WO2015086866A1 - Use of 12-deoxyphorbols for stimulating the expansion of neural stem cells - Google Patents

Use of 12-deoxyphorbols for stimulating the expansion of neural stem cells Download PDF

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WO2015086866A1
WO2015086866A1 PCT/ES2014/000180 ES2014000180W WO2015086866A1 WO 2015086866 A1 WO2015086866 A1 WO 2015086866A1 ES 2014000180 W ES2014000180 W ES 2014000180W WO 2015086866 A1 WO2015086866 A1 WO 2015086866A1
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neural
stem cells
proliferation
neural stem
precursors
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French (fr)
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Carmen Castro Gonzalez
Noelia Geribaldi Doldan
Maria Isabel Murillo Carretero
Jesús DOMINGUEZ RISCART
Purificación ORTIZ CUELLAR
Francisco GARCIA BERNAL
Cristina VERASTEGUI ESCOLANDO
Maria Eugenia FLORES GIUBI
Rosario HERNANDEZ GALAN
Antonio José MACIAS SANCHEZ
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Universidad De Cádiz (Otri)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)

Definitions

  • This invention is related to the use of 12-deoxiforboles or a pharmacologically active salt of compounds of this family, to favor the proliferation of neural precursors or neural stem cells in culture. Additionally, it is also related to the use of the same family of compounds for the preparation of a pharmaceutical composition useful in the treatment of diseases of the central nervous system that present with neuronal loss.
  • CNS Central Nervous System
  • DG dentate gyrus of the hippocampus
  • SVZ subventricular zone
  • NPC highly proliferative undifferentiated neural precursors
  • Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. Journal of Neuroscience 1997, 17, 3727-3738), giving rise to the production of new neuroblasts that gradually migrate in the direction of the injured area.
  • the capacity of neuronal regeneration in the injured areas is very low, a neuronal replacement of between 0.2 and 10% has been described, depending on the area affected and the type of lesion (Arvidsson , A., et al. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nature Medicine 2002, 8, 963-970; Nakatomi, et al.
  • an objective of current research in the field of neurodegenerative diseases and injuries is to establish which molecules favor or prevent neurogenesis to, by inserting the first and / or eliminating the latter, try to generate a neurogenic niche Where necessary.
  • the neurogenic niche there is therefore a set of local extracellular signals that allow the continuous generation of new neurons (Alvarez-Buylla, A., and Lint, DA For the long run: maintaining germinal niches in the adult brain. Neuron 2004, 41 , 683-686; Lee, C, et al. The molecular profiles of neural stem cell niche in the adult subventricular zone. PLoS One 2012, 7, e50501).
  • these factors affect intracellular signaling pathways.
  • both extracellular factors and proteins that make up intracellular signaling pathways can be acted upon.
  • stem cell transplantation One of the alternatives that could contribute to solving the clinical problems posed by diseases that occur with neuronal loss is stem cell transplantation.
  • stem cells of embryonic origin Cao, Q. et al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage.
  • Exp Neurol 2001 167, 48 -58 stem cells neurals of adult animals
  • Pluchino, S., et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis.
  • the present invention faces the following problem: although the stem cells that are implanted in the neurogenic areas of the brain (for example, the subventricular zone or the olfactory bulb in rodents) differentiate to neuroblasts and become neurons mature, when the transplant is performed in other areas, they begin the route of glial differentiation. On the other hand, in the non-neurogenic areas of the brain, parenchymal glia cells are activated after an injury and give rise to neural precursors, but most of them differ from glia cells and very few to neurons.
  • the neurogenic areas of the brain for example, the subventricular zone or the olfactory bulb in rodents
  • the present invention also faces the problem of choosing the source of the precursors to be transplanted when performing this type of transplant.
  • Embryonic stem cells differentiated in vitro to neural stem cells, or fetal neural precursors are from the most abundant sources. However, it is not easy to find donors of embryonic or fetal precursors, so it would be very useful to be able to isolate adult brain neural precursors, grow them and expand them in vitro for later use in transplants. Thus, identify agents that favor expansion of the adult brain neural precursors in culture will be very useful in the development of regenerative therapies of the nervous system.
  • Figure 1 Effect of prostratin on in vitro neural precursor cultures.
  • Neurosphere test Representative contrast microscopy images of cultures of neural precursor cultures grown respectively in the absence (A), presence of prostratin (5 ⁇ ) (B). The white line represents a distance of 200 ⁇ .
  • C and D Effect of different concentrations of prostratin on the area and number of neurospheres respectively.
  • E and F Feasibility test: Effect of prostratin (5 ⁇ ) on the percentage of non-viable cells (E) and on the total number of living cells at 24, 48 and 72 hours of treatment (F). In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p ⁇ 0.05 in T-Student test in relation to control).
  • Figure 2 Effect of prostratin on the proliferation of neural precursors.
  • A Representative fluorescence microscopy images of cultures of neural precursors grown respectively in the absence and presence of prostratin (5 ⁇ ) in which the cell cycle cell marker protein, Ki67 and the core marker, DAPI has been detected. The white line represents a distance of 100 ⁇ .
  • B Quantification of the effect of prostratin on the percentage of Ki67 + cells.
  • C Effect of prostratin on the percentage of picnotic nuclei. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p ⁇ 0.05).
  • Figure 3 Effect of prostratin on cyclin expression in NPC cultures.
  • FIG. 4 Effect of 12-deoxifor bowls on NPC cultures.
  • A Chemical structure of the 12-deoxiforbol trees evaluated in the neurosphere test.
  • B Representative images of phase contrast microscopy of cultures of neural precursors grown in the absence (1-2) and presence of the 12-deoxy-trees (1 ⁇ ): ER271 (3), ER272 (4), ER14 (5) , ER2 (6), ER3 (7) and ER8 (8). The white line represents a distance of 200 ⁇ .
  • C and D Effect of different concentrations of 12-deoxiforboles on the area of the neurospheres (% of control). In all cases, proliferation was stimulated by adding the bFGF growth factor to the culture medium (* p ⁇ 0.05 in T-Student test in relation to the control).
  • Figure 5 Effect of ER271 on cell proliferation in neural precursor cultures in vitro.
  • a and B Representative images of phase contrast microscopy of cultures of neural precursors grown respectively in the absence and presence of ER271 (1 ⁇ ). The white line represents a distance of 200 ⁇ .
  • C and D Effect of different concentrations of ER271 on the area and number of neurospheres, respectively.
  • E Effect of ER271 (1 ⁇ ) on the percentage of non-viable cells at 24, 48 and 72 hours of treatment. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p ⁇ 0.05).
  • Figure 6. Effect of ER272 on cell proliferation in in vitro neural precursor cultures.
  • FIG. 1 Representative images of phase contrast microscopy of cultures of neural precursors grown respectively in the absence and presence of ER272 (1 ⁇ ). The white line represents a distance of 200 ⁇ .
  • C and D Effect of different concentrations of ER272 on the area and number of neurospheres, respectively.
  • E Effect of ER271 (1 ⁇ ) on the percentage of non-viable cells at 24, 48 and 72 hours of treatment. In all cases, proliferation was stimulated by adding the growth factor bFGF to the culture medium (* p ⁇ 0.05).
  • Figure 7 Effect of ER271 treatment on the percentage of cells in the cell cycle in cultures of neural precursors.
  • A Representative fluorescence microscopy images of neural precursor cultures grown respectively in the absence and presence of ER271 (1 ⁇ ) in which the cell cycle cell marker protein, Ki67 and the nucleus marker, DAPI has been detected. The white line represents a distance of 100 ⁇ .
  • B Quantification of the effect of ER271 on the percentage of K ⁇ 67 + cells. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p ⁇ 0.05).
  • A Representative fluorescence microscopy images of cultures of neural precursors grown respectively in the absence and presence of ER272 (1 ⁇ ) in which the cell cycle cell marker protein, Ki67 and the core marker, DAPI has been detected. The white line represents a distance of 100 ⁇ .
  • B Quantification of the effect of ER272 on the percentage of K ⁇ 67 + cells. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p ⁇ 0.05). DETAILED DESCRIPTION OF THE INVENTION
  • the present invention solves the problem of generating neurogenic niches in both neurogenic and non-neurogenic areas of the Central Nervous System, based on the use of compounds of the family of 12-deoxyfor trees
  • a first aspect of the present invention comprises the use of compounds of the family 12-deoxyforbol trees or pharmacologically active salts of said compounds, for the preparation of a medicament (hereinafter pharmaceutical composition of the present invention) for the treatment of diseases or injuries that occur with neuronal loss.
  • pharmaceutical composition of the present invention for the treatment of diseases or injuries that occur with neuronal loss.
  • R ⁇ can be hydrogen or acetyl (COCH 3 )
  • R 2 and R 3 can be hydrogen or alkyl and R 4 can be hydrogen, methyl (CH 3 ) or phenyl (Ph).
  • prostratin 13-0-Acetyl-12-deoxiforbol with CAS number 60857-08-1 (hereinafter prostratin) whose chemical structure is as follows:
  • ER272 13-0-angeloil-12-deoxiforbol with CAS number 28152-96-7 (hereinafter ER271) whose chemical structure is as follows:
  • ER2 20-O-acetyl-13-O-Isobutyroyl-12-deoxyforbol (hereinafter ER3) and whose chemical structure is shown below.
  • the first aspect of the present invention relates to compounds of the family of 12-deoxiforbol trees such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 or pharmacologically active salts thereof for use in the treatment of diseases or injuries that occur with neuronal loss.
  • the pharmaceutical composition of the present invention further comprises neural precursors or neural stem cells.
  • the pharmaceutical composition of the present invention comprises at least one pharmaceutically acceptable excipient.
  • neural precursors or neural stem cells are understood as those stem cells isolated from adult or fetal neural tissue, which have the capacity to self-replicate, but have limited potential, since they can only be differentiated into the three subtypes of cells of the neural lineage: neurons, astrocytes and oligodendrocytes.
  • the diseases or lesions that occur with neuronal loss are those selected from the list consisting of:
  • Neurodegenerative diseases They occur as a result of early neuronal death. The most frequent are Alzheimer's disease, with neuronal loss in the hippocampus and cerebral cortex, Parkinson's disease, with selective death of neurons in the black substance, or amyotrophic lateral sclerosis (ALS), with deficit of neurons in the spinal cord.
  • Alzheimer's disease with neuronal loss in the hippocampus and cerebral cortex
  • Parkinson's disease with selective death of neurons in the black substance
  • ALS amyotrophic lateral sclerosis
  • Attic cranioencephalic trauma it is a traumatic injury that affects the skull, with cerebral involvement.
  • the damage can be focal - limited to a single area of the brain - or involve more than one area of the brain.
  • head trauma can cause brain damage due to neuronal death that could be treated by therapies aimed at promoting neuronal regeneration.
  • hypoxia-ischemia Reduction of cerebral blood flow to levels that are insufficient to maintain the metabolism necessary for normal brain function and structure.
  • ischemia is mainly caused by cerebrovascular accidents, which can be focal (of ischemic, hemorrhagic or mixed origin), or multiple (as in multi-infarcted dementia).
  • said hypoxia / ischemia is mainly due to fetal or perinatal suffering.
  • hypoxia-ischemia is a condition that produces cellular suffering due to the lack of oxygen supply to brain tissue that in most cases produces neuronal death.
  • 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 due to the cerebral edema they cause, and can cause neuronal death.
  • - Epilepsy Chronic disease characterized by one or several neurological disorders that leaves a predisposition to generate recurrent seizures, which usually lead to neurobiological, cognitive and psychological consequences.
  • the diseases or injuries that occur with neuronal loss are focused cerebral ischemia, head trauma with neuronal damage, Parkinson's, epilepsy and amyotrophic lateral sclerosis.
  • a second aspect of the present invention relates to the in vitro use of compounds of the family of 12-deoxy-trees such as, for example, prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 to favor the proliferation of neural precursors or stem cells Neurals
  • a third aspect of the present invention relates to a method (hereinafter method of the present invention) for the expansion of neural stem cells or neural precursors in vitro comprising:
  • neural stem cells or neural precursors with a concentration range from 1 pmol / L to 40 ⁇ / L of 12-deoxy-trees, in a medium that does not prevent the proliferation of neural stem cells or neural precursors and that includes the factor epidermal growth (EGF) and / or the basic fibroblast growth factor (bFGF); and b. subsequently harvest the cells.
  • EGF factor epidermal growth
  • bFGF basic fibroblast growth factor
  • the compound of the family of 12-deoxiforbol trees such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 in an in vitro culture it is preferable to dissolve them prior to their use in a solution that can dissolve both the compound and its salt pharmaceutically accepted.
  • the solvent may be dimethyl sulfoxide, water or the like. Additionally this compound may be dissolved in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the compound of the 12-deoxyforwood family such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 in a concentration range is added to the neural stem cells from 1 pmol / L to 40 ⁇ / L.
  • Stem cells are grown in flotation at a density of 20 to 200 x 10 6 cells / L.
  • the compound is added to a static culture at 37 ° C for 1 to 14 days in an atmosphere of 5% C0 2 , changing the medium totally or partially every two days.
  • the medium in which the cells are grown can be any medium that does not prevent the proliferation of neural stem cells, as an example, a Dulbecco's modified Eagle's medium (DMEM) / F-12 (1: 1) medium can preferably be used, which contain 2% of supplement B27 (Invitrogen), 2mM L-glutamine and 2 ⁇ / ⁇ 1 of gentamicin. Additionally, the medium must contain either the epidermal growth factor (EGF), preferably at a concentration of 20 ⁇ g / L, the basic fibroblast growth factor (bFGF), preferably at a concentration of 10 ⁇ g / L, or a mixture of both.
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • a fourth aspect of the present invention relates to a culture medium (hereinafter culture medium of the present invention), suitable for the proliferation of neural stem cells or neural precursors, comprising a compound of the family of 12- deoxiforboles such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14, in a concentration range of 1 pmol / L to 40 ⁇ / L.
  • culture medium of the present invention suitable for the proliferation of neural stem cells or neural precursors, comprising a compound of the family of 12- deoxiforboles such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14, in a concentration range of 1 pmol / L to 40 ⁇ / L.
  • a fifth aspect of the present invention relates to the use of the culture medium of the present invention for the expansion of neural stem cells or neural precursors.
  • a sixth aspect of the invention relates to a population of neural stem cells or neural precursors obtainable by the method of the present invention.
  • a seventh aspect of the present invention relates to the use of the population of neural stem cells or neural precursors obtainable by the method of present invention for the preparation of a medicament for use in the treatment of diseases or injuries that occur with neuronal loss.
  • neurosphere test The biological activity of the prostratin compound on the neural stem cells extracted from the subventricular area of 7-day postnatal mice has been investigated. The so-called “neurosphere test” has been used. The rationale for this test is that neural precursors when grown under floating conditions and are not adhered to the substrate of the material on which the culture is carried out, proliferate forming aggregates called neurospheres formed by proliferating cells and their progeny.
  • the lateral walls of the lateral ventricles of the subventricular zone of 7-day postnatal mice were enzymatically extracted and dissociated in CSF Ca 2 under high Mg 2 (5 mM KC1, 124 mM NaCl, 3.2 mM MgCl 2 , 100 uM CaCl 2 , 26 mM NaHC03, and 10 mM glucose) supplemented with 1 mg / ml trypsin and 0.2 mg / ml quinurenic acid preheated at 37 ° C for 15 minutes. It was incubated at 37 ° in an oven for 13 minutes.
  • Mg 2 5 mM KC1, 124 mM NaCl, 3.2 mM MgCl 2 , 100 uM CaCl 2 , 26 mM NaHC03, and 10 mM glucose
  • the tissue was centrifuged 5 minutes and resuspended in normal CSF (5 mM KC1, 124 mM NaCl, 1.3 mM MgCl 2 , 2 mM CaCl 2 , 26 mM NaHC03, and 10 mM glucose). It was incubated at 37 ° C for 5 minutes and centrifuged under the same conditions. The cells were then resuspended in Dulbecco's modified Eagle's medium (DMEM) / F-12 (1: 1) supplemented with 0.7 mg / ml ovomucoid and mechanically disintegrated with a Pasteur pipette with the diameter reduced by half.
  • DMEM Dulbecco's modified Eagle's medium
  • neurospheres were then centrifuged again and resuspended in 6 ml of defined neurosphere medium (45 ml of (DMEM) / F-12, 900 ⁇ , of supplement B 27 , 2 mM L-glutamine and 2 ⁇ g / ml of gentamicin) supplemented with 6 ⁇ L of 20ng / ml EGF and 10ng / ml bFGF, and maintained at 37 ° in 5% atmosphere C0 2 . After 1-2 days under these conditions, cell aggregates called neurospheres are formed.
  • defined neurosphere medium 45 ml of (DMEM) / F-12, 900 ⁇ , of supplement B 27 , 2 mM L-glutamine and 2 ⁇ g / ml of gentamicin
  • the subcultures were passed every 3-4 days by centrifugation of neurospheres and mechanical dissociation of the cells in 1 ml of defined medium of neurospheres; then the cell suspension was grown in new bottles with fresh medium to obtain new neurospheres.
  • the experiments were carried out between passes 3 and 5.
  • the neurospheres were centrifuged and the cells were resuspended and disintegrated in a defined medium of neurospheres and seeded 20 l cells to which the bFGF growth factor (10 ng / mL) was added.
  • Prostratin was added at the same time by placing a different concentration in each well. 5 wells were used for the different concentrations (Control, ⁇ g / ml, 5 ⁇ g / ml, and 10 ⁇ g / ml) and in triplicate. The experiments were carried out so that the person who takes the images and performs the quantification does not know the conditions of each of the cultures. The number of new formed neurospheres was counted 72h later on the Olympus 1X70 inverted phase contrast microscope. To measure the area of the neurospheres, images of 50 neurospheres per well were obtained and analyzed using the Image J analysis system.
  • the Ki67 marker that detects those nuclei that are cell cycle was detected by immunocytochemistry. The analysis was performed in control cultures and in cultures treated with prostratin 5 ⁇ .
  • the cells were fixed for 20 minutes at room temperature with 4% paraformaldehyde (PFA) prepared in 0.1 M phosphate buffer. The wells were subsequently washed 3 times with PBS. The cells were permeabilized by incubating 20 minutes at -20 ° C with an ethanol / acetic solution (95% absolute ethanol, and 5% glacial acetic acid). It was washed 3 times with PBS and subsequently blocked with a solution of 1.5% BSA (bovine serum albumin) in PBS; They were incubated for 30 minutes. The anti Ki67 antibody was added and left overnight at 4 ° C in blocking solution.
  • PFA paraformaldehyde
  • EXAMPLE 4 Effect of 12-deoxiforbol trees isolated from Euphorbia resiniferous on the formation of neurospheres in neural precursor cultures.
  • Treatment with ER2 increased the size of the neurospheres in a dose-dependent manner, reaching a 200% increase in the size of the neurospheres at a concentration of 10 ⁇ .
  • Treatment with ER3 increased proliferation as much as ER2, although the concentration needed to see the maximum effect was 1 ⁇ .
  • ER8 treatment only increased the size of the neurospheres by 20% and only at the concentration of 1 ⁇ compared to the control.
  • EXAMPLE 5 Detailed study of the proliferative effect of ER271 and ER272 on the proliferation of neural precursors.
  • the ER271 and ER272 were selected arbitrarily to study this effect more closely.
  • the 12-deoxyforbol trees 13-0-angeloyl-12-deoxyforbol or ER271, 13-0- isobutyroyl-12-deoxyforbol or ER272, 13- ⁇ 9-phenylacetyl-12-deoxyforbol or ER14, 20-O-acetyl-13-O -angeloyl-12-deoxiforbol or ER2, 20-O-acetyl-13-O-isobutyroyl-12-deoxyforbol or ER3 and 2O-0-acetyl-13-0-phenylacetyl-12-deoxyforbol or ER8 were obtained by isolation and purification from the resin Euphorbia latex collected in Tanant, Sydney (December, 2007).

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Abstract

The invention relates to the use of compounds of the 12-deoxyphorbol family or a pharmacologically active salt of said compounds, for stimulating the proliferation of neural precursors or neural stem cells in a culture. The invention also relates to the use of said family of compounds for developing a pharmaceutical composition that can be used in the treatment of diseases of the central nervous system with neuron loss.

Description

USO DE 12-DESOXIFORBOLES PARA PROMOVER LA EXPANSIÓN DE CÉLULAS MADRE NEURALES.  USE OF 12-DEOXIFORBOLS TO PROMOTE THE EXPANSION OF NEURAL MOTHER CELLS.
SECTOR DE LA TÉCNICA SECTOR OF THE TECHNIQUE
Esta invención está relacionada con el empleo de 12-desoxiforboles o una sal farmacológicamente activa de compuestos de esta familia, para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. Adicionalmente, también se relaciona con el empleo de la misma familia de compuestos para la elaboración de una composición farmacéutica útil en el tratamiento de enfermedades del sistema nervioso central que cursen con pérdida neuronal. This invention is related to the use of 12-deoxiforboles or a pharmacologically active salt of compounds of this family, to favor the proliferation of neural precursors or neural stem cells in culture. Additionally, it is also related to the use of the same family of compounds for the preparation of a pharmaceutical composition useful in the treatment of diseases of the central nervous system that present with neuronal loss.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
La reposición neuronal dentro del Sistema Nervioso Central (en adelante SNC) se lleva a cabo a partir de células madre neurales. Éstas son células indiferenciadas que mediante divisiones asimétricas pueden dar lugar a cada uno de los tipos celulares propios del tejido nervioso: neuronas, astrocitos y oligodendrocitos. Las células madre neurales se distribuyen a lo largo de diversas regiones del cerebro adulto, aunque en condiciones fisiológicas la mayoría de estas células permanecen quiescentes y no producen neuronas. Sólo existe generación de nuevas neuronas en dos regiones del cerebro adulto: el giro dentado del hipocampo (en adelante DG) y la zona subventricular (en adelante SVZ), en las que estas células madre neurales no están quiescentes sino que se dividen con una baja frecuencia y generan precursores neurales indiferenciados (en adelante NPC) altamente proliferativos que, posteriormente pueden dar lugar a precursores ya comprometidos con la estirpe neuronal, llamados neuroblastos, cuya diferenciación final da lugar a neuronas maduras (Goldman, S. Glia as neural progenitor cells. Trends in Neuroscience 2003 26, 590-596; Alvarez-Buylla, A., and Garda-Verdugo, J. M. Neurogenesis in adult subventricular zone. Journal of Neuroscience 2002, 22, 629-634). Neural replacement within the Central Nervous System (hereinafter CNS) is carried out from neural stem cells. These are undifferentiated cells that by means of asymmetric divisions can give rise to each of the cell types of nerve tissue: neurons, astrocytes and oligodendrocytes. Neural stem cells are distributed throughout various regions of the adult brain, although under physiological conditions most of these cells remain quiescent and do not produce neurons. There is only generation of new neurons in two regions of the adult brain: the dentate gyrus of the hippocampus (hereinafter DG) and the subventricular zone (hereinafter SVZ), in which these neural stem cells are not quiescent but divide with a low frequency and generate highly proliferative undifferentiated neural precursors (hereinafter NPC) that can subsequently lead to precursors already compromised with the neuronal line, called neuroblasts, whose final differentiation gives rise to mature neurons (Goldman, S. Glia as neural progenitor cells. Trends in Neuroscience 2003 26, 590-596; Alvarez-Buylla, A., and Garda-Verdugo, JM Neurogenesis in adult subventricular zone. Journal of Neuroscience 2002, 22, 629-634).
En distintos tipos de lesiones cerebrales experimentales (isquemia transitoria global o focal, epilepsia, entre otras) se ha observado un aumento de la proliferación de precursores neurales en el giro dentado y en la zona subventricular (Liu, J. et al. Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. Journal of Neuroscience 1998 18, 7768- 7778; Jin, K., et al. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proceedings of the National Academy of Sciences U S A 2001 98, 4710-4715; Parent, J. M. et al. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. Journal of Neuroscience 1997, 17, 3727-3738), dando lugar a la producción de nuevos neuroblastos que, paulatinamente, migran en dirección a la zona lesionada. Sin embargo, a pesar de este incremento en la neurogenesis, la capacidad de regeneración neuronal en las zonas lesionadas es muy escasa, habiéndose descrito una reposición neuronal de entre 0,2 y 10%, según el área afectada y el tipo de lesión (Arvidsson, A., et al. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nature Medicine 2002, 8, 963-970; Nakatomi, et al. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 2002 110, 429-441; Teramoto, T., et al. EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia. The Journal of clinical investigation 2003, 111, 1125-1132). Por otra parte, las lesiones cerebrales inducen la aparición localizada de células con características de precursores neurales que proliferan y que generan nuevas células gliales pero no neuronas (Seidenfaden, R et al, Glial conversión of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain. Mol Cell Neurosci 200632, 187-198). Todos estos resultados indican que en el tejido cerebral lesionado se dan las condiciones que favorecen la diferenciación glial, pero no neuronal, de las células madre neurales, ya sean propias o transplantadas. Esto se debe a que el conjunto de interacciones de citoquinas y de contactos celulares que se genera en una zona de lesión tisular (expresión de nuevos factores de crecimiento y sus correspondientes receptores, etc.), difiere notablemente de las condiciones existentes en las regiones neurogénicas. Se trataría por tanto de modificar el nicho no-neurogénico de la zona lesionada y convertirlo en un nicho neurogénico en el que tanto las células madre endógenas como las transplantadas pudieran dar lugar a neuronas maduras y funcionales. Por ello, un objetivo de la investigación actual en el campo de las enfermedades y lesiones neurodegenerativas es establecer qué moléculas favorecen o impiden la neurogénesis para, mediante la inserción de las primeras y/o la eliminación de las segundas, tratar de generar un nicho neurogénico donde sea necesario. En el nicho neurogénico por tanto existen un conjunto de señales extracelulares locales que permiten la continua generación de nuevas neuronas (Alvarez-Buylla, A., and Lint, D. A. For the long run: maintaining germinal niches in the adult brain. Neuron 2004, 41, 683-686; Lee, C, et al. The molecular profiles of neural stem cell niche in the adult subventricular zone. PLoS One 2012, 7, e50501). Además, estos factores inciden sobre rutas de señalización intracelulares. De este modo, a la hora de favorecer la neurogénesis, se puede actuar tanto sobre los factores extracelulares como sobre las proteínas que conforman las rutas de señalización intracelular. In different types of experimental brain lesions (transient global or focal ischemia, epilepsy, among others) an increase in the proliferation of neural precursors has been observed in the dentate gyrus and in the subventricular zone (Liu, J. et al. Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils Journal of Neuroscience 1998 18, 7768-7777; Jin, K., et al. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proceedings of the National Academy of Sciences USA 2001 98, 4710-4715; Parent, JM et al. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. Journal of Neuroscience 1997, 17, 3727-3738), giving rise to the production of new neuroblasts that gradually migrate in the direction of the injured area. However, despite this increase in neurogenesis, the capacity of neuronal regeneration in the injured areas is very low, a neuronal replacement of between 0.2 and 10% has been described, depending on the area affected and the type of lesion (Arvidsson , A., et al. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nature Medicine 2002, 8, 963-970; Nakatomi, et al. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 2002 110, 429-441; Teramoto, T., et al. EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia. The Journal of clinical investigation 2003, 111, 1125-1132). On the other hand, brain lesions induce the localized appearance of cells with characteristics of proliferating neural precursors that generate new glial cells but not neurons (Seidenfaden, R et al, Glial conversion of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain Mol Cell Neurosci 200632, 187-198). All these results indicate that in the injured brain tissue the conditions that favor the glial, but not neuronal, differentiation of the neural stem cells, whether own or transplanted, occur. This is due to the fact that the set of interactions of cytokines and cellular contacts that are generated in a zone of tissue injury (expression of new growth factors and their corresponding receptors, etc.), differs markedly from the conditions existing in the neurogenic regions . It would therefore be a matter of modifying the non-neurogenic niche of the injured area and converting it into a neurogenic niche in which both endogenous and transplanted stem cells could give rise to mature and functional neurons. Therefore, an objective of current research in the field of neurodegenerative diseases and injuries is to establish which molecules favor or prevent neurogenesis to, by inserting the first and / or eliminating the latter, try to generate a neurogenic niche Where necessary. In the neurogenic niche there is therefore a set of local extracellular signals that allow the continuous generation of new neurons (Alvarez-Buylla, A., and Lint, DA For the long run: maintaining germinal niches in the adult brain. Neuron 2004, 41 , 683-686; Lee, C, et al. The molecular profiles of neural stem cell niche in the adult subventricular zone. PLoS One 2012, 7, e50501). In addition, these factors affect intracellular signaling pathways. Thus, when it comes to promoting neurogenesis, both extracellular factors and proteins that make up intracellular signaling pathways can be acted upon.
El hecho de que haya una respuesta neurogénica a la lesión hace pensar que la formación de nuevas neuronas sea un mecanismo eficaz en la reparación de pequeñas lesiones, que permanecen silentes precisamente porque las neuronas que hacen apoptosis son reemplazadas, al menos en parte, por neuronas de nueva formación. Un estudio reciente demuestra que en los animales que han sufrido un traumatismo craneal, se incrementa la neurogénesis en el giro dentado del hipocampo y posteriormente estos animales recuperan su capacidad de realizar tareas de memoria espacial. Sin embargo, si en estos animales se eliminan selectivamente las células madre que comienzan a dividirse en el hipocampo como consecuencia del daño inducido por el traumatismo, estos animales no pueden recuperar la capacidad para realizar tareas de memoria espacial (Blaiss CA, et al. Temporally specified genetic ablation of neurogenesis impairs cognitive recovery after traumatic brain injury. J Neurosci. 2011 31:4906-4916), demostrando así que la reposición neuronal en estas regiones tiene un efecto sobre la recuperación de la memoria tras un traumatismo craneal. Adicionalmente, la aparición de células madre neurales activadas en lesiones cerebrales se ha demostrado también en humanos. Concretamente se ha podido observar que en muestras de cerebro de pacientes sometidos a cirugía tras una lesión por traumatismo craneal, aparecen en la zona de la corteza cerebral alrededor de la lesión, células que expresan marcadores de precursores neurales (Zheng, W., et al. Neurogenesis in adult human brain after traumatic brain injury. J Neurotrauma 2013 30, 1872-1880). The fact that there is a neurogenic response to the lesion 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 do apoptosis are replaced, at least in part, by neurons. of new formation. A recent study shows that in animals that have suffered cranial trauma, neurogenesis is increased in the dentate gyrus of the Hippocampus and subsequently these animals recover their ability to perform spatial memory tasks. However, if these animals selectively remove stem cells that begin to divide in the hippocampus as a result of trauma-induced damage, these animals cannot regain the ability to perform spatial memory tasks (Blaiss CA, et al. Temporally specified genetic ablation of neurogenesis impairs cognitive recovery after traumatic brain injury. J Neurosci. 2011 31: 4906-4916), thus demonstrating that neuronal replacement in these regions has an effect on memory recovery after head trauma. Additionally, the appearance of activated neural stem cells in brain lesions has also been demonstrated in humans. Specifically, it has been observed that in brain samples of patients undergoing surgery after a head injury, cranial areas around the lesion appear in the area of the cerebral cortex, expressing markers of neural precursors (Zheng, W., et al. Neurogenesis in adult human brain after traumatic brain injury. J Neurotrauma 2013 30, 1872-1880).
Sin embargo, las lesiones graves o las experimentales con mayor pérdida neuronal no pueden ser resueltas, a menos que se descubran medidas terapéuticas eficaces para incrementar de forma significativa el proceso neurogénico. La principal causa del fallo en la reposición neuronal es que los precursores neurales en las regiones lesionadas tienden a diferenciarse en células de glía, mientras la mayoría de los neuroblastos mueren antes de diferenciarse a neuronas maduras dentro de las regiones lesionadas. However, serious or experimental lesions with greater neuronal loss cannot be resolved, unless effective therapeutic measures are discovered to significantly increase the neurogenic process. The main cause of the failure in neuronal replacement is that neural precursors in the injured regions tend to differentiate into glia cells, while most neuroblasts die before differentiating into mature neurons within the injured regions.
Una de las alternativas que podría contribuir a resolver los problemas clínicos que plantean las enfermedades que cursan con pérdida neuronal es el transplante de células madre. Varios laboratorios han intentado esta estrategia en modelos animales transplantados con células madre de origen embrionario (Cao, Q. et al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol 2001 167, 48-58), células madre neurales de animales adultos (Pluchino, S., et al. Injection of adult neurospheres induces recovery in a chronic model of múltiple sclerosis. Nature 2003 422, 688- 694), o bien con células procedentes de células madre sometidas a diferentes grados de diferenciación in vitro. El fenómeno más generalmente observado es que, si bien las células madre que se implantan en las zonas neurogénicas del cerebro se diferencian a neuroblastos y se convierten en neuronas maduras, aquellas que se implantan en zonas del cerebro no neurogénicas inician la ruta de diferenciación glial (Herrera, D. G., et al, A. Adult-derived neural precursors transplanted into múltiple regions in the adult brain. Ann Neurol 1999, 46, 867- 877; Mligiliche, N. L., et al. Survival of neural progenitor cells from the subventricular zone of the adult rat after transplantation into the host spinal cord of the same strain of adult rat. Anat Sci Int 2005, 80, 229-234). One of the alternatives that could contribute to solving the clinical problems posed by diseases that occur with neuronal loss is stem cell transplantation. Several laboratories have tried this strategy in animal models transplanted with stem cells of embryonic origin (Cao, Q. et al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol 2001 167, 48 -58), stem cells neurals of adult animals (Pluchino, S., et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 2003 422, 688-694), or with cells from stem cells subjected to different degrees of differentiation in vitro The most generally observed phenomenon is that, although stem cells that are implanted in neurogenic areas of the brain differentiate into neuroblasts and become mature neurons, those that are implanted in non-neurogenic areas of the brain initiate the glial differentiation pathway ( Herrera, DG, et al, A. Adult-derived neural precursors transplanted into multiple regions in the adult brain. Ann Neurol 1999, 46, 867-877; Mligiliche, NL, et al. Survival of neural progenitor cells from the subventricular zone of the adult rat after transplantation into the host spinal cord of the same strain of adult rat. Anat Sci Int 2005, 80, 229-234).
Por lo tanto, la presente invención se enfrenta al siguiente problema: si bien las células madre que se implantan en las zonas neurogénicas del cerebro (por ejemplo, la zona subventricular o el bulbo olfativo en roedores) se diferencian a neuroblastos y se convierten en neuronas maduras, cuando el transplante se realiza en otras zonas, inician la ruta de diferenciación glial. Por otro lado, en las zonas no neurogénicas del cerebro, las células de glía parenquimal se activan tras una lesión y dan lugar a precursores neurales, pero la mayoría de ellos se diferencian a células de glía y muy pocos a neuronas. Therefore, the present invention faces the following problem: although the stem cells that are implanted in the neurogenic areas of the brain (for example, the subventricular zone or the olfactory bulb in rodents) differentiate to neuroblasts and become neurons mature, when the transplant is performed in other areas, they begin the route of glial differentiation. On the other hand, in the non-neurogenic areas of the brain, parenchymal glia cells are activated after an injury and give rise to neural precursors, but most of them differ from glia cells and very few to neurons.
Adicionalmente y sin perjuicio de lo anterior, la presente invención también se enfrenta al problema de elegir la fuente de los precursores a trasplantar a la hora de realizar este tipo de trasplantes. Las células madre embrionarias diferenciadas in vitro a células madre neurales, o los precursores neurales fetales, son de las fuentes más abundantes. Sin embargo, no es fácil encontrar donantes de precursores embrionarios o fetales, por lo que sería de gran utilidad poder aislar precursores neurales de cerebro adulto, cultivarlos y expandirlos in vitro para su posterior uso en trasplantes. Así, identificar agentes que favorezcan la expansión de los precursores neurales de cerebro adulto en cultivo resultará de gran utilidad en el desarrollo de terapias regenerativas del sistema nervioso. Additionally and without prejudice to the foregoing, the present invention also faces the problem of choosing the source of the precursors to be transplanted when performing this type of transplant. Embryonic stem cells differentiated in vitro to neural stem cells, or fetal neural precursors, are from the most abundant sources. However, it is not easy to find donors of embryonic or fetal precursors, so it would be very useful to be able to isolate adult brain neural precursors, grow them and expand them in vitro for later use in transplants. Thus, identify agents that favor expansion of the adult brain neural precursors in culture will be very useful in the development of regenerative therapies of the nervous system.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Figura 1. Efecto de prostratina sobre cultivos de precursores neurales in vitro. Ensayo de neuroesferas: Imágenes representativas de microscopía de contraste de fases de cultivos de precursores neurales crecidos respectivamente en ausencia (A), presencia de prostratina (5 μΜ) (B). La línea blanca representa una distancia de 200 μπι. (C y D). Efecto de diferentes concentraciones de prostratina sobre el área y número de neuroesferas respectivamente. (E y F) Ensayo de viabilidad: Efecto de prostratina (5 μΜ) sobre el porcentaje de células no viables (E) y sobre el número total de células vivas a las 24, 48 y 72 horas del tratamiento (F). En todos los casos la proliferación se estimuló mediante la adición del factor de crecimiento bFGF al medio de cultivo. (* p < 0.05 en test T-Student en relación al control).  Figure 1. Effect of prostratin on in vitro neural precursor cultures. Neurosphere test: Representative contrast microscopy images of cultures of neural precursor cultures grown respectively in the absence (A), presence of prostratin (5 μΜ) (B). The white line represents a distance of 200 μπι. (C and D). Effect of different concentrations of prostratin on the area and number of neurospheres respectively. (E and F) Feasibility test: Effect of prostratin (5 μΜ) on the percentage of non-viable cells (E) and on the total number of living cells at 24, 48 and 72 hours of treatment (F). In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p <0.05 in T-Student test in relation to control).
Figura 2. Efecto de prostratina sobre la proliferación de precursores neurales. (A) Imágenes representativas de microscopía de fluorescencia de cultivos de precursores neurales crecidos respectivamente en ausencia y presencia de prostratina (5 μΜ) en los que se ha detectado la proteína marcadora de células en ciclo celular, Ki67 y el marcador de núcleos, DAPI. La línea blanca representa una distancia de 100 μιη. (B) Cuantificación del efecto de prostratina sobre el porcentaje de células Ki67+.(C) Efecto de prostratina sobre el porcentaje de núcleos picnóticos. En todos los casos la proliferación se estimuló mediante la adición del factor de crecimiento bFGF al medio de cultivo. (* p < 0.05). Figura 3. Efecto de prostratina sobre la expresión de ciclinas en cultivos de NPCs. (A) Imágenes de autorradiografías obtenidas después de la inmunodetección de las ciclinas A, E y DI bajo las condiciones de cultivo indicadas, y de α-tubulina como control de la carga proteica total. (B-D) Cuantificación de la inmunodetección de ciclinas A, E y DI expresada en relación a los valores de α-tubulina. (* p < 0.05, en test T-Student en relación al control sin tratamiento). Figure 2. Effect of prostratin on the proliferation of neural precursors. (A) Representative fluorescence microscopy images of cultures of neural precursors grown respectively in the absence and presence of prostratin (5 μΜ) in which the cell cycle cell marker protein, Ki67 and the core marker, DAPI has been detected. The white line represents a distance of 100 μιη. (B) Quantification of the effect of prostratin on the percentage of Ki67 + cells. (C) Effect of prostratin on the percentage of picnotic nuclei. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p <0.05). Figure 3. Effect of prostratin on cyclin expression in NPC cultures. (A) Images of autoradiographs obtained after immunodetection of cyclines A, E and DI under the indicated culture conditions, and of α-tubulin as a control of the total protein load. (BD) Quantification of the immunodetection of cyclines A, E and DI expressed in relation to the values of α-tubulin. (* p <0.05, in T-Student test in relation to the control without treatment).
Figura 4. Efecto de 12-desoxifor boles sobre cultivos de NPCs. (A) Estructura química de los 12-desoxiforboles evaluados en el ensayo de neuroesferas. (B) Imágenes representativas de microscopía de contraste de fase de cultivos de precursores neurales crecidos en ausencia (1-2) y presencia de los 12- desoxiforboles (1 μΜ): ER271 (3), ER272 (4), ER14 (5), ER2 (6), ER3 (7) y ER8 (8). La línea blanca representa una distancia de 200 μπι. (C y D) Efecto de diferentes concentraciones de 12-desoxiforboles sobre el área de las neuroesferas (% del control). En todos los casos la proliferación se estimuló mediante la adición del factor de crecimiento bFGF al medio de cultivo (* p < 0.05 en test T- Student en relación al control). Figure 4. Effect of 12-deoxifor bowls on NPC cultures. (A) Chemical structure of the 12-deoxiforbol trees evaluated in the neurosphere test. (B) Representative images of phase contrast microscopy of cultures of neural precursors grown in the absence (1-2) and presence of the 12-deoxy-trees (1 μΜ): ER271 (3), ER272 (4), ER14 (5) , ER2 (6), ER3 (7) and ER8 (8). The white line represents a distance of 200 μπι. (C and D) Effect of different concentrations of 12-deoxiforboles on the area of the neurospheres (% of control). In all cases, proliferation was stimulated by adding the bFGF growth factor to the culture medium (* p <0.05 in T-Student test in relation to the control).
Figura 5. Efecto de ER271 sobre la proliferación celular en cultivos de precursores neurales in vitro. (A y B) Imágenes representativas de microscopía de contraste de fase de cultivos de precursores neurales crecidos respectivamente en ausencia y presencia de ER271 (1 μΜ). La línea blanca representa una distancia de 200 μπι. (C y D) Efecto de diferentes concentraciones de ER271 sobre el área y el número de neuroesferas, respectivamente. (E) Efecto de ER271 (1 μΜ) sobre el porcentaje de células no viables a las 24, 48 y 72 horas del tratamiento. En todos los casos la proliferación se estimuló mediante la adición del factor de crecimiento bFGF al medio de cultivo. (* p < 0.05). Figura 6. Efecto de ER272 sobre la proliferación celular en cultivos de precursores neurales in vitro. (A y B) Imágenes representativas de microscopía de contraste de fase de cultivos de precursores neurales crecidos respectivamente en ausencia y presencia de ER272 (1 μΜ). La línea blanca representa una distancia de 200 μηι. (C y D) Efecto de diferentes concentraciones de ER272 sobre el área y el número de neuroesferas, respectivamente. (E) Efecto de ER271 (1 μΜ) sobre el porcentaje de células no viables a las 24, 48 y 72 horas del tratamiento. En todos los casos la proliferación se estimulaba mediante la adición del factor de crecimiento bFGF al medio de cultivo (* p < 0.05). Figure 5. Effect of ER271 on cell proliferation in neural precursor cultures in vitro. (A and B) Representative images of phase contrast microscopy of cultures of neural precursors grown respectively in the absence and presence of ER271 (1 μΜ). The white line represents a distance of 200 μπι. (C and D) Effect of different concentrations of ER271 on the area and number of neurospheres, respectively. (E) Effect of ER271 (1 μΜ) on the percentage of non-viable cells at 24, 48 and 72 hours of treatment. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p <0.05). Figure 6. Effect of ER272 on cell proliferation in in vitro neural precursor cultures. (A and B) Representative images of phase contrast microscopy of cultures of neural precursors grown respectively in the absence and presence of ER272 (1 μΜ). The white line represents a distance of 200 μηι. (C and D) Effect of different concentrations of ER272 on the area and number of neurospheres, respectively. (E) Effect of ER271 (1 μΜ) on the percentage of non-viable cells at 24, 48 and 72 hours of treatment. In all cases, proliferation was stimulated by adding the growth factor bFGF to the culture medium (* p <0.05).
Figura 7. Efecto del tratamiento con ER271 sobre el porcentaje de células en ciclo celular en cultivos de precursores neurales. (A) Imágenes representativas de microscopía de fluorescencia de cultivos de precursores neurales crecidos respectivamente en ausencia y presencia de ER271 (1 μΜ) en los que se ha detectado la proteína marcadora de células en ciclo celular, Ki67 y el marcador de núcleos, DAPI. La línea blanca representa una distancia de 100 μπι. (B) Cuantificación del efecto de ER271 sobre el porcentaje de células KÍ67+. En todos los casos la proliferación se estimulaba mediante la adición del factor de crecimiento bFGF al medio de cultivo. (* p < 0.05). Figure 7. Effect of ER271 treatment on the percentage of cells in the cell cycle in cultures of neural precursors. (A) Representative fluorescence microscopy images of neural precursor cultures grown respectively in the absence and presence of ER271 (1 μΜ) in which the cell cycle cell marker protein, Ki67 and the nucleus marker, DAPI has been detected. The white line represents a distance of 100 μπι. (B) Quantification of the effect of ER271 on the percentage of KÍ67 + cells. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p <0.05).
Figura 8. Efecto del tratamiento con ER272 sobre el porcentaje de células en ciclo celular en cultivos de precursores neurales. (A) Imágenes representativas de microscopía de fluorescencia de cultivos de precursores neurales crecidos respectivamente en ausencia y presencia de ER272 (1 μΜ) en los que se ha detectado la proteína marcadora de células en ciclo celular, Ki67 y el marcador de núcleos, DAPI. La línea blanca representa una distancia de 100 μηι. (B) Cuantificación del efecto de ER272 sobre el porcentaje de células KÍ67+. En todos los casos la proliferación se estimulaba mediante la adición del factor de crecimiento bFGF al medio de cultivo. (* p < 0.05). DESCRIPCIÓN DETALLADA DE LA INVENCIÓN Figure 8. Effect of ER272 treatment on the percentage of cells in cell cycle in cultures of neural precursors. (A) Representative fluorescence microscopy images of cultures of neural precursors grown respectively in the absence and presence of ER272 (1 μΜ) in which the cell cycle cell marker protein, Ki67 and the core marker, DAPI has been detected. The white line represents a distance of 100 μηι. (B) Quantification of the effect of ER272 on the percentage of KÍ67 + cells. In all cases the proliferation was stimulated by adding the growth factor bFGF to the culture medium. (* p <0.05). DETAILED DESCRIPTION OF THE INVENTION
La presente invención resuelve el problema de generación de nichos neurogénicos tanto en zonas neurogénicas como no neurogénicas del Sistema Nervioso Central, basándose en el empleo de compuestos de la familia de los 12-desoxiforboles The present invention solves the problem of generating neurogenic niches in both neurogenic and non-neurogenic areas of the Central Nervous System, based on the use of compounds of the family of 12-deoxyfor trees
Estos compuestos promueven la proliferación de los cultivos celulares de precursores neurales y su perfusión en el cerebro adulto lesionado favorece la proliferación de precursores neurales que posteriormente se diferenciarán a neuronas y repararán el tejido dañado. These compounds promote the proliferation of cell cultures of neural precursors and their perfusion in the injured adult brain favors the proliferation of neural precursors that will subsequently differentiate to neurons and repair damaged tissue.
Estos nuevos fármacos basados en 12-desoxiforboles permiten superar la inhibición de la neurogénesis fuera de regiones neurogénicas y favorecer la generación de nuevas neuronas en estas regiones a partir de precursores neurales, bien endógenos o bien trasplantados. Por lo tanto, a la hora de regenerar lesiones, en la región no-neurogénica de la zona lesionada el tratamiento con 12- desoxiforboles incrementa la probabilidad de que las células madre endógenas como las trasplantadas puedan dar lugar a neuronas maduras y funcionales.  These new drugs based on 12-deoxiforboles allow to overcome the inhibition of neurogenesis outside neurogenic regions and favor the generation of new neurons in these regions from neural precursors, either endogenous or well transplanted. Therefore, when it comes to regenerating lesions, in the non-neurogenic region of the injured area, treatment with 12-deoxyforests increases the likelihood that endogenous stem cells such as transplanted cells can give rise to mature and functional neurons.
Por lo tanto, un primer aspecto de la presente invención, comprende el uso de compuestos de la familia 12-desoxiforboles o de sales farmacológicamente activas de dichos compuestos, para la elaboración de un medicamento (de aquí en adelante composición farmacéutica de la presente invención) para el tratamiento de enfermedades o lesiones que cursen con perdida neuronal. La estructura química de estos compuestos se describirá a continuación para las estructuras genéricas la y Ib: Estructura la: Therefore, a first aspect of the present invention comprises the use of compounds of the family 12-deoxyforbol trees or pharmacologically active salts of said compounds, for the preparation of a medicament (hereinafter pharmaceutical composition of the present invention) for the treatment of diseases or injuries that occur with neuronal loss. The chemical structure of these compounds will be described below for the generic structures la and Ib: Structure the:
Figure imgf000011_0001
Figure imgf000011_0001
Donde R¡ puede ser hidrógeno ó acetilo (COCH3), R2 y R3 pueden ser hidrógeno ó alquilo y R4 puede ser hidrógeno, metilo (CH3) ó fenilo (Ph). Where R¡ can be hydrogen or acetyl (COCH 3 ), R 2 and R 3 can be hydrogen or alkyl and R 4 can be hydrogen, methyl (CH 3 ) or phenyl (Ph).
Estructura Ib: Ib Structure:
Figure imgf000011_0002
Figure imgf000011_0002
Son ejemplos de compuestos de esta familia: Examples of compounds of this family are:
13-0-Acetil-12-desoxiforbol con número CAS 60857-08-1 (en adelante prostratina) cuya estructura química es la siguiente:  13-0-Acetyl-12-deoxiforbol with CAS number 60857-08-1 (hereinafter prostratin) whose chemical structure is as follows:
Estructura II:  Structure II:
Figure imgf000012_0001
Figure imgf000012_0001
Prostratina  Prostratin
13-0-Isobutiroil-12-desoxiforbol con número CAS 25090-74-8 (en adelante ER272), cuya estructura química es la siguiente: 13-0-Isobutiroyl-12-deoxiforbol with CAS number 25090-74-8 (hereinafter ER272), whose chemical structure is as follows:
Estructura III:  Structure III:
Figure imgf000012_0002
Figure imgf000012_0002
ER272 13-0-angeloil-12-desoxiforbol con número CAS 28152-96-7 (en adelante ER271) cuya estructura química es la siguiente: ER272 13-0-angeloil-12-deoxiforbol with CAS number 28152-96-7 (hereinafter ER271) whose chemical structure is as follows:
Estructura IV:  Structure IV:
Figure imgf000013_0001
Figure imgf000013_0001
ER271  ER271
20-O-acetil-13-O-angeloil-12-desoxiforbol con número CAS 25090-72-6 (en adelante ER2) cuya estructura química es la siguiente: 20-O-acetyl-13-O-angeloil-12-deoxiforbol with CAS number 25090-72-6 (hereinafter ER2) whose chemical structure is as follows:
Estructura V:  Structure V:
Figure imgf000013_0002
Figure imgf000013_0002
ER2 20-O-acetil-13-O-Isobutiroil-12-desoxiforbol (en adelante ER3) y cuya estructura química se muestra a continuación. ER2 20-O-acetyl-13-O-Isobutyroyl-12-deoxyforbol (hereinafter ER3) and whose chemical structure is shown below.
Estructura VI:  Structure VI:
Figure imgf000014_0001
Figure imgf000014_0001
ER3  ER3
13-0-Fenilacetil-12-desoxiforbol (en adelante ER14) y cuya estructura química se muestra a continuación. 13-0-Phenylacetyl-12-deoxiforbol (hereinafter ER14) and whose chemical structure is shown below.
Estructura VII:  Structure VII:
Figure imgf000014_0002
ó 20-O-Acetil-13-O-fenilacetil-12-desoxiforbol (en adelante ER8) y cuya estructura química se muestra a continuación.
Figure imgf000014_0002
or 20-O-Acetyl-13-O-phenylacetyl-12-deoxyforbol (hereinafter ER8) and whose chemical structure is shown below.
Estructura VIH:  HIV structure:
Figure imgf000015_0001
Figure imgf000015_0001
ER8  ER8
Alternativamente, el primer aspecto de la presente invención se refiere a compuestos de la familia de los 12-desoxiforboles como son prostratina, ER272, ER271, ER2, ER3, ER8 y ER14 o sales farmacológicamente activas de los mismos para su uso en el tratamiento de enfermedades o lesiones que cursen con pérdida neuronal. Alternatively, the first aspect of the present invention relates to compounds of the family of 12-deoxiforbol trees such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 or pharmacologically active salts thereof for use in the treatment of diseases or injuries that occur with neuronal loss.
En un aspecto concreto de la presente invención, la composición farmacéutica de la presente invención además comprende precursores neurales o células madre neurales. En un aspecto aún más concreto, la composición farmacéutica de la presente invención comprende al menos un excipiente farmacéuticamente aceptable.  In a particular aspect of the present invention, the pharmaceutical composition of the present invention further comprises neural precursors or neural stem cells. In an even more specific aspect, the pharmaceutical composition of the present invention comprises at least one pharmaceutically acceptable excipient.
En el contexto de la presente invención, se entiende por precursores neurales o células madre neurales a aquellas células madre aisladas del tejido neural adulto o fetal, que presentan capacidad de auto-replicarse, pero una potencialidad limitada, pues sólo pueden diferenciarse hacia los tres subtipos de células del linaje neural: neuronas, astrocitos y oligodendrocitos. En un aspecto particular de la presente invención, las enfermedades o lesiones que cursan con pérdida neuronal son las seleccionadas de la lista que consiste en: In the context of the present invention, neural precursors or neural stem cells are understood as those stem cells isolated from adult or fetal neural tissue, which have the capacity to self-replicate, but have limited potential, since they can only be differentiated into the three subtypes of cells of the neural lineage: neurons, astrocytes and oligodendrocytes. In a particular aspect of the present invention, the diseases or lesions that occur with neuronal loss are those selected from the list consisting of:
Enfermedades neurodegenerativas: Se producen como consecuencia de la muerte neuronal precoz. Las más frecuentes son la enfermedad de Alzheimer, con pérdida neuronal en el hipocampo y corteza cerebral fundamentalmente, la enfermedad de Parkinson, con muerte selectiva de neuronas en la sustancia negra, o la esclerosis lateral amiotrófica (ELA), con déficit de neuronas en la médula espinal.  Neurodegenerative diseases: They occur as a result of early neuronal death. The most frequent are Alzheimer's disease, with neuronal loss in the hippocampus and cerebral cortex, Parkinson's disease, with selective death of neurons in the black substance, or amyotrophic lateral sclerosis (ALS), with deficit of neurons in the spinal cord.
- Traumatismo craneoencef ático: es una lesión de origen traumático que incide sobre el cráneo, con afectación cerebral. El daño puede ser focal— limitado a una sola área del cerebro— o involucrar a más de un área del cerebro. En el contexto de esta invención el traumatismo craneoencefálico puede producir daño cerebral por muerte neuronal que podría ser tratado mediante terapias dirigidas a favorecer la regeneración neuronal.  - Attic cranioencephalic trauma: it is a traumatic injury that affects the skull, with cerebral involvement. The damage can 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, head trauma can cause brain damage due to neuronal death that could be treated by therapies aimed at promoting neuronal regeneration.
- Lesión hipóxico-isquémica: Reducción del flujo sanguíneo cerebral hasta niveles que son insuficientes para mantener el metabolismo necesario para la normal función y estructura del cerebro. En los adultos, la isquemia es causada fundamentalmente por accidentes cerebro-vasculares, que pueden ser focales (de origen isquémico, hemorrágico o mixto), o múltiples (como en la demencia multiinfarto). En el recién nacido, dicha hipoxia/isquemia se debe fundamentalmente a sufrimiento fetal o perinatal. En el contexto de la presente invención la hipoxia-isquemia es una condición que produce sufrimiento celular debido a la falta de aporte de oxígeno al tejido cerebral que en la mayoría de los casos produce muerte neuronal.  - Hypoxic-ischemic lesion: 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 mainly caused by cerebrovascular accidents, which can be focal (of ischemic, hemorrhagic or mixed origin), or multiple (as in multi-infarcted dementia). In the newborn, said hypoxia / ischemia is mainly due to fetal or perinatal suffering. 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 that in most cases produces neuronal death.
- Infecciones del SNC: Afectación cerebral por distintos agentes infecciosos que originan meningitis, encefalitis o meningoencefalitis. En el contexto de esta invención, las infecciones del SNC originan sufrimiento celular bien directamente o indirectamente por el edema cerebral que originan, pudiendo causar muerte neuronal. - Epilepsia: Enfermedad crónica caracterizada por uno o varios trastornos neurológicos que deja una predisposición para generar convulsiones recurrentes, que suele dar lugar a consecuencias neurobiológicas, cognitivas y psicológicas. - 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 due to the cerebral edema they cause, and can cause neuronal death. - Epilepsy: Chronic disease characterized by one or several neurological disorders that leaves a predisposition to generate recurrent seizures, which usually lead to neurobiological, cognitive and psychological consequences.
En un aspecto aún más particular de la presente invención, las enfermedades o lesiones que cursan con pérdida neuronal son isquemia cerebral focalizada, traumatismo craneoencefálico con daño neuronal, Parkinson, epilepsia y esclerosis lateral amiotrófica. In an even more particular aspect of the present invention, the diseases or injuries that occur with neuronal loss are focused cerebral ischemia, head trauma with neuronal damage, Parkinson's, epilepsy and amyotrophic lateral sclerosis.
Un segundo aspecto de la presente invención, se refiere al uso in vitro de compuestos de la familia de 12-desoxiforboles como son por ejemplo prostratina, ER272, ER271, ER2, ER3, ER8 y ER14 para favorecer la proliferación de precursores neurales o células madre neurales.  A second aspect of the present invention relates to the in vitro use of compounds of the family of 12-deoxy-trees such as, for example, prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 to favor the proliferation of neural precursors or stem cells Neurals
Un tercer aspecto de la presente invención se refiere a un método (de aquí en adelante método de la presente invención) para la expansión de células madre neurales o precursores neurales in vitro que comprende: A third aspect of the present invention relates to a method (hereinafter method of the present invention) for the expansion of neural stem cells or neural precursors in vitro comprising:
a. poner en contacto células madre neurales o precursores neurales con un rango de concentración desde 1 pmol/L a 40 μπιοΙ/L de 12-desoxiforboles, en un medio que no impida la proliferación de las células madre neurales o precursores neurales y que comprenda el factor de crecimiento epidérmico (EGF) y/o el factor básico de crecimiento de fibroblastos (bFGF); y b. cosechar posteriormente las células.  to. contacting neural stem cells or neural precursors with a concentration range from 1 pmol / L to 40 μπιοΙ / L of 12-deoxy-trees, in a medium that does not prevent the proliferation of neural stem cells or neural precursors and that includes the factor epidermal growth (EGF) and / or the basic fibroblast growth factor (bFGF); and b. subsequently harvest the cells.
Para utilizar compuestos de la familia de 12-desoxiforboles como por ejemplo prostratina, ER272, ER271, ER2, ER3, ER8 y ER14 en un cultivo in vitro es preferible disolverlos previo a su utilización en una solución que pueda disolver tanto el compuesto como su sal farmacéuticamente aceptada. Ejemplos del disolvente pueden ser dimetilsufóxido, agua o similares. Adicionalmente este compuesto puede estar disuelto en tampón fosfato salino (PBS). En un aspecto particular de la invención, para cultivar las células madre neurales con 12-desoxiforboles se añade el compuesto de la famila de 12-desoxiforboles como por ejemplo prostratina, ER272, ER271, ER2, ER3, ER8 y ER14 en un rango de concentración desde 1 pmol/L a 40 μπιοΙ/L. Las células madre se cultivan en flotación a una densidad de 20 a 200 x 106 células/L. El compuesto se añade a un cultivo estático a 37°C durante 1 a 14 días en una atmósfera de 5% C02, cambiando el medio de forma total o parcial cada dos días. In order to use compounds of the family of 12-deoxiforbol trees such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 in an in vitro culture it is preferable to dissolve them prior to their use in a solution that can dissolve both the compound and its salt pharmaceutically accepted. Examples of the solvent may be dimethyl sulfoxide, water or the like. Additionally this compound may be dissolved in phosphate buffered saline (PBS). In a particular aspect of the invention, the compound of the 12-deoxyforwood family, such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14 in a concentration range is added to the neural stem cells from 1 pmol / L to 40 μπιοΙ / L. Stem cells are grown in flotation at a density of 20 to 200 x 10 6 cells / L. The compound is added to a static culture at 37 ° C for 1 to 14 days in an atmosphere of 5% C0 2 , changing the medium totally or partially every two days.
El medio en el que se cultivan las células puede ser cualquier medio que no impida la proliferación de las células madre neurales, como ejemplo se puede utilizar preferentemente un medio Dulbecco's modified Eagle's médium (DMEM)/F-12 (1:1), que contenga 2% del suplemento B27 (Invitrogen), 2mM L- glutamina y 2μ§/ηι1 de gentamicina. Adicionalmente el medio debe contener bien el factor de crecimiento epidérmico (EGF), preferiblemente a una concentración 20μg/L, el factor de básico de crecimiento de fibroblastos (bFGF), preferiblemente a una concentración de 10μg/L, o una mezcla de ambos.  The medium in which the cells are grown can be any medium that does not prevent the proliferation of neural stem cells, as an example, a Dulbecco's modified Eagle's medium (DMEM) / F-12 (1: 1) medium can preferably be used, which contain 2% of supplement B27 (Invitrogen), 2mM L-glutamine and 2μ§ / ηι1 of gentamicin. Additionally, the medium must contain either the epidermal growth factor (EGF), preferably at a concentration of 20μg / L, the basic fibroblast growth factor (bFGF), preferably at a concentration of 10μg / L, or a mixture of both.
Un cuarto aspecto de la presente invención se refiere a un medio de cultivo (de aquí en adelante medio de cultivo de la presente invención), adecuado para la proliferación de células madre neurales o precursores neurales, que comprenda un compuesto de la famila de 12-desoxiforboles como son por ejemplo prostratina, ER272, ER271, ER2, ER3, ER8 y ER14, en un rango de concentración de 1 pmol/L a 40 μηιοΙ/L.  A fourth aspect of the present invention relates to a culture medium (hereinafter culture medium of the present invention), suitable for the proliferation of neural stem cells or neural precursors, comprising a compound of the family of 12- deoxiforboles such as prostratin, ER272, ER271, ER2, ER3, ER8 and ER14, in a concentration range of 1 pmol / L to 40 μηιοΙ / L.
Un quinto aspecto de la presente invención se refiere al uso del medio de cultivo de la presente invención para la expansión de células madre neurales o precursores neurales.  A fifth aspect of the present invention relates to the use of the culture medium of the present invention for the expansion of neural stem cells or neural precursors.
Un sexto aspecto de la invención se refiere a una población de células madre neurales o precursores neurales obtenibles por el método de la presente invención.A sixth aspect of the invention relates to a population of neural stem cells or neural precursors obtainable by the method of the present invention.
Un séptimo aspecto de la presente invención se refiere al uso de la población de células madre neurales o precursores neurales obtenibles por el método de la presente invención para la elaboración de un medicamento para su uso en el tratamiento de enfermedades o lesiones que cursen con perdida neuronal. A seventh aspect of the present invention relates to the use of the population of neural stem cells or neural precursors obtainable by the method of present invention for the preparation of a medicament for use in the treatment of diseases or injuries that occur with neuronal loss.
Los siguientes ejemplos son meramente ilustrativos de la presente invención y en ningún lugar han de entenderse como limitativos de la misma. The following examples are merely illustrative of the present invention and are nowhere to be construed as limiting thereof.
EJEMPLO 1. Efecto de prostratina sobre las células madre neuronales EXAMPLE 1. Effect of prostratin on neuronal stem cells
Se ha investigado la actividad biológica del compuesto prostratina sobre las células madre neurales extraídas de la zona subventricular de ratones postnatales de 7 días. Se ha utilizado el denominado "ensayo de neuroesferas". El fundamento de este ensayo es que los precursores neurales cuando se cultivan en condiciones de flotación y no están adheridos al sustrato del material sobre el cual se realiza el cultivo, proliferan formando unos agregados llamados neuroesferas formados por las células proliferantes y su progenie. The biological activity of the prostratin compound on the neural stem cells extracted from the subventricular area of 7-day postnatal mice has been investigated. The so-called "neurosphere test" has been used. The rationale for this test is that neural precursors when grown under floating conditions and are not adhered to the substrate of the material on which the culture is carried out, proliferate forming aggregates called neurospheres formed by proliferating cells and their progeny.
Para realizar este ensayo, las paredes laterales de los ventrículos laterales de la zona subventricular de ratones posnatales de 7 días (P7), fueron extraídas y disociadas enzimáticamente en LCR Ca2 bajo Mg2 alto (5 mM KC1, 124 mM NaCl, 3.2 mM MgCl2, 100 uM CaCl2, 26 mM NaHC03, and 10 mM glucosa) suplementado con 1 mg/ml de tripsina y 0.2 mg/ml de ácido quinurénico calentado previamente a 37°C durante 15 minutos. Se incubó a 37° en estufa 13 minutos. El tejido fue centrifugado 5 minutos y resuspendido en LCR normal (5 mM KC1, 124 mM NaCl, 1.3 mM MgCl2, 2 mM CaCl2, 26 mM NaHC03, y 10 mM glucosa). Fue incubado a 37°C durante 5 minutos y centrifugado en las mismas condiciones. Luego, las células fueron resuspendidas en Dulbecco's modified Eagle's médium (DMEM)/F-12 (1 :1) suplementado con 0.7 mg/ml de ovomucoide y disgregadas mecánicamente con una pipeta Pasteur con el diámetro reducido a la mitad. Después fueron centrifugadas nuevamente y resuspendidas en 6 mi de medio definido de neuroesferas (45 mi de (DMEM)/F-12, 900 μΐ, de suplemento B27, 2mM L-glutamina y 2μg/ml de gentamicina) suplementado con 6 μL· de EGF 20ng/ml y bFGF 10ng/ml, y mantenido a 37° en atmósfera con 5% C02. Después de 1-2 días en estas condiciones se forman los agregados celulares llamados neuroesferas. Los subcultivos fueron pasados cada 3-4 días por centrifugación de neuroesferas y disociación mecánica de las células en 1 mi de medio definido de neuroesferas; después la suspensión de células fue cultivada en nuevos frascos con medio fresco para obtener nuevas neuroesferas. Los experimentos se llevaron a cabo entre los pases 3 y 5. To perform this test, the lateral walls of the lateral ventricles of the subventricular zone of 7-day postnatal mice (P7) were enzymatically extracted and dissociated in CSF Ca 2 under high Mg 2 (5 mM KC1, 124 mM NaCl, 3.2 mM MgCl 2 , 100 uM CaCl 2 , 26 mM NaHC03, and 10 mM glucose) supplemented with 1 mg / ml trypsin and 0.2 mg / ml quinurenic acid preheated at 37 ° C for 15 minutes. It was incubated at 37 ° in an oven for 13 minutes. The tissue was centrifuged 5 minutes and resuspended in normal CSF (5 mM KC1, 124 mM NaCl, 1.3 mM MgCl 2 , 2 mM CaCl 2 , 26 mM NaHC03, and 10 mM glucose). It was incubated at 37 ° C for 5 minutes and centrifuged under the same conditions. The cells were then resuspended in Dulbecco's modified Eagle's medium (DMEM) / F-12 (1: 1) supplemented with 0.7 mg / ml ovomucoid and mechanically disintegrated with a Pasteur pipette with the diameter reduced by half. They were then centrifuged again and resuspended in 6 ml of defined neurosphere medium (45 ml of (DMEM) / F-12, 900 μΐ, of supplement B 27 , 2 mM L-glutamine and 2 μg / ml of gentamicin) supplemented with 6 μL of 20ng / ml EGF and 10ng / ml bFGF, and maintained at 37 ° in 5% atmosphere C0 2 . After 1-2 days under these conditions, cell aggregates called neurospheres are formed. The subcultures were passed every 3-4 days by centrifugation of neurospheres and mechanical dissociation of the cells in 1 ml of defined medium of neurospheres; then the cell suspension was grown in new bottles with fresh medium to obtain new neurospheres. The experiments were carried out between passes 3 and 5.
Una vez obtenidas las neuroesferas se analizó el efecto de prostratina sobre las células madre neurales a distintas concentraciones. Para ello las neuroesferas fueron centrifugadas y las células fueron resuspendidas y disgregadas en medio definido de neuroesferas y sembradas 20 células l iL a la que se le añadió el factor de crecimiento bFGF (10 ng/mL). Prostratina se añadió al mismo tiempo poniendo en cada pocilio una concentración diferente. Se utilizaron 5 pocilios para las diferentes concentraciones (Control, ^g/ml, 5μg/ml, y 10 μg/ml) y por triplicado. Los experimentos se llevaron a cabo de modo que la persona que toma las imágenes y realiza la cuantificación no conoce las condiciones de cada uno de los cultivos. El número de nuevas neuroesferas formadas fue contada 72h después en el microscopio invertido y de contraste de fase Olympus 1X70. Para medir el área de las neuroesferas se obtuvieron imágenes de 50 neuroesferas por pocilio y se analizaron empleando el sistema de análisis Image J.  Once the neurospheres were obtained, the effect of prostratin on neural stem cells at different concentrations was analyzed. To this end, the neurospheres were centrifuged and the cells were resuspended and disintegrated in a defined medium of neurospheres and seeded 20 l cells to which the bFGF growth factor (10 ng / mL) was added. Prostratin was added at the same time by placing a different concentration in each well. 5 wells were used for the different concentrations (Control, ^ g / ml, 5μg / ml, and 10 μg / ml) and in triplicate. The experiments were carried out so that the person who takes the images and performs the quantification does not know the conditions of each of the cultures. The number of new formed neurospheres was counted 72h later on the Olympus 1X70 inverted phase contrast microscope. To measure the area of the neurospheres, images of 50 neurospheres per well were obtained and analyzed using the Image J analysis system.
Se observó cómo el área de las neuroesferas aumentaba en los cultivos tratados con prostratina de manera dosis-dependiente hasta la concentración de ΙΟμΜ. Se observó además que el mayor efecto se obtenía a la concentración 5μΜ pues inducía un mayor aumento del tamaño de las neuroesferas (Figura 1). El incremento en el tamaño de las neuroesferas coincidía con un incremento en el número total de células en los cultivos tratados con prostratina 5 μΜ, apoyando la hipótesis de que el incremento en el tamaño de las neuroesferas se debía a una estimulación de la proliferación celular. No se observó muerte celular inducida por prostratina. EJEMPLO 2. Efecto de prostratina sobre el porcentaje de núcleos en ciclo celular. Inmunocitoquímica frente a Ki67. It was observed how the area of the neurospheres increased in the prostratin-treated cultures in a dose-dependent manner to the concentration of ΙΟμΜ. It was also observed that the greatest effect was obtained at the 5μΜ concentration because it induced a greater increase in the size of the neurospheres (Figure 1). The increase in the size of the neurospheres coincided with an increase in the total number of cells in the cultures treated with prostratin 5 μΜ, supporting the hypothesis that the increase in the size of the neurospheres was due to a stimulation of cell proliferation. No prostratin-induced cell death was observed. EXAMPLE 2. Effect of prostratin on the percentage of nuclei in the cell cycle. Immunocytochemistry against Ki67.
Con el fin de confirmar si el incremento en el tamaño de las neuroesferas se debía a un aumento en la proliferación de los precursores neurales se detectó mediante inmunocitoquímica el marcador Ki67 que detecta aquellos núcleos que están ciclo celular. El análisis se realizó en cultivos control y en cultivos tratados con prostratina 5 μΜ. In order to confirm whether the increase in the size of the neurospheres was due to an increase in the proliferation of neural precursors, the Ki67 marker that detects those nuclei that are cell cycle was detected by immunocytochemistry. The analysis was performed in control cultures and in cultures treated with prostratin 5 μΜ.
Para ello, una vez pasadas las 48 horas de tratamiento, se procedió a fijar las células durante 20 minutos a temperatura ambiente con paraformaldehido (PFA) al 4% preparado en tampón fosfato 0.1 M. Posteriormente se lavaron los pocilios 3 veces con PBS. Se permeabilizaron las células incubando 20 minutos a -20 °C con una solución de etanol/acético (95% etanol absoluto, y 5% de ácido acético glacial). Se lavó 3 veces con PBS y posteriormente se bloqueó con una solución de BSA al 1.5% (albúmina sérica bovina) en PBS; se incubaron durante 30 minutos. Se añadió el anticuerpo anti Ki67 y se dejó durante toda la noche a 4°C en solución de bloqueo. Al día siguiente se lavaron con PBS, se bloquearon nuevamente durante 20 minutos y se incubaron con los anticuerpos secundarios durante 40 minutos en solución de bloqueo en oscuridad. El montaje se realizó con una solución de Electron Microscopy Science y se añadió DAPI en el momento del montaje. Se utilizó un microscopio de epifluorescencia Olympus BX60 para la cuantificación.  To do this, after 48 hours of treatment, the cells were fixed for 20 minutes at room temperature with 4% paraformaldehyde (PFA) prepared in 0.1 M phosphate buffer. The wells were subsequently washed 3 times with PBS. The cells were permeabilized by incubating 20 minutes at -20 ° C with an ethanol / acetic solution (95% absolute ethanol, and 5% glacial acetic acid). It was washed 3 times with PBS and subsequently blocked with a solution of 1.5% BSA (bovine serum albumin) in PBS; They were incubated for 30 minutes. The anti Ki67 antibody was added and left overnight at 4 ° C in blocking solution. The next day they were washed with PBS, blocked again for 20 minutes and incubated with the secondary antibodies for 40 minutes in dark blocking solution. The assembly was performed with an Electron Microscopy Science solution and DAPI was added at the time of assembly. An Olympus BX60 epifluorescence microscope was used for quantification.
Se observó que el tratamiento con prostratina (5 mM) aumentaba el porcentaje de células en ciclo celular con núcleos Ki67+, así como el número total de células (núcleos totales teñidos con DAPI). En las células tratadas con prostratina el porcentaje de núcleos Ki67 positivos (Figura 2) era significativamente mayor que en el control, indicando que prostratina activa la proliferación de los precursores neurales. EJEMPLO 3. Efecto de prostratina sobre la expresión de ciclinas. Las ciclinas son proteínas que determinan la entrada de la célula en distintas fases del ciclo celular y su expresión es indicadora de proliferación. La expresión de ciclinas tipo D está regulada por señales mitogénicas, de esa manera, estas proteínas son los principales sensores de un microambiente que favorece el crecimiento celular. Con el fin de evaluar el efecto de los 12-desoxiforboles sobre la expresión de ciclinas, se llevó a cabo el tratamiento de las células en presencia y ausencia de prostratina con y sin bFGF. Transcurrido el tiempo de incubación y tratamiento, las células fueron Usadas y procesadas para realizar un western-blot. Como se indican los resultados en la figura 3, la prostratina presenta un leve efecto sobre la expresión de ciclina A, cuando las células fueron tratadas con bFGF.Por otro lado, el aumento de la cantidad de ciclina DI y E es mayor en presencia de prostratina, siendo más del doble con respecto a sus controles. Cuando las células crecieron en medio con prostratina y bFGF los valores correspondientes a las cantidades de proteínas son notablemente más altos que en el resto de las condiciones. Indicando que prostratina incrementa la concentración de ciclinas, proteínas que determinan la entrada en ciclo celular. It was observed that treatment with prostratin (5 mM) increased the percentage of cells in the cell cycle with Ki67 + nuclei, as well as the total number of cells (total nuclei stained with DAPI). In the cells treated with prostratin the percentage of Ki67 positive nuclei (Figure 2) was significantly higher than in the control, indicating that prostratin activates the proliferation of neural precursors. EXAMPLE 3. Effect of prostratin on cyclin expression. Cyclines are proteins that determine the entry of the cell into different phases of the cell cycle and its expression is indicative of proliferation. The expression of type D cyclins is regulated by mitogenic signals, in this way, these proteins are the main sensors of a microenvironment that favors cell growth. In order to evaluate the effect of 12-deoxiforboles on cyclin expression, the treatment of the cells was carried out in the presence and absence of prostratin with and without bFGF. After the incubation and treatment time, the cells were used and processed to perform a western blot. As the results are indicated in Figure 3, prostratin has a slight effect on the expression of cyclin A, when the cells were treated with bFGF.On the other hand, the increase in the amount of cyclin DI and E is greater in the presence of prostratin, being more than double with respect to its controls. When the cells grew in medium with prostratin and bFGF the values corresponding to the amounts of protein are markedly higher than in the rest of the conditions. Indicating that prostratin increases the concentration of cyclines, proteins that determine cell cycle entry.
EJEMPLO 4. Efecto de 12-desoxiforboles aislados de Euforbia resinífera sobre la formación de neuroesferas en cultivos de precursores neurales. EXAMPLE 4. Effect of 12-deoxiforbol trees isolated from Euphorbia resiniferous on the formation of neurospheres in neural precursor cultures.
Se ha investigado la actividad biológica de compuestos de la familia de los 12- desoxiforboles sobre cultivos de células madre neurales extraídas de la zona subventricular de ratones postnatales de 7 días. Siguiendo la metodología indicada en el ejemplo 2, se analizó el efecto de ER271, ER272, ER2, ER3, ER8, y ER14 sobre la formación de neuroesferas en cultivos de precursores neurales. The biological activity of compounds of the family of 12-deoxy-trees on cultures of neural stem cells extracted from the subventricular area of 7-day postnatal mice has been investigated. Following the methodology indicated in example 2, the effect of ER271, ER272, ER2, ER3, ER8, and ER14 on the formation of neurospheres in neural precursor cultures was analyzed.
Se seleccionaron seis 12-desoxiforboles aislados en E. Resinífera (ER271, ER272, ER2, ER3, ER8 y ER14). Los resultados se exponen en la figura 4. Los seis 12- desoxiforboles fueron evaluados mediante ensayos de neuroesferas tratados con concentraciones crecientes de los mismos (1, 5, 10 μΜ). Se observó que el tratamiento con todos los 12-desoxiforboles evaluados incrementaban el área de neuroesferas a, al menos, una de las concentraciones evaluadas. Los 12- desoxiforboles ER271, ER272 y ER14 fueron capaces de incrementar el tamaño de las neuroesferas con un máximo de actividad a la concentración ΙμΜ. El tratamiento con ER2 aumentaba el tamaño de las neuroesferas de forma dependiente de dosis, alcanzando un aumento del 200% en el tamaño de las neuroesferas a la concentración 10 μΜ. El tratamiento con ER3 aumentaba la proliferación tanto como ER2, aunque la concentración necesaria para ver el efecto máximo era 1 μΜ. El tratamiento con ER8 sólo aumentaba el tamaño de las neuroesferas en un 20% y sólo a la concentración de 1 μΜ comparando con el control. Six isolated 12-deoxyfoam trees were selected in E. Resiniferous (ER271, ER272, ER2, ER3, ER8 and ER14). The results are shown in Figure 4. The six 12-deoxyfor trees were evaluated by tests of neurospheres treated with increasing concentrations thereof (1, 5, 10 μΜ). It was observed that the Treatment with all 12-deoxyforlocks evaluated increased the area of neurospheres to at least one of the concentrations evaluated. The 12-deoxiforbol trees ER271, ER272 and ER14 were able to increase the size of the neurospheres with a maximum activity at the concentration ΙμΜ. Treatment with ER2 increased the size of the neurospheres in a dose-dependent manner, reaching a 200% increase in the size of the neurospheres at a concentration of 10 μΜ. Treatment with ER3 increased proliferation as much as ER2, although the concentration needed to see the maximum effect was 1 μΜ. ER8 treatment only increased the size of the neurospheres by 20% and only at the concentration of 1 μΜ compared to the control.
EJEMPLO 5: Estudio detallado del efecto proliferativo de ER271 y ER272 sobre la proliferación de los precursores neurales. EXAMPLE 5: Detailed study of the proliferative effect of ER271 and ER272 on the proliferation of neural precursors.
De modo arbitrario se seleccionaron el ER271 y ER272 para estudiar este efecto más detenidamente. The ER271 and ER272 were selected arbitrarily to study this effect more closely.
En primer lugar utilizando el ensayo de neuroesferas estimulados con bFGF, se analizó el efecto de concentraciones menores de ER271 y ER272 (0.1, 0.25, 0.5 y 1 μΜ) y se midieron los efectos sobre área y número de neuroesferas comparándolas con el control sin tratamiento. Tanto con ER271 como con ER272 se observó un aumento en el área de las neuroesferas a las concentraciones 0.25 y 0.5 μΜ (Figuras 5 y 6). Este aumento era similar al obtenido con la concentración de 1 μΜ. No se observaron cambios significativos en el número de neuroesferas. En el ensayo de viabilidad celular se observó que tanto ER271 como ER272 incrementaban de forma significativa la supervivencia de las células (Figuras 7 y 8). Los resultados de la inmunodetección de Ki67 en cultivos tratados con ER271 y ER272 indicaron que ambos compuestos eran capaces de aumentar de forma significativa el porcentaje de células Ki67+. Obtención de 12-desoxiforboles de Euphorbia resinífera. First, using the bFGF stimulated neurosphere test, the effect of lower concentrations of ER271 and ER272 (0.1, 0.25, 0.5 and 1 μΜ) was analyzed and the effects on area and number of neurospheres were measured compared to the untreated control . With both ER271 and ER272, an increase in the area of the neurospheres was observed at concentrations 0.25 and 0.5 μΜ (Figures 5 and 6). This increase was similar to that obtained with the concentration of 1 μΜ. No significant changes were observed in the number of neurospheres. In the cell viability test it was observed that both ER271 and ER272 significantly increased cell survival (Figures 7 and 8). The results of Ki67 immunodetection in cultures treated with ER271 and ER272 indicated that both compounds were able to significantly increase the percentage of Ki67 + cells. Obtaining 12-deoxyforboles from Euphorbia resinífera.
Los 12-desoxiforboles 13-0-angeloil-12-desoxiforbol o ER271, 13-0- isobutiroil-12-desoxiforbol o ER272, 13-<9-fenilacetil-12-desoxiforbol o ER14, 20-O-acetil-13-O-angeloil-12-desoxiforbol o ER2, 20-O-acetil-13-O-isobutiroil- 12-desoxiforbol o ER3 y 2O-0-acetil-13-0-fenilacetil-12-desoxiforbol o ER8 fueron obtenidos por aislamiento y purificación a partir del látex de Euphorbia resinífera colectada en Tanant, Marruecos (Diciembre, 2007). La obtención de los productos purificados se realizó a partir del extracto metanólico del látex de la planta y sometido a un fraccionamiento posterior mediante cromatografía en gel de sílice y cromatografía líquida de alta eficacia (HPLC); este fraccionamiento fue controlado mediante cromatografía en capa fina. Los productos fueron identificados mediante técnicas espectroscópicas y espectrométricas. Los datos espectroscópicos de los productos obtenidos en E. resinífera coinciden plenamente con los datos reportados previamente en la literatura. The 12-deoxyforbol trees 13-0-angeloyl-12-deoxyforbol or ER271, 13-0- isobutyroyl-12-deoxyforbol or ER272, 13- <9-phenylacetyl-12-deoxyforbol or ER14, 20-O-acetyl-13-O -angeloyl-12-deoxiforbol or ER2, 20-O-acetyl-13-O-isobutyroyl-12-deoxyforbol or ER3 and 2O-0-acetyl-13-0-phenylacetyl-12-deoxyforbol or ER8 were obtained by isolation and purification from the resin Euphorbia latex collected in Tanant, Morocco (December, 2007). Purification of the purified products was made from the methanolic extract of the plant latex and subjected to a subsequent fractionation by silica gel chromatography and high efficiency liquid chromatography (HPLC); This fractionation was controlled by thin layer chromatography. The products were identified by spectroscopic and spectrometric techniques. The spectroscopic data of the products obtained in E. resinífera fully coincide with the data previously reported in the literature.

Claims

Uso in vitro de compuestos de la familia de los 12-desoxiforboles tales como los detallados en las estructuras la y Ib o una sal farmacológicamente activa de los mismos, para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of compounds of the family of 12-deoxyfor trees such as those detailed in structures la and Ib or a pharmacologically active salt thereof, to favor the proliferation of neural precursors or neural stem cells in culture.
Uso in vitro del compuesto según la reivindicación 1 denominado 13-O- acetil-12-desoxiforbol para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of the compound according to claim 1 called 13-O-acetyl-12-deoxiforbol to favor the proliferation of neural precursors or neural stem cells in culture.
Uso in vitro del compuesto según la reivindicación 1 denominado 13-O- Isobutiroil-12-desoxiforbol para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of the compound according to claim 1 called 13-O-Isobutyroyl-12-deoxyforbol to favor the proliferation of neural precursors or neural stem cells in culture.
Uso in vitro del compuesto según la reivindicación 1 denominado 13-O- angeloil-12-desoxiforbol para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of the compound according to claim 1 called 13-O-angeloyl-12-deoxyforbol to favor the proliferation of neural precursors or neural stem cells in culture.
Uso in vitro del compuesto según la reivindicación 1 denominado 20-O- acetil-13-O-angeloil-12-desoxiforbol para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of the compound according to claim 1 called 20-O-acetyl-13-O-angeloyl-12-deoxyforbol to favor the proliferation of neural precursors or neural stem cells in culture.
Uso in vitro del compuesto según la reivindicación 1 denominado 20-O- acetil-13-O-Isobutiroil-12-desoxiforbol para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of the compound according to claim 1 called 20-O-acetyl-13-O-Isobutyroyl-12-deoxyforbol to favor the proliferation of neural precursors or neural stem cells in culture.
Uso in vitro del compuesto según la reivindicación 1 denominado 20-O- acetil-13-O-fenilacetil-12-desoxiforbol para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of the compound according to claim 1 called 20-O-acetyl-13-O-phenylacetyl-12-deoxyforbol to favor the proliferation of neural precursors or neural stem cells in culture.
Uso in vitro del compuesto según la reivindicación 1 denominado 13-O- fenilacetil-12-desoxiforbol para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. In vitro use of the compound according to claim 1 called 13-O-phenylacetyl-12-deoxyforbol to favor the proliferation of neural precursors or neural stem cells in culture.
HOJA DE REEMPLAZO (REGLA 26) REPLACEMENT SHEET (RULE 26)
9. Método para la proliferación de células madre neurales o precursores neurales in vitro que comprende: a. Disolver un compuesto de la familia de 12-desoxiforboles o cualquiera de los compuestos recogidos en las reivindicaciones 2-8 o una sal farmacológicamente activa de los mismos en una solución que pueda disolver tanto el compuesto como su sal farmacéuticamente aceptada; y b. Poner en contacto células madre neurales o precursores neurales con la disolución descrita en (a), en un medio adecuado para la proliferación de las células madre neurales o precursores neurales y que comprenda el factor de crecimiento epidérmico (EGF) y/o el factor básico de crecimiento de fibroblastos (bFGF); y donde los compuestos de la familia de 12-desoxiforboles o cualquiera de los compuestos recogidos en las reivindicaciones 2-8 o una sal farmacológicamente activa de dichos compuestos se hallen en el medio en un rango de concentración de 1 pmol/L a 40 μπιοΙ/L. 9. Method for proliferation of neural stem cells or neural precursors in vitro comprising: a. Dissolving a compound of the family of 12-deoxiforbol trees or any of the compounds set forth in claims 2-8 or a pharmacologically active salt thereof in a solution that can dissolve both the compound and its pharmaceutically accepted salt; and b. Contacting neural stem cells or neural precursors with the solution described in (a), in a suitable medium for the proliferation of neural stem cells or neural precursors and comprising the epidermal growth factor (EGF) and / or the basic factor fibroblast growth (bFGF); and wherein the compounds of the family of 12-deoxiforbol trees or any of the compounds set forth in claims 2-8 or a pharmacologically active salt of said compounds are in the medium in a concentration range of 1 pmol / L at 40 μπιοΙ / L.
10. Medio de cultivo adecuado para la proliferación de células madre neurales o precursores neurales in vitro, que comprenda un compuesto de la familia de 12-desoxiforboles o cualquiera de los compuestos recogidos en las reivindicaciones 2-8 y/o una sal farmacológicamente activa de dichos compuestos. 10. Culture medium suitable for the proliferation of neural stem cells or neural precursors in vitro, comprising a compound of the family of 12-deoxyforests or any of the compounds set forth in claims 2-8 and / or a pharmacologically active salt of said compounds.
11. Medio de cultivo según la reivindicación 10, que además comprenda el factor de crecimiento epidérmico (EGF) y/o el factor básico de crecimiento de fibroblastos (bFGF) y donde los compuestos de la familia de 12-desoxiforboles o cualquiera de los compuestos recogidos en las reivindicaciones 2-8 y/o la sal farmacológicamente activa del mismo se hallen en el medio en un rango de concentración de 1 pmol/L a 40 μπιοΙ/L. 11. Culture medium according to claim 10, further comprising the epidermal growth factor (EGF) and / or the basic fibroblast growth factor (bFGF) and wherein the compounds of the family of 12-deoxyfor trees or any of the compounds collected in claims 2-8 and / or the pharmacologically active salt thereof are in the medium in a concentration range of 1 pmol / L to 40 μπιοΙ / L.
HOJA DE REEMPLAZO (REGLA 26) REPLACEMENT SHEET (RULE 26)
12. Uso del medio de cultivo según cualquiera de las reivindicaciones 10 o 11, para favorecer la proliferación de precursores neurales o células madre neurales en cultivo. 12. Use of the culture medium according to any of claims 10 or 11, to promote the proliferation of neural precursors or neural stem cells in culture.
13. Precursores neurales o células madre neurales obtenibles por el método de la reivindicación 9. 13. Neural precursors or neural stem cells obtainable by the method of claim 9.
14. Uso de compuesto de la familia de 12 desoxiforboles o cualquiera de los compuestos recogidos en las reivindicaciones 2-8 y/o una sal farmacológicamente activa de dichos compuestos, para la elaboración de un medicamento para su uso en el tratamiento de enfermedades o lesiones que cursen con perdida neuronal seleccionadas del grupo que consiste en: isquemia cerebral focalizada, traumatismo craneoencefálico con daño neuronal, Parkinson, epilepsia y esclerosis lateral amiotrófíca. 14. Use of a compound of the family of 12 deoxiforbol trees or any of the compounds set forth in claims 2-8 and / or a pharmacologically active salt of said compounds, for the preparation of a medicament for use in the treatment of diseases or injuries with neuronal loss selected from the group consisting of: focused cerebral ischemia, craniocerebral trauma with neuronal damage, Parkinson's, epilepsy and amyotrophic lateral sclerosis.
15. Uso según la reivindicación 14, que además comprende uno o más excipientes farmacéuticamente aceptables.  15. Use according to claim 14, further comprising one or more pharmaceutically acceptable excipients.
16. Uso según cualquiera de las reivindicaciones 14 o 15, que además comprende una población de precursores neurales o células madre neurales.  16. Use according to any of claims 14 or 15, further comprising a population of neural precursors or neural stem cells.
17. Uso según la reivindicación 16, donde dicha población de precursores neurales o células madre neurales es la población de la reivindicación 13.  17. Use according to claim 16, wherein said population of neural precursors or neural stem cells is the population of claim 13.
HOJA DE REEMPLAZO (REGLA 26) REPLACEMENT SHEET (RULE 26)
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SAMARA C ET AL.: "Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 107, no. 43, 26 October 2010 (2010-10-26), pages 18342 - 18347 *

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