Lectin proteins for the treatment and prevention of neurodegenerative diseases

Abstract

The present invention relates to a lectin protein for the treatment and prevention of neurodegenerative diseases.The present invention further relates to a recombinant lectin protein derived from the lectin of Sclerotium rolfsii, having a sequence with 60% homology to SEQ ID NO: 4, for the treatment and prevention of neurodegenerative diseases.The present invention particularly relates to a lectin protein derived from the lectin of Sclerotium rolfsii, having a sequence with 60% homology to SEQ ID NO: 4, and variants thereof, for the treatment or prevention of Parkinson's disease, Alzheimer's disease, dementia and dementia symptoms.

Description

Technical Field

[0001] The present invention relates to proteins for the treatment and prevention of neurodegenerative diseases.

Background Art

[0002] Neurodegenerative diseases are conditions that cause progressive and irreversible damage to the nervous system. As a result, neurodegenerative diseases lead to either ataxia or dementia. The pathogenesis of neurodegenerative diseases is characterized by extracellular and intercellular deposition of amyloid-beta protein (Aβ) peptides and hyperphosphorylation of tau protein, which result in plaques and neurofibrillary changes, respectively, leading to oxidative stress and causing neuroinflammation; post-translational modifications of α-synuclein, such as phosphorylation, ubiquitination, and nitration, are widely involved in the α-synuclein aggregation process that leads to Lewy body formation and dopamine neuron death. In addition, any abnormality in any one of the following pathways causes neurodegenerative diseases: intracellular mechanisms (e.g., apoptosis, autophagy, mitochondrial dysfunction, oxidative DNA damage and repair, ubiquitin-proteasome system); local tissue environment (e.g., cell adhesion, endocytosis, neurotransmission, prions, and transmissible factors); systemic environment (inflammation and immune dysfunction, lipids, metabolic endocrine factors, vascular changes) and development and aging (e.g., epigenetic changes, neurotrophic factors, telomeres), etc.

[0003] Dementia is defined as cognitive impairment in one or more cognitive domains, characterized by a loss of intelligence of a degree sufficient to impair occupational functioning, normal social activities, or interpersonal relationships, either without significant confusion of consciousness or with motor impairment (Sharma and Singh et al., 2010 Indian Journal of Pharmacology, Vol. 42, No. 3; pp. 164-167). Cognitive domains involved in dementia include language (aphasia), motor (apraxia), agnosia (cognitive impairment), and executive function (abstract reasoning, judgment, and planning) (Parris M. Kidd; Alternative Medicine Review, 2008, Vol. 13, No. 2, pp. 85-115, 31p). There are many types of dementia, including Alzheimer's disease, vascular dementia and dementia with Lewy bodies, frontotemporal dementia and Parkinson's disease, dementia associated with Huntington's disease, hydrocephalus, dementia due to Wernicke-Korsakoff syndrome, and Creutzfeldt-Jakob disease dementia (Husband, A. and Worsley, A 2006, The Pharmaceutical Journal, 277(7426), pp. 579-582).

[0004] Aging societies face a significant increase in the incidence of age-related neurodegenerative diseases (Rasalan and Kee 2013; Genes & Genomics, Vol. 35, pp. 425-440). Therapeutic and non-therapeutic approaches such as lifestyle changes or interventions, or appropriate diet (Gurjit et al., 2019 Front Aging Neurosci. 2019;11:369) are available recourses for the treatment of neurodegenerative diseases. Therapeutic approaches commonly used and approved for the treatment or prevention of neurodegenerative diseases include cholinesterase inhibitors (galantamine, donepezil, rivastigmine), memantine, istradefylline, dopamine agonists (pramipexole, apomorphine), levodopa / carbidopa, monoclonal antibodies such as daclizumab, natalizumab, alemtuzumab, and immunomodulators such as teriflunomide. These medications can cause severe side effects, such as convulsions, nausea, dizziness, bradycardia, falls, and even death.

[0005] Therefore, there is an urgent need to develop new, potent therapeutic agents that possess essential properties to alleviate learning and memory impairments, restore normal expression levels of nerve growth factor (NGF) and acetylcholinesterase (AChE), and exert protective effects on the brain against diseases such as Alzheimer's disease and Parkinson's disease.

[0006] Lectins are carbohydrate-binding proteins that are ubiquitous in plants, animals, and microorganisms. Their aggregation properties have enhanced their applications in cutting-edge medical research. The therapeutic potential of lectins in neurodegenerative diseases is also well-studied.

[0007] U.S. Patent Application No. 20200017578 discloses the use of sialic acid-binding specific lectins, such as aglutinin I from the yellow slug (Limax flavus) (LFA), aglutinin from the horseshoe crab (Limulus polyphemus) (LPA), aglutinin from Paecilomyes japonica (PJA), aglutinin I from lobster, or lectins from the prawn (Penaeus monodin), for the treatment of neurodegenerative disorders such as Alzheimer's disease.

[0008] U.S. Patent No. 8916387 describes a method for the prevention, treatment, and diagnosis of Alzheimer's disease based on the glycosylation pattern of amyloid-beta peptides in body fluids and tissues. Lectins derived from mistletoe, Japanese pagoda tree (Maackia amurensis), and willow mushroom (Agrocybe cylindracea) are disclosed as useful pharmaceutical or diagnostic agents for the prevention and treatment of cortical atrophy, neuronal loss, region-specific amyloid deposition, senile plaques, and neurofibrillary tangles. Lectins are further disclosed to be used to treat or prevent diseases involving amyloid-beta protein plaque deposition, where the diseases are selected from the group consisting of cerebral amyloid angiopathy and Alzheimer's disease or HIV-related neurocognitive disorders.

[0009] Tetranectin is a human homotrimeric 21KD protein belonging to the C-type lectin family. Tetranectin levels in cerebrospinal fluid are dramatically reduced in Parkinson's disease patients compared to normal controls, and tetranectin has been found to act as a neuroprotective agent by inhibiting apoptosis and autophagy in 1-methyl-4-phenylpyridine-induced neurotoxicity (Qiang Xie et al., 2018 World Neurosurgery, Vol. 122, pp. e375-e382).

[0010] The treatment of neurodegenerative diseases using synthetic drugs or peptides is well-established by conventional techniques, and lectins are used as cell surface binding agents or delivery agents. There is very limited information regarding the use of lectins as therapeutic agents in the treatment and prevention of neurodegenerative diseases. In most of the above studies, the following lectins were explored for the treatment or prevention of neurological diseases: plant lectins, e.g., Japanese pagoda tree; animal lectins, e.g., prawn-yellow slug (garden slug); horseshoe crab; and fungal lectins, willow mushroom.

[0011] The use of several commensal lectins as conjugates or delivery agents for active pharmaceutical agents is known and has been reported for the treatment of neurodegenerative diseases (e.g., US20070243132). However, the efficacy of lectins as therapeutic agents in the treatment or prevention of neurodegenerative diseases has not been studied in detail. Therefore, there is a need to search for and identify potent lectins that alleviate cognitive impairment, restore the expression levels of neurotrophic factors and cholinesterases, and provide high therapeutic efficacy against neurodegenerative diseases.

[0012] Sclerotium rolfsii lectin (SRL) is a lectin isolated from the sclerotial body of the soil-transmitting plant pathogenic fungus S. rolfsii. SRL is specific to the Thomsen-Friedenreich (TF) antigen and the Tn antigen. The TF antigen is a disaccharide (Galβ1→3GalNAc-α-Ser / Thr) overexpressed on the cell surface of various human cancer cells. The Tn antigen is a monosaccharide (GalNAc-α-Ser / Thr). WO2010 / 095143 discloses recombinant lectin mutants Rec-2 and Rec-3 derived from the native SRL sequence, with substitutions of 3 or 5 amino acids, respectively. The crystal structures of these mutants have been reported (Peppa et al., Molecules. June 12, 2015; 20(6): 10848~65). WO2014 / 203261 discloses recombinant lectin variants derived from the native SRL sequence by 12 amino acid substitutions. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] U.S. Patent Application No. 20200017578 [Patent Document 2] U.S. Patent No. 8916387 [Patent Document 3] US20070243132 [Patent Document 4] WO2010 / 095143 [Patent Document 5] WO2014 / 203261 [Patent Document 6] WO2020 / 044296 [Patent Document 7] WO / 2020 / 074977 [Patent Document 8] Indian Patent Application No. 201921027358 [Non-patent literature]

[0014] [Non-Patent Document 1] Sharma and Singh et al., 2010 Indian Journal of Pharmacology, Vol. 42, No. 3; pp. 164-167. [Non-Patent Document 2] Parris M. Kidd; Alternative Medicine Review. 2008, Vol. 13, No. 2, pp. 85-115. 31p [Non-Patent Document 3] Husband, A. and Worsley, A. 2006, The Pharmaceutical Journal, 277(7426), pp. 579-582. [Non-Patent Document 4] Rasalan and Kee 2013; Genes & Genomics, Vol. 35, pp. 425-440. [Non-Patent Document 5] Gurjit et al., 2019 Front Aging Neurosci.2019;11:369

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0015] The problem of the present invention is to develop a new method for the prevention and treatment of neurodegenerative diseases. The new method means a method including a new therapeutically effective agent for the prevention and treatment of neurodegenerative diseases. Therefore, it is one problem to establish the use of a new therapeutic agent in the method for the treatment and prevention of neurodegenerative diseases, and the new therapeutic agent is a recombinant lectin.

[0016] Another problem of the present invention is to provide a recombinant lectin for the treatment and prevention of neurodegenerative diseases. Therefore, the problem is to provide a recombinant lectin for the treatment and prevention of diseases causing dementia, and the problem is also to particularly provide a recombinant lectin for the treatment and prevention of Alzheimer's disease and Parkinson's disease.

[0017] Yet another problem of the present invention is to provide a composition containing a recombinant lectin for the treatment and prevention of neurodegenerative diseases. The use of a composition containing a recombinant lectin for the treatment and prevention of neurodegenerative diseases is also one problem of the present invention. [Means for solving the problem]

[0018] The present invention relates to recombinant lectin proteins derived from Sclerotium rolfsii lectins for the treatment or prevention of neurodegenerative diseases.

[0019] The present invention further relates to a pharmaceutical composition for the treatment or prevention of neurodegenerative diseases, comprising a therapeutically effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin and a pharmaceutically acceptable excipient.

[0020] The present invention also relates to a method for treating or preventing neurodegenerative diseases, comprising administering an effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin to a subject.

[0021] The present invention relates to the use of recombinant lectin proteins derived from Sclerotium rolfsii lectins for the treatment or prevention of neurodegenerative diseases.

[0022] In yet another aspect, the present invention relates to recombinant lectin proteins derived from Sclerotium rolfsii lectin for the treatment or prevention of neurodegenerative diseases in subjects, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment and symptoms associated with dementia.

[0023] In yet another aspect, the present invention relates to recombinant lectin proteins derived from Sclerotium rolfsey lectin for the treatment or prevention of neurodegenerative diseases in subjects, wherein the neurodegenerative disease is selected from Huntington's disease, prion diseases such as Creutzfeldt-Jakob disease, Lewy body disease, diffuse Lewy body disease (DLBD), polyglutamine (PolyQ) repeat disease, cerebral degenerative diseases, spinal and bulbar muscular atrophy (SBMA), ataxia, Pick's disease, primary progressive aphasia, multiple system atrophy, pantothenate kinase-associated neurodegeneration (PANK), spinal degenerative diseases / motor neuron degenerative diseases, hippocampal sclerosis, corticobasal degeneration, and Batten disease.

[0024] In yet another aspect, the present invention relates to recombinant lectin proteins derived from Sclerotium rolfsii lectins for the treatment or prevention of neurodegenerative diseases in subjects, wherein the neurodegenerative disease is selected from motor neuron disease-like amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy, and hereditary spastic paraplegia, which are variants of ALS.

[0025] In yet another aspect, the present invention provides a method for treating or preventing a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment and dementia-related symptoms.

[0026] In yet another aspect, the present invention provides a method for treating or preventing a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of recombinant lectin protein derived from Sclerotium rolfsey's lectin, wherein the neurodegenerative disease is selected from Huntington's disease, prion diseases such as Creutzfeldt-Jakob disease, Lewy body disease, diffuse Lewy body disease (DLBD), polyglutamine (PolyQ) repeat disease, cerebral degenerative diseases, spinal and bulbar muscular atrophy (SBMA), ataxia, Pick's disease, primary progressive aphasia, multiple system atrophy, pantothenate kinase-associated neurodegeneration (PANK), spinal degenerative diseases / motor neuron degenerative diseases, hippocampal sclerosis, corticobasal degeneration, and Batten disease.

[0027] In yet another aspect, the present invention provides a method for treating or preventing a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin, wherein the neurodegenerative disease is selected from motor neuron disease-like amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy and hereditary spastic paraplegia, which are variants of ALS.

[0028] According to another aspect of the present invention, a pharmaceutical composition for the treatment or prevention of a neurodegenerative disease is provided, comprising a therapeutically effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin and a pharmaceutically acceptable excipient, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment and symptoms associated with dementia.

[0029] In yet another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of a neurodegenerative disease, comprising a therapeutically effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin and a pharmaceutically acceptable excipient, wherein the neurodegenerative disease is selected from Huntington's disease, prion diseases such as Creutzfeldt-Jakob disease, Lewy body disease, diffuse Lewy body disease (DLBD), polyglutamine (PolyQ) repeat disease, cerebral degenerative diseases, spinal and bulbar muscular atrophy (SBMA), ataxia, Pick's disease, primary progressive aphasia, multiple system atrophy, pantothenate kinase-associated neurodegeneration (PANK), spinal degenerative diseases / motor neuron degenerative diseases, hippocampal sclerosis, corticobasal degeneration, and Batten disease.

[0030] In yet another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of a neurodegenerative disease, comprising a therapeutically effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin and a pharmaceutically acceptable excipient, wherein the neurodegenerative disease is selected from motor neuron disease-like amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy and hereditary spastic paraplegia, which are variants of ALS.

[0031] According to yet another aspect of the present invention, the use of recombinant lectin proteins derived from Sclerotium rolfsii lectins for the treatment or prevention of neurodegenerative diseases in subjects is provided, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment and symptoms associated with dementia.

[0032] In yet another embodiment, the use of recombinant lectin proteins derived from Sclerotium rolfsey lectins for the treatment or prevention of neurodegenerative diseases in subjects is provided, wherein the neurodegenerative disease is selected from Huntington's disease, prion diseases such as Creutzfeldt-Jakob disease, Lewy body disease, diffuse Lewy body disease (DLBD), polyglutamine (PolyQ) repeat disease, cerebral degenerative diseases, spinal and bulbar muscular atrophy (SBMA), ataxia, Pick's disease, primary progressive aphasia, multiple system atrophy, pantothenate kinase-associated neurodegeneration (PANK), spinal degenerative diseases / motor neuron degenerative diseases, hippocampal sclerosis, corticobasal degeneration, and Batten disease.

[0033] In yet another embodiment, the use of recombinant lectin proteins derived from Sclerotium rolfsey lectins for the treatment or prevention of neurodegenerative diseases in subjects is provided, wherein the neurodegenerative disease is selected from motor neuron disease-like amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy and hereditary spastic paraplegia, which are variants of ALS.

[0034] According to one aspect of the present invention, a method for inducing neuronal outgrowth is provided, comprising administering an effective amount of recombinant lectin protein derived from Sclerotium rolfsey lectin to a target.

[0035] According to the above-described aspects of the present invention, recombinant lectin protein is i) Sequence ID 4, or ii) An amino acid sequence having at least 70% identity with SEQ ID NO: 4 It contains an amino acid sequence selected from the following.

[0036] According to the above-described aspects of the present invention, the recombinant lectin protein contains an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with respect to SEQ ID NO: 4.

[0037] According to the above-described aspects of the present invention, the effective amount of recombinant lectin protein administered for the treatment or prevention of neurodegenerative diseases is within the range of 0.01 mg / kg to 1000 mg / kg of body weight.

[0038] According to one particular aspect of the present invention, a recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 is provided for the treatment or prevention of neurodegenerative diseases.

[0039] According to another specific aspect of the present invention, a method for treating or preventing a neurodegenerative disease is provided, comprising administering an effective amount of recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 to a subject.

[0040] According to yet another specific aspect of the present invention, a composition is provided for the treatment or prevention of neurodegenerative diseases in a subject, comprising a therapeutically effective amount of recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0041] According to another specific aspect of the present invention, the use of a recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 is provided for the treatment or prevention of neurodegenerative diseases in a subject.

[0042] According to yet another specific aspect of the present invention, the use of a composition comprising a therapeutically effective amount of recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 is provided for the treatment or prevention of neurodegenerative diseases in a subject.

[0043] In particular aspects of the present invention, recombinant lectin proteins having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 are provided for the treatment or prevention of neurodegenerative diseases in a subject, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, and symptoms associated with dementia.

[0044] In another particular aspect of the present invention, a method is provided for the treatment or prevention of a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, and symptoms associated with dementia.

[0045] In yet another particular aspect of the present invention, a pharmaceutical composition for the treatment or prevention of a neurodegenerative disease is provided, comprising a therapeutically effective amount of recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 and a pharmaceutically acceptable excipient, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, and symptoms associated with dementia.

[0046] In yet another specific aspect of the present invention, the use of recombinant lectin proteins having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 for the treatment or prevention of neurodegenerative diseases in a subject is provided, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, and symptoms associated with dementia.

[0047] One aspect of the present invention provides a method for inducing neuronal proliferation, comprising administering an effective amount of recombinant lectin protein having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0048] definition As used herein, the term "lectin" refers to a carbohydrate-binding protein.

[0049] As used herein, the term "protein" refers to a polymer of amino acid residues.

[0050] As used herein, the term “amino acid” refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and mimetic compounds that have similar functions to naturally occurring amino acids. Naturally occurring amino acids are those encoded in the genetic code and include those that make up proteins. Naturally occurring amino acids also include those that have been post-translationally modified in cells. Synthetic amino acids include non-standard amino acids, such as selenocysteine ​​and pyrrolidine. Typically, synthetic amino acids are not amino acids that make up proteins.

[0051] The term “nerve” or “neuron” refers to cells present in the brain, central nervous system, and peripheral nervous system, including, but not limited to, nerve cells, glial cells, oligodendrocytes, microglia, or neural stem cells.

[0052] The terms disease or disorder are used interchangeably unless otherwise specified. “Disease” is a health condition in an animal that is unable to maintain homeostasis, and if the disease does not improve, the animal’s health continues to deteriorate. “Disorder” in animals is a health condition in which the animal is able to maintain homeostasis, but the animal’s health condition is less desirable than that without disorder. The terms “disease” and “disorder” are used interchangeably and may also refer to any change in the condition of the body or any organ that interferes with or disrupts the performance of a function and / or causes symptoms such as discomfort, dysfunction, pain, or even death in the person suffering or in contact with the person suffering. Disease or disorder may also relate to distemper, ailing, ailment, disease, disorder, pathological condition, illness, complaint, inder-disposion, or affectation.

[0053] The term “neurodegenerative disease” encompasses both neurodegenerative diseases and disorders and is defined as a gradual and progressive loss of function or structure and / or function of nerve tissue. This leads to impaired nerve cell function and / or increased nerve cell death. Possible causes of this disease or disorder may include aging, genetic abnormalities, or exposure to toxins, chemicals, or viruses. Sometimes, the cause may be a medical condition such as alcoholism, tumors, or stroke. Typically, neurodegenerative diseases cause problems with physical activity, such as movement (known as ataxia), mental function (known as dementia), balance, speech, and breathing. In certain cases, this may also affect cardiac function. Neurodegenerative diseases according to the present invention include, but are not limited to, conditions in which neurons are dysfunctional and / or degenerated.Non-exclusive examples of such diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, dementia or more neurodegenerative diseases listed herein, symptoms of dementia, frontotemporal dementia (FTD), FTD caused by mutations in the progranulin protein or tau protein (e.g., progranulin deficiency FTLD), frontotemporal dementia with inclusion body myopathy (IBMPFD), frontotemporal dementia with motor neuron disease, and amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease). ), amyotrophic lateral sclerosis (ALSD) with dementia, primary lateral sclerosis (PLS), spinal muscular atrophy (SMA), multiple sclerosis (MS), prion diseases, such as Creutzfeldt-Jakob disease, Lewy body dementia, diffuse Lewy body disease (DLBD), polyglutamine (PolyQ) repeat disease, trinucleotide repeat disease, neurodegenerative diseases, presenile dementia, senile dementia, parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), progressive bulbar palsy (PBP), pseudobulbar palsy, spinal and bulbar muscular atrophy (SBMA), free tracheal palsy Ich's ataxia, cerebellar ataxia, Pick's disease, primary progressive aphasia, corticobasal dementia, HIV-related dementia, Parkinson's disease with dementia, dementia with Lewy bodies, multiple system atrophy, spinal muscular atrophy, e.g., Werdnig-Hoffmann disease, Kugelberg-Welander disease or congenital SMA with joint contractures, progressive bulbar spinal muscular atrophy, e.g., Kennedy disease, spinocerebellar ataxia, pantothenate kinase-associated neurodegeneration (PANK), spinal degenerative diseases / motor neuron degenerative diseases, upper motor neuron disorders, lower motor neuron disorders, Haller's disease Holden-Spats syndrome, amyotrophic lateral sclerosis-parkinsonism-dementia, parkinsonism-dementia in Guam, hippocampal sclerosis, corticobasal degeneration, Alexander disease, Alpers disease, Krabbe disease, neuroborreliosis, neurosyphilis, Sandhoff disease, Schilder disease, Batten disease, Cockaine syndrome, Kahns-Sayer syndrome, Gerstmann-Streusler-Scheinker syndrome, hereditary spastic paraplegia, Leigh syndrome, demyelinating diseases, other brain disorders such as bipolar disorder, epilepsy, schizophrenia, depression, mania, autism, ADHD, traumatic brain injury, and stroke.

[0054] The "neurodegenerative diseases" listed above are, in a very broad sense, "dementia" including, for example, Alzheimer's disease, frontotemporal dementia (Pick's disease), Lewy body dementia, neurofibrillary tangle dementia and related symptoms, Creutzfeldt-Jakob disease (which has clinical symptoms similar to Alzheimer's), hippocampal sclerosis, Schilder's disease, vascular (multiple cerebral infarct) dementia, Huntington's disease, Parkinson's disease related to AD, diffuse Lewy body disease (DLBD), and other diseases; "Parkinson's disease and parkinson's disease" It is well understood by those skilled in the art that "Kinsonian diseases" can be categorized as, for example, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), and corticobasal degeneration (CBD); and "motor neuron diseases" can include diseases affecting the upper / lower motor neuron regions, such as amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), and variants of ALS such as progressive bulbar palsy (PBP), pseudobulbar palsy, and hereditary spastic paraplegia. These categories may be based on the cause or mechanism of the disease, the organs, body parts, or bodily functions affected by these diseases, or the relationship between two diseases.

[0055] The terms “neuroprotective” and “cytoprotective” are used interchangeably and refer to the protection or prevention of abnormalities in nerve cells caused by aging, genetic abnormalities, and external factors, such as neurotoxins, as well as the restoration of normal or healthy neuronal function.

[0056] In this specification, interchangeable therapeutic agents or therapeutically effective agents mean agents administered to a subject to reduce or eliminate one or more symptoms of a disease or disorder, the agents according to the present invention are recombinant lectins, and the disease or disorder is a neurodegenerative disease.

[0057] In this specification, the terms "neuronal proliferation" or "neurite" as used interchangeably mean projections from the cell body of a neuron, including, for example, axons or dendrites.

[0058] As used herein, the term "modulation" refers to altering or regulating the physiological mechanisms of an organelle (e.g., membrane potential).

[0059] As used herein, the term “therapeutic dose” means an amount sufficient to produce a desired therapeutic benefit, where the therapeutic benefit means an effect in the treatment or prevention of one or more neurodegenerative diseases. The effect is such that the subject is either free from one or more diseases, or the symptoms of one or more diseases are controlled or reduced, or the onset or progression of one or more diseases is delayed. A therapeutic dose may be administered in one or more doses. In the present invention, a therapeutic dose of recombinant protein is an amount sufficient to alleviate, improve, stabilize, reverse, prevent, slow or delay the progression of a disease.

[0060] As used herein, the terms “homology” or “homological” refer to two or more referenced entities that share at least partial identity across a given region or portion. An area, region, or domain of homology or identity refers to a portion of two or more referenced entities that share homology or are identical. Thus, if two sequences are identical across one or more sequence regions, they share identity across those regions. Substantial homology refers to a molecule that is structurally or functionally conserved such that it has, or is expected to have, at least partial structure or function (e.g., biological function or activity) of one or more of the structure or function (e.g., biological function or activity) of a reference molecule or a related / corresponding region or portion of a reference molecule that shares homology.

[0061] In one embodiment, the "homology" percentage between two sequences is determined using the BLASTP algorithm with default parameters (Altschu et al., Nucleic Acids Res. 1997 / 9 / 1;25(17):3389~402). In particular, the BLAST algorithm can be accessed via the internet using URL:https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. In an alternative embodiment, for global sequence alignment, the homology percentage between two sequences is determined using the EMBOSS Needle algorithm with default parameters. In particular, the EMBOSS Needle algorithm can be accessed via the internet using URL:https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / .

[0062] Unless otherwise indicated, the term “homology” shall be used interchangeably with the term “sequential identity” in this specification. [Brief explanation of the drawing]

[0063] [Figure 1] This is a graphical representation of the effect of Sequence ID No. 1 on neurite formation in a basic model neuron (pc12). [Figure 2] This is a graphical representation of the effect of Sequence ID No. 1 on neurite formation in nerve cells (pc12) in response to MPP+-induced injury. [Figure 3] This is a histopathological image (H&E staining; 100×). [Modes for carrying out the invention]

[0064] Table 1 - Cytoprotective effect of Sequence ID No. 1 in nerve cells (SH-SY5Y) against neurotoxic (MPP+)-induced injury. Table 2 - Anti-apoptotic effect of Sequence ID No. 1 in neurons (SH-SY5Y) via mitochondrial membrane potential restoration against MPP+ iodide damage. Table 3 - Anti-apoptotic effect of SEQ ID NO: 1 in neurons (SH-SY5Y) against MPP+ iodide injury via reduction of annexin-positive cell population. Table 4 - Anti-apoptotic effect of Sequence ID No. 1 in neurons (SH-SY5Y) via reduction of sub(G0 / G1) cell population against MPP+ iodide-induced injury. Table 5: Effect of Sequence ID No. 1 on neurite formation in the basic model nerve cell (pc12) Table 6: Protective effect of Sequence ID No. 1 on neurite formation in nerve cells (PC12) against MPP+-induced injury. Table 7: Effect of Sequence ID No. 1 on the expression of biomarkers associated with Alzheimer's disease in the neuronal cell line (SH-SY5Y). Table 8: Effect of Sequence ID No. 1 on the expression of Parkinson's disease-related biomarkers in the neuronal cell line (SH-SY5Y). Table 9: Animal Allocation Table 10: Average Transfer Latency Time (seconds) Table 11: Effect of Sequence ID No. 1 on nerve growth factor (NGF) (pg / ml) Table 12: Effect of Sequence ID No. 1 on brain acetylcholinesterase (mU / ml) Table 13: Effect of Sequence ID No. 1 on brain TNF-alpha (pg / ml) Table 14: Histopathological examination (mean score)

[0065] Array display: Sequence ID 4: Represents the amino acid sequence of the native S. rolfsee lectin (reported as Sequence ID 1 in WO2010 / 095143), and has the following sequence:

[0066] [ka]

[0067] Sequence ID 1: Represents a variant of the amino acid sequence of S. rolfsee's lectin (reported as Rec-2 in WO2010 / 095143), and has the following sequence:

[0068] [ka]

[0069] Sequence ID 2: Represents a variant of the amino acid sequence of S. rolfsee's lectin (reported as Rec-3 in WO2010 / 095143), and has the following sequence:

[0070] [ka]

[0071] Sequence ID 3: Represents a variant of the amino acid sequence of S. rolfsee's lectin (reported in WO2014 / 203261) and has the following sequence:

[0072] [ka]

[0073] Detailed description of the invention In a first aspect, the present invention provides recombinant lectins derived from Sclerotium rolfsii (S. rolfsii) lectins for the treatment or prevention of neurodegenerative diseases.

[0074] In one embodiment of the present invention, the lectin is derived from a group consisting of fungi and plants, but is not limited. In some embodiments, the lectin is derived from soil-transmitting plant pathogenic fungi such as S. rolfsii.

[0075] Please understand that "derived from" means that the lectin can be isolated from its native environment, or that the lectin contains an amino acid sequence that is identical to or similar to the natural sequence.

[0076] Lectins may contain amino acid sequences having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to the native sequence. Native lectins can be isolated from S. rolfsee.

[0077] The lectin may contain an amino acid sequence having at least 60% homology to SEQ ID NO: 4. In some embodiments, the lectin may contain an amino acid sequence having at least 60% homology to SEQ ID NO: 1, 2, or 3. In some embodiments, the amino acid sequence may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 4. In some embodiments, the amino acid sequence may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 1, 2, or 3.

[0078] Sequence ID 1 has 98% homology to Sequence ID 4. Sequence ID 2 has 96% homology to Sequence ID 4. Sequence ID 3 has 91% homology to Sequence ID 4.

[0079] According to any one of the embodiments described above, the recombinant lectin is a modified lectin protein (i.e., a recombinant lectin protein having at least one amino acid modification in the molecule, preferably in the carbohydrate-binding site) as defined in WO2020 / 044296, which is incorporated herein by reference.

[0080] According to some specific embodiments of the present invention, the lectin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0081] According to a particular embodiment of the present invention, the lectin protein of the present invention is preferably synthesized using recombinant technology. Methods for preparing recombinant proteins are well known to those skilled in the art. In one embodiment, a cloned nucleotide sequence encodes a modified lectin protein that is close to the amino acid sequence of the native lectin but provides different properties. Alternatively, the nucleotide sequence encoding the recombinant lectin protein can be synthesized using chemical or recombinant means and expressed in a suitable host to obtain the recombinant protein. Suitable host cells include prokaryotic cells and both lower and higher eukaryotic cells. The introduction of the recombinant molecule into the host cell can be achieved using methods known in the art. In an exemplary embodiment of the present invention, the suitable host is a microbial cell. In a preferred embodiment, the microbial cell is selected from the group consisting of, but not limited to, yeast cells, Escherichia coli, insect cell lines, or mammalian cell lines. Furthermore, the recombinant protein can be obtained by isolation as an expression product from the recombinant host. In one embodiment, the recombinant protein of the present invention is purified by conventional techniques, typically by chromatographic methods. For example, the recombinant lectin protein of the present invention can be prepared by the method disclosed in the applicant's previous application WO / 2020 / 074977.

[0082] In another embodiment of the present invention, the molecular weight of the recombinant lectin, as determined by SDS-PAGE and mass spectrometry, is approximately 16,000 daltons.

[0083] According to one embodiment, the present invention provides recombinant lectins for the treatment or prevention of neurodegenerative diseases, wherein treatment includes reducing, eliminating, decreasing, or mitigating the signs and symptoms of neurodegenerative diseases, and prevention includes suppressing, controlling, or delaying the onset or development of neurodegenerative diseases or disease-related symptoms.

[0084] According to some embodiments, neurodegenerative diseases include diseases or disorders caused by the degeneration of neurons or nerve cells within and around the brain or central nervous system.

[0085] Neurodegenerative diseases can be caused by increased or decreased levels of biomarkers such as ICAM-1 / CD54, dopamine, serotonin, S100b, Park7 / DJ-1, calbindin D, B-NGF, RAGE, MPO, Tau, GDNF, α-synuclein, amyloid-beta protein, acetyline cholinesterase, periostin, angiostatin-converting enzyme (ACE), thrombosporin-1, plasma amyloid-beta protein, VE-cadherin, LGALS3BP (lectin galactoside-linked soluble triglyceride protein), and TNF-α (tumor necrosis factor-alpha). Increases or decreases in the normal levels of these biomarkers may indicate the onset or presence of neurodegenerative disease in the body under laboratory conditions.

[0086] For example, S100b is a calcium-binding protein that plays a crucial role in the pathogenesis of Parkinson's disease. While the normal level of S100b in healthy individuals without Parkinson's disease is thought to be between 10 pg / mL and 150 pg / mL, individuals with Parkinson's disease are thought to have levels exceeding 200 pg / mL. Therefore, it is likely that S100b levels are elevated in individuals with Parkinson's disease.

[0087] According to one aspect of the present invention, recombinant lectin proteins can reduce the level of S100b.

[0088] Similarly, according to one embodiment, the recombinant lectin protein of the present invention can modulate the level of a biomarker in accordance with the requirements of the body's cells, thereby treating or preventing the progression of disease.

[0089] In one aspect of the present invention, the markers listed above may be involved in the cause of one or more neurodegenerative diseases. The recombinant lectin proteins of the present invention can modulate the levels of these biomarkers and are therefore effective in controlling one or more diseases or the progression or onset of a disease.

[0090] In one embodiment of the present invention, a recombinant lectin having SEQ ID NO: 1 helps normalize the levels of the biomarkers dopamine and serotonin in neurotoxically damaged cells, thereby restoring cognitive health of the brain. Dopamine and serotonin hormones can transmit signals to nerve cells and are therefore involved in maintaining sleep cycles, muscle contraction, mood function, movement, and autonomic nervous system function. Cognitive health refers to the overall health of the brain, tissues, and blood supply, as well as their ability to function properly under various conditions. Good cognitive health is crucial for the brain to collectively perform all psychological processes known as cognition, including, but not limited to, learning, intuition, judgment, language, attention, arousal, concentration, and memory (both long-term and short-term). Poor cognitive health due to aging, disease, and / or other cognitive loss reduces the brain's ability to function properly, resulting in a significant decline in cognitive function and ability. Some cognitive health-related disorders include panic disorder, obsessive-compulsive disorder (OCD), attention deficit hyperactivity disorder (ADHD), seasonal affective disorder (SAD), sleep disorders, memory loss or confusion, stress, and depressed mood. During the pathogenesis of Parkinson's disease, the normal synthesis of serotonin and dopamine is severely affected, which leads to cognitive health impairment.

[0091] In some embodiments of the present invention, a method for treating or preventing neurodegenerative diseases is provided, comprising administering an effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin to a subject.

[0092] The subjects may be mammals. In some embodiments, the subjects are humans. In particular, the subjects may be humans who have a neurodegenerative disease or who are seeking prevention of a neurodegenerative disease.

[0093] In one embodiment, a method for treating or preventing a neurodegenerative disease comprises administering a therapeutically effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin, where the therapeutically effective amount of lectin may be in a dose range of 0.01 mg / kg to 1000 mg / kg of body weight. In some embodiments, the dose range may be 0.1 mg / kg to 500 mg / kg, 0.5 mg / kg to 100 mg / kg, or 1 mg / kg to 50 mg / kg. Determining the amount of lectin administered according to the condition being treated and the nature of the subject is within the capabilities of those skilled in the art.

[0094] In some embodiments, the recombinant lectin protein of the present invention can be administered either on its own or in the form of a pharmaceutical composition.

[0095] Accordingly, the present invention also provides a pharmaceutical composition for the treatment or prevention of neurodegenerative diseases, comprising a recombinant lectin protein derived from Sclerotium rolfsii lectin and a pharmaceutically acceptable excipient. Exemplary excipients include sterile water, physiological saline, and / or a pharmaceutically acceptable buffer.

[0096] The composition may further include protein stabilizers, polymers, solubilizers, cryoprotectants, lioprotectants, fillers / diluents, or mixtures thereof. The composition may also include excipients listed in Applicant's concurrently pending Indian application No. 201921027358, which is incorporated in whole by reference in this application.

[0097] Administration of lectin proteins or compositions may be by any suitable route understood by those skilled in the art, including, but not limited to, injection (e.g., intravenous (bolus or infusion), intra-arterial, intraperitoneal, subcutaneous (bolus or infusion), intraventricular, intramuscular, or subarachnoid), oral ingestion (e.g., tablets, gels, lozenges, or liquids), inhalation, topical, transmucosal (e.g., oral, nasal, or rectal mucosa), spray, tablet, transdermal patch, subcutaneous implant, or suppository.

[0098] In some embodiments, lectins (e.g., lectins having the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4) or the pharmaceutical compositions described herein are administered to a subject enterally, parenterally, or topically. Lectins or pharmaceutical compositions can be administered in dosage forms such as solids (e.g., tablets or capsules), lyophilized powders, liquids (e.g., solutions or suspensions), semi-solids, or any other form known to those skilled in the art. Lectins or pharmaceutical compositions can be administered to a subject by depot injection of solutions or suspensions intravenously, intramuscularly, intraperitoneally, subcutaneously, or intradermally, or they can be administered intrathecally, percutaneously, sublingually, or orally, topically, or by inhalation.

[0099] As will be understood by those skilled in the art, the appropriate form of a composition can be determined by the route of administration. Therefore, appropriate forms of a composition may include, but are not limited to, injections for intravenous (bolus or infusion), intra-arterial, intraperitoneal, subcutaneous (bolus or infusion), intraventricular, intramuscular, or subarachnoid routes; tablets, capsules, gels, lozenges, or liquids for oral administration; solutions, suspensions, or aerosols as sprays for inhalation; gels, sprays, or creams for topical application; transmucosal compositions for administration via the mucous membranes of the oral cavity, nose, or rectum; and delivery in the form of transdermal patches, subcutaneous implants, or suppositories. Lectin proteins can also be formulated into rectal compositions such as suppositories or retained enemas. For buccal administration, the composition may take the form of tablets or lozenges. The composition may be a vesicular drug delivery system, such as, but not limited to, bilosomes, liposomes, niosomes, transferosomes, ethosomes, sphingosomes, pharmacosomes, multilamellar vesicles, microspheres, etc.

[0100] The composition of the present invention can be formulated according to the understanding and knowledge of those skilled in the art.

[0101] In one embodiment, the present invention provides the use of recombinant lectin proteins derived from Sclerotium rolfsii lectins for the treatment or prevention of neurodegenerative diseases.

[0102] According to this embodiment, use may be of the recombinant lectin protein itself, or in the form of a composition comprising the lectin protein and a pharmaceutically acceptable excipient.

[0103] In one embodiment, the present invention provides a method for inducing neuronal proliferation by administering an effective amount of recombinant lectin protein derived from Sclerotium rolfsii lectin.

[0104] According to the present invention, "induction of neuronal proliferation" refers to the induction of neurite growth, where neurites are projections extending from the cell body of a neuron. The lectin proteins of the present invention can induce neurite growth in neurons when administered to targets that require them.

[0105] In one specific embodiment of the present invention, the recombinant lectin protein can be selected from lectins having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0106] In one embodiment, the neurodegenerative diseases treated or prevented according to the present invention are those listed herein above.

[0107] In certain embodiments, neurodegenerative diseases include, but are not limited to, dementia, e.g., Alzheimer's disease, frontotemporal dementia (Pick's disease), Lewy body dementia, neurofibrillary tangle dementia and dementia-related symptoms, Creutzfeldt-Jakob disease (which has clinical symptoms similar to Alzheimer's), hippocampal sclerosis, Schilder's disease; Parkinson's disease, Parkinsonian diseases, e.g., progressive supranuclear palsy (PSP), multiple system atrophy (MSA), and corticobasal degeneration (CBD); ataxia, cognitive impairment, motor disorders Amyotrophic lateral sclerosis (ALS) with neuronal disease-like characteristics, primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy and hereditary spastic paraplegia (variants of ALS); aneurysms, epilepsy and Huntington's disease, pantothenate kinase-associated neurodegeneration (PKAN), stroke, Batten disease, Gerstmann-Streussler-Scheinker syndrome, CADASIL (autosomal dominant cerebral arteriovenous disease with subcortical infarction and leukoencephalopathy), cerebellar hypoplasia, cerebral arteriosclerosis, hypoxic encephalopathy (Cerebral arteriosclerosis) Hypoxia), chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), corpus callosum agenesis, globular glial tauopathies, primary age-related tauopathies, chronic traumatic encephalopathy (CTE), age-related tauastrogliopathies, Leigh syndrome, Lewy body-like diffuse Lewy body disease (DLBD), genetic disorders causing neuronal state / loss, such as spinal muscular atrophy (SMA), a trinucleotide repeat inheritance disorder, congenital SMA with joint contractures, a rare form of SMA, spinal and bulbar muscular atrophy (SBMA), polyglutamine (PolyQ) repeat disease, primary progressive aphasia (PPA), Alexander disease, Alpers disease, Krabbe disease, Sandhoff disease, Haller-Holden-Spats syndrome, and Kugelberg-Welander disease.

[0108] In very specific embodiments, neurodegenerative diseases may be selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment and dementia-related symptoms, amyotrophic lateral sclerosis (ALS), Lewy body dementia, spinal muscular atrophy, and Huntington's disease.

[0109] In another very specific embodiment, the neurodegenerative disease may be selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, and symptoms associated with dementia.

[0110] The present invention relates to a method for treating or preventing neurodegenerative diseases using recombinant lectin proteins having the sequence of SEQ ID NO: 4 or its homologous sequence. In vitro studies of recombinant lectins having the sequence of SEQ ID NO: 1 have shown a significant positive effect on nerve cell lines in the presence of neurotoxins. Recombinant lectins protected nerve cells from neurotoxins. Similarly, when the effects of recombinant lectins having the sequence of SEQ ID NO: 1 on biomarkers associated with neurodegenerative diseases were studied, it was shown that lectins modulated the biomarkers from abnormal to normal ranges in pathological conditions.

[0111] In particular, we conducted in vitro studies on several cell lines for Parkinson's disease and Alzheimer's disease or dementia.

[0112] The cytoprotective effect of recombinant lectin protein containing SEQ ID NO: 1 was elucidated by determining the cell viability of the human neuronal cell line SH-SY5Y in the presence of the neurotoxin 1-methyl-4-phenylpyridinium iodide (MPP + iodide). Cell viability and recovery of cell viability against neurotoxin-induced cytotoxicity were determined. SEQ ID NO: 1 at concentrations of 0.001 μg / mL to 50 μg / mL showed cytoprotection of approximately 41% to 76%, compared to 40% to 86% at concentrations of the positive control deprenyl (1 μM to 100 μM).

[0113] First, the cytoprotective effect was studied by treating SH-SY5Y cell lines with a non-cytotoxic concentration of recombinant lectin protein containing SEQ ID NO: 1, and then exposing the cell lines to the neurotoxin MPP + iodide. Deprenyl was used as a positive control. Compared to the positive control deprenyl, which showed a cell viability range of 79.3%–91.3%, an increase in cell viability between 78.8% and 94.9% was observed for Parkinson-inducible SH-SY5Y cells treated with recombinant lectin containing SEQ ID NO: 1.

[0114] To study the anti-apoptotic effect in human neuron (SH-SY5Y) cell lines, the effect of SEQ ID NO: 1 was evaluated. Parkinson's disease was induced in SH-SY5Y cell lines using 1-methyl-4-phenylpyridinium iodide (MPP+iodide) as a neurotoxin. The in vitro anti-apoptotic effect of recombinant lectins containing SEQ ID NO: 1 was determined using three different studies, the Annexin V staining technique, and sub-G0 / G1 staining by PI.

[0115] The anti-apoptotic effect of the recombinant lectin protein SEQ ID NO: 1 was evaluated by restoring mitochondrial membrane potential in SH-SY5Y cells following MPP+ iodide-induced injury. Recombinant lectin proteins with SEQ ID NO: 1 showed anti-apoptotic effects accompanied by a 22.7%–115.9% increase in mitochondrial membrane potential in the concentration range of 1 μg / mL to 50 μg / mL. Positive controls showed a 40%–95% increase in mitochondrial membrane potential at concentrations of 1 μM to 100 μM.

[0116] Further reductions in apoptotic cells were determined using annexin-positive staining for neurotoxins, MPP + iodide. Recombinant lectin proteins, at concentrations of 0.001 μg / mL to 1 μg / mL, showed a reduction of 23.5% to 70.4% of apoptotic cells per population compared to cells not treated with lectins and exposed to neurotoxins. The positive control deprenyl, at concentrations of 10 μM to 100 μM, showed a reduction of 27.2% to 38.3% of the population.

[0117] The anti-apoptotic effect of SEQ ID NO: 1 was further confirmed by evaluating the sub(G0 / G1) cell population in human neuron (SH-SY5Y) cell lines in response to the neurotoxin MPP + iodide. This study showed that apoptosis in the sub(G0 / G1) cell population was reduced by 19% to 35% at concentrations ranging from 0.001 μg / mL to 1 μg / mL compared to cells that were not treated with the lectin and were exposed to the neurotoxin. The positive control deprenyl showed a reduction of 13% to 42% of the population at concentrations of 10 μM to 100 μM. The neuroprotective efficacy of recombinant lectins containing SEQ ID NO: 1 will be further investigated by evaluating cognitive health using a neurite proliferation assay with nerve cell PC12 (rat pheochromocytoma cells). Cells were treated with nerve growth factor (NGF), recombinant lectin containing SEQ ID NO: 1, and MPP + iodide, and their neuroprotective effects were tested. This study demonstrated that recombinant lectins with Sequence ID No. 1 possess potent neuroprotective properties, assisting neurite growth recovery by 7.3% to 78.2% in MPP+iodide-induced cell damage at concentrations of 0.0001 μg / mL to 5 μg / mL. In contrast, cells treated with NGF and MPP+iodide alone showed 0% protection in neurite formation.

[0118] The above research has revealed that recombinant lectin proteins having SEQ ID NO: 1 have a cytoprotective effect that protects nerve cells from neurotoxins and significantly reduces cellular apoptosis. Therefore, the recombinant lectin proteins of the present invention prevent the onset of disease in cells, and thus in the body, by helping nerve cells survive when exposed to neurotoxins.

[0119] Further studies were conducted to evaluate the effect of recombinant lectin proteins having SEQ ID NO: 1 on neurite growth. PC12 cells were treated with recombinant lectin having SEQ ID NO: 1 at concentrations ranging from 0.001 μg / mL to 5 μg / mL. The lectin showed a 19% to 55% increase in neurite number compared to untreated cells. The recombinant lectin of the present invention exhibits considerable neurite proliferation and can be used for the aforementioned purposes in subjects requiring such treatment.

[0120] The mechanism of action of recombinant lectin containing Sequence ID No. 1 in Parkinson's disease was determined by multiplex analysis and ELISA, and the expression levels of biomarkers were evaluated. Test samples were prepared by treating human neuron (SH-SY5Y) cells with recombinant lectin protein containing Sequence ID No. 1 at various concentrations from 0.001 μg / mL to 1 μg / mL for 24 hours, and then further treating with the neurotoxin MPP + iodide to induce neuronal damage.

[0121] This study reveals that recombinant lectins with SEQ ID NO: 1 restore Parkinson's disease-affected neurons by activating neurotransmitters and neuroprotective signaling cascade proteins, including dopamine (22.9%–82.5% increase), serotonin (5.1%–23.5% increase), and calbindin D (1.7%–9.7% increase), compared to cells treated with MPP + iodide. On the bright side, recombinant lectins with SEQ ID NO: 1 also exhibit significant inhibitory effects on inflammatory cell adhesion proteins, such as ICAM-1 / CD54 levels (6.2%–41.3% decrease) and calcium-binding protein S100b (6.5%–11.4% decrease), compared to cells treated with MPP + iodide. However, no significant modulation was observed for α-synuclein.

[0122] The mechanism of action of recombinant lectin with SEQ ID NO: 1 in Alzheimer's disease was elucidated through multiplex analysis. Biomarkers evaluated to describe the mechanism of action were β-NGF, RAGE, MPO, and Tau. The mechanism of action in Alzheimer's disease was determined by treating human neuron (SH-SY5Y) cells with recombinant lectin protein containing SEQ ID NO: 1 at concentrations ranging from 0.01 μg / mL to 25 μg / mL for 24 hours, followed by further treatment with the neurotoxin scopolamine to induce neuronal damage.

[0123] The effects of recombinant lectin protein with SEQ ID NO: 1 in suppressing Alzheimer's disease were evaluated by activation and inhibition of certain biomarkers, such as β-NGF, a neuronal growth factor biomarker, whose levels increased from 41% to 89% compared to scopolamine-treated cells; MPO (metalloperoxidase) and RAGE (advanced glycation end product receptor) were inhibited by SEQ ID NO: 1, showing reductions in levels of 46% to 64% and 3% to 19%, respectively. Inhibition of MPO and RAGE demonstrates the significant therapeutic effect of recombinant lectin on Alzheimer's disease. RAGE is a multiligand surface molecule of the immunoglobulin superfamily and acts as a receptor for amyloid-beta protein, thus playing an important role in the prognosis of Alzheimer's disease. Increased RAGE expression due to genetic abnormalities leads to neuroinflammation accompanied by the generation of reactive oxygen species, which contribute to oxidative stress. MPO is an immunomodulatory protein that plays a crucial role in cytokine induction, primarily catalyzing the conversion of hydrogen peroxide to hypochlorous acid in the presence of chlorides. This reaction with biological species leads to the formation of oxidative adducts. Abnormalities in the MPO gene result in overexpression of MPO in the prefrontal cortex of the brain, leading to regeneration.

[0124] In vitro studies have suggested the safety and therapeutic efficacy of recombinant lectin proteins for neurodegenerative diseases. The therapeutic effects of recombinant lectin proteins were further investigated by determining their in vivo anti-dementia activity using scopolamine-induced dementia in Swiss albino mice, through measuring movement waiting times in passive avoidance tests, changes in brain biomarker levels, and histopathological examination of the hippocampal region of the brain.

[0125] The cognitive enhancement activity of recombinant lectin protein with SEQ ID NO: 1 against scopolamine-induced memory impairment in mice was determined using a passive avoidance test. Animals injected with scopolamine, group G2, showed a significant reduction in movement waiting time (p<0.001) (i.e., 56.93±5.12 seconds) compared to the normal control group G1 (i.e., 135.70±5.11 seconds), indicating short-term memory impairment in mice. Animals treated with donepezil (G3, 2.5 mg / kg) showed a significant increase in movement waiting time (p<0.001) (i.e., 110.61±7.02 seconds) compared to G2, indicating that scopolamine-induced short-term memory impairment was reversed by the cholinesterase inhibitor donepezil. In animals treated with recombinant lectin protein containing SEQ ID NO: 1 at three dose levels, group G4 (0.5 mg / kg) showed a significant increase (p<0.05) (85.26±6.52). Compared to G2, G5 (0.25 mg / kg) showed an increase in memory time of 80.58±9.08 seconds, and G6 (0.125 mg / kg) showed an improvement in memory time of 75.81±7.75 seconds.

[0126] The percentage increase in migration waiting time was 48.5% for animals treated with donepezil compared to untreated animals. Animals G4, G5, and G6 treated with recombinant lectin protein having SEQ ID NO: 1 at different doses showed increases in migration waiting time of 33.2%, 29.4%, and 24.9%, respectively, compared to G2.

[0127] Further brain biomarkers, such as brain NGF (nerve growth factor), TNF-alpha, and acetylcholinesterase (AChE) levels, were estimated and evaluated by ELISA.

[0128] NGF (nerve growth factor) plays a central role in neuronal plasticity and neurogenesis by inhibiting the phosphorylation of cAMP response element-binding protein (CREB, where cAMP is cyclic adenosine monophosphate). Impairment of CREB phosphorylation is a known pathological factor in neurodegenerative disorders, inducing neuronal loss in the hippocampus and cortex through pro-apoptotic processes. CREB inhibition impairs behavioral ability in various memory tests. Conversely, CREB overexpression promotes neuronal survival and improves cognitive impairment through the cholinergic system. Treatment with the neurotoxin scopolamine significantly reduces brain NGF expression (p<0.001) compared to levels observed in a normal control group. However, the groups treated with recombinant lectin protein having SEQ ID NO: 1 at variable doses of 0.5 mg / kg and 0.25 mg / kg showed a significant (p<0.001) increase in NGF levels (71.6 pg / mL and 62 pg / mL, respectively) compared to the dementia group treated with scopolamine (34.5 pg / mL). The group treated with donepezil (positive control) showed an increase of 57 pg / mL. The results indicate that recombinant lectin protein having SEQ ID NO: 1 protects or improves learning and memory impairment by activating neurotrophic factors and preventing neuronal apoptosis. Further AChE activity in the brain was evaluated in the study groups. This study revealed that the scopolamine group significantly increased AChE activity in the brain (p<0.001), suggesting that the observed cognitive impairment was induced by cholinergic dysfunction. However, pretreatment with recombinant lectin protein containing Sequence ID No. 1, 0.5 mg / kg, significantly (p<0.001) attenuated these scopolamine-induced dysfunctions. AChE is a well-known enzyme that plays a central role in learning and memory. Choline acetyltransferase (ChAT) is an important cholinergic marker involved in acetylcholine (Ach) synthesis. While Ach maintenance is essential for normal function, excessive AChE activity leads to disruption of Ach levels in the brain.Acetylcholine signaling ultimately leads to the phosphorylation of cAMP (cyclic adenosine monophosphate) response element-binding protein (CREB), which then translocates to the nucleus to regulate the transcription of target genes. CREB is well known to play a crucial role in neuronal growth, proliferation, differentiation, and survival. Numerous studies have also highlighted the interrelationship between CREB transcriptional activity and hippocampus-dependent memory formation.

[0129] The study group of animals treated with the neurotoxin scopolamine showed an AChE level of 6.74 mU / mL, while the positive control group, treated with both scopolamine and donepezil, showed an AChE level of 4.39 mU / mL. The study group treated with both scopolamine and SEQ ID NO: 1 showed AChE levels of 4.47 mU / mL at 0.5 mg / kg, 6.27 mU / mL at 0.25 mg / kg, and 6.59 mU / mL at 0.125 mg / kg. In the untreated group, the AChE level was 4.19 mU / mL.

[0130] Further studies showed that scopolamine-induced dementia increased brain TNF-α levels, indicating mild to moderate neuroinflammation. The scopolamine group showed a significant (p<0.01) increase in brain TNF-α levels (144 pg / mL); on the other hand, recombinant lectin with SEQ ID NO: 1 at dose levels of 0.5 mg / kg (121.7 pg / mL), 0.25 mg / kg (67.65 pg / mL), and 0.125 mg / kg (79.53 pg / mL) significantly reduced brain TNF-α content, suggesting anti-inflammatory activity. The normal group without scopolamine treatment had a TNF-α level of 61.02 pg / mL, while the positive control group treated with donepezil and scopolamine showed a TNF-α level of 156 pg / mL.

[0131] Detailed histological investigations were performed on H&E (hematoxylin-eosin) stained brain tissue from the cerebral cortex and hippocampus (CA1, CA3, and DG) regions. The results showed no significant changes in the cerebral cortex and hippocampal regions excluding the CA3 region in any of the treatment groups or the scopolamine injection group. In group G2, the scopolamine disease control group which showed a mean score of 2.0 compared to the normal control group (0), significant (p<0.01) damage was found in the CA3 region of the hippocampus. No significant reduction was observed in group G3, the group treated with donepezil (1.33), compared to G2. Furthermore, group G5, treated with recombinant lectin protein having SEQ ID NO: 1 at 0.25 mg / kg, showed a significant (p<0.05) decrease in value, i.e., 0.60, while groups G4 (SEQ ID NO: 1 at 0.5 mg / kg) and G6 (SEQ ID NO: 1 at 0.125 mg / kg) showed decreases in mean score values, i.e., 0.67 and 1.00, respectively. It is well known to those skilled in the art that the hippocampus is a central region of the brain for learning and memory. Neuronalization in the adult hippocampus plays a crucial role in memory formation; therefore, impairments in neurogenesis and neuronal integration are considered pathological features of neurodegenerative disorders. Accordingly, treatment with recombinant lectin protein having SEQ ID NO: 1 significantly reversed the scopolamine-induced inhibition of neurogenesis in the hippocampal structure of CA3. This neurogenesis is known to depend on the activity of both neurotrophins and their receptors.

[0132] According to one embodiment of the present invention, a recombinant lectin protein having SEQ ID NO: 1, derived from Sclerotium rolfsee lectin, prevents nerve cells from apoptosis due to excitotoxicity and restores neuronal function through nerve growth in disease-inducible animal models. Excitotoxicity can arise from endogenous or exogenous harmful substances and is an important mechanism in neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's disease, amyotrophic lateral sclerosis (ALS), and dementia. Excitotoxicity is the overactivation of neurotransmitters that primarily causes severe damage to nerve cells by affecting mitochondrial function, which then leads to oxidative stress. Excessive influx of calcium ions may also be one of the mechanisms involved in nerve cell loss. As a result, nerve cells lose their function and are degenerated by apoptosis. Therefore, recombinant lectin proteins with Sequence ID No. 1 derived from Sclerotium rolfsee lectin have therapeutic efficacy for the treatment or prevention of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's disease, amyotrophic lateral sclerosis (ALS), and dementia, by restoring mitochondrial membrane potential; regulating the expression levels of certain biomarkers that play a crucial role in neuronal function; and further promoting neuronal growth, thereby preventing neuronal cells from apoptosis.

[0133] In further embodiments of the present invention, lectin proteins derived from Sclerotium rolfsii lectins exhibited therapeutic efficacy and were effective in the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and dementia or dementia-related symptoms. Lectins further demonstrated efficacy in neurite proliferation and cognitive function recovery in disease-induced models. [Examples]

[0134] The following examples are provided to illustrate the best embodiments of the present invention. The examples do not limit the present invention in any way.

[0135] SH-SY5Y cells are derived from a subcloned cell line of SK-N-SH human neuroblastoma cells. Because these cells can be modified into various types of functional neurons by adding specific compounds, they serve as a model for neurodegenerative disorders. Therefore, this property of the SH-SY5Y cell line makes it a suitable model for experimental neurological studies, including the analysis of neuronal differentiation, metabolism, and function related to neurodegenerative processes, neurotoxicity, and neuroprotection. The human neuronal cell line SH-SY5Y was obtained from the National Centre for Cell Science (NCCS), Pune.

[0136] SH-SY5Y cell lines were maintained in DMEM:Ham F-12 (1:1) + 10% FBS growth medium under growth conditions of 5% CO2, 37°C, and 95% humidity. The cell lines were subcultured by dividing the cell suspension into new flasks and replenishing with fresh culture medium.

[0137] PC-12 cells, or the adrenal pheochromocytoma (PC12) cell line, were first isolated from the adrenal medulla of rats. The ability of PC12 cells to synthesize and conserve dopamine and to resemble sympathetic ganglion neurons during differentiation mediated by nerve growth factor (NGF) made them a suitable model for Parkinson's disease. Rat pheochromocytoma cells PC12 were obtained from the American Cell Culture and Cell Lineage Preservation Center (ATCC) USA.

[0138] The PC12 cell line was maintained in Ham F12 + 10% FBS growth medium under growth conditions of 5% CO2, 37°C, and 95% humidity. The cell line was subcultured by trypsin treatment and by dividing the cell suspension into new flasks and replenishing with fresh culture medium.

[0139] An aqueous solution of recombinant lectin protein containing the sequence with SEQ ID NO: 1 was prepared. The stock solution was diluted in serum-free medium (SFM) to achieve the final concentration.

[0140] (Example 1) Evaluation of the cytoprotective effect of Sequence ID No. 1 in nerve cells regarding beneficial effects in Parkinson's disease. The cytoprotective effect of Sequence ID No. 1 in SH-SY5Y neurons was examined using the following assay.

[0141] Human neuron SH-SY5Y cells were counted using a hemocytometer, plated at a density of 25,000 cells / well into 96-well plates, and incubated at 37°C for 24 hours. After incubation, cells were treated for 24 hours with recombinant lectin protein containing the sequence of Sequence ID No. 1 at concentrations ranging from 0.001 μg / ml to 50 μg / ml. After 24 hours of treatment, cells were exposed to a neurotoxin (MPP + iodide, 1 mM) for 24 hours. Cells treated with MPP + iodide alone were included as negative controls. Untreated cells were included as controls. Cells treated with deprenyl (1 μM to 100 μM) were treated as positive controls. After treatment, the cytoprotective effect of SEQ ID NO: 1 on cell viability was evaluated by a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay: Plates were removed and 20 μl of 5 mg / ml MTT 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide solution was added to all wells. SH-SY5Y cells were incubated at 37°C for 3 hours. The supernatant was aspirated and 150 μl of DMSO was added to each well to dissolve the formazan crystals. The absorbance of each well was read at 540 nm using a Synergy HT microplate reader. The protective effect of SEQ ID NO: 1 on the viability of SH-SY5Y cells against MPP+ iodide-induced damage was determined as follows: Cell viability was determined as follows: Cell viability%=(100-cytotoxicity%); In the formula, the cytotoxicity percentage corresponding to each treatment is calculated as follows: Cytotoxicity%=[(RX) / R]*100 X = MPP + iodide / absorbance of cells treated with recombinant lectin containing SEQ ID NO: 1 + MPP + iodide R = Absorbance of control cells (untreated) The protection percentage was calculated as follows: [(Absorbance of SEQ ID NO: 1 + MPP + Iodide) - (Absorbance of MPP + Iodide) / (Absorbance of untreated material) - (Absorbance of MPP + Iodide alone)] * 100

[0142] [Table 1]

[0143] (Example 2) Evaluation of the anti-apoptotic effect of recombinant lectin with SEQ ID NO: 1 in nerve cells, regarding beneficial effects in Parkinson's disease. The anti-apoptotic effect of recombinant lectin with SEQ ID NO: 1 in nerve cells, which is relevant to beneficial effects in Parkinson's disease, was determined using the following assay.

[0144] (Example 2a) Determination of the effect of recombinant lectins with SEQ ID NO: 1 on mitochondrial membrane potential. Cells were counted using a hemocytometer and plated into 96-well plates with black wells at a density of 25,000 cells / well in complete growth medium. The plated cells were incubated overnight at 37°C in a 5% CO2 incubator. After 24 hours, the cells were treated for 24 hours with recombinant lectin protein having SEQ ID NO: 1 at concentrations ranging from 1 μg / ml to 50 μg / ml. After 24 hours of treatment, the cells were exposed to damage (MPP + 1 mM iodide) for 24 hours. Cells treated with MPP + iodide alone were included as negative controls. Untreated cells were included as controls. Cells treated with deprenyl were treated as positive controls. After exposure to MPP + iodide, the protective effect of recombinant lectin protein having SEQ ID NO: 1 on mitochondrial membrane potential was evaluated using the JC-1 assay as follows: After incubation, the supernatant was discarded and 100 μl of JC1-dye solution (prepared by diluting 1 mM DMSO stock to 10 μM in 1 × PBS) was added to each well. The cells were then incubated with the dye at 37°C for 15 minutes in a CO2 incubator. After 15 minutes of incubation, the supernatant was removed and the cells were washed twice with 1 × PBS (phosphate-buffered saline). Finally, 100 μl of 1 × PBS was added to each well. Red fluorescence (excitation 550 nm, emission 600 nm) and green fluorescence (excitation 485 nm, emission 535 nm) were measured using a Biotek Synergy HT plate reader. Mitochondrial membrane potential (Δψm) was calculated as the ratio of the intensity of red fluorescence to the intensity of green fluorescence, as described below: Δψm = Red fluorescence intensity / Green fluorescence intensity

[0145] The percentage increase in MPP + recovery of mitochondrial membrane potential in response to iodide damage was calculated as follows: Increase % = [(RX) / R] * 100 In the formula, X = Sequence ID 1 + MPP + Δψm corresponding to iodide-treated cells. R = Δψm corresponding to control cells (MPP + iodide damage alone)

[0146] [Table 2]

[0147] (Example 2b) Determination of the effect of recombinant lectin protein with SEQ ID NO: 1 on annexin-V staining. After incubation, cells were treated with recombinant lectin protein having SEQ ID NO: 1 at concentrations ranging from 0.001 μg / ml to 1 μg / ml for 24 hours. After 24 hours of treatment, cells were exposed to damage (MPP + 1 mM iodide) for 24 hours. Cells treated with MPP + iodide alone were included as negative controls. Untreated cells were included as controls. Cells treated with deprenyl were treated as positive controls. After treatment, cells were harvested by trypsin treatment and processed for the Annexin V assay as follows: Cells were gently collected in pre-labeled sterile centrifuge tubes and centrifuged at 300 × g for 5-7 minutes. After 24 hours of treatment, cells were exposed to damage (MPP + 1 mM iodide) for 24 hours. Cells treated with MPP + iodide alone were included as controls. Untreated cells were included as negative controls. Cells treated with deprenyl were treated as positive controls. After processing, cells were collected by trypsin treatment and processed for the Annexin-V assay as follows: Cells were gently collected in pre-labeled sterile centrifuge tubes and centrifuged at 300×g for 5-7 minutes. The supernatant was discarded and the pellet was resuspended in 200 μl of fresh culture medium. 100 μl of the cell suspension was transferred to pre-labeled sterile centrifuge tubes. 100 μl of Annexin-V reagent was added to each tube and incubated in the dark at room temperature for 30 minutes. The Annexin-V stained cells were then transferred to 96-well plates and data were obtained using a flow cytometer (Guava Technologies) (acquired). The percentage of Annexin-V positive cells was determined.

[0148] Percentage of apoptotic cell inhibition = [(Annexin-positive cells in MPP + iodide alone) - (Annexin-positive cells in recombinant lectin with SEQ ID NO: 1 + MPP + iodide) / Annexin-positive cells in MPP + iodide alone] * 100

[0149] [Table 3]

[0150] (Example 2c) Anti-apoptotic effect of recombinant lectin protein SEQ ID NO: 1 in neurons (SH-SY5Y) via reduction of sub(G0 / G1) cell population against MPP+ iodide-induced injury. After incubation similar to that in Examples 2a and 2b, cells were harvested by trypsin treatment and processed for a cell cycle assay as follows: The cell cycle reagent included PI staining, which stains the DNA of cells at different stages of the cell cycle: sub(G0 / G1), G1, S, G2, and M. Cells in the sub(G0 / G1) stage correspond to apoptotic cells.

[0151] Cells were gently collected in pre-labeled centrifuge tubes and centrifuged at 450 g for 5 minutes at room temperature (low brake). The supernatant was carefully removed and discarded. 1 ml of 1×PBS was added to the pellet and gently resuspended to a homogeneous suspension. Cells were centrifuged at 450 g for 5 minutes at room temperature (low brake). The supernatant was carefully removed, leaving approximately 100 μl of PBS. Cells were gently but completely resuspended in the remaining PBS. Cells were fixed by adding 100 μl of ice-cold 70% ethanol dropwise to the cells in each tube while vortexing at a low speed. Cells were stored at 4°C for 24 hours before staining.

[0152] Cells fixed with ethanol were centrifuged at 450g for 5 minutes at room temperature (low brake), and the supernatant was carefully removed (without touching the pellet) and discarded. 1 ml of 1×PBS was added to the pellet and gently resuspended. Cells were incubated at room temperature for 1 minute. Cells were centrifuged at 450g for 5 minutes at room temperature (low brake). (Washing step) The supernatant was carefully removed, leaving approximately 20 μl to 50 μl of PBS. 200 μl of cell cycle reagent was added to each tube. Cells were gently resuspended and mixed. Cells were incubated at room temperature in the dark for 30 minutes. Stained samples were transferred to 96-well plates and data was acquired using a flow cytometer (Guava Technologies). The percentage of cells in the sub(G0 / G1) phase was determined.

[0153] Percentage of apoptotic cell inhibition = [(Sub(G0 / G1) cells with MPP + iodide alone) % - (Sub(G0 / G1) cells with SEQ ID NO: 1 + MPP + iodide %) / Sub(G0 / G1) cells with MPP + iodide alone %] * 100

[0154] [Table 4]

[0155] (Example 3) Evaluation of the effect of Sequence ID No. 1 on cognitive health in Parkinson's disease using an in vitro neurite proliferation assay. We studied cognitive health related to neurite proliferation in Parkinson's disease using the following methods.

[0156] (Example 3a) Effect of Sequence ID No. 1 on neurite formation in the basic model neuron (pc12) Use a hemocytometer to count cells, 1 × 10 4Cells were plated into 24-well plates at a density equivalent to 500 μl of growth medium per well. The cells were then incubated for 48 hours in a 5% CO2 incubator at 37°C. The effect of recombinant lectin containing SEQ ID NO: 1 on neurite formation was evaluated in both the basic model and the MPP+ injury-inducible model.

[0157] For the basic model, the percentage increase in neurite formation was determined as follows. Increase % = [(Number of neurites in treated cells - Number of neurites in untreated cells) / Number of neurites in untreated cells] × 100

[0158] [Table 5]

[0159] (Example 3b) Protective effect of Sequence ID No. 1 on neurite formation in nerve cells (pc12) against MPP+-induced injury. The percentage of protection in neurite formation against MPP+-induced injury was determined as follows: {(AB) / (CB)}*100 During the ceremony, A = Number of neurites in cells treated with NGF + Sequence ID 1 + MPP Number of neurites in cells treated with B=NGF+MPP+ Number of neurites in cells treated with C=NGF alone

[0160] [Table 6]

[0161] (Example 4) Elucidation of the mechanism of action of recombinant lectin protein containing SEQ ID NO: 1 in Parkinson's disease and Alzheimer's disease through multiplex analytical culture and maintenance of cell lines. A. Estimation and evaluation of markers by multiple analysis / ELISA After incubation overnight as in previous examples, cells were treated with recombinant lectin protein having SEQ ID NO: 1 for 24 hours at various concentrations ranging from 0.001 μg / ml to 1 μg / ml for Parkinson's disease, and for 24 hours at various concentrations ranging from 0.01 μg / ml to 25 μg / ml for Alzheimer's disease. • Parkinson's disease: Cells were pretreated with recombinant lectin protein containing Sequence ID No. 1 for 24 hours, followed by a further 24-hour exposure (MPP+). Cells treated with MPP+ iodide were included as a control. Cells treated with deprenyl were included as a positive control. • Alzheimer's disease: Cells were pretreated with recombinant lectin protein containing SEQ ID NO: 1 for 24 hours, followed by further exposure to scopolamine (4 mM) for 24 hours. Cells treated with scopolamine were included as a control. Cells treated with galantamine were included as a positive control.

[0162] Marker levels were determined by multiplex analysis as follows: Cell culture supernatant was diluted with calibrator diluent (1:2). 50 μl of standard substance or sample was added per well. 50 μl of microparticle cocktail was added to each well of the microplate and covered with a foil plate sealer. The plate was incubated at room temperature for 2 hours on a horizontal orbit microplate shaker. The plate was washed using a magnetic device designed for microplates. Washing was performed by applying a magnet to the bottom of the microplate, allowing it to stand for 1 minute before removing the liquid, filling each well with washing buffer (100 μl), allowing it to stand for 1 minute before removing the liquid again. 50 μl of diluted biotin-antibody cocktail was added to each well. The plate was covered with a foil plate sealer and incubated at room temperature for 1 hour on a shaker. The washing process was repeated. 50 μl of diluted streptavidin-PE was added to each well. The plate was tightly covered with a foil plate sealer and incubated at room temperature for 30 minutes on a shaker. The washing process was repeated. Microparticles were resuspended by adding 100 μl of washing buffer to each well. The plates were incubated on a shaker for 2 minutes and read within 90 minutes using a Magpix® multiplex instrument. Levels of biomarkers for Parkinson's disease and Alzheimer's disease were estimated and evaluated using a Magpix® multiplex instrument.

[0163] For Parkinson's disease, the modulation percentage for each sample was determined as follows: [{Concentration of biomarker in MPP + iodide-treated cells (pg / ml)) - Concentration of biomarker in control cells (MPP + iodide alone) (pg / ml)} / Concentration of biomarker in control cells (MPP + iodide alone) (pg / ml)] * 100

[0164] For Alzheimer's disease, the modulation percentage for each sample was determined as follows: Change % = [(Concentration of biomarker in control cells - Concentration of biomarker in control cells) / Concentration of analyte in control cells] × 100

[0165] result:

[0166] [Table 7]

[0167] [Table 8]

[0168] In vivo research:

[0169] (Example 5) Recombinant lectin protein having SEQ ID NO: 1 The required amount of recombinant lectin protein having SEQ ID NO: 1 was diluted in sterile Tris buffered saline (TBS) to the desired final concentrations, i.e., 0.1 mg / mL, 0.05 mg / mL, and 0.025 mg / mL, respectively, at doses of 0.5 mg / kg, 0.25 mg / kg, and 0.125 mg / kg. The formulation was prepared fresh daily.

[0170] The reference drug, donepezil hydrochloride, was suspended in 0.5% Na-CMC to obtain a final concentration of 0.25 mg / mL (dose: 2.5 mg / kg; dose volume: 10 mL / kg).

[0171] TBS buffer and 0.5% CMC were used as vehicles for preparing the recombinant lectin containing the test item, SEQ ID NO: 1, and the reference item formulation, respectively.

[0172] Male mice (Mus musculus) (Swiss albino) aged 8-10 weeks were obtained from GENTOX Bio services GmbH, Hyderabad. The animals were divided into six groups and allowed to acclimate for two weeks. Animals were identified by cage labeling and tail marking, and then randomized based on body weight.

[0173] Healthy male Swiss albino mice (n=48) were selected and randomized into six groups based on body weight (n=8 per group), as described in Table No. 1. Control group G1 was treated intravenously daily on a vehicle until completion of the experiment. Group G2 was considered a negative control and was treated on a vehicle. Group G3 was orally treated with the standard compound donepezil hydrochloride at a dose of 2.5 mg / kg. Animals in groups G4, G5, and G6 were treated with the test item, namely recombinant lectin protein with SEQ ID NO: 1, at doses of 0.5 mg / kg, 0.25 mg / kg, and 0.125 mg / kg, respectively. All test items were administered intravenously (iv) daily at a dose volume of 5 ml / kg for 14 days. Body weight was recorded daily throughout the entire experimental period. All animals were observed for clinical signs throughout the study. One hour after the final dose of the test product (day 14), all animals except the normal control group G1 received an intravenous injection of scopolamine hydrobromide at a dose of 2.5 mg / kg. A passive avoidance test was performed 30 minutes after the scopolamine injection.

[0174] [Table 9]

[0175] (Example 5a) Passive avoidance test This learning and memory test was conducted in two chambers containing identically sized square boxes placed side-by-side as a lit chamber and a dark chamber. A lamp was placed above one of the chambers for illumination. Each test consisted of two separate trials: a training trial and an examination trial.

[0176] For the training trial, mice were initially placed in the illuminated chamber. When a mouse entered the dark chamber, it received a 3-second electric shock (0.5mA) through a stainless steel rod. The waiting time after the mouse returned from the illuminated compartment to the dark compartment was recorded using a built-in timer. The test trial followed the training trial for 24 hours, measuring the waiting time before re-entering the dark chamber up to a maximum of 5 minutes.

[0177] [Table 10]

[0178] (Example 5b) Brain collection and estimation / evaluation of brain biomarkers After a passive avoidance test to screen memory models, the animals were humanely slaughtered. The entire brain was carefully removed from the skull and weighed. The brain was dissected and divided into two parts.

[0179] A part of the brain: A 10% w / v brain homogenate (100 mg / mL) was prepared by homogenization in ice-cold phosphate buffer. Subsequently, the homogenate was centrifuged using a cooled centrifuge at 3000 rpm for 10 minutes, and the supernatant was separated and used for biochemical estimation.

[0180] The following biomarkers were estimated and evaluated using an ELISA kit according to the manufacturer's instructions. • Brain NGF (Nerve Growth Factor), CUSABIO, Catalog Number CSB-E04684m • Acetylcholinesterase, CUSABIO, catalog number CSB-E17521m TNF Alpha, CUSABIO Corporation, catalog number CSB-E04741m

[0181] [Table 11]

[0182] [Table 12]

[0183] [Table 13]

[0184] (Example 5c) Histopathological examination Mouse brains were collected after humane slaughter and fixed in 10% neutral buffered formalin. Brain tissue was then excised, processed, and embedded in paraffin. 5-micron sections were prepared on slides for hematoxylin-eosin (H&E) staining. Hippocampal lesions were evaluated microscopically at 100x magnification.

[0185] result: Detailed histological investigations were performed on H&E-stained brain tissue. Scoring was performed according to neuronal damage. The results of the histopathological evaluation showed no significant changes in the cerebral cortex and hippocampal region excluding the CA3 region in all treatment groups and the scopolamine injection group. The criteria for classifying the histopathological examination scores were as follows: normal or no injury - 0; rare neuronal injury (<5 clusters) - 1; occasional neuronal injury (5-15 clusters) - 2; frequent neuronal injury (>15 clusters) - 3; generalized neuronal injury - 4.

[0186] [Table 14]

Claims

1. A pharmaceutical composition for the treatment or prevention of neurodegenerative diseases comprising a recombinant lectin protein, wherein the recombinant lectin protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90% identity with SEQ ID NO: 1, and has a cytoprotective effect on nerve cells.

2. The pharmaceutical composition according to claim 1, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, symptoms associated with dementia, Huntington's disease, prion disease, Creutzfeldt-Jakob disease, Lewy body disease, diffuse Lewy body disease (DLBD), polyglutamine (PolyQ) repeat disease, cerebral degenerative disease, spinal and bulbar muscular atrophy (SBMA), ataxia, Pick's disease, primary progressive aphasia, multiple system atrophy, pantothenate kinase-associated neurodegeneration (PANK), spinal degenerative disease / motor neuron degenerative disease, hippocampal sclerosis, corticobasal degeneration, Batten's disease, motor neuron-like amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), primary lateral sclerosis (PLS), progressive bulbar palsy (PBP), pseudobulbar palsy, and hereditary spastic paraplegia, which are variants of ALS.

3. The pharmaceutical composition according to claim 2, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, dementia, cognitive impairment, and symptoms associated with dementia.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the effective amount of recombinant lectin protein administered for the treatment or prevention of neurodegenerative diseases is within the range of 0.01 mg / kg to 1000 mg / kg of target body weight.

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