, burns, infections leading to overproduction of inflammatory cytokines induced by bacteria, viruses, fungi, parasites, hemodialysis, chronic fatigue syndrome, stroke, cancer, cardiovascular diseases associated with overproduction of inflammatory cytokines. Heart disease, cardiopulmonary bypass, ischemia-reperfusion injury, ischemia-reperfusion associated with overproduction of inflammatory cytokines, toxic shock syndrome, adult respiratory distress syndrome, cachexia, myocarditis, autoimmune diseases, eczema. Psoriasis, heart failure, dermatitis, hives, cerebral ischemia, systemic lupus erythematosus, AIDS, AIDS dementia, chronic neurodegenerative diseases, chronic pain, priapism, cystic fibrosis, amyotrophic lateral sclerosis, schizophrenia, depression. Premenstrual syndrome, anxiety, poisoning, migraine, Huntington's disease, epilepsy, gastrointestinal motility disorders, obesity, dysphagia, neuroblastoma, malaria, blood cancer, myelofibrosis, lung injury, graft-versus-host disease, head injury central nervous system trauma, hepatitis, renal failure, chronic hepatitis C, paraquat poisoning, transplant rejection and preservation, enhanced reproductive ability, bacterial translocation, circulatory shock, traumatic shock, hemodialysis, hangover, and combinations of two or more thereof.

[0027] Preferably, the cPG compound can be used for the restoration of axonal myelination in mammals with myelin depletion due to nerve damage or disease.

[0028] Preferably, the cPG compound can be used for the restoration of myelination depleted due to trauma, toxin exposure, asphyxia or hypoxia-ischemia, perinatal hypoxia-ischemia injury, injury or disease to the central nervous system white matter, acute brain injury, chronic neurodegenerative diseases including multiple sclerosis, and demyelinating diseases and disorders including acute disseminated encephalomyelitis, optic neuritis, polymyositis, Devic's disease, leukodystrophy. Diseases such as non-inflammatory lesions, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

[0029] Preferably, the cPG compound can be administered in combination with IGF-1 or interferon.

[0030] Another related aspect is that the present invention relates to a method for treating or preventing cell damage or cell death in response to injury and disease by administering at least one cPG compound.

[0031] Preferably, the cPG compound can be administered at a dose between about 1 μg and about 150 mg per kilogram of body weight. Suitable dosages for the administration of cPG can be, for example, preferably between about 0.1 mg and about 100 mg per kilogram of body weight, between about 1 mg and about 100 mg per kilogram of body weight, between about 5 mg and about 70 mg per kilogram of body weight, between about 10 mg and about 50 mg per kilogram of body weight, or between about 20 mg and about 40 mg per kilogram of body weight, but are not limited thereto. The dosage, route of administration, and dosing regimen of cPG may vary depending on the disease, disorder, and condition. As an example, in mild cognitive impairment, the dosage using the same or different routes of administration may be lower than in the case of Alzheimer's disease.

[0032] For example, the typical dosage for a patient with mild cognitive impairment can be administered orally between about 0.2 mg and about 1 mg per day (e.g., take 1 capsule of 20 mg or 2 capsules per day based on a doctor's prescription). On the other hand, patents with more severe Alzheimer's disease or severe traumatic brain injury can be administered intravenously in the range of about 50 mg to about 300 mg per day. The determination of the dosage, administration route, and regimen for a particular disease, disorder, or condition can generally or can be evaluated for a particular subject or patient.

[0033] A further aspect of the present invention relates to a method for restoring axonal myelination in a mammal in need of remyelination due to nerve injury or disease, the method comprising administering a therapeutic amount of a cPG compound, where the cPG compound consists of cPG. Biologically active cPG analogs such as c(PG)3 and cGAL, biologically active cPG peptidomimetics, compounds that increase the concentration of cPG, or compounds that increase the concentration of cPG analogs, which are effective in restoring axonal myelination in a mammal. In one aspect of the present invention, the method for restoring axonal myelination comprising administering a therapeutic amount of a cPG compound consists of stimulating astrocytes to promote remyelination. In another aspect of the present invention, the method for restoring axonal myelination comprising administering a therapeutic dosage of a cPG compound consists of stimulating oligodendrocytes to produce myelin.

[0034] In yet another aspect of the present invention, a method of restoring myelination of axons further comprises administering a therapeutic amount of a cPG compound in combination with a compound selected from IGF-I or interferon to a mammal in need of restored myelination. In one aspect of the present invention, a method of restoring myelination of axons comprises administering a therapeutic dose of a cPG compound in combination with IGF-I or interferon to stimulate astrocytes to promote remyelination. In another aspect of the present invention, a method of restoring myelination of axons comprises administering a therapeutic dose of a cPG compound in combination with IGF-I or interferon to stimulate oligodendrocytes to produce myelin. In a preferred embodiment, the interferon consists of interferon beta 1b (Betaseron). In a further most preferred embodiment, the interferon consists of consensus interferon (Infergen®, interferon alphacon-1).

[0035] In yet another aspect of the present invention, a method of treating or preventing cell damage and death in response to injury and disease comprises administering a therapeutic dose of a cPG compound, preferably an amount of cPG between about 10 μg and about 150 mg per kg of mammalian body weight, but not limited thereto. Suitable amounts for administration of cPG are, for example, preferably between about 0.1 mg and about 100 mg per kg of body weight, between about 1 mg and about 100 mg per kg of body weight, between about 5 mg and about 70 mg per kg of body weight, between about 10 mg and about 50 mg per kg of body weight, or between about 20 mg and about 40 mg per kg of body weight, but not limited to these. The dosage, route of administration, and dosing regimen of cPG may vary depending on the disease, disorder, or condition. As an example, in mild cognitive impairment, the dosage may be lower, using the same or different routes of administration, than in the case of Alzheimer's disease. For example, a typical dosage for a patient with mild cognitive impairment may be about 10 mg to about 50 mg per day (1 capsule of 20 mg taken 1 capsule per day or 2 capsules per day as prescribed by a physician) by oral administration. On the other hand, patents for more severe Alzheimer's disease or severe traumatic brain injury can be administered intravenously in the range of about 50 mg to about 300 mg per day. Details of the dosage, route of administration, and regimen for a particular disease, disorder, or condition can generally be, or can be evaluated for a particular subject or patient.

[0036] In yet another aspect of the present invention, a method of restoring myelination of axons in a mammal in need thereof comprises administering to the mammal a therapeutically effective amount of a cPG compound in combination with interferon of about 1 mg to about 10 mg of IGF-I per kg of body weight of the mammal, or about 1.0 μg to about 10 μg of IGF-I per kg of body weight of the mammal. In a preferred embodiment, the interferon is interferon β. Suitable dosages for administration of cPG can be, for example, preferably about 0.1 mg to about 100 mg per kg of body weight, about 1 mg to about 100 mg per kg of body weight, about 5 mg to about 70 mg per kg of body weight, about 10 mg to about 50 mg per kg of body weight, or about 20 mg to about 40 mg per kg of body weight, but are not limited thereto. The dosage, route of administration, and dosing regimen of cPG may vary depending on the disease, disorder, or condition. As an example, mild cognitive impairment may be able to have a lower dosage using the same or different routes of administration than in the case of Alzheimer's disease. For example, a typical dosage for a patient with mild cognitive impairment may be about 10 mg to about 40 mg per day administered orally (taking 1 or 2 capsules of 20 mg capsules per day, as prescribed by a physician). On the other hand, patents for more severe Alzheimer's disease or severe traumatic brain injury can be administered intravenously in the range of about 50 mg to about 300 mg per day. The determination of the dosage, route of administration, and regimen for a particular disease, disorder, or condition can generally or can be evaluated for a particular subject or patient.

[0037] In a further preferred embodiment of a method of treating or preventing cell damage and death in response to injury and disease, which consists of administering a cPG compound, the cPG compound is administered to the mammal via a shunt to the ventricle of the mammal.

[0038] In a further preferred embodiment of a method of treating or preventing cell damage and death in response to injury and disease, which consists of administering a cPG compound, the cPG compound is administered to the mammal by peripheral administration.

[0039] The present invention provides a method of stimulating mature astrocytes to promote myelin production after hypoxic-ischemic injury, the method comprising increasing the active concentration of cPG and / or the concentration of an analog of cPG in the mammalian CNS.

[0040] Most preferably, it is an effective amount of IGF-I itself increased within the mammalian CNS. This can be effected by direct administration of a cPG compound such as cPG, c(PG)3 or cGAL or cGMeP, and in practice this is preferred. However, it is not possible to exclude administration of compounds that indirectly increase the effective amount of IGF-I (for example, prodrugs that are cleaved within the patient to release cPG).

[0041] The active compound (IGF-I or an analog or mimetic thereof) can be administered alone or, preferably, as part of a pharmaceutical composition.

[0042] The composition can be administered directly to the CNS. The latter route of administration can include, for example, lateral cerebral vein injection, local injection, or a shunt surgically inserted into the lateral cerebral vein of the patient's brain.

[0043] Advantageously, in the prevention or treatment of demyelinating diseases such as multiple sclerosis, administration of a cPG compound promotes the stimulation and promotion of myelin production in oligodendrocytes and the support, stimulation and promotion of remyelination by mature astrocytes.

[0044] As generally described in U.S. Patent Publication No. US20100247483A1, which is hereby incorporated by reference in its entirety, cyclic prolylglycine ("cyclic PG" or "cPG") has the following structure.

Chemical formula

[0045] The present invention includes novel diketopiperazines structurally related to cPG.

[0046] One aspect of the present invention provides a novel cyclic compound having the structural formulas and substituents described below.

Chemical formula

[0047] In the formula, R can be "alkyl" which refers to a saturated branched, straight-chain or cyclic hydrocarbon radical. Exemplary alkyl groups include methyl, ethyl, isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, hexyl, and the like.

[0048] In the formula, R can be allyl referring to a group, a substituent having the structural formula H2C=CH-CH2R, where R is the remainder of the molecule.

[0049] When R is methyl, one aspect of the present invention comprising cycloglycyl-2-alkylproline is (8as)-methyl-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, which is referred to as cycloglycyl-2-methylprolym or cyclogMeP or cGMeP.

[0050]

Chemical formula

[0051] Generally, c(PG)3 and cGAL can be prepared by methods well known to those of ordinary skill in the art of peptide and modified peptide synthesis. See, for example, Bodanzsky. See Principles of Peptide Synthesis, Berlin, New York. Springer-Verlag 1993. The synthesis of the diketopiperazine compounds of the present invention may be by solid-phase synthesis as discussed in the examples, or Merrifield et al. (1963 J.Amer.Chem.Soc.:85,2149-2156. Specific examples of diketopiperazine synthesis are described in Fischer, 2003, J.Peptide Science.9:9-35 and the references cited therein. Those of ordinary skill in the art will have no difficulty in developing one or more suitable synthetic methods for the compounds of the present invention in view of the techniques and available knowledge and the present disclosure.

[0052] In the present application, although the names, structures, and abbreviations of the compounds are not particularly limited in this section where they are provided, various compounds can be used in all aspects of the present invention included in this specification. For example, if cPG is described in this specification, all other compounds in this section (and throughout this application), such as cPG compounds and related derivatives such as cGAL, are not limited to the treatment methods for various conditions described in this specification, but are included in the parts and other descriptions thereof, which are not limited to the treatment methods for various conditions described in this specification.

[0053]

Chemical Structure

[0054] The chemical synthesis of cyclo(glycyl-L-prolyl-L-prolyl) was carried out as described by Charles M. Deber and Erkan R. Boute in Israel Journal of Chemistry, Vol. 12, Nos. 1-2, 1974, pp. 15-29 “CYCLIC Peptides VII: The Synthesis and Characterization of Cyclic Peptides with Repeating Pro-Gly Sequences”.

[0055] Synthesis of Cyc / o(glycyl-L-prolyl-glycyl-L-prolyl) A solution of p-nitrophenyl ester hydrochloride (500 mg) dissolved in dimethylformamide (DMF) (20 mL, dried over sodium sulfate) was added dropwise with stirring to 500 mL of reagent grade pyridine at room temperature over 6 hours. The bright yellow mixture was stirred continuously at room temperature for 48 hours. The solvent was removed by a rotary evaporator high vacuum pump system at 45°. The residue was washed with 20 ml of acetone, which dissolved p-nitrophenol and pyridine hydrochloride but left the peptide fraction insoluble. The insoluble material and acetone were transferred to a flask and the acetone was evaporated at 45°. The material was then dissolved in a minimum of DMF. The white microcrystalline precipitate was shown to be cyclo(glycyl-L-prolyl-glycyl-L-prolyl) (155 mg, 28% yield) which had formed a complex with DMF. Crystallization of 100 mg of this material from methanol-ether gave crystalline cyclo(Pro-Gly)3 (55 mg) free of DMF. Chemical analysis C 21 H 30 Calculated for C6H2O6N6. C, 52.49; H, 6.71; N, 17.49. Elemental analysis found C, 52.60; H, 6.81; N, 17.38.

[0056] In yet other aspects, the present invention provides a pharmaceutical composition for treating a disease, disorder, or condition, including but not limited to related conditions such as Alzheimer's disease and cognitive impairment, the composition comprising a pharmaceutically acceptable excipient or carrier, and a therapeutically effective amount of a cyclic GP or an analog thereof having the structural formula given above.

[0057] In a further aspect, the present invention provides a method for treating an animal having cognitive impairment, the method comprising administering to the animal an effective amount of a composition comprising a cyclic GP or an analog thereof. In a further aspect, the animal to be treated is a human.

[0058] One aspect of the present invention is generally directed to the therapeutic treatment of neurological diseases and injuries. Expressly not wishing to be limited to any mechanism of action, and merely proposing any mechanism of action, the inventor(s) propose that the present invention can be based, in part, on inducing neurogenesis, particularly the proliferation of neural stem cells, or progenitor cells. According to one aspect of the present invention, cyclic prolylglycine and its analogs ("cPG compounds") act as important regulators of neurogenesis that promote and induce proliferation and / or differentiation in nerve cells.

[0059] "Neurogenesis" is defined herein to mean the proliferation, differentiation, migration, or survival of nerve cells in vivo or in vitro. In a preferred aspect of the present invention, the nerve cells can be adult, fetal, or embryonic neural stem cells or progenitor cells. Neural progenitor cells also refer to a net increase in cell number or a net increase in cell survival rate. As used herein, "NSC" will include at least all brain stem cells, all brain progenitor cells, and all brain precursor cells.

[0060] Heretofore, cAMP and / or Ca 2+It has been shown that an increase in the level induces the proliferation of adult neural stem cells. In some cases, this induction follows the activation of G protein-coupled receptors (GPCRs). cAMP and / or Ca 2+ increasing the level can induce the proliferation of adult neural stem cells.

[0061] G protein-coupled receptors (GPCRs), also known as G protein-linked receptors (GPLRs), constitute a large family of receptors that detect extracellular molecules, activate intracellular signaling pathways, and ultimately activate cellular responses.

[0062] Ligands that bind to and activate these receptors include photosensitive compounds, odors, pheromones, hormones, neurotransmitters, etc., and their sizes vary from small molecules to peptides and large proteins. GPCRs in the mammalian brain bind several different neurotransmitters such as serotonin, dopamine, GABA, and glutamate. G protein-coupled receptors are involved in many diseases and are also the targets of approximately 34% of all modern pharmaceuticals.

[0063] One aspect of the present invention is not limited by the proposed mechanism and includes that cPG and its analogs can act as neurogenic regulators that regulate the intracellular levels of cAMP and / or Ca 2+ , where cPG can increase cAMP (e.g., by increasing synthesis or decreasing degradation) and / or Ca 2+ (e.g., by increasing influx or decreasing efflux), as has been chemically and biologically shown to be possible.

[0064] One aspect of the present invention describes a novel method for promoting the regeneration of damaged neural tissue, the method comprising administering an effective amount of cyclopropylglycine (cPG) and its analogs, which can reduce the growth rate of glial cells to promote the growth of neural tissue.

[0065] Neurons are closely surrounded by glial cells or astrocytes. One of the difficulties in achieving neuron regeneration after a neuron has been damaged or severed is that glial cells proliferate and form a barrier to the regenerating neurons. As a result, further movement of the neurons to the expected adhesion sites is inhibited and the regeneration of structure and function ceases. The formation of scars in astrocytes and connective tissue and progressive necrosis have been observed to have an adverse effect on the functional regeneration of nerve cells.

[0066] Accordingly, one aspect of the present invention is a method for promoting the regeneration of damaged nerve tissue in a mammal (e.g., a human), comprising administering an effective amount of a cPG compound (cPG and its analogs) to the site of injury.

[0067] Some of the purposes of the experiments provided herein are to provide an enabling for methods of regenerating neurons and glial cells, or of repairing damaged neurons and glial cells as claimed.

[0068] One aspect of the present invention involves cyclic prolylglycine (cPG) and its pharmaceutically active analogs acting as regulators of nerve cells for treating depression and other psychological disorders. The N-methyl-D-aspartic acid receptor (the "NMDA receptor") is a glutamate receptor and ion channel protein found in nerve cells. The NMDA receptor is one of three ionotropic glutamate receptors, the other receptors being the AMPA receptor and the kainate receptor. The NMDA receptor is activated when glutamate and glycine bind, and when activated, positively charged ions flow through the cell membrane. [Furukawa, Hiroyasu; Singh, Satinder K; Mancussol, Romina; Gouaux, Eric (November 2005). “Subunit arrangement and function in NMDA. Receptors”. Nature. 438 (7065): 185-92. doi: 10.1038 / nature04089. PMID 16281028.]

[0069] The NMDA receptor channel plays an important role in synaptic plasticity and synaptogenesis during the development of the central nervous system (CNS). When the NMDA receptor is over-activated, Ca 2+ flows in excessively, causing excitotoxicity, which has been suggested to be involved in neurodegenerative diseases. Therefore, blocking the NMDA receptor is theoretically considered useful for treating such diseases.

[0070] The NMDA receptor is an ion channel protein receptor that is activated when glycine and glutamate bind. The receptor is a heteromeric complex that interacts with multiple intracellular proteins through three different subunits. It is a heteromeric complex that interacts with multiple intracellular proteins through three subunits: NR1, NR2, and NR3. There are eight different subunits of NR1 generated by alternative splicing from one gene. There are four different NR2 subunits (A-D) of the NR2 subunit, and NR3A and NR3B subunits have been reported. Six separate genes encode NR2 and NR3. [Loftis J. M., Janowsky A. (2003). “The N-methyl-D-aspartate receptor subunit NR2B: localization, functional properties, regulation, and clinical implications”. Pharmacol Ther. 97 (1): 55-85. doi: l 0. 016 / sO 163-7258(02)00302-9.]。

[0071] Agonists or allosteric modulators of the NMDA receptor, particularly channels containing the NR2B subunit, are being studied as therapeutic agents for major depressive disorders (G. Sanacora, 2008, Nature Rev. Drug Disc. 7: 426-437). The NR2B subunit is involved not only in regulating activities such as learning, memory, processing, and feeding behavior, but also in the number of human disorders. The basic structure and function associated with the NMDA receptor are thought to be due to the NR2B subunit.

[0072] In addition, an allosteric and non-competitive binding site has been identified in the N-terminal domain of NR2B. The NR2 subunit functions as a binding site for glutamate, one of the major excitatory neurotransmitter receptors in the mammalian brain. [Yoshimura Y, Ohmura T, Komatsu Y (July 2003). “Two forms of synaptic plasticity with distinct dependence on age, experience, and NMDA receptor subtype in rat visual cortex”. The Journal of Neuroscience. 23 (16): 6557-66. PMID 12878697].

[0073] NR2B is associated with plasticity that depends on age and visual experience in the rat neocortex, and an increase in the NR2B / NR2A ratio is directly correlated with the strength of excitatory LTP in young animals. This is thought to contribute to the experience-dependent refinement of developing cortical circuits.

[0074] The role of the NR2B subunit of the NMDA receptor has been demonstrated in the actions of various antidepressants. [Poleszak E, Wlaz P, Szewczyk B, Wlaz A, Kasperek R, Wrobel A, Nowak G (2011) A complex interaction between glycine / NMDA receptors and serotonergic / noradrenergic antidepressants in the forced swim test in mice. J Neural Transm 1 18: 1535-1546].

[0075] G protein-coupled receptors (GPCRs), also known as G protein-linked receptors (GPLRs), constitute a large family of proteins that detect extracellular molecules, activate intracellular signaling pathways, and ultimately activate cellular responses.

[0076] GPCRs in the mammalian brain bind several different neurotransmitters such as serotonin, dopamine, GABA, and glutamate. G protein-coupled receptors are involved in many diseases, and approximately 34% of all modern pharmaceuticals target them.

[0077] One aspect of the invention involves cyclic prolylglycine (cPG) and its pharmaceutically active analogs acting as neuronal regulators for treating depression and other psychological disorders. Without explicitly limiting to any mechanism, one possible mechanism is the regulation of the intracellular levels of cAMP and / or Ca 2+ Here, cPG has been shown chemically and biologically to be able to increase cAMP (e.g., by increasing synthesis or decreasing degradation) and / or Ca 2+ by increasing influx or decreasing efflux).

[0078] Also, cyclic prolylglycine and its pharmaceutically active analogs have been shown to selectively bind to the N-terminal domain of NR2B, which may sustain an antidepressant response in humans.

[0079] The present invention provides the technical advantages of cyclic prolylglycine (“cPG”) and its pharmaceutically active analogs, which together are known as cPG compounds that are ligands for the NR2B receptor and may be useful for treating various disorders of the central nervous system. Further, the cPG compounds offer advantages for pharmaceutical use with respect to, for example, one or more of their mechanism of action, binding, inhibitory potency, target selectivity, solubility, safety profile, or bioavailability.

[0080] In practicing the method of the present invention, a patient with depression is administered a combination of substances at a pharmaceutically effective dosage level using a suitable route of administration and regimen. The substances may be administered in the form of a single dosage unit in which the active substance is combined with a suitable carrier, or in separate dosage units in which the active substances are separately combined with a suitable carrier. When administered separately, the administration may be simultaneous or at selected time intervals.

[0081] The administration is preferably oral administration, and the carrier or carriers are selected in consideration thereof. Such being the case, other modes of administration of both substances, and modes of mixing with the individual materials, are considered to be part of the present invention.

[0082] The dosage levels of the materials will vary depending on the particular materials used and the severity of the condition of the patient being treated. Cyclic prolylglycine (cPG) is used in an amount of about 0.1 mg to about 10 mg per kg of body weight. Oral administration is recommended at a dosage of about 20 mg to about 80 mg per day, and in some severe cases, up to a dosage of about 100 mg per day can be administered according to a physician's prescription order.

[0083] The pharmaceutical composition of the present invention is prepared using active ingredients associated with conventionally employed pharmaceutical carriers. The composition of the present invention is generally intended to be administered orally to achieve an antidepressant effect. This may be in any of the dosage forms of tablets, capsules, powders, suspensions, solutions, syrups, etc., including sustained-release formulations. The term dosage form as used herein and in the claims refers to physically discrete units administered in single or multiple dosages, each unit containing a predetermined amount of the active substance in association with the necessary diluent, carrier or vehicle. The amount of the active substance is an amount calculated to produce the desired therapeutic effect upon administration of one or more of such units.

[0084] The powder is prepared by grinding the active substance to a suitable container size and mixing it with a similarly ground pharmaceutical carrier of a diluent, such as an edible carbohydrate material like starch. Sweeteners, flavors, preservatives, dispersants, and colorants may also be present.

[0085] The capsules are manufactured by preparing the powder mixture as described above and filling the formed gelatin sheaths. Lubricants such as talc, magnesium stearate, calcium stearate, etc. may be added to the powder mixture as adjuvants before the filing operation, lubricants such as colloidal silica may be added to improve fluidity, and disintegrants or solubilizers may be added to improve the effectiveness of the drug when the capsule is ingested.

[0086] Tablets are prepared by preparing the powder mixture, granulating or slugging it, adding lubricants and disintegrants, and compressing it into tablets. The powder mixture is prepared by mixing a suitably ground active substance with a diluent or base such as starch, sucrose, kaolin, dicalcium phosphate, etc. The powder mixture can be prepared by wetting it with a binder such as syrup, starch paste, acacia mucilage, or a solution of a cellulose-based material or a polymer-based material and forcing it through a screen to granulate it. As an alternative to granulation, the powder mixture can be passed through a tablet machine and the resulting incompletely formed slug can be crushed into granules. The granules can be lubricated by the addition of stearic acid, stearates, talc, or mineral oil to prevent adhesion to the tablet-forming die. The lubricated mixture is then compressed into tablets.

[0087] The drug can also be directly compressed into tablets in combination with a free-flowing inert carrier without going through the steps of granulation or slugging. A protective coating consisting of a shellac sealing coat, a coating of sugar or a polymer material, and a wax polish coat can be provided. Dyes can be added to these coatings to distinguish different unit doses.

[0088] Oral liquid preparations such as syrups and elixirs can be prepared in unit dose form such that a predetermined amount, for example one teaspoonful, contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution of sucrose while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be prepared by dispersing the drug in a non-toxic vehicle which is insoluble.

[0089] In one important embodiment of the invention, pharmaceutically acceptable non-toxic acid addition salts of the active drug are used, particularly for the preparation of solid pharmaceutical formulations. Such pharmaceutically acceptable non-toxic acid addition salts include those derived from both organic and inorganic acids, for example, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, acetic acid, lactic acid, succinic acid, malic acid, maleic acid, aconitic acid, phthalic acid, tartaric acid, embonic acid, enenenic acid and such acids.

[0090] Although the invention is mainly envisaged for oral administration, other modes are certainly not excluded. Ampoules for parenteral application can be prepared, preferably containing the water-soluble salts of the active substance and possible buffer substances in an aqueous solution.

[0091] In liquid compositions designed for oral or parenteral administration in which the active substance is incorporated, care must be taken to ensure the stability of the active substance.

[0092] When the active substances are administered separately, the individual compositions are prepared in the manner described above. These individual compositions can then be administered in such a way, while maintaining their separate identities, for example in a multi-layer tablet or a single capsule containing both components in a plurality of discrete particles, or in combination in a single dosage unit.

[0093] A better understanding of the invention will be obtained with reference to the following examples and drawings.

Brief Description of the Drawings

[0094]

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Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Best Mode for Carrying Out the Invention

[0095] The present invention is described with reference to its specific embodiments. Other aspects of the present invention can be understood with reference to the drawings. The drawings are also provided in the above text and the accompanying drawings, and are further provided below, where their descriptions are provided.

[0096] The following examples are given for illustrative purposes only and should not be taken as limiting the scope of the present invention.

[0097] It has been surprisingly found that the process by which IGF1 is metabolized to the tripeptide GPE and des IGF is only a part of it.

[0098] The cis isomer of GPE can be further decomposed to form cyclic prolylglycine and glutamic acid. This is shown in Figure 1.

[0099] The cyclic PG structure is small enough to cross the blood-brain barrier.

[0100] Also, as shown in Figure 2, it can provide ligands for binding metal ions such as Mg 2+ , Ca 2+ , Co 2+ and has a molecular structure that can function as a chelating agent.

[0101] The possible role of cPG as an agent is further supported by the companion degradation product, glutamic acid.

[0102] Glutamate is known to be associated with brain diseases. (Johnston, G.A.R. in Roberts P.J. et al Editors, Glutamate: Transmitter in the Central Nervous System, John Wiley & Sons, 1981, pp.77-87).

[0103] As used herein, a cPG compound is a compound having a biological activity similar or identical to that of cPG, and cPG compounds consist of cPG, biologically active cPG analogs, biologically active cPG mimetics, and compounds that increase the concentration of cPG and cPG analogs in mammals. CPG compounds include cPG molecules such as truncated portions of IGF-I compounds, and other chemical and biological analogs and mimetics.

[0104] As used herein, "cPG analog" means any analog, naturally occurring cPG analog, or variant thereof, of cPG that can effectively bind to mGluR receptors in the CNS and promote an equivalent neuroprotective effect on CNS neurons. Examples of CPG analogs include c(PG)3 and cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline.

[0105] The term "cPG molecule" includes peptide fragments and truncated portions of longer IGF-I compounds, as well as other chemical and biological analogs and mimetics, and cPG compounds can be used for the treatment of mammals suffering from neutral injury or disease. In particular, cPG compounds can be used for the treatment of human patients suffering from nerve injury or disease. More generally, the compositions and methods of the present invention are found to be used for the treatment of mammals such as human patients suffering from nerve injury or potential apoptosis and / or necrotic cell death resulting from injuries or diseases such as septic shock, ischemia, etc. Administration of cytokines, overexpression of cytokines, ulcers, gastritis, ulcerative colitis, Crohn's disease, diabetes, rheumatoid arthritis, asthma, Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, cirrhosis, transplant rejection, transplant rejection, encephalomyelitis. Meningitis, pancreatitis, peritonitis, vasculitis, lymphocytic choriomeningitis, glomerulonephritis, uveitis, glaucoma, blepharitis, carradion, allergic eye diseases, corneal ulcers, keratitis, cataracts, retinal disorders, age-related macular degeneration, optic neuritis ileus. Inflammation due to overproduction of inflammatory cytokines, hemorrhagic shock, anaphylactic shock, burns, infectious diseases leading to overproduction of inflammatory cytokines by bacteria, viruses, fungi, parasites, hemodialysis, chronic fatigue syndrome, stroke cancer, cardiovascular diseases associated with overproduction of inflammatory cytokines, heart diseases, cardiopulmonary bypass, ischemia-reperfusion injury, ischemia-reperfusion associated with overproduction of inflammatory cytokines, toxic shock syndrome, adult respiratory distress syndrome cachexia, myocarditis, autoimmune diseases, eczema, psoriasis, heart failure, dermatitis, hives, cerebral ischemia, systemic lupus erythematosus, AIDS, AIDS dementia, chronic neurodegenerative diseases, chronic pain, priapism, cystic fibrosis, amyotrophic lateral sclerosis. Schizophrenia, depression, premenstrual syndrome, anxiety, addiction, migraine, Huntington's disease, epilepsy, gastrointestinal motility disorders, obesity, dysphagia, neuroblastoma, malaria, blood cancer, myelofibrosis, lung injury, graft-versus-host disease head trauma, central nervous system trauma, hepatitis, renal failure, chronic hepatitis C, paraquat poisoning, transplant rejection and preservation, enhanced fertility, bacterial translocation, circulatory shock, traumatic shock, hemodialysis, hangover, and combinations of two or more of them.

[0106] Furthermore, cPG and its analogs, such as c(PG)3 and cGMeP, can be used, but are not limited to, treating mammals suffering from white matter insult as a result of acute brain injury such as perinatal hypoxic-ischemic injury. Or, from neurodegenerative diseases such as chronic nerve injury or multiple sclerosis, or other demyelinating diseases and disorders involving inflammatory involvement such as acute disseminated encephalomyelitis, optic neuritis, polymyositis, Devic's disease, leukodystrophy, non-inflammatory involvement, progressive multifocal leukoencephalopathy, central pontine myelinolysis. Patients suffering from such diseases and injuries can benefit greatly from treatment protocols that can initiate remyelination.

[0107] The present invention is applicable to the induction of myelin production following injury in the form of trauma, toxin exposure, asphyxia or hypoxic-ischemia, and to the treatment or prevention of apoptosis in response to injury or disease in the form of cancer, viral infections, autoimmune diseases, nerve diseases and injuries, cardiovascular diseases.

[0108] Treatment with cPG or its analogs, including but not limited to c(PG)3 and cGAL, can be performed (not only modified) prior to injury, such as before a selective surgery. Examples of related selective procedures include neurosurgery where retraction of a lobe of the brain can lead to cerebral edema, or cardiac procedures such as valve replacement where a small embolism that cannot be avoided is said to lead to a detectable impairment of brain function in some 75% of cases.

[0109] Pharmacology and efficacy cPG can act as anti - necrotic and anti - apoptotic during the process of cell death. Its anti - apoptotic activity and anti - necrotic activity in vivo can be measured by cell number. cPG can also be measured in vitro (Gudasheva T.A. et al. FEBS Letters, Vol. 391, Issues 1 - 2, 5 August 1996, pp. 149 - 152). CNS injury can be clinically measured, for example, by the degree of permanent neurological deficit, cognitive function, and / or the tendency of seizure disorder. (Rakic L.J et al, in Rakic L.J et al Peptide and Amino Acid Transport Mechanisms in The Central Nervous System, 1988, The MacMillan Press Ltd.Press Ltd. (London) pp.167 - 181).

[0110] Pharmaceutical composition and its administration cGP itself, as part of the present invention, can be used to prevent or treat cell injury, programmed death, and the induction of myelin production. Usually, this is done by direct administration of cGP to the patient. If desired, a combination of cPG compounds and their analogs can be administered in a pharmaceutically acceptable composition.

[0111] Those skilled in the art will understand that the applicant does not intend to exclude the administration of other forms of cPG and its analogs. By way of example, administering a prodrug of cPG consisting of cPG and a carrier can increase the effective amount of cPG in the CNS, and the cPG and the carrier are linked by a linkage that is easily cleaved or digested within the patient. Any suitable linker that is cleaved or digested to release cPG after administration can be employed.

[0112] Furthermore, it is envisioned that cPG levels can be increased via an implant containing cell lines capable of expressing active cPG within the patient's CNS.

[0113] Prodrugs of cPG and its analogs can also be administered. In that case, the prodrug is metabolized or otherwise changed within the subject to form cPG. Examples of CPG and its analogs include, but are not limited to, c(PG)3 and cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylprolym, etc., and can be administered as part of a pharmaceutical or pharmaceutical formulation. This includes combining cPG with a pharmaceutically suitable carrier, adjuvant or excipient. The choice of carrier, adjuvant or excipient will of course usually depend on the route of administration employed.

[0114] The routes of administration are diverse and can be any suitable route of administration. The advantage of cPG is that it can be administered peripherally. That is, in order to exert an effect in the CNS, it is not necessary to administer directly to the patient's CNS.

[0115] Any peripheral route known in the art can be employed. These can include, for example, non-parenteral routes such as injection into the peripheral circulation, subcutaneous injection, intraorbital injection, ophthalmic injection, intrathecal injection, intravesical injection, local injection, infusion (using, for example, a mini pump such as a controlled release device or an osmotic pump or a skin patch), transplantation, aerosol, inhalation, scarification, intraperitoneal injection, intracapsular injection, intramuscular injection, intranasal injection, oral injection, buccal injection, pulmonary injection, rectal injection or vaginal injection, etc., but are not limited thereto. The compositions of the present invention can be formulated for parenteral administration to humans or other mammals in a therapeutically effective amount (e.g., an amount that eliminates or alleviates the patient's condition).

[0116] Preferred routes of administration include, but are not limited to, subcutaneous injection (e.g., dissolved in 0.9% sodium chloride) or oral administration (in capsules).

[0117] It will also be understood that in some cases it may be desirable to administer the cPG compound directly to the patient's CNS. Again, this can be accomplished by any suitable direct administration route. Examples include administration by lateral cerebral vein injection or via a shunt surgically inserted into the lateral cerebral vein of the patient's brain.

[0118] Calculation of the effective amount of the cPG compound to be administered is within the skill of the ordinary skilled artisan in the art and will be routine to one skilled in the art. Needless to say, the ultimately administered amount depends on the administration route and the nature of the neuropathy or condition to be treated. Preferably, the cPG compound is administered at between about 1 μg and about 100 mg per kg of body weight when the dose is administered centrally. Suitable doses for administration of cPG are, for example, between about 0.1 mg and about 10 mg per kg of body weight, or between about 1 mg and about 5 mg per kg of body weight.

[0119] For inclusion in a pharmaceutical, the cPG compound can be obtained from a suitable commercial source such as Bachem AG of Bubendorf, Switzerland. Alternatively, cPG can be prepared by the stepwise solid-phase synthesis method of Merrifield et al. (1963 J. Amer. Chem. Soc.: 85, 2149-2156). Alternative syntheses can involve the use of a commercially available peptide synthesizer such as the Applied Biosystems model 430A.

[0120] cGAL can be prepared by methods well known to those of ordinary skill in the synthesis of peptides and analogs. Example. by "Principles of Peptide Synthesis", published by Springer-Verlag in 1993.

[0121] C(PG)3 can be prepared by the method disclosed in Israel Journal of Chemistry, Vol. 12, Nos. 1-2, 1974, pp. 15-29, “CYCLIC Peptides VII: The Synthesis and Characterization of Cyclic Peptides with Repeating Pro-Gly Sequences” by Charles M. Deber and Erkan R. Blout.pu.

[0122] As a general proposition, the total pharmaceutically effective amount of a cPG compound administered parenterally per single dose will be in the range measurable by a dose-response curve. One can administer increasing amounts of the cPG compound to a patient and confirm the patient's serum levels for cPG. The amount of the cPG compound to be employed can be calculated on a molar basis based on these serum levels of the cPG.

[0123] Specifically, one method for determining the appropriate dosage of a compound involves measuring the cPG levels in a biological fluid such as a body fluid or a blood fluid. Measurement of such levels can be done by any means including RIA and ELISA. After measuring the cPG levels, the fluid is contacted with the compound using a single dose or multiple doses. After this contacting step, the cPG levels in the fluid are re-measured. If the cPG levels in the fluid have decreased by an amount sufficient to bring about the desired effectiveness where the molecule is to be administered, the dosage of the molecule can be adjusted to bring about maximum effectiveness. This method can be carried out in vitro or in vivo. Preferably, this method is carried out in vivo, i.e., a fluid is extracted from a mammal, the cPG levels are measured, then the compounds of the present specification are administered to the mammal using a single dose or multiple doses (i.e., the contacting step is achieved by administration to the mammal), and then the cPG levels are re-measured from the fluid extracted from the mammal.

[0124] The compounds of the present invention may also preferably be administered by a sustained release composition. Suitable examples of sustained release compositions include shaped articles such as films, or semipermeable polymer matrices in the form of microcapsules. Sustained release matrices include polylactide (U.S. Patent No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., 1983), poly(2-hydroxyethyl methacrylate) (Langer et al., 1981), ethylene vinyl acetate (Langer et al., supra), or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained release compositions also include compounds encapsulated in liposomes. Liposomes containing the compounds are prepared by methods known per se. DE Patent 3,218,121; Epstein et al., 1985; Hwang et al., 1980; EP Patent 52,322; EP Patent 36,676; EP Patent 88,046; EP 143,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,485,045; and EP 102,324. Usually, the liposomes are small (from about 200 angstroms to about 800 angstroms) unilamellar type with a cholesterol lipid content of about 30 mol percent or more, and the selected ratio is adjusted for the most effective treatment.

[0125] PEGylated peptides having a longer lifespan can also be employed, for example, based on the conjugation techniques described in WO95 / 32003 issued on November 30, 1995.

[0126] When parenteral administration is preferred, the compounds are generally formulated in unit dosage injectable form (solution, suspension, or emulsion), each at the desired concentration, by mixing with a pharmaceutically or parenterally acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosage and concentration employed and compatible with the other components of the formulation.

[0127] Generally, a formulation is prepared by contacting a compound with a liquid carrier or a subdivided solid carrier or both. Then, it is shaped into the desired formulation as needed. Preferably, the carrier is a parenteral carrier, and more preferably, a solution isotonic with the recipient's blood. Examples of such carrier vehicles include water, physiological saline, Ringer's solution, buffer solutions, glucose solutions, and the like. Also, non-aqueous vehicles such as fixed oils and ethyl oleate may be used.

[0128] The carrier may further contain additives such as substances that enhance isotonicity and chemical stability. Such substances are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, succinates, acetates, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, e.g., polyarginine or tripeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrofluorophosphate salts such as proteins; polyarginine or tripeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine; amino acids such as glutamic acid, aspartic acid, histidine or arginine. Monosaccharides, disaccharides, and other carbohydrates such as cellulose or its derivatives, glucose, mannose, trehalose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; nonionic surfactants such as polysorbate, poloxamer, or polyethylene glycol (PEG); and / or neutral salts such as, for example, the following. g., NaCl, KCl, MgCl2, CaCl2, etc.

[0129] cPG compounds are typically formulated in such vehicles at a pH between about 5.5 and about 8.0. Typical adjuvants that may be incorporated into tablets, capsules, etc. include binders such as acacia, corn starch, gelatin; excipients such as microcrystalline cellulose; disintegrants such as corn starch and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose and lactose; flavors such as peppermint, wintergreen, cherry, etc. When the dosage form is a capsule, in addition to the above materials, it may also contain a liquid carrier such as a fatty oil. Additionally, various types of materials may be used as coating agents or as modifiers of the physical form of the dosage unit. Syrups or elixirs may contain the active compound, a sweetening agent such as sucrose, a preservative such as propylparaben, a coloring agent, and a flavor such as cherry. Sterile injectable compositions can be formulated according to conventional pharmaceutical practices. For example, dissolution or suspension of the active compound in a vehicle such as water or a naturally occurring vegetable oil such as sesame oil, peanut oil, or cottonseed oil, or a synthetic fatty vehicle such as ethyl oleate may be desired. Buffers, preservatives, antioxidants, etc. can be incorporated according to accepted pharmaceutical practices.

[0130] Compounds used for therapeutic administration must be sterile. Sterility is readily achieved by filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Therapeutic compositions are generally placed in a container having a sterile access port, e.g., an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle.

[0131] Compounds are usually stored in unit dose or multi-dose containers, e.g., sealed glass ampoules or vials, as an aqueous solution or a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 1% (w / v) aqueous solution of the compound (5 mL) is aseptically filtered and filled into a 10 mL vial, and the resulting mixture is lyophilized. Infusions are prepared by reconstituting the lyophilized compound with bacteriostatic water for injection.

[0132] Combination therapies of the cPG compounds of this specification with one or more other suitable reagents that increase total cPG in the blood or enhance the effect of cPG are also contemplated. These reagents generally enable the release of cPG produced by the cPG compounds of this specification.

[0133] Furthermore, in one aspect of the invention, for treating a mammal, when the cPG compound is a peptide, it includes using gene therapy with a nucleic acid encoding the cPG compound. Generally, gene therapy is used to increase (or overexpress) cPG levels in a mammal. Nucleic acids encoding cPG peptides can be used for this purpose. Given the amino acid sequence, multiple nucleic acid molecules can be generated using the degeneracy of the genetic code, and one can be selected for use in gene therapy.

[0134] There are two main approaches for introducing nucleic acids (optionally contained in a vector) into a patient's cells for gene therapy purposes: in vivo and ex vivo. In in vivo introduction, the nucleic acid is injected directly into the patient, usually at the site that requires the cPG compound. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient, either directly or encapsulated, for example, within a porous membrane transplanted into the patient. See, for example, U.S. Pat. Nos. 4,892,538 and 5,283,187.

[0135] There are various techniques available for introducing nucleic acids into germline cells. The techniques vary depending on whether the nucleic acid is introduced into cultured cells in vitro or in vivo into the cells of the intended host. Techniques suitable for introducing nucleic acids into mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE - dextran, calcium phosphate precipitation, etc. A commonly used vector for ex vivo delivery of this gene is a retrovirus.

[0136] Currently preferred in vivo nucleic acid introduction techniques include transfection by viral vectors (e.g., adenovirus, herpes simplex type I virus, or adeno-associated virus, etc.), and lipid-based systems (useful lipids for lipid-mediated introduction of genes are, for example, DOTMA, DOPE, and DC-Chol). In some situations, it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as a cell surface membrane protein or an antibody specific to the target cells, a ligand for a receptor on the target cells, etc. When using liposomes, in order to promote targeting and / or uptake to proteins that bind to cell surface membrane proteins related to endocytosis, for example, a capsid protein or a fragment thereof that is tropic to a specific cell type, an antibody to a protein that undergoes internalization during cycling, a protein that targets intracellular localization and increases the intracellular half-life, etc. can be used. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., 1987; Wagner et al., 1990). See Anderson 1992 for a review of currently known gene marking and gene therapy protocols. Also see WO93 / 25673 and the references cited therein.

[0137] The present invention also contemplates kits. A typical kit will consist of a container, preferably a vial, containing a cPG compound formulation consisting of a cPG compound in a pharmaceutically acceptable buffer, and instructions such as a product insert or label that instructs the user on how to use the formulation.

[0138] Certain aspects of the present invention include the use of cPG in the treatment of age-associated cognitive impairment with neurodegenerative conditions or in situations where cognitive impairment is found without obvious neurodegeneration.

[0139] Such other agents can be selected from the non-limiting group consisting of, for example, growth factors and related derivatives such as insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), growth hormone, nerve growth factor, growth hormone binding protein, and / or IGF binding protein.

[0140] Application to treatment The compositions and methods of the present invention find use in the treatment of animals such as human patients suffering from cognitive impairment. More generally, the compositions and methods of the present invention find use in the treatment of mammals such as human patients and subjects suffering from dementia including dementia due to memory impairment, mild cognitive impairment, Alzheimer's disease, Lewy body disease, frontotemporal lobar degeneration, vascular dementia, dementia due to brain atrophy associated with head trauma, Huntington's disease, Parkinson's disease, or Down syndrome, but are not limited thereto.

[0141] Pharmaceutical composition and its administration The cyclic PG compound can be administered as part of a medicament or pharmaceutical formulation. This can include combining the compounds of the present invention with any pharmaceutically suitable carrier, adjuvant or excipient. The choice of carrier, adjuvant or excipient will, of course, normally depend on the route of administration employed.

[0142] In general, the compounds of the present invention are administered in a therapeutically effective amount by any of the usual modes known in the art, either alone or in combination with other conventional therapeutic agents for the disease to be treated. The therapeutically effective amount can vary depending on the disease or injury to be treated, its severity, the age and relative health of the animal to be treated, the potency of the compound(s), and other factors. The therapeutically effective amount of cyclic prolylglycine can range from 0.01 to 10 milligrams per kilogram of the animal's mass, and low doses such as 0.01 to 0.1 mg / kg are suitable for administration via cerebrospinal fluid, such as intracerebral venous administration, while high doses such as 0.1 to 10 mg / kg are suitable for administration by methods such as oral administration, systemic administration (e.g., transdermal administration), or parenteral administration (e.g., intravenous administration). Those of ordinary skill in the art, considering their art and this disclosure, will be able to determine the therapeutically effective amount of the compounds of the present invention for a given disease or injury without undue experimentation.

[0143] Cyclic prolylglycine and cPG compounds can be administered orally or peripherally via any peripheral route known in the art. These can include, but are not limited to, parenteral routes such as injection into the peripheral circulation, subcutaneous injection, intraorbital injection, ophthalmic injection, intrathecal injection, intravesical injection, local injection, intravenous injection, aerosol injection, inhalation injection, scar injection, intraperitoneal injection, intracapsular injection, intramuscular injection, nasal injection, buccal injection, transdermal injection, pulmonary injection, rectal injection, or vaginal injection.

[0144] For the convenience of the patient, the cyclic prolylglycine compounds and cPG compounds of the present invention can be administered orally. The amount of the compound of the present invention in the composition can vary widely depending on the type of composition, the size of the unit dose, the type of excipient, and other factors well known to those of ordinary skill in the art. Generally, the final composition may consist of 5 mg to 50 mg of cPG per typical adult weighing 50 to 120 kg, or 1×10 -5 % to 3×10 -4 % by weight (%w), with the remainder being excipient or excipients.

[0145] Other convenient routes of administration include subcutaneous injection or intravenous infusion (e.g., the active cPG is dissolved in a physiologically compatible carrier such as 0.9% sodium chloride or dextrose), or direct administration to the CNS. Using a stereotaxic apparatus and an accurate map of the animal's CNS, the compound can be injected directly into the site of nerve injury.

[0146] The effective amount of the compound in the central nervous system can be increased by administration of the compound in prodrug form, which consists of the compound of the invention and a carrier, where the carrier is attached to the compound of the invention by a linkage that is readily cleaved or digested in the patient. Any suitable linker that is cleaved or digested after administration can be employed.

[0147] However, the applicant does not intend to exclude other administrative forms.

[0148] In other embodiments of the present invention, restoration of nerve function in an animal can include administering a therapeutic amount of cyclic prolylglycine or a cPG compound, for example, in combination with another neuroprotective agent selected from, for example, growth factors and related derivatives (insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), transforming growth factor-β1, activin, growth hormone, nerve growth factor, growth hormone binding protein, IGF binding protein, keratinocyte growth factor, androgen-induced growth factor. Additional members of the FGF family include, for example, fibroblast growth factor homolog-1 (FHF-1), FHF-2, FHF-3 and FHF-4, keratinocyte growth factor 2, brain-derived growth factor, neurotrophin 3, and neurotrophin 4). Neuroprotective therapeutic agents of other embodiments include crometazole, kynurenic acid, semax, tacrolimus; glutamate agonists such as NPS1506, GV1505260, MK-801, GV150526; AMPA aryls such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX). Anti-MAdCAM-1 mAb MECA-367 (ATCC accession number HB-9478) and the like can be mentioned.)

[0149] The cyclic prolylglycine compound can preferably be administered by a sustained release composition. Suitable examples of sustained release compositions include, for example, semipermeable polymer matrices in the form of shaped articles, films, or microcapsules.

[0150] For parenteral administration, in one embodiment, the cyclic prolylglycine or cPG compound is generally formulated by mixing each in an injectable form (solution, suspension, or emulsion) of unit dose, to the desired degree of purity, with a pharmaceutically or parenterally acceptable carrier, for example, a carrier that is non-toxic to the recipient at the employed doses and concentrations and is compatible with the other components of the formulation.

[0151] Generally, a formulation is prepared by bringing a cyclic prolylglycine or cPG compound into intimate and uniform contact with a liquid carrier or a subdivided solid carrier or both. Then, if desired, it is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier and the vehicle is preferably a solution isotonic with the recipient's blood. Examples of such carrier vehicles include water, saline, Ringer's solution, buffer solutions, glucose solutions, and the like. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein.

[0152] The cyclic prolylglycine or cPG compound is typically formulated in such vehicles at a pH from 4.5 to about 8. It will be understood that the particular use of the excipients, carriers, or stabilizers described above may result in the formation of salts of the compounds. The final preparation may be a stable liquid or a lyophilized solid.

[0153] Formulations of cyclic prolylglycine or cPG compounds in pharmaceutical compositions can also contain adjuvants. Representative adjuvants that can be incorporated into tablets, capsules, etc. include binders such as acacia, corn starch, gelatin; excipients such as microcrystalline cellulose; disintegrants such as corn starch and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose and lactose; flavors such as peppermint, wintergreen, cherry, etc. When the dosage form is a tablet, the cyclic prolylglycine or cPG compound and the composition can contain a binder and optionally, a smooth coating. When the dosage form is a capsule, in addition to the above materials, a liquid carrier such as a fatty oil may be included. Various other types of materials may be used as coatings or as modifiers of the physical form of the dosage form. Syrups or elixirs may contain the active compound, a sweetening agent such as sucrose, a preservative such as propylparaben, a coloring agent, and a flavor such as cherry. Sterile compositions for injection can be formulated according to conventional pharmaceutical practices. For example, dissolution or suspension of the active compound in a vehicle such as water or a naturally occurring vegetable oil such as sesame oil, peanut oil, or cottonseed oil, or a synthetic fatty vehicle such as ethyl oleate may be desired.

[0154] For injection, intravenous administration, and other invasive routes of administration, the cyclic prolylglycine or cPG compound is preferably sterile. Sterilization can be achieved by any method known in the art, for example, by filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Therapeutic compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle.

[0155] Preferred embodiments of the present invention The first aspect of the present invention includes a method for regenerating the loss of neurons and glial cells as a result of injury or disease damage, and includes the following steps. a) providing a subject in need of the loss of said regenerating neurons and glial cells; b) administering to said subject cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof) in an amount effective to regenerate new neurons and glia. Here, in said subject, neurons are regenerated and the loss of glial cells is regenerated; here, further, said cPG compound functions as a neurogenic agent in the central nervous system; and, a further method in which the loss of neurons and glial cells as a result of injury or disease damage is regenerated.

[0156] Neuronal regeneration generally means the regeneration or repair of neurons, glia, axons, myelin, or synapses. Researchers are developing new tools to effectively control the process of nerve injury and degeneration, enhance the natural repair ability, and create a microenvironment that enhances the effectiveness of other regenerative strategies such as nerve cell replacement and nerve rehabilitation. Experiments 7 and 8 describe methods for regenerating nerve cells and glial cells lost as a result of injury or disease damage.

[0157] Another aspect of the present invention includes the case where the administration is in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier.

[0158] A further aspect of the present invention includes that the effective amount of the cPG compound is about 1 μg to about 100 mg per kg of body weight.

[0159] A further aspect of the present invention includes the case where the administration is carried out in combination with artificial cerebrospinal fluid.

[0160] Another aspect of the present invention includes the case where the administration is intravenous administration.

[0161] A further aspect of the invention includes the case where the administration is combined with a neuroprotective agent, insulin-like growth factor-II (IGF-I) or insulin-like growth factor-III (IGF-II).

[0162] A further aspect of the invention where the administration is combined with an anti-inflammatory agent, an anti-integrin α4 subunit reagent.

[0163] A second aspect of the invention includes a method of repairing the loss of neurons and glial cells damaged as a result of injury or disease, and includes the following steps. a) Providing a subject in need of regeneration of said neuronal and glial cell loss; and b) Administering to said subject cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or Cyclic glycyl-2-methylproline (cPMeG) , collectively referred to as cPG compounds or combinations thereof) in an amount effective to regenerate new neurons and glia; wherein the neurons are regenerated and the loss of glial cells is regenerated in said subject; wherein said cPG functions as a neurorescue agent in the central nervous system, and further wherein the loss of neurons and glial cells damaged as a result of injury or disease is repaired.

[0164] Repairing damaged neurons generally refers to reconstructive techniques or processes for repairing damaged neurons to prevent loss of neurons and glia. The present invention describes a method capable of repairing neurons and glia damaged following traumatic, anoxic, infectious, and immunological side effects. The old doctrine that axons cannot be regenerated and dead neurons cannot be replaced is no longer applicable. In particular, in Experiments 7 and 8, the repair processes by nerve cells, glia, and pharmacological intervention are described.

[0165] Another aspect of the present invention herein includes that cyclic prolylglycine (cPG) or its analogs (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof) are from about 1 μg to about 100 mg per kg of body weight.

[0166] A further aspect of the present invention includes the case where the administration is in the form of a pharmaceutical composition comprising its pharmaceutically acceptable carrier.

[0167] A further aspect of the present invention includes the case where the administration is carried out in combination with artificial cerebrospinal fluid.

[0168] Another aspect of the present invention includes the case where the administration is combined with a neuroprotective agent, insulin-like growth factor-I (IGF-I) or insulin-like growth factor-III (IGF-II).

[0169] A further aspect of the present invention herein includes that the administration is combined with an anti-inflammatory agent.

[0170] A third aspect of the present invention includes a method for alleviating or reducing cognitive dysfunction resulting from a disease, injury or condition in a mammal in need thereof, and includes the following steps: a) providing a mammal in need of alleviation or reduction of cognitive dysfunction caused by a disease, injury or condition; and b) administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (collectively referred to as cPG compounds or combinations thereof)). Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration, and the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia. The injury is selected from the group consisting of neurotoxic injury, cerebral hypoxia / ischemia, traumatic brain injury, coronary artery bypass surgery, and when the condition is normal aging, memory loss associated with aging, memory impairment, cholinergic hypofunction, stenosis or occlusion of intracerebral blood vessels, neuroinflammation, mild cognitive impairment, brain atrophy, frontotemporal lobe degeneration, Pick's disease, HIV infection, Down's syndrome, and loss of synaptic plasticity. Further, the mammal is for alleviating or reducing cognitive impairment caused by a disease, injury or condition including Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0171] The alleviation of cognitive impairment generally refers to ways that can alleviate symptoms related to memory, thinking, language, and other thought processes. Additionally, it may also be helpful for mood, feelings of anxiety, and other behavioral problems.

[0172] Another aspect of the present invention herein involves the cyclic prolylglycine (cPG) or its analogs (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds, or combinations thereof) being included in an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers, or adjuvants.

[0173] A further aspect of the present invention involves the cyclic prolylglycine (cPG) or its analogs (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds), or combinations thereof, further including one or more excipients, carriers, additives, adjuvants, or binders in tablets or capsules.

[0174] A further aspect of the present invention herein includes cases where the disorder is mild cognitive impairment, Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy, or corticobasal degeneration.

[0175] Another aspect of the present invention includes cases where cyclic prolylglycine (cPG) or its analogs (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof) are administered by routes of administration including oral, intraperitoneal, intravascular, peripheral circulation, subcutaneous, intraorbital, intraocular, intraspinal, intravesical, topical, drip, implantation, aerosol, inhalation, scarring, intracapsular, intramuscular, nasal, buccal, transdermal, pulmonary, rectal, vaginal, or combinations thereof.

[0176] Another aspect of the present invention includes that the pharmaceutically effective amount has a lower limit of about 0.001 milligrams and an upper limit of about 100 mg / kg per kilogram mass (mg / kg) of the mammal.

[0177] A further aspect of the present invention includes cases where the cognitive impairment is caused by cholinergic hypofunction.

[0178] A further aspect of the present invention includes that the cognitive impairment is caused by a decrease in glutamate receptors in the granule cell layer (CA1) of the hippocampus of the mammal.

[0179] Another aspect of the present invention includes that cyclic prolylglycine (cPG) or its analogs (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof) cause an increase in AMPA receptors in the granule cell layer (CA1) of the hippocampus of the mammal.

[0180] A further aspect of the present invention herein includes cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG)). Collectively referred to as the cPG compound or a combination thereof, it causes an increase in neuronal plasticity caused by the cPG compound in the granule cell layer (CA1) and pyramidal cell layer (CA3) regions of the mammalian hippocampus.

[0181] A further aspect of the present invention includes cerebral hypoxia / ischemia caused by traumatic brain injury.

[0182] In another aspect of the present invention herein, it includes that the cognitive impairment is caused by multi-infarct dementia.

[0183] A further aspect of the present invention herein includes that the cognitive impairment is caused by coronary artery bypass grafting (CABG).

[0184] A further aspect of the present invention herein includes cognitive impairment due to Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0185] A fourth aspect of the present invention includes a method of preventing, in a mammal in need thereof, symptoms of mild cognitive impairment caused by or associated with a disease, injury, or condition, comprising: a) providing a mammal in need of prevention of cognitive impairment caused by a disease, injury or condition; b) administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof). Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0186] Preventing symptoms of mild cognitive impairment generally refers to actions taken to prevent the symptomatic stage and mainly reduce the risk of disease. Generally, prevention in dementia is referred to as primary prevention, secondary prevention, and tertiary prevention.

[0187] In degenerative dementia, the secondary prevention stage corresponds to the stage of mild cognitive impairment. At this stage, there are symptoms, but they are not severe enough to constitute dementia. Therefore, the treatment of cPG compounds in subjects with MCI is considered a secondary prevention trial.

[0188] Tertiary prevention refers to a treatment method aimed at stopping the progression of a disease that has already occurred. It aims to reduce the disability of people with mild cognitive impairment and improve the long-term prognosis.

[0189] A fifth aspect of the present invention includes a method of treating symptoms of mild cognitive impairment caused by or associated with a disease, injury, or condition in a mammal in need of treatment, comprising: a) providing a mammal in need of treatment for cognitive dysfunction caused by a disease, injury or condition; b) administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (collectively referred to as cPG compounds or combinations thereof, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG)). Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0190] Treating symptoms of mild cognitive impairment generally means treating cognitive function, i.e., the process of thinking, including but not limited to the abilities of learning, reading comprehension, conversation, writing, etc. Patients with mild cognitive impairment (MCI) retain these important cognitive abilities necessary to manage daily activities, but have difficulty remembering recent events or recently acquired information. Long-term memory tends to remain intact. In particular, see Experiment 12, a clinical trial conducted on Alzheimer's type dementia patients with mild cognitive impairment.

[0191] Neurons are among the oldest of all specialized animal cells. Their structure is not like that of other classes of cells. The central challenge of neural growth and development is how to grow axons and dendrites, find appropriate partners, and selectively form synapses with them to build a functional network.

[0192] The sixth aspect of the present invention includes a method for increasing neuron growth or synapse formation in a mammal caused by or associated with a disease, injury, or in need thereof, comprising: a) providing a mammal in need of increasing neuron growth or synapse formation; b) administering to the mammal a pharmaceutically effective amount of a composition consisting of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG) or a combination thereof. Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration

[0193] A seventh aspect of the present invention includes a method of providing a mammal in need of a method of reducing or alleviating a cognitive impairment caused by a disease, injury, or condition, the method including: a) providing a mammal in need of reducing or alleviating a cognitive impairment caused by a disease, injury or condition; and b) administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (collectively referred to as cPG compounds or combinations thereof, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG)). Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration. The injury is selected from the group consisting of neurotoxic injury, cerebral hypoxia / ischemia, traumatic brain injury, coronary artery bypass surgery, and when the condition is normal aging, memory loss associated with aging, memory impairment, cholinergic hypofunction, stenosis or occlusion of intracerebral blood vessels, neuroinflammation, mild cognitive impairment, brain atrophy, frontotemporal lobe degeneration, Pick's disease, AIDS infection, Down's syndrome, and loss of synaptic plasticity. Further, the mammal is for the alleviation or reduction of cognitive impairment caused by a disease, injury or condition including Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0194] Another aspect of the present invention herein includes that cyclic prolylglycine (cPG) or an analog thereof (collectively called cPG compounds, or combinations thereof, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG)) is composed of an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers, or adjuvants.

[0195] A further aspect of the present invention includes that cyclic prolylglycine (cPG) or an analog thereof (collectively called cPG compounds, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG)), or combinations thereof further comprises one or more excipients, carriers, additives, adjuvants or binders in tablets or capsules.

[0196] A further aspect of the present invention herein includes cases where the disorder is mild cognitive impairment, Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0197] Another aspect of the present invention includes cases where cyclic prolylglycine (cPG) or an analog thereof (collectively called cPG compounds, or combinations thereof, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG)) is administered by oral, intraperitoneal, intravascular, peripheral circulation, subcutaneous, intraorbital, intraocular, intraspinal, intravesical, topical, drip, implantation, aerosol, inhalation, scarring, intracapsular, intramuscular, nasal, buccal, transdermal, pulmonary, rectal, vaginal, or combinations thereof.

[0198] A further aspect of the invention includes that the pharmaceutically effective amount has a lower limit of about 0.001 milligrams and an upper limit of about 100 mg / kg per kilogram mass (mg / kg) of the mammal.

[0199] A further aspect of the invention includes the case where the cognitive impairment is caused by cholinergic hypofunction.

[0200] Another aspect of the invention includes the case where the cholinergic hypofunction is caused by scopolamine.

[0201] A further aspect of the invention is that here, the cognitive impairment is age-related memory loss, cognitive impairment, MCI ("mild cognitive impairment"), Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0202] A further aspect of the invention includes that the cognitive impairment is caused by a decrease in glutamate receptors in the granule cell layer (CA1) of the hippocampus of the mammal.

[0203] Another aspect of the invention includes that the cPG compound causes an increase in AMPA receptors in the granular cell layer (CA1) of the hippocampus of the mammal.

[0204] A further aspect of the invention is that here, cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG)) is included. It is collectively referred to as a cPG compound or a combination thereof, and causes an increase in neuronal plasticity caused by the cPG compound in the granule cell layer (CA1) and pyramidal cell layer (CA3) regions of the hippocampus of the mammal.

[0205] A further aspect of the present invention includes cerebral hypoxia / ischemia caused by traumatic brain injury.

[0206] In another aspect of the present invention, here, including that the cognitive impairment is caused by multi-infarct dementia.

[0207] A further aspect of the present invention, here, includes that the cognitive impairment is caused by coronary artery bypass grafting (CABG).

[0208] A further aspect of the present invention, here, includes cognitive impairment, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration resulting from Alzheimer's disease.

[0209] Eight aspects of the present invention include a method of preventing symptoms of mild cognitive impairment caused by or associated with a disease, injury, or condition in a mammal in need thereof, including the following. a) Providing a mammal in need of preventing cognitive dysfunction caused by a disease, injury or condition; and b) Administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG, or a combination thereof). Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0210] A ninth aspect of the present invention includes a method of treating symptoms of mild cognitive impairment caused by or associated with a disease, injury, or condition in a mammal in need of treatment, comprising: a) providing a mammal in need of treatment for cognitive dysfunction caused by a disease, injury or condition; and b) administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof). Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0211] A tenth aspect of the present invention includes a method of increasing neuron growth or synapse formation in a mammal caused by or associated with a disease, injury, or condition, or in need thereof, comprising: a) providing a mammal in need of increasing neuron growth or synapse formation; and b) administering to the mammal an effective amount of a composition comprising a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof). Here, the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, dementia of mixed vascular origin, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration.

[0212] The eleventh aspect of the present invention includes a method of regulating neurogenesis in the nerve tissue of a patient exhibiting at least one symptom of a central nervous system disorder that is a neurodegenerative disorder, an ischemic disorder, a neurological trauma, a learning and memory disorder, or a combination thereof, by administering cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline). Or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds, or a combination thereof, or a functional or active analog, variant, derivative, and combination thereof having the same, substantially the same, or similar function as cPG, wherein the agent regulates neurogenesis in the patient, thereby regulating neurogenesis in the nerve tissue of the patient.

[0213] Neurogenesis is the process by which new neurons are generated, mature, specialize, and become functional within a neuronal network from neural stem cells and progenitor cells. Regulation of neurogenesis in nerve tissue generally refers to a method of regulating or controlling the growth of nerve cells in the human brain.

[0214] Another aspect of the present invention includes cases where the disorder of the nervous system is Alzheimer's disease, Parkinson's disease and Parkinsonian disorders, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body type, spinal cord ischemia, ischemic stroke, cerebral infarction, spinal cord injury, cancer-related brain and spinal cord injury, multi-institutional dementia, senile dementia, mild cognitive impairment, depression, and traumatic injury.

[0215] A further aspect of the present invention includes that the regulation of the neurogenesis is performed by activation of a GPCR receptor in the nerve tissue.

[0216] A further aspect of the present invention, in a further aspect of the present invention, the agent is in an amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds, or combinations thereof) from about 0.1 mg to about 10 mg / kg / day, from about 0.5 mg to about 20 mg / kg / day, from about 0.2 mg to about 40 mg / kg / day, from about 5 mg to about 50 mg / kg / day, or from about 10 micrograms to about 100 mg / kg / day.

[0217] A twelfth aspect of the present invention includes a method of regulating neurogenesis in the neural tissue of a patient exhibiting at least one symptom of a central nervous system disorder that is a neurodegenerative disorder, an ischemic disorder, a neurological trauma, a learning and memory disorder, or a combination thereof. By administering to the patient an amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-2-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof) sufficient to increase the patient's adult neural stem cells, at least one symptom of the disorder can be alleviated in the patient.

[0218] Another aspect of the present invention, wherein the disorder includes Alzheimer's disease, Parkinson's disease and Parkinsonian disorders, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease, spinal cord ischemia, ischemic stroke, cerebral infarction, spinal cord injury, cancer-related brain and spinal cord injury, multi-bed dementia, senile dementia, cognitive impairment, depression, traumatic injury, or combinations thereof.

[0219] Another aspect of the present invention herein involves administering cyclic prolylglycine (cPG) or an analog thereof (collectively referred to as cPG compounds, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or combinations thereof), in an amount of from about 0.01 mg / kg to about 100 mg per kilogram of body weight per day.

[0220] A thirteenth aspect of the present invention includes a method for treating depression or other psychological disorders in a subject, comprising: a) providing a subject in need of treatment for depression or other psychological disorder; and b) administering to the subject a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (collectively referred to as cPG compounds, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), or combinations thereof); wherein the subject is characterized by receiving treatment for depression or other psychological disorder.

[0221] Symptoms of depression vary from mild to severe and include: feeling sad or down, loss of interest or pleasure in activities once enjoyed, changes in appetite (weight loss or gain unrelated to dieting), sleep disturbances or oversleeping, loss of energy or increased fatigue, thinking difficulties, decreased concentration, decreased decision-making ability, thoughts of death or suicide.

[0222] Another aspect of the present invention herein involves cyclic prolylglycine (cPG) or an analog thereof (collectively referred to as cPG compounds, such as cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-2-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or combinations thereof) being composed of an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers, or adjuvants.

[0223] A further aspect of the present invention further includes that cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds), or a combination thereof, consists of one or more excipients, carriers, additives, adjuvants or binders in a tablet or capsule.

[0224] A further aspect of the present invention includes the case where cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or a combination thereof) is administered by an administration route such as oral, intraperitoneal, intravascular, peripheral circulation, subcutaneous, intraorbital, intraocular, intraspinal, intravesical, topical, drip, implantation, aerosol, inhalation, scarring, intracapsular, intramuscular, nasal, buccal, transdermal, pulmonary, rectal, vaginal, or a combination thereof.

[0225] Another aspect of the present invention includes that the pharmaceutically effective amount has a lower limit of about 0.1 milligram per kilogram mass (mg / kg) of the mammal and an upper limit of about 10 milligrams / kg.

[0226] A further aspect of the present invention includes that the pharmaceutically effective amount is between about 20 mg and about 80 mg per day when administered orally, and in some severe cases can be up to about 100 mg per day by a doctor's prescription order.

[0227] The 13th aspect of the present invention includes a method of prevention for a subject in need of preventing symptoms of depression or other psychological disorders, and includes the following: a) providing a subject in need of preventing depression or other psychological disorders; b) administering to the subject a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof); wherein preventing the subject from developing symptoms of depression or other psychological disorders is included.

[0228] Preventing symptoms of depression generally means preventing a medical condition that adversely affects the way a person feels, thinks, and behaves. Depression causes feelings of sadness and loss of interest in activities that were once enjoyable. Depression can cause a variety of emotional and physical problems and can reduce functioning at work and at home. In particular, see Experiment 15.

[0229] The 14th aspect of the present invention includes a method of treating symptoms of depression or other psychological disorders in a subject in need of treating depression or other psychological disorders, and includes the following: a) providing a subject in need of treating depression or other psychological disorders; b) administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof); wherein treating the subject for symptoms of depression or other psychological disorders is included.

[0230] The symptoms of depression vary from mild to severe and include the following: feeling sad or down, losing interest or pleasure in activities once enjoyed, changes in appetite (weight loss or gain unrelated to dieting), sleep disturbances or excessive sleeping, loss of energy or increased fatigue, thinking difficulties, reduced concentration, reduced decision-making ability, thoughts of death or suicide, etc.

[0231] The 15th aspect of the present invention includes a method for treating a patient in a mentally depressed state, including the following. a) Providing a subject in need of treatment for depression or other mental disorders. b) Administering to a patient suffering from mental depression a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (collectively referred to as cPG compounds or combinations thereof), for example, not limited to 20 mg to 80 mg per day, but not limited to these); where the subject is capable of receiving treatment for mental depression.

[0232] In the treatment of patients in a mentally depressed state, it was generally common to use commercially available antidepressants that increase the risk of suicidal thoughts and behaviors in children, adolescents, and young adults in short-term trials. However, cPG of the present invention has been demonstrated to be an effective antidepressant with few side effects. In the present invention, it has been demonstrated that cPG functions as an effective antidepressant with few or no side effects. See Experiment 15.

[0233] The sixteenth aspect of the present invention includes a method for reducing or alleviating the symptoms of depression or other psychological disorders, and is a method for reducing or alleviating the symptoms of a subject in need of depression or other psychological disorders, including the following. a) Providing a subject in need of reducing or alleviating the symptoms of depression or other psychological disorders; b) Administering to a mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof); wherein the subject includes alleviating the symptoms of depression or other psychological disorders.

[0234] The seventeenth aspect of the present invention includes a method for intervening in or preventing a subject in need of a cascade of depression or other psychological disorders, including the following. a) Providing a subject in need of intervening in or preventing depression or other psychological disorders; b) Administering to a mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG) or an analog thereof (cyclic (tri(prolylglycine)) or cyclic glycyl-2-allylproline, cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), collectively referred to as cPG compounds or combinations thereof); wherein the subject includes preventing the symptoms of depression or other psychological disorders.

[0235] Although treatment methods for depression have been established, it is presumed that the majority of depression patients are not receiving appropriate care. Timely intervention is an important element of the continuity model of care for people with major depressive episodes. In particular, refer to Experiment 15.

Example

[0236] The following in vitro and in vivo studies demonstrate the effectiveness of cyclic prolylglycine in reducing cognitive impairment. These are not intended to be limiting, and other compositions and methods of the present invention can be developed without undue experimentation. All of the following experiments were conducted using protocols developed under guidelines approved by the Animal Ethics Committee and the Institutional Review Board.

[0237] Cyclic prolylglycine is available from commercial suppliers such as Bachem (Torrance, CA) and Sigma (St. Louis, MO, USA).

[0238] Experiment 1. Cyclic PG prevents glutamate-induced neuronal cell death in vitro in a dose-related manner. Materials and methods : Preparation and coating of cerebellar cell culture coverslips Ten cover slips were placed in a large petri dish, washed with 70% alcohol for 5 minutes, and then washed with Millipore H2O. The cover slips were air-dried, coated with poly-D-lysine (1 mg / ml stock solution in PBS, 90 - 100 μl), and incubated at 34 °C for 2 hours.

[0239] Extraction Wistar rats on the 4th day after birth were used. The rats were placed in ice for 1 minute, decapitated, and the cerebellum was removed on ice. The cerebellar tissue was placed in 1 ml of 0.65% glucose-supplemented PBS (10 μl of 65% stock D(+) glucose / 1 ml PBS) in a large petri dish, minced into smaller pieces, triturated with a 1 ml insulin syringe through a 23G (0.4 mm) needle, and returned to the glucose solution on the large petri dish for aspiration. The tissue was sieved (125 μm pore size sieve), centrifuged twice (60 g for 2 minutes), and the medium was changed to serum-free BSA-supplemented START V medium (Biochrom). The second centrifugation step was performed with 1 ml of STARTART V medium. Microexplosions were reconstituted in 500 μl of START V medium and placed on ice.

[0240] Culture and immobilization of cerebellar cells Two hours after the PDL coating, the slides were washed with Millipore H2O and air-dried. Each slide was placed in a small 35 mm Petri dish, and 40 μl of the V / cell suspension was added. The tissue was incubated at 34 °C (sedimentation period) for 2 hours. Then, Start V (1 ml) was added to the Petri dish and cultured at 34 °C / 5% CO2 / 100% humidity for 48 hours. The cells were washed with PBS and fixed with increasing concentrations of paraformaldehyde (500 μl of 0.4% PFA was applied, then 1.2% PFA, then 3% PFA, and finally 4% PFA - all fixation solutions contained 0.2% glutaraldehyde) for 2 - 3 minutes. Finally, the microexplants were rinsed with PBS.

[0241] Application of drugs For Test 1, 10 μl of the toxin (in L-glutamic acid - 100 Millipore water) was applied simultaneously with cPG (manufactured by Bachem, prepared from a 10 mM stock in PBS and diluted to a final concentration between 1 - 100 nM). For Test 2, the administration of cPG was delayed by 6 hours after the glutamic acid treatment.

[0242] Results: Test 1: Glutamic acid treatment resulted in an 85% loss of cerebellar neurons. Cyclic PG significantly reduced glutamate-induced neuronal death in a dose-responsive manner when administered simultaneously with glutamate (Figure 3). Treatments with lower doses of cPG (10 - 100 nM) showed a significant recovery from glutamate-induced neurotoxicity.

[0243] Test 2: Cyclic PG showed a significant recovery from glutamate-induced neurotoxicity in the dose range of 1 - 100 nM when administered 6 hours after the glutamic acid treatment, compared to the vehicle-treated group (Figure 4).

[0244] Furthermore, when even lower doses of cPG were administered, the number of neurons increased significantly compared to the normal control group, suggesting a role of cPG in neuronal growth and differentiation.

[0245] Conclusion Excess glutamate can potentially cause excitotoxicity of neurons by active NMDA receptors. cPG acts as a direct or indirect NMDA agonist and, when administered immediately after glutamate treatment or 6 hours later, was able to completely prevent glutamate-induced neurotoxicity. Considering that cPG can act as an agonist for the mGlu2 / 3 receptors that can inhibit NMDA activity. GPE, the precursor hormone of cPG, has been shown to be a partial NMDA receptor in the promotion of pCREB, probably due to its antagonistic effect on the mGlu2 / 3 receptors. Since cPG is effective as a delayed treatment and can promote the proliferation of neurons, it can be involved in the prevention of neurons undergoing apoptosis.

[0246] Experiment 2. Cyclic (tri(prolylglycyl) or c(PG)3 suppresses glutamate-induced neuronal cell death in vitro in a dose-related manner. Materials and methods :(See the above for Experiment 1 incorporated herein by reference.)

[0247] Application of drugs 10 μl of the toxin (L-glutamate - 100 mM, Millipore water) was applied simultaneously with cyclic (tri(prolylglycyl)). A 10 mM stock obtained from (Neurobiomed, San Jose, California) was prepared in PBS and diluted to a final concentration of 1 - 100 nM for use in Test 1. For Test 2, the administration of cyclic (tri(prolylglycyl)) was delayed 6 hours after glutamate treatment.

[0248] Results: Test 1: 85% of cerebellar neurons disappeared after glutamate administration. Cyclic (Tris(ProlylGlycyl)) significantly reduced glutamate-induced neuronal death by 57% in a dose-responsive manner when administered simultaneously with glutamate. Treatment with lower doses of cyclic (Tris(ProlylGlycyl)) (10 - 100 nM) showed significant recovery from glutamate-induced neurotoxicity.

[0249] Test 2: When cyclic (Tris(ProlylGlycyl)) was administered 6 hours after glutamate treatment, it showed a significant recovery of approximately 43% from glutamate-induced neurotoxicity in the dose range of 1 - 100 nM, and there was an improvement compared to the vehicle-treated group.

[0250] Even when lower doses of cyclic (Tris(ProlylGlycyl)) were administered, the number of neurons increased significantly compared to the normal control group, suggesting the role of cPG in neuronal growth and differentiation.

[0251] Experiment 3 Cyclic glycyl-2-methylproline prevents glutamate-induced neuronal cell death in a dose-related manner. Materials and methods : For Experiments 1 and 2 incorporated herein by reference, see the above reference

[0252] Application of drugs For Test 1, 10 μl of the toxin (L-glutamate - 100 mM in Milli-Q water) was applied simultaneously with cyclic glycyl-2-methylproline (obtained from Neurobiomed, San Jose, California), prepared as a 10 mM stock in PBS and diluted to a final concentration between 1 - 100 nM). For Test 2, the administration of cPG was delayed 6 hours after glutamate treatment.

[0253] Results: Test 1: Glutamate treatment resulted in an 85% loss of cerebellar neurons. Cyclic glycyl-2-methylproline significantly decreased glutamate-induced neuronal death in a dose-responsive manner by 63% when administered simultaneously with glutamate. Treatments with lower doses of cyclic glycyl-2-methylproline (10 - 100 nM) showed significant recovery from glutamate-induced neurotoxicity.

[0254] Test 2: When cyclic glycyl-2-methylproline was administered 6 hours after glutamate treatment, an improvement of approximately 58% significant recovery from glutamate-induced neurotoxicity in the dose range of 1 - 100 nM was shown compared to the vehicle-treated group.

[0255] Even when lower doses of cyclic glycyl-2-methylproline were administered, the number of neurons was significantly increased compared to the normal control group, suggesting the role of cPG in neuronal growth and differentiation.

[0256] Conclusion Excessive glutamate may cause excitotoxicity of neurons through active NMDA receptors. Cyclic PG analogs, cyclic (tri(prolylglycyl)) and cyclic glycyl-2-methylproline, significantly prevented glutamate-induced neurotoxicity when administered immediately after or 6 hours after glutamate treatment by acting as direct or indirect NMDA agonists.

[0257] cPG compounds are effective as a delayed treatment and can promote neuronal growth, so they can be used for the prevention of neurons undergoing apoptosis, but are not limited to these. For example, cyclic PGs such as cyclic (tri(prolylglycyl)) and cyclic glycyl-2-methylproline and their analogs can be used.

[0258] Experiment 4. Effect of cPG after lesion induced by 6-HDA. Materials and methods Twenty male Wistar rats (280 - 310 grams) were used. After exposing the skull, 6 - OHDA (8 μg in 2 μl of 0.9% saline base containing 1% ascorbic acid) was administered to the right medial forebrain bundle (MFB) using coordinates AP + 4.7 millimeters, R 1.6 mm, V - 8 millimeters under 3% halothane anesthesia. 6 - OHDA was injected via a 25G needle connected to a Hamilton syringe with a polyethylene catheter through a 100 μl volume. 6 - OHDA was injected with a microdialysis infusion pump at a rate of 0.5 μl / min. The needle remained in the brain for an additional 3 minutes before being slowly withdrawn. The skin was sutured with 2.0 silk, and the rats were allowed to recover from anesthesia. The rats were housed in a holding room with free access to food and water at all times except during the behavioral tests.

[0259] cPG was dissolved in saline. Four different doses of cPG (0, 0.1, 0.5, 1 mg / kg, Sigma) were administered intraperitoneally 2 hours after the lesion.

[0260] Seven days later, rats were injected with 0.1 mg / kg of apomorphine, and the number of contralateral rotations per hour was recorded and calculated using a computerized rotameter (San Diego Instruments). The experimenter was blinded to the treatment groups.

[0261] Results: The group administered 1 mg of cPG (n = 5, 154 ± 64) showed a tendency for a decrease in the number of rotations compared to the vehicle - administered group (n = 5, 290 ± 18). (Figure 5).

[0262] Experiment 5 Effect of cyclic (tri(prolylglycyl) after gliosis induced by 6-HDA Materials and methods (See above for Experiments 1, 2, 3, and 4).

[0263] Cyclic (Tris(ProlylGlycyl)) was dissolved in physiological saline. Four different doses of cyclic (Tris(ProlylGlycyl)) 0, 0.1, 0.5, 1 mg / kg, Neurobiomed) were intraperitoneally administered 2 hours after the lesion.

[0264] In the group treated with 1 mg of cyclohexyl (Tris(ProlylGlycyl)) (n = 5, 172 ± 69), there was a tendency for the rotation number to decrease compared to the vehicle-treated group (n = 5, 290 ± 18), suggesting the role of cyclic (Tris(ProlylGlycyl)) in the improvement of functional recovery in 6-OHDA-induced ganglionic lesions.

[0265] Conclusion Cyclic (Tris(ProlylGlycyl)) dose-dependently improved the functional recovery of neuro-angualar lesions after 6-OHDA induction.

[0266] This data indicates that cyclic (Tris(ProlylGlycyl)) can be used as a therapeutic agent for Parkinson's disease and other neurological disorders.

[0267] Experiment 6. Effect of cyclic glycyl-2-methylproline after gliosis induced by 6-HDA Materials and methods (See Experiments 1 to 5 above).

[0268] Cyclic Glycyl-2-methylproline (cGMeP) was dissolved in physiological saline. Four doses of cGMeP (0, 0.1, 0.5, 1 mg / kg, Neurobiomed) were intraperitoneally administered 2 hours after the lesion.

[0269] The group administered 1 mg of cGMeP (n = 5, 134 ± 69) showed a tendency for a significant decrease in the rotation number compared to the group treated with vehicle (n = 5, 292 ± 21), suggesting the role of cGMeP in the improvement of functional recovery in 6-OHDA-induced ganglionic lesions.

[0270] Conclusion Cyclic glycyl-2-methylproline improved the functional recovery of nerve-bevel lesions after 6-HDA induction in a dose-related manner.

[0271] This data indicates that cycloglycyl-2-methylproline is effective as a therapeutic agent for Parkinson's disease.

[0272] Experiment 7. The Morris water maze (MWM) model of learning and memory is used to evaluate the effect of cyclic prolylglycine on cognitive function. Administration of cPG to animals with scopolamine-induced cognitive dysfunction results in clinical improvements similar to those observed in people suffering from cholinergic hypofunction. Scopolamine is commonly used in animal models of cholinergic functional decline associated with Alzheimer's disease. The functional impairments observed after scopolamine treatment include those seen in human Alzheimer's disease patients. Thus, scopolamine treatment can reasonably predict the cognitive impairments seen in human diseases. Furthermore, scopolamine treatment mimics cognitive impairment in humans without neurodegenerative diseases.

[0273] The aim of this study was to investigate cyclic prolylglycine and evaluate its effects on cognitive deficits and emotional state (anxiety).

[0274] Methods The first part of the study was involved in an acute test of cyclic prolylglycine in the Morris water maze memory model. The MWM test is one of the most frequently used tests to evaluate spatial memory in rats and is generally well known to accurately predict the effects of diseases and treatments on spatial memory. Therefore, the MWM test reflects the effects of diseases and treatments in human subjects.

[0275] It followed the standard procedure of MWM. A circular pool (water depth 80 cm × diameter 150 cm) filled with opaque water was used, and the temperature was maintained at 20 °C. A platform was hidden 1 cm below the water surface, and a white flag (10 cm × 10 cm) was placed either 20 cm above the platform for visual cues or at the 3 o'clock position in relation to the starting position for spatial cues. From the 1st day to the 4th day of the experiment, the rats received memory acquisition trials in 6 trials (60 seconds each) on each day of the test (habituation phase). The latency to reach the platform was recorded, and the daily decrease in the average latency was used to measure the ability to learn where the hidden platform was located.

[0276] On the 5th day of the experiment, normal, non - aged Wistar rats were grouped and either saline (N = 12) or scopolamine (0.5 mg / kg, i.p., N = 12) was administered to induce memory impairment. Scopolamine was administered 30 minutes before the start of the probe test.

[0277] Ten minutes after scopolamine treatment, cyclic prolylglycine was orally administered at 10 mg / kg (n = 16), and diluent was orally administered to vehicle - treated animals using the same treatment protocol (n = 15).

[0278]

Table 1

[0279] Next, the acute effect of cPG was tested using animals with scopolamine - induced memory impairment and control animals matched for age without memory impairment to determine the direct pharmacological effect on memory processing. The experimental groups are detailed in Table 1 below.

[0280] On the 5th day, the probe MWM test was conducted with the platform removed. The test was performed 6 times, the maximum duration of each test was 60 seconds, and at least a 5-minute break was taken between tests. The time the rats spent swimming near the platform served as an indicator of how much they relied on visual and spatial cues to identify the position of the platform, as opposed to using non-spatial strategies. Data were collected and analyzed using Any-maze (v4.2) software.

[0281] Data generated from the behavioral tests were analyzed using one-way ANOVA to determine differences between age groups. To examine the course of the results of the behavioral tests, two-way ANOVA with the time point treated as a dependent factor was used. GraphPad Prism version 3.02 was used for data analysis.

[0282] Results Treatment with scopolamine significantly impaired the acquisition of spatial memory in the treated animals (the time to the platform on the 4th day was approximately 208% of that of the control). Cycloprolylglycine (20 mg / kg; daily) significantly reversed the cognitive impairment induced by scopolamine (Figure 6).

[0283] From these results, it was confirmed that NA-831 showed a cholinergic effect in the retrieval of the skill (spatial memory) of finding the learned submerged platform and was also effective in patients with mild cognitive impairment.

[0284] Experiment 8. Determination of neurogenesis by testing cPG and its analogs c(PG)3 and cGMeP using bromodeoxyuridine Purpose The purpose of this experiment was to examine the effects of intraperitoneal administration of cPG and its analogs c(PG)3 and cGMeP in rats co-administered with BrdU in highly neurogenic regions including the subventricular zone and the dentate gyrus of the hippocampus.

[0285] Experimental method 。 Male Wistar rats weighing approximately 250 - 270 g (not neonatal) were used. All animal experiments were conducted in accordance with national and international guidelines. Care was taken to minimize the suffering of the animals. The animals could be acclimatized for 1 week before the start of the study. The animals were housed under standardized conditions in a normal light - dark cycle and groups of 5 animals per cage. The animals had free access to food and water during the study period.

[0286] Male Wistar rats (N = 10) were separately intraperitoneally administered with three types of neurogenic modulators, cPG, c(PG)3, and cGMeP, at a dose of 10 mg / kg in 0.1% rat serum albumin (RSA). The negative control (n = 12), the vehicle group, was injected with saline (in 0.1% RSA). Bromodeoxyuridine (BrdU; 50 mg / kg) was co - administered with the compounds. Intraperitoneal injections were performed at 12 - hour intervals for 7 days. The animals were perfused on the 8th day. The rats were housed on a 12 - hour light - dark regimen. For perfusion, the animals were perfused transcardially with 50 ml of ice - cold phosphate - buffered saline (PBS), followed by perfusion with 100 ml of 4% paraformaldehyde in PBS. The brains were removed and fixed in 4% paraformaldehyde in PBS at 4°C for at least 3 days before sectioning. The transcardial injection procedure consisted of the following steps.

[0287] Animals were weighed in 0.1 gram increments and administered pentobarbital sodium and ketamine / xylazine. The animals were placed in a heated cage for 10 - 15 minutes. Rats were fixed in a supine position (lying on their backs with their faces up) with their forelimbs and hindlimbs secured to the foamed styrene work surface within a chemical fume hood. Incisions were made through the skin with surgical scissors just below the scaphoid process of the clavicle along the midsternal line of the thorax. Two additional skin incisions were made laterally from the rostral process along the base of the ventral thorax. Carefully reflect the two flaps of skin rostrally and laterally and confirm complete exposure of the thoracic field. Grasp the cartilage of the anastomotic process with a blunt instrument and slightly elevate it to insert a pointed-tip scissors. Incise the pectoralis muscle and thorax between the sternum and the medial rib insertion and extend the incision to the level of the clavicle for anastomosis. The diaphragm from both sides of the thoracic wall was separated by a scissor cut. The reflected thorax was taped or pinned with an 18G needle laterally to expose the heart and other thoracic organs. The pericardium was gently grasped with a blunt instrument and completely opened and torn. The beating heart was fixed with a blunt forceps and a 1 - 2 mm incision was made in the left ventricle. A 24G X 25.4 mm animal feeding needle Barnus tip (Harvard apparatus cat. The feeding needle was threaded to the base of the aortic arch using a dissecting microscope. The base of the needle was clamped to the left ventricle over the incision site using a hemostatic plug. The right atrium was immediately incised with scissors and the infusion of heparinized saline was initiated at the first sign of blood flow (Stage 1 perfusate) and continued until the fluid emerging from the right atrium became completely clear. The saline perfusate was changed to an aldehyde-based fixative (Stage 2 perfusate) and the total amount of fixative injected into the animal was changed to 20 - 30 ml. The animal was decapitated with large surgical scissors. The brain was removed and embedded in paraffin. Sections were prepared using a cryostat microtome and stored in a cryoprotectant at -20°C prior to immunostaining for BrdU. Sections were immunostained for BrdU with mouse anti-BrdU paired with biotinylated goat anti-mouse IgG and visualized using an ABC Elite kit (Vectorlab, following the manufacturer's instructions).

[0288] Using standard light microscopy techniques, the total number of BrdU-positive cells in each section and related regions of the brain was counted. Analyses and quantification were performed on the proliferative brain regions, the subventricular zone, and the dentate gyrus of the hippocampus. Other experimental details not described herein are known to those skilled in the art, for example, as described in Pencea V et al. J. Neurosci Sep. 1 (2001). 21 (l 7):6706-17.

[0289] Results Notably, when rats were intraperitoneally injected with 10 mg / kg of cPG, c(PG)3, and cGMeP dissolved in 0.1% RSA twice a day in co-administration with BrdU, the number of newborn cells (BrdU-positive) in highly neurogenic regions including the subventricular zone and the dentate gyrus of the hippocampus increased significantly (non-parametric one-way ANOVA) (Figs. 7 and 8).

[0290] Conclusion From the above, it was revealed that cPG, c(PG)3, and cGMeP exhibit a proliferative effect on neural stem cells.

[0291] Experiment 9 Determination of regeneration of damaged nerve tissue using cPG Ten postpartum day 4 Wistar rats were used in the study. The rats were divided into two groups, one group of 5 rats treated with the chemical solution and the other group of 5 rats treated with a 0.9% sodium chloride (physiological saline) solution. The chemical solution containing cPG (manufactured by Bachem, a 10 mM solution prepared in 0.9% sodium chloride (physiological saline) solution) was administered 4 times a day in a volume of 0.75 ml, and the injury site was thoroughly soaked in water.

[0292] Two days after the spinal cord was crushed, the dura mater of the rats was opened, and the polyethylene tube was sutured to the soft tissue adjacent to the vertebral spine such that the opening at one end lay directly over the injured part of the spinal cord. The tube was brought through a subcutaneous tunnel such that the other end emerged at the base of the skull. A syringe adapter was attached to the external opening for injecting the drug. All experiments were conducted in a double-blind manner in two groups: one group of five animals was treated with a 10 mM cPG solution in saline, and the other group was treated with a 0.9% sodium chloride (saline) solution. Treatment of all animals continued for 14 days after the animals were killed and histological sections were prepared.

[0293] Results In the drug-treated animals, there was greater infiltration of the lesion by nerve fibers compared to the saline-treated animals. In the drug-treated animals, nerve fibers had entered the lesion in large numbers and no longer changed direction longitudinally but grew randomly in all directions. The fibers were often sinuous and venous and were frequently small bundles containing 3 to 6 axons. The axons were very fine, and most of them had a diameter of 3 to 7 microns. When the slides were coded and randomized, there was no difficulty in distinguishing between specimens from drug-treated and saline-treated animals. The most robust nerve growth was in the animals treated with cPG.

[0294] Conclusion This example demonstrated that nerve regeneration was promoted by sufficiently bathing or otherwise contacting the composition at the injury site. When the composition was administered directly to the injury site, regeneration of the damaged nerve tissue was promoted.

[0295] Experiment 10 Regeneration of damaged nerve tissue of cyclic (glycyl-L-prolyl-L-prolyl) Ten Wistar rats on the fourth day after parturition were used in the experiment. The rats were divided into two groups, one group of 5 rats treated with a drug solution and the other group of 5 rats treated with a 0.9% sodium chloride (physiological saline) solution. A drug solution containing 10 mM of cyclic (glycyl-L-prolyl-L-prolyl) or c(PG)3 obtained from Neurobiomed (San Jose, California) was prepared with a 10 mM solution of 0.9% sodium chloride (physiological saline) and administered 4 times a day in a volume of 0.75 ml, and the injury site was thoroughly soaked in water.

[0296] Two days after the spinal cord was crushed, the dura mater of the rats was opened, and the polyethylene tube was sutured to the soft tissue adjacent to the vertebral spine so that the opening at one end lay directly over the injured part of the spinal cord. The tube was brought through a subcutaneous tunnel so that the other end emerged at the base of the skull. A syringe adapter was attached to the external opening for injecting the drug. All experiments were based on double-blind in two groups: one group of 5 animals was treated with a drug solution of 10 mM c(PG)3 with physiological saline, and the other group of 5 animals was treated with a 0.9% sodium chloride (physiological saline) solution. The treatment of all animals continued for 14 days after the animals were killed and histological sections were prepared.

[0297] Results The results were the same as in the above experiment using cPG. In the animals treated with c(PG), the nerve fibers were no longer oriented longitudinally, but rather grew in large numbers to the lesion site as if growing disorderly in all directions. The fibers often became serpentine and venous, and often formed small bundles containing 3 to 6 axons. The axons were very thin in diameter, and most of them were 3 to 7 microns in diameter.

[0298] When the slides were coded and randomized, there was no difficulty in distinguishing between the specimens of drug-treated animals and physiological saline-treated animals. The most vigorous nerve growth was in the animals treated with c(PG)3.

[0299] By sufficiently immersing the composition in the damaged area, nerve regeneration is promoted. The composition promotes the regeneration of damaged nerve tissue by being administered to the damaged area, including direct administration to the injury site.

[0300] Experiment 11 Regeneration of damaged nerve tissue of cyclic glycyl-2-methylproline For the experiment, 10 Wistar rats on the 4th day after parturition were used. The rats were divided into two groups, one group of 5 rats treated with a chemical solution and the other group of 5 rats treated with a 0.9% sodium chloride (physiological saline) solution. A chemical solution containing 10 mM of cyclic glycyl-2-methylproline, that is, cGMeP (manufactured by Neuro Biomed Co., a 10 mM solution prepared in a 0.9% sodium chloride (physiological saline) solution), was administered 4 times a day in a volume of 0.75 ml, and the injury site was thoroughly soaked.

[0301] Two days after crushing the spinal cord, the dura mater of the rat was opened, and the polyethylene tube was sutured to the soft tissue adjacent to the vertebral spine so that one end opening directly lay on the injured part of the spinal cord. The tube was brought through a subcutaneous tunnel so that the other end came out at the base of the skull. The syringe adapter was attached to the external opening for injecting the drug. All experiments were conducted based on double-blind in two groups: one group of 5 animals was treated with a chemical solution of 10 mM cGMeP with physiological saline, and the other group of 5 animals was treated with a 0.9% sodium chloride (physiological saline) solution. The treatment of all animals continued for 14 days after killing the animals and preparing histological sections.

[0302] Results The results are the same as those of the above experiment using cPG. In the animals treated with cGMeP, the nerve fibers no longer oriented longitudinally, but rather grew in large numbers to the lesion site in a disorderly manner in all directions. The fibers often curved and became vein-like, and were often in small bundles containing 3 to 6 axons. The axons were very thin in diameter, and most of them had a diameter of 3 to 7 microns.

[0303] When the slides were coded and randomized, there was no difficulty in distinguishing between specimens from drug-treated animals and saline-treated animals. The animals treated with cGMeP had the most active nerve growth.

[0304] Nerve regeneration is promoted, but not limited to, by contacting the composition to the site of injury. When the composition is directly administered to the site of injury, it promotes the regeneration of damaged nerve tissue.

[0305] Experiment 12 Clinical trial for Alzheimer's disease patients with mild cognitive impairment. Mild cognitive impairment (MCI) refers to a state in which cognitive function is reduced more than expected according to age and educational background. Mild cognitive impairment refers to a condition in which there is a disorder in cognitive functions such as memory, which is beyond the norm for age, but does not reach the level of dementia, which is a characteristic of dementia.

[0306] In population-based studies, mild cognitive impairment is 3% to 15% in adults 65 years of age and older. More than half of MCI patients progress to dementia within 5 years.

[0307] The detection of mild cognitive impairment is important because treatment at this stage is more effective than treatment after the onset of dementia.

[0308] Materials and methods A randomized clinical trial of NA-831 (alias: cyclic prolylglycine) was conducted on Alzheimer's disease patients with vascular-derived mild cognitive impairment. NA-831 was orally administered at a dose of 10 mg once a day for 12 weeks. There were 32 Alzheimer's disease patients who participated in the trial.

[0309] Inclusion criteria - Males or females aged 55 to 85 years (inclusive) at the time of screening. - Self-reported memory dissatisfaction, supported by a spouse or companion as needed. - Age - adjusted Logical Memory II score of the Wechsler Memory Scale - III (WMS - III) ≤ 5. - Mini - Mental State Examination (MMSE) ≥ 24. - Epidemiologic Research Center - Depression (CES - D) score < 27. - Normal thyroid function (TSH, TS3, and T4 are defined as being within the normal range). - Agree not to consume alcoholic beverages within 8 hours prior to each study visit. - Sign an informed consent and have the willingness and ability to complete CTB and all other procedures described in the tests and protocols. - Female subjects must be surgically sterile or post - menopausal for at least 2 years. If less than 2 years post - menopause, follicle - stimulating hormone (FSH) ≥ 40 mIU / mL must be obtained.

[0310] Exclusion criteria - Subjects with major untreated mental or central nervous system diseases (such as schizophrenia, Parkinson's disease, stroke, etc.) that may interfere with the evaluation and procedures of the study or pose additional risks. - Subjects with a history of uncomplicated depression may participate under the following conditions. - If they are in remission and have been taking stable antidepressants for at least 2 months. - A history of persistent neurological abnormalities or known brain structural abnormalities following a major head injury. - Those who have had a stroke, transient ischemic attack (TIA), or loss of consciousness of unknown cause in the past year. - A history of unstable angina, myocardial infarction, chronic heart failure, or clinically significant conduction abnormalities within 1 year prior to the first screening visit. - A history of alcohol or substance abuse or dependence within the past year. - Acute infectious rhinitis - History or presence of abnormalities of the external or internal structures of the nose or nasopharynx, except for correction by septal surgery, or correction by septal or "broken nose" surgery more than 2 years ago, or correction by cleft palate surgery before the age of 30. - Use of medications known to cause a frank blunting of cognition - Use of Alzheimer's disease treatment or investigational drugs within 3 months of screening - History of systemic diseases or current serious diseases that are judged to interfere with the evaluation of the trial or raise concerns about safety. - Individuals with untreated sleep apnea or a history of sleep apnea treatment of less than 3 months. - If there is a clinically significant systemic disease or serious infection within 30 days before or during the screening period - Among the medications permitted for chronic diseases, use at an unstable dose for at least 4 weeks before the first screening visit, or use of an AD treatment drug at an unstable dose for at least 8 weeks before the first screening visit. - Abnormal clinical test values, specifically in the following cases. Alanine transaminase (ALT) or aspartate transaminase (AST) > 2 x the upper limit of normal (ULN), hematological test values less than 80% of the lower limit of normal, creatinine ≥ 2 mg / dL, or other clinical test values or vital signs are judged by the principal investigator of the trial to be clinically significant. - Use of investigational drugs, biological products, or devices within 30 days before screening. - Surgery requiring general anesthesia within the past 3 months, or planning surgery requiring general anesthesia during the trial period. - Contraindications to the trial procedure - Use of drugs that may contribute to cognitive impairment and increase the risk of adverse events (AE), or use of drugs that may impair the ability to perform cognitive tests or complete the trial procedure.

[0311] Evaluation 1) A scale for evaluating the severity of symptoms based on the "Unified Evaluation of the Clinical Pharmacological Effects of Psychotropic Drugs in Patients with Organic Disorders," which can objectively and quantitatively grasp the treatment dynamics of psychopathological symptoms and the characteristics of the psychotropic effects of drugs. 2) The Mini-Mental State Examination (MMSE) consists of neuropsychological tests that evaluate cognitive functions in each item of attention, memory, gnosis, speech ability, praxis, and counting. 3) The Brief Cognitive Rating Scale (BCRS) for evaluating the severity of individual components of cognitive impairment. 4) The Cognitive Capacity Screening Examination (CCSE) consists of tests that evaluate cognitive functions such as orientation, memory, counting, and the ability to infer or group objects. 5) The Clinical Global Impression Scale is a scale that investigates severity, degree of "overall improvement," treatment effect, presence or absence of side effects during treatment, severity, etc., and quantitatively evaluates the treatment effect of drugs together with tolerance and safety. 6) Laboratory analysis consisting of general blood tests, including biochemical tests (AST, ALT), and urine tests. 7) ECG traces.

[0312] Results The mental state of patients with vascular-derived mild cognitive impairment was defined by evaluations including the Mini-Mental State Examination (MMSE), Brief Cognitive Rating Scale (BCRS), and Cognitive Capacity Screening Examination (CCSE), but is not limited to this.

[0313] The therapeutic effects of NA-831 in patients include a reduction in neurotic symptoms and cognitive impairment.

[0314] The results are shown in Table 2.

Table 2

[0315] W is the Wilcoxon signed-rank test, a non-parametric test corresponding to the dependent t-test. It is used to compare two sets of scores from the same participants. This occurs when one wants to investigate the change in scores from one point in time to another, or when an individual is exposed to two or more conditions.

[0316] Clinicians used the Wilcoxon signed-rank test to understand whether there were differences in the patient's condition regarding fatigue before and after the drug treatment period.

[0317] It should be noted that NA-831 has been shown to significantly improve the following functions at p < 0.01: - Reduction of fatigue - Reduction of anxiety - Reduction of irritability - Improvement of mobility - Reduction of disruptions during waking up at night - Reduction of daytime sleepiness - Improvement of nocturnal sleep duration

[0318] The analysis of the therapeutic effect of NA-831 was carried out on all patients participating in this trial using evaluation methods including the Mini-Mental State Examination (MMSE), Brief Cognitive Rating Scale (BCRS), and Cognitive Capacity Screening Examination (CCSE).

[0319] Clinical data regarding the effectiveness of NA-831 in Alzheimer's disease patents are summarized and emphasized as follows.

[0320] NA-831 was found to improve the patient's concentration and counting ability. In the graph, when the measurements of the Brief Cognitive Rating Scale, which are statistical data from the 42nd day to the 84th day, were plotted against time, the p-value was 0.01 or less. This means that there is a significant effect on the improvement of the patient's concentration and counting ability. Please refer to Figure 9.

[0321] It is a common symptom that the short-term memory of patients with Alzheimer's disease deteriorates steadily. NA-831 improves not only short-term memory but also long-term memory. Please refer to Figure 10.

[0322] In addition, the long-term memory of patients with Alzheimer's disease deteriorates steadily. NA-831 improves not only short-term memory but also long-term memory. Please refer to Figure 11.

[0323] Patients with Alzheimer's disease often lose their sense of direction even at home in the early stages of the disease. NA-831 improves the sense of direction and restores the sense of time and place. Please refer to Figure 12.

[0324] As the disease progresses, patients with Alzheimer's disease become unable to take care of themselves. This drug improves the patient's daily living activities and self-care ability. Please refer to Figure 13.

[0325] The Mini-Mental State Examination (MMSE) is a 30-item questionnaire widely used in clinical and research settings to measure cognitive impairment. It was shown that the MMSE improved significantly from an average of 23.5 (mild impairment) on the first day to an average of 29.75 (normal) on the 84th day, indicating the impact of NA-831 on emotions. Please refer to Figure 14.

[0326] NA-831 has been shown to be highly effective in patients with mild cognitive impairment. Significant improvement was observed in 92.5% of all patients, while little improvement was observed in 7.5% of the patients. Please refer to Figure 15.

[0327] Conclusion Cyclic prolylglycine has been shown to reduce or prevent glutamate-induced neurotoxicity, have a neuroprotective effect, and be able to suppress neuronal degeneration or cell death.

[0328] Experiment 13 Assay for examining the relationship between cPG and NR2B-subtype receptor in animals after intravenous administration In this experiment, male CD-1 mice (n = 12) at 8 - 10 weeks of age were intravenously administered with a vehicle consisting of 10% dimethylacetamide, 40% PEG - 400, 30% hydroxypropyl - betacyclodextrin, and 30% water with cyclic prolylglycine (0.10 mg / ml) added. Fifteen minutes after administration, the forebrain was collected by decapitation. Brain samples were immediately frozen and stored at - 80°C.

[0329] The next day, the administered brain samples were thawed on ice for 20 - 30 minutes, and then homogenized for 10 seconds using a Polytron in a cold homogenization buffer consisting of 50 mM KH2PO4 (pH adjusted to 7.4 with KOH), 1 mM EDTA, 0.005% Triton X - 100, and protease inhibitor cocktail (Fischer Scientific). The crude homogenate was further homogenized using a Dounce homogenizer (Thomas Scientific), and the homogenized membrane aliquots from whole animals were flash - frozen and stored at - 80°C until further use. The entire homogenization process was carried out on ice.

[0330] To determine the occupancy, the membrane homogenate was thawed on ice and needle - homogenized using a 25 - gauge needle. The homogenized membrane (6.4 mg / ml) was added to a 96 - well plate, and then 3 H Ro25 - 6981 (6 nM) was added. The reaction mixture was incubated on a shaker at 4°C for 5 minutes and then collected onto a GF / B fractionation plate (treated with 0.5% PEI at room temperature for 1 hour). The partitioning plate was dried at 50°C for 30 minutes, incubated with MicroScint 20 for 15 minutes, and read using a tabletop microplate scintillation and luminescence counter (TopCount NXT model from PerkinElmer). Each dosing group or compound group consisted of 4 animals. Animals in the control group were administered with vehicle only. Membranes from each animal were added to the assay plate in three aliquots. Nonspecific binding was determined by adding 10 μM Ro25 - 6981 to wells containing membrane homogenate from vehicle - administered animals.

[0331] Using the following formula, the specific count per minute was converted to the occupancy rate at each dose of the compound for each animal: [Number]

[0332] Using this method, the cPG compound showed a 93% occupancy rate of the NR2B receptor at an intravenous dose of 3 mg / Kg. The drug concentration was measured by mass spectrometry. As a result, the drug concentration in plasma was 1074 nM, and the drug concentration in brain tissue was 1632 nM.

[0333] As a result, in animals after intravenous administration, it was revealed that cPG occupied the resident-type NR2B subtype receptor in the brain, and that this compound and its analogs were pharmacologically effective.

[0334] Experiment 14: Mouse forced swimming test (mFST) The forced swim test (FST) is an animal model used to evaluate antidepressant compounds in preclinical trials. The FST was performed with modifications similar to the method of Porsolt et al. (Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 1977; 229:327-36).

[0335] In this experiment, mice were forced to swim in an inescapable cylinder filled with water. Under this condition, mice initially try to escape and eventually adopt an immobile behavior, which is interpreted as a passive stress coping strategy or a behavior similar to a depressive state. A swim tank was placed inside a plastic box. Each water tank was partitioned by an opaque plastic sheet up to the height of the cylinder. Three mice were tested at a time. Mice were placed in a glass cylinder (height 46 cm × diameter 20 cm) filled with water (maintained at a water depth of 20 cm and a temperature of 24 - 25 °C) and allowed to swim for 6 minutes. At this water level, the mice's tails do not touch the bottom of the container. Immobility was determined when the mouse floated passively without struggling in the water and made only the movements necessary to keep its nose and head above the water surface. The duration of immobility was evaluated during the total 6 minutes of the test and expressed as the duration of immobility (sec). Each mouse was tested only once. At the end of each session, the mice were dried with a dry cloth and returned to their home cages placed on a warming blanket to prevent hypothermia. The water was changed after each test.

[0336] The results obtained in the FST were shown as the arithmetic mean value (given in seconds) of the immobility time of the animals in each experimental group ± the standard error of the mean (SEM).

[0337] To avoid the risk of false positives and false negatives in the FST due to the potential effect of the test drug on motor activity, spontaneous motor activity was measured using an animal activity meter, OptoVarimex-4 AutoTrack (Columbus Instruments, USA). This device consists of a set of 4 transparent cages with lids (43 × 43 × 32 cm), 4 infrared emitters (each emitter has 16 laser beams), and 4 detectors for monitoring the movement of the animals. Each mouse was placed in a cage for 10 minutes. The amount of spontaneous movement was evaluated from the 2 minute corresponding to the time interval analyzed in the FST 6 to the

[0338] Furthermore, all test sessions were recorded with a video camera (Sony Handicam, Model: DCR-HC38E; PAL), and scoring was performed using Forced Swim Scan, Version 2.0 software (Clever Systems Inc., Reston, Va., USA; see Hayashi E, Shimamura M, Kuratani K, Kinoshita M, Hara H. Automated experimental system capturing three behavioral components during murine forced swim test. Life Sci. 2011 Feb. 28; 88(9-10):41 1-7; and Yuan P, Tragon T, Xia M, Leclair C A, Skoumbourdis A P, Zheng W, Thomas C J, Ffuang R, Austin C P, Chen G, Guitart X. Phosphodiesterase 4 inhibitors enhance sexual pleasure-seeking activity in rodents. Pharmacol Biochem Behav. 2011; 98(3):349-55).

[0339] Thirty minutes before swimming, 12 mice were intravenously administered cyclopropylglycine (concentration 0.10 mg / ml), and the immobility time for 6 minutes was recorded. After the FST was completed, the mice were euthanized by rapid decapitation, and plasma and brain samples were collected and stored at -80°C. In the mouse forced swimming test, the cPG compound was intravenously administered at a dose of 5 mL / kg in a physiological saline (0.90% sodium chloride) vehicle. The compound showed a statistically significant decrease in immobility time at 1 mg / kg under these conditions. The drug concentration at this dose was 237+ / -128 nM in plasma and 632+ / -173 nM in the brain. The NR2B receptor occupancy was determined as reported above and was determined to be 73%. The cPG analog showed a statistically significant decrease in immobility time at 1 mg / kg under these same conditions. The drug concentration was 215 nM in plasma. The NR2B receptor occupancy was determined to be 68%.

[0340] As a result, it was found that the cyclic prolylglycine (NA-831) compound exhibits an antidepressant effect.

[0341] Experiment 15 Clinical trial: Double-blind, randomized, placebo-controlled, active reference trial of cyclic prolylglycine (NA-831) for major depressive disorder patients Introduction Cyclic prolylglycine (NA-831) is a novel compound under development as an antidepressant. From the data of preclinical trials, these affinities are clinically useful and are thought to be involved in the mechanism of action at therapeutic doses. NA-831 increases the extracellular levels of serotonin (5-HT), noradrenaline, dopamine, acetylcholine, and histamine in the prefrontal cortex and hippocampus of rats, and increases the extracellular levels of serotonin (5-HT), noradrenaline, dopamine, acetylcholine, and histamine in the prefrontal cortex and hippocampus of rats. The purpose of this clinical trial was to examine the efficacy, safety, and tolerability of two fixed doses of NA-831 (20 mg / d and 40 mg / d) and placebo after 6 weeks of administration in adult patients with major depressive disorder (MDD). Venlafaxine XR was used as the active comparator.

[0342] Methods In this randomized double-blind, fixed-dose, placebo-controlled, active reference trial, 32 randomized patients were recruited in accordance with the principles of Good Clinical Practice [ICH (1996) Harmonized Tripartite Guideline E6: Guidelines for Good Clinical Practice (http: / / www.fda.gov / downloads / drugs / guidancecomplianceregulatoryinformation / guidances / ucm073122.pdf)] and the Declaration of Helsinki [WMA (1964). Ethical Principles for Medical Research Involving Human Subjects (http: / / www.wma.net / en / 30publications / 10policies / b3 / ) World Medical Association]. Local ethics committees approved the study design, and eligible patients provided written informed consent prior to participation.

[0343] Eligible patients were randomly assigned equally (1:1:1:1:1) to one of four treatment groups during a 6-week double-blind treatment period. Randomized patients were given a one-week wallet card at each visit and instructed to take 2 capsules daily at the same time each day (preferably in the morning). NA-831 was administered at 20 mg / day or 40 mg / day for 6 weeks, and venlafaxine was administered at 75 mg / day for 6 weeks. Efficacy and tolerability were evaluated at screening, baseline, and after 1, 2, 3, 4, 5, and 6 weeks. Patients were contacted for safety follow-up 4 weeks after the end of dosing.

[0344] Main inclusion criteria Patients with MDD presenting a current major depressive episode according to the DSM-IV-TR criteria were outpatients of either sex, aged 20 to 65 years (mean = 39.7 ± 8.5) (if the total score on the Montgomery-Åsberg Depression Rating Scale (MADRS) (Montgomery & Åsberg, 1979) was ≥ 30 at the baseline visit, they were included in the study [Montgomery S Asberg M (1979). A new depression scale designed to be sensitive to change. British Journal of Psychiatry 134, 382-389. https: / / doi.org / 10.1192 / bjp.134.4.382].

[0345] Patients were evaluated using the Mini International Neuropsychiatric Interview [Sheehan DV Lecrubier Y Sheenan KH Amorim P et al. (1998). The Mini-International Neuropsychiatric Interview (M.I.N.I.). Journal of Clinical Psychiatry 59 (Suppl. 20), 22-33, quiz 34-57] to exclude those with current mental disorders other than MDD as defined in the Diagnostic and Statistical Manual of Mental Disorders, 4 th Edition (DSM-IV-TR), or a current or past history of bipolar or hypomanic episodes, schizophrenia or other psychotic disorders including major depressive disorder with psychotic features, mental retardation, organic mental disorders, or mental disorders due to a general medical condition, substance use disorder within the past 6 months, the presence or history of clinically significant neurological disorders (including epilepsy), neurodegenerative diseases, or the presence or history of an Axis II disorder that might interfere with the study.

[0346] In addition, patients with a high risk of suicide based on the clinical judgment of the researcher, patients with a score of ≥ 5 on the 10-item (suicidal ideation) of the MADRS scale, patients receiving formal behavioral therapy or systematic psychotherapy, pregnant or lactating patients, those known to be allergic, unresponsive to venlafaxine, or whose current depressive symptoms were judged by the researcher to be resistant to two appropriate antidepressant treatments at least twice within 6 weeks, or those who had been previously exposed to NA-831 were also excluded.

[0347] In addition, those who had taken the following psychotropic drugs within 2 weeks before baseline or during the test period were also excluded. Reversible or irreversible monoamine oxidase inhibitors, SSRI (fluoxetine within 5 weeks), SNRI, tricyclic antidepressants, psychotropic traditional Chinese medicines, drugs used to enhance antidepressant effects or other antidepressants, oral antipsychotics and mood stabilizers, or dopamine agonists, any anxiolytics (including benzodiazepines). And any anticonvulsants, serotonin agonists, narcotic analgesics or cough suppressants, antiarrhythmics, oral anticoagulants, proton pump inhibitors, steroids, cisapride, macrolide antibiotics, antifungal drugs, antihypertensive drugs, all anti-inflammatory drugs, anti-migraine drugs, pseudoephedrine, hypolipidemic drugs, and episodic use of insulin. Occasional use of zolpidem, zopiclone, zaleplon for insomnia was permitted.

[0348] If a patient became pregnant during the trial, if the principal investigator judged that it was in the best interest of the patient from the perspective of safety / efficacy, if the clinical test values were outside the normal range and clinically significant, if a serious risk of suicide was judged, if the score on item 10 (suicidal ideation) of the MADRS was 0, if the randomization code of the patient was broken, if the consent to participate was withdrawn, if the test drug was not taken continuously for more than 6 days, or if the patient dropped out of the follow-up survey, the patient was withdrawn from the trial. If a serious adverse event (SAE) occurred, the patient might be withdrawn from the trial. If an adverse event (AE) contributed to the discontinuation of the study, it was always regarded as the main reason for the discontinuation of the study.

[0349] Efficacy evaluation Patients were evaluated using the MADRS from baseline to week 6. Rater training was conducted to enhance inter-rater reliability and was supervised by experienced principal investigators of the clinical trial. Only principal investigators of the clinical trial who actively participated in rater training before enrolling patients in the study were able to evaluate the patients. Patient evaluations were performed by the same investigator at each visit whenever possible.

[0350] Allocation to treatment The drug was administered as capsules of the same appearance. Patients who met the selection criteria at the baseline visit were assigned to double-blind treatment according to a computer-generated randomization list. Details of the randomization list were unknown to the researchers and were contained in sealed opaque envelopes. At each study site, sequentially enrolled patients were assigned the lowest available randomization number from among four blocks. All researchers, study personnel, and participants were blinded to treatment assignment throughout the study period.

[0351] Analysis set All safety analyses were performed based on the all-patient treated set (APTS) of randomized patients who had taken the investigational drug at least once. All efficacy analyses were performed based on the modified intent-to-treat set (ITT), i.e., the full analysis set (FAS), which consisted of all patients included in the APTS who had received at least one valid MADRS total score assessment after baseline.

[0352] Primary efficacy analysis Four hypotheses were included in the primary efficacy analysis, and multiplicity was fully adjusted using a hierarchical test method with a significance level of 5% as long as the previous hypothesis was rejected. The order of the tests was that there was no difference between the 20 mg dose and placebo at week 6, no difference between the 40 mg dose and placebo at week 6, no difference between the 20 mg dose and placebo at week 1, and no difference between the 40 mg dose and placebo at week 1. The statistical model was an analysis of covariance (ANCOVA) of the change from baseline in the MADRS total score (FAS, LOCF) with treatment and site as fixed factors and the baseline MADRS score as a covariate. The primary efficacy analysis was repeated for the observed case (OC) data using both ANCOVA and MMRM (mixed model for repeated measures).

[0353] Evaluation of the tolerance range All adverse events (AEs) (including changes in concomitant diseases and new diseases) observed by the principal investigator of the clinical trial or spontaneously reported by the patients were recorded. The AEs were coded using the lowest level of terms compliant with the Medical Dictionary for Regulatory Activities (Medical Dictionary for Regulatory Activities version 10.0). As a post hoc analysis, the safety database was searched at the preferred term and verbatim term levels for AEs potentially related to suicide as described by the FDA [Laughren T (2006) Memorandum on suicidality (http: / / www.fda.gov / ohrms / dockets / ac / 06 / briefing / 2006-4272b1-01-fda.pdf)].

[0354] Results Patient baseline characteristics The APTS consisted of 32 patients (placebo, 8; venlafaxine, 8; 20 mg NA-831, 8; 40 mg NA-831, 8). No clinically relevant or statistically significant differences were observed among treatment groups in baseline patient demographics or clinical characteristics (Table 3). The mean age (±S.D.) of the patients was 39.7 ± 8.5 years, and 59.4% were female. The mean baseline MADRS total score was 34.0, indicating a group of patients with severe depression, and was consistent with a mean CGI-S score of 5.1. Patients were diagnosed with their first MDE approximately 10 years prior to enrollment. Between 74% and 80% of patients in each treatment group had previously had an MDE, and the current episode had started approximately 5 months prior to enrollment.

[0355]

Table 3

[0356] Withdrawal from the study Only 4 patients discontinued the study for various reasons: 1 in the placebo group, 1 in the 40 mg NA-831 group, and 2 in the venlafaxine group. Over 87% of patients completed the study.

[0357] Efficacy In the efficacy evaluation, which is a predefined primary evaluation item, both doses of NA-831 were statistically significantly superior (p<0.0001) to placebo in the mean change rate from baseline in the MADRS total score at week 6 (FAS, LOCF) in the multiple-treatment comparison analysis. The mean treatment differences from placebo were 7.7 points (20 mg) and 8.5 points (40 mg) (Table 4). Also, venlafaxine was statistically significantly superior (p<0.0001) to placebo at week 6, and the mean treatment difference from placebo was 7.2 points (LOCF). The estimated treatment differences and nominal p-values at week 6 obtained from the analysis using MMRM were the same as those obtained from the ANCOVA analysis: 5.7±1.3 (20 mg NA-831), 7.8±1.3 (40 mg NA-831), 5.6±1.3 (venlafaxine), all with p<0.0001 (Table 2).

[0358]

Table 4

[0359] Acceptability and safety During the 6-week treatment period, AEs were observed in approximately 60% of the patients in the placebo group and 75% of the patients in the venlafaxine group. Only 12.5% of the patients in the 20 mg NA-831 group and 12.5% of the patients in the 40 mg NA-83 group developed AEs.

[0360] A total of 10 patients (33.3%) withdrew due to AEs, with 1 patient (10%) in the placebo group, 1 patient (12.5%) in the 20 mg NA-831 group, and 2 patients (25%) in the venlafaxine group. There were no withdrawals.

[0361] The most common AEs reported in the active NA-831 treatment group were mild headache and dry mouth. In the venlafaxine treatment group, the majority of patients reported nausea, severe headache, loss of strength, blurred vision, chest pain, rapid and irregular heartbeat, suicidal thoughts, etc.

[0362] Conclusion The objective of this trial was to evaluate the efficacy, safety, and tolerability of NA-831 in patients with MDD. For the purpose of verifying the validity of the trial method and patient population, the active reference drug venlafaxine XR was included, and efficacy was recognized in the primary efficacy analysis. Both doses of NA-831 showed significant improvement compared to placebo in the primary efficacy analysis.

[0363] The difference between active treatment and placebo on the MADRS is a clinically meaningful difference in response rate of 32.5% units compared to the average of 16% units of antidepressants approved by the European authorities [Melander H, Salmonson T, Abadie E, van Zwieten-Boot B (2008). A regulatory apologia - A review of placebo-controlled studies in regulatory submissions of new-generation antidepressants. European Neuropsychopharmacology 18, 623-627. https: / / doi.Org / l0.l016 / j.euroneuro.2008.06.003].

[0364] In conclusion, the 6-week administration of NA-831 in this trial was highly tolerable and effective in reducing depressive and anxiety symptoms in patients with MDD.

[0365] The present invention is described with reference to its specific embodiments. Other features and other embodiments of the present invention can be made by those skilled in the art without undue experimentation and without a reasonable possibility of success. All of those and other embodiments are considered to be part of the present invention.

[0366] Advantages of the present invention Some of the advantages provided by the present invention using cyclic peptides, particularly cyclic peptides exceeding IGF-I, include the following.

[0367] The active ingredient is easy to synthesize both in vitro and by other means such as recombinant technology.

[0368] Since the peptide is of low molecular weight, it can easily diffuse in the body and between compartments (blood-brain barrier, mucosa, etc.), improving the choice of administration method and the accessibility to the injured site.

[0369] cPG, c(PG)3, and cGMeP are very stable molecules and are less likely to pose problems to the immune system, so they may be administered for a long time or prophylactically.

[0370] The present invention provides a novel treatment method that prevents brain disorders and degenerative diseases and brings about a long-term brain recovery effect by regulating mGluRs, particularly 2 / 3.

[0371] cPG, which plays a role in regulating IGF-1 induction, provides additional neuroprotection with less potential for growth side effects.

[0372] Conclusion Cyclic prolylglycine has been shown to reduce or prevent glutamate-induced neurotoxicity, have a neuroprotective effect, and suppress the degeneration or cell death of nerve cells.

[0373] The present invention is described with reference to its specific embodiments. Other features and other embodiments of the present invention can be manufactured by those skilled in the art without undue experimentation and with a reasonable possibility of success. All of those and other embodiments are considered to be part of the present invention.

[0374] Although various embodiments of the present invention have been described above, it should be understood that they are presented by way of example only and not by way of limitation. Those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the present invention as defined in the appended claims. Accordingly, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined in accordance with the following claims and their equivalents.

[0375] All publications, including patent documents and scientific papers, referred to in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference separately. All headings are for the convenience of the reader and should not be used to limit the meaning of the text following the heading unless specifically designated.

Claims

1. A pharmaceutical composition for use in a method of alleviating or reducing mild cognitive impairment by regenerating neurons lost as a result of damage due to injury or disease, the composition comprising cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, wherein the method comprises: a) providing a subject having mild cognitive impairment and in need of regeneration of said neurons; and b) administering to the subject an effective amount of cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof to regenerate new neurons; comprising, wherein the subject has mild cognitive impairment, in the subject, the neurons are regenerated, the cPG compound functions as a neurogenic agent in the central nervous system, and in the subject, neurons lost as a result of damage due to injury or disease are regenerated, thereby alleviating or reducing the mild cognitive impairment of the subject, said pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, comprising a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 1, wherein the effective amount of the cPG compound is from about 1 μg to about 100 mg per kg of body weight.

4. The pharmaceutical composition according to claim 1, wherein the administration is in combination with artificial cerebrospinal fluid.

5. The pharmaceutical composition according to claim 1, wherein the administration is intravenous administration.

6. The pharmaceutical composition according to claim 1, wherein the administration is combined with a neuroprotective agent, insulin-like growth factor-I (IGF-I) or insulin-like growth factor-II (IGF-II).

7. The pharmaceutical composition according to claim 1, wherein the administration is combined with an anti-inflammatory agent, an anti-integrin α4 subunit reagent.

8. A pharmaceutical composition for use in a method of reducing or alleviating mild cognitive impairment caused by a required mammalian disease, injury, or condition, comprising cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, wherein the method comprises a) providing a mammal in need of reducing or alleviating mild cognitive impairment caused by a disease, injury, or condition; b) administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof; comprising wherein the disease is selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, mixed vascular dementia, degenerative dementia, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration, wherein the injury is selected from the group consisting of neurotoxic injury, cerebral hypoxia / ischemia, traumatic brain injury, coronary artery bypass surgery, and the condition is selected from the group consisting of normal aging, age-related memory loss, memory impairment, cholinergic hypofunction, intracranial vascular stenosis or occlusion, neuroinflammation, mild cognitive impairment, brain atrophy, frontotemporal lobe degeneration, Pick's disease, HIV infection, Down's syndrome, and loss of synaptic plasticity, wherein the mammal has the mild cognitive impairment caused by a disease, injury or condition including Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, mixed vascular dementia, degenerative dementia, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration alleviated or reduced by the pharmaceutical composition. **Claim 9** The cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, is the pharmaceutical composition according to claim 8, which comprises an aqueous solution and one or more pharmaceutically acceptable excipients, additives, carriers or adjuvants.

10. The cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, further comprises one or more excipients, carriers, additives, adjuvants or binders in tablets or capsules, and is the pharmaceutical composition according to claim 8.

11. The disorder is mild cognitive impairment, Alzheimer's disease, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, mixed vascular dementia, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration, and is the pharmaceutical composition according to claim 8.

12. The cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, is administered by an administration route of oral, intraperitoneal, intravascular, peripheral circulation, subcutaneous, intraorbital, intraocular, intrathecal, intravesical, topical, infusion, implantation, aerosol, inhalation, scarring, intracapsular, intramuscular, nasal, buccal, transdermal, pulmonary, rectal, vaginal, or a combination thereof, and is the pharmaceutical composition according to claim 8.

13. The pharmaceutically effective amount has a lower limit of about 0.001 milligram (mg / kg) per kilogram mass of the mammal and an upper limit of about 100 mg / kg of the mammal, and is the pharmaceutical composition according to claim 8.

14. The mild cognitive impairment is caused by cholinergic hypofunction, and is the pharmaceutical composition according to claim 8.

15. The cholinergic hypofunction is caused by scopolamine, and is the pharmaceutical composition according to claim 8.

16. The pharmaceutical composition according to claim 8, wherein the mild cognitive impairment is caused by a decrease in glutamate receptors in the granule cell layer (CA1) of the hippocampus of the mammal.

17. The pharmaceutical composition according to claim 8, wherein the cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof causes an increase in AMPA receptors in the granule cell layer (CA1) of the hippocampus of the mammal.

18. The pharmaceutical composition according to claim 8, wherein the cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof increases the plasticity of nerve cells caused by the cPG compound in the granule cell layer (CA1) and the pyramidal cell layer (CA3) regions of the hippocampus of the mammal.

19. The pharmaceutical composition according to claim 8, wherein the cerebral hypoxia / ischemia is caused by traumatic brain injury.

20. The pharmaceutical composition according to claim 8, wherein the mild cognitive impairment is caused by multi-infarct dementia.

21. The pharmaceutical composition according to claim 8, wherein the mild cognitive impairment is caused by coronary artery bypass grafting (CABG).

22. A pharmaceutical composition comprising cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, for use in a method of preventing symptoms of mild cognitive impairment resulting from or associated with a disease, injury or condition in a mammal in need thereof, wherein the method comprises a. providing a mammal in need of prevention of mild cognitive impairment resulting from a disease, injury or condition; b. Administering to the mammal a pharmaceutically effective amount of cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof; comprising said disease being selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, mixed vascular dementia, degenerative dementia, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration, said pharmaceutical composition. [

23. ] A pharmaceutical composition comprising cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, for use in a method of alleviating or reducing mild cognitive impairment by increasing the growth or synaptogenesis of nerve cells in a mammal resulting from or associated with a disease, injury, or condition in the mammal in need thereof, wherein said mammal has mild cognitive impairment, wherein said method comprises a. Providing a mammal having mild cognitive impairment and in need of an increase in nerve cell growth or synaptogenesis; b. Administering to the mammal an effective amount of a composition comprising a pharmaceutically effective amount of cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof; comprising said disease being selected from the group consisting of Alzheimer's disease, Huntington's disease, Lewy body disease, dementia, and multi-infarct dementia, memory loss, attention deficit symptoms associated with Alzheimer's disease, neurodegeneration associated with Alzheimer's disease, mixed vascular dementia, dementia of degenerative origin, pre-senile dementia, senile dementia, dementia associated with Parkinson's disease, progressive supranuclear palsy or corticobasal degeneration, Here, the growth or synaptogenesis of nerve cells in the mammal is increased, thereby alleviating or reducing the mild cognitive impairment of the mammal. The pharmaceutical composition.

24. For use in a method of modulating neurogenesis in the nerve tissue of a patient having mild cognitive impairment exhibiting at least one symptom of a central nervous system disorder that is a neurodegenerative disorder, an ischemic disorder, a nerve injury, a learning and memory disorder, or a combination thereof, by administering cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline, or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, a pharmaceutical composition comprising cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof, wherein the agent modulates neurogenesis in the patient, thereby modulating neurogenesis in the patient's nerve tissue and thereby reducing or alleviating the patient's mild cognitive impairment. The pharmaceutical composition.

25. The pharmaceutical composition according to claim 24, wherein the nervous system disorder is Alzheimer's disease, Parkinson's disease and Parkinsonian disorders, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body disease, spinal ischemia, ischemic stroke, cerebral infarction, spinal cord injury, cancer-related brain and spinal cord injury, multi-institutional dementia, senile dementia, mild cognitive impairment, depression, traumatic injury.

26. The pharmaceutical composition according to claim 24, wherein the modulation of neurogenesis is effected by activation of a GPCR receptor in the nerve tissue.

27. The pharmaceutical composition according to claim 24, wherein the agent is administered in an amount of from about 0.1 mg to about 10 mg / kg / day, from about 0.5 mg to about 20 mg / kg / day, from about 0.2 mg to about 40 mg / kg / day, from about 5 mg to about 50 mg / kg / day, or from about 10 mg to about 100 mg / kg / day of cyclic prolylglycine (cPG), or cyclic (tri(prolylglycine)), or cyclic glycyl-2-allylproline, or cyclic glycyl-alkylproline or cyclic glycyl-2-methylproline (cPMeG), or a combination thereof.

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