Treatment of degenerative disc disease and stimulation of protein glycan synthesis by fibroblast conditioned medium and formulations thereof

Abstract

Methods and compositions related to promoting intervertebral disc regeneration and / or repair in an individual are provided.SOLUTION: In some cases, conditioned media is obtained from fibroblasts stimulated with one or more opioid receptor antagonists and one or more toll-like receptor agonists. Conditioned medium from fibroblasts may be provided in an effective amount to an individual in need thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (Reference to Related Application) This application claims priority to U.S. Provisional Patent Application No. 62 / 901,164, filed September 16, 2019, which is incorporated herein by reference in its entirety.

[0002] (Technical field) Embodiments of the present disclosure include at least the fields of cell biology, molecular biology, and medicine. [Background technology]

[0003] Intervertebral discs are made up of a highly organized matrix of collagen, water, and proteoglycans. Proteoglycan production in the disc is thought to occur by differentiated chondrocytes. Each disc contains a central, highly hydrated, gelatinous nucleus pulposus (core) surrounded by an elastic, highly fibrous annulus fibrosus (annulus). Cartilaginous endplates provide attachment to the vertebrae below and above the disc. This cushioned arrangement within the disc allows the disc to dissipate fluid pressure through the spine while facilitating movement and flexibility within the spine.

[0004] As a result of aging, mechanical stress, and / or other environmental and / or genetic changes, the intervertebral disc may begin to degenerate. With aging, the disc matrix is ​​known to undergo substantial structural, molecular, and mechanical changes. The present disclosure fulfills a long-felt need in the art for compositions and methods for the treatment of intervertebral disc degeneration. Summary of the Invention

[0005] The present disclosure is directed to methods and compositions related to promoting intervertebral disc regeneration and / or repair in an individual. Disclosed herein are methods for promoting intervertebral disc regeneration and / or repair in an individual using one or more components derived from stimulated fibroblasts. In certain embodiments, the disclosed compositions comprise one or more components derived from fibroblasts cultured with one or more opioid receptor antagonists and one or more toll-like receptor (TLR) agonists. Some embodiments relate to the isolation of fibroblast regenerative cells from a cell population, optionally using components in the medium in which the cultured fibroblasts are present for therapeutic purposes.

[0006] In some embodiments, provided herein are compositions comprising one or more components derived from fibroblasts cultured with one or more opioid receptor antagonists and one or more TLR agonists. The one or more components may be derived from a medium derived from a culture of the fibroblasts. The one or more components may include one or more growth factors, such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1, FGF-2, FGF-5, FGF-15, insulin-like growth factor (IGF), placenta growth factor, and hepatocyte growth factor (HGF), and in certain embodiments, the one or more growth factors are derived from fibroblasts cultured under specific conditions. In some embodiments, the one or more components comprise exosomes. The exosomes may include one or more markers (e.g., CD9). In some embodiments, the exosomes may bind to dendritic cells and / or mesenchymal stem cells. In a further embodiment, the one or more components were derived from fibroblasts cultured with one or more opioid receptor antagonists and one or more TLR agonists in proliferative conditions for the cells.

[0007] In some embodiments, the opioid receptor antagonist is naltrexone, 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, alvimopan, diprenorphine, nalorphine, nalorphine dinicotinate, levallorphan, samidorphan, nalodain, naloxegol, axelopran, bebenoplan, methylsamidorphan, naldemedine, or a combination thereof. In some embodiments, the TLR agonist is Pam3CSK4, LPS, CpG DNA, Poly(ic), flagellin, MALP-2, imiquimod, resmiquimod, zymosan, or a combination thereof. In some embodiments, the fibroblasts express a marker selected from the group consisting of Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD344, Stella, and combinations thereof. In further embodiments, the fibroblasts express a marker selected from the group consisting of CD10, CD13, CD44, CD73, CD90, CD141, PDGFr-α, HLA-A, HLA-B, HLA-C, and combinations thereof. In some cases, the fibroblasts do not express a marker from the group consisting of MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, CD90, and combinations thereof. In some cases, the fibroblasts do not express a marker selected from the group consisting of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, HLA-DQ, and combinations thereof.

[0008] In a further aspect, a method for promoting intervertebral disc regeneration in an individual is provided, the method comprising providing to the individual an effective amount of one or more components derived from fibroblasts cultured with one or more opioid receptor antagonists and one or more toll-like receptor (TLR) agonists. In some cases, the method for promoting intervertebral disc regeneration comprises providing to the individual an effective amount of fibroblasts (and / or components derived therefrom) previously cultured with one or more opioid receptor antagonists and one or more TLR agonists. The fibroblasts cultured with one or more opioid receptor antagonists and one or more TLR agonists, and / or one or more components therefrom (e.g., one or more regenerative factors), can be provided to the individual by any suitable delivery route, including at least locally (e.g., intradiscally) or systemically.

[0009] In some embodiments, provided herein are methods for improving the efficacy of tolerogenic therapy, the methods comprising: (a) providing the tolerogenic therapy to an individual; and (b) providing the individual with one or more opioid receptor antagonists in an amount sufficient to enhance the efficacy of the tolerogenic therapy. The tolerance induction therapy can include administering an autoantigen, which can be administered intravenously and / or orally. In some embodiments, the autoantigen administration includes providing immature antigen-presenting cells comprising the autoantigen, providing tolerogenic presenting cells comprising the autoantigen, providing mesenchymal stem cells comprising the autoantigen, providing hematopoietic stem cells comprising the autoantigen, and / or providing allogeneic mesenchymal stem cells. The tolerogenic antigen-presenting cells can optionally be dendritic cells. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows epidermal growth factor (EGF) production from neonatal foreskin cells cultured with naltrexone and the indicated Toll-like receptor (TLR) agonists. DETAILED DESCRIPTION OF THE INVENTION

[0011] I. Definition example In accordance with long-standing patent law convention, the terms "a" and "an," as used herein, in conjunction with words including claims, mean "one or more." As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "nucleic acid" includes a plurality of nucleic acids (including mixtures thereof). Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments can be combined.

[0012] As used herein, the term "about" or "approximately" refers to an amount, level, number, frequency, percentage, size, percentage, dimension, amount, weight, or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, number, frequency, percentage, dimension, amount, weight, or length. In certain embodiments, when the term "about" or "approximately" precedes a numerical value, it indicates a value that is within a range of plus or minus 15%, 10%, 5%, or 1%. With respect to biological systems or processes, the term can mean within an order of magnitude of a value, preferably within 5-fold, and more preferably within 2-fold. Unless otherwise specified, the word "about" refers to a range of tolerance for the particular numerical value.

[0013] The terms "administered" or "administering," as used herein, refer to any method of providing a composition to an individual so that the composition has its intended effect on the patient. For example, one method of administration is by an indirect mechanism using a medical device such as a catheter, applicator gun, syringe, etc., but is not limited to these. A second exemplary method of administration is by a direct mechanism, such as local tissue administration, oral ingestion, transdermal patch, topical, inhalation, suppository, etc.

[0014] As used herein, "allogeneic" refers to tissues or cells or other materials derived from one or more individuals of the same species, but from another body, that are or may be immunologically incompatible in their natural setting.

[0015] As used herein, the term "allograft" refers to the transplantation of organs, tissues, and / or cells from a donor to a recipient, where the donor and recipient are different individuals but of the same species. Tissues transplanted by such procedures are called allografts or allotransplants.

[0016] As used herein, the terms "allostimulatory" and "alloresponsive" refer to the stimulation and response of the immune system in response to alloantigens, or "alloantigens" or cells expressing a foreign HLA haplotype.

[0017] As used herein, "autologous" refers to tissues or cells or other materials that originate from or are transferred from the body of the same individual (ie, autologous blood donation, autologous bone marrow transplant).

[0018] As used herein, the term "autologous transplantation" refers to the transplantation of organs, tissues, and / or cells from one part of an individual's body to another part of the same individual (i.e., the donor and recipient are the same individual). Tissues transplanted by such "autologous" procedures are called autografts or autotransplants.

[0019] The term "biologically active" refers to any molecule that has a structural, regulatory, or biochemical function. For example, biological activity can be determined by, for example, restoration of wild-type growth in cells lacking protein activity. Cells lacking protein activity can be produced by a number of methods (i.e., point mutations and frameshift mutations), and complementation can be achieved by transfecting cells lacking protein activity with an expression vector that expresses the protein, its derivative, or a portion thereof. In other cases, a fragment of a gene product (e.g., a protein) can be considered biologically active (or referred to as functionally active) if it retains the activity of the full-length gene product, but at a detectable reduced level of the activity of the full-length gene product.

[0020] A "cell culture" is an artificial in vitro system containing living cells, whether quiescent, senescent, or (actively) dividing. In cell culture, cells are grown and maintained at a suitable temperature, typically 37°C, in an atmosphere typically containing oxygen and CO2, although these may be varied in other cases. Culture conditions can vary widely for each cell type, but variations in conditions for a particular cell type can result in different phenotypes being expressed. The factor most commonly varied in a culture system is the growth medium. Growth medium can vary in nutrients, growth factor concentrations, and the presence of other components. Growth factors used to supplement the medium are often derived from animal blood, such as bovine serum.

[0021] Throughout this specification, unless the context requires otherwise, the terms "comprises," "comprises," and "comprises" will be understood to refer to the inclusion of a recited step or element, or group of steps or elements, but not to the exclusion of other steps or elements, or group of steps or elements. "Consisting of" means including, but not limited to, what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements are present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0022] As used herein, the terms "drug," "agent," or "compound" refer to any pharmacologically active substance that can be administered to achieve a desired effect. A drug or compound can be synthetic or naturally occurring, a non-peptide, a protein or peptide, an oligonucleotide, or a nucleotide (DNA and / or RNA), a polysaccharide, or a sugar.

[0023] As used herein, the term "individual" refers to a human or animal who may or may not be housed in a medical facility, or who may be treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the internet. An individual may include humans or non-human animals of any age, and thus includes both adults and infants (i.e., children) and babies. The term "individual" is not intended to imply the need for medical treatment; thus, an individual may be voluntarily or involuntarily part of an experiment, whether clinical or supporting basic science research. The term "subject" or "individual" refers to any living organism or animal subject that is the subject of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), farm animals (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals.

[0024] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0025] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can mean "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z," and it is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0026] As used herein, the terms "pharmaceutically" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans.

[0027] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents or dispersion media including, but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, as well as vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercially available detergents, etc. Supplementary biologically active ingredients can also be incorporated into such carriers.

[0028] When referring to the occurrence of any symptom in an untreated subject compared to a treated subject, the terms "reduce," "inhibit," "reduce," "reduce," "prevent," and grammatical equivalents (including "lower," "less than," etc.) mean that the amount and / or magnitude of the symptom in the treated subject is lower than the amount and / or magnitude of the symptom in the treated subject by any amount recognized as clinically relevant by any medically trained personnel. In one embodiment, the amount and / or magnitude of the symptom in the treated subject is at least 10% lower, at least 25% lower, at least 50% lower, at least 75% lower, and / or at least 90% lower than the amount and / or magnitude of the symptom in the untreated subject.

[0029] A "therapeutic agent" and amount of therapeutic agent sufficient to have "therapeutic efficacy" in modulating angiogenesis and / or wound healing is said to be an "angiogenesis-modulating amount" if administration of that amount of therapeutic agent is sufficient to cause significant modulation (i.e., increase or decrease) of angiogenic activity when administered to a subject (e.g., an animal model or a human patient) in need of modulation of angiogenesis.

[0030] As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective amount," and / or "effective dose," and refers to an amount of a compound that elicits the biological, cosmetic, or clinical response sought by a practitioner in an individual in need thereof. As an example, an effective amount is an amount sufficient to reduce the immunogenicity of a cell population. As a non-limiting example, an effective amount is an amount sufficient to promote the formation of a blood supply sufficient to support the transplanted tissue. As another non-limiting example, an effective amount is an amount sufficient to promote the formation of new blood vessels and associated vasculature (angiogenesis) and / or promote the repair or remodeling of existing blood vessels and associated vasculature. The appropriate effective amount to be administered for a particular application of the disclosed methods can be determined by one of skill in the art using the guidance provided herein. For example, effective amounts can be extrapolated from in vitro and in vivo assays, as described herein. Those skilled in the art will recognize that an individual's condition can be monitored throughout the course of treatment, and that the effective amount of the compounds or compositions disclosed herein administered can be adjusted accordingly.

[0031] "Treatment," "treatment," or "treatment" refers to an approach to reducing the effects of a disease or condition. Treatment can also refer to a method of alleviating the disease or condition itself, rather than just the symptoms. Treatment can be any reduction from pre-treatment levels and cannot be limited to complete ablation of the disease, condition, or symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or disease progression, including a reduction in the severity of at least one symptom of the disease. For example, the disclosed methods for reducing the immunogenicity of a cell are considered to be treatment if there is a detectable decrease in the immunogenicity of the cell when compared to pre-treatment levels in the same subject or a control subject. Thus, the decrease can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to native or control levels. It is understood and contemplated herein that "treatment" does not necessarily refer to a cure of a disease or condition, but rather to an improvement in the outlook for a disease or condition. In certain embodiments, treatment refers to a decrease in the severity or extent of at least one symptom, and may alternatively or additionally refer to a delay in the onset of at least one symptom.

[0032] II. Embodiment Stimulated fibroblast-derived medium and its components In some embodiments, the present disclosure relates to components obtained from fibroblast culture. Such components may be, or may be obtained from, cell culture medium from fibroblasts cultured with one or more compounds that stimulate the production of regenerative factors from the fibroblasts. Components obtained from fibroblast culture may be useful in one or more methods disclosed herein, including, for example, promoting intervertebral disc regeneration or repair, treating degenerative disc disease, and stimulating proteoglycan synthesis.

[0033] In one embodiment, disclosed herein is the use of culture medium or components present therein obtained from tissue culture of fibroblasts. In certain embodiments, for use within the disclosed methods, the fibroblasts in culture are in a proliferative state, which is described as cells not reaching confluence. In some embodiments, the fibroblasts are growing at 25% to 75% confluence. One or more components (e.g., regenerative factors) can be obtained from fibroblasts growing in a proliferative state.

[0034] In some embodiments, the use of opioid receptor antagonists as modulators of immune responses to autoantigens by stimulating regulated cell proliferation through alteration of fibroblast activity is disclosed. Stimulation of fibroblast production of regenerative factors can be achieved through treatment of fibroblasts with one or more opioid receptor antagonists. Opioid receptor antagonists include, but are not limited to, naltrexone, 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, alvimopan, diprenorphine, nalorphine, nalorin dinicotinate, levallorphan, samidorphan, nalodein, naloxegol, axelopran, benoplan, methylsamidorphan, and / or naldemedine. It is demonstrated herein that treatment of fibroblasts with opioid receptor antagonists (e.g., naltrexone) induces the production of various growth factors, including EGF. Furthermore, upregulation of regenerative factor production is further demonstrated by the combination of administering an opioid receptor antagonist (e.g., naltrexone) along with an agonist of the Toll-like receptor (TLR) family, such as the Toll-like receptor 2 agonist Pam3CSK4, the Toll-like receptor 4 agonist lipopolysaccharide (LPS), and the Toll-like receptor 9 agonist CpG. In some embodiments, a TLR-4 antagonist is used to stimulate fibroblasts instead of an opioid receptor antagonist. Example TLR-4 antagonists that can be used to stimulate fibroblasts include LPS and lipid A from Rhodobacter sphaeroides, LOS from Bartonella Quintana, LPS from Oscillatoria Planktothrix FP1, curcumin from Curcuma longa, sulforaphane and iberin from cruciferous vegetables, xanthohumol from hops and beer, and celastrol from Tripterygium wilfordii.

[0035] TLRs can bind damage-associated molecular patterns (DAMPs), which are produced under stress, tissue injury, or cell apoptosis. TLRs are thought to bridge the gap between innate immunity and autoimmunity. There are five adaptors for TLRs, including MyD88, TRIF, TIRAP / MAL, TRAM, and SARM. Upon activation, TLRs recruit specific adaptors to initiate downstream signaling pathways leading to the production of proinflammatory cytokines and chemokines. Under certain circumstances, TLR ligation can drive aberrant activation and uncontrolled inflammatory responses, thereby contributing to the persistence of inflammation in autoimmune diseases. While most research in the past has focused on intracellular TLRs, such as TLR3, TLR7, and TLR9, recent studies have revealed that cell surface TLRs, particularly TLR2 and TLR4, also play essential roles in the pathogenesis of autoimmune diseases and provide multiple therapeutic targets [Clin Rev Allergy Immunol. 47(2):136-47(2014)]. TLR4 is associated with hepatocytes and nonparenchymal cells, including Kupffer cells, myeloid dendritic cells, stellate cells, T cells, NK cells, and sinusoidal endothelial cells. Recently, several lines of evidence suggest that TLR4 may mediate the pathogenesis and progression of autoimmune liver disease (AILD) (He et al. 2006; Longhi et al. 2009; Mencin et al. 2009). When monocytes from PBC patients are challenged with various ligands, particularly those signaling through TLR4 and TLR5, levels of proinflammatory cytokines such as IL-1β and IL-6 increase (Mao et al. 2005). Endogenous DAMPs are released following tissue injury. Ligands for TLR-2 and TLR-4, such as heat shock proteins, HMGB1, hyaluronan, fibronectin, heparan sulfate, and biglycan, are produced to mediate sterile inflammation (Moreth et al. 2014).The biological properties, signaling mechanisms, negative regulators of the TLR2 / 4 pathway, and the important functions of TLR2 / 4 in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, psoriasis, multiple sclerosis, and autoimmune diabetes, were recently reviewed by Liu Y et al. [Clin Rev Allergy Immunol. 47(2):136-47(2014)].

[0036] The compositions of the present disclosure can be obtained from isolated fibroblasts or populations thereof (including from cultures thereof) capable of proliferation and differentiation into ectoderm, mesoderm, or endoderm. In some embodiments, the isolated fibroblasts express at least one of Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD344, or Stella markers. In some embodiments, the isolated fibroblasts do not express at least one of MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, or CD90 cell surface proteins. Such isolated fibroblasts can be used as a source of conditioned medium. The cells can be cultured alone or in the presence of other cells to further upregulate the production of growth factors in the conditioned medium.

[0037] Fibroblasts can be cultured in growth medium (or components thereof can be used) to obtain a conditioned medium that can be expanded and utilized by administration itself or used thereafter. The term growth medium generally refers to a medium sufficient for the culture of fibroblasts. In particular, one particular medium for the culture of the cells disclosed herein comprises Dulbecco's Modified Essential Medium (DMEM). In a specific embodiment, it is DMEM-Low Glucose (also referred to herein as DMEM-LG) (Invitrogen®, Carlsbad, CA). DMEM-low glucose can be supplemented with 15% (v / v) fetal bovine serum (e.g., defined fetal bovine serum, Hyclone®, Logan, UT), antibiotic / antimycotic (e.g., penicillin (100 units / milliliter), streptomycin (100 milligrams / milliliter), and amphotericin B (0.25 micrograms / milliliter) (Invitrogen®, Carlsbad, CA), and 0.001% (v / v) 2-mercaptoethanol (Sigma®, St. Louis, MO). In some cases, different growth media are used or different supplements are provided, and these are usually referred to as supplements to the growth media. Standard growth conditions also refer to growth at 37°C, 5% CO₂, and 10% CO₂. This refers to culturing cells in a standard atmosphere containing CO2, where the relative humidity is maintained at about 100%. While the aforementioned conditions are beneficial for culture, it is understood that such conditions can be varied by altering the options available to one skilled in the art for culturing cells, such as body temperature, CO2, relative humidity, oxygen, growth medium, etc.

[0038] In certain embodiments, the fibroblasts used in the disclosed methods for obtaining conditioned medium and / or regenerative factors may undergo at least 25, 30, 35, or 40 doublings before reaching a senescent state. 14 Methods are provided for inducing cells capable of doubling to reach more than about 10 cells. 14 , 10 15 , 10 16 , or 10 17 More than 10 cells were cultured3 from about 10 6 cells / cm 2 This method derives cells that can double sufficiently to produce up to 1000 cells when seeded. In certain cases, these cell numbers are produced within 80, 70, or 60 days. In one embodiment, the fibroblasts used to produce the conditioned medium are isolated and expanded and have one or more markers selected from the group consisting of CD10, CD13, CD44, CD73, CD90, CD141, PDGFr-α, HLA-A, HLA-B, HLA-C, and combinations thereof. In some embodiments, the fibroblasts do not produce one or more of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, or HLA-DQ.

[0039] In some cases, fibroblasts are obtained from a biopsy, and the donor providing the biopsy may be different from the individual being treated (autologous) or different from the individual being treated (allogeneic). When allogeneic fibroblasts are utilized for an individual, the fibroblasts may be derived from one or more donors.

[0040] The fibroblasts may be obtained from a source selected from the group consisting of skin fibroblasts, placental fibroblasts, adipose fibroblasts, bone marrow fibroblasts, foreskin fibroblasts, umbilical cord fibroblasts, hair follicle-derived fibroblasts, nail-derived fibroblasts, endometrium-derived fibroblasts, keloid-derived fibroblasts, and combinations thereof.

[0041] In some embodiments, the components obtained from the fibroblast culture include one or more regenerative factors. Regenerative factors produced by fibroblasts cultured with an opioid receptor antagonist and a TLR agonist include, for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-1, FGF-2, FGF-5, FGF-15, insulin-like growth factor (IGF), placenta growth factor, and hepatocyte growth factor (HGF). The regenerative factors can be isolated from the cell culture medium before use in the disclosed methods (e.g., stimulating intervertebral disc regeneration or repair). Alternatively, the cell culture medium can be used without isolating the regenerative factors.

[0042] In some embodiments, components obtained from fibroblast culture include exosomes. Fibroblasts can produce exosomes containing one or more regenerative factors (e.g., growth factors), which can be used in the disclosed methods (e.g., promoting intervertebral disc regeneration). Exosomes can be isolated from fibroblasts cultured with one or more opioid receptor antagonists and one or more TLR agonists, thereby obtaining one or more regenerative factors. Exosomes can be purified and concentrated from fibroblast culture medium. In some embodiments, exosomes obtained from fibroblasts are 60 to 200 nanometers in size. In some embodiments, exosomes obtained from fibroblasts stimulated with one or more opioid receptor antagonists and one or more TLR agonists can induce the production of anti-inflammatory mediators (e.g., IL-10, IL-20, TGF-β, etc.) from dendritic cells. In some embodiments, exosomes obtained from fibroblasts stimulated with an opioid receptor antagonist and a TLR agonist can bind to mesenchymal stem cells and, in at least some cases, induce the production of TGF-β from the mesenchymal stem cells.

[0043] The conditioned medium obtained from the fibroblasts can be concentrated by filtration and / or desalting means. In one embodiment, an Amicon® filter with a specific molecular weight cutoff, or a substantially equivalent method, is utilized. The cutoff can be selected to be greater than 1 kDa to 50 kDa.

[0044] Alternatively, cell culture supernatants may be concentrated using means known in the art, such as solid-phase extraction using C18 cartridges (Mini-Speed ​​C18-14%, SPELimited, Concord, ON). The cartridges are prepared by washing with methanol followed by deionized distilled water. Up to 100 ml of stem or progenitor cell supernatant can be passed through each of these specific cartridges before elution, although those skilled in the art will understand that larger cartridges can be used. After washing the cartridges, adsorbed material is eluted with 3 ml of methanol, evaporated under a stream of nitrogen, redissolved in a small volume of methanol, and stored at 4°C.

[0045] Before testing the eluate for in vitro activity, the methanol is evaporated under nitrogen and replaced with culture medium. C18 cartridges are used to adsorb small hydrophobic molecules from stem cell or progenitor cell culture supernatants, allowing for the removal of salts and other polar contaminants. However, it may be desirable to use other adsorption means to purify specific compounds from the fibroblast supernatant. The fibroblast-enriched supernatant can be directly evaluated for biological activity useful in the practice of the present invention or can be further purified. In one embodiment, the supernatant of a fibroblast culture is evaluated for its ability to stimulate proteoglycan synthesis using an in vitro bioassay. This allows for quantification and identification of which molecular weight fractions of the supernatant are biologically active. Bioassays for testing the ability to stimulate proteoglycan synthesis are known in the art. The production of various proteoglycans can be assessed by analysis of protein content using techniques including mass spectrometry, column chromatography, immuno-based assays such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and flow cytometry.

[0046] Further purification can be achieved, for example, by gel filtration using a Bio-Gel P-2 column (Bio-Rad®, Richmond, CA) with a nominal exclusion limit of 1800 Da. The column can be washed, pre-swollen in 20 mM Tris-HCl buffer, pH 7.2 (Sigma®), and degassed by gentle swirling under reduced pressure. The Bio-Gel P-2 material can be packed into a 1.5 x 54 cm glass column and equilibrated with three column volumes of the same buffer. The amniotic fluid stem cell supernatant concentrate extracted by the C18 cartridge can be dissolved in 0.5 ml of 20 mM Tris buffer, pH 7.2, and passed through the column. Fractions can be collected from the column and analyzed for biological activity. Other purification, fractionation, and identification methods are known to those skilled in the art and include anion exchange chromatography, gas chromatography, high-performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry.

[0047] III. Usage Examples Embodiments of the present invention include a means of enhancing regeneration of discs undergoing one or more degenerative processes through the introduction of components from fibroblasts stimulated with one or more opioid receptor antagonists (e.g., naltrexone) alone and / or together with one or more Toll-like receptor (TLR) agonists. The components from fibroblasts may include conditioned medium from a culture of fibroblasts with one or more opioid receptor antagonists and one or more TLR agonists. The conditioned medium may be used as a source of regenerative factors. The conditioned medium may be concentrated. The fibroblast-derived components may be administered to an individual in need of disc regeneration or repair (e.g., an individual with degenerative disc disease). The components may be administered intradiscally or systemically. In some embodiments, microvesicles and / or exosomes derived from stimulated fibroblasts are used as a source of regenerative factors.

[0048] In some aspects, the present disclosure relates to methods for treating or preventing pathological disc disorders by delivering (e.g., secreting, releasing, etc.) one or more components from fibroblasts stimulated with one or more opioid receptor antagonists and one or more TLR agonists. The stimulated fibroblasts are suitable for administration to an intervertebral disc and are capable of producing one or more regenerative factors capable of stimulating disc regeneration. In some embodiments, one or more of these regenerative factors are provided to an individual in need thereof, in some cases together in a medium. Alternatively or additionally, stimulated fibroblasts capable of producing regenerative factors can be delivered directly to an individual.

[0049] Embodiments of the present disclosure encompass certain conditioned media, including therapeutic applications. In certain embodiments, the conditioned media are useful for stimulating disc regeneration or repair in individuals, including those suffering from or at risk for degenerative disc disease (e.g., individuals over the age of about 40, 45, 50, 55, 60, 65, 70, 75, 80, etc., individuals who are or were athletes, individuals whose occupations require physical activity, individuals with spinal injuries, or combinations thereof). Thus, in certain embodiments, methods encompassed by the present disclosure may be used to prevent disc degeneration, or to delay the onset and / or reduce the severity of disc degeneration.

[0050] The conditioned medium can be generated by stimulation of fibroblasts, which can be any type of fibroblast. In some embodiments, such stimulation includes treatment with an effective amount of one or more opioid receptor antagonists and one or more TLR agonists sufficient to stimulate the production of regenerative factors (e.g., one or more growth factors) by the fibroblasts. The fibroblasts can be stimulated with any opioid receptor antagonist, including, for example, naltrexone, 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, alvimopan, diprenorphine, nalorphine, nalorline dinicotinate, levallorphan, samidorphan, nalodain, naloxegol, axelopran, benoprand, methylsamidorphan, or naldemedine. In some embodiments, the fibroblasts are stimulated with naltrexone. Fibroblasts can be stimulated with any TLR agonist, including, for example, Pam3CSK4, LPS, CpG DNA, Poly(ic), flagellin, MALP-2, imiquimod, resmiquimod, zymosan, or combinations thereof. Conditioned medium produced from fibroblast stimulation can be obtained, optionally concentrated, and provided to an individual in need thereof.

[0051] In some embodiments, fibroblast conditioned medium is utilized as part of a formulation with other therapeutic compounds, where the formulation is administered intradiscally or systemically to an individual to induce proteoglycan production from the intervertebral disc. The compound can be vitamin A, vitamin C, vitamin E, vitamin K, folic acid, choline, vitamin B1, vitamin B2, vitamin B5, vitamin B6, biotin, nicotinamide, beta-carotene, coenzyme Q, selenium, superoxide dismutase, glutathione peroxide, uridine, creatine succinate, pyruvate, dihydroxyacetone, acetyl-L-carnitine, alpha-lipoic acid, cardiolipin, omega fatty acids, lithium carbonate, lithium citrate, calcium, or any combination thereof. In some aspects, the compound is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, alpha lipoic acid, alpha tocopherol, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anilorac, anitrazafen, apazone, ascorbic acid, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelain, broperamol, budessonide, carprofen, chlorogenic acid, cycloprofen, synthasone, criplofen, clobetasol propionate, clobetasone butyrate, clopirac, clotiasone propionate, cormetasone acetate, cortodoxun, deflazacort, desonide, desoximetasone, dexamethasone dipropionate, diclofenac potassium, diclofenac sodium, dichlorazonidine. acetate, diflumidone sodium, diflunisal, difluprednate, diphthalone, dimethyl sulfoxide, drocinonide, ellagic acid, endrisone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamol, fenbufen, fenclofenac, fenclorac, fendosal, fenpiparone, fentiazac, furazaron, fluazacort, flufenamic acid, flumisole, flunisolide acetate, flunixin, flunixin meglumine, flucortine butyl, fluoromesolone acetate, fluquazone,Flurbiprofen, Fluretofen, Fluticasone propionate, Furaprofen, Flobufen, Glutathione, Halcinonide, Halobetasol propionate, Halopredone acetate, Hesperin, Ibufenac, Ibuprofen, Ibuprofen aluminum, Ibuprofen piconol, Ilonidap, Indoprofen, Indomethacin, Indomethacin sodium, Indoprofen, Indoxol, Intrazole, Isoflupredone acetate, Isoxepac, Isoxicam, Ketoprofen, Lofemizole hydrochloride, Lomoxicam, Loteprednol etabonate, Lycopene, Meclofenamate sodium, Meclofenamic acid, Mechrysolide, Mefenamic acid, Mesalamine, Meseclazone, Methylprednisolone Suleptanic acid, morniflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone, oleuropein, olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone, para oleuropein, olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, pycnogenol, polyphenols, prednazate, preferon, prodolic acid, proquazone, proxazole, proxazole citrate, quercetin, resveratrol, rimexolone, romazarith, In some embodiments, the compound is one or more of: sumaric acid, rutin, sarcorex, salnadecin, salsalate, sanguinarium chloride, seclazone, selmetacin, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, tetrahydrocurcumin, totridamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, zidometacin, zomepirac sodium. In some embodiments, the compound is a growth factor, a cytokine, an antibody, an antibody fragment,and / or organic molecules with a mass of less than 5,000 daltons. The compound can be administered simultaneously with the composition of the present disclosure. Alternatively, the compound can be administered before and / or after the composition is administered to the subject.

[0052] IV. Obtaining fibroblast regenerative cells Embodiments of the present disclosure include methods for obtaining or isolating regenerative fibroblasts. Obtaining regenerative fibroblasts can include enriching a population of regenerative fibroblasts from tissue with regenerative activity. In some embodiments, regenerative fibroblasts are obtained from tissue with regenerative activity by enriching for cells approximately 6-12 μm in size that express at least one of Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD344, and Stellar, and do not express at least one of MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, or CD90 cell surface proteins. In some embodiments, cell types such as granulocytes, T cells, B cells, NK cells, erythrocytes, or any combination thereof, are separated from the regenerative fibroblasts. In some aspects, cell type separation is performed by cell depletion. In some embodiments, fibroblast regenerative cells are enriched by flow cytometry.

[0053] Further embodiments of the present disclosure relate to methods for identifying fibroblast regenerative cells. In some embodiments, a vector containing a fibroblast-specific promoter linked to at least one selectable marker gene is introduced into cells. The selectable marker gene is expressed from the cell-specific promoter in the cells and detected, thereby identifying fibroblast regenerative cells. In some embodiments, the fibroblast regenerative cells do not express at least one of MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD66b, CD73, CD105, and CD90 cell surface proteins. In some embodiments, the fibroblast regenerative cells express at least one of Oct-4, Nanog, Sox-2, Rex-1, GDF-3, Stella, FoxD3, or Polycomb embryonic transcription factor. In some embodiments, the fibroblast regenerative cells do not express CD13, CD44, CD90, or a combination thereof.

[0054] In some embodiments, the vector is a retroviral vector. In some embodiments, the selectable marker gene encodes a fluorescent protein (e.g., green fluorescent protein (GFP)). In some embodiments, the vector comprises two selectable marker genes, wherein the two selectable marker genes comprise fluorescent proteins, proteins sensitive to drug selection, cell surface proteins, or any combination thereof. In some embodiments, the fibroblast-specific promoter is an Oct-4 promoter, a Nanog promoter, a Sox-2 promoter, a Rex-1 promoter, a GDF-3 promoter, aStella promoter, a FoxD3 promoter, a Polycomb repressor complex 2 promoter, or an aCTCF promoter. In some embodiments, the fibroblast-specific promoter is flanked by loxP sites.

[0055] In some embodiments, the fibroblast regenerative cells can differentiate into mesoderm, ectoderm, and / or endoderm. In some aspects, the fibroblast regenerative cells further comprise rhodamine 123 efflux activity. In further aspects, the fibroblast regenerative cells have enhanced expression of GDF-11 compared to a control. In some embodiments, the disclosed methods include transfecting the fibroblast regenerative cells with a transcription factor that can enhance the regenerative activity of the fibroblast regenerative cells. In some embodiments, the fibroblast regenerative cells are transfected with OCT-4 transcription factor. In some embodiments, the regenerative fibroblasts are fused with cells having pluripotent potential, thereby generating fibroblasts with enhanced regenerative activity.

[0056] In some embodiments, the disclosed methods include isolating fibroblast regenerative cells from a mammal. In some embodiments, the fibroblast regenerative cells are derived from a mammalian bodily fluid. In some embodiments, the fibroblast regenerative cells are derived from a mammalian tissue. In some embodiments, the mammal is human. In some embodiments, the fibroblasts are enriched by contacting the cells with a detectable compound that enters the cells, the compound being selectively detectable in proliferating and non-proliferating cells, and proliferating cells are enriched based on detection of the compound. In some embodiments, the detectable compound is carboxyfluorescein diacetate, succinimidyl ester, or Aldefluor®. In some cases, fibroblasts expressing one or more markers can be selected. In some embodiments, fibroblasts expressing CD105 and / or CD117 are selected. Fibroblasts expressing CD105 and / or CD117 may be transfected with the NANOG gene.

[0057] Cells expressing cell surface markers or MHC proteins can be separated or depleted from a population of fibroblasts, thereby isolating a population of stem cells. In some embodiments, the cells to be depleted express MHC class I, CD66b, glycophorin a, or glycophorin b. Cells can be transfected with a stem cell-specific promoter operably linked to a reporter or selection gene. The stem cell-specific promoter can be, for example, the Oct-4, Nanog, Sox-9, GDF3, Rex-1, or Sox-2 promoter.

[0058] V. Disclosure Kit Any cellular and / or non-cellular composition described herein or similar thereto can be included in the kit. In a non-limiting example, one or more reagents for use in a method for preparing fibroblasts can be included in the kit. Such reagents can include cells, vectors, one or more growth factors, one or more costimulatory factors, media, enzymes, buffers, nucleotides, salts, primers, compounds, etc. Kit components are provided in suitable container means.

[0059] Some components of the kit may be packaged either in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container into which the components may be placed, and preferably appropriately aliquoted. Where more than one component is present in the kit, the kit will also generally include a second, third, or other additional container into which the additional components may be separately placed. However, various combinations of components may be included in a single vial. The kits of the present invention will also include a means for containing the components in a confined manner, typically for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials are retained.

[0060] When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may be the container itself, a syringe, pipette, and / or other such device, or may be a substrate having multiple compartments for the desired reactions.

[0061] Some components of the kit may be provided as a dry powder. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kit may also comprise a second container means for containing a sterile acceptable buffer and / or other diluent.

[0062] In certain embodiments, the reagents and materials include primers for amplifying the desired sequence, nucleotides, appropriate buffers or buffering reagents, salts, etc., and in some cases, the reagents include equipment or reagents for the isolation of specific desired cells.

[0063] In certain embodiments, one or more devices suitable for extracting one or more samples from an individual are present in the kit. The device may be a syringe, a fine needle, a scalpel, or the like. [Example]

[0064] The following examples are included to demonstrate specific embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the disclosed methods, and therefore can be considered to constitute preferred modes for their practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the present disclosure.

[0065] Example 1 Stimulation of regenerative growth factor production in fibroblasts by naltrexone in combination with toll-like receptor agonists This example characterizes the use of naltrexone and TLR agonists to stimulate EGF production in foreskin fibroblasts as an example type of fibroblast.

[0066] Neonatal foreskin fibroblasts were obtained from ATCC and cultured in typical DMEM culture medium containing 10% fetal bovine serum and antibiotics. After 3 days of culture, fibroblasts were plated into 12-well plates and cultured at 50% confluence. Naltrexone (Sigma-Aldrich®) and the indicated TLR agonists were added for 12 hours of culture. Pam3CSK4 was added at a total concentration of 1 μg / ml. LPS was added at 0.5 μg / ml. CpG was added at 0.2 μg / ml. EGF concentrations were assessed using ELISA (R&D Systems). The results are shown in Figure 1.

Claims

1. A composition for promoting intervertebral disc regeneration in an individual, the composition being used to provide to the individual an effective amount of fibroblasts pre-cultured with one or more opioid receptor antagonists and one or more toll-like receptor (TLR) agonists.

2. 2. The composition of claim 1, wherein the opioid receptor antagonist is 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, alvimopan, diprenorphine, nalorphine, nalorphine dinicotinate, levallorphan, samidorphan, nalodein, naloxegol, axelopran, benoprand, methylsamidorphan, naldemedine, or a combination thereof.

3. 3. The composition of claim 1 or 2, wherein the TLR agonist is Pam3CSK4, LPS, CpG DNA, poly(ic), flagellin, MALP-2, imiquimod, resmiquimod, zymosan, or a combination thereof.

4. (a) the fibroblasts were cultured in a proliferative state with an opioid receptor antagonist and a TLR agonist; (b) the fibroblasts express a marker selected from the group consisting of Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD344, Stella, and combinations thereof; (c) the fibroblasts express markers selected from the group consisting of CD10, CD13, CD44, CD73, CD90, CD141, PDGFr-α, HLA-A, HLA-B, HLA-C, and combinations thereof; (d) the fibroblasts do not express a marker selected from the group consisting of MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, CD90, and combinations thereof; and / or (e) the fibroblasts do not express a marker selected from the group consisting of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, HLA-DQ, and combinations thereof; The composition according to any one of claims 1 to 3.

5. The composition of any one of claims 1 to 4, wherein the fibroblasts are administered intradiscally or systemically to an individual.

6. A pharmaceutical composition for producing one or more components from fibroblasts, the composition comprising culturing the fibroblasts with one or more opioid receptor antagonists and one or more Toll-like receptor (TLR) agonists.

7. 7. The composition of claim 6, wherein the opioid receptor antagonist is 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, alvimopan, diprenorphine, nalorphine, nalorphine dinicotinate, levallorphan, samidorphan, nalodein, naloxegol, axelopran, benoprand, methylsamidorphan, naldemedine, or a combination thereof.

8. 8. The composition of claim 6 or 7, wherein the TLR agonist is Pam3CSK4, LPS, CpG DNA, poly(ic), flagellin, MALP-2, imiquimod, resmiquimod, zymosan, or a combination thereof.

9. (a) the fibroblasts were cultured in a proliferative state; (b) the fibroblasts express a marker selected from the group consisting of Oct-4, Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, CD105, CD117, CD344, Stella, or a combination thereof; (c) the fibroblasts express markers selected from the group consisting of CD10, CD13, CD44, CD73, CD90, CD141, PDGFr-α, HLA-A, HLA-B, HLA-C, and combinations thereof; (d) the fibroblasts do not express a marker selected from the group consisting of MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, CD90, and combinations thereof; and / or (e) the fibroblasts do not express a marker selected from the group consisting of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, HLA-DQ, and combinations thereof; The composition according to any one of claims 6 to 8.

10. The composition of any one of claims 6 to 9, wherein the fibroblasts do not express a marker selected from the group consisting of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, HLA-DQ, and combinations thereof.

11. The composition of any one of claims 6 to 10, further comprising isolating the fibroblasts from a biological sample from the individual prior to culturing.

12. 1. A pharmaceutical composition for improving the efficacy of tolerogenic therapy, comprising using said composition for: (a) providing tolerance therapy to the individual; and (b) providing the individual with an effective amount of one or more opioid receptor antagonists sufficient to enhance the effectiveness of the tolerance-inducing therapy.

13. The composition of claim 12 , wherein the tolerogenic treatment comprises autoantigen administration.

14. The composition of claim 13 , wherein the autoantigen administration comprises intravenous administration and / or oral administration.

15. 15. The composition of claim 13 or 14, wherein the autoantigen administration comprises providing to an individual: (a) an immature antigen-presenting cell containing an autoantigen; (b) tolerogenic antigen-presenting cells containing an autoantigen; (c) mesenchymal stem cells containing an autoantigen; (d) hematopoietic stem cells containing an autoantigen; (e) allogeneic mesenchymal stem cells, or (f) Combinations of these.

16. The composition of claim 15, wherein the tolerogenic antigen-presenting cells are dendritic cells.

17. 17. The composition of any one of claims 12 to 16, wherein the opioid receptor antagonist is naltrexone, 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, alvimopan, diprenorphine, nalorphine, nalorline dinicotinate, levallorphan, samidorphan, nalodein, naloxegol, axelopran, benoprand, methylsamidorphan, naldemedine, or a combination thereof.

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