Cannabidiol adjunct therapy for the treatment of degenerative disc disease

Abstract

The use of cannabidiol for use as an adjuvant in the treatment of degenerative disc disease, either by systemic and / or local administration, is disclosed. In some embodiments, cannabidiol is used to enhance the therapeutic and / or regenerative activity of fibroblasts. In other embodiments, cannabidiol is used to increase the regenerative effects of other regenerative cells, including monocytes, mesenchymal stem cells, and / or hematopoietic stem cells. In some embodiments, cannabidiol is used in the culture medium of regenerative cells prior to administration of the cells.

Description

Technical Field

[0001] (Reference to Related Applications) This application claims priority to U.S. Provisional Patent Application No. 62 / 914,523, filed October 13, 2019, which is hereby incorporated by reference in its entirety. (Technical Field) Embodiments of the present disclosure include at least the fields of molecular biology, cell biology, and medicine.

Background Art

[0002] Low back pain is caused by inflammatory events that occur in at least the majority of patients in conjunction with lumbar intervertebral disc degeneration. The association between inflammation and pain has been established in studies showing that radiologically degenerated intervertebral discs are not associated with pain in a large number of subjects. In contrast, the presence of local inflammation can be associated with intervertebral disc degeneration, as exemplified by the presence of granulation tissue, which is thought to be a cause of nociception, and the symptoms of chronic and refractory low back pain (1,2).

[0003] Intervertebral disc degeneration continues to have a major and growing impact worldwide, but current treatment options do not address the underlying causes. Current treatments include bed rest, non-steroidal anti-inflammatory drugs in the early stages of the disease, and surgical procedures such as late-stage discectomy, arthroplasty (joint replacement), injection of artificial nucleus pulposus, and fixation, after pain improvement has not been achieved with previous approaches. Approaches such as those mentioned above are not only unpredictable, but also do nothing to change the disease process itself, as they only address almost end-stage clinical symptoms. Furthermore, procedures such as vertebral fixation lead to an increased incidence of intervertebral disc degeneration in adjacent intervertebral discs due to changes in the biomechanical distribution of the workload.

[0004] In recent years, advances in biotechnology and a better understanding of the biochemical composition and environment of intervertebral discs have increased interest in the degenerative process, raising the potential for developing novel therapies directly aimed at disc preservation. Certain genes that have been shown to significantly influence matrix synthesis and catabolism within the disc have provided targets for scientists seeking to alter the balance between the two. For this purpose, much attention in recent years has centered on gene therapy, and these efforts have yielded promising preclinical results regarding its use in the treatment of intervertebral disc degeneration (3). Unfortunately, none of these approaches are close to clinical implementation at the time of writing. Furthermore, it is important to note that even in situations where only disc regeneration can be achieved through gene therapy or other interventions, the underlying processes that originally caused the degeneration must be addressed to prevent recurrence.

[0005] Currently, there are no widely available biological therapies for intervertebral disc degeneration. However, many different molecules with potential therapeutic benefits are being studied. The focus of molecular therapy has been on preventing or reversing one or more aspects of these changes in the extracellular matrix of the intervertebral disc. At least four different classes of molecules may be effective in intervertebral disc repair. These include anti-catabolics, mitogens, chondroplastic morphogens, and intracellular regulators (4-6).

[0006] Characteristic features of intervertebral disc degeneration include the loss of proteoglycans, water, and type II collagen in the intervertebral disc matrix. Furthermore, qualitative changes in the matrix, including the loss of high molecular weight proteoglycans and other changes that are more difficult to quantify (such as collagen crosslinking and proteoglycan organization), are not very clear. A key process in intervertebral disc degeneration appears to be a shift in the differentiated chondrocyte phenotype in the nucleus pulposus to a more fibrous phenotype. These changes in the intervertebral disc matrix, when combined, ultimately lead to anatomical changes in the intervertebral disc and vertebrae associated with the pathological condition (7, 8).

[0007] Matrix loss is an imbalance between matrix synthesis and degradation; therefore, it is possible to increase the disk matrix by increasing synthesis or by decreasing degradation. One approach is to prevent matrix loss by inhibiting degrading enzymes.

[0008] Degenerated discs show elevated concentrations of matrix metalloproteinases (MMPs). Within the matrix, MMP activity is normally inhibited by tissue inhibitors of MMPs (TIMPs) (9-11). Wallach et al. investigated in vitro whether one of these anti-catabolic molecules, TIMP-1, could increase the accumulation of matrix proteoglycans. The researchers found that TIMP-1 expression in intervertebral disc cells did indeed increase accumulation and also increased the "measuring synthesis rate" of proteoglycans (11). Inflammatory processes are well known to be associated with degenerative disc disease. Multiple studies have shown elevated levels of inflammatory cytokines such as interleukin-1 (12-16), TNF-α (17-20), and interleukin-6 (21) in patients with intervertebral disc degeneration. Inflammatory cytokines help accelerate degeneration through several mechanisms, including stimulation of matrix metalloproteinases (22,23) that disrupt the extracellular matrix (17,24), and stimulation of nitric oxide production that induces apoptosis in nucleus pulposus cells (25,26). It is interesting that cells in degenerated intervertebral discs are hypersensitive to inflammatory cytokines, not only producing inflammatory cytokines themselves, but also producing larger amounts of inflammatory cytokines in response to other inflammatory cytokines. This suggests the existence of a self-amplifying feedback loop (27). The effect of inflammatory cytokines on the production of extracellular matrix proteins is also thought to contribute to degeneration by reducing regenerative activity (28). For example, in one study, researchers generated nucleus pulposus tissue in vitro and treated it with the inflammatory cytokine TNF-α (up to 50 ng / mL) for 48 hours. The tissue was evaluated for histological appearance, proteoglycan and collagen content, and proteoglycan and collagen synthesis. The effect of TNF-α on NP cell gene expression was measured using reverse transcriptase polymerase chain reaction. Proteoglycan degradation was evaluated by immunoblotting.At doses of 1–5 ng / mL, TNF-α induced multiple cellular responses, including: decreased expression of both aggrecan and type II collagen genes; decreased accumulation and overall synthesis of aggrecan and collagen; increased expression of MMP-1, MMP-3, MMP-13, ADAM-TS4, and ADAM-TS5; and induction of ADAM-TS-dependent proteoglycan degradation. Within 48 hours, these cellular responses resulted in NP tissue with only 25% of its original proteoglycan content. These results strongly support the ability of TNF-α to potently inhibit the function of nucleus pulposus cells and block their regenerative capacity (29).

[0009] The importance of inflammatory cytokines in intervertebral disc degeneration is highlighted not only by their direct cause of somatic pathology but also by studies demonstrating their direct pain-inducing effects. For example, Harii et al. used retrograde neuronal tracking and immunohistochemistry to investigate the effects of the TNF-α inhibitor etanercept on calcitonin gene-related peptide (CGRP) expression in dorsal root ganglion (DRG) neurons innervating rat intervertebral discs, a molecular model of pain. Their idea was that lumbar disc degeneration is the cause of low back pain. TNF-α in the intervertebral disc may be a major contributing factor to disc pain. They evaluated the effects of TNF-α inhibition on CGRP expression in DRG neurons. Gold fluoride-labeled neurons innervating the L4 / 5 intervertebral disc were distributed throughout the L1-L6 DRG in all regions. Among FluoroGold-labeled neurons, the proportion of CGRP immunoreactive neurons was 21%±4% in the sham surgery control group, 32%±7% in the puncture + saline group, and 23%±4% in the puncture + etanercept group. The proportion of CGRP immunoreactive neurons was significantly higher in the puncture + saline group compared to the sham control and puncture + etanercept groups. The authors concluded that in this model, CGRP is upregulated in DRG neurons innervating the injured intervertebral disc, and that direct application of etanercept into the intervertebral disc immediately after disc puncture suppresses CGRP expression in DRG neurons innervating the injured intervertebral disc (30). Other studies have reported that, in addition to TNF-α, other inflammatory cytokines such as IL-1 (31) are associated with the induction of direct pain (32-43).

[0010] Regenerative cells, including mesenchymal stem cells, fibroblasts, hematopoietic stem cells, and even immune cells, all require a suitable environment when transplanted to perform their healing functions. This disclosure provides the use of cannabidiol as a means of reducing inflammation and stimulating the efficacy of regenerative cells and / or growth factors when administered to children with intervertebral disc degeneration. [Overview of the project]

[0011] This disclosure relates to methods and compositions for the treatment of degenerative disc disease. Certain embodiments relate to the administration of one or more compositions to an individual who has, is suspected of having, or is at risk of having degenerative disc disease. In some embodiments, the composition administered to the individual comprises any form of cannabidiol (CBD) for therapeutic intervention for degenerative disc disease, which in some embodiments includes being combined with at least one therapeutic or prophylactic intervention for degenerative disc disease. The therapeutic intervention for degenerative disc disease may, for example, include a therapeutically effective dose of fibroblasts.

[0012] Specific embodiments relate to the administration of cannabidiol to an individual. Cannabidiol may be administered to an individual systemically and / or topically (e.g., to the intervertebral disc). The administration of cannabidiol may be given to an individual in a dose and frequency sufficient to reduce apoptosis of cells in the nucleus pulposus, including endogenous and / or exogenous cells in the nucleus pulposus. Exogenous cells in the nucleus pulposus may include, for example, fibroblasts, fibroblasts differentiated into notochord cells, and / or fibroblasts differentiated into chondrocytes, and may include therapeutic interventions for degenerative disc diseases as encompassed herein. Endogenous cells in the nucleus pulposus may include chondrocytes, notochord cells, notochord progenitor cells, chondrocyte progenitor cells, or combinations thereof. Cannabidiol may be administered to an individual daily in any dosage required for a therapeutic effect (including dosages between 50 mg and 500 mg, or any induced range within that range). Cannabidiol may be administered at any frequency required for therapeutic effect, including daily, twice daily, weekly, bi-weekly, monthly, bi-monthly, etc. Cannabidiol may contain (-)-cannabidiol or consist solely of (-)-cannabidiol. Cannabidiol may be included in gel formulations that include penetration-enhancing gels. In some embodiments, cannabidiol may be administered with a localization composition that may contain one or more extracellular matrix molecules, including hyaluronic acid and / or long-chain hyaluronic acid molecules.

[0013] Fibroblasts incorporated herein, including fibroblasts administered to or not administered to an individual, may be manipulated or not. In some embodiments, fibroblasts differentiate into non-fibroblasts such as notochord cells or chondrocytes. In some embodiments, fibroblasts are contacted with cannabidiol, hCG, oxytocin, or a combination thereof, at least in certain cases, to enhance immunomodulatory activity. hCG may be contacted or administered to cells at concentrations of 1 nM to 1 μM, 10 nM to 100 nM, or any induced range thereof per million fibroblasts. Oxytocin may be contacted or administered to cells at concentrations of 1 nM to 10 μM, 100 nM to 1 μM, or any induced range thereof. Fibroblasts may be from one or more tissues selected from the group consisting of adipose tissue, skin tissue, bone marrow, peripheral blood, Wharton's jelly, placenta, amniotic membrane, recruited peripheral blood, and combinations thereof. The fibroblasts described herein can proliferate at a rate of 14 to 21 hours per cell proliferation. The fibroblasts described herein can secrete 0.1 to 77 pg of interleukin-1 per million fibroblast cultures at 75% surface confluence. The fibroblasts described herein can secrete 1 to 500 pg of FGF-1 per million fibroblast cultures at 75% surface confluence. In some embodiments, the fibroblasts can substantially reduce the ability of responsive T cells to proliferate in a mixed lymphocyte response by, for example, more than about 20%.

[0014] In some embodiments, fibroblasts are brought into contact with cannabidiol to increase their regenerative activity. Contact between fibroblasts and cannabidiol can enhance the production of IGF-1 and / or EGF-1 in at least some cases. The cannabidiol may include (-)-cannabidiol or consist solely of (-)-cannabidiol.

[0015] The above provides a fairly broad overview of the features and technical advantages of this disclosure so that the following detailed explanation may be better understood. Additional features and advantages that form the subject matter of the claims of this specification are described below. It should be understood by those skilled in the art that the disclosed concepts and particular embodiments may readily be used as a basis for modifying or designing other structures to accomplish the same purpose of this design. It should also be understood by those skilled in the art that such equivalent configurations will not deviate from the spirit and scope set forth in the appended claims. Novel features that are considered to be features of the designs disclosed herein, along with further purposes and advantages, will be better understood from the following description when considered in conjunction with the appended drawings, both in terms of the configuration and method of operation. However, it should be clearly understood that each drawing is provided for illustrative and explanatory purposes only and is not intended as a definition of the limitations of this disclosure. [Brief explanation of the drawing]

[0016] To fully understand this disclosure, please refer to the following description in conjunction with the attached drawings.

[0017] [Figure 1] Figure 1 shows the stimulation of IGF-1 from fibroblasts by CBD (cannabidiol) at various concentrations. In the three-bar grouping, the middle bar at each concentration represents 100,000 fibroblasts co-cultured with CBD at the indicated concentration. The right bar at each concentration represents 200,000 fibroblasts co-cultured with CBD at the indicated concentration. The left bar at each concentration represents 0 fibroblasts co-cultured with CBD at the indicated concentration.

[0018] [Figure 2]Figure 2 shows the stimulation of EGF-1 from fibroblasts by CBD at various concentrations. In the three-bar grouping, the middle bar at each concentration represents 100,000 fibroblasts co-cultured with CBD at the indicated concentration. The right bar at each concentration represents 200,000 fibroblasts co-cultured with CBD at the indicated concentration. The left bar at each concentration represents 0 fibroblasts co-cultured with CBD at the indicated concentration. [Modes for carrying out the invention]

[0019] In accordance with long-standing patent law treaties, the terms “a” and “an,” as used herein, mean “one or more,” including the claims, and some embodiments of the disclosure may consist of, or essentially consist of, one or more elements, method steps, and / or methods of the disclosure. Any method or composition described herein may be carried out in relation to any other method or composition described herein, and different embodiments may be combined.

[0020] Throughout this specification, unless the context requires otherwise, the terms “to include,” “to include,” and “to include” shall mean the inclusion of the described step or element, or group of steps or elements, but not the exclusion of other steps or elements, or groups of steps or elements. “Consisting of” means, but is not limited to, what follows the term “consisting of.” Thus, the term “consisting of” indicates that the enumerated elements are required or essential, and that other elements are absent. “To be essentially from” means to include any elements enumerated after the phrase, but is limited to other elements that do not interfere with or contribute to the activity or action identified in the disclosure of the enumerated elements. Thus, the term “to be essentially from” indicates that the enumerated elements are required or essential, but other elements are not optional and may or may not be present depending on whether they affect the activity or action of the enumerated elements.

[0021] 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 connection with 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.

[0022] I. Cannabidiol

[0023] As used herein, "cannabidiol" or "CBD" refers to cannabidiol; a cannabidiol prodrug; a pharmaceutically acceptable derivative of cannabidiol (including pharmaceutically acceptable salts of cannabidiol, cannabidiol prodrugs, and cannabidiol derivatives). CBD includes 2-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol, and its pharmaceutically acceptable salts, solvates, metabolites (e.g., skin metabolites), and metabolic precursors. The synthesis of CBD is described, for example, in Petilka et al., Helv. Chim. Acta, 52:1102 (1969), and Mechoulam et al., J. Am. Chem. Soc., 87:3273 (1965), which are incorporated herein by reference.

[0024] Certain embodiments of this disclosure relate to the use of cannabidiol (CBD) as an adjuvant for regenerative cell activation or regenerative cell therapy. In certain embodiments, cannabidiol is administered together with regenerative cells for the treatment of intervertebral disc degeneration or degenerative disc disease. Administration of cannabidiol can be systemic and / or topical. In some embodiments, CBD is used to activate regenerative cells in vitro or ex vivo before transplantation. In certain embodiments, CBD is administered systemically, which may increase the suitability of the microenvironment for maintaining regenerative cell activity, suppress inflammation, and / or directly stimulate regenerative cells to perform healing functions.

[0025] CBD may be synthetic or not. CBD may be purified. CBD may be plant-derived. CBD may be administered transdermally to the upper arm and shoulder of the subject. In some embodiments, CBD is administered transdermally to the thigh or back of the subject. CBD may be administered directly to the intervertebral disc, whether or not it shows signs of degeneration. Topical administration of an effective amount of cannabidiol (CBD) in certain cases may reduce the intensity of at least one adverse event or side effect compared to oral administration of CBD. At least one adverse event or side effect may be a gastrointestinal (GI) adverse event. At least one adverse event or side effect may be an adverse event or side effect affecting liver function. In some embodiments, at least one adverse event is somnolence. In some embodiments, the frequency and intensity of somnolence are reduced as an adverse event by transdermal administration.

[0026] In some embodiments, CBD comprises (-)-CBD or consists of (-)-CBD. The effective amount of CBD (e.g., a therapeutically effective amount) can be from about 50 mg to about 500 mg per day. In some embodiments, the effective amount of CBD that can be administered to an individual is initiated at about 50 mg / day and titrated up to about 500 mg / day. In some embodiments, the effective amount of CBD that can be administered to an individual is initiated at about 50 mg per day and titrated up to about 250 mg per day. In some embodiments, the effective amount of CBD that can be administered to an individual is initiated at 250 mg / day. In some embodiments, the effective amount of CBD that can be administered to an individual is initiated at 500 mg per day. In some embodiments, a daily dose of 500 mg is administered to patients with a body weight over 35 kg. CBD may be administered at a frequency useful for a therapeutic effect, including once-a-day administration or twice-a-day administration. In some embodiments, the effective amount of CBD can be 390 mg in divided daily doses.

[0027] As used herein, the term “transdermally” means, in the context of administration, bringing CBD into contact with the skin of a patient or subject under conditions that are effective for CBD to penetrate the skin. In certain embodiments, CBD is formulated as a gel or oil. In some embodiments, CBD is formulated as a penetration-enhancing gel. The gel may contain 1% (wt / wt) to 7.5% (wt / wt) of CBD. In some embodiments, the gel contains 4.2% (wt / wt) of CBD. In some embodiments, the gel contains 7.5% (wt / wt) of CBD. In some embodiments, the transdermal preparation may be a cream, ointment, or cream. CBD can be delivered by bandage, pad, or patch. CBD may be in gel form and may be pharmaceutically produced as a clear, penetration-enhancing gel designed to provide transdermally controlled drug delivery with once or twice daily dosing. CBD gels may contain between 1% (wt / wt) CBD and 7.5% (wt / wt) CBD, or any achievable range within that range. For example, a CBD gel could have 4.2% (wt / wt) CBD or 7.5% (wt / wt) CBD. The CBD gel may be applied topically by the patient or caregiver to the patient's upper arm and shoulder, back, thigh, or any combination thereof. The CBD gel may contain diluents and carriers, as well as other conventional excipients such as wetting agents, preservatives, and suspending and dispersing agents.

[0028] CBD gels may contain solubilizers, osmosis enhancers, antioxidants, thickeners, and / or pH adjusters. A composition of a CBD gel may, for example, consist of cannabidiol present in an amount of about 0.1% to about 20% (wt / wt) of the composition; a lower alcohol having 1 to 6 carbon atoms present in an amount of about 15% to about 95% (wt / wt) of the composition; a first osmosis enhancer present in an amount of about 0.1% to about 20% (wt / wt) of the composition; and a sufficient amount of water to bring the composition to a total of 100% (wt / wt). Other formulations of CBD gels can be found in International Publication No. WO2010 / 127033, the full contents of which are incorporated herein by reference.

[0029] In some embodiments, cannabinoids (e.g., cannabidiol or prodrugs of cannabidiol) are delivered systemically to achieve therapeutically effective plasma concentrations in an individual. However, oral cannabinoid formulations, including those containing cannabiol, must overcome several obstacles to achieving therapeutically effective systemic concentrations. Firstly, cannabinoids are generally highly lipophilic. Their limited water solubility thereby restricts the amount of cannabinoid available for absorption in the gastrointestinal tract. Secondly, cannabidiol, like other cannabinoids, undergoes substantial first-pass metabolism once absorbed from the human gastrointestinal tract. Finally, if the patient avoids taking the oral medication or suffers nausea or vomiting because the oral dosage form does not remain in the gastrointestinal tract long enough to release the full dose and achieve therapeutic concentrations, the oral bioavailability of any product is further reduced. Therefore, considering the above, it is desirable to deliver a therapeutically effective amount of cannabinoid, such as cannabinol or cannabinol prodrug, systemically to the mammal in need, via a route of administration that does not depend on absorption from the mammalian gastrointestinal tract, for the treatment of one or more medical conditions that respond to cannabinoids (including pain, degenerative disc disease, nausea, or appetite stimulation).

[0030] One parenteral administration route for systemic delivery of cannabidiol is transdermal administration. In addition, the epidermis and dermis of many mammals, such as humans and guinea pigs, contain enzymes that can metabolize active drugs that pass through the stratum corneum. The metabolic processes occurring in the skin of mammals such as humans can be utilized to deliver pharmaceutically effective amounts of cannabinoids, such as cannabinol, into the systemic circulation of mammals that need it. Cannabinoid prodrugs (e.g., cannabidiol prodrugs) and compositions containing cannabinoid prodrugs that can be administered transdermally to mammals such as humans, and as a result, metabolites resulting from metabolism in the skin are cannabinoids that are systemically available for the treatment of medical conditions that respond to cannabinoids, are included herein. Unfortunately, due to its highly lipophilic nature, cannabidiol is hardly absorbed through membranes such as the skin of mammals, including humans. Therefore, the success of administering therapeutically effective amounts of cannabidiol transdermally to mammals that need it, within a reasonable timeframe and over an appropriate surface area has been substantially limited.

[0031] In some embodiments, cannabidiol is administered topically to an individual, for example, to the intervertebral disc of the individual. Cannabidiol may be administered topically to an individual at any dosage and frequency, including the dosages and frequencies incorporated herein. Topically administered cannabidiol may be administered together with one or more other compositions, including cell therapies incorporated herein. In some embodiments, cannabidiol administered to the intervertebral disc in an individual reduces apoptosis of cells including the intervertebral disc of the individual. The cells including the disc may be endogenous (including notochord cells, notochord progenitor cells, chondrocytes, and / or chondrocyte progenitor cells) and / or exogenous cells including any cell therapy administered to the individual.

[0032] In some embodiments, the administration of cannabidiol to an individual is carried out in conjunction with other agents that inhibit inflammation. In one embodiment, the administration of interleukin-1 (IL-1) receptor antagonists, including proteins, genes, and / or mRNA, is carried out to reduce several aspects of inflammation, which unexpectedly synergizes with the administration of cannabidiol.

[0033] In some embodiments, one or more treatments for degenerative disc disease target Cannabidiol or cannabidiol with intervention is administered together with a localization composition. The localization composition may be one or more extracellular matrix molecules containing hyaluronic acid and / or long-chain hyaluronic acid.

[0034] In some embodiments, cannabidiol is brought into contact with, cultured with, or administered to cells such as fibroblasts that can increase regenerative activity in the cells. Cells, including fibroblasts, that have been brought into contact with, cultured with, or administered cannabidiol (containing (-)-cannabidiol or cannabidiol consisting of (-)-cannabidiol) may increase the production of IGF-1 and / or EGF-1. Fibroblasts that have been brought into contact with, cultured with, or administered cannabidiol (containing (-)-cannabidiol or cannabidiol consisting of (-)-cannabidiol) may be used for methods incorporated herein. Fibroblasts that have been brought into contact with, cultured with, or administered cannabidiol (containing (-)-cannabidiol or cannabidiol consisting of (-)-cannabidiol) may be administered topically (including intradiscally) and / or systemically to individuals, including individuals with or at risk of developing degenerative disc disease.

[0035] II. Treatment for degenerative intervertebral disc disease target intervention

[0036] Certain embodiments of this disclosure may include one or more cell therapies for degenerative disc disease, including a variety of cell therapies, including regenerative cells, such as fibroblasts, mesenchymal stem cells, hematopoietic stem cells, and other cells having the ability to heal tissue. target Regarding the use of the intervention: In certain embodiments, fibroblasts are administered to the individual together with cannabidiol. The fibroblasts may be of any autologous, allogeneic, or heterogeneous source. The fibroblasts may be from tissues selected from the group consisting of adipose tissue, skin tissue, bone marrow, peripheral blood, Wharton's jelly, placenta, amniotic membrane, mobilized peripheral blood, and combinations thereof.

[0037] In certain embodiments, fibroblasts, including engineered or unengineered fibroblasts, are delivered topically to individuals, such as those requiring treatment for degenerative disc disease, for example, to the intervertebral disc. The fibroblasts can be administered as a formulation containing a hydrogel. The hydrogel may consist, for example, of platelet-rich plasma (PRP) and hyaluronic acid (HA) blended with batroxobin (BTX) as a gelling agent. The fibroblasts may be encapsulated in the PRP / HA / BTX hydrogel and cultured for up to 21 days in both growth medium and medium containing or not containing TGF-β1. In one embodiment, the hydrogel is gelled at 20°C for 15 minutes and 37°C for 3 minutes to maintain high cell viability and proliferation of the fibroblasts. In one embodiment, the fibroblasts are used for intradiscal injection in patients with degenerative disc disease. In such embodiments, the fibroblasts are cultured with means to enhance GAG ​​production, which can be achieved by culture with cytokines (e.g., TGF-β). A methodology for the proliferation of mesenchymal stem cells, which may be modified for fibroblast-specific use, is provided below and is incorporated by reference (44).

[0038] Certain embodiments relate to the activation of fibroblasts prior to therapeutic use and / or the administration of a drug acting as a “regenerative adjuvant” for fibroblasts. Cells in the formulation may exhibit typical fibroblast morphology when grown in a culture monolayer. Specifically, cells may exhibit an elongated spindle-shaped or spindle-shaped appearance with elongated extensions, or cells may appear as larger, flatter astrocytes with a cytoplasmic anterior margin. Mixtures of these morphologies can also be observed. Cells may express proteins characteristic of normal fibroblasts, including the fibroblast-specific marker, CD90(Thy-1), a 35kDa cell surface glycoprotein, and extracellular matrix proteins, including collagen. Fibroblast dosing formulations may be autologous cell therapeutic products consisting of a suspension of autologous fibroblasts grown from biopsies of each individual’s own skin using standard tissue culture procedures. In certain embodiments, fibroblasts may also be used to produce other cell types for tissue repair or regeneration.

[0039] Fibroblasts incorporated herein may be generated by proliferation from a biopsy of skin (in the case of an autologous preparation) of an individual receiving the incorporated herein or skin (in the case of an allogeneic preparation) of a healthy donor. While skin-derived fibroblasts are disclosed as an example, fibroblasts incorporated herein may originate from any source or tissue. In some embodiments, fibroblasts are used from young donors. In another embodiment, fibroblasts are transfected with genes that enable enhanced proliferation and overcoming the Hayflick limit, following induction of cell proliferation in culture using standard cell culture techniques. The techniques include expansion in a liquid medium, and the use of media such as RPMI 1640, DMEM, OPTI-MEM, or AIM-V. Standard techniques for tissue culture are well known in the art and include the need to dissociate cells. In some embodiments, dissociation includes treatment with trypsin or collagen to enable cell passage. Skin tissue (dermis and epidermal layers) can be biopsied from the postauricular region of a subject. In one embodiment disclosed for the purpose of illustrating a non-limiting example, the starting material consists of three 3 mm punch skin biopsy materials collected using a standard sterile practice. The biopsy materials are collected by persons skilled in the art and placed in vials containing sterile phosphate-buffered saline (PBS). The biopsy materials are shipped at 2–8°C. The refrigerated shipper returns to the manufacturing facility. In one embodiment, after arrival at the manufacturing facility, the biopsies are inspected and, upon acceptance, transferred directly to the manufacturing area. At the start of the process, the biopsy tissue is then washed before enzymatic digestion. After washing, liberase digestion enzyme solution is added without mincing, and the biopsy tissue is incubated at 37.0+ / -2.0°C for 1 hour. The time of biopsy tissue digestion is a critical process parameter that can affect the viability and growth rate of cells in culture. Liberase is a collagenase / neutral protease enzyme cocktail prescribed by Lonza Walkersville, Inc. (Walkersville, Md) and obtained unprescribed from Roche Diagnostics Corp. (Indianapolis, Ind.).Alternatively, other commercially available collagenases (e.g., Serva Collagenase NB6 (Helidelburg, Germany)) may be used. After digestion, neutralize the enzyme by adding initiation growth medium (IMDM, GA, 10% fetal bovine serum (FBS)), pelletize the cells by centrifugation, and resuspend them in 5.0 mL of initiation growth medium. Alternatively, centrifugation may be omitted, and complete inactivation of the enzyme may occur by adding only the initiation growth medium. To initiate cell growth and growth, add the growth initiation medium before seeding the cell suspension into a T-175 cell culture flask. T-75, T-150, T-185, or T-225 flasks can be used instead of T-75 flasks. Incubate the cells at 37+ / 2.0°C. Feed with fresh complete grow media at 5.0+ / 1.0% CO2 approximately every 3-5 days. All feeding in this process is done by removing half of the complete growth medium and replacing it with the same volume of fresh medium. Alternatively, a complete supply can be provided. Cells should not remain in the T-175 flask until approximately 30 days before subculturing. Confluence is monitored throughout the process to ensure an appropriate seeding density during culture division. If cell confluence is 40% or higher in the T-175 flask, the cells are subculturised by removing the used medium, washing the cells, and treating them with trypsin-EDTA to release adherent cells in the flask into solution. The cells are then trypsinized and seeded into a T-500 flask to continue cell proliferation. Alternatively, one or two T-300 flasks, one layer cell stack (1CS), one layer cell factory (1CF), or two layer cell stacks (2CS) can be used instead of a T-500 flask.

[0040] Morphology is evaluated before each passage and harvest to monitor culture purity throughout the process. Morphology is evaluated by comparing the observed sample to a visual standard for morphological testing of cell cultures. When cells grow in a cultured monolayer, they exhibit typical fibroblast morphology. Cells may appear as elongated spindle-shaped or spindle-shaped cells with elongated extensions, or as larger, flatter astrocytes that may have an anterior cytoplasmic margin. Mixtures of these morphologies can also be observed. Fibroblasts in non-confluent regions may have similar shapes but may be randomly oriented. The presence of keratinocytes in the cell culture is also evaluated. Keratinocytes appear round and irregular in shape, and at higher confluence, they appear organized into cobblestone formations. At lower confluence, keratinocytes are observable in small colonies. Cells are incubated at 37+ / 2.0°C. Cells were subcultured in T-500 flasks every 3–5 days and in 10-layer cell stacks (10CS) every 5–7 days at 5.0+ / 1.0% CO2. Cells should not remain in T-500 flasks for more than 10 days prior to subculturing. Quality control (QC) release testing for the safety of bulk active pharmaceutical ingredient (cells) includes sterility testing and endotoxin testing. If the cell confluence in the T-500 flask is >95%, the cells are subcultured into 10CS culture vessels. Alternatively, two 5-layer cell stacks (5CS) or 10-layer cell factories (10CF) can be used instead of 10CS. Passage to 10CS is performed by removing used medium, washing the cells, and treating them with trypsin-EDTA to release adherent cells in the flask into solution. The cells are then transferred to 10CS. Add additional complete growth medium to neutralize the trypsin, and pipette the cells from the T-500 flasks into a 2L bottle containing fresh complete growth medium. Transfer the contents of the 2L bottle to 10CS and seed all layers. Then incubate the cells at 37+ / 2.0°C. Feed with fresh complete grow media every 5-7 days at 5.0+ / 1.0% CO2. Cells should not remain in 10CS for more than 20 days before subculturing.In one embodiment, passaged dermal fibroblasts are incubated in a protein-free medium for a certain period of time to substantially eliminate immunogenic proteins present in the culture medium, and cells are harvested when the cell density in the primary harvest (10 CS) is 95% or higher. The harvest is performed by removing the used medium, washing the cells, treating them with trypsin-EDTA to release adherent cells into solution, and neutralizing the trypsin by adding additional complete growth medium. The cells are collected by centrifugation, resuspended, and then subjected to in-process QC testing to determine the total viable cell count and cell viability.

[0041] In some embodiments, if a large number of cells are required after receiving the cell counting results from the primary 10CS collection, further subculturing into multiple cell stacks (up to 4 10CS) is performed. For further subculturing, cells from the primary collection are added to a 2L medium bottle containing fresh complete growth medium. The resuspended cells are added to multiple cell stacks and incubated at 37+ / 2.0°C in 5.0+ / 1.0% CO2. The cell stacks are supplied and collected as described above, except that the cell density must be 80% or more before cell collection. The collection procedure is the same as that described for the primary collection above. Mycoplasma samples are collected from the cells and used medium, and cell counting and viability are performed as described for the primary collection above. This method reduces or eliminates immunogenic proteins by avoiding the introduction of immunogenic proteins from animal-derived reagents. To reduce process residues, cells are cryopreserved in protein-free freezing medium, then thawed, washed, and then pre-treated for final injection to further reduce residues. After further cell collection and cryopreservation from subsequent passages are complete, if additional drug material is needed, thaw the aliquot vials of the frozen drug material and use them to seed into 5CS or 10CS culture vessels. Alternatively, instead of 5CS or 10CS, a 4-layer cell factory (4CF), two 4CFs, or two 5CS can be used. Thaw the frozen vials of cells, wash them, add them to a 2L medium bottle containing fresh complete growth medium, culture as described above, harvest, and cryopreserve. The cell density in the cell suspension should be 80% or higher before cell collection.

[0042] Upon completion of culture and growth, the cells are harvested, washed, and then 2.2 × 10⁶ 7 100,000 cells with a target of cells / mL or more ~2.7×10 7Formulate to contain cells / mL or more. Alternatively, the target can be adjusted within the formulation range to adapt to different dosages. The active pharmaceutical ingredient contains a population of viable autologous human fibroblasts suspended in cryopreservation medium consisting of Iscove's Modified Dulbecco's Medium (IMDM) and Profreeze-CDM® (Lonza, Walkerville, Md) + 7.5% dimethyl sulfoxide (DMSO). Alternatively, a lower DMSO concentration may be used instead of 7.5%, or CryoStor® CS5 or CryoStor® CS10 (BioLife Solutions, Bothell, WA) may be used instead of IMDM / Profreeze / DMSO. In addition to cell count and viability, purity / identity of the drug substance must be performed to ensure that the suspension contains 98% or more fibroblasts. Common cell contaminants include keratinocytes. The purity / identification assay uses fluorescently tagged antibodies against CD90 and CD104 (cell surface markers for fibroblasts and keratinocytes, respectively) to quantify the percentage purity of the fibroblast population. CD90 (Thy-1) is a 35 kDa cell surface glycoprotein. Antibodies against the CD90 protein have been shown to exhibit high specificity for human fibroblasts. CD104, an integrin β4 chain, is a 205 kDa transmembrane glycoprotein that binds to an integrin α6 chain (CD49f) to form an α6 / β4 complex. This complex has been shown to act as a molecular marker for keratinocytes (Adams and Watt 1991).

[0043] Antibodies against the CD104 protein bind to 100% of human keratinocyte cells. Cell count and viability are determined by incubating the sample with Viacount Dye Reagent and analyzing the sample using the Guava PCA system. This reagent consists of two dyes: a membrane-permeable dye that stains all nucleated cells, and a membrane-impermeable dye that stains only damaged or dead cells. The use of this dye combination allows the Guava PCA system to estimate the total number of cells present in the sample and to determine which cells are viable, apoptotic, or dead. This method was specifically custom-developed for use in determining the purity / identity of autologous cultured fibroblasts.

[0044] Alternatively, cells can be subcultured from either a T-175 flask (or its substitute) or a T-500 flask (or its substitute) into a spinner flask containing microcarriers as a cell growth surface. Microcarriers are small, bead-like structures used as a growth surface for anchorage-dependent cells in suspension culture. They are designed to yield large cell yields with small quantities. In this setup, 50 mL to 300 mL of complete growth medium is added to a 500 mL, IL, or 2 L sterile disposable spinner flask. Sterile microcarriers are added to the spinner flask. The culture medium is allowed to stand for a short period (1-24 hours) in a 37+ / -2.0°C, 5.0+ / - 0.0% CO2 incubator, or placed on a starting plate at a low rotation speed (15-30 RRM) to allow cells to attach to the carriers. After the attachment period, the rotation speed of the spin plate is increased (30-120 RPM). Cells are supplied with new complete culture medium every 1-5 days, or when the medium color changes and it appears to be used. Cells are collected at regular intervals by sampling microcarriers, separating cells, and performing cell count and viability analysis. The cell concentration per carrier is used to determine when to scale up the culture. Once sufficient cells have been produced, wash with PBS, collect cells from the microcarriers using trypsin-EDTA, and reseed into spinner flasks with a larger volume of microcarriers and a larger volume of complete growth medium (300 mL-2 L). Alternatively, additional microcarriers and complete growth medium can be added directly to a spinner flask containing an existing microcarrier culture, allowing cells to be transferred directly from bead to bead without trypsin treatment and reseeding. Alternatively, if sufficient cells have been produced from the initial T-175 or T-500 flask, the cells can be directly seeded into the scaled-up volume of microcarriers. After the adhesion period, increase the spin plate speed (30-120 RPM). Supply the cells with new Complete Growth Media every 1 to 5 days, or when the color of the medium changes and it is no longer needed.Once the concentration reaches the cell count required for the target indication, wash with PBS and collect the cells using trypsin-EDTA. Microcarriers used in disposable spinner flasks may be made from polyblendes such as BioNOC II® (Cesco Bioengineering, Bellco Biotechnology, Vineland, NJ) or FibraCel® (New Brunswick Scientific, Edison, NJ), gelatin such as Cultispher-G (Percell Biolytica, Astrop, Sweden), cellulose such as Cytopore® (GE Healthcare, Piscataway, NJ), or coated / uncoated polystyrene such as 2D MicroHex® (Nunc, Weisbaden, Germany), Cytodex® (GE Healthcare, Piscataway, NJ), or Hy-Q Sphere® (Thermo Scientific Hyclone, Logan, Utah).

[0045] In another embodiment, instead of a spinner flask apparatus, an automated bellows system such as FibraStage® (New Brunswick Scientific, Edison, NJ) or BelloCell® (Cesco Bioengineering, Distributed by Bellco Biotechnology, Vineland, NJ) can be used to process cells on polyblend 2D microcarriers such as BioNOC II® or FibraCel®. Cells in a T-175 (or alternative) or T-500 (or alternative) flask are subcultured into a bellows bottle containing microcarriers with an appropriate amount of complete growth medium and placed in the system. The system repeatedly supplies medium onto the microcarriers and drains the medium to supply oxygen in a fixed cycle. Cells are monitored, supplied, washed, and harvested in the same order as described above. Alternatively, cells can be processed using automated equipment. After digestion of biopsy tissue or after the first passaging is complete (T-175 flask or alternative), cells can be seeded into automated equipment. One method is the ACE (Automated Cellular Expansion) system, which connects a series of commercially available or custom-made components to form a cell proliferation platform in which cells can be grown without human intervention. Cells are expanded in a cell tower consisting of stacks of discs that support anchorage-dependent cell adhesion. After cell proliferation is complete, the culture medium is automatically circulated and the cells are harvested by trypsin treatment.

[0046] Alternatively, the ACE system can be reduced to a single-lot unit version consisting of disposable components comprising a cell growth surface, delivery tubes, culture medium and reagents, and a permanent base housing mechanisms and computer processing capabilities for heating / cooling, medium transfer and execution of automated programming cycles. Upon receipt, each sterile irradiation ACE disposable unit is opened from its packaging, loaded with culture medium and reagents by suspending a pre-filled bag and connecting the bag to an existing tube via a sterile connector. This process proceeds as follows: a) Inside a biological safety cabinet (BSC), a suspension of cells from an enzymatically digested biopsy is introduced into a “pre-growth chamber” (a small unit at the top of the cell tower) already filled with initiation growth medium containing antibiotics. From the BSC, the disposables are transferred to the permanent ACE unit, which is already in place; b) After approximately 3 days, the cells in the pre-growth chamber are trypsinized and introduced into the cell tower itself, which is pre-filled with complete growth medium. Here, the "foaming effect" caused by the CO2 injection circulates the medium at a rate such that the cells rotate spirally downwards and settle on the surface of the disk in a uniformly distributed manner; c) The cells are able to grow for about 7 days. At this time, confluence is checked (method of writing is unknown) to confirm that the culture is growing. Also at this point, the complete growth medium is replaced with fresh complete growth medium. The CGM is changed every 7 days for 3-4 weeks. At the end of the culture period, confluence is checked again to confirm that there is enough growth to probably produce the desired amount of cells for the intended processing; d) If the culture is sufficiently confluent, it is collected. The used medium (supernatant) is drained from the container. PBS (FBS from cells to wash the medium) is then pumped into the container and drained almost immediately. Trypsin-EDTA is pumped into the container to separate the cells from the growth surface. The trypsin / cell mixture is drained from the container and enters the spindle separator. The cryopreservation agent is pumped into the container to rinse off any remaining cells from the surface of the disc and also into a spin separator. The spin separator collects the cells and then uniformly resuspends them in the transport / injection medium.From the spin separator, the cells are transported through an in-line automated cell counting device or through a sample collected for cell counting and viability testing by laboratory analysis. Once a certain number of cells have been counted and the appropriate cell concentration has been reached, the collected cells are delivered to a collection vial from which the sample can be aliquoted for cryopreservation.

[0047] In some embodiments, fibroblasts are cultured with or administered with hCG and / or oxytocin, which can enhance immunomodulatory activity. Fibroblasts may be cultured with or administered with hCG at concentrations of 1 nM to 1 μM, 10 nM to 100 nM, or any induced range within that range. Fibroblasts may be cultured with or administered with oxytocin at concentrations between 1 nm and 10 μM, or between 100 nm and 1 μM, or any induced range within that range. [Examples]

[0048] The following embodiments are included to illustrate preferred specific examples of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in carrying out the present invention and can therefore be considered to constitute preferred forms for carrying out the present invention. However, those skilled in the art will understand that, considering the disclosure of the present invention, many modifications are possible in the specific embodiments disclosed and that yield similar results without departing from the spirit and scope of the present invention.

[0049] Example 1 Cannabidiol stimulates fibroblast production of IGF-1 and EGF-1. Skin fibroblasts were obtained from Allcells, Inc. and cultured in CBD at the specified concentration for 48 hours. As shown in Figure 1, an increase in IGF-1 was observed. As shown in Figure 2, an increase in EGF-1 was observed. The culture was performed in OPTI-MEM medium containing 10% fetal bovine serum. References All publications, patents, and patent applications referenced herein are incorporated by reference in whole, as if each individual publication, patent, or patent application were specifically and individually indicated so as to be incorporated by whole. 1. Hwang, GJ, Suh, JS, Na, JB, Lee, HM, and Kim, NH (1997) Contrast enhancement pattern and frequency of previously unoperated lumbar discs on MRI. J Magn Reson Imaging 7, 575-578 2. Peng, B., Wu, W., Hou, S., Li, P., Zhang, C., and Yang, Y. (2005) The pathogenesis of discogenic low back pain. J Bone Joint Surg Br 87, 62-67 3. 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[0050] While the present disclosure and its merits have been described in detail, it should be understood that various modifications, substitutions, and changes can be made herein without departing from the spirit and scope of the design defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to specific embodiments of the processes, machines, manufactures, compositions, means, methods, and steps described herein. As will be readily apparent to those skilled in the art from this disclosure, existing or subsequently developed processes, machines, manufactures, compositions of materials, means, methods, or steps that perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with this disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions, means, methods, or steps.

Claims

1. A composition for treating intervertebral disc degenerative disease in an individual, comprising cannabidiol and a therapeutically effective amount of dermal-derived fibroblasts.

2. The composition according to claim 1, wherein cannabidiol and dermal-derived fibroblasts are administered to an individual simultaneously or at different times.

3. The composition according to claim 2, wherein cannabidiol is administered before, during, or after administration of dermal-derived fibroblasts.

4. The composition according to any one of claims 1 to 3, wherein dermal-derived fibroblasts are administered in a therapeutically effective amount.

5. The composition according to any one of claims 1 to 4, wherein the dermal fibroblasts are differentiated into notochord cells, chondrocytes, or a mixture thereof.

6. The composition according to any one of claims 1 to 5, wherein the dermal fibroblasts are derived from skin tissue.

7. The composition according to any one of claims 1 to 6, wherein cannabidiol is administered systemically.

8. The composition according to any one of claims 1 to 6, wherein cannabidiol is administered topically to the intervertebral disc of an individual.

9. The composition according to any one of claims 1 to 8, wherein cannabidiol is administered in a dose and frequency sufficient to reduce apoptosis of cells in the nucleus pulposus.

10. The composition according to claim 9, wherein the cells in the nucleus pulposus are selected from the group consisting of chondrocytes, notochord cells, notochord progenitor cells, chondrocyte progenitor cells, and combinations thereof.

11. The composition according to claim 9, wherein the cells in the nucleus pulposus include cells administered exogenously.

12. The composition according to any one of claims 9 to 11, wherein the cells in the nucleus pulposus include dermal-derived fibroblasts.

13. The composition according to any one of claims 4 to 12, wherein the dermal-derived fibroblasts are proliferating at a rate of 14 to 21 hours per cell proliferation.

14. The composition according to any one of claims 4 to 13, wherein dermal-derived fibroblasts secrete 0.1 pg to 77 pg of interleukin-1 per 1 million fibroblast cultures at a surface confluence of 75%.

15. The composition according to any one of claims 4 to 13, wherein dermal-derived fibroblasts secrete 1 pg to 500 pg of FGF-1 per 1 million fibroblast cultures at a surface confluence of 75%.

16. The composition according to any one of claims 4 to 15, wherein dermal-derived fibroblasts have reduced ability to respond to proliferating T cells in a mixed lymphocyte reaction.

17. The composition according to claim 16, wherein the reduction in proliferation includes a reduction of more than 20% compared to a control mixed lymphocyte response in which dermal-derived fibroblasts were not added.

18. A composition according to any one of claims 4 to 17, comprising treating dermal-derived fibroblasts with human chorionic gonadropine (hCG) to increase immunomodulatory activity.

19. The composition according to claim 18, wherein the hCG is administered to cells at a concentration of 1 nM to 1 μM.

20. The composition according to claim 18, wherein the hCG is administered to cells at a concentration of 10 nM to 100 nM.

21. The composition according to any one of claims 4 to 20, wherein the fibroblasts are treated with oxytocin to enhance immunomodulatory activity.

22. The composition according to claim 21, wherein the oxytocin is administered to cells at a concentration of 1 nM to 10 μM.

23. The composition according to claim 21, wherein oxytocin is administered to cells at a concentration of 100 nM to 1 μM.

24. The composition according to any one of claims 1 to 23, comprising cannabidiol (-)-cannabidiol.

25. The composition according to any one of claims 1 to 23, wherein the cannabidiol comprises (-)-cannabidiol.

26. The composition according to any one of claims 1 to 25, wherein the effective amount of cannabidiol is approximately 50 mg to approximately 500 mg / day in total.

27. Cannabidiol is included in the gel formulation, in the composition according to any one of claims 1 to 26.

28. The composition according to claim 27, wherein the gel formulation comprises a penetration-enhancing gel.

29. The composition according to any one of claims 1 to 28, comprising administering cannabidiol and / or dermal-derived fibroblasts to an individual once daily.

30. The composition according to any one of claims 1 to 28, comprising administering cannabidiol and / or dermal-derived fibroblasts to an individual twice daily.

31. The composition according to any one of claims 1 to 30, wherein the administration of cannabidiol and / or dermal-derived fibroblasts comprises the transdermal administration of cannabidiol to an individual.

32. The composition according to any one of claims 1 to 31, wherein the administration of cannabidiol includes intradiscal administration of cannabidiol to an individual.

33. The composition according to any one of claims 1 to 32, wherein the cannabidiol is administered together with the localization composition.

34. The composition according to claim 33, wherein the localized composition comprises an extracellular matrix capable of acting to provide sustained release.

35. The composition according to claim 34, wherein the extracellular matrix is ​​hyaluronic acid.

36. The composition according to claim 34, wherein the hyaluronic acid comprises long-chain hyaluronic acid molecules.

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