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    • 专利摘要:
    Composition capable of promoting the formation of neurons in culture and in brain lesions. This invention is related to the use of the compound 7,8,12-tri-O-acetyl-3-O- (4-methoxyphenyl) acetylingol (hereinafter EOF2) or a pharmacologically active salt of said compound, to promote the differentiation to neurons of neural precursors or neural stem cells in culture and in brain lesions. Additionally, it is also related to the use of the same compound for the elaboration of a pharmaceutical composition useful in the treatment of CNS injuries that occur with neuronal loss. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2757606A1
    申请号:ES201800246
    申请日:2018-10-29
    公开日:2020-04-29
    发明作者:Gonzalez Carmen Castro；Galan Rosario Hernandez；Garcia Samuel Dominguez；Doldan Noelia Geribaldi；Carretero María Isabel Murillo；Sanchez Antonio José Macias；Vinuela Manuel Carrasco；Alloza Mónica Garcia
    申请人:Universidad de Cadiz；
    IPC主号:
    专利说明:

    [0001]
    [0002] Composition capable of promoting the formation of neurons in culture and in brain lesions.
    [0003]
    [0004] Technique sector
    [0005]
    [0006] This invention is related to the use of compound 7, 8,12-tri-0-acetyl-3-0- (4-methoxyphenyl) acetylingol (hereinafter EOF2) or a pharmacologically active salt of said compound, to promote the differentiation to neurons of neural precursors or neural stem cells in culture and in brain lesions. Additionally, it is also related to the use of the same compound for the elaboration of a pharmaceutical composition useful in the treatment of lesions of the central nervous system that occur with neuronal loss.
    [0007]
    [0008] Background of the Invention
    [0009]
    [0010] Brain injuries of different causes and origins are associated with irreversible loss of neurons and currently lack effective treatment. We can highlight among this type of injuries those caused by strokes, neurodegenerative diseases or trauma. These injuries involve cognitive disturbances, as well as disturbances in the motor and somatosensory systems, or behavioral and personality disturbances ( Blennow K., et al. 2012. The neuropathology and neurobiology of traumatic brain injury. Neuron 886-899; Xiong Y ., et al. 2013. Animal models of traumatic brain injury. Nature reviews. Neuroscience 128-142). Currently, the search for therapeutic options that allow to cure or at least alleviate the consequences of this type of pathology is a field of research of special relevance ( Grade S. and Gotz M. 2017. Neuronal replacement therapy: previous achievements and challenges ahead. NPJ Regen Med 29). Despite the fact that for a long time the treatment of these types of injuries has focused on rehabilitation therapies by means of which it is facilitated that other regions of the central nervous system (CNS) resume the functions of the damaged regions, recently and after the discovery of Neurogenesis in the adult brain, considerable efforts are being focused on the development of regenerative-type therapies that aim to facilitate the replacement of dead neurons from neural stem cells (NSCs). Most of the information available so far on lesion regeneration has been obtained from animal models. In rodents, it seems well established that under physiological conditions, neurogenesis exists mainly in two areas of the adult brain, the dentate gyrus of the hippocampus (DG), which generates neurons that are integrated into the hippocampal circuits, and the subventricular zone (SVZ) ( Doetsch F ., et al. 1997. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 5046-5061.; Gage FH, et al. 1995. Isolation, characterization, and use of stem cells from the CNS Annu Rev Neurosci 159-192) where neuroblasts that migrate to the olfactory bulb are produced. In the human brain, the precursors of adult SVZ also migrate to the striatum, causing mature neurons in this region ( Dayer AG, et al. 2005. New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors. The Journal of cell biology 415 427; Ernst A., et al. 2014. Neurogenesis in the striatum of the adult human brain. Cell 1072 1083; Luzzati F., et al. 2014. Quiescent neuronal progenitors are activated in the juvenile guinea pig lateral striatum and give rise to transient neurons. Development 4065-4075).
    [0011]
    [0012] Brain injuries alter the homeostasis of these neurogenic niches, activate neural stem cells in the environment of the injury ( Llorens-Bobadilla E., et al. 2015. Single-Cell Transcriptomics Reveal a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury. Cell Stem Cell 329-340) and modify the migratory routes of their progeny (reviewed in ( Grade S. and Gotz M. 2017. Neuronal replacement therapy: previous achievements and challenges ahead. NPJ Regen Med 29)), so that after damage Cells with multipotent neural precursor (NPC) characteristics appear in the vicinity of the lesion that can generate neurons in response to the lesion. In addition to this mobilization of cells from neurogenic niches, activation of NSC also occurs locally in the lesion in an attempt to generate new neurons ( Ohira K., et al. 2010. Ischemia-induced neurogenesis of neocortical yesterday 1 progenitor cells. Nat Neurosci 173-179). Unfortunately, the regenerative capacity of the CNS in injured regions is very limited in the absence of treatment ( Jin K., et al. 2001. Neurogenesis in dentate subgranular zone and rostral subventricular zone añer focal cerebral ischemia in the rat. Proc Nati Acad Sci USA 4710 -4715; Liu J., et al. 1998. Increased neurogenesis in the dentate gyrus add transient global ischemia in gerbils. J Neurosci 7768-7778; Moraga A., et al. 2014. Toll-like receptor 4 modulated cell migration and cortical neurogenesis add focal cerebral ischemia.FASEB J 4710-4718; Parent JM, et al. 1997. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus.J Neurosci 3727-3738; Romero-Grimaldi C ., et al. 2011. ADAM-17 / Tumor Necrosis Factor-alpha-Converting Enzyme Inhibits Neurogenesis and Promotes Gliogenesis from Neural Stem Cells. Stem Cells 1628-1639; Saha B., et al. 2013. Cortical lesion stimulates adult subvent ricular zone neural progenitor cell proliferation and migration to the site of injury. Stem Cell Res 965 977). This failed repair attempt may be due to two main elements: on the one hand, the inflammatory process induced by the damage, which favors the formation of a glial scar, and prevents the survival of neuroblasts that migrate from neurogenic regions and, on the other, the microenvironment Gliogenic / non-neurogenic that is generated around the lesion that prevents stem cells that are activated after damage to produce neuroblasts that differentiate themselves from mature neurons. Therefore, when it comes to regenerating brain lesions, through strategies aimed at promoting endogenous neurogenesis, there are two challenges: on the one hand, counteracting the non-neurogenic environment of the lesion and, on the other hand, facilitating the migration of neuroblasts from neurogenic regions and their survival and differentiation to functional neurons that integrate into pre-existing circuits ( Grade S. and Gotz M.
    [0013] 2017. Neuronal replacement therapy: previous achievements and challenges ahead. NPJ Regen Med 29).
    [0014]
    [0015] Whatever its origin, regeneration capacity is very low, with a neuronal replacement between 0.2 and 10% having been described, depending on the area affected and the type of injury ( Jin K., et al. 2001. Neurogenesis in dentate subgranular zone and rostral subventricular zone add focal cerebral ischemia in the rat Proc Nati Acad Sci USA 4710-4715; Liu J., et al. 1998. Inere ased neurogenesis in the dentate gyrus add transient global ischemia in gerbils. J Neurosci 7768-7778 ; Moraga A., et al. 2014. 7o // - like receptor 4 modulates cell migration and cortical neurogenesis add focal brain ischemia. FASEB J 4710-4718; Parent JM, et al. 1997. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus.J Neurosci 3727-3738; Romero-Grimaldi C., et al.
    [0016] 2011. ADAM-17 / Tumor Necrosis Factor-alpha-Converting Enzyme Inhibits Neurogenesis and Promotes Gliogenesis from Neural Stem Cells. Stem Cells 1628-1639; Saha B., et al. 2013. Cortical injury stimulates adult subventricular zone neural progenitor cell proliferation and migration to the site of injury. Stem Cell Res 965-977). The fact that there is a neurogenic response to the injury suggests that the formation of new neurons is an effective mechanism in the repair of small lesions, which remain silent precisely because the neurons that make apoptosis are replaced, at least in part, by neurons newly formed. It has been shown that in animals that have suffered a head injury, neurogenesis is increased in the dentate gyrus of the hippocampus and subsequently these animals regain their ability to perform spatial memory tasks. However, if stem cells that begin to divide in the hippocampus are selectively removed as a consequence of trauma-induced damage, these animals cannot regain the ability to perform spatial memory tasks ( Blaiss CA, et al. 2011. Temporally specified genetic ablation of neurogenesis impairs cognitive recovery after traumatic brain injury. J Neurosci 4906-4916) thus demonstrating that neuronal replacement in these regions has an effect on memory recovery after head trauma.
    [0017]
    [0018] Additionally, the appearance of neurogenesis in brain injuries has also been demonstrated in humans, specifically it has been observed that in samples of the brain of patients who underwent surgery after an injury due to head injury, they appear in the area of the cerebral cortex around the injury, proliferating neural stem cells, and neuroblasts ( Zheng W, et al. 2013. Neurogenesis in adult human brain add traumatic brain injury. J Neurotrauma 1872-1880)
    [0019]
    [0020] However, severe or experimental lesions with increased neuronal loss cannot be resolved unless effective therapeutic measures are established to significantly increase the neurogenic process. One of the alternatives that could contribute to solving the clinical problems posed by diseases that occur with neuronal loss is the transplantation of stem cells that can subsequently generate new neurons. Several laboratories have attempted this strategy in animal models to which stem cells of embryonic origin have been transplanted ( Cao et al. Pluripotent stem cells engrafted into the normal orlesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol. 167: 48 -58, 2001), neural stem cells from adult animals ( Pluchino, S. et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422: 688-694, 2003), or cells derived from cells mother subjected to different degrees of differentiation in vitro. The most generally observed phenomenon is that stem cells that are implanted in the neurogenic zones of the brain (for example, the subventricular zone or the olfactory bulb in rodents) differentiate into neuroblasts and become mature neurons, but this same phenomenon does not occur. in the non-neurogenic areas of the brain. ( Herrera, DG et al. Adult-derived neural precursors transplanted into multiple regions in the adult brain. Ann Neurol. 46: 867-77, 1999; ( Mligiliche NL, etol. 2005. Survivol of neural progenitor cells f rom the subventricular zone of the adult rat after transplantation and rite the host spinal chorus of the same strain of adult rat. AnatSci Int 229-234).
    [0021]
    [0022] Therefore one of the problems with the present invention is that, although the stem cells that are implanted in the neurogenic zones of the brain (for example, the subventricular zone or the olfactory bulb in rodents) differ from neuroblasts and become mature neurons, when the transplantation occurs in other areas, they initiate the glial differentiation route. Proliferating neural precursors appear in lesions that occur in non-neurogenic areas and could later differentiate, and the endogenous source of neural precursors in these regions is the parenchymal glia. These glial cells are activated upon injury and give rise to neural precursors, most of which differ from glial cells and very few to neurons.
    [0023]
    [0024] Taking into account the state of the art, it is necessary to find molecules with pharmacological activity that promote neurogenesis in brain lesions.
    [0025]
    [0026] Description of the Invention
    [0027]
    [0028] Brief description of the figures
    [0029]
    [0030] Figure 1: EOF2 favors differentiation to neurons and reduces differentiation to glial cells of NPCs in vitro, while EOF3 does not exert this effect.
    [0031]
    [0032] Disintegrated cells derived from neurospheres were seeded under differentiation conditions, in the absence (Control), presence of compound EOF2 (5 pM) or presence of compound EOF3 (5 pM) for 72 h.
    [0033] A. Representative fluorescence microscopy images of processed neural precursor cultures for immunohistochemical detection of the glial cell marker GFAP and the neuronal marker p-lll-tubulin. The nuclei were counterstained with DAPI.
    [0034]
    [0035] B. Effect of EOF2 and EOF3 on the percentage of GFAP + cells with respect to the control.
    [0036] C. Effect of EOF2 and EOF3 on the percentage of p-lll-tubulin + cells with respect to the control.
    [0037]
    [0038] D. Effect of compounds EOF2 and EOF3 on apoptosis related to the control. Each bar represents the mean ± standard error of at least three independent experiments. Statistics: * p <0.05 compared to the control by means of the Student's t test.
    [0039]
    [0040] Figure 2. EOF2 over promotes cell proliferation and neuroblast generation in injured cerebral cortex.
    [0041]
    [0042] Mechanical lesions were made in the primary cerebral cortex of adult mice, and they were divided into two experimental groups: through controlled release osmotic mini-pumps, some were treated locally in the lesion with the compound EOF2 (5 pM) and others received only vehicle (saline). Treatment lasted for 14 days post injury, and mice received intraperitoneal injections of BrdU (100 mg / ml) on days 12, 13, and 14 post injury. Animals were perfused and their brains processed for immunohistochemical detection. A. Fluorescence microscopy images of coronal sections of the injured cerebral cortex, processed for immunohistochemical detection of the BrdU proliferation marker and the doublecortin neuroblast marker (DCX). The calibration bar represents 50 pm and the dotted line represents the limit of the lesion. BC. The graphs represent the number of BrdU-positive cells (B) and the number of DCX marker-positive neuroblasts (C) per mm3 of injured tissue. Each bar represents the mean ± standard error of at least 4 animals per condition. Statistics: * p <0.05 compared to the vehicle using the Student's t test.
    [0043] Figure 3. EOF2 reduces the generation of glial cells after mechanical cortical injury. Mechanical lesions were made in the cerebral cortex of adult mice. The experimental groups correspond to the same ones explained in figure 2. The treatment lasted for 14 days after the injury, and the mice received intraperitoneal injections of BrdU (100 mg / ml) on days 12, 13 and 14 post injury. Animals were perfused and their brains processed for immunohistochemistry.
    [0044]
    [0045] A. Fluorescence microscopy images of coronal sections of injured cerebral cortex, processed for immunohistochemical detection of the BrdU proliferation marker and the glial cell marker GFAP. The calibration bar represents 50 pm and the dotted line represents the limit of the lesion.
    [0046]
    [0047] B. The graph shows the percentage of BrdU + cells that co-express the GFAP marker.
    [0048] C. Percentage of the injured area showing expression of the GFAP marker. Each bar represents the mean ± standard error of at least 4 animals per condition. Statistics: * p <0.05 compared to the vehicle using the Student's t test.
    [0049]
    [0050] Figure 4. EOF2 has no effect on undifferentiated cells in mechanical cortical injury.
    [0051] Mechanical lesions were performed in the cerebral cortex of adult mice. The experimental groups correspond to those explained in figure 2. The treatment lasted for 14 days after the injury, and the mice received intraperitoneal injections of BrdU on days 12, 13 and 14 post injury. Animals were perfused and their brains processed for immunohistochemistry.
    [0052] A. Fluorescence microscopy images of coronal sections of injured cerebral cortex, processed for immunohistochemical detection of the BrdU proliferation marker and the undifferentiated precursor marker nestin. The calibration bar represents 50 pm and the dotted line represents the limit of the lesion.
    [0053] B. The graph represents the number of nestin * cells per mm3.
    [0054] C. Percentage of BrdU cells expressing the undifferentiated precursor marker nestin. Each bar represents the mean ± standard error of at least 4 animals per condition. Statistics: * p <0.05 compared to the vehicle using the Student's t test.
    [0055] Figure 5. Effect of intranasal administration of EOF2 on the migration of neuroblasts from SVZ to injury.
    [0056] A. Experimental procedure to exclusively label proliferating cells with BrdU in neurogenic niches and not in the lesion.
    [0057] B. Confocal microscopy images of the peri-lesion area of mice to which EOF2 or vehicle has been administered. Tissues have been processed by immunohistochemistry to detect the BrdU proliferation marker and the DCX neuroblast marker. Calibration bar 50 pm. The dotted lines define the lesion. C. The graph shows the number of BrdU + cells / mm3 in the perimeter of the lesion.
    [0058] D. The graph shows the number of DCX + cells / mm3 at the perimeter of the lesion. No DCX + cells could be found in the vehicle treated animals.
    [0059] E. The graph shows the number of BrdU + / DCX + doubly labeled cells. No doubly labeled cells could be found at the perimeter of the lesion from vehicle treated animals.
    [0060] Detailed description of the invention
    [0061] The present invention solves the problem of generating neurogenic niches in both neurogenic and non-neurogenic areas of the CNS, based on the use of the compound 7, 8,12-tri-0-acetyl-3-0- (4-methoxy phenyl) acetyl ingol (hereafter EOF2)
    [0062]
    [0063] This compound favors neuronal differentiation from neural stem cells and, perfused into the injured brain (eg, via intranasal administration), promotes neuronal differentiation of activated NSCs in the perilesion region in response to damage and can facilitate the repair of damaged tissue.
    [0064] This new drug allows to overcome the non-neurogenic environment that is generated in injured regions of the adult brain and favor the generation of new neurons in these regions from neural precursors, either endogenous or transplanted. Therefore, when regenerating lesions, in the non-neurogenic region of the injured area, EOF2 increases the probability that endogenous stem cells such as transplanted ones can give rise to mature and functional neurons.
    [0065] Therefore, a first aspect of the present invention comprises the use of the compound EOF2 with CAS number 944799-47-7 and whose chemical structure was previously shown, or pharmacologically active salts thereof or an enantiomeric mixture that includes the compound of the formula I or its salts, and where the compound of formula I is in a percentage greater than or equal to 50% of the total, for the preparation of a medicine (hereinafter the pharmaceutical composition of the present invention), where preferably Said drug or pharmaceutical composition is used for the treatment of diseases or injuries that cause neuronal loss. Alternatively, the present invention claims the compound EOF2 or pharmacologically active salts thereof or an enantiomeric mixture including the compound of formula I or its salts, and where the compound of formula I is in a percentage greater than or equal to 50% of the total, for use in therapy, preferably in the treatment of diseases or injuries that occur with neuronal loss.
    [0066] In an even more concrete aspect of the present invention, the pharmaceutical composition of the present invention comprises at least one pharmaceutically acceptable excipient.
    [0067] In the context of the present invention, neural precursors or neural stem cells are understood as those neural stem cells isolated from adult or fetal tissue, which have the capacity to self-replicate, but a limited capacity for differentiation, since they can only be differentiated towards three subtypes of cells of the neural lineage: neurons, astrocytes, and oligodendrocytes.
    [0068] In a particular aspect of the present invention, the diseases or injuries that occur with neuronal loss are selected from the list consisting of:
    [0069] - Neurodegenerative diseases: They occur as a consequence of neuronal death. The most frequent are dementias, among which Alzheimer's disease stands out, with neuronal loss in the hippocampus and the cerebral cortex, and vascular dementia, with neuronal loss associated with small glass disease and; Parkinson's disease, with selective death of neurons in the substantia nigra; amyotrophic lateral sclerosis - with neuronal deficits in the spinal cord; vascular dementia.
    [0070]
    [0071] - Cranioencephalic trauma: it is a traumatic injury that affects the skull, with brain involvement. The damage may be focal — limited to a single area of the brain — or involve more than one area of the brain. In the context of this invention, traumatic brain injury can produce brain damage due to neuronal death that could be treated by therapies aimed at promoting neuronal regeneration.
    [0072] - Hypoxic-ischemic injury: Reduction of cerebral blood flow to levels that are insufficient to maintain the metabolism necessary for normal brain function and structure. In adults, ischemia is primarily caused by strokes, which can be focal (of ischemic, hemorrhagic, or mixed origin), or multiple (as in multi-infarct dementia). In the newborn, said hypoxia / ischemia is primarily due to fetal or perinatal distress. In the context of the present invention, hypoxia-ischemia is a condition that produces cellular suffering due to the lack of oxygen supply to brain tissue, which in most cases produces neuronal death.
    [0073]
    [0074] - CNS infections: Brain involvement by different infectious agents that cause meningitis, encephalitis or meningoencephalitis. In the context of this invention, CNS infections cause cellular suffering either directly or indirectly from the brain edema they cause, and can cause neuronal death.
    [0075]
    [0076] - Epilepsy: Chronic disease characterized by one or more neurological disorders that leaves a predisposition to generate recurrent seizures, which usually leads to neurobiological, cognitive and psychological consequences.
    [0077]
    [0078] - Huntington's disease: Huntington's disease (HD) is a neurodegenerative disorder of the CNS characterized by involuntary choreic movements, behavioral and psychiatric disorders, and dementia. It is caused by an expansion of CAG triplet repeats on the short arm of chromosome 4 (4p16.3) in the huntingtin gene, HTT. The greater the expansion of CAG repeats, the earlier the disease appears.
    [0079]
    [0080] In an even more particular aspect of the present invention, the diseases or injuries that occur with neuronal loss are focused cerebral ischemia, traumatic brain injury with neuronal damage, Parkinson's, epilepsy and amyotrophic lateral sclerosis. More particularly, the pharmaceutical composition of the present invention is administered non-invasively trying to avoid the blood-brain barrier, such as intranasal administration.
    [0081] A second aspect of the present invention relates to a method (hereinafter the method of the present invention) for obtaining neurons from stem cells or neural precursors to neurons in vitro comprising:
    [0082]
    [0083] to. contacting neural stem cells or neural precursors with a concentration range from 1 pmol / to 40 pM EOF2, in a medium that does not prevent the differentiation of neural stem cells or neural precursors; and preferably between 1-10 pM. more preferably between 3.5 and 6.5 pM; and optionally
    [0084]
    [0085] b. subsequently harvest the obtained neurons.
    [0086] To use EOF2 in an in vitro culture, it is preferable to dissolve it before use in a solution that can dissolve both the compound and its pharmaceutically accepted salt. Examples of the solvent can be dimethyl sulfoxide, water or the like. Additionally this compound can be dissolved in phosphate buffered saline (PBS).
    [0087]
    [0088] In a particular aspect of the invention, to cultivate neural stem cells with EOF2, EOF2 is added in a concentration range from 1 pM to 40 pM. Stem cells are grown adhered to a substrate at a density of 20 to 200 x 106 cells / L. The compound is added to a static culture at 37 ° C for 1 to 14 days in an atmosphere of 5% C02, changing the medium totally or partially every other day.
    [0089]
    [0090] The medium in which the cells are grown can be any medium that does not impede the differentiation of neural stem cells, as an example Dulbecco's modified Eagle's medium (DMEM) / F-12 (1: 1) medium can be preferably used, which contain 2% of the B27 supplement (Invitrogen), 2mM L-glutamine and 2pg / ml gentamicin.
    [0091]
    [0092] A fourth aspect of the present invention relates to a culture medium (hereinafter culture medium of the present invention), suitable for differentiation to neurons from stem cells or neural precursors, comprising EOF2 in a concentration range from 1 pM to 40 pM preferably between 1-10 pM, more preferably between 3.5 and 6.5 pM
    [0093]
    [0094] A fifth aspect of the present invention relates to the use of the culture medium of the present invention for differentiation to neurons from stem cells or neural precursors.
    [0095] A sixth aspect of the invention relates to a population of neurons obtainable by the method of the present invention.
    [0096]
    [0097] A seventh aspect of the present invention refers to the use of the population of neural stem cells or neural precursors obtainable by the method of the present invention for the preparation of a medicine for use in the treatment of diseases or injuries that cause neuronal loss. .
    [0098]
    [0099] Lastly, the inventors of the present invention have found that compounds structurally similar to EOF2 as the compound EOF3 (Figure 2) do not have any activity that favors the differentiation of neural precursors. , 12-di-0-acetyl-8-0-methyl-3-0-phenylacetylingol (EOF3) with CAS number 944799-48-8 and the chemical structure shown below is not capable of promoting neuron differentiation of neural precursors.
    [0100]
    [0101]
    [0102]
    [0103]
    [0104] Formula 2
    [0105] The following examples are merely illustrative of the present invention and are not to be understood as limiting it in any way.
    [0106]
    [0107] Examples
    [0108]
    [0109] EXAMPLE 1 Effect of natural compounds EOF2 and EOF3 on the differentiation of neurospheres in vitro
    [0110]
    [0111] In order to check whether EOF2 and EOF3 favored neurosphere differentiation, cells from the neurospheres were disaggregated and cultured adhered to a PLO substrate in the absence of growth factors to promote their differentiation. These cells were cultured in the presence and absence of EOF2 or EOF3 for 72 h.
    [0112]
    [0113] To analyze the differentiation of the neural precursors isolated from SVZ in vitro, the cells were seeded in glass slides, each with an area of 0.8 cm2. Wells were pretreated with poly-L-ornithine (PLO) (25 pg / ml) in 0.1 M borate buffer pH 8.4. 300 µl of PLO was added to each well and incubated overnight at 37 ° C; the next day it was washed with sterile water and allowed to dry for at least two hours under sterile conditions. Once the plate was dry, the neurospheres were disaggregated and seeded in defined medium in the absence of growth factors at a density of 40,000 cells per well in 300 µl final. Compounds EOF2 and EOF3 were added at a concentration of 5 pM. The absence of growth factors favors the exit of the cell cycle and the differentiation of neural precursors to neurons, astrocytes and oligodendrocytes, allowing the in vitro study of this process.
    [0114]
    [0115] Subsequently, the cellular phenotypes present in the culture were analyzed by immunocytochemistry. Under these conditions, cells from neurosphere cultures differentiate into different neural phenotypes (glial or neuronal). The percentage of neuroblasts and neurons (plll-tubulin + cells) and glial astrocytes or progenitors (GFAP + cells) were quantified. It was observed that in the presence of EOF2 the percentage of pllltubulin + cells was approximately twice that of the control cultures (Figure 2 A, C). A small tendency to decrease in the percentage of GFAP + cells was also observed, which, however, was not statistically significant. It is also appreciated that the compound EOF2 significantly reduces cell death with respect to the control and EOF3 has no effect on it compared to the control (Figure 2). These results indicate that EOF2 is able to induce differentiation of neurospheres to neuroblasts at the expense of differentiation to glioblasts and suggest that their use in brain lesions could reverse the glyogenic / non-neurogenic environment of these lesions and transform it into a neurogenic environment where new neurons could be generated. For this reason, the compound EOF2 was postulated as a candidate for the treatment of brain lesions, as a compound capable of modifying the gliogenic niche of the lesion turning it into a neurogenic niche. Thus, we decided to analyze whether this compound infused in vivo into brain lesions was actually capable of promoting neurogenesis in the lesion. Compound EOF3 had no effect on the differentiation of neurospheres to neurons or glial cells.
    [0116]
    [0117] EXAMPLE 2. The compound EOF2 has no effect on proliferation but favors the generation of neuroblasts in the injured cerebral cortex
    [0118]
    [0119] To determine the effect of EOF2 on brain lesions, controlled mechanical lesions were performed in the primary motor cortex of adult mice, restricted to gray matter. Previous work has shown that neural stem cells are activated in these types of lesions but are not capable of generating neuroblasts or neurons due to the glyogenic environment that is created in the lesion ( Romero-Grimaldi C., et al. 2011. ADAM-17 / necrosis tumor factor-alpha-converting enzyme inhibits neurogenesis and promotes gliogenesis from neural stem cells. Stem Cells 1628-1639).
    [0120]
    [0121] Adult mice were anesthetized with a combination of ketamine (100 mg / kg; Imalgene® 500, Merial) and xylazine (10 mg / kg; Rompún® 2%, Bayer). Animals were placed in a stereotaxic apparatus and an anteroposterior incision was made in the skin, in the midline, leaving the skull visible. Once the periosteum was removed, Bregma was located as a reference point. A 1.4 mm rostral and 1.5 mm right lateral craniotomy was performed on Bregma. The right primary motor cortex was injured by inserting a drill bit 0.7 mm in diameter to a depth of 1 mm.
    [0122]
    [0123] The injured animals were divided into two groups of 5 animals each. Immediately after making the lesions, infusion cannulas connected to mini osmotic pumps were implanted locally, capable of releasing their content locally and sustained for 14 days. Half of the injured animals were connected to mini pumps that released vehicle (saline), and the other half were connected to mini pumps that released the EOF2 compound at a concentration of 5pm until the time of sacrifice. Fourteen days after injury, the mice were sacrificed and their brains were removed for further analysis. All mice received BrdU injections (120 mg / kg) on days 12, 13 and 14 post injury, and were euthanized 2 hours after the last injection.
    [0124]
    [0125] Bromodeoxyuridine or BrdU (Sigma-Aldrich) is a synthetic nucleotide analogous to deoxythymidine, which is incorporated into the DNA of cells while the replication of their genetic material occurs (S phase of the cell cycle) to divide, so that the cells that proliferate during exogenous administration of this compound are marked.
    [0126]
    [0127] The brains were removed and serial sections were performed. In the sections containing the injured area, the number of proliferating cells, the number of neuroblasts and the number of astrocytes and glioblasts were analyzed by immunohistochemistry.
    [0128]
    [0129] Immunodetection of BrdU in the injured tissue showed the existence of BrdU + nuclei in the perimeter of the lesion, thus indicating the presence of cells that are actively dividing during the 9 hours prior to the sacrifice of the animal. By analyzing the effect of EOF2 treatment on injured cortical tissue, we were able to observe that, in animals treated with EOF2 during the 14 days after injury, the number of BrdU + proliferating cells was not different from that of control animals, corroborating that As observed in vitro, EOF2 also did not promote proliferation of NSC or NPC in vivo (Figure 2 AB). As a marker of the presence of neuroblasts or neuronal precursors, the DCX protein was detected by immunohistochemistry in both control animals and those treated with EOF2. In control animals, DCX + cells only appeared sporadically in some animals and in very few numbers, not in all. However, in the lesions treated with EOF2, an abundant and quantifiable quantity of DCX + neuroblasts could be observed in all the lesions of all the animals analyzed. That is, EOF2 treatment dramatically increased, and significantly compared to control (Figure 2 A, C), the number of neuroblasts in the area peripheral to the lesion.
    [0130]
    [0131] EXAMPLE 3. Compound EOF2 decreases glial cells in the environment of mechanical cortical injury.
    [0132]
    [0133] Using the methodology of Example 2, glial cells in the environment of the lesion were analyzed. To quantify the number of astrocytes and glioblasts in control and EOF2-treated lesions, the number of GFAP + cells was quantified. Our results showed that, in the lesions of mice treated with EOF2, the area occupied by GFAP + cells decreased statistically significantly with respect to the lesions of the control animals (Figure 3 A, C). The analysis of the percentage of proliferating cells (BrdU +), which co-located with the GFAP marker, also showed that in the mice that received the treatment with EOF2, the percentage of proliferating glial cells was lower than in the control mice, such and as can be seen in figure 3 (Figure 3 A, B).
    [0134]
    [0135] EXAMPLE 4. Effect of compound EOF2 on the generation of undifferentiated precursors after mechanical cortical injury
    [0136]
    [0137] Using the methodology explained in Example 2, the number of undifferentiated precursors in the lesion (nestin + cells) at the perimeter of the lesion was also studied in both groups of animals (Figure 4).
    [0138]
    [0139] The results indicated that in the perimeter of the lesion there are undifferentiated neurospheres in both groups of animals that constitute around 45% of all the cells that incorporate BrdU (Figure 4). However, although there are no significant differences in this parameter between both groups, the trends show that in the presence of EOF2 there are fewer undifferentiated precursors. Therefore, the increase in neuroblasts observed after EOF2 treatment was not due to an increase in the number of undifferentiated precursors, but to a higher rate of neuronal differentiation at the expense of less glial differentiation.
    [0140]
    [0141] EXAMPLE 5: Treatment with EOF2 intranasally allows the migration of neuronal precursors from the SVZ to the lesion.
    [0142]
    [0143] To determine the effect of EOF2 on brain lesions, controlled mechanical lesions were performed in the primary motor cortex of adult mice, restricted to gray matter. Previous work has shown that neural stem cells are activated in these types of lesions but are not capable of generating neuroblasts or neurons due to the glyogenic environment that is created in the lesion ( Romero-Grimaldi C., et al. 2011. ADAM-17 / tumor necrosis factor-alpha-converting enzyme inhibits neurogenesis and promotes gliogenesis from neural stem cells. Stem Cells 1628-1639).
    [0144]
    [0145] Six days prior to injury, mice were injected with BrdU to mark proliferating SVZ and DG cells. It was waited three days for the animal's body to eliminate all BrdU and lesions were performed as explained in Example 2. The mice were subsequently divided into two groups of 5, one group (EOF2) received EOF2 intranasally for 14 days. while another group received only the vehicle, saline. Intranasal drug administration is currently being widely used to deliver drugs to the nervous system. Recent publications have shown that intranasal administration of biological substances allows the blood-brain barrier to be crossed and the distribution of these substances to different regions of the brain, including DG and SVZ, such as, for example, IGF or insulin. Intranasal administration was performed manually in animals without anesthesia while the animal is held supine with the neck in extension as previously described ( Francis GJ, et al. 2008. Intranasal insulin prevent cognitive decline, cerebral atrophy and white matter changes in murine type I diabetic encephalopathy. Brain 3311-3334). 18 pL of a 5 pM solution of EOF2 and or vehicle were administered over the two nostrils alternately adding 3pL each time with a Gilson p10 micropipette. The mouse was held in that position for an additional 10 s to ensure that all fluid had been inhaled.
    [0146]
    [0147] Finally, after 14 days of treatment, as explained in figure 5, the animals were sacrificed and BrdU + and DCX + cells were analyzed in them as explained in example 2.
    [0148] The results show that only in the EOF2-treated mice do neurons appear on the perimeter of the lesion that cannot be seen in the control animals (Figure 5).
    权利要求:
    Claims (10)
    [1]
    1. In vitro use of the compound of formula I (EOF2):

    [2]
    2. Method for neuronal differentiation of neural precursors or neural stem cells to cultured neurons comprising:
    to. Dissolving the compound of formula I or a pharmacologically active salt thereof in a solution that can dissolve both the compound and its pharmaceutically accepted salt or an enantiomeric mixture including the compound of formula I or its salts, and where the compound of formula I is in a percentage greater than or equal to 50% of the total; and
    b. Bringing neural stem cells or neural precursors into contact with the solution described in (a), in a medium suitable for differentiating neurons from neural stem cells or neural precursors; and where the compound of formula I or a pharmacologically active salt of the same compound or an enantiomeric mixture that includes the compound of formula I or its salts, where the compound of formula I is in a percentage greater than or equal to 50% of the total, is in the medium in a concentration range from 1 pM to 40 pM. preferably between 1-10 pM. more preferably between 3.5 and 6.5 pM.
    [3]
    3. Culture medium suitable for neuronal differentiation of neural precursors or neural stem cells to cultured neurons, comprising the compound of formula I or a pharmacologically active salt thereof, or an enantiomeric mixture including the compound of formula I or its salts, and where the compound of formula I is in a percentage greater than or equal to 50% of the total.
    [4]
    4. Culture medium according to claim 3, wherein the compound of formula I or the pharmacologically active salt thereof, are in the medium in a concentration range from 1 pM to 40 pM, preferably between 1-10 pM, more preferably between 3.5 and 6.5 pM.
    [5]
    5. Use of the culture medium according to any of claims 3 or 4, to promote neuronal differentiation of neural precursors or neural stem cells to cultured neurons.
    [6]
    6. Pharmaceutical composition comprising the compound of formula I or a pharmacologically active salt thereof, or an enantiomeric mixture including the compound of formula I or its salts, where the compound of formula I is in a greater or equal percentage to 50% of the total.
    [7]
    7. Compound of formula I or a pharmacologically active salt thereof or an enantiomeric mixture that includes the compound of formula I or its salts, where the compound of formula I is in a percentage greater than or equal to 50% of the total ., for use in therapy.
    [8]
    8. Composition comprising the compound of formula I or a pharmacologically active salt thereof or an enantiomeric mixture including the compound of formula I or its salts, where the compound of formula I is in a percentage greater than or equal to 50% of the total, for use in the treatment of diseases or injuries that occur with neuronal loss selected from the group consisting of: focused cerebral ischemia, traumatic brain injury with neuronal damage, Parkinson's, epilepsy, and amyotrophic lateral sclerosis.
    [9]
    9. Pharmaceutical composition according to claim 6, wherein said composition is suitable for intranasal administration.
    [10]
    10. Composition for use according to claim 7 or 8, wherein said composition is administered intranasally.
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    US20140056995A1|2012-08-27|2014-02-27|Mackay Memorial Hospital|Use of compounds isolated from euphorbia neriifolia for treating cancer and/or thrombocytopenia|
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