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    • 专利摘要:
    Pharmaceutical composition for the treatment and/or prevention of degenerative retinal diseases. The present invention relates to a composition comprising mesenchymal stem cells (mscs), preferably derived from adipose tissue (ad-mscs), combined with vasoactive intestinal peptide (vip) and nicotinamide (nic), and more preferably also combined with retinoic acid (atra) and with supernatant of a retinal pigment epithelial cell culture (epr). It also relates to the use of this composition for the treatment and/or prevention of retinal damage, preferably degenerative diseases of the retina, for example, but without limitation, degenerative diseases of rpe, photoreceptors and neuroretina. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2539329A1
    申请号:ES201331743
    申请日:2013-11-29
    公开日:2015-06-29
    发明作者:Amar Kumar SINGH；Girish Kumar SRIVASTAVA；Iván FERNÁNDEZ BUENO；Manuel José GAYOSO RODRÍGUEZ；José Carlos PASTOR JIMENO
    申请人:Universidad de Valladolid；
    IPC主号:
    专利说明:

    The present invention falls within the field of cellular therapy for the regeneration of ocular tissues, specifically within pharmaceutical compositions comprising mesenchymal stem cells useful for tissue regeneration of the damaged retina, preferably of the pigment epithelium of the
    10 retina (EPR), photoreceptors or neuroretin affected by a degenerative retinal disease.
    STATE OF THE TECHNIQUE
    15 The retina is a tissue with great complexity, consisting of several clearly differentiated layers: the retinal pigment epithelium (RPE), the photoreceptors (PRs), the internal and external limiting membranes (ILM and OLM), the internal nuclear layers and external (INL and ONL), the internal and external plexiform layers (IPL and OPL), the ganglion cell layer (GeL) and the fiber layer of the
    20 optic nerve (NFL). The integrity of these layers is essential for the normal functioning of the retina. Thus, any structural or functional alteration in them causes a retinal disease. Retinal degenerations are responsible for alterations in retinal structure and function. Such degenerations eventually induce retinal diseases that result in a
    25 visual dysfunction and / or loss of vision. There are a large number of eye diseases related to retinal degeneration that do not currently have adequate treatment, such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), Stargardt's disease and acute anterior ischemic optic neuropathy non-arteritic (NOIANA).
    30 DMAE is the leading cause of blindness in industrialized countries (Europe and North America) among people over 65. This pathology has two forms, dry and wet. The dry form of AMD E constitutes between 60 and 90% of cases, while the wet form (also called CNV) of the
    35 English choroidal neovascufarization) represents between 10 and 40% of total cases of AMD. The dry AMD encompasses a market of 25-30 billion
    dollars between the United States and Europe and has a negative annual impact of around 30 billion dollars on the gross domestic product of the United States. The population called "elderly" will increase considerably in the coming decades in industrialized countries, which should alert the 5 health systems as it will increase the economic and social burden of the system. Therefore, the DMAE is considered a major public health problem, which can have a devastating effect on the quality of life of patients and also entail significant financial consequences for the economy of the industrialized world. Current AMD therapies have only shown
    10 small advances, especially in the CNV, but in many cases they have only managed to slow down the course of the disease. In the case of the dry form of AMD there is no clinically acceptable and / or effective treatment today.
    RP is caused by a progressive dysfunction of photoreceptors, which
    15 subsequently extends to other retinal layers. The PR has a prevalence of 1 case per 4000 individuals, the population affected in the world being 1.5 million. This pathology affects patients of school age and also working age. Currently, there is no treatment, although it has a great socio-health impact on the affected population.
    20 In addition to the two mentioned pathologies, there are a large number of retinal diseases caused by the degeneration of the different retinal cells, and for most of them there is no adequate treatment.
    25 The EPR plays an extremely important role in the development of a normal visual function, as well as in the maintenance of the structure and integrity of the retina. RPE is considered as one of the main responsible for the pathogenesis of various retinal pathologies, due to the structural and biochemical changes that occur throughout life in these cells. All these harmful changes
    30 progressively affect retinal health. Thus, the neural retina, mainly the photoreceptors, begin to degenerate gradually and synchronously with the functional deterioration of the RPE. In this case, it seems logical that if the damaged EPR could be replaced by healthy and new EPR, especially in the initial state of the pathology when the photoreceptors still function properly, it could be
    35 prevent or slow down its degeneration. There are some examples where researchers have tried to transplant EPR and iris pigment epithelium from autologous, homologous and heterologous sources. However, in the case of homologous and heterologous cells, important immunological rejections occurred. On the contrary, autologous sources showed adequate immune tolerance, but did not achieve an improvement in visual function, because the cells have the
    5 same genetic and environmental footprints.
    The three structures that are mainly affected in retinal pathologies are Bruch's membrane (BM), EPR and photoreceptors. The objective of advanced therapies is to be reconstructed jointly, by means of tissue engineering with tissue / cellular and non-tissue / cellular components, such as a substrate that acts as the basement membrane on which RPE cells can grow. It is clearly known that stem cells have the ability to regenerate themselves and differentiate into any adult cell using the right conditions. Therefore, stem cells can be a potential therapy in cell replacement, such as EPR, photoreceptors or even the entire retina. Thus, stem cells have created enormous hope in the regeneration of retinal cells. There are significant advances in obtaining fresh and functional EPR cells, including photoreceptors, from endothelial stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Several clinical trials for the transplantation of RPE cells derived from ESCs in patients with degenerative retinal diseases are being developed. In Japan, the Riken Center for Development Biology has launched a pilot study to assess the safety and feasibility of autologous transplantation of RPE cell layers derived from iPSCs in patients with wet AMD (Sipp, Douglas and Takahashi,
    25 Masayo. Pilot Clinical Study into iPS Cell Therapy for Eye Disease Starts in Japan, 30th July, 2013, hrlp: //www.riken.jp/en/pr/press/2013/20130730_1/). However, the use of both ESCs and iPSCs has some difficulties, due to ethical problems and the risk of tumor development.
    30 On the other hand, there are several growth factors that play an important role in maintaining neuroretinal health, and also of RPE, such as PEDF, BDNF, VEGF, CNTF, HGF, NGF, UF, etc. The EPR supplies the nutrients, coming from the choroidal circulation, to the outermost layers of the neural retina, recycles the external segments of the photoreceptors and secretes different factors of
    35 growth and neurotrophic factors, such as PEDF (in English, pigment epithelium-derived factor), BDNF (in English, brain ~ erived neurotrophic factor) and VEGF (in English,
    vascular endothelial growth factor). These factors are essential for themaintenance of neuroretinal homeostasis. Some recent clinical trials(Kauper K, et al. Two-year intraocular delivery of ciliary neurotrophic factor byencapsulated cell technology implants in patients with chronic retinal degenerative5 diseases Invest Ophthalmol Vis Sei. 2012 Nov 1; 53 (12): 7484-91; Zhang K, al.Ciliary neurotrophic factor delivered by encapsulated cell intraocular implants forlrealmenl of geographic alrophy in age-related macular degeneration. Proc Natl AcadSci. USA. 2011 Apr 12; 108 (15): 6241-5; Sieving PA, et al. Ciliary neurotrophic factor(CNTF) for human retinal degeneration: phase I trial of CNTF delivered by10 encapsulated cell intraocular implants. Proc Natl Acad Sci. USA. 2006 Mar 7; 103 (10): 3896-901; Thanos eG, et al. Sustained secrelion of ciliary neurotrophic factor lo the vitreous, using the encapsulated cell therapy-based NT-501 intraocular device.
    Tissue Eng. 2004 Nov-Dee; 10 (11-12): 1617-22) have studied the paracrine properties of genetically modified EPR cells (human NTC-201 derived from the ARPE19 cell line) encapsulated in an intraocular implant, in degenerative retinal diseases (such as RP and geographical atrophy or AG).
    However, the development of safe and effective therapies for the treatment and / or prevention of retinal degenerative diseases 20 that improve the quality of life of affected patients remains a necessity.
    DESCRIPTION OF THE INVENTION
    The present invention relates to a composition comprising stem cells
    25 mesenchymal (MSCs), preferably from adipose tissue (AD-MSCs), combined with vasoactive intestinal peptide (VIP) and nicotinamide (NIC), and more preferably also combined with retinoic acid (ATRA) and with the supernatant of a culture of EPR cells. This composition is useful for the treatment and / or prevention of tissue damage caused in the retina, preferably
    30 as a consequence of a retinal degenerative disease, including degeneration of RPE, photoreceptors and neuroretin.
    Thus, the composition described in the present invention can be used for the maintenance of the retina if it is decided to transplant it to the damaged tissue; or it can be used in the preparation of medicaments, preferably cell therapy,
    intended for the treatment and / or prevention of retinal degenerative diseases, including but not limited to inflammatory or genetic diseases.
    Chimeric stem cells, preferably derived from adipose tissue, in
    5 combination with the VIP and NIC biomolecules, and preferably also with ATRA and with factors secreted by the EPR, protect against degeneration of the EPR, photoreceptors and neuroretin, which can be used in the treatment of degenerative diseases of the retina in, for example, but not limited to humans. As the examples of the present invention show, the AD-MSCs, the
    10 factors secreted by the EPR or the VIP, NIC and ATRA biomolecules used individually show lower effectiveness than those observed when used in the combinations proposed here.
    Therefore, a first aspect of the invention relates to a pharmaceutical composition, hereinafter "composition of the invention", comprising mesenchymal stem cells, vasoactive intestinal peptide and nicolinamide.
    "Mesenchymal stem cells", "stromal stem cells" or "MSC", are multipotent cells, with fibroblastoid morphology, originating from the mesodermal germ layer 20, with the ability to differentiate into various cell types. Preferably, the MSCs express at least one of the specific surface antigens CD105, CD73 and CD90, and do not express the CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA class 11 antigens. These MSCs are able to differentiate, by example, but not limited to osteoblasts, adipocytes and chondroblasts under 25 standard in vitro differentiation conditions. MSCs can be isolated from various tissues, including, but not limited to, the bone marrow (BM), adipose tissue (lipoaspirates) (AD), liver, spleen, testicles, menstrual blood, amniotic fluid, pancreas, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, trabecular bone, umbilical cord, lung, dental pulp or peripheral blood. 30 Specifically, adipose tissue has different advantages over other sources, such as bone marrow, due to the ease of obtaining stem cells and the availability of a large number of cells, without large differences in properties. of BM-MSCs and AD-MSCs. Therefore, in a preferred embodiment of the composition of the invention, the MSCs come from
    35 adipose tissue.
    The use of both endothelial stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in cell therapy presents some difficulties, due to ethical problems and the risk of tumor development. However, in the case of using a source of adult stem cells, such as cells
    5 mesenchymal mother, these problems are significantly minimized. In addition, the usefulness of MSCs in the treatment / prevention of retinal degenerative diseases is not only due to their ability to differentiate into RPE cells or photoreceptors, but also to the neurotrophic, immunomodulatory or anti-inflammatory effect of the factors secreted by these cells.
    10 In addition, the use of MSCs could be an advantage compared to genetically modified EPR cells that have also been previously used in cell therapy for tissue regeneration of degenerating retina.
    On the other hand, the "vasoactive intestinal peptide" or "VIP" (from English, vasoactive intestinal peptide) is a neuropeptide belonging to the glucagon growth hormone VIP, of 28 amino acids (His-Ser-Asp-Ala-Val-Phe- Thr-Asp-Asn-Tyr-Thr-Arg-Leu-ArgLys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-lIe-Leu-Asn, SEO ID NO: 1) And with the empirical formula C'47H238N «042S (molecular weight 3325.9). It belongs to the superfamily of secretins, which are found in the retina (amacrine cells) and choroids in the eyeball, as well as in the cerebral cortex, the pituitary, the adrenal glands, the nerve endings of the respiratory system, the gastrointestinal tract and the reproductive system. This peptide promotes the growth and differentiation of multiple cell types in tissue cultures. The effect of VIP on proliferation, differentiation and neuroprotection is dose dependent. Its neuroprotective effect in vivo is mediated by BDNF. VIP is a strong secretagogue that promotes the release of factors derived from astroglia (chemokines), such as IL-1 (from English, interJeukin 1), IL-6, NT-3 (from English, neurotrophin-3), PN-1 (from English, 30 protease nexin-1), RANTES (from English, regulated upon activation normal T cell expressed and presumably secreted), MIP-1 (from English, macrophage inflammatory protein-1), ADNF (from English, activity dependent neuroprotective factor) and ADNP (activity dependent neuroprotective protein). VIP receptors (VPAC-1, VPAC-2 and PAC-1) occur in EPR cells, neurons and cells
    35 glials The expression of the VPAC-1 receptor increases in pathological conditions, as in ischemic damage.
    "Nicotinamide" or "NIC" (in English, nicotinamide) or "vitamin B", also called niacinamide or nicotinic acid amide, is a water-soluble amide originated from nicotinic acid (vitamin B3 / niacin). The chemical name is pyrido-3-carboxamide and its empirical formula is CsHsN20. The NIC is the first precursor of nicotinamide adenine dinucleotide (NAD +) and its phosphorylated derivative (NADP +), an essential coenzyme for mitochondrial production of ATP and the only substrate for the nuclear enzyme PARP-1 (English, poJy- ADP-ribose polymerase-1). The NIC acts as an inhibitor of the PARP enzyme, which plays an important role in the repair of cellular AON, also in retinal cells, in case of cell degeneration. The NIC also inhibits histone-dependent NAO deacetylase, which plays an important role in silencing and differentiating gene expression, in genomic stability, in the transduction signal and in cell growth and death. The NIC has various effects on cell cultures, including the inhibition of PARP, which protects cells from oxidative stress. 15 There are also scientific papers that support the neuroprotective role of the NIC. Retinal damage in vivo and in vitro extends beyond the injured area itself, covering the different layers of the retina. PARP activation influences some of the apoptotic pathways and it has been suggested that overregulation is involved in some types of retinal cell death. PARP activation is
    20 dependent on NAO + and during the poly-AOP ribosylation process the PARP dependent mechanism is activated and leads to apoptosis. These multiple effects of the NIC may be due to the fact that it is an essential substrate for NADH and NADPH.
    In a more preferred embodiment of the composition of the invention, it further comprises retinoic acid (ATRA).
    ~ Or "UATRA" retinoic acid (from English, all-trans-renoic acid) is also known as vitamin A acid. Its empirical formula is C2QH280 2 (molecular weight 300.4). 30 It is a lipophilic molecule that can easily cross the cell membrane. It is synthesized in vivo in the neural tissue from the retino !. ATRA is involved in the differentiation of the optic vesicle in EPR. ATRA is the natural retinoic acid metabolite and plays an important role in growth and differentiation. It is used in the differentiation of embryonic stem cells to different cell types, such as neurons, heart cells, smooth muscle or germ cells. ATRA is also associated with neuroprotection, both in vivo and in vitro. It has been shown to reduce oxidative stress in embryonic neurons by increasing the activity of the enzymes superoxide dismutase, cata lasa and glutathione reductase. It has also been described that modulates proliferation, differentiation and apoptosis, both in normal and altered cells, in vitro. It potentiates the neuroprotective effect of NGF. Influence early developmental stages, such as neurogenesis or axonal growth, by binding to RARs (in English, nuclear retinoic acid receptors) and RXRs (in English, retinoid X receptors) receptors, which regulate gene transcription. It has been described as having a potent antiapoptotic effect in the spontaneous death of eosinophils. Plays an important role
    10 in the differentiation of stem cells by altering epigenetic changes in the AON, as well as in histone proteins. It has been described that it has a neuroprotective effect in different experiments in vivo and in vitro and that it induces pigmentation in melanoma cell lines.
    In a more preferred embodiment, the composition of the invention further comprises the supernatant of a retinal pigment epithelial cell culture.
    The "pigment epithelium of the retina" or UEPR "is the layer of pigmented cells located on the outside of the retina that interacts closely with the photoreceptor cells (cones and rods) in maintaining visual function. It is firmly anchored to the underlying choroid by the Bruch membrane The retinal pigmentary epithelium is composed of a layer of hexagonal cells that are densely packed with pigment granules Seen in section, each cell consists of an un pigmented outer part in which a nucleus is located With a large oval shape and a pigmented inner portion that extends a series of straight filiform processes between the rods, it serves as a transport limiting factor that maintains the retina's environment, supplying small molecules such as amino acids, ascorbic acid and O-glucose, while representing a narrow barrier for substances carried by the choroid's blood. itelio
    The pigment of the retina also has the function of phagocytosis of the external segments of the photoreceptor cells and regeneration of the photopigment.
    "EPR cells" means any cell type present in said epithelium, preferably epithelial cells. EPR cells can be cultured in the presence of culture media and conditions known in the technical field for epithelial cell culture. Thus, the culture medium may comprise, for example, but not limited to, fetal bovine (FBS) or human serum, antibiotics, antifungals, growth factors, etc. The base medium that can be used in the culture medium could be any of those known in the state of the art for in vitro cell culture, such as, but not limited to, basal medium 5 "Eagle", CRCM-30, CMRL-1066, "Dulbecco's Modified Eagle's Medium" (DMEM), "Fischer's Medium", "Glasgow Minimum Essential Medium", Ham's F-10, Ham's F-12 (F12), "High Cell Density Medium", "Iscove's Modified Dulbecco's Medium ", Leibovitz's L-15, McCoy's 5A, medium 199," Minimum Essential Medium Eagle "," Alpha Minimum Essenlial Medium ", CnT20, NCTC 109, NCTC 135, RPMI-1640," William's Medium E ", 10 Waymouth's MB 75211 , Waymouth's MB 70511, "Keratinocyte serum-free medium" (KSFM), or any combination thereof. Preferably, the culture medium comprises a DMEM / F12 base medium. In addition, the culture conditions may be, for example, but not limited to, in the presence of between 5 and 10% CO2, between 36 and 38 ° C and for 24 to 48 hours. Preferably, said culture is carried out.
    15 as described below in the examples of the present invention.
    As the examples of the present invention show, neuroretinal explants that had undergone progressive degeneration during culture were significantly better conserved when co-cultured with AD-MSCs in
    20 presence of the supernatant of a culture of EPR and VIP + NIC or VIP + NIC + ATRA cells. Therefore, in an even more preferred embodiment, the composition of the invention comprises AD-MSCs, VIP, NIC, ATRA and the supernatant of an EPR cell culture.
    The MSCs and EPR cells referred to in the present invention can be derived from any animal, preferably mammal, more preferably human. In another preferred embodiment, the MSCs and EPR cells come from a human.
    In addition, both MSCs and EPR cells can be of autologous or heterologous origin. The possibility that said cells are of autologous origin allows that subsequent transplantation or administration of the composition of the invention for tissue regeneration of the damaged retina can be performed without the need for immunosuppression of the treated subject. For eyl, in a more preferred embodiment, the
    35 MSCs are autologous in origin. "Autologous origin" means any origin of the sample, taken from the tissues or cells of an individual or patient, which is the same in the donor and the recipient thereof when they are administered after treatment or transplanted after modification. In an even more preferred embodiment, the MSCs are of autologous origin and the EPR cells are of heterologous origin.
    In another preferred embodiment, the composition of the invention further comprises a pharmaceutically acceptable carrier. In addition, said composition may comprise one or more excipients.
    The term "excipient" refers to a substance that aids the absorption of the elements of the composition of the invention, stabilizes said elements, activates or aids the preparation of the composition in the sense of giving it consistency. Thus, the excipients may have the function of keeping the ingredients together, such as starches, sugars or cellulose, for example, the function of
    Dye, the function of protection of the composition, such as to isolate it from air and / or moisture, the filling function of a tablet, capsule or any other form of presentation, the disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Thus, the term excipient includes, for example, although
    20 without limitation, binders, dispersing agents, lubricants, glidants, etc.
    The "pharmaceutically acceptable carrier", like the excipient, is a substance that is used in the composition to dilute any of the components included therein to a certain volume or weight. The vehicle
    Pharmaceutically acceptable is an inert substance or action analogous to any of the elements included in the composition of the present invention. The function of the vehicle is to facilitate the incorporation of other elements, allow a better dosage and administration or give consistency and form to the composition.
    Preferably, the composition of the invention comprises MSCs, VIP and NIC, and more preferably also ATRA and the supernatant of an EPR cell culture, in a therapeutically effective amount, "therapeutically effective amount" being understood as the level, amount or concentration of those elements that
    35 produce the desired effect by treating and / or preventing damage or degeneration of the retina without causing adverse effects. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, the
    age, weight, sex, disease or tolerance of the individual who is going to be
    administered the composition of the invention.
    The composition of the present invention can be formulated for administration in a variety of ways known in the state of the art. Examples of preparations include any solid composition (tablets, pills, capsules, tablets, pearls, granules, pastes, peliets, etc.), semi-solid (gels, creams, ointments, etc.) or liquid (solutions, suspensions, osmotic device or
    10 emulsions) for oral, topical or parenteral administration. The composition of the present invention may also be in the form of sustained release formulations of drugs or any other conventional release system, so it may be contained, but not limited to, in nanoparticles, liposomes or nanospheres, in a polymeric material, in a polymeric material. biodegradable or non-biodegradable implant or in
    15 biodegradable microparticles, such as biodegradable microspheres.
    Such a composition and / or its formulations can be administered to an animal, including
    a mammal and, therefore, man, in a variety of ways, including, but not
    limited, intraperitoneal, intravenous, intradermal, intraspinal, intrastromal,
    20 intraarticular, intrasynovial, intrathecal, intralesional, intraarterial, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intravitreal, intracamerular, intraretinal, subretinal, intracapsular, topical, using transdermal patches, percutaneous, nasal spray, surgical implant, surgical paint, internal surgical pump Infusion
    In an even more preferred embodiment, the composition of the invention is formulated for ophthalmic administration. The term "formulated for ophthalmic administration" refers to a formulation that allows the composition of the invention to be administered ocularly, for example although without
    30 to limit ourselves, topically or intraocularly (including intravitreal, intracamerular, intraretinal, subretinal and others) without such administration negatively affecting the properties, for example structural and / or physiological, of the eye. Examples of the composition of the invention formulated for ophthalmic administration are, but not limited to, said composition associated with water, salts,
    A polymeric or semi-solid liquid carrier, a phosphate buffer or any other ophthalmic liquid vehicle that is acceptable to those known in the state of the art.
    In another preferred embodiment, the composition of the invention further comprises another active ingredient. As used herein, the term "active ingredient ~," active substance "," pharmaceutically active substance ~, "active ingredient ~ or" pharmaceutically active ingredient "refers to any component that potentially provides a pharmacological activity or other different effect in the cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.The term includes those components that promote a chemical change in the preparation of the drug and are present in it in a planned modified manner that provides the specific activity or the
    10 effect
    In another preferred embodiment, the composition of the invention is a "preparation
    combined "or also called" juxtaposition ~, which means that
    components of the combined preparation need not be present as
    15 union, for example in a composition, to be available for separate or sequential application. In this way, the expression "juxtaposed" implies that it is not necessarily a true combination, in view of the physical separation of the components. Thus, the components included in the composition of the invention can be administered simultaneously or sequentially.
    As the examples of the present invention show, the composition of the invention is capable of improving the conservation status of both neuroretinal explants and RPE cells cultured ex vivo that have undergone a degenerative process. Therefore, another aspect of the invention relates to the use of the composition of the
    Invention for the preparation of a medicament, or alternatively, to the composition of the invention for use as a medicament.
    The "medicaments" referred to in the present invention may be for human or veterinary use. The "medicine for human use" is any substance or
    A combination of substances that are presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct or modify physiological functions by exerting a pharmacological action, immunological
    or metabolic The "veterinary medicinal product" is any substance or combination of
    35 substances that are presented as having curative or preventive properties with respect to animal diseases or that can be administered to the animal in order to restore, correct or modify its physiological functions by exerting a pharmacological, immunological or metabolic action.
    Another aspect of the invention relates to the use of the composition of the invention for
    5 the development of a medicine for the treatment and / or prevention ofdegenerative retinal diseases, or alternatively to the composition of theinvention for use in the treatment and / or prevention of retinal diseasesdegenerative
    The term "treatment", as understood in the present invention, refers to combating the effects caused as a result of the disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
    (i) inhibit the disease or pathological condition, that is, stop its development;
    15 (ii) to alleviate the disease or the pathological condition, that is, to cause the regression of the disease or the pathological condition or its symptomatology;
    (iii) stabilize the disease or pathological condition.
    The term "prevention", as understood in the present invention, consists of
    20 to prevent the onset of the disease, that is, to prevent the disease or the pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particular, when said subject is predisposed to the pathological condition, but still He has not been diagnosed to have it.
    25 "Degenerative retinal disease" means any disease or pathology that triggers or causes a tissue degeneration of the retina or retinal degeneration, meaning "retina" any of its layers: retinal pigment epithelium, photoreceptors, internal limiting membranes and external, internal and external nuclear layers, internal and external plexiform layers,
    30 ganglion cells and optic nerve fiber layer. In a more preferred embodiment, degenerative retinal disease is a degenerative disease of RPE, photoreceptors or any of the other neurons or retinal cells, that is, of any other neuroretinal layer.
    35 A "degenerative disease of RPE" is any disease or pathology that triggers or progresses with a degeneration of the retinal pigment epithelium.
    Examples of such diseases are, but not limited to, AMD, pathological myopia and pigmentary retinosis, among others.
    "Photoreceptors" are specialized light-sensitive neurons, located in the
    5 external retina of vertebrates. Cones and rods are one of the cellsmore specialized and complex body. They convert the light intonerve impulses that the brain transforms into images. The photoreceptorsthey contain several zones where specific functions are performed: an external segment,an internal segment, a cell body and a synaptic terminal. Segments
    10 external of the rods are formed by an accumulation of membranous discs in the form of coin stacks surrounded by the cell membrane, where the phenomenon of phototransduction is carried out and the photosensitive pigment, rhodopsin is found. In the cones, the discs are formed by folds of the plasma membrane itself and the photosensitive molecules are the opsins.
    15 Separating the outer segment from the inner segment is an internal connecting cilia that has a structure similar to the cilia or flagella of other cells. The cilia connector region serves as a passage of vesicles between the external and internal segment. In the internal segment two parts are distinguished: the ellipsoid and the myoid; in the first one a large accumulation of mitochondria is located and in the second it is
    20 where the protein synthesis machinery of the cell resides.
    A "degenerative photoreceptor disease" is any disease or
    pathology that triggers or involves a degeneration of the photoreceptors of the
    retina. Examples of such diseases are, but not limited to, dystrophy.
    25 of cones and canes, Stargardt's disease, fundus flavimaculatus or pattern dystrophies, among others.
    In an even more preferred embodiment, the disease is DMAE, RP, disease ofStargardt or some of the optic nerve diseases such as optic neuropathy
    30 ischemic, preferably NOlANA.
    "Macular degeneration" or "age-related macular degeneration" or "AMD" is a disease caused by degeneration, damage or deterioration of the macula. The macula is a yellowish layer of light sensitive tissue found in the back of the eye, in the center of the retina. This area provides visual acuity, allowing you to perceive fine and small details. When the macula does not
     29-11 -201 3
    It works correctly, the areas of the center of the visual field begin to lose sharpness, becoming cloudy, blurred. The macular degeneration referred to in the present invention includes both dry and wet AMD.
    5 1O "Retinitis pigmentosa", "retinosis pigmentosa" or "RP" is not a single disease, but a set of chronic eye diseases of genetic origin and degenerative character that are grouped under this name. It is characterized by a progressive degeneration of the retina, which gradually loses the cones and rods. It produces as main symptoms a slow but progressive decrease in visual acuity that predominantly affects night vision and the peripheral field in the early stages, while maintaining central vision.
    fifteen Stargardt disease (also known as "Stargardt macular dystrophy") is an inherited eye disease characterized by macular degeneration. The onset of symptoms occurs in childhood or adolescence, and is manifested by loss of visual acuity The most common symptoms of this disease are blurred vision, blind areas in the visual field or scotomas, difficulty adapting to the dimness and sensitivity to light
    20 25 The "non-arteritic anterior ischemic optic neuropathy" or "NOlANA, is defined as an infarction in the head of the optic nerve, that is, an interruption of the blood supply, after which a certain degree of ischemia occurs with its consequent associated visual loss. It is characterized by a unilateral, sudden and painless episode of vision loss, although it is generally considered that the etiology is systemic so the condition can be bilateral.It represents a group of optic nerve disorders characterized by a lack of adequate vascular flow and which determines the death and subsequent disappearance of retinal ganglion cells.This entity constitutes a papilla infarction.
    30 In an even more preferred embodiment, retinal disease affects a human.
    35 Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the
    invention. The following examples and figures are provided by way of illustration, and
    They are not intended to be limiting of the present invention.
    DESCRIPTION OF THE FIGURES
    Fig. 1. Cell proliferation of EPR cells (ARPE19) treated with mitomycin C (MMC), cultured with VIP, NIC or ATRA and their combinations (VIP + NIC = VN, VIP + ATRA = VA, NIC + ATRA = NA and VIP + NIC + ATRA = VNA) evaluated by the alamarBlue® test. T indicates treatment with MMC (50 ~ lg / ml) of the
    10 cells of EPR. The statistical significance is set in psO, 01 (**) / (+) and PsO, 001 (+++). "+" Signs have been used to indicate the significant difference between 3 and 5 days. The error bar is indicated as ± SD (n = 3).
    Fig. 2. Cell proliferation of EPR cells (ARPE19) treated with
    15 mitomycin C (MMC) in indirect culture with AD-MSCs, evaluated by the alamarBlue® test, in the presence of factors (VIP, NIC or ATRA) and their combinations (VIP + NIC = VN, VIP + ATRA = VA, NIC + ATRA = NA and VIP + NIC + ATRA = VNA). T indicates treatment with MMC (50) .Ig / ml) of EPR cells. The statistical significance is set at p: S0.5 (*), pSO, 01 (**) and pSO, 001
    20 (***) / (+). The error bar is indicated as ± SD (n = 3).
    Fig. 3. Evaluation of neuroretinal explants co-cultivated with AD-MSCs, conditioned medium EPR (RCM) and factors (VIP, NIC or ATRA) or their combinations at 7 days of culture, using toluidine blue (first
    25 column) and immunohistochemical markers (second and third column). Calbindin, CRALBP, GFAP and synaptophysin represent cones, Müller cells, reactive gliosis and synapses, while the nuclei are marked with DAPI. Scale bar: 50 ~ m_
    30 Fig. 4. Evaluation of neuroretinal explants co-cultivated with AD-MSCs, conditioned medium EPR and factors (VIP, NIC or ATRA) or their combinations at 7 days of culture, by immunohistochemistry. Rhodopsin occurs in sticks and nuclei are identified with DAPI. Control I (A), Control II (B), Control 111 (C), VIP (D), NIC (E), ATRA (F), VIP + NIC (G), VIP + ATRA (H), NIC + ATRA
    35 (1) and VIP + NIC + ATRA (J). Scale bar: 50 ~ m.
    Fig. 5. Evaluation of degeneration of neuroretinal explants basedin the histological and immunohistochemical study. The state assessmentneuroretinal samples grown in all experiments was taken tocarried out by three subject matter experts individually, so that5 numerically assessed the neuroretinal conservation status from 0 to 10. So, oneO assessment represents total disorganization and retinal degeneration, whilethat 10 corresponds to a well preserved retina. The statistical significance isset to psO, 05 (*) / (+). Where the "+" sign indicates significant difference between theControl 11 and the experiments, and the "* ~ indicates significant difference between Control 11 1y
    10 the experiments. The error bar is indicated as ± SD (n = 3 to 6).
    EXAMPLES
    The invention will be illustrated below by tests carried out by the
    15 inventors who demonstrate the effectiveness of the composition of the invention. Thus, the following examples illustrate the use of AD-MSCs cells together with factors secreted by the EPR and different biomolecules (VIP, NIC and ATRA) to create an environment that allows differentiation of AD-MSCs to EPR cells, which simultaneously can protect EPR and neuroretin in
    20 degeneration For this purpose, several methods were developed under standard culture conditions of 5% CO2 at 37 ° C in humidified atmosphere, with changes of the culture medium every 2-3 days, which are described below.
    EXAMPLE 1. Co-culture of EPR cells that have undergone a process of
    25 cell degeneration and AD-MSCs in the presence of VIP, NIC, ATRA and their combinations.
    AD-MSCs and EPR cells were co-cultivated in a 2: 1 ratio, in 6-well plates (Falcon OS), with OMEM / F12 medium supplemented with 10% FSS, 1%
    30 antibiotics (penicillin / streptomycin) and 1% antifungal (amphotericin). Both cell types were physically separated by a porous membrane (Falcon OS). Previously, EPR cells (60,000 cells / cm2) were grown in the bottom of 24-well plates for 24 hours, and treated for 2 hours with an optimal concentration of mitomycin e (50 IJg / ml dissolved in the culture medium) for
    35 induce a process of cell degeneration (RPE cells under stress conditions).
    AD-MSCs were cultured (30,000 cells / cm2) for 24 hours in the upper part of the membranes of Transwell-type wells (tissue culture lreated tracketched polyethylene terephthalate (PET); membrane with pore size of 0.41Jm and pore density 2.0 ± 0.2x106 / cm 2) for adhesion and growth.
    For the co-culture, the growth medium was supplemented with VIP (5 IJM), NIC (10 mM), ATRA (5 ~ M) or its 4 combinations VIP + NIC, VIP + ATRA, NIC + ATRA and VIP + NIC + ATRA, to evaluate the effect of these biomolecules and their combinations in the presence of AD-MSCs on the cells of the RPE under stress. The
    The protective capacity of AD-MSCs alone, as well as in the different combinations with biomolecules, was evaluated at 3 and 5 days by the alamarBlue® method. 1.1. Results
    15 Regarding the evaluation of the protection capacity, VIP, NIC and ATRA individually and their combinations showed a suppressive effect on the proliferation of stress-prone RPE cells (Fig. 1). However, in the presence of AD-MSCs some of the combinations with these factors (VIP,
    20 ATRA, VIP + NIC, NIC + ATRA or VIP + NIC + ATRA) stimulated the proliferation of EPR cells. This was not observed in the combinations with NIC and VIP + ATRA (Fig. 2).
    EXAMPLE 2. Co-culture of human neuroretinal explants that suffer from
    25 progressive degeneration with AD-MSCs, medium conditioned with EPR and VIP, NIC, ATRA and their combinations.
    AD-MSCs cells were cocultured with human neuroretinal explants that spontaneously undergo progressive degeneration during culture. For him
    Co-culture 6-well Transwell plates were used, so that the retinal tissue and AD-MSCs remained physically separated by the membrane of the Transwell wells. DMEM / F12 culture medium supplemented with 10% FaS, 1% antibiotics, 1% antifungal and 50% medium conditioned with RPE was used.
    Previously, AD-MSCs (30,000 cells / cm2) were cultured for 24 hours in the bottom of 24-well plates.
    Neuroretinal explants (7x7 mm) were obtained from the central retinal area and placed on the membrane of the Transwell wells (tissue culture treated polycarbonate (eg) membrane diameter 24 mm, thickness 10¡Jm, and pore size 0.4 .Jm with a density of 1x108 pores / cm2) with the photoreceptors in contact with the
    5 well membrane.
    The medium conditioned with EPR was prepared by culturing EPR cells up to 90% confluence, at which time the medium was replaced and the culture was continued for 48 hours. Finally, the medium was collected, centrifuged at 1000 rpm
    10 for 10 minutes to discard possible cell debris and the supernatant (EPR conditioned medium) was stored at -80 ° C until use.
    For the co-culture of AD-MSCs with neurorretin explants, the culture medium was supplemented with VIP (5¡.JM), NIC (10mM), ATRA (5¡.JM) or its 4 combinations
    15 VIP + NIC, VIP + ATRA, NIC + ATRA and VIP + NIC + ATRA, to evaluate the effect of these biomolecules and their combinations on AD-MSCs and neuroretin in degeneration.
    The protective capacity of AD-MSCs alone, as well as in the different
    20 combinations with the biomolecules, was evaluated at 7 days of coculture by studying retinal morphology (staining with toluidine blue) and cell immunoexpression of different proteins, calbindin (CS), synaptophysin (SYP), rhodopsin (RHO) ), cell retinaldehyde binding protein (CRALBP) and acidic fibrillar glial protein (GFAP). Anti-CB and -RHO antibodies were used to evaluate the
    25 integrity of the cones and rods, respectively; anti-SYP to evaluate synapses between different retinal neurons at the level of OPL and IPL; the antiCRALBP to evaluate the functional state of Müller cells and differentiate them from astrocytes (GFAP + and CRALP-); and anti-GFAP to evaluate the process of reactive gliosis of retinal tissue. DAPI staining was used to identify the nuclei
    30 cell phones 2.1. Results
    As controls, initial retinal explants (uncultivated) were used (Control 1) 35 (Figs. 3A, B, C and 4A), explants grown for 7 days with EPR conditioned medium (Control 11) (Figs. 3D, E, FY 48 ) And with conditioned medium EPR + AD-MSCs (Control 111) (Fig. 3G, H, IY 4C).
    Control I (Hare Neuroretinal Explants) (Fig. 3A B C and 4A)
    These neuroretinal explants showed initial degenerative changes in retinal cells, mainly in photoreceptors (Fig. 3A), and slight cellular vacuolization, probably due to post-mortem ischemic damage (lack of cellular nutrient supply) and manipulation of explants for 10 the crop. However, the general structure of the retina was adequately preserved, where the external and internal segments of the photoreceptors were adequately preserved (Fig. 38, white arrows), which are the structures most sensitive to damage, the nuclei of these cells are presented slightly edematous (INL); the OLM was perfectly identified as well as the synaptic connections between the pedicles of the cones and the horizontal and bipolar cone cells at the level of the OPL (Fig. 38, fluorescence band, due to the combination of labeling with calbindin (CS) and synaptophysin (SYP)); the cytoplasm of integration neurons appeared slightly edematous (ONL); the neural processes of the IPL appeared slightly edematous and the dense cytoplasm could be differentiated from the 20 Müller cells; ganglion cells appeared adequately conserved while the NFL was edematized; the ILM was clearly defined.
    Control 11 (Neuroretinal explants cultured 7 days in the presence of EPR conditioned medium) (Fig. 3D E F Y 48)
    25 The retinal structure appeared disorganized and cell degeneration processes were shown. The photoreceptors appeared unstructured and in a small number; cellular edematization, picnosis, cariorrexis and karyolysis were observed in retinal cells; the OPL disappeared, which overlapped the ONL and the INL; the
    30 ganglion cells were degenerated; a seemingly intact ILM was observed. In many of the samples studied, retinal architecture was completely disorganized.
    Control 111 (Neuroretinal explants cultured 7 days in the presence of conditioned medium EPR + AD-MSCs) (Fig. 3G H IV 4C)
    The general retinal structure was better preserved compared to Control H. In the external retinal layers, some areas where the photoreceptors formed rosettes could be seen, but maintained their external and internal segments, with some picnotic nuclei, in cariorrexis and karyolysis, their cytoplasm
    5 appeared edematous and contained nuclear remains; the OPL had disappeared and the INL showed picnosis, cariorrexis and karyolysis; IPL was clearly differentiated but neuronal processes appeared almost degenerate; NFL showed a high degree of degeneration and thickened prolongations of Müller cells were appreciated; the ILM was clearly defined.
    10 Experiments (Neuroretinal Explants cultured 7 days in the presence of EPR + AD-MSCs conditioned medium with the different factors or their combinations) (Figs. 3J-AD 4D-J)
    15 In these samples different degrees of degeneration could be seen in comparison with the Control lit, several variations appeared in the state of the retinal explants depending on the initial state of Control 1.
    The culture with VIP showed a marked reactive gliosis, determined by the intense
    20 GFAP labeling (Fig. 3L), where Müller cells appeared hypertrophic extending to fill the space left by degenerating neurons. The VIP did not seem to improve retinal status compared to Control 111, and even in some cases the state of neuroretinal degeneration was greater. However, NIC and ATRA showed better neuroretinal conservation, which may
    25 considered as a protective effect on the general conservation of human neuroretin explants. The samples with VIP + N IC showed some external segments of the apparently intact cones (Fig. 3T, arrows). In these samples and in the VIP + NIC + ATRA a good conservation of the retinal structure was observed in comparison with the rest of the groups of the experiment.
    30 In immunohistochemical analysis, neuroretinal explants in the presence of AD-MSCs appeared better preserved than in the absence of these cells. NIC and ATRA seem to have a positive effect for conservation of the retinal structure, but that is not significantly better than AD-MSCs individually, at
    35 contrary to what was observed in the case of VIP + NIC and VIP + NIC + ATRA compared to the other experimental groups.
    The evaluation of the neuroretinal state of the samples cultured in all the experiments was carried out by three experts in the field in an individualized manner, so that the state of
    ° to
    neuroretinal conservation Thus, a valuation of ° represents the total5 disorganization and retinal degeneration, while 10 corresponds to a retinawell preserved, as would be the case with Controls 1.
    The evaluation of the effects of each factor is summarized and represented in Figure 5. Neuroretinal explants with AD-MSCs (Control 111) were clearly better conserved (p <0.05) than with EPR conditioned medium (Control 11). Explants cultured with AD-MSCs and biomolecules (N IC and ATRA) did not show a significantly better degree of preservation than with AD-MSCs (Control 111). Explants cultured only with VIP and AD-MSCs did not conserve neuroretin, developing a reactive gliosis (GFAP immunoexpression). Neuroretinal explants with medium
    15 conditioned EPR, AD-MSCs and combinations of biomolecules (VIP + NIC and VIP + NIC + ATRA) were significantly better preserved (p <0.05) than in any other treatment.
    Thus, taking into account the results obtained, it can be concluded that:
    20 -VIP, NIC and ATRA individually and their combinations do not show a significantly effective conservation on degenerating EPR cells. However, some combinations of these biomolecules show a positive effect in the presence of AD-MSCs.
    25 -VIP, NIC and ATRA And their combinations, together with AD-MSCs and the factors secreted by the EPR, show a potential capacity for conservation of the neuroretinal structure.
    30 -In all cases the combinations of these biomolecules have always shown better effects to conserve the EPR in degeneration and the neuroretinal structure than VIP, NIC and ATRA individually.
    权利要求:
    Claims (11)
    [1]
    one. Pharmaceutical composition comprising mesenchymal stem cells, vasoactive intestinal peptide (VIP) and nicotinamide (NIC).
    [2]
    2. Pharmaceutical composition according to claim 1, further comprising retinoic acid (ATRA).
    [3]
    3. Pharmaceutical composition according to any one of claims 1 or 2, which
    10 also comprises the supernatant of a retinal pigment epithelial cell culture.
    [4]
    Four. Pharmaceutical composition according to any one of claims 1 to 3, wherein the mesenchymal stem cells are derived from adipose tissue.
    [5]
    5. Pharmaceutical composition according to any one of claims 1 to 4, comprising mesenchymal stem cells from adipose tissue, VIP, NIC, ATRA and the supernatant of a retinal pigment epithelial cell culture.
    [6]
    6. Pharmaceutical composition according to any one of claims 1 to 5, wherein the mesenchymal stem cells and retinal pigment epithelium cells come from a human.
    7. Pharmaceutical composition according to any of claims 1 to 6, wherein the mesenchymal stem cells are of autologous or heterologous origin.
    [8]
    8. Pharmaceutical composition according to any of claims 1 to 7, further comprising a pharmaceutically acceptable carrier.
    [9]
    9. Use of the pharmaceutical composition according to any of claims 1 to 8, for the preparation of a medicament.
    [10]
    10. Use according to claim 9, wherein the medicament is for the treatment and / or prevention of degenerative retinal diseases.
    [11 ]
    eleven . Use according to claim 10, wherein the degenerative retinal disease is a degenerative disease of the retinal pigment epithelium or any other neuroretinal layer.
    12. Use according to any of claims 10 or 11, wherein the disease is macular degeneration, retinitis pigmentosa (RP), Stargardt disease or non-arteritic anterior ischemic optic neuropathy (NOlANA).
    [13]
    13. Use according to any of claims 10 to 12, wherein degenerative retinal disease affects a human.
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