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WO2015006474A1 - Méthodes de traitement d'un patient atteint d'un dysfonctionnement cognitif au moyen de cellules souches neuronales provenant de la moelle épinière - Google Patents

Méthodes de traitement d'un patient atteint d'un dysfonctionnement cognitif au moyen de cellules souches neuronales provenant de la moelle épinière Download PDF

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Publication number
WO2015006474A1
WO2015006474A1 PCT/US2014/045993 US2014045993W WO2015006474A1 WO 2015006474 A1 WO2015006474 A1 WO 2015006474A1 US 2014045993 W US2014045993 W US 2014045993W WO 2015006474 A1 WO2015006474 A1 WO 2015006474A1
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Prior art keywords
spinal cord
neural stem
stem cells
subject
derived neural
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Inventor
Karl K. Johe
Charles L. LIMOLI
Thomas G. Hazel
Michael HEFFERAN
Munjal M. ACHARYA
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University of California Irvine
Palisade Bio Inc
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Neuralstem Inc
University of California Irvine
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/99—Serum-free medium
    • 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
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/32—Polylysine, polyornithine
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates generally to methods for treating a subject with a cognitive dysfunction by introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of the subject's brain.
  • Debilitating and progressive cognitive dysfunctions are commonly associated with neurological diseases and can also be an unintended side effect of various therapeutic regimens such as radiotherapy.
  • Such cognitive dysfunctions encompass a broad range of cognitive domains, including memory, communication, perception and concentration, problem-solving and decision-making, among others.
  • the present disclosure provides methods for treating a subject with a cognitive dysfunction including, for example, a subject that has received radiotherapy for the treatment of a primary or secondary brain tumor. Such methods may be useful for treating symptoms resulting from exposure to radiation or symptoms of neurological diseases.
  • the present disclosure provides methods for treating a subject with a cognitive dysfunction, for example, a human, by introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of the subject's brain (e.g., hippocampus).
  • the present disclosure also provides methods of treating a subject (e.g., a human) with a cognitive dysfunction by obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain (e.g., hippocampus).
  • a subject e.g., a human
  • a cognitive dysfunction by obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain (e.g., hippocampus).
  • the present disclosure also provides methods of augmenting neural cell numbers, increasing synaptic connections and/or providing paracrine support in a subject's brain, the methods comprising: obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain (e.g., hippocampus).
  • a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain (e.g., hippocampus).
  • the present disclosure also provides methods for treating a subject with a brain cancer by administering radiation therapy to the brain of the subject, and introducing a therapeutically effective amount of spinal cord-derived neural stem cells to the subject's brain, wherein the therapeutically effective amount of spinal cord-derived neural stem cells is effective to treat a cognitive dysfunction.
  • the spinal cord-derived neural stem cells are embryonic spinal cord-derived neural stem cells.
  • the spinal cord-derived neural stem cells are fetal spinal cord-derived neural stem cells, wherein the fetal spinal cord-derived neural stem cells are obtained from a fetus being a gestational age of about 5 to about 20 weeks.
  • the spinal cord-derived neural stem cells are human spinal cord-derived neural stem cells.
  • the spinal cord-derived neural stem cells are expanded to form an expanded spinal cord-derived neural stem cell population.
  • expanding the spinal cord-derived neural stem cells includes culturing the spinal cord-derived neural stem cells in the absence of serum.
  • expanding the spinal cord-derived neural stem cells includes exposing the spinal cord-derived neural stem cells to at least one growth factor.
  • the growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof.
  • the spinal cord-derived neural stem cells differentiate into neurons that engraft in vivo into the brain tissue of the subject. In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the spinal cord-derived neural stem cells are capable of generating neurons in brain tissue of the subject.
  • the cognitive dysfunction is induced by prior exposure of the subject to radiation.
  • the cognitive dysfunction is associated with traumatic brain injury, diabetes, dementia, depression, aging, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, Multiple Sclerosis, epilepsy, cerebrovascular disease or substance abuse.
  • the cognitive dysfunction is dementia, delirium or amnesia.
  • the dementia is vascular dementia, dementia with Lewy bodies, mixed dementia, frontotemporal dementia.
  • the cognitive dysfunction includes speech impairment, confusion, disorientation, loss of memory, learning, perception, judgment, initiative, attention, planning, multitasking, spatial or analytical skills, reasoning ability, or combinations thereof.
  • introducing the therapeutically effective amount of spinal cord-derived neural stem cells includes injecting at least a portion of the therapeutically effective amount of spinal cord-derived neural stem cells into a plurality of areas of the brain tissue of the subject including, for example, the cerebral hemispheres, cerebral cortex, subcortex, motor cortex, basal ganglia, striatum, internal capsule, thalamus, hypothalamus, hippocampus, corpus callosum, midbrain, substantia nigra, brainstem and cerebellum.
  • the subject may be administered one or more immunosuppressive drugs.
  • the spinal cord-derived neural stems are injected into the subject's hippocampus.
  • Figure 1A-B Figure 1A shows a schematic of the research design. Two month old athymic nude rats receiving 10Gy head-only ⁇ -irradiation were transplanted two days later with human fetal-derived neural stem cells (NSI566 NSCs). At 1 -month posttransplantation surgery, animals were administered a novel place recognition and fear conditioning tasks. Three weeks later, after cognitive testing, animals were euthanized for immunohistochemical analysis. Non-irradiated control and irradiated animals receiving sterile hibernation buffer served as sham surgery groups.
  • Figure 1 B shows immunocytochemical analysis of NSI566 NSCs and following in vitro differentiation.
  • Undifferentiated NSI566 NSCs (far left panel) stained with DAPI show strong expression and co-localization of the multipotent marker Nestin. Following in vitro differentiation, NSI566 NSCs express both neuronal (MAP2- and SMI312-positive) and astrocytic (GFAP) cells (right panels). (Scale bars: Nestin, 10 ⁇ ; MAP2, SMI312 and GFAP, 50 ⁇ ).
  • Figure 2A-D shows that transplantation of NSI566 NSCs improves radiation- induced cognitive impairments at 1-month post-transplantation.
  • Figure 2A shows NSI566 NSC-transplanted animals (IRR+NSI) explore more than controls (CON) and irradiated- sham (IRR) animals during the initial familiarization phase of NPR task (p ⁇ 0.001 , post hoc). Exploration ratios were calculated as, timenovei/time n ovei + timefamiiiar, for the first minute of 5min ( Figure 2B) and 24h (Figure 2C) test sessions in the NPR task.
  • Figure 2B shows that for the 5 minute NPR test, IRR animals spent a significantly lower proportion of time exploring the novel place (p ⁇ 0.001 vs. CON and vs. IRR+NSI, post hoc), while CON and IRR+NSI animals did not differ. IRR animals did not spend more time exploring the novel place than expected by chance (dashed line at 50%).
  • Figure 2C shows that for the 24h test, after the initial familiarization phase, when animals were presented the same two objects, with one moved to a new spatial location, none of the groups spent more time exploring the novel place than expected by chance.
  • FIG 3 shows survival and location of transplanted NSI566 NSCs.
  • NSI566 NSCs are located near the injection (cells displayed as white) (Nt, needle track; Tc, transplant core; Figure 3A-E, 5 to 60x magnification) and CA1 and corpus callosum (CC) areas.
  • Transplanted NSI566 NSCs did not show extensive migration patterns in the host hippocampus (DG, dentate gyrus; DH, dentate hilus, CA3 subfields).
  • Transplanted NSI566 NSCs were detected with human specific nuclear antigen (Ku80) and counterstained with nuclear dye (TOTO-3).
  • the insert in Figure 3E represents orthogonal reconstruction of confocal Z-stacks. (Scale bars: A-B, 100 ⁇ ; C, 50 ⁇ ; D, 20 ⁇ ; E, 10 ⁇ and E-insert, 5 ⁇ ).
  • Figure 4 shows differentiation of transplanted NSI566 NSCs in the irradiated hippocampus.
  • Ku80-positive (human specific nuclear antigen) NSI566 NSCs differentiated into immature (doublecoritin, DCX, Figure 4A and a) and mature (neuron specific nuclear antigen, NeuN, Figure 4B and b) neurons as visualized by dual labeling of neuron-specific markers with Ku80.
  • immature glial fibrillary acidic protein, GFAP, Figure 4C and c
  • mature (S100 protein, Figure 4D and d) astrocytes were a similar pattern of differentiation.
  • FIG. 4A-D Confocal z-stack orthogonal reconstructions of dual-labeled cells are shown for neuronal (NeuN, a; DCX, b) and astrocytic (GFAP, c; S100, d) phenotypes ( Figure 4a-d).
  • DG dentate gyrus
  • CC corpus callosum. Scale bars: A-D, 50 ⁇ and a-d, 10 ⁇ ).
  • the disclosed methods relate to the treatment of a subject with a cognitive dysfunction and may be used to ameliorate complex learning and memory deficits.
  • Cognitive dysfunctions are commonly associated with neurological disorders and can also be unintended side effects of various therapeutic regimens such as radiotherapy ⁇ e.g., radiotherapy administered for the treatment of a brain tumor).
  • the types of dysfunctions encompass a broad range of cognitive domains, including memory, communication, perception and concentration, problem-solving and decision-making, among others.
  • the present application provides methods for the treatment of a subject with a cognitive dysfunction by introducing spinal cord-derived neural stems cells to the subject's brain.
  • spinal cord-derived neural stems cells Prior to this invention, it was uncertain whether spinal cord-derived neural stems cells could survive and differentiate at a site in the brain that has been subjected to ionizing radiation (e.g., radiotherapy). This disclosure surprisingly demonstrates that spinal cord-derived neural stems cells differentiate into neurons and astrocytes in the radiation- treated brain, increase synaptic connectivities, and ameliorate any cognitive dysfunctions that arise as a result of treatment of a subject with radiation.
  • ionizing radiation e.g., radiotherapy
  • the present disclosure also provides methods for treating a subject with a brain cancer by: administering radiation therapy or chemotherapy to the subject, obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain ⁇ e.g., hippocampus).
  • hippocampus hippocampus
  • NSCs neural stem cells
  • NSCs can also refer to neural or neuronal progenitors, or neuroepithelial precursors. NSCs can be functionally defined according to their capacity to differentiate into each of the three major cell types of the CNS: neurons, astrocytes, and oligodendrocytes.
  • the present disclosure provides methods of treating a subject with a cognitive dysfunction (e.g., a human) by obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population (e.g., injecting 70,000 NSI566 NSCs in 1 ⁇ _ of cell suspension) to one or more areas of the subject's brain (e.g., hippocampus).
  • a cognitive dysfunction e.g., a human
  • the present disclosure also provides methods of augmenting neural cell numbers, increasing synaptic connections and/or providing paracrine support in a subject's brain, the methods comprising: obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population (e.g., injecting 70,000 NSI566 NSCs in 1 ⁇ _ of cell suspension) to one or more areas of the subject's brain (e.g., hippocampus).
  • a therapeutically effective amount of the expanded neural stem cell population e.g., injecting 70,000 NSI566 NSCs in 1 ⁇ _ of cell suspension
  • the present disclosure also provides methods for treating a subject with a brain cancer by administering radiation therapy to the brain of the subject, and introducing a therapeutically effective amount of spinal cord-derived neural stem cells (e.g., injecting 70,000 NSI566 NSCs in 1 ⁇ _ of cell suspension) to the subject's brain, wherein the therapeutically effective amount of spinal cord-derived neural stem cells is effective to treat a cognitive dysfunction.
  • a therapeutically effective amount of spinal cord-derived neural stem cells e.g., injecting 70,000 NSI566 NSCs in 1 ⁇ _ of cell suspension
  • "treating" or "treatment” of a disease, disorder, or condition includes at least partially: (1 ) preventing the disease, disorder, or condition, i.e. causing the clinical symptoms of the disease, disorder, or condition not to develop in a mammal that is exposed to or predisposed to the disease, disorder, or condition but does not yet experience or display symptoms of the disease, disorder, or condition; (2) inhibiting the disease, disorder, or condition, i.e., arresting or reducing the development of the disease, disorder, or condition or its clinical symptoms; or (3) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, or condition or its clinical symptoms.
  • an "effective amount” refers to the amount of spinal cord-derived neural stem cells that is required to confer a therapeutic effect on the subject.
  • a “therapeutically effective amount,” as used herein, refers to a sufficient amount spinal cord-derived neural stem cells being administered which will relieve to some extent one or more of the symptoms of the disease, disorder, or condition being treated.
  • the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • an "effective amount" for therapeutic uses is the amount of the spinal cord-derived neural stem cells required to provide a clinically significant decrease in disease symptoms without undue adverse side effects.
  • an appropriate "effective amount” in any individual case is determined using techniques, such as a dose escalation study.
  • the term "therapeutically effective amount” includes, for example, a prophylactically effective amount.
  • an "effective amount” of spinal cord-derived neural stem cells is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects.
  • an effect amount or "a therapeutically effective amount” varies from subject to subject, due to variation in metabolism, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.
  • Cognitive dysfunction may include amnesia, dementia, and delirium.
  • amnesia refers to a deficit in memory.
  • the term “dementia” refers to a loss of global cognitive ability in a previously unimpaired person, which may be mild to severe in degree. It is understood in the field that dementia is not a single disease, but a non-specific syndrome generally describing diseases associated with loss of memory and/or other mental abilities severe enough to interfere with daily life.
  • dementia The most common type of dementia is Alzheimer's disease, but other types of dementia include, but are not limited to, vascular dementia, dementia with Lewy bodies, mixed dementia, Creutzfeldt-Jakob disease, Huntington's disease, or diseases associated with cerebrovascular disease and substance abuse such as normal pressure hydrocephalus and Wernicke-Korsakoff syndrome, respectively.
  • vascular dementia dementia with Lewy bodies
  • mixed dementia Creutzfeldt-Jakob disease
  • Huntington's disease Huntington's disease
  • diseases associated with cerebrovascular disease and substance abuse such as normal pressure hydrocephalus and Wernicke-Korsakoff syndrome, respectively.
  • the term "delirium” refers to sudden severe confusion and disorientation, which develops with a relative rapid onset and fluctuates in intensity.
  • the term "associated with”, when used in the context to a condition or disease associated with cognitive impairment, means that the condition or disease may lead to cognitive impairment, may result from cognitive impairment, may be characterized by cognitive impairment or may otherwise be associated with or involve cognitive impairment in any way.
  • the cognitive impairment associated with the condition or disease may be of any degree, for example mild, moderate or severe.
  • the association between the condition or disease and cognitive impairment may be direct or indirect and it should be understood that cognitive impairment need not be the predominant feature of the condition or disease, nor necessarily be a feature of the condition or disease in every individual.
  • neural stem cell refers to a multipotential stem cell that can be functionally defined according to their capacity to differentiate into each of the three major cell types of the central nervous system (CNS): neurons, astrocytes, and oligodendrocytes.
  • stem cell refers to an undifferentiated cell that is capable of self-renewal, meaning that with each cell division at least one daughter cell will also be a stem cell. NSCs can also refer to neural or neuronal progenitors, or neuroepithelial precursors.
  • the NSCs are multipotent such that each cell has the capacity to differentiate into a neuron, astrocyte or oligodendrocyte.
  • the NSCs are bipotent such that each cell has the capacity to differentiate into two of the three cell types of the CNS.
  • the NSCs include at least bipotent cells generating both neurons and astrocytes in vitro and include at least unipotent cells generating neurons in vivo.
  • the NSCs are isolated from the CNS.
  • isolated refers to a cell that is in an environment different from that which the cell naturally occurs (e.g. where the cell naturally occurs in an organism) and the cell is removed from its natural environment.
  • NSCs may be isolated from an area which is naturally neurogenic for a desired population of neurons and from embryonic, fetal, post-natal, juvenile or adult tissue.
  • the desired population of cells may include the cells of a specific neuronal phenotype which can replace or supplement such phenotype lost or inactive in the course of disease progression.
  • the NSCs are isolated from the subventricular zone (SVZ) or from the subgranular zone of the dentate gyrus (DG).
  • DG dentate gyrus
  • the NSCs are isolated from the spinal cord in which neurogenesis of ventral motor-neurons is substantial and obtained at a gestational age of human fetal development during which neurogenesis of ventral motor-neurons is substantial.
  • NSCs are isolated from the spinal cord at a gestational age of about 6.5 to about 20 weeks.
  • NSCs are isolated from the spinal cord at a gestational age of about 7 to about 9 weeks.
  • the NSCs are isolated from embryonic spinal cord tissue.
  • neural stem cells are isolated from a human. It should be appreciated that the proportion of the isolatable NSC population can vary with the age of the donor. Expansion capacity of the cell populations can also vary with the age of the donor.
  • the NSCs of the ventral midbrain are distinct from the NSCs obtained from the spinal cord at the same gestational stage.
  • the NSCs from the ventral midbrain exclusively give rise to tyrosine-hydroxylase-expressing dopaminergic neurons, whereas NSCs from the spinal cord exclusively generate acetylcholine-producing cholinergic neurons.
  • Both cell types however, simultaneously generate the more ubiquitous glutamate- and GABA-producing neurons. Therefore, in an embodiment, the disclosed methods include obtaining NSCs from the spinal cord to treat conditions ameliorated or attenuated, at least in part, by the implantation of acetylcholine-producing cholinergic neurons.
  • NSCs can also be isolated from post-natal and adult tissues. NSCs derived from post-natal and adult tissues are quantitatively equivalent with respect to their capacity to differentiate into neurons and glia, as well as in their growth and differentiation characteristics. However, the efficiency of in vitro isolation of NSCs from various post-natal and adult CNS can be much lower than isolation of NSCs from fetal tissues which harbor a more abundant population of NSCs. Nevertheless, as with fetal-derived NSCs, the disclosed methods enable at least about 30% of NSCs derived from neonatal and adult sources to differentiate into neurons in vitro. Thus, post-natal and adult tissues can be used as described above in the case of fetal-derived NSCs.
  • human fetal spinal tissue is dissected under a microscope. A region of tissue corresponding to the lower cervical/upper thoracic segments is isolated.
  • the NSCs are isolated, pooled, and expanded on poly-D-lysine coated culture vessels in a media containing fibronectin and basic fibroblast growth factor (bFGF; FGF-2). Cells are expanded and then concentrated to the desired target cell density of about 10,000 cells per microliter in a medium free of preservative and antibiotics. Concentrated cells may be used fresh for implantation or frozen for later use.
  • bFGF basic fibroblast growth factor
  • the NSCs are derived from embryonic stem cells or induced pluripotent stem cells.
  • embryonic stem cell refers to a stem cell isolated from the developing embryo which can give rise to all of the cells of the body ⁇ e.g., cells of the ecto-, meso-, and/or endo-dermal cell lineages).
  • induced pluripotent stem cell refers to a stem cell derived from a somatic cell (e.g., a differentiated somatic cell) that has a higher potency than the somatic cell.
  • Embryonic stem cells and induced pluripotent stem cells are capable of differentiation into more mature cells (e.g., neural stem cells or neural progenitor cells).
  • Methods employed for growing and differentiating embryonic or induced pluripotent stem cells into NSCs in vitro can, for example, be such as those described in Daadi et al., PLoS One. 3(2):e1644 (2008).
  • the NSCs can be diluted with an acceptable pharmaceutical carrier.
  • pharmaceutical carrier refers to a diluent, adjuvant, excipient, or vehicle with which the cells of the disclosure are administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • the neural stem cells and pharmaceutically acceptable carriers can be sterile.
  • Water is a useful carrier when the cells are administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use.
  • suitable carrier is within the skill of the ordinary artisan.
  • neuronal subtypes can be obtained from manipulation of embryonic stem cells expanded in culture.
  • specific neuronal subtypes based on the disclosed methods, can be isolated and purified from other irrelevant or unwanted cells to improve the result, as needed, and can be used for treatment of cognitive dysfunction.
  • the NSCs in the disclosed methods can be derived from one site and transplanted to another site within the same subject as an autograft. Furthermore, the NSCs in the disclosed methods can be derived from a genetically identical donor and transplanted as an isograft. Still further, the NSCs in the disclosed methods can be derived from a genetically non-identical member of the same species and transplanted as an allograft. Alternatively, NSCs can be derived from non-human origin and transplanted as a xenograft. With the development of powerful immunosuppressants, allograft and xenograft of non-human neural precursors, such as neural precursors of porcine origin, can be grafted into human subjects.
  • a sample tissue can be dissociated by any standard method.
  • tissue is dissociated by gentle mechanical trituration using a pipette and a divalent cation-free buffer (e.g. saline) to form a suspension of dissociated cells.
  • a divalent cation-free buffer e.g. saline
  • Sufficient dissociation to obtain largely single cells is desired to avoid excessive local cell density.
  • stem cells can be cultured according to the methods set forth in U.S. 8,460,651 , U.S. 8,236,299, U.S. 7,691 ,629, U.S. 5,753,506, U.S. 6,040, 180, or U.S. 7,544,51 1 , the entireties of which are incorporated by reference herein.
  • the NSCs of the disclosed methods can include pre- differentiated cells for transplantation. For maximum yield of the cells and for simplicity of the procedure, a confluent culture is harvested for transplantation which comprises primarily a population of undifferentiated cells. It should be appreciated, however, that a minor population of cells just starting to differentiate spontaneously can also exist due to the increased cell density.
  • the NSCs are concentrated in a solution such as the clinically usable, hibernation or freezing solutions described above.
  • the NSCs are concentrated to an appropriate cell density which can be the same or different from the cell density for administration of the cells.
  • the cell density for administration can vary from about 1 ,000 cells per microliter to about 1 ,000,000 cells per microliter depending upon factors such as the site of the injection, the minimum dose necessary for a beneficial effect, and toxicity side-effect considerations.
  • the NSCs are concentrated to a density of about 1 ,000 to about 1 ,000,000 cells per microliter. In one embodiment, the NSCs are concentrated to a density of about 2,000 to about 80,000 NSCs per microliter. In another embodiment, about 5,000 to about 50,000 NSCs per microliter have been used for effective engraftment. In another embodiment, about 10,000 to 30,000 NSCs per microliter are used. In a preferred embodiment, the NSCs are concentrated to a density of about 70,000 NSCs per microliter.
  • the NSCs are concentrated to a density of about
  • the NSCs are concentrated to a density of about 100,000 to about 200,000 cells per microliter, about 200,000 to about 300,000 cells per microliter, about 300,000 to about 400,000 cells per microliter, about 400,000 to about 500,000 cells per microliter, about 500,000 to about 600,000 cells per microliter, about 600,000 to about 700,000 cells per microliter, about 700,000 to about 800,000 cells per microliter, about 800,000 to about 900,000 cells per microliter, about 900,000 to about 1 ,000,000 cells per microliter.
  • the NSCs can be delivered to a treatment area suspended in an injection volume of less than about 100 microliters per injection site.
  • an injection volume of 0.1 and about 100 microliters per injection site can be used.
  • the NSCs can be delivered to a treatment area suspended in an injection volume of about 1 microliter per injection site.
  • the disclosed methods include injecting NSCs at a cell density of about 1 ,000 to about 10,000 cells per microliter, about 10,000 to about 20,000 cells per microliter, about 20,000 to about 30,000 cells per microliter, about 30,000 to about 40,000 cells per microliter, about 40,000 to about 50,000 cells per microliter, about 50,000 to about 60,000 cells per microliter, about 60,000 to about 70,000 cells per microliter, about 70,000 to about 80,000 cells per microliter, about 80,000 to about 90,000 cells per microliter, or about 90,000 to about 100,000 cells per microliter into to one or more areas of the brain of the subject.
  • the disclosed methods include injecting NSCs at a cell density of about 100,000 to about 200,000 cells per microliter, about 200,000 to about 300,000 cells per microliter, about 300,000 to about 400,000 cells per microliter, about 400,000 to about 500,000 cells per microliter, about 500,000 to about 600,000 cells per microliter, about 600,000 to about 700,000 cells per microliter, about 700,000 to about 800,000 cells per microliter, about 800,000 to about 900,000 cells per microliter, or about 900,000 to about 1 ,000,000 cells per microliter into to one or more areas of the brain of the subject.
  • the disclosed methods include injecting NSCs at a cell density of about 5,000 to about 50,000 cells per microliter. In preferred embodiments, the disclosed methods include injecting NSCs at a cell density of about 70,000 cells per microliter.
  • the disclosed methods include multiple injections of NSCs at a total cell number of about 4,000 to about 40,000 cells, about 40,000 to about 80,000 cells, about 80,000 to about 120,000 cells, about 120,000 to about 160,000 cells, about 160,000 to about 200,000 cells, about 200,000 to about 240,000 cells, about 240,000 to about 280,000 cells, about 280,000 to about 320,000 cells, about 320,000 to about 360,000 cells, or about 360,000 to about 400,000 cells introduced into one or more areas of the brain of the subject.
  • the disclosed methods include multiple injections of
  • NSCs with a total cell number of about 400,000 to about 800,000 cells, about 800,000 to about 1 ,200,000 cells, about 1 ,200,000 to about 1 ,600,000 cells, about 1 ,600,000 to about 2,000,000 cells, about 2,000,000 to about 2,400,000 cells, about 2,400,000 to about 2,800,000 cells, about 2,800,000 to about 3,200,000 cells, about 3,200,000 to about 3,600,000 cells, or about 3,600,000 to about 4,000,000 cells introduced into one or more areas of the brain of the subject.
  • the volume of media in which the expanded NSCs are suspended for delivery to a treatment area can be referred to herein as the injection volume.
  • the injection volume depends upon the injection site and the degenerative state of the tissue. More specifically, the lower limit of the injection volume can be determined by practical liquid handling of viscous suspensions of high cell density as well as the tendency of the cells to cluster.
  • the upper limit of the injection volume can be determined by limits of compression force exerted by the injection volume that are necessary to avoid injuring the host tissue, as well as the practical surgery time.
  • Any suitable device for injecting the cells into a desired area can be employed in the disclosed methods.
  • a syringe capable of delivering sub-microliter volumes over a time period at a substantially constant flow rate is used.
  • the cells can be loaded into the device through a needle or flexible tubing or any other suitable transfer device.
  • the cells are injected at between about 2 and about
  • the cells are injected at between about 5 and about 10 sites in the brain. In an embodiment, the cells are injected at between about 10 to about 30 sites in the brain. In an embodiment, the cells are injected at between about 10 to about 50 sites in the brain. At least two of the sites can be separated by a distance of approximately 100 microns to about 5,000 microns. In an embodiment, the distance between injection sites is about 400 to about 600 microns.
  • the distance between injections sites is about 100 to about 200 microns, about 200 to about 300 microns, about 300 to about 400 microns, about 400 to about 500 microns, about 500 to about 600 microns, about 600 to about 700 microns, about 700 to about 800 microns, about 800 to about 900 microns, or about 900 to about 1 ,000 microns. In an embodiment, the distance between injection sites is about 1 ,000 to about 2,000 microns, about 2,000 to about 3,000 microns, about 3,000 to about 4,000 microns, or about 4,000 to about 5,000 microns.
  • the distance between injections sites can be determined based on generating substantially uninterrupted and contiguous donor cell presence throughout the spinal cord tissue and based on the average volume of injections demonstrated to achieve about 2-3 month survival in animal models such as rats or pigs.
  • the actual number of injections and distance between injections in humans can be extrapolated from results in animal models.
  • the NSCs of the disclosed methods can generate large numbers of neurons in vivo.
  • the NSCs When the NSCs are not overtly pre-differentiated prior to transplant, the NSCs can proliferate up to two to four cell divisions in vivo before differentiating, thereby further increasing the number of effective donor cells.
  • the neurons Upon differentiation, the neurons secrete specific neurotransmitters.
  • the neurons secrete into the milieu surrounding the transplant in vivo growth factors, enzymes and other proteins or substances which are beneficial for different conditions. Accordingly, a variety of conditions can be treated by the disclosed methods because of the ability of the implanted cells to generate large numbers of neurons in vivo and because the cognitive dysfunction may be caused by or result in missing elements including neuron-derived elements. Therefore, subjects suffering from cognitive dysfunctions due to lack of such neuron-derived elements, such as growth factors, enzymes and other proteins, can be treated effectively by the disclosed methods.
  • the composition comprising an amount of NSCs may be administered to a subject in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intravenous, subcutaneous, intraarticular, intrasynovial, or intrathecal routes.
  • intravenous administration e.g., as a bolus or by continuous infusion over a period of time
  • intramuscular, intraperitoneal, intracerebrospinal, intravenous, subcutaneous, intraarticular, intrasynovial, or intrathecal routes intramuscular, intraperitoneal, intracerebrospinal, intravenous, subcutaneous, intraarticular, intrasynovial, or intrathecal routes.
  • Intracerebrospinal, intrathecal, intravenous, intraperitoneal, or subcutaneous administration of the cells is preferred, with intracerebrospinal, intrathecal
  • introducing the therapeutically effective amount of the NSCs includes injecting at least a portion of the therapeutically effective amount into a plurality of areas of the brain of a subject.
  • compositions of the NSCs of the invention are formulated as an injectable formulation and comprise, for example, an aqueous solution or suspension of the active ingredient suitable for intracerebrospinal delivery.
  • a continuous phase can be present that comprises an aqueous solution of tonicity modifiers, buffered to a pH below about 7, or below about 6, for example about 2 to about 7, about 3 to about 6 or about 3 to about 5.
  • the tonicity modifiers can comprise, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other pharmaceutical agents that render osmotic pressure of the formulation isotonic with blood.
  • a larger quantity of the tonicity modifier is used in the formulation, it can be diluted prior to injection with a pharmaceutically acceptable diluent to render the mixture isotonic with blood.
  • the composition comprising NSCs is administered once.
  • administration of an initial dose the composition comprising NSCs is followed by the administration of one or more subsequent doses.
  • dosing regimens e.g., an interval between the first dose and one or more subsequent doses
  • dosing regimens include an interval of about once every week to about once every 12 months, an interval of about once every two weeks to about once every 6 months, an interval of about once every month to about once every 6 months, an interval of about once every month to about once every 3 months, or an interval of about once every 3 months to about once every 6 months.
  • administration is monthly, every two months, every three months, every four months, every five months, every six months, or upon disease recurrence.
  • the NSCs are injected at between about 5 and about 50 sites. In an embodiment, the NSCs are injected at between about 10 to about 30 sites. At least two of the sites can be separated by a distance of approximately 100 microns to about 5000 microns. In an embodiment, the distance between injection sites is about 400 to about 600 microns. The actual number of injections in humans can be extrapolated from results in animal models.
  • the methods of the present disclosure may include administration of one or more immunosuppressive drugs prior to, concurrent with, or after the injection of the NSCs.
  • the NSCs and immunosuppressive drug may be coadministered.
  • the NSCs and immunosuppressive drug which make up the therapy may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration.
  • the NSCs and immunosuppressive drug may also be administered sequentially, with either the NSCs or immunosuppressive drug being administered by a regimen calling for multiple step administration.
  • a regimen may call for sequential administration of the NSCs and immunosuppressive drug with spaced- apart administration of the separate, active agents.
  • the time period between the multiple administration steps may range from, for example, a few minutes to several hours to days, depending upon the properties of the NSCs and immunosuppressive drug such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the therapeutic compound, as well as depending upon the effect of food ingestion and the age and condition of the subject. Circadian variation of the target molecule concentration may also determine the optimal dose interval.
  • the NSCs and immunosuppressive drug whether administered simultaneously, substantially simultaneously, or sequentially, may involve a regimen calling for administration of the NSCs by intravenous route and the immunosuppressive drug by an oral route, a percutaneous route, an intravenous route, an intramuscular route, or by direct absorption through mucous membrane tissues, for example.
  • each such therapeutic compound will be contained in a suitable pharmaceutical formulation of pharmaceutically-acceptable excipients, diluents or other formulations components.
  • immunosuppressive drug denotes any drug aimed at decreasing or preventing activity of the subject's immune response.
  • Immunosuppressive drugs act to inhibit the proliferation and activity of all or a substantial portion of the immune cells within the body. Many immunosuppressive drugs function by inhibiting a step in the interleukin 2 (IL-2) signaling pathway.
  • IL-2 is a cytokine that regulates the growth, proliferation, and activation of lymphocytes.
  • the immunosuppressive drug tacrolimus considered one of the most potent immune system suppressors, is a calcineurin-dependent inhibitor that blocks IL-2 production and reduces proliferation of T-cells.
  • Another immunosuppressive drug sirolimus acts in a calcineurin- independent fashion to inhibit the response of T- and B-cells to IL-2.
  • immunosuppressive drugs such as mycophenolate mofetil and prednisolone, function by inhibiting key enzymes required for T- and B-cell growth or by binding to glucocorticoid receptors, respectively. It is well-recognized in the field that many immunosuppressive drugs have high inter- and intra-patient variability and require routine dosage adjustments to maintain appropriate trough levels for therapeutic concentrations.
  • the immunosuppressive drug comprises methylprednisolone.
  • an effective amount can range from about 4 to 1 ,000 mg per dose.
  • a preferred dosage of methylprednisolone may be about 125 mg administered intravenously immediately prior to surgery.
  • Methylprednisolone also goes by the trade names Medrol® and Solu-Medrol®. Effective dosages will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether methylprednisolone is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient. Determining the appropriate dosage of an immunosuppressive drug are customary methods to physicians skilled in the art.
  • the immunosuppressive drug comprises prednisone.
  • an effective amount can range from about 5 to 70 mg per dose.
  • a preferred dosage of prednisone may be 60 mg delivered orally and tapered to 0 mg over 1 month. Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the prednisone is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.
  • the immunosuppressive drug comprises basiliximab.
  • an effective amount can range from about 10 to 20 mg per dose.
  • a preferred dosage of basiliximab may be 20 mg delivered intravenously, one dose given during transplantation and one given on postoperative day 4.
  • Basiliximab also goes by the trade name Simulect®. Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the basiliximab is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.
  • the immunosuppressive drug comprises tacrolimus.
  • an effective amount can range from about 0.03 to 0.3 milligrams per kilogram per dose.
  • Tacrolimus also goes by FK-506, fujimycin or trade names Prograf®, LCP-TacroTM, Advagraf®, and Protopic®.
  • Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the tacrolimus is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.
  • a toxic dose of tacrolimus is administered to a subject.
  • Trough concentrations of tacrolimus are assessed to establish the appropriate dosing regimen.
  • Therapeutic doses of tacrolimus have been reported to be 10- 20 ng/mL while doses greater than 20 ng/mL are associated with neurotoxicity.
  • a preferred dosage of tacrolimus may maintain trough concentrations of about 4 to 8 ng/mL delivered orally twice a day.
  • the immunosuppressive drug comprises mycophenolate mofetil.
  • mycophenolate mofetil an effective amount can range from about 1000 to 2000 milligrams per dose.
  • a preferred dosage of mycophenolate mofetil may be 1 ,000 mg given orally twice a day.
  • Mycophenolate mofetil also goes by mycophenolic acid and the trade name CellCept®.
  • the salt mycophenolate sodium may also be used.
  • Mycophenolate sodium goes by the trade name Myfortic®.
  • Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the mycophenolate mofetil is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.
  • the immunosuppressive drug comprises sirolimus.
  • sirolimus an effective amount can range from about 1 to 20 milligrams per dose.
  • Sirolimus also goes by the name rapamycin and the trade name Rapamune®.
  • Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the sirolimus is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.
  • the dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the compositions may be given as a bolus dose, to maximize the circulating levels for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
  • compositions used in the methods described herein further comprise a pharmaceutically acceptable excipient.
  • excipient refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • the compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • the pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known in the art. "Administration" is not limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, or oral (for example, in capsules (e.g., as, powder, granules, microtablet, micropellets, etc.), suspensions, or tablets).
  • parenteral including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection
  • rectal topical
  • transdermal or oral
  • oral for example, in capsules (e.g., as, powder, granules, microtablet, micropellets, etc.), suspensions, or tablets).
  • an immunosuppressive drug is administered daily (or 1 to 5 times daily), weekly, or monthly.
  • the composition is administered three times a week for five weeks and then weekly for an additional five weeks.
  • a dosage and dosage regimen may be administered to provide the optimal desired response (e.g., therapeutic response).
  • the dose of an immunosuppressive drug may be measured in units of mg/kg of patient body weight.
  • the dose of an immunosuppressive drug is measured in units of mg/kg of patient lean body weight (e.g., body weight minus body fat content), in units of mg/m 2 of patient body surface area, or in units of mg per dose (e.g., a fixed dose) administered to a patient.
  • Any measurement of dose can be used in conjunction with the compositions and methods of the invention and dosage units can be converted by means standard in the art.
  • the method comprises the administration of an immunosuppressive drug of the present invention to a subject in need thereof.
  • the dosage regimen of immunosuppressive drug corresponds to once-a-day or twice-a-day dosages, and can include, for example, about 0.0001 mg/kg, about 0.0005 mg/kg, about 0.001 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80, mg/kg, about 90 mg/kg, about 100 mg/kg, about 1 10 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg, about 150 mg/kg, about 160 mg/kg, about 170 mg/kg, about 180 mg/kg, about 190
  • mg/kg amounts can vary, for example, from about 0.01 % to about 20% or more, depending on the application and desired therapeutic result. Other factors include the type of subject, the age, weight, sex, diet, and medical condition of the subject and the severity of the disease. Thus, the dosage regimen actually employed can vary widely and therefore deviate from the dosage regimen set forth above.
  • An immunosuppressive drug for use in any of the aforementioned methods may be administered in one or more doses (e.g., an initial dose optionally followed by one or more subsequent doses).
  • doses are generally higher and/or frequency of administration greater for initial treatment as compared with maintenance regimens.
  • two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more or eleven or more subsequent doses of the antibody are administered.
  • the aforementioned dosage amounts refer to mg (immunosuppressive drug)/kg (weight of the individual to be treated).
  • An immunosuppressive drug thereof for use in any of the aforementioned methods may also be administered as a fixed dose, independent of a dose per subject weight ratio.
  • the immunosuppressive drug is administered in one or more fixed doses of about 1000 mg or less, 500 mg or less, or 250 mg or less, 100 mg or less, 90 mg or less, 80 mg or less, 70 mg or less, 60 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, or 10 mg or less of immunosuppressive drug.
  • the immunosuppressive drug is administered in one or more doses of at least 0.01 mg, at least 0.5 mg of immunosuppressive drug, at least 1 mg of immunosuppressive drug, or at least 10 mg of immunosuppressive drug.
  • the immunosuppressive drug thereof is administered in one or more doses of 1 mg to 100 mg of immunosuppressive drug.
  • the fixed dose immunosuppressive drug is from about 1 mg to about 10 mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 25 mg to about 50 mg, about 25 mg to about 100 mg, about 50 mg to about 100 mg, about 50 mg to about 150 mg, about 100 mg to about 150 mg, about 100 mg to about 200 mg, about 150 mg to about 200 mg, about 150 mg to about 250 mg, about 200 mg to about 250 mg, about 200 mg to about 300 mg, about 250 mg to about 300 mg, about 250 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 400 mg to about 600 mg, about 500 mg to about 750 mg, about 600 mg to about 750 mg, about 700 mg to about 800 mg, or about 750 mg to about 1000 mg. In some embodiments, the fixed dose of immunosuppressive drug thereof is less than 100 mg.
  • dosage units of the present invention contain, for example, about 1 ng to about 2000 mg, about 0.001 mg to about 750 mg, about 0.01 mg to about 500 mg, about 0.1 mg to about 300 mg or about 1 mg to about 100 mg of an immunosuppressive drug of the present invention.
  • such unit dosage forms can contain about 0.001 mg, or about 0.01 mg, or about 0.1 mg, or about 1 mg, or about 2 mg, or about 5 mg, or about 10 mg, or about 15 mg, or about 20 mg, or about 30 mg, or about 40 mg, or about 50 mg, or about 60 mg, or about 70 mg, or about 80, mg, or about 90 mg, or about 100 mg, or about 1 10 mg, or about 120 mg, or about 130 mg, or about 140 mg, or about 150 mg, or about 160 mg, or about 170 mg, or about 180 mg, or about 190 mg, or about 200 mg, or about 300 mg, or about 400 mg, or about 500 mg, or about 750 mg, or about 1 ,000 mg of an immunosuppressive drug of the present invention.
  • dosage units each contain about 0.01 mg, about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 40 mg, about 80 mg, about 100 mg, about 250 mg, about 500 mg, or about 1000 mg of an immunosuppressive drug of the present invention.
  • the dosage unit form can be selected to accommodate the desired frequency of administration used to achieve the specified daily dosage.
  • a composition of the invention will be administered to a subject in an amount sufficient to about 0.1 to about 15 mg, about 0.5 to about 10 mg, and or about 1 to about 5 mg of the active agent, for example methylprednisolone, prednisone, sirolimus, etc.
  • administration of an initial dose of immunosuppressive drug is followed by the administration of one or more subsequent doses.
  • dosing regimens e.g., an interval between the first dose and one or more subsequent doses
  • dosing regimens include an interval of about once every week to about once every 12 months, an interval of about once every two weeks to about once every 6 months, an interval of about once every month to about once every 6 months, an interval of about once every month to about once every 3 months, or an interval of about once every 3 months to about once every 6 months.
  • administration is monthly, every two months, every three months, every four months, every five months, or every six months.
  • the disclosure also provides dosing regimens for use in any of the aforementioned methods, wherein the dosing regimens comprise more than one dosing interval for administration of the immunosuppressive drug.
  • the dosage regimen comprises at least two (e.g., two, three, four, five, six) different dosing intervals for administration of the immunosuppressive drug.
  • the dosage regimen comprises two different dosing intervals for administration of the immunosuppressive drug.
  • the dosing regimen comprises two different dosing intervals for administration of the immunosuppressive drug, wherein a first dosing interval comprises administration of one or more doses of immunosuppressive drug thereof and a second dosing interval comprises administration of one or more doses of the immunosuppressive drug thereof, and wherein the first dosing interval is shorter in time than the second dosing interval.
  • the first dosing interval may be days or weeks, and the second dosing interval may be months.
  • the first dosing interval is about 5 days to about 28 days, about 7 days to about 21 days, about 12 days to about 16 days, or about 14 days.
  • the second dosing interval is about 1 month to about 3 months, about 1 month to about 2 months, or about 1 month.
  • the dose can be escalated or reduced to maintain a constant dose in the blood or in a tissue.
  • the dose is escalated or reduced by about 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% in order to maintain a desired level of the immunosuppressive drug.
  • the immunosuppressive drug is administered to a subject such that the interval between doses is a time sufficient to maintain a plasma concentration of said immunosuppressive drug in the subject at a level of at least about 0.1 ⁇ g/mL, at least about 0.3 ⁇ g/mL, at least about 1 ⁇ g/mL or at least about 2 ⁇ g/mL.
  • these plasma concentration values refer to values obtained for an individual that is treated with the immunosuppressive drug in accordance with the disclosure herein.
  • administering is followed by the administration of one or more subsequent doses, and wherein said one or more subsequent doses are in an amount that is approximately the same or less than the initial dose.
  • administering is followed by the administration of one or more subsequent doses, and wherein at least one of the subsequent doses is in an amount that is more than the initial dose.
  • an immunosuppressive drug is administered, wherein administration of an initial dose of the immunosuppressive drug is followed by the administration of one or more subsequent doses, and wherein the plasma concentration of said immunosuppressive drug in the human is permitted to decrease below a level of about 0.1 ⁇ g/mL, about 0.07 ⁇ g/mL, about 0.05 ⁇ g/mL, about 0.03 ⁇ g/mL or about 0.01 ⁇ g/mL for a period of time greater than about 1 week and less than about 6 months between administrations during a course of treatment with said initial dose and one or more subsequent doses.
  • the plasma concentration values refer to values obtained for an individual that is treated with immunosuppressive drug in accordance with the disclosure herein.
  • the amount of immunosuppressive drug necessary to elicit a therapeutic effect can be experimentally determined based on, for example, the absorption rate of the immunosuppressive drug into the blood serum or the bioavailability of the immunosuppressive drug. It is understood, however, that specific dose levels of the immunosuppressive drug of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject (including, for example, whether the subject is in a fasting or fed state), the time of administration, the rate of excretion, the drug combination, the severity of the diabetes mellitus and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy.
  • dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for subject administration.
  • Studies in animal models generally may be used for guidance regarding effective dosages for treatment of diabetic disorders or diseases in accordance with the present invention.
  • the dosage to be administered will depend on several factors, including the particular immunosuppressive drug that is administered, the route administered, the condition of the particular subject, etc.
  • one will desire to administer an amount of the immunosuppressive drug for a period of time that elicits a desired therapeutic effect for example, lowering blood glucose level to acceptable levels, or improvement or elimination of symptoms, and other indicators as are selected as appropriate measures by those skilled in the art. Determination of these parameters is well within the skill of the art.
  • the composition comprising the immunosuppressive drug may be administered to a subject in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
  • intravenous administration e.g., as a bolus or by continuous infusion over a period of time
  • intramuscular, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes Intravenous, intraperitoneal, or oral administration of the immunosuppressive drug is preferred, with intravenous or oral routes being particularly preferred.
  • an immunosuppressive drug of the invention is formulated as an injectable formulation and comprises, for example, an aqueous solution or suspension of the active ingredient suitable for intravenous delivery.
  • a continuous phase can be present that comprises an aqueous solution of tonicity modifiers, buffered to a pH below about 7, or below about 6, for example about 2 to about 7, about 3 to about 6 or about 3 to about 5.
  • the tonicity modifiers can comprise, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other pharmaceutical agents that render osmotic pressure of the formulation isotonic with blood.
  • a larger quantity of the tonicity modifier is used in the formulation, it can be diluted prior to injection with a pharmaceutically acceptable diluent to render the mixture isotonic with blood.
  • the immunosuppressive drug of the present invention is administered by intravenous (IV) infusion or intra-arterial administration over a desired period (for example, bolus injection, 5 min, 15 min, 30 min, 1 hr, 2 hr, 3 hr, 6 hr, 24 hr, 48 hr, 72 hr or 96 hour infusions).
  • IV intravenous
  • intra-arterial administration over a desired period (for example, bolus injection, 5 min, 15 min, 30 min, 1 hr, 2 hr, 3 hr, 6 hr, 24 hr, 48 hr, 72 hr or 96 hour infusions).
  • the period of administration is no greater than about 3 hours.
  • fibronectin 1 mg/ml fibronectin for 18 hours at 37°C. was used.
  • Culture media consisting of N2 (DMEM/F12 plus insulin, transferrin, selenium, putrescine, and progesterone) was supplemented with 1 human recombinant basic fibroblast growth factor (bFGF).
  • bFGF basic fibroblast growth factor
  • a range of 0.1 ng/ml-100 ng/ml can be used.
  • 10 ng/ml of bFGF was used.
  • the resulting initial culture consists of post-mitotic neurons and proliferative NSCs in a monolayer. Subsequently, after approximately five to about twenty days in culture, the dividing, nestin-positive, NSCs dominate the culture over the non-dividing neurons or the slowly-dividing glia. Under these culture conditions, NSCs are selectively favored for expansion.
  • the expanding NSC population was passaged by mild enzymatic treatment, such as using trypsin.
  • the cells were then cultured in media free of serum or substantially free of serum. Although low concentration of serum may be tolerated by the cells, it is best to avoid exposing the cells to serum since serum contains many cytokines such as LIF and CNTF which promote glial differentiation of the NSCs.
  • human NSCs can be expanded beyond 10 18 -fold increase in population while maintaining their growth and differentiation properties. During the expansion, almost all cells express nestin, the in vivo marker of mitotic neuroepithelial cells, and are absent of antigens of differentiated neurons and glia such as type 3-beta tubulin and GFAP.
  • the cells were also negative by immunostaining for PSA-NCAM, a possible marker of committed neuronal progenitors, 04 and GalC, markers of oligodendrocytes, and RC2, a marker of radial glia.
  • PSA-NCAM a possible marker of committed neuronal progenitors
  • GalC a possible marker of committed neuronal progenitors
  • RC2 markers of oligodendrocytes
  • RC2 a marker of radial glia.
  • the cultures can be differentiated by withdrawal of the mitogen in the culture such as bFGF. Differentiation of NSCs ensues within about 1-3 days after the removal of mitogen, and distinct heterogeneous cell morphologies are apparent.
  • the mitogen in the culture such as bFGF.
  • Differentiation of NSCs ensues within about 1-3 days after the removal of mitogen, and distinct heterogeneous cell morphologies are apparent.
  • neuron-specific antigens such as MAP2c, tau, and type III beta-tubulin, can be visualized by immunostaining.
  • MAP2c neuron-specific antigens
  • tau elongated, fasciculated axonal processes are evident throughout the culture along with clear polarization of subcellular protein trafficking.
  • synaptic proteins such as synapsin and synaptophysin
  • synapsin and synaptophysin localize into axon terminals, appearing as punctate staining.
  • Additional feeder layer of astrocytes can be provided to further promote long-term maturation of the neurons.
  • Differentiation of human spinal NSCs generates mixed cultures of neurons and glia wherein the neurons robustly express neuron-specific antigens such as tau, MAP2ab and type3 beta tubulin and comprises approximately 50% of the culture. Additionally, the culture spontaneously generates long, bundled, axon cables that stretch for several centimeters. A significant proportion of the neurons are GABAergic with cholinergic motor neurons also being present in the culture.
  • the human spinal NSCs may be expanded with or without further phenotype-enhancing conditions, harvested, and injected into a neural area of deficiency.
  • spinal cord-derived NSCs hereon referred to as, "NSI566 NSCs"
  • NSCs spinal cord-derived NSCs
  • Robust expression of the neural stem cell marker nestin validated the undifferentiated state of the NSI566 NSCs ( Figure 1 B).
  • Additional in vitro differentiation analysis demonstrated the capability of NSI566 NSCs to generate cell types positive for the neuronal neurofilament proteins MAP2 and SMI312 and astocytic GFAP following growth factor deprivation for 7 days ( Figure 1 B).
  • a neuronal stem cell may be isolated, expanded in vitro and then introduced (e.g., transplanted) to one or more areas in a subject (e.g., a subject's brain) afflicted with cognitive dysfunction.
  • constitutively immunodeficient athymic nude (ATN) rats (stain 0N01 , Cr:NIH-rnu) were injected with NSI566 NSCs. All rats were maintained in an animal facility for the duration of the experiments and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). Male ATN rats (2 months old, purchased from Frederick National Laboratory, NCI, MD, USA) were maintained in sterile housing conditions (20°C ⁇ 1 °C; 70% humidity; 12 hour each light and dark cycle) and had free access to sterilized diet and water.
  • ATN athymic nude
  • a 137 Cs irradiator J.L. Shepard, Mark I, CA, USA
  • the NSI566 NSCs were transplanted in irradiated rats as represented in the schematic in Figure 1A.
  • each rat received bilateral, intra- hippocampal transplantation of 70,000 NSI566 NSCs (IRR+NSI) in 1 ⁇ _ of cell suspension using a 33-gauge microsyringe at an injection rate 0.25 ⁇ _/ ⁇ .
  • Both hippocampi received 4 distinct injections (total 2.8 x 10 ⁇ 5 live NSI566 NSCs per hemisphere) using precise stereotaxic coordinates, as described in Acharya et al., Proc. Natl. Acad. Sci. (2009) 106(45): 19150-19155.
  • Sham-operated control (CON) and irradiated (IRR) rats received sterile vehicle (hibernation buffer) at the same stereotaxic coordinates.
  • Tissues were processed in a sucrose gradient (10-30%) and 30 ⁇ -thick sections cut coronally through the hippocampus using a cryostat (Leica Microsystems, Wetzlar, Germany) were then stored in phosphate buffered saline (PBS) with 0.02% sodium azide (Sigma-Aldrich, MO, USA).
  • PBS phosphate buffered saline
  • the secondary antibodies and detection reagents included biotinylated horse anti-goat IgG (1 :200, Vector Labs, CA, USA), donkey anti-mouse and anti-rabbit conjugated with Alexa Fluor 488 or 594 (1 :200, Invitrogen, CA, USA) and TOTO- 3 iodide (infrared nuclear counterstain, Invitrogen, CA, USA).
  • Free floating sections were first rinsed in TBS followed by Tris-A (TBS with 0.1 % Triton-X-100, Sigma-Aldrich, MO, USA), blocked with 10% normal donkey serum (NDS with Tris-A, Sigma-Aldrich, MO, USA) and incubated overnight in a mouse anti-Ku80 solution (1 :100) prepared in 3% NDS and Tris-A. The next day, the sections were treated with donkey anti-mouse IgG conjugated with Alexa Fluor 594 (1 :200) made with Tris-A and 3% NDS for 1 h. The sections were light-protected, washed with Tris-A and blocked in serum and primary antibodies for neuronal and glial markers.
  • a multicenter, randomized, double-blind, placebo- controlled study is undertaken to evaluate treatment with a weight-based or fixed dose of spinal cord-derived neural stem cells (NSCs) in human subjects diagnosed with cognitive dysfunction. More specifically, a clinical study was performed to examine the efficacy and safety of introducing a therapeutically effective amount of spinal cord-derived NSCs to at least one area of the brain of the human subject.
  • the composition is effective to treat cognitive dysfunction.
  • the NSCs are present in the afflicted individual, they engraft and differentiate and thereby help treat the cognitive dysfunction.
  • One advantage of this method is that it may be repeated, as needed, and thereby alleviate some or all of the cognitive dysfunction in the human subject.
  • cells may be differentiated into appropriate cell types in vitro before transplantation.
  • aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments.
  • methods for treating a disease or disorder associated with cognitive dysfunction comprising: introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of brain tissue of the subject.
  • methods of treating cognitive dysfunction in a subject comprising: obtaining at least one neural stem cell from spinal cord tissue of a human; expanding the at least one neural stem cell to form an expanded neural stem cell population; concentrating the expanded neural stem cell population; and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of brain tissue of the subject.
  • the spinal cord-derived neural stem cells are embryonic spinal cord-derived neural stem cells.
  • the spinal cord-derived neural stem cells are fetal spinal cord-derived neural stem cells.
  • the fetal spinal cord-derived neural stem cells are obtained from a fetus being a gestational age of about 5 to about 20 weeks.
  • the spinal cord-derived neural stem cells are human spinal cord-derived neural stem cells.
  • expanding the spinal cord-derived neural stem cells includes culturing the spinal cord-derived neural stem cells in the absence of serum.
  • expanding the spinal cord-derived neural stem cells includes exposing the spinal cord-derived neural stem cells to at least one growth factor.
  • the growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof.
  • the spinal cord-derived neural stem cells differentiate into neurons that engraft in vivo into the brain tissue of the subject.
  • the spinal cord-derived neural stem cells are capable of generating neurons in brain tissue of the subject.
  • the subject is human.
  • the cognitive dysfunction is associated with Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, Multiple Sclerosis, cerebrovascular disease or substance abuse.
  • the cognitive dysfunction is dementia, delirium or amnesia.
  • the dementia is vascular dementia, dementia with Lewy bodies, mixed dementia, frontotemporal dementia.
  • the cognitive dysfunction includes speech impairment, confusion, disorientation, loss of memory, learning, perception, judgment, initiative, attention, planning, multitasking, spatial or analytical skills, reasoning ability, or combinations thereof.
  • introducing the therapeutically effective amount of spinal cord-derived neural stem cells includes injecting at least a portion of the therapeutically effective amount of spinal cord-derived neural stem cells into a plurality of areas of the brain tissue of the subject
  • the areas of the brain tissue include cerebral hemispheres, cerebral cortex, subcortex, motor cortex, striatum, internal capsule, thalamus, hypothalamus, hippocampus, midbrain, brainstem and cerebellum.

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Abstract

L'invention concerne, de manière générale, des méthodes de traitement d'un patient atteint d'un dysfonctionnement cognitif par administration au cerveau dudit patient d'une quantité efficace de cellules souches neurales provenant de la moelle épinière. La présente invention concerne des méthodes de traitement d'un patient atteint d'un dysfonctionnement cognitif, y compris, par exemple, d'un patient ayant été soumis à une radiothérapie pour le traitement d'une tumeur cérébrale primitive ou secondaire. De telles méthodes peuvent être utiles pour le traitement de symptômes résultant de l'exposition à des rayonnements, ou de symptômes de maladies neurologiques. Selon un aspect, la présente invention fournit des méthodes de traitement d'un patient atteint d'un dysfonctionnement cognitif, par exemple, d'un être humain, par l'introduction d'une quantité thérapeutiquement efficace de cellules souches neurales provenant de la moelle épinière dans au moins une zone du cerveau du patient (p.ex., l'hippocampe).
PCT/US2014/045993 2013-07-09 2014-07-09 Méthodes de traitement d'un patient atteint d'un dysfonctionnement cognitif au moyen de cellules souches neuronales provenant de la moelle épinière Ceased WO2015006474A1 (fr)

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US9867853B2 (en) 2014-05-30 2018-01-16 International Cell Technologies Inc. Method of providing cellular based immune enhancement for restoring immunity and preventing age related diseases
US20180104240A1 (en) * 2016-10-18 2018-04-19 Neuralstem, Inc. Amelioration of radiation-induced cognitive dysfunction
WO2018075667A1 (fr) * 2016-10-18 2018-04-26 Neuralstem, Inc. Amélioration d'un dysfonctionnement cognitif induit par radiation

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