Treatment of intervertebral disc degenerative disease, stimulation of protein glycan synthesis using fibroblast conditioning medium, and preparation thereof.

Abstract

In some embodiments, disclosed are methods and compositions for intervertebral disc regeneration and / or repair using one or more components from conditioned medium from fibroblasts. In some cases, the conditioned medium is obtained from fibroblasts stimulated with one or more opioid receptor antagonists and one or more Toll-like receptor agonists. The conditioned medium from the fibroblasts can be provided in an effective amount to an individual in need thereof.

Description

Technical Field

[0001] (Reference to Related Applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 901,164, filed on September 16, 2019, which is hereby incorporated 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 Art

[0003] Intervertebral discs are made from a highly organized matrix of collagen, water, and proteoglycans. Proteoglycan production in intervertebral discs is thought to occur by differentiated chondrocytes. Each intervertebral disc includes a central, highly hydrated gelatinous nucleus pulposus (nucleus) surrounded by an elastic and highly fibrous annulus fibrosus (ring). The cartilaginous endplates provide connection to the vertebrae below and above the intervertebral disc. This buffered arrangement within the intervertebral disc allows the intervertebral disc to facilitate movement and flexibility within the spine while dissipating hydraulic pressure through the spine.

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

Summary of the Invention

[0005] This disclosure relates to methods and compositions for promoting intervertebral disc regeneration and / or repair in an individual. Methods for promoting intervertebral disc regeneration and / or repair in an individual using one or more components derived from stimulated fibroblasts are disclosed herein. In certain embodiments, the compositions of this disclosure include 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 regenerating cells from a cell population and, if necessary, use components in a culture medium in which fibroblasts cultured for therapeutic purposes are present.

[0006] In some embodiments, compositions are provided herein comprising one or more components derived from fibroblasts cultured with one or more opioid receptor antagonists and one or more TLR agonists. One or more components may be derived from a culture medium derived from the fibroblast culture. One or more components may comprise 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), placental growth factor, and hepatocyte growth factor (HGF), and in certain embodiments, one or more growth factors are derived from fibroblasts cultured under specific conditions. In some embodiments, one or more components comprise exosomes. Exosomes may comprise one or more markers (e.g., CD9). In some embodiments, exosomes may bind to dendritic cells and / or mesenchymal stem cells. In a further embodiment, one or more components were derived from fibroblasts cultured with one or more opioid receptor antagonists and one or more TLR agonists in a proliferative state relative to the cells.

[0007] In some embodiments, opioid receptors Antagonist (opioid receptor) Antagonist )The agonist is naltrexone, 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, albimopan, dyprenorphine, nalorphine, nalorphine dinicotinate, levallorphan, samidolphan, nalodeine, naloxegol, axeroplan, bebenoplan, methylsamidolphan, naldemedine, or a combination thereof. In some embodiments, the TLR agonist is Pam3CSK4, LPS, CpG DNA, Poly(ic), flagellin, MALP-2, imiquimod, resmiquimod, thymosan, 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, 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. In some cases, fibroblasts do not express markers derived from MHC class I, MHC class II, CD45, CD13, CD49c, CD66b, CD73, CD105, CD90, and combinations thereof. In some cases, fibroblasts do not express markers selected from the group consisting of CD31, CD34, CD45, CD117, CD141, HLA-DR, HLA-DP, HLA-DQ, and combinations thereof.

[0008] In a further embodiment, a method is provided for promoting intervertebral disc regeneration in an individual, the method comprising providing the individual with 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 one or more opioid receptor antagonists and one or more TLR agonists In advanceThe process includes the step of providing an effective amount of cultured fibroblasts (and / or components derived therefrom) to an individual. Fibroblasts cultured with one or more opioid receptor antagonists and one or more TLR agonists and / or one or more components derived therefrom (e.g., one or more regenerative factors) can be provided to an individual via 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 tolerance-induced therapy, the methods comprising the steps of (a) providing a tolerance-induced 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 tolerance-induced therapy. Tolerance-inducing therapy may include autoantigen administration, which can be administered intravenously and / or orally. In some embodiments, autoantigen administration includes providing immature antigen-presenting cells containing the autoantigen, providing tolerance-induced cells containing the autoantigen, providing mesenchymal stem cells containing the autoantigen, providing hematopoietic stem cells containing the autoantigen, and / or providing allogeneic mesenchymal stem cells. Tolerogenic antigen-presenting cells may optionally be dendritic cells. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows epidermal growth factor (EGF) production from neonatal foreskin cells cultured with naltrexone and the indicated Toll-like receptor (TLR) agonists. [Modes for carrying out the invention]

[0011] I. Definition example In accordance with the long-standing patent law treaties, the terms “a” and “an,” as used herein, together with the words containing the claims, mean “one or more.” As used herein and in the claims, the singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. For example, the term “nucleic acid” includes multiple nucleic acids (including mixtures thereof). Some embodiments of the Disclosure may consist of one or more elements, method steps, and / or methods of the Disclosure, or may consist essentially of them. Any method or composition described herein may be implemented in relation to any other method or composition described herein, and different embodiments may be combined.

[0012] As used herein, the terms “about” or “approximately” refer to a quantity, level, number, frequency, percentage, dimension, volume, weight, or length that varies by approximately 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the reference quantity, level, number, frequency, percentage, size, percentage, dimension, volume, weight, or length. In certain embodiments, when the terms “about” or “approximately” precede a numerical value, it indicates a value that is plus or minus a range of 15%, 10%, 5%, or 1%. With respect to biological systems or processes, the term may mean within the order of the value, preferably within five times, more preferably within two times. Unless otherwise specified, the word “about” means within a tolerance range relative to a particular numerical value.

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

[0014] As used herein, “homogeneous” means tissues, cells, or other substances of another body that originate from one or more individuals of the same species but are immunologically incompatible 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. The tissue transplanted by such a procedure is called an allograft or allograft.

[0016] As used herein, the terms “allo-irritant” and “allo-reactive” mean an allo-antigen, or an “allo-antigen” or cell expressing a heterogeneous HLA haplotype, which is a stimulus and reactant of the immune system in response to such an allo-antigen.

[0017] As used herein, “autologous” means tissue, cells, or other material that originates from or is transferred from the body of the same individual (i.e., autologous blood donation, autologous bone marrow transplant).

[0018] As used herein, the term “autotransplantation” means the transplantation of an organ, tissue, and / or cells from one part of the body in an individual to another part in the same individual (i.e., the donor and recipient are the same individual). Tissue transplanted by such an “auto” procedure is called an autograft or autotransplant.

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

[0020] A "cell culture" is an artificial in vitro system containing viable cells, whether quiescent, senescent, or actively dividing. In cell culture, cells are grown and maintained at a suitable temperature, typically 37°C, and typically in an atmosphere containing oxygen and CO2, although these conditions may be altered in other cases. Culture conditions can vary widely for each cell type, and variations in conditions for a particular cell type can result in different phenotypes being expressed. The most commonly changing factor in a culture system is the growth medium. Growth media can vary in terms of nutrients, the concentration of growth factors, 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 "comprise", "comprising", and "comprises" are understood to imply the inclusion of a stated step or element, or group of steps or elements but not the exclusion of any other step or element, or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are essential or mandatory, and that no other elements are present. "Consisting essentially of" means including any elements listed after the phrase, limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that other elements are not optional and may or may not be present depending on whether they affect the activity or action of the listed 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, and can be a non-peptide, protein or peptide, oligonucleotide, or nucleotide (DNA and / or RNA), polysaccharide or sugar.

[0023] As used herein, the term "subject" refers to a human or an animal, who may or may not be housed in a medical facility and may be treated as an outpatient in a medical facility. A subject may receive one or more pharmaceutical compositions via the Internet. A subject can include any age of a human or non-human animal, and thus includes both adults and infants (i.e., children) as well as neonates. The term "subject" is not intended to mean a need for medical treatment, and thus, an individual may be part of an experiment, either voluntarily or involuntarily, whether clinical or in support of basic science research. The terms "subject" or "individual" refer to any living organism or animal subject that is the subject of a method or material, including mammals such as humans, experimental animals (e.g., primates, rats, mice, rabbits), livestock (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", "related embodiments", "specific embodiments", "additional embodiments", or "further embodiments", or combinations thereof, mean that the particular features, structures, or characteristics described in relation to the embodiment are included in at least one embodiment of the invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the 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 used to combine a plurality of components or to describe them mutually exclusively. 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 specifically, it is 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, when administered to animals or humans, do not produce harmful, allergic, or other adverse reactions.

[0027] As used herein, the term “pharmaceutically acceptable carrier” includes water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and any and all solvents or dispersion media, including but not limited to vegetable oils, coatings, isotonic and absorption retardants, liposomes, and commercially available cleaning agents. Auxiliary bioactive components may 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 “reduced,” “suppressed,” “decrease,” “reduction,” “prevention,” and grammatical equivalents (including “lower,” “smaller,” 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%, at least 25%, at least 50%, at least 75%, and / or at least 90% lower than the amount and / or magnitude of the symptom in the untreated subject.

[0029] A therapeutic agent and its dosage that have therapeutic efficacy in regulating angiogenesis and / or wound healing are said to be an angiogenesis-modulating dose if the dose is sufficient to cause a significant regulation (i.e., increase or decrease) of angiogenesis when administered to a subject (e.g., an animal model or a human patient) that requires regulation of angiogenesis.

[0030] As used herein, the term “therapeutic effective dose” is synonymous with “effective dose,” “therapeutic effective dose,” and / or “effective dose,” and refers to the amount of a compound that elicits a biological, cosmetic, or clinical response in an individual requiring it, as determined by a practitioner. For example, an effective dose is an amount sufficient to reduce the immunogenicity of a group of cells. In a non-limiting example, an effective dose is an amount sufficient to promote the formation of a sufficient blood supply to support transplanted tissue. In another non-limiting example, an effective dose is an amount sufficient to promote the formation of new blood vessels and associated vascular systems (angiogenesis), and / or sufficient to promote the repair or remodeling of existing blood vessels and associated vascular systems. An appropriate effective dose to be administered for a particular application of the disclosed method can be determined by a person skilled in the art using the guidance provided herein. For example, an effective dose can be extrapolated from in vitro and in vivo assays as described herein. A person skilled in the art will recognize that the condition of the individual can be monitored throughout the course of treatment, and that the effective dose of the compound or composition disclosed herein administered can be adjusted accordingly.

[0031] "Treatment," "therapy," or "treatment" refers to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself, rather than merely the symptoms. Treatment can be any reduction from a pre-treatment level and is not limited to the complete ablation of the disease, condition, or symptoms of the disease or condition. Accordingly, 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, a disclosed method for reducing cellular immunogenicity is considered to be a treatment in which there is a detectable reduction in cellular immunogenicity compared to a pre-treatment level in the same subject or control subject. Accordingly, the reduction may be a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, compared to the natural or control level. "Treatment" is understood and intended herein to mean an improvement in the prognosis of a disease or condition, rather than necessarily a cure for the disease or condition. In certain embodiments, treatment may mean a reduction in the severity or degree of at least one symptom, and alternatively or additionally, a delay in the onset of at least one symptom.

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

[0033] In one embodiment, disclosed herein is the use of a culture medium obtained from a tissue culture of fibroblasts or components present therein. In certain embodiments, for use in the disclosed method, the fibroblasts in the culture are in a proliferative state, said proliferative state is described as cells that have not reached confluence. In some embodiments, the fibroblasts are proliferating in a 25% to 75% confluent state. One or more components (e.g., regenerative factors) may be obtained from fibroblasts proliferating in a proliferative state.

[0034] In some embodiments, the use of opioid receptor antagonists as modulators of the immune response to autoantigens by stimulating regulatory cell proliferation through modification 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. AntagonistExamples include, but are not limited to, naltrexone, 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, albimopan, dyprenorphine, nalorphine, nalolin dinicotinate, levallorphan, samidolphan, nalodeine, naloxegol, axeroplan, benoplan, methylsamidolphan, and / or naldemedine. Treatment of fibroblasts with opioid receptor antagonists (e.g., naltrexone) is demonstrated herein to induce the production of various growth factors, including EGF. Furthermore, the upregulation of regenerative factor production is further disclosed by the combination of administration of opioid receptor antagonists (e.g., naltrexone) with agonists 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, TLR-4 antagonists are used to stimulate fibroblasts instead of opioid receptor antagonists. Examples of TLR-4 antagonists that may 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 ivelin from cruciferous vegetables, xanthohumol from hops and beer, and celastrol from Tripterygium wilfordii.

[0035] TLRs can bind to damage-associated molecular patterns (DAMPs) produced under stress, tissue injury, or cellular apoptosis. TLRs are thought to bridge the gap between innate and autoimmune immunity. There are five adapters for TLRs, including MyD88, TRIF, TIRAP / MAL, TRAM, and SARM. When activated, TLRs recruit specific adapters to initiate downstream signaling pathways that lead to the production of inflammatory cytokines and chemokines. Under certain circumstances, TLR binding drives abnormal activation and uncontrolled inflammatory responses, thereby contributing to the persistence of inflammation in autoimmune diseases. While past research has mostly 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 development of autoimmune diseases and offer multiple therapeutic targets [Clin Rev Allergy Immunol. 47(2):136-47 (2014)]. TLR4 is associated with hepatocytes and non-parenchymal cells, including Kupffer cells, bone marrow dendritic cells, astrocytics, T cells, NK cells, and sinusoidal endothelial cells. Recently, some evidence suggests a possible mediation of TLR-4's role in the pathogenesis and progression of autoimmune liver disease (AILD) (He et al. 2006, Longhi et al. 2009, Mencin et al. 2009). Monocytes from PBC patients, when challenged with various ligands, particularly TLR4 and TLR5-mediated signaling molecules, show increased levels of pro-inflammatory cytokines such as IL-1β and IL-6 (Mao et al. 2005). Endogenous DAMP is released following tissue injury. Ligands of TLR-2 and TLR-4, such as heat shock proteins, HMGB1, hyaluronic acid, fibronectin, heparan sulfate, and biglycan, are produced to mediate sterile inflammation (Moreth et al. 2014).The biological characteristics, signaling mechanisms, negative regulators of the TLR2 / 4 pathway, and 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 this disclosure can be obtained from isolated fibroblasts or populations thereof (including from cultures thereof) that can proliferate and differentiate 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 conditional media. The cells may be cultured alone or in the presence of other cells to further upregulate the production of growth factors in the conditional media.

[0037] Fibroblasts can be grown and utilized by administration itself, or cultured in growth medium to obtain a condition medium that can be subsequently used (or its components can be used). The term growth medium generally refers to a medium sufficient for culturing fibroblasts. In particular, one specific medium for culturing cells in the disclosure herein is Dulbecco's Modified Essential Medium (DMEM). In certain embodiments, it is DMEM-Low Glucose (also known here as DMEM-LG) (Invitrogen®, Carlsbad, CA). DMEM-low glucose can be supplemented with 15% (v / v) fetal bovine serum (e.g., Standard Fetal Bovine Serum, Hyclone®, Logan, Utah), antibiotics / antifungal agents (e.g., penicillin (100 units / ml), streptomycin (100 mg / ml), and amphotericin B (0.25 micrograms / ml) (Invitrogen®, Carlsbad, California), 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 indicated as supplements to the growth medium. Also, standard growth conditions are 37°C, 5% This refers to culturing cells in standard air containing CO2, where relative humidity is maintained at approximately 100%. While the aforementioned conditions are beneficial for cell culture, it should be understood that such conditions can be altered by changing the body temperature, CO2, relative humidity, oxygen, growth medium, etc., which are options available to those skilled in the art for culturing cells.

[0038] In certain embodiments, fibroblasts used in the disclosed method for obtaining a conditioning medium and / or regenerating factors may undergo at least 25, 30, 35, or 40 doubling cycles before reaching a senescent state. 14 A method is provided for inducing cells that can double to reach a number of cells greater than or equal to 10. In particular, at least about 10 14 , 10 15 , 10 16 , or 10 17 The above cells were cultured for approximately 103 From about 10 6 Cells / cm 2 This is a method for deriving cells that can be sufficiently doubled to produce when seeded up to a certain number. In specific cases, these cell numbers are produced within 80, 70, or 60 days. In one embodiment, fibroblasts used to produce the leveling medium are isolated and grown to 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 collected from a biopsy, and the donor providing the biopsy may be the same individual being treated (autologous) or a different individual being treated (allogeneic). When allogeneic fibroblasts are used in an individual, the fibroblasts may originate from one or more donors.

[0040] Fibroblasts can be obtained from sources selected from the group consisting of cutaneous fibroblasts, placental fibroblasts, adipose fibroblasts, bone marrow fibroblasts, foreskin fibroblasts, umbilical cord fibroblasts, hair follicle-derived fibroblasts, nail-derived fibroblasts, endometrial-derived fibroblasts, keloid-derived fibroblasts, and combinations thereof.

[0041] In some embodiments, the components obtained from fibroblast culture include one or more regenerative factors. Regenerative factors produced from fibroblasts cultured with opioid receptor antagonists and TLR agonists 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), placental growth factor, and hepatocyte growth factor (HGF). The regenerative factors may be isolated from the cell culture medium before use in the disclosed method (e.g., stimulation of intervertebral disc regeneration or repair). Alternatively, the cell culture medium may be used without isolating the regenerative factors.

[0042] In some embodiments, components obtained from fibroblast culture include exosomes. Fibroblasts may produce exosomes containing one or more regenerative factors (e.g., growth factors), which can be used in disclosed methods (e.g., promoting intervertebral disc regeneration). Exosomes may 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 may be purified and concentrated from fibroblast culture medium. In some embodiments, the exosomes obtained from fibroblasts are 60–200 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 opioid receptor antagonists and TLR agonists can bind to mesenchymal stem cells and, in at least some cases, induce TGF-β production from mesenchymal stem cells.

[0043] The culture medium obtained from fibroblasts can be concentrated by filtration and / or desalting. In one embodiment, an Amicon® filter having a specific molecular weight cutoff, or a substantially equivalent method, is used. The cutoff can be selected to have a molecular weight higher than 1 kDa to 50 kDa.

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

[0045] Before testing the eluate for in vitro activity, methanol is evaporated under nitrogen and replaced with culture medium. The C18 cartridge is used to adsorb small hydrophobic molecules from stem cell culture supernatant or progenitor cell culture supernatant, 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 for carrying out the invention, or it 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. The in vitro bioassay allows for the quantification and knowledge of which molecular weight fractions of the supernatant have biological activity. Bioassays for testing the ability to stimulate proteoglycan synthesis are known in the art. The production of various proteoglycans can be evaluated by analysis of protein content using techniques including mass spectrometry, column chromatography, immuno-based assays such as enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, and flow cytometry.

[0046] Further purification can be performed, 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-swelled in 20 mM Tris-HCl buffer, pH 7.2 (Sigma®), and degassed by gentle swirling under reduced pressure. The Bio-Gel P-2 material is packed into a 1.5 × 54 cm glass column and equilibrated with three columns 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. The fraction 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. Examples of Usage Embodiments of the present invention include means for enhancing the regeneration of one or more degenerated discs 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 fibroblast-derived components may include a conditioning medium from a culture of fibroblasts having one or more opioid receptor antagonists and one or more TLR agonists. The conditioning medium can be used as a source of regeneration factors. The conditioning medium can be concentrated. The fibroblast-derived components can be administered to individuals requiring intervertebral disc regeneration or repair (e.g., individuals with degenerative disc disease). The components can be administered intradiscally or systemically. In some embodiments, microvesicles and / or exosomes derived from stimulated fibroblasts are used as a source of regeneration factors.

[0048] In some embodiments, the disclosure relates to a method for treating or preventing pathological disc disorders by delivering (e.g., secreted, released, 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 the intervertebral disc and can produce one or more regenerative factors that can stimulate disc regeneration. In some embodiments, one or more of these regenerative factors are, in some cases, combined in a medium and delivered to an individual in need. Alternatively, stimulated fibroblasts capable of producing regenerative factors can be delivered directly to the individual.

[0049] Embodiments of this disclosure encompass specific modified culture media, including therapeutic applications. In specific embodiments, the modified culture media are useful for stimulating disc regeneration or repair in individuals, including individuals with or at risk of developing degenerative disc disease (individuals at risk are, for example, individuals over the age of approximately 40, 45, 50, 55, 60, 65, 70, 75, 80, athletes, individuals who are or were athletes, individuals who perform physically demanding work, individuals with spinal cord injuries, or a combination thereof). Thus, in specific embodiments, methods encompassed by this disclosure may be used to prevent or delay the onset and / or reduce the severity of degenerative disc disease.

[0050] The prepared medium can be generated by stimulating fibroblasts, which may be any type of fibroblast. In some embodiments, such stimulation involves treatment with one or more opioid receptor antagonists and one or more TLR agonists in an effective amount sufficient to stimulate the production of regenerative factors (e.g., one or more growth factors) by the fibroblasts. The fibroblasts may be any opioid receptor, including, for example, naltrexone, 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, albimopan, dyprenorphine, nalorphine, nalolin dinicotinate, levallorphan, samidorphan, nalodeine, naloxegol, axeroplan, benoplan, methylsamidorphan, or naldemedine. Antagonist They can be stimulated with naltrexone. In some embodiments, 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. The prepared medium generated from the stimulation of fibroblasts can optionally be concentrated and provided to individuals requiring it.

[0051] In some embodiments, fibroblast conditioning medium is used as part of a formulation with other therapeutic compounds, which are administered intradiscally or systemically to an individual to induce proteoglycan production from the intervertebral disc. The compounds may 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, ω-fatty acids, lithium carbonate, lithium citrate, calcium, or any combination thereof. In some embodiments, the compounds are anti-inflammatory agents. In one aspect, anti-inflammatory agents include alclofenac, alclomethasone dipropionate, algestone acetonide, α-amylase, α-lipoic acid, α-tocopherol, amsinaphal, amsinafid, amfenac sodium, ammyprilose hydrochloride, anakinra, anilorac, anitrazafen, apazon, ascorbic acid, valsalazid disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelain, budesonide, carprofen, lorogenic acid, cycloprofen, syntazone, cliprofen, clobetasol propionate, clobetazone butyrate, clopirac, clotitazone propionate, colmetasone acetate, cortodoxon, deflazacort, desonide, desoxymethasone, dexamethasone dipropionate, diclofenac potassium, diclofenac sodium, and dichlorazon. Cetate, diflumidone sodium, diflunisal, difluprednate, diphthalone, dimethyl sulfoxide, drocinonide, ellagic acid, endrison, enlimomab, enolicum sodium, epirizole, etodolac, etofenamate, felbinac, phenamol, fenbufen, fenclofenac, fenchlorac, fendosal, fenpiparone, fentiazac, flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin, flunixin meglumine, flucortin butyl, fluoromesolone acetate, flucasone,Flurbiprofen, Fluretofen, Fluticasone propionic acid, Flaprofen, Flobufen, Glutathione, Halcionide, Halobetasol propionate, Halopredone acetate, Hesperin, Ibufenac, Ibuprofen, Ibuprofen aluminum, Ibuprofen piconol, Ilonidap, Indoprofen, Indomethacin, Indomethacin sodium, Indoprofen, Indoxol, Intrazol, Isoflupredone acetate, Isoxepak, Isoxicam, Ketoprofen, Lofemizole hydrochloride, Romoxicam, Loteprednol etabonate, Lycopene, Meclofenamate sodium, Meclofenamic acid, Mechryso, Meclofenamate sodium, Meclofenamic acid, Meclolyso, Mefenamic acid, Mesalamine, Mesecrazone, Methylprednisolone Sleptanoic acid, morniflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazon, oleuropein, olsalazine sodium, olgothain, olpanoxin, oxaprozin, oxyfenbutazone, paraoleuropein, olsalazine sodium, olgothain, olpanoxin, oxaprozin, oxyfenbutazone, paraniline hydrochloride, pentosan polysulfate sodium, fenbutazone sodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, pycnogenol, polyphenols, prednazate, priferon, prodolic acid, procazone, proxazole, proxazole citrate, quercetin, resveratrol, rimexolone, romazalit, ro The compound includes one or more of the following: sumaric acid, rutin, sarcorex, sarnadesine, sarsalate, sanguinalium chloride, secrazone, celmethacin, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebuferon, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, tetrahydrocurcumin, totridamine, thiopinac, thixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, didomemethacin, zomepirac sodium. In some embodiments, the compound is a growth factor, cytokine, antibody, antibody fragment,and / or a bioactive compound containing, but not limited to, organic molecules with a mass of less than 5000 daltons. The compound may be administered simultaneously with the composition of this disclosure, or the compound may be administered before and / or after the composition is administered to the subject.

[0052] IV. Obtaining fibroblast regenerative cells Embodiments of this disclosure include methods for obtaining or isolating regenerating fibroblasts. Obtaining regenerating fibroblasts may involve enriching a population of regenerating fibroblasts from tissue having regenerative activity. In some embodiments, regenerating fibroblasts are obtained from tissue having regenerative activity by enriching 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 Stella, 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 isolated from the fibroblast regenerating cells. In some embodiments, cell type isolation is performed by cell depletion. In some embodiments, fibroblast regenerating cells are enriched by flow cytometry.

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

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

[0055] In some embodiments, fibroblast regenerating cells can differentiate into mesoderm, ectoderm, and / or endoderm. In some embodiments, fibroblast regenerating cells further contain rhodamine 123 efflux activity. In further embodiments, fibroblast regenerating cells exhibit enhanced GDF-11 expression compared to a control. In some embodiments, the disclosed method comprises the step of transfecting fibroblast regenerating cells with a transcription factor that can enhance the regenerative activity of the fibroblast regenerating cells. In some embodiments, fibroblast regenerating cells are transfected with the OCT-4 transcription factor. In some embodiments, regenerating fibroblasts are fused with pluripotent cells to generate fibroblasts with enhanced regenerative activity.

[0056] In some embodiments, the disclosed method includes isolating fibroblast regenerative cells from a mammal. In some embodiments, the fibroblast regenerative cells are derived from mammalian body fluids. In some embodiments, the fibroblast regenerative cells are derived from mammalian tissues. 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 the proliferating cells being enriched based on the detection of this compound. In some embodiments, the detectable compound is carboxyfluorescein diacetate, succinimidyl ester, or Aldefluor®. In some cases, fibroblasts expressing one or more markers may 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 isolated 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. The cells can be transfected with a stem cell-specific promoter functionally linked to a reporter or selected gene. The stem cell-specific promoter may be, for example, Oct-4, Nanog, Sox-9, GDF3, Rex-1, or Sox-2 promoters.

[0058] V. Disclosure Kit Any cellular and / or non-cellular compositions described herein or similar thereto may be included in the kit. In non-limiting examples, one or more reagents for use in a method for preparing fibroblasts may be included in the kit. Such reagents may include cells, vectors, one or more growth factors, one or more costimulators, culture media, enzymes, buffers, nucleotides, salts, primers, compounds, and the like. The kit components are provided in appropriate container means.

[0059] Some components of the kit may be packaged in either an aqueous medium or a lyophilized form. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe, or other container in which the components may be arranged and preferably appropriately divided. If the kit contains two or more components, the kit also generally includes a second, third, or other additional container in which the additional components may be arranged separately. However, various combinations of components may be included in a single vial. The kit of the present invention also typically includes means for containing the components in a sealed state for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held.

[0060] If the components of the kit are provided in one or more liquid solutions, the liquid solutions are aqueous solutions, and sterile aqueous solutions are particularly useful. In some cases, the container means may be itself, a syringe, a pipette, and / or other such instrument, or a substrate having multiple compartments for the desired reaction.

[0061] Some components of the kit may be provided as dry powders. If reagents and / or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent. It is assumed that the solvent is also provided in a separate container. The kit may also include a second container for containing sterile, acceptable buffers and / or other diluents.

[0062] In certain embodiments, the reagents and materials include primers, nucleotides, suitable buffers or buffering reagents, salts, etc., for amplifying a desired sequence, and in some cases, the reagents include apparatus or reagents for isolating specific desired cells.

[0063] In certain embodiments, the kit contains one or more devices suitable for extracting one or more samples from an organism. These devices may include syringes, fine needles, scalpels, and the like. [Examples]

[0064] The following embodiments are included to demonstrate specific embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in carrying out the methods of the present disclosure, and can therefore be considered to constitute preferred forms for its implementation. However, those skilled in the art should understand that many modifications can be made in light of the present disclosure to the specific embodiments disclosed, and that similar or analogous results can still be obtained 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 a Toll-like receptor agonist. This embodiment characterizes the use of naltrexone and TLR agonists to stimulate EGF production in precipitous fibroblasts as an example of fibroblast type.

[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, the fibroblasts were plated in 12-well plates and cultured under 50% confluence conditions. Addition of naltrexone (Sigma-Aldrich®) and the indicated TLR agonist was performed for 12 hours of culture. Pam3CSK4 was added at a total concentration of 1 ug / ml. LPS was added at 0.5 ug / ml. CpG was added at 0.2 ug / ml. EGF concentration was evaluated using ELISA (R&D Systems). The results are shown in Figure 1.

Claims

1. A composition for promoting intervertebral disc regeneration in an individual, comprising one or more components derived from fibroblasts pre-cultured with one or more opioid receptor antagonists and one or more Toll-like receptor (TLR) agonists, Herein, the composition comprises one or more opioid receptor antagonists containing naltrexone, and one or more TLR agonists containing Pam3CSK4, LPS, and / or CpG DNA.

2. The composition according to claim 1, further comprising one or more components derived from a culture medium derived from a fibroblast culture.

3. The composition according to claim 1 or 2, further comprising one or more growth factors.

4. The composition according to claim 3, wherein the growth factor is 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), placental growth factor, hepatocyte growth factor (HGF), or a combination thereof.

5. The composition according to any one of claims 1 to 4, further comprising exosomes.

6. The composition according to claim 5, wherein the exosome comprises CD9.

7. The composition according to claim 5 or 6, wherein the exosomes can bind to dendritic cells and / or mesenchymal stem cells.

8. The composition according to any one of claims 1 to 7, wherein the one or more opioid receptor antagonists further comprises 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, albimopane, dyprenorphine, nalorphine, nalolin dinicotinate, levallorphan, samidolphan, nalodeine, naloxegol, axeroplan, benoplan, methylsamidolphan, naldemedine, or a combination thereof.

9. The composition according to any one of claims 1 to 8, wherein the one or more TLR agonists further comprises poly(ic), flagellin, MALP-2, imiquimod, resmiquimod, zymosan, or a combination thereof.

10. (a) The fibroblasts were cultured in advance in a proliferative state together with one or more opioid receptor antagonists and one or more TLR agonists. (b) The fibroblasts express markers 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 any 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 markers 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 9.

11. A pharmaceutical composition for promoting intervertebral disc regeneration in an individual, wherein the composition is used to provide 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 to the individual, Herein, the composition comprises one or more opioid receptor antagonists containing naltrexone, and one or more TLR agonists containing Pam3CSK4, LPS, and / or CpG DNA.

12. The composition according to claim 11, wherein one or more of the components are derived from a culture medium derived from fibroblasts.

13. The composition according to claim 11 or 12, wherein one or more of the components include exosomes.

14. The composition according to claim 13, wherein the exosome contains CD9 and / or can bind to dendritic cells and / or mesenchymal stem cells.

15. The composition according to any one of claims 12 to 14, wherein the one or more opioid receptor antagonists further comprises 6B-naltrexol, nalmefene, naloxone, N-methylnaltrexone, albimopane, dyprenorphine, nalorphine, nalolin dinicotinate, levallorphan, samidolphan, nalodeine, naloxegol, axeroplan, benoplan, methylsamidolphan, naldemedine, or a combination thereof.

16. The composition according to any one of claims 12 to 15, wherein the TLR agonist is Pam3CSK4, LPS, CpG DNA, poly(ic), flagellin, MALP-2, imiquimod, resmiquimod, zymosan, or a combination thereof.

17. (a) The fibroblasts were cultured in a proliferative state together with one or more opioid receptor antagonists and one or more TLR agonists. (b) The fibroblasts express markers 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 any 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 markers 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 12 to 16.

18. The composition according to any one of claims 12 to 17, wherein one or more of the components are administered to an individual intradiscally or systemically.

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