WO2011021916A2 - Utilisation du peptide à domaine de type egf de l'héréguline bêta 1 - Google Patents

Utilisation du peptide à domaine de type egf de l'héréguline bêta 1 Download PDF

Info

Publication number
WO2011021916A2
WO2011021916A2 PCT/KR2010/005589 KR2010005589W WO2011021916A2 WO 2011021916 A2 WO2011021916 A2 WO 2011021916A2 KR 2010005589 W KR2010005589 W KR 2010005589W WO 2011021916 A2 WO2011021916 A2 WO 2011021916A2
Authority
WO
WIPO (PCT)
Prior art keywords
memory
cells
preventing
hrgβe
composition
Prior art date
Application number
PCT/KR2010/005589
Other languages
English (en)
Korean (ko)
Other versions
WO2011021916A3 (fr
Inventor
김윤희
유민주
허훤
Original Assignee
경희대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 경희대학교 산학협력단 filed Critical 경희대학교 산학협력단
Publication of WO2011021916A2 publication Critical patent/WO2011021916A2/fr
Publication of WO2011021916A3 publication Critical patent/WO2011021916A3/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the use of the EGF-like domain peptide of hiregulin beta1 to improve memory or to prevent or treat memory impairment diseases.
  • Mammalian brain has a complex function by securing a number of cells by proliferation of neural precursor cells and generating a systematic neural network through a series of processes such as differentiation, survival, death and synapse formation. Will be able to perform Recently, neural progenitor cells have been identified in adults, and the process of stem cell proliferation and differentiation (neurogenesis) in the adult brain can be called regeneration (Identification of a neural stem cell in the adult mammalian). central nervous system (1999), Cell 96, 25-34). Neural progenitor cells are found mainly in the subventricular zone of the striatum, which is in contact with the lateral ventricle, and also in the subventricular zone in the dentate gyrus of the hippocampus.
  • Neuronal neurogenesis in the adult hippocampus (2002) Nature 415, 1031-1034.
  • neuronal progenitor cells proliferate in some parts of the brain, such as the hippocampus, to replace dead cells.
  • Neuronal cell proliferation occurs in areas affected by degenerative brain disease or brain damage, which promotes neurogenesis. Should be.
  • the adult brain produces many substances necessary for nerve growth, grows axons and dendrites, and continuously remodels synaptic connections and neural networks with new learning and memory. Continues.
  • the proliferation and differentiation of neural progenitor cells in the adult brain occurs mainly in the hippocampus, the hippocampus that forms spatial perception and memory, and dementia patients initially have entorhinal coetex and cholinergic neurons and glutamate neurons in the hippocampus. (Glutamatergic neurons), GABAergic neurons (regulating their activity) are killed by the loss of spatial memory. In addition, nerve cell death caused by cramps and ischemia in the hippocampus causes memory loss in epilepsy and stroke.
  • nerve cells die when they do not receive a target-derived survival factor, such as nerve growth factors.
  • a target-derived survival factor such as nerve growth factors.
  • Cell death by stress and cytotoxic agents is degenerative. It is a major cause of brain disease.
  • Degenerative brain diseases in which death of neuronal cells is a major cause include various diseases such as dementia, Parkinson's disease, and Alzheimer's disease. Most degenerative brain diseases are accompanied by memory impairment (memory loss), and memory deterioration phenomenon is appearing in the general public due to deterioration of living environment such as stress and pollution caused by complicated social activities.
  • the most likely candidate for neuroregeneration is the neurotrophic factor. Therefore, it is possible to promote neuronal regeneration by securing the number of cells by promoting the proliferation of neural progenitor cells using nerve growth factors and promoting their differentiation.Based on this, the survival and death of neurons, migration, synapse formation, etc. Modulation may help to restore memory and improve memory.
  • Nerve growth factors present in the nervous system generally induce neuronal responses by activating cell membrane receptors to regulate signaling.
  • neuroregulin is a structurally related gene family and is expressed in over 30 different forms by alternative splicing (Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system (1993) Nature 362, 312-318; Neuregulins: functions, forms, and signaling strategies (2003) Exp. Cell Res. 284, 14-30).
  • Neuregulin is divided into three types, Type I, Type II, and Type III, among which Type I Neuregulin is called NDF (Neu differentiation factor), HRG (Heregulin), ARIA (Acetylcholin receptor inducing activity), etc.
  • Type I nuregulin is collectively called hiregulin).
  • Type II nuregulin is called GGF (glial growth factor) and is mainly expressed in the late development of nervous system, and type III nuregulin is called sensory and motor neuron derived facto (SMDF) and Cytoin rich domain (CRD) nuregulin.
  • GGF glial growth factor
  • CMD Cytoin rich domain
  • Hiregulin is divided into hiregulin ⁇ or ⁇ by the ⁇ or ⁇ form domain next to the EGF-sexual domain, where ⁇ is found in non-neuronal cells and ⁇ is in nerve cells.
  • Hiregulin induces the death and proliferation of Schwann cells and glial cells in the central and peripheral nervous system, survival and migration and differentiation of neurons, and axon growth (Role of neuregulins in glial cell development (2000) Glia 29, 104- 111; GDNF family ligands activate multiple events during axonal growth in mature sensory neurons (2004) Mol. Cell. Neurosci. 25, 453-459).
  • neural progenitor cells In the disease of the nervous system, the overall research of neuroscience about the study of brain function is still in a poor state, and the development of therapeutic drugs for various chronic nervous system diseases is also in trouble.
  • the use of neural progenitor cells is a revolutionary new treatment of the medical community that treats diseases in a completely different perspective, and can promote differentiation into neuronal cell types suitable for transplantation.
  • direct injection of gene therapy that promotes the proliferation and differentiation of neuronal progenitor cells could make this treatment a reality and popularization.
  • the present invention provides a composition for improving memory or preventing or treating memory impairment disorders comprising a nucleic acid encoding the EGF-like domain peptide of hyregulin beta1.
  • the present invention provides a composition for improving memory or preventing or treating memory impairment disorders comprising the EGF-like domain peptide of hiregulin beta1.
  • the present invention also provides a DNA sequence encoding the EGF- sex domain peptide of hyregulin beta1; And a recombinant adenovirus comprising a DNA sequence encoding a secretory indication peptide, the composition for improving memory or preventing or treating a memory damage disease.
  • the present invention also provides a DNA sequence encoding the EGF- sex domain peptide of hyregulin beta1; And a mesenchymal stem cell incorporating a recombinant adenovirus containing a DNA sequence encoding a secreted indication peptide, and a composition for preventing or treating memory impairment.
  • EGF-domain domain peptide of the hiregulin beta 1 can improve memory by inducing differentiation into neurons necessary for the proliferation and memory formation of neural progenitor cells and can be used for the prevention or treatment of memory damage diseases. .
  • Figure 1 is a graph quantified by analyzing the amount of HRG ⁇ E released extracellularly after introduction of HRG ⁇ E-Ad to human bone marrow mesenchymal stem cells by ELISA.
  • FIG. 2 is a micrograph showing cell proliferation after treatment with LacZ, sHRG ⁇ E, and HRG ⁇ E neutralizing antibodies using BrdU immunostaining
  • FIG. 3 is a graph showing the quantification thereof.
  • Figure 4 is a schematic diagram showing the process of experimenting the proliferation and regeneration effect of the endogenous neuronal progenitor cells after transplanting the adenovirus-infected mesenchymal stem cells in the animal model of memory impairment.
  • 5 is a diagram showing the schematic diagram of the mesenchymal stem cell transplantation site and the site and shape of the human mesenchymal stem cell transplanted by fluorescent labeling.
  • 6 and 7 show the results of proliferation of endogenous neural progenitor cells after transplanting adenovirus-infected mesenchymal stem cells into a memory impaired animal model.
  • 6 is a micrograph showing cell proliferation by BrdU immunostaining
  • FIG. 7 is a graph showing the quantification thereof.
  • FIGS. 8 and 9 show the results of early neuronal differentiation of endogenous neural progenitor cells proliferated after transplantation of adenovirus infected mesenchymal stem cells.
  • Figure 8 is a micrograph showing early neuronal differentiation
  • Figure 9 is a table showing the measurement of the number of cells expressing each marker.
  • 10 and 11 show the results of observing the survival and differentiation of neural progenitor cells after 4 weeks of proliferating endogenous neural progenitor cells after transplantation of adenovirus infected mesenchymal stem cells.
  • 10 shows that proliferated neuronal progenitor cells move to each hippocampal region and survive.
  • FIG. 11 shows neurotransmitters of neural progenitor cells proliferated as a result of performing fluorescent tissue staining with markers for each neuronal cell type. It has been shown to differentiate into a glutamate- or gaba-like neuron type secreting the substance glutamate or gaba.
  • FIG. 12 is a schematic diagram showing a process of performing differentiation and memory test of mesenchymal stem cells 10 weeks after transplanting human mesenchymal stem cells infected with adenovirus into a memory impaired animal model.
  • Figures 13 and 14 show the results of observing the differentiation of mesenchymal stem cells 10 weeks after transplanting human mesenchymal stem cells infected with adenovirus into the animal model of memory impairment.
  • Figure 13 is a micrograph showing the results of staining with markers for each neuronal cell type
  • Figure 14 is cholinergic and Gabbasi to secrete acetylcholine, GABA, which is a neurotransmitter necessary for memory formation of transplanted mesenchymal stem cells Neuronal cell differentiation.
  • Figures 15 and 16 show the results of memory recovery after transplantation of adenovirus infected human mesenchymal stem cells.
  • 15 is a graph showing the results of the Y-shaped maze test for testing the short-term spatial memory
  • Figure 16 is a graph showing the passive avoidance response results for testing long-term memory.
  • the present invention provides a composition for improving memory or preventing or treating memory impairment diseases, including a nucleic acid encoding an EGF-like domain peptide of hyregulin beta1 (hereinafter referred to as HRG ⁇ E).
  • HRG ⁇ E EGF-like domain peptide of hyregulin beta1
  • HRG ⁇ E has an effect on the migration, differentiation, neurite outgrowth and regeneration of peripheral nervous system neurons, and thus it is effective in the treatment of peripheral nervous system neuropathy diseases. 0765496).
  • HRG ⁇ E has the effect of inducing the proliferation, survival, and differentiation of neuronal progenitor cells of the central nervous system, and more surprisingly, HRG ⁇ E has the effect of improving memory.
  • mammalian brains perform complex functions by obtaining a number of nerve cells and generating systematic neural networks through a series of processes, such as movement, differentiation, survival and death, and synapse formation, to appropriate locations within organs.
  • HRG ⁇ E has a therapeutic effect of memory enhancement and memory impairment disease is the discovery of surprising effects that could not be predicted at all from previous studies.
  • the present inventors transplanted mesenchymal stem cells into which a recombinant adenovirus containing a DNA sequence encoding HRG ⁇ E (hereinafter referred to as HRG ⁇ E-Ad) into the brain of rats through the following examples, to proliferate neuronal progenitor cells, It was confirmed that there was an effect of promoting differentiation and improving memory. That is, as a result of HRG ⁇ E-Ad infection of human bone marrow mesenchymal stem cells, HRG ⁇ E-Ad synthesized HRG ⁇ E and secreted it extracellularly, and co-cultured HRG ⁇ E-Ad-infected mesenchymal stem cells with neuronal progenitor cells.
  • HRG ⁇ E-Ad recombinant adenovirus containing a DNA sequence encoding HRG ⁇ E
  • HRG ⁇ E induces proliferation of neural progenitor cells.
  • transplantation of the HRG ⁇ E-Ad-infected mesenchymal stem cells into the brain of a rat model of brain disease resulted in proliferation of endogenous neural progenitor cells, survival of proliferating cells, and differentiation into neurons. Differentiation into glutamate, Gaba and cholinergic cells required for neuronal cell types.
  • the memory test results of the brain injury model rats completed the present invention by obtaining a result that the memory is improved.
  • the nucleic acid encoding the HRG ⁇ E may be a nucleic acid having a nucleotide sequence of SEQ ID NO: 1, but not limited to.
  • the nucleotide sequence of SEQ ID NO: 1 or more than 90% homology includes a nucleic acid encoding a variant peptide having an activity of inducing the proliferation of neural precursor cells, for example, GGF (glial growth factor), NDF ( Nucleic acids encoding other types of rat or human hyregulin alpha / beta EGF-like domain peptides such as Neu differentiation factor (ARIA), Acetylcholin receptor inducing activity (ARIA), or sensory and motor neuron derived factor (SMDF).
  • GGF glial growth factor
  • NDF Nucleic acids encoding other types of rat or human hyregulin alpha / beta EGF-like domain peptides such as Neu differentiation factor (ARIA), Acetylcholin receptor inducing activity (ARIA), or sensory and motor
  • the present invention also provides a composition for improving memory or preventing or treating memory impairment diseases, including HRG ⁇ E.
  • the HRG ⁇ E may be a peptide having an amino acid sequence of SEQ ID NO: 4, but not limited to.
  • a variant peptide having an activity that induces the proliferation of neural precursor cells for example, GGF (glial growth factor), NDF (Neu differentiation factor) , EGF-like domain peptides of other types of rat or human hyregulin alpha / beta such as Acetylcholin receptor inducing activity (ARIA), and sensory and motor neuron derived factor (SMDF).
  • GGF glial growth factor
  • NDF Neuro differentiation factor
  • ARIA Acetylcholin receptor inducing activity
  • SMDF sensory and motor neuron derived factor
  • the present invention also provides a composition for improving memory or preventing or treating memory impairment disorders comprising a recombinant adenovirus (HRG ⁇ E-Ad) comprising a DNA sequence encoding HRG ⁇ E.
  • a recombinant adenovirus HRG ⁇ E-Ad
  • the recombinant adenovirus may further comprise a DNA sequence encoding a secretion signal peptide (signal peptide).
  • the secretion indicator peptide may be a peptide having an amino acid sequence of SEQ ID NO: 5
  • the DNA sequence encoding the secretion indicator peptide may have a sequence of SEQ ID NO: 2, but is not limited thereto It is not intended to be construed as including variants or fragments thereof having substantially the same activity.
  • the recombinant adenovirus of the present invention may have a nucleotide sequence in which the DNA sequence encoding the HRG ⁇ E and the DNA sequence encoding the secretion indicating peptide. More specifically, but not limited thereto, the nucleotide sequence of the DNA sequence encoding the HRG ⁇ E and the DNA sequence encoding the secretion indicator peptide may be the nucleotide sequence of SEQ ID NO: 3, HRG ⁇ E and secretion indicator peptide is fused The peptide may have an amino acid sequence of SEQ ID NO: 6, but is not limited thereto, and may be interpreted to include variants or fragments thereof having substantially the same activity.
  • the recombinant adenovirus of the invention may further comprise a viral promoter capable of expressing a DNA sequence, wherein the viral promoter may be a CMV promoter.
  • the recombinant adenovirus of the present invention may be deficient in parts of the genome required for replication in a target cell, wherein the deficient part is E1, E3, E4, and L1. It may be selected from the -L5 gene, preferably may be lacking the E1 gene.
  • the recombinant adenovirus used in the embodiment of the present invention is a replication defective viral vector in which the E1B region has been deleted, and has recently been studied to obtain a therapeutic effect on a disease site by injecting a therapeutic gene into a neurological disease ( Viral vectors for modulating expression in neurons (1996) Curr. Opin. Neurobiol. 6, 576-583; Adenovirus vectors for gene delivery (1999) Curr. Opin. Biotech .. 10, 440-447).
  • adenovirus is that finally even possible to introduce different types of cells differentiated as nerve or muscle cells, unlike retroviruses to be introduced only in dividing cells (Gene delivery into the brain using virus vector (1993) Nature genetics 3 , 187-189; Gene therapy for cerebral vascular disease (1997), Stroke 27, 534-539).
  • the cytotoxicity is low compared to other viral mediators, it is safe and can be purified at a high concentration, it has a relatively large capacity of 8kb and is easy to produce.
  • the recombinant adenovirus used in the present invention is 100% introduced into mesenchymal stem cells and disappears after 3 months because it does not intervene in the chromosome, and the low risk of insertional mutagenesis is also a great advantage. .
  • the present inventors reported that recombinant LacZ adenovirus is well introduced into HiB5 neuroprogenitor cells, is well introduced into the rat nervous system, and expressed over 6 weeks (Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy of peripheral nerve injury (2000) Mol. cells . 10, 540-545). Therefore, the recombinant adenovirus HRG ⁇ E-Adv used in the present invention can be introduced and transplanted into mesenchymal stem cells as described below, or directly used for regeneration of degenerative brain diseases involving memory disorders.
  • the present invention also provides a composition for improving memory or preventing or treating memory impairment diseases, including mesenchymal stem cells incorporating a nucleic acid encoding HRG ⁇ E.
  • the introduction of the nucleic acid may be performed using a microinjection method, an electrointroduction method, a direct injection method, a particle injection method, a liposome method or an expression vector.
  • Microinjection is not limited to this, for example, can be used stereotek microinjection, there is an advantage that it is well inserted into the genome and delivered to the offspring, regardless of the size of the DNA (50-200kb).
  • the introduction of nucleic acid by electroporation is a method commonly used in ES cells, etc. The principle is that foreign DNA enters the cell suspension due to a high voltage applied to the cell suspension for a short time and its permeability changes. .
  • Particle bombardment is a method of injecting DNA into metal cells together with metal particles by applying a desired DNA to the surface of microparticles (about 0.5-10 ⁇ m in diameter) such as gold and tungsten and spraying them into cells or tissues at high speed.
  • the liposome method is a method in which DNA is fused to a cell membrane using liposomes.
  • Expression vectors may be, but are not limited to, plasmids or viruses, preferably recombinant adenoviruses.
  • the recombinant adenovirus (HRG ⁇ E-Ad) is as described above.
  • Mesenchymal stem cells commonly known as hematopoietic stem cells in the bone marrow, have the ability to differentiate into a variety of mesodermal cells, including bone, cartilage, fat, and muscle cells. The maintenance costs and effort are relatively low compared to other undifferentiated cells.
  • mesodermal stem cells are known to have the potential to differentiate into glial cells in the brain (Kofen et al. 1999; Azizi et al. 1998), treatment of central nervous system diseases using mesenchymal stem cells as cell therapy It is presented as possible.
  • the method of delivering HRG ⁇ E using mesenchymal stem cells is not only HRG ⁇ E but also the effect of neuronal regeneration by the growth factor secreted by mesenchymal stem cells and the effect of replacing mesenchymal stem cells by neural cells. It is advantageous to have.
  • HRG ⁇ E-induced mesenchymal stem cells were more differentiated into neurons after brain transplantation, and GABAergic neurons, glutamatergic neurons, which are the types of neurons necessary for the transplantation site.
  • Cholinergic neurons cholinergic neuron
  • differentiation In particular, neurons expressing glutamic acid dehydrogenase (GAD), an enzyme that produces GABA, a neurotransmitter secreted early in neuronal differentiation, differentiated more.
  • GABA glutamic acid dehydrogenase
  • the mesenchymal stem cells can be effectively prevented or treated for memory decay due to neuronal death by differentiating into glutamate, gabb, and cholinergic neurons, which are important for memory formation in the hippocampus.
  • the mesenchymal stem cells may be, but is not limited to, bone marrow mesenchymal stem cells or umbilical cord blood mesenchymal stem cells, preferably bone marrow mesenchymal stem cells, more preferably Mesenchymal stem cells extracted from the bone marrow of the subject to which the composition is to be administered.
  • the subject includes, but is not limited to, humans, orangutans, chimpanzees, mice, rats, dogs, cattle, chickens, pigs, goats, sheep, and the like.
  • Mesenchymal stem cells extracted from the bone marrow of the subject can be proliferated through cell division, and thus, a large amount of cells can be easily obtained without the help of an oncogene.
  • compositions for improving memory or preventing or treating memory impairment diseases of the present invention can be used as a pharmaceutical composition.
  • the present invention provides the use of mesenchymal stem cells infected with nucleic acids, HRG ⁇ E peptides, HRG ⁇ E-Ad or HRG ⁇ E-Ad encoding HRG ⁇ E for the manufacture of a medicament for the prevention or treatment of amelioration or memory impairment disease;
  • Pharmaceutical compositions for preventing or treating memory enhancement or memory impairment diseases including mesenchymal stem cells infected with a nucleic acid encoding HRG ⁇ E, an HRG ⁇ E peptide, HRG ⁇ E-Ad or HRG ⁇ E-Ad; And mesenchymal stem cells infected with a therapeutically effective amount of a nucleic acid encoding HRG ⁇ E, HRG ⁇ E peptide, HRG ⁇ E-Ad or HRG ⁇ E-Ad to a subject.
  • the memory impairment disease may be a disease such as, but not limited to, dementia, Parkinson's disease, Alzheimer's disease, epilepsy, stroke, schizophrenia or traumatic brain injury.
  • diseases exhibiting memory impairment due to impaired function of neurons are well known in the art.
  • the HRG ⁇ E peptide promotes the proliferation of neuronal progenitor cells and differentiation into neurons and consequently improves or restores memory
  • those skilled in the art will recognize nucleic acids encoding the HRG ⁇ E for the prevention or treatment of diseases related to memory damage.
  • Mesenchymal stem cells infected with HRG ⁇ E peptide, HRG ⁇ E-Ad or HRG ⁇ E-Ad may be used.
  • An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to achieve the effect of improving memory or preventing or treating memory impairment disease.
  • the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently.
  • 0.1ng / kg to 10g / kg for HRG ⁇ E peptide, 0.01 for mesenchymal stem cells infected with nucleic acid encoding HRG ⁇ E, HRG ⁇ E-Ad or HRG ⁇ E-Ad when administered once or several times a day for adults, 0.1ng / kg to 10g / kg for HRG ⁇ E peptide, 0.01 for mesenchymal stem cells infected with nucleic acid encoding HRG ⁇ E, HRG ⁇ E-Ad or HRG ⁇ E-Ad. It can be administered at a dose of ng / kg ⁇ 10g / kg.
  • the 'object' includes, but is not limited to, humans, orangutans, chimpanzees, mice, rats, dogs, cattle, chickens, pigs, goats, sheep, and the like.
  • the pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants. Or solubilizers such as flavoring agents can be used.
  • the pharmaceutical composition of the present invention can be preferably formulated into a pharmaceutical composition by containing one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration.
  • Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.
  • compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. Can be.
  • compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered.
  • HRG ⁇ E-Ad used in the examples of the present invention was prepared according to the method described in the examples of Korean Patent No. 10-0765496.
  • the sequence of SEQ ID NO: 1 was used for the DNA sequence encoding HRG ⁇ E
  • the sequence of SEQ ID NO: 2 was used for the DNA sequence encoding the secretion indicating peptide.
  • the rats used for the experiment were 6 to 7 week old (190-240 g) Sprague-Dawley (CrjBgi: CD (SD) IGS, Orient, Korea), and were used in the experimental animal breeding room (20-23 ° C, day / night (12h / 12h) 1 to 2 weeks after the cycle was used in the brain transplant experiment.
  • hMSCs were labeled with fluorescent 1,1-dioctadecyl-3,3,3,3-tetra-methyllindocarbocyanine perchlorate (DiI-C18- (3)) for 2 hours at room temperature in the dark. Fluorescently labeled hMSCs were cultured in 37 ° C., 10% FBS ⁇ -MEM medium and treated for 2 hours to differentiate in 0.5% FBS ⁇ -MEM, a serum deficient condition.
  • HMSC cells were injected in a stereotactic manner using a 2.0 ⁇ l automicrosjector. After the recovery phase, the memory test was performed, and after 10 weeks, the brain was removed by perfusion.
  • Proliferating cells were labeled by intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine, Sigma) at 50 mg / kg for 3 days after transplantation of mesenchymal stem cells. Twenty four hours after the last intraperitoneal injection, brain tissue was obtained through perfusion.
  • BrdU 5-bromo-2-deoxyuridine
  • the rats that were killed by injecting carbon dioxide for about 5 minutes were opened, and about 400 ml of 4% PFA (paraformaldehyde, Sigma, USA) PBS at pH 7.25 was injected into the right ventricle.
  • An injection needle with a diameter of 1 mm connected with a peristaltic pump (manhatan company, South Korea) was used to inject the needle into the lower ventricle to allow it to pass through the artery. It burst the right atrium, preventing blood from entering the heart at the same time, while removing the blood flowing through the perfusion.
  • the brain was extracted and placed in 4 ° C., 4% PFA solution for 4 hours, and then placed in a fixed solution (30% sucrose 1 ⁇ PBS) for 10 hours to be fixed and dehydrated.
  • a fixed solution (30% sucrose 1 ⁇ PBS) for 10 hours to be fixed and dehydrated.
  • OCT compound Sakura co. Japan
  • Cryostat Cryostat
  • the storage buffer was stored in 24well and stored at 4 ° C. containing the beginning to the end of the hippocampus. Since DiI, a fluorescent dye, diffuses from the cell membrane from the moment of death of the living body, all subsequent experiments were performed within 48 hours from the time of the perfusion method.
  • Frozen sections were stored in acryl 24well, washed three times with PBS and then reacted with a 0.5% Triton X-100 in PBS solution in a stirrer for 25 minutes. After transferring the tissue to 2N HCl solution of 37 °C and reacted for 30 minutes at 37 °C. The tissues were then washed three times with PBS, followed by a 1 hour blocking step for non-specific immune responses in 10% normal serum in 3% Bovine Serum Albumin (BSA) with 0.1% Triton X-100 in PBS solution. The primary antibody was then attached at 4 ° C. for 16 hours. The antibody was used diluted with 1% BSA PBS.
  • BSA Bovine Serum Albumin
  • the antibody used at this time is BrdU (BD bioscience, 1: 500), Nestin (1: 1000), Tuj1 (Sigma, 1: 1500), GFAP (Chemicon, 1: 700), ChAT (Chemicon, 1: 500), GAD67 (Chemicon, 1: 2000), VGluT1 (Chemicon, 1: 500), and TH (Chemicon, 1: 700).
  • the secondary antibody complementary to the primary antibody was then attached for 45 minutes at room temperature. Secondary antibodies used at this time are fluorescently labeled Cy3 (Jackson, 1: 500), FITC (Chemicon, 1: 500), and Alexa488 (Molecular probe, 1: 700).
  • the number of BrdU cells was measured by counting the cells in the nucleus of the counterstained signal, and BrdU was round in the shape of a nucleus and did not count nonspecific signals at the edges or tears of tissues. Data analysis was performed using one-way batch analysis (Anova).
  • culture medium was taken 2 days after the introduction and analyzed using standard ELISA method to analyze the secretion amount of sHRG ⁇ E.
  • human bone marrow mesenchymal stem cells were planted in a 35 mm culture vessel at 3 x 10 5, and then introduced with LacZ-Ad or HRG ⁇ E-Ad for 2 hours in a medium containing 2% FBS for one hour, followed by 10% FBS medium. After 12 hours of exchange, it was finally exchanged again with 1 mL of alpha-MEM. After 24 hours, the medium was obtained and stored at ⁇ 70 ° C. until used with the protease inhibitor.
  • An ELISA kit (R & D Systems, Germany) was used to measure the amount of sHRG ⁇ E in the medium.
  • the HRG ⁇ E peptide (6.5kDa) is a very small molecular weight compared to the molecular weight of the total HRG ⁇ protein (45kDa), it can be seen that the total protein was produced at 5.6 nM, which is about 5 times when produced with peptides compared to 1.17nM.
  • neuronal progenitor cells obtained from mesenchymal stem cells and embryonic day 14 were co-cultured.
  • Human bone marrow mesenchymal stem cells were planted in a cover glass and introduced with LacZ-Ad or sHRG ⁇ E-Ad for 2 hours in a medium containing 2% FBS the next day and then exchanged with 10% FBS medium.
  • cortical neural stem cells isolated from embryonic day 14 of rats were planted in mesenchymal stem cells and incubated for 12 hours in N2 medium containing FGF, followed by N2 medium.
  • BrdU was treated with 10 uM.
  • the cells were fixed with 4% paraformaldehyde and immunostained with BrdU antibody and HuNu (human nuclear protein) antibody.
  • Neural progenitor cells require substrates such as polyornithine and polylysine and extracellular matrix proteins such as fibronectin and laminin on the surface when cultured.
  • substrates such as polyornithine and polylysine and extracellular matrix proteins such as fibronectin and laminin
  • the neural progenitor cells grew by seating the mesenchymal stem cells as a support without the stroma and extracellular matrix protein, and the nerves by HRG ⁇ E The effect of proliferating progenitor cells was observed.
  • the human mesenchymal stem cells were divided into a control group in which LacZ-Ad was introduced and a test group in which HRG ⁇ E-Ad was introduced.
  • BrdU was intraperitoneally injected for 3 days from 11 days after brain transplantation to label endogenous neural progenitor cells.
  • the brains of rats were extracted after perfusion and detected with BrdU-labeled proliferative cells by immunohistochemistry (FIG. 4).
  • Figure 5 shows a schematic diagram of the transplantation site of the mesenchymal stem cells and compared to the seated sites of the mesenchymal stem cells transplanted by fluorescent labeling. The placement and shape of the transplanted mesenchymal stem cells indicate that they are implanted at the correct site.
  • BrdU-labeled proliferating cells are mainly present in the subgranular zone (SGZ) and hippocampal white plate region of the CA1 region of the hippocampus.
  • SGZ subgranular zone
  • FIG. 7 the number of proliferating cells of neural progenitor cells transplanted with HRG ⁇ E-Ad infected mesenchymal stem cells increased more than twice in the dentate gyrus and CA1 region of the hippocampus.
  • FIG. 8 the expression of Nestin was increased and the expression of GFAT was decreased in BrdU labeled cells of the HRG ⁇ E-Ad group.
  • Figure 9 shows the quantitative expression of the differentiation cells, the number of cells expressing the nestin, a neuronal marker in the HRG ⁇ E-Ad group was increased, while the number of cells expressing GFAP expressing astrocytes in the control group Rather reduced. Therefore, it can be seen that HRG ⁇ E promotes differentiation of neural progenitor cells into early neurons, while inhibiting differentiation into glial cells.
  • the BrdU-labeled cells shown in FIG. 10 are cells that divide at 2 weeks and survived for 4 weeks, and in the HRG ⁇ E-Ad group, a large number of cells survive compared to the control group, so (stratum oriens), py ( stratum pyramidale; pyramidal layer), sr (stratum radiatum), etc. are moved to exist and can be seen that the proliferation of cells proliferated compared to the control group in the gcl (granule cell layer) of the dentate gyrus (DG) region. ..
  • VGluT1 vesicular glutamate transporter 1
  • GAD67 glutmic acid decarboxylase 67
  • choline acetyl as a marker for cholinergic neurons using acetylcholine as a neurotransmitter
  • TH tyrosine hydroxylase
  • HRG ⁇ E endogenous neural progenitor cells proliferated by HRG ⁇ E induce differentiation into glutamate neurons, GABA neurons, cholinergic neurons, and dopaminergic neurons, which are neurons that regulate memory formation in the hippocampus.
  • a blocking step for nonspecific immune responses in 15% normal serum in 3% Bovine Serum Albumin (BSA) with 0.1% Triton X-100 in PBS solution was then performed on a shaker for 2 hours and then the primary antibody was attached for 4 hours. (3 hours 4 ° C., 1 hour room temperature). At this time, the antibody was diluted with 1% BSA in PBS.
  • the antibody used was ChAT (Chemicon, 1: 500), GAD65 / 67 (Chemicon, 1: 500), GAD67 (Chemicon, 1: 2000), VGluT1 (Chemicon, 1: 500), NeuN (Chemicon, 1: 700). ), GFAP (Chemicon, 1: 700), O4 (Chemicon, 1: 700).
  • the secondary antibody complementary to the primary antibody was then attached for 45 minutes at room temperature.
  • the secondary antibodies used were fluorescently labeled Cy2 (Jackson, 1: 500) and FITC (Chemicon, 1: 500) to set different wavelength bands from Rhodamine of DiI. After the whole process was mounted and observed and photographed by confocal laser scanning microscope (LSM510, Carl Zeiss, Germany).
  • the number of transplanted hMSCs labeled with DiI on the double-photographed slides was counted, and the number of FITC cells of each of the differentiation markers ChAT, GAD65 / 67, VGluT1, NeuN, GFAP, and O4 was counted.
  • the number of differentiation markers in the hippocampus was percentaged by DiI number. At this time, the number of FITCs in the shape and position of the cells and the shape of the cells was counted.
  • FIG. 12 is a schematic diagram illustrating a process of performing differentiation experiments and memory test of mesenchymal stem cells 10 weeks after transplantation of human mesenchymal stem cells into which HRG ⁇ E-Ad or LacZ-Ad is introduced into the hippocampus of a memory impaired animal model. to be.
  • Figure 13 is a photograph showing the differentiation of transplanted mesenchymal stem cells into neurons of each type as a result of performing fluorescent tissue staining with differentiation markers for each neuron type. These cells express markers of cells constituting the central nervous system such as ChAT, GAD65 / 67, TH, NeuN, GFAP, O4 in both the HRG ⁇ E-Adv group and the control group, indicating that they differentiate into neural cells.
  • Figure 14 is a graph showing the quantitative analysis of the differentiation degree for each neuron type.
  • the differentiation of ChAT-labeled cells increased by 1.5-fold
  • the differentiation of GAD65 / 67-labeled cells by 1.8-fold and 1.3-fold in NeuN.
  • the differentiation of GFAP-labeled cells decreased by 1.8-fold and 1.5-fold in the case of O4.
  • the expression of TH a marker of dopaminergic neurons, was not significantly different between the experimental and control groups.
  • HRG ⁇ E-Ad-infected mesenchymal stem cells were observed to differentiate into cholinergic neurons and GABA neurons, which are neurons necessary for memory formation in the hippocampus when transplanted into the brain, and the number of cells differentiated into neurons (NeuN-expressing cells). The number) increases but the differentiation into astrocytic and oligodendrocytes is rather reduced.
  • Y-shaped maze experiments were performed 6 weeks after brain transplantation.
  • a Y-shaped maze is placed in a dark space where objects can be identified, and visual signs are installed around it.
  • the test rats were moved to the space, and then adjusted to the space for about 1 hour or more.
  • Each branch passage was randomly labeled, and after 8 minutes, 10 minutes, and 12 minutes, the order and number of passages into each branch passage by the rats were recorded. They give a score when entering a branch passage other than the branch passage that was selected in the previous stage. When another branch passage is selected three times in a row, the score is given and expressed as a percentage of the total selection. Rats remember the visual signs installed around them to search for a new basin. The higher the number, the higher the probability of alteration.
  • Training was the avoidance reaction to light, blocking the passage of the two distinct chambers, turning on the lights in the chamber containing the rats, and opening the passage to record the time of movement to the opposite dark chamber.
  • the training was conducted 4-6 times a day, and after 24 hours of training, the training was performed again, and the experiment was conducted when the average travel time was about 20 seconds for 4-5 days.
  • the experiment was conducted by light avoidance and moved to another chamber, giving an electric shock (1mA) for 3 seconds. After 24 hours of electric shock, they were placed in the chamber under the same conditions and the lamps were turned on, and the time when they moved to the opposite chamber was recorded. The higher this number, the more you can compare the degree of memory recovery for the stimulus.
  • HRG ⁇ E secreted from them induces proliferation of human mesenchymal stem cells and differentiation into functional neurons. It can be seen that it replaces apoptosis neurons and establishes neuronal connections and consequently contributes to memory recovery.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Psychiatry (AREA)
  • Plant Pathology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hospice & Palliative Care (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention porte sur une utilisation d'un peptide à domaine de type EGF de l'héréguline bêta 1 pour améliorer la mémoire ou pour prévenir ou traiter des maladies de défaillance de la mémoire. Le peptide à domaine de type EGF de l'héréguline bêta 1 de la présente invention induit la prolifération de cellules précurseurs neurales et la différenciation de cellules nerveuses qui produisent des neurotransmetteurs, et pourrait ainsi améliorer la mémoire et être utilisé dans la prévention ou le traitement de maladies de défaillance de la mémoire.
PCT/KR2010/005589 2009-08-21 2010-08-23 Utilisation du peptide à domaine de type egf de l'héréguline bêta 1 WO2011021916A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2009-0077802 2009-08-21
KR1020090077802A KR101186218B1 (ko) 2009-08-21 2009-08-21 히레귤린 베타1의 ｅｇｆ-성 도메인 펩타이드의 용도

Publications (2)

Publication Number Publication Date
WO2011021916A2 true WO2011021916A2 (fr) 2011-02-24
WO2011021916A3 WO2011021916A3 (fr) 2011-07-07

Family

ID=43607511

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2010/005589 WO2011021916A2 (fr) 2009-08-21 2010-08-23 Utilisation du peptide à domaine de type egf de l'héréguline bêta 1

Country Status (2)

Country Link
KR (1) KR101186218B1 (fr)
WO (1) WO2011021916A2 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018976A1 (fr) * 1997-10-14 1999-04-22 Cambridge Neuroscience, Inc. Methode therapeutiques comportant l'utilisation d'une neureguline
US20020168350A1 (en) * 2000-11-10 2002-11-14 Brazelton Timothy R. Methods for treating disorders of neuronal deficiency with bone marrow-derived cells
US20060029602A1 (en) * 1996-07-12 2006-02-09 Genentech, Inc. Gamma-heregulin
KR20070060721A (ko) * 2005-12-09 2007-06-13 경희대학교 산학협력단 히레귤린 베타1의 ｅｇｆ-성 도메인 펩타이드를 암호화하는ｄｎａ 서열을 포함하는 재조합 아데노바이러스 및 이를포함하는 신경세포 분화 및 재생용 약학 조성물
WO2007113361A1 (fr) * 2006-04-05 2007-10-11 Consejo Superior De Investigaciones Científicas Traitement de maladies causées par des altérations du développement axonal des neurones

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060029602A1 (en) * 1996-07-12 2006-02-09 Genentech, Inc. Gamma-heregulin
WO1999018976A1 (fr) * 1997-10-14 1999-04-22 Cambridge Neuroscience, Inc. Methode therapeutiques comportant l'utilisation d'une neureguline
US20020168350A1 (en) * 2000-11-10 2002-11-14 Brazelton Timothy R. Methods for treating disorders of neuronal deficiency with bone marrow-derived cells
KR20070060721A (ko) * 2005-12-09 2007-06-13 경희대학교 산학협력단 히레귤린 베타1의 ｅｇｆ-성 도메인 펩타이드를 암호화하는ｄｎａ 서열을 포함하는 재조합 아데노바이러스 및 이를포함하는 신경세포 분화 및 재생용 약학 조성물
WO2007113361A1 (fr) * 2006-04-05 2007-10-11 Consejo Superior De Investigaciones Científicas Traitement de maladies causées par des altérations du développement axonal des neurones

Also Published As

Publication number Publication date
KR101186218B1 (ko) 2012-10-08
WO2011021916A3 (fr) 2011-07-07
KR20110020090A (ko) 2011-03-02

Similar Documents

Publication Publication Date Title
US6812027B2 (en) Discovery, localization, harvest, and propagation of an FGF2 and BDNF-responsive population of neural and neuronal progenitor cells in the adult human forebrain
US7303912B2 (en) Cultures of GFAP nestin cells that differentiate to neurons
US20050003531A1 (en) Lineage-restricted neuronal precursors
US20090087851A1 (en) Lineage-Restricted Neuronal Precursors
JPH06509225A (ja) 試験管内で増殖し得る新規な成長因子応答性の前駆細胞
JP2005514926A (ja) 新規ほ乳類多分化能幹細胞および組成物、その調製方法および投与方法
Cunningham et al. Nerve growth factor released by transgenic astrocytes enhances the function of adrenal chromaffin cell grafts in a rat model of Parkinson's disease
US9649358B2 (en) Methods for use of neural stem cell compositions for treatment of central nervous system lesions
CN111484977A (zh) 重编程产生功能性去甲肾上腺素能神经元的方法
KR20120003098A (ko) ＦｏｘＡ2 및 Ｎｕｒｒ1 도입을 이용한 중뇌 도파민성 신경세포로의 분화방법
US20040115808A1 (en) Enteric nervous system derived stem and progenitor cells and uses thereof
WO2011021916A2 (fr) Utilisation du peptide à domaine de type egf de l'héréguline bêta 1
US7943376B2 (en) Platelet derived growth factor (PDGF)-derived neurospheres define a novel class of progenitor cells
WO2019190175A2 (fr) Méthode pour la différenciation de neurones moteurs à partir de cellules souches mésenchymateuses dérivées de tonsil
US20040147020A1 (en) Dopamine neurons from human embryonic stem cells
Aharoni et al. Transplanted myogenic progenitor cells express neuronal markers in the CNS and ameliorate disease in experimental autoimmune encephalomyelitis
WO2015080376A1 (fr) Procédé pour différencier des cellules nerveuses et des cellules ciliées et des cellules souches mésenchymateuses dérivées du chorion placentaire ou de la gelée de wharton
US20120308530A1 (en) Materials Composition and Methods to Control Neural Progenitor and Stem Cell Attachment, Proliferation and Guide Cell Differentiation
EP1231263A1 (fr) O-2A progeniteurs cellules multipotentes de neurohypophysis
구교연 Transplantation of Ngn2-expressing human neural stem/progenitor cells into hypoxic-ischemic injured neonatal mouse brain
AU2002228196A1 (en) Enteric nervous system derived stem and progenitor cells and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10810224

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10810224

Country of ref document: EP

Kind code of ref document: A2