WO2022186946A1 - Compositions dérivées de cordon ombilical humain et leurs utilisations pour le traitement de la neuropathie - Google Patents

Compositions dérivées de cordon ombilical humain et leurs utilisations pour le traitement de la neuropathie Download PDF

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Publication number
WO2022186946A1
WO2022186946A1 PCT/US2022/015256 US2022015256W WO2022186946A1 WO 2022186946 A1 WO2022186946 A1 WO 2022186946A1 US 2022015256 W US2022015256 W US 2022015256W WO 2022186946 A1 WO2022186946 A1 WO 2022186946A1
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Prior art keywords
collagen
huc
composition
collagenase
forming agent
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PCT/US2022/015256
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English (en)
Inventor
Kiri K. HAMAKER
Tracy LOVE
Alec SIMON
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Axogen Corporation
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Priority claimed from US17/461,830 external-priority patent/US20220280573A1/en
Application filed by Axogen Corporation filed Critical Axogen Corporation
Priority to AU2022229090A priority Critical patent/AU2022229090A1/en
Priority to KR1020237030006A priority patent/KR20230166076A/ko
Priority to CA3210411A priority patent/CA3210411A1/fr
Priority to JP2023553116A priority patent/JP2024508876A/ja
Priority to IL305497A priority patent/IL305497A/en
Priority to CN202280032294.9A priority patent/CN117320734A/zh
Priority to EP22705956.5A priority patent/EP4284399A1/fr
Publication of WO2022186946A1 publication Critical patent/WO2022186946A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction

Definitions

  • the present disclosure relates generally to the field of neurobiology, medicine, and medical procedures. More particularly, it concerns improved biomaterials extracted from human umbilical cord (hUC) material that have improved inflammatory/anti-inflammatory profiles and their use in the treatment of neuropathy.
  • hUC human umbilical cord
  • Painful neuropathy is largely treated non-surgically, often with systemic administration of analgesics or NSAIDs to treat pain.
  • analgesics and NSAIDs may resolve neuropathic pain depending on the type of injury; however, if pain does not resolve with analgesic or NSAID treatment, secondary treatment options become more invasive or risky such as treatment with anti-inflammatory steroid medication (e.g., corticosteroids), anti-convulsant medication (e.g., pregabalin), or surgery become options.
  • anti-inflammatory steroid medication e.g., corticosteroids
  • anti-convulsant medication e.g., pregabalin
  • the present disclosure provides a physiologically buffered human umbilical cord (hUC) extract composition comprising micronized particles of ECM- degrading protease-treated hUC tissue.
  • the hUC tissue may comprise, consist of, or consist essentially of hUC membrane, hUC stroma, or a combination of hUC membrane and hUC stroma.
  • the composition may further comprise one or more gel forming agents, crosslinkers, biological molecules, enzymes, and/or buffers.
  • the composition may comprise an in situ polymerizing gel forming agent.
  • the in situ polymerizing gel forming agent may be present at about 0.1 to 8 mg/ml.
  • the composition may further comprise a crosslinker, such as genipin or transglutaminase, among other suitable crosslinkers/crosslinking agents.
  • the gel forming agent e.g., polymerizing gel forming agent, e.g., a thermally polymerizing gel forming agent, may be or comprise one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, or collagen XXVII.
  • the ECM-degrading protease-treated hUC tissue comprises hUC membrane and the thermally polymerizing gel forming agent is not fibrin.
  • the composition may be a saline-based suspension buffered at about pH 7.2 to 7.4.
  • the composition may further comprise one or more biological molecules, such as hyaluronic acid, chondroitin sulfate, chitosan, PEG, collagen VI, collagen VII, collagen IX, collagen XII, collagen XIII, collagen XIV, collagen XV, collagen XVI, collagen XVII, collagen XVIII, collagen XIX, collagen XX, collagen XXI, collagen XXIII, collagen XXVI and/or collagen XXVIII.
  • the composition does not comprise chondroitin sulfate.
  • the composition may comprise micronized particles. For example, a majority of the micronized particles may have a diameter of between about 140 nm and about 160 nm.
  • the method may produce a human umbilical cord (hUC) extract, the method comprising (a) providing hUC membrane and/or stroma; (b) mechanically bombarding said hUC membrane and/or stroma to produce micronized particles; and (c) treating with an ECM-degrading protease, one or more of (i) the composition of step (a) prior to mechanical bombardment; (ii) the composition of step (b) during mechanical bombardment; and (iii) the micronized particles of resulting from step (b).
  • the methods herein may further comprise inactivating the protease.
  • an in situ gel forming agent e.g., polymerizing gel forming agent
  • the method may further comprise polymerizing the gel forming agent. Polymerizing may occur in the presence of a crosslinker, such as genipin or transglutaminase.
  • the gel forming agent may be or comprise one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, or collagen XXVII.
  • the in situ gel forming agent e.g., polymerizing gel forming agent, may be present at about 0.1 mg/ml to 8 mg/ml.
  • About 0.5-1.0 cm 2 ofhUC tissue comprising hUC membrane and/or stroma may be provided in step (a).
  • the hUC membrane and/or stroma provided in step (a) may be dispersed in a saline-based suspension buffered at between about pH 6.0 and 8.0.
  • the mechanical bombardment may be performed for between 1 and about 5 cycles. Further, for example, the mechanical bombardment may be performed with about a 60 second duration per cycle, at speeds ranging from, e.g., about 3400 RPM to about 3700 RPM.
  • the methods herein may further comprise centrifuging the micronized particles prior to ECM- degrading protease treatment, such as at a speed of about 5000 x g.
  • the protease e.g., ECM-degrading protease
  • MMP matrix metalloproteinase
  • One or more of hyaluronic acid, chondroitin sulfate, chitosan, PEG, collagen VI, collagen VII, collagen IX, collagen XII, collagen XIII, collagen XIV, collagen XV, collagen XVI, collagen XVII, collagen XVIII, collagen XIX, collagen X, collagen XXI, collagen XXII, collagen XXIII, collagen XXVI and/or collagen XXVIII may be added to the composition, e.g., after inactivation of the ECM-degrading protease.
  • the mechanical bombardment may provide a majority of micronized particles having a diameter of between about 140 nm and about 160 nm, e.g., the particle size distribution having an average diameter ranging from about 140 nm to about 150 nm.
  • the present disclosure also includes methods of treating peripheral neuropathy comprising administering, e.g., injecting, a composition as described herein into a subject at or proximate a site of peripheral neuropathy.
  • the method of treating peripheral neuropathy may comprise injecting a composition made by a process as defined herein into a subject at or proximate a site of peripheral neuropathy.
  • the subject may be a human, a primate, a non-human mammal, or another vertebrate or animal.
  • the method may further comprise treating said subject with a second therapy such as analgesic therapy, NSAID treatment, and/or anti-convulsant medication.
  • the methods herein may further comprise administering, e.g., injecting, said composition into said subject at least a second, third, fourth or fifth time, or on an ongoing or permanent, chronic, basis.
  • FIGS. 1A-1B show preparation of hUC membrane extract as discussed in Example 1.
  • FIGS. 1A-1B show preparation of hUC membrane extract as discussed in Example 1.
  • FIG. 1A Pre-treatment hUC in tubes loaded at about 0.7 cm 2 of debrided hUC with 700 pL IX PBS.
  • FIG. IB homogenized hUC when homogenized for 3 cycles at 6 m/s speeding setting for 60 sec in CoolPrep ® sample holder followed by centrifuging at 5000 xG for 10 minutes.
  • FIG. 2 shows a hUC membrane extract expelled from a syringe with a 26G needle as discussed in Example 2.
  • FIG. 3 shows a collagen gel and hUC membrane extract polymerization as discussed in Example 2, including a 30 minute incubation at 37°C in circular molds.
  • FIG. 4 shows a collagen gel and hUC membrane extract polymerization as discussed in Example 2.
  • a pre-polymerized gel extract mixture was expelled from a syringe with a 26G needle followed by 60 minutes incubation at 37 °C.
  • FIGS. 5-6 show polymerization profiles as discussed in Example 3. hUC extract-loaded gels polymerized at 37°C within 10 minutes without the addition of a gel crosslinker and within 8 minutes with a crosslinker added, indicating fast-acting in situ gel polymerization.
  • FIG. 7 shows gel degradation profiles as discussed in Example 2. Polymerized gels retained over 50% of their mass for a period of 1 day in physiological conditions without the addition of a crosslinker and for up to 36 days with the addition of a gel crosslinker.
  • FIGS. 8A-8B show particle size distributions as discussed in Example 3.
  • hUC extracts contained a monodisperse particle suspension with a predominant fraction of particles ranging between 160 and 180 nm in diameter.
  • FIG. 8B Extracts contained a variable particle dispersity and size distribution profile depending on the preparation conditions.
  • FIG. 9 shows results of an immune modulation assay as discussed in Example 4. hUC extracts were observed to modulate the immune response of human peripheral blood mononuclear cells in vitro during an inflammatory challenge by changing their secretion response of immune biomarkers: IL-lb, IL-10, and MMP-9.
  • FIGS. 10A-10B show immune response modulation in human U-937 cell-line derived macrophage-like cells as discussed in Example 4.
  • hUC extracts modulated the immune response of human U937 cell-line derived macrophage-like cells in vitro during an inflammatory challenge by altering their secretion response of immune biomarkers: IL-Ib and IL-10.
  • FIG. 11 shows collagenase disruption of collagen polymer formation as discussed in Example 5. The concentration of disrupted collagen polymer fragments was decreased by treatment with collagenase.
  • FIG. 12 shows an exemplary schematic for preparing a hUC gel composition.
  • hUC compositions may be prepared by mechanical processing of hUC tissue to obtain an extract, purifying the extract (e.g., removing cellular debris), treating the purified extract with a protease to reduce inflammatory components, and formulating the treated/purified hUC extract as a hydrogel suitable for injection.
  • FIG. 13 shows reduced expression of decorin as discussed in Example 6. hUC compositions treated with MMP-7 exhibited reduced expression of decorin (DCN) as compared to an untreated control.
  • FIG. 14A shows immune response modulation in human U-937 cell-line derived macrophage-like cells as discussed in Example 7.
  • FIG. 14B reports total protein content for treated and untreated hUC extracts, as discussed in Example 7.
  • FIG. 15 shows an exemplary schematic for performing an immunomodulation assay, as discussed in Example 7.
  • FIGS. 16A-16B show results of an immunomodulation assay of protease- treated hUC extracts, as discussed in Example 7.
  • FIG. 17 shows reduced expression of decorin in protease-treated hUC extracts, as discussed in Example 7
  • FIGS. 18A-18B show results of another immunomodulation assay of protease- treated hUC extracts, as discussed in Example 7.
  • the use of amniotic/birth materials is a promising avenue for treatment of tissue regeneration, including in the area of neuropathy.
  • the present disclosure provides methods for generating improved biomaterials and methods of use therefore.
  • the disclosure is directed to a hUC material-derived saline-based suspension created by mechanical bombardment (e.g., bead-beating homogenization) of human umbilical cord (hUC) membrane and hUC stroma sections.
  • This fabrication technique is designed to release a rich volume of soluble bioactive components relevant to inflammation modulation and restoration of healthy tissue into the saline-based suspension while reducing the soluble content of inflammatory components of the hUC membrane and stroma such as DNA, cytosolic DAMPs (damage associated molecular patterns), and ECM protein fragments in the same suspension.
  • This suspension may be processed further to reduce inflammatory protein fragments by incubation with ECM-protease enzymes (e.g., collagenase or matrix metalloproteinase (MMP) such as MMP-7).
  • ECM-protease enzymes e.g., collagenase or matrix metalloproteinase (MMP) such as MMP-7
  • This suspension may also be supplemented with collagen gel at a range of concentrations to enhance sustained delivery of therapeutic agents to the local tissue.
  • This hUC membrane- and stroma-derived saline-based suspension may be delivered to a site of tissue injury (e.g., neuropathy
  • Neuropathy refers generally to nerve damage.
  • Peripheral neuropathy describes damage to nerves other than those of the central nervous system, that is, damage to nerves other than those in the brain and spinal cord.
  • peripheral neuropathy encompasses damage to sensory and motor nerves connecting the brain and spinal cord to the rest of the body. (Damage to peripheral nerves can impair sensation, movement, and functionality, depending on the extent of damage and the peripheral nerves affected.
  • Peripheral neuropathy encompasses damage that is reversible or permanent, where effects can be acute with sudden onset, rapid progress or chronic with symptoms that begin subtly and progress over time.
  • causes of peripheral neuropathy can be genetic or idiopathic (no known cause) and may accompany other medical conditions or prescribed medications.
  • compositions herein are derived from umbilical cord tissue, including the membrane and/or stroma.
  • Umbilical cord extracts may have benefits in treating neuropathy over materials derived from other types of tissues. Without being bound by theory, it is believed that the compositions herein derived from umbilical cord tissue may lead to reduced immune response, e.g., because umbilical cord tissue is deficient in human leukocyte antigen (HLA). Further, umbilical cord tissues comprise higher levels of bioactive growth factors, stem cells, free floating proteins, and glucosamine that may be beneficial in promoting nerve repair.
  • the compositions described herein can be prepared from human umbilical cord materials as described herein. These materials can be obtained from any suitable source. For example, at least one of the components can be obtained from human tissues. The components can be also obtained from commercial sources. The components can be purified, substantially purified, partially purified, or non-purified.
  • Human umbilical cord tissue can be obtained, for example, from commercial sources or from hospitals or surgical/matemity centers.
  • the tissue is typically obtained in either a fresh or frozen state.
  • the tissue can be washed to remove excess storage buffer, blood, or contaminants.
  • the excess liquid can be removed, for example, using a brief centrifugation step, or by other means.
  • the tissue can be frozen, using, for example, liquid nitrogen or other cooling means, to facilitate the subsequent homogenization.
  • the source of the umbilical cord tissue can be a human umbilical cord (hUC).
  • Whole hUC material may be debrided to remove materials extraneous to the membrane and/or stroma, e.g., through the use of surgical cutting tools, manual operated cutting machine, or automated cutting machine.
  • the hUC may also be processed to produce an extract through the use of mechanical bombardment, such as “bead-beating” techniques. Such processing may remove cellular debris.
  • Bead-beating is a laboratory-scale mechanical method for processing biological samples using, for example, glass, ceramic or steel beads, mixed with a sample suspended in aqueous media. The sample and bead mix is subjected to agitation by, for example, stirring or shaking. Beads collide with the tissue material, mechanically disrupting the tissue to release therapeutic bioactive molecules. It has the advantage over other mechanical processing methods of being able to generate a micronized extract with a unique distribution of bioactive molecules and small particles, process many samples at a time with no cross-contamination concerns, and does not release potentially harmful aerosols in the process.
  • an amount of beads e.g., an equal volume of beads as compared to the amount of tissue
  • the sample is vigorously mixed on a laboratory vortex mixer. While processing times may be relatively slow, taking 3-10 times longer than that in specialty shaking machines, it effectively processes tissue and is inexpensive. Scale up procedures with larger volumes and faster processing times are contemplated.
  • Successful bead-beating may depend not only on design features of the shaking machine (which take into consideration shaking oscillations per minute, shaking throw or distance, shaking orientation and vial orientation), but also the selection of correct bead size, bead composition (glass, ceramic, steel) and bead load in the vial.
  • High energy bead-beating machines typically warm the sample due to frictional collisions of the beads during homogenization. Cooling of the sample during or after bead beating may be necessary to prevent damage to heat-sensitive proteins such as enzymes.
  • Sample warming can be controlled by bead-beating for short time intervals and/or with cooling on ice/dry ice between each interval, by processing samples in pre-chilled aluminum vial holders, by circulating gaseous coolant through the machine during bead-beating.
  • samples in the processing chamber are cooled with dry ice, e.g., using a device from MP Biomedicals.
  • a different bead-beater configuration suitable for larger sample volumes, uses a rotating fluorocarbon rotor inside a 15-, 50- or 200-ml chamber to agitate the beads.
  • the chamber can be surrounded by a static cooling jacket.
  • large commercial machines are available to process many liters of cell suspension. Currently, these machines are limited to processing monocellular organisms such as yeast, algae and bacteria.
  • the hUC extract may comprise particles have a monomodal or bimodal particle size distribution, depending on the processing conditions.
  • the composition (before and/or after treatment with a protease as discussed further below) may have an average diameter ranging from about 50 nm to about 500 nm, such as from about 70 nm to about 250 nm, from about 80 nm to about 180 nm, from about 120 nm to about 350 nm, from about 150 nm to about 300 nm, from about 165 nm to about 200 nm, from about 140 nm to about 160 nm, from about 200 nm to about 275 nm, from about 175 nm to about 325 nm, or from about 250 nm to about 450.
  • particles above a given threshold may be removed (e.g., removing large proteoglycans and/or large tissue fragments, etc. released during homogenization), resulting in a desired monomodal or bimodal particle size distribution.
  • Particle size may be measured, for example, by nanoparticle tracking analysis (NT A) or Multi Angle Dynamics Light Scattering (MADLS).
  • the tissue optionally can be frozen prior to the bombardment process.
  • the freezing step can occur by any suitable cooling process.
  • the tissue can be flash- frozen using liquid nitrogen.
  • the material can be placed in an isopropanol/dry ice bath or can be flash-frozen in other coolants. Commercially available quick-freezing processes can be used.
  • the material can be placed in a freezer and allowed to equilibrate to the storage temperature more slowly, rather than being flash-frozen.
  • the tissue can be stored at any desired temperature. For example, -20 °C or -80 °C or other temperatures can be used for storage. Disruption of the tissue while frozen, rather than prior to freezing, is one optional method for preparing the tissue.
  • hUC preparations can be in a liquid, suspension, or dry (including, but not limited to, lyophilized) forms.
  • Antimicrobial agents such as antibiotics or anti-fungal agents may be added.
  • the material can be packaged and stored, for example, at room temperature, or for example, at -20 °C or -80 °C prior to use.
  • the hUC material used to prepare the compositions e.g., gel compositions, herein is present as a dry formulation.
  • a dry formulation can be stored in a smaller volume and may not require the same low temperature storage requirements to keep the formulation from degrading over time.
  • a dry formulation can be stored and reconstituted prior to use.
  • the dry formulation can be prepared, for example, by preparing the freeze- morselized hUC as described herein, then removing at least a portion of the water in the composition. Water can be removed from the preparation by any suitable means. An exemplary method of removing the water is by use of lyophilization using a commercially available lyophilizer or freeze-dryer.
  • Suitable equipment can be found, for example, through Virtis, Gardiner, N.Y.; FTS Systems, Stone Ridge, N.Y.; and SpeedVac (Savant Instruments Inc., Farmingdale, N.Y.).
  • the water content of the dry formulation will be less than about 20%, down to about 10%, down to about 5% or down to about 1% by weight of the formulation. In some embodiments, substantially all of the water is removed.
  • the lyophilized composition can then be stored.
  • the storage temperature can vary from less than about -196 °C, -80 °C, -50 °C, or -20 °C to more than about 23 °C. If desired, the composition can be characterized (weight, protein content, etc.) prior to storage.
  • the lyophilized composition can be reconstituted in a suitable solution or buffer prior to use.
  • exemplary solutions include but are not limited to phosphate buffered saline (PBS), Dulbecco's Modified Eagle's medium (DMEM), and balanced salt solution (BSS).
  • PBS phosphate buffered saline
  • DMEM Dulbecco's Modified Eagle's medium
  • BSS balanced salt solution
  • the pH of the solution can be adjusted as needed.
  • the concentration of the hUC can be varied as needed. In some examples herein a more concentrated hUC solution is useful, whereas in other examples, a solution with a low concentration of hUC is useful.
  • Additional compounds can be added to the solution.
  • Exemplary compounds that can be added to the reconstituted formulation include but are not limited to pH modifiers, buffers, collagen, hyaluronic acid (HA), antibiotics, surfactants, stabilizers, proteins, and the like (discussed further below).
  • compositions as described above. These compositions may be further treated or supplemented with other materials as described herein, including, but not limited to, one or more gel forming agents, crosslinkers, biological molecules, enzymes, and/or buffers.
  • the compositions herein may be formulated for administration to a subject, such as in solution or gel form.
  • compositions herein may include one or more gel-forming agents.
  • Suitable gel forming agents may be thermally -polymerizable at temperatures found in the human body, around 37 °C (98-99 °F).
  • Exemplary gel forming agents include, but are not limited to, Collagens I, II, III, IV, V, VIII, X, XI, XXIV, and XXVII; polyethylene glycol (PEG); poly(lactic co-glycolic acid) (PLGA); poly(ethylene glycol) diacrylate (PEGDA); gelatin methacryloyl (GelMA); and methacrylated hyaluronic acid (MeHA); as well as fibrin.
  • Fibrin also called Factor la, is a fibrous, non-globular protein involved in the clotting of blood.
  • the amount of gel-forming agent generally may range from about 0.1 g/mL to about 8 mg/mL, such as about 0.5 g/mL to about 5 mg/mL, about 1 mg/mL to about 4 mg/mL, or about 3.5 mg/mL to about 4.5 mg/mL.
  • the composition comprising hUC extract may include one or more collagen types. Fibril-forming or network-forming collagens including type I, II, III, IV, V, VIII, X, XI, XXIV, or XXVII may be employed as in situ polymerizing gel-forming agents (discussed below). Other collagens may be included in the composition as well.
  • composition comprising hUC extract may include one or more collagen types, none of which are fibril-forming or network-forming.
  • Collagen is the main structural protein in the extracellular matrix in the various connective tissues in the body. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole-body protein content. Collagen is made of amino acids bound together to form a triple helix of elongated fibril known as a collagen helix. It is mostly found in fibrous tissues such as tendons, ligaments, and skin. [0058] Any one or more of the following may be included in the composition of the hUC extract:
  • Fibrillar Type I, II, III, V, XI, XXIV, XXVII
  • FACIT Fibril Associated Collagens with Interrupted Triple Helices
  • Type I skin, tendon, vasculature, organs, bone (main component of the organic part of bone), Type II (cartilage; main collagenous component of cartilage), Type III (reticulate; main component of reticular fibers; commonly found alongside Type I), Type IV (forms basal lamina, the epithelium-secreted layer of the basement membrane) and Type V (cell surfaces, hair, and placenta).
  • collagen is understood to perform some or all of the following functions in wound healing:
  • Collagen fibers serve to guide fibroblasts. Fibroblasts migrate along a connective tissue matrix.
  • Chemotactic properties The large surface area available on collagen fibers can attract fibrogenic cells, which help in healing.
  • Collagen in the presence of certain neutral salt molecules, can act as a nucleating agent causing formation of fibrillar structures.
  • a collagen wound dressing might serve as a guide for orienting new collagen deposition and capillary growth.
  • Hemostatic properties Blood platelets interact with the collagen to make a hemostatic plug.
  • compositions herein additionally or alternatively may comprise a crosslinker, also referred to herein as a crosslinking agent.
  • Crosslinkers generally provide a bond that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural polymers (such as proteins).
  • cross-linking usually refers to the use of cross-links to promote a change in the polymers' physical properties. When “crosslinking” is used in biology, it generally refers to the use of an agent to link proteins together to check for protein-protein interactions or to create a strengthening of the overall biological material.
  • Exemplary crosslinkers useful for the present disclosure include, but are not limited to, genipin, transglutaminases, the imidoester crosslinker dimethyl suberimidate, the N-hydroxysuccinimide-ester crosslinker BS3, and formaldehyde.
  • the compositions herein may comprise one or more photo-crosslinkable components, e.g., wherein UV light may be used to initiate crosslinking.
  • the crosslinkers dimethyl suberimidate, the N-hydroxysuccinimide-ester crosslinker BS3, and formaldehyde generally form a bond by inducing nucleophilic attack of the amino group of lysine and subsequent covalent bonding via the crosslinker.
  • the zero-length carbodiimide crosslinker EDC functions by converting carboxyls into amine-reactive isourea intermediates that bind to lysine residues or other available primary amines.
  • SMCC or its water- soluble analog, Sulfo-SMCC may be used to prepare antibody -hapten conjugates for antibody development.
  • Genipin is a chemical compound found in gardenia fruit extract. It is an aglycone derived from an iridoid glycoside called geniposide present in fruit of Gardenia jasminoides. Genipin is a natural cross-linker for proteins, collagen, gelatin, and chitosan cross- linking. It has a low acute toxicity, with LD 50 i.v. 382 mg/kg in mice, therefore, much less toxic than glutaraldehyde and many other commonly used synthetic cross-linking reagents. Furthermore, genipin can be used as a regulating agent for drug delivery and as the intermediate for alkaloid syntheses. In vitro experiments have shown that genipin blocks the action of the enzyme uncoupling protein 2.
  • Transglutaminase isolated from Streptomyces mobaraensis bacteria for example, is a calcium-independent enzyme. Mammalian transglutaminases among other transglutaminases require Ca 2+ ions as a cofactor.
  • Transglutaminases form extensively cross-linked, generally insoluble protein polymers. These biological polymers are indispensable for an organism to create barriers and stable structures. Examples are blood clots (coagulation factor XIII), as well as skin and hair. The catalytic reaction is generally viewed as being irreversible and must be closely monitored through control mechanisms.
  • the amount of crosslinker/crosslinking agent may range from about 0.1 mM to about 5 mM, such as about 0.5 mM to about 3 mM, about 3 mM to about 4 mM, about 1.5 mM to about 2.5 mM, or about 1 mM to about 2 mM.
  • the hUC compositions may further include one or more components such as, e.g., hyaluronic acid, chondroitin sulfate, chitosan, and/or polyethylene glycol (PEG).
  • one or more components such as, e.g., hyaluronic acid, chondroitin sulfate, chitosan, and/or polyethylene glycol (PEG).
  • Hyaluronic acid also called hyaluronan
  • hyaluronan is an anionic, non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. It is unique among gly cos aminogly cans in that it is non-sulfated, forms in the plasma membrane instead of the Golgi apparatus, and can be very large: human synovial HA averages about 7 million Da per molecule, or about twenty thousand disaccharide monomers, while other sources mention 3-4 million Da. As one of the chief components of the extracellular matrix, hyaluronan contributes significantly to cell proliferation and migration, and may also be involved in the progression of some malignant tumors.
  • Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) that includes a chain of alternating sugars (N-acetylgalactosamine and glucuronic acid). It is usually found attached to proteins as part of a proteoglycan.
  • a chondroitin chain can have over 100 individual sugars, each of which can be sulfated in variable positions and quantities.
  • the composition comprises a reduced amount of one or more types of proteoglycans (e.g., biglycan, decorin, versican, etc.) and/or sulfated GAGs associated with native hUC tissue.
  • the composition does not include one or more types of proteoglycans and/or sulfated GAGs associated with native hUC tissue.
  • the compositions herein may have a reduced amount of biglycan, decorin, and/or versican as compared to the native hUC tissue.
  • compositions herein do not contain (e.g., have a level below detection) one or more proteoglycans present in the native hUC tissue, such as, e.g., biglycan, decorin, and/or versican.
  • Chitosan is a linear polysaccharide composed of randomly distributed b-(1 4)- linked D-glucosamine (deacetylated unit) and /V-acetyl-D-glucosamine (acetylated unit) obtained from chitin. It is made by treating the chitin shells of shrimp and other crustaceans with an alkaline substance, like sodium hydroxide.
  • PEG Polyethylene glycol
  • PEO polyethylene oxide
  • POE polyoxyethylene
  • the resulting hUC extract may comprise disrupted protein fragments such as protein monomers and ECM fragments.
  • Such components may cause or lead to adverse effects when administered to a patient.
  • certain components of the hUC extract may provoke or be associated with an inflammatory response and/or immune response when injected at the site of nerve damage.
  • the compositions herein may be prepared by treatment with a protease to selectively remove, reduce, or inactivate certain components while simultaneously retaining bioactive components useful in promoting nerve repair.
  • Components that may be at least partially, substantially, or completely removed may include, for example, native proteoglycans such as decorin, biglycan, and/or versican.
  • the protease treatment may lead to degradation of or reduced expression of proteoglycans like decorin present in ECM associated with inflammation via the TLR4 pathway.
  • decorin is believed to affect inflammatory signaling events, being recognized by inflammatory cells as a DAMP. Removing or reducing the amount of proteoglycans like decorin may reduce the risk of inflammatory response by a subject when administered the compositions herein.
  • Embodiments of the present disclosure may effectively guide the immune system to facilitate healing while reducing or preventing the potentially damaging aspects of the immune response.
  • the making of the hUC compositions herein may include treatment with one or more ECM-degrading proteases.
  • the protease may be a collagenase, such as Collagenase I, II, III, IV, V, VI or VII, or other suitable protease including, but not limited to, MMPs, such as MMP-2, MMP-3, or MMP-7.
  • the enzyme treatment may precede the introduction of a gel-forming agent (discussed above) and/or may precede further introduction of one or more other components, including types of collagen and/or other gel forming agents as described above.
  • Collagenases are enzymes that break the peptide bonds in collagen. They assist in destroying extracellular structures in the pathogenesis of bacteria such as Clostridium. They are considered a virulence factor, facilitating the spread of gas gangrene. They normally target the connective tissue in muscle cells and other body organs. Collagen, a key component of the animal extracellular matrix, is made through cleavage of pro-collagen by collagenase once it has been secreted from the cell. This stops large structures from forming inside the cell itself. In addition to being produced by some bacteria, collagenase can be made by the body as part of its normal immune response. This production is induced by cytokines, which stimulate cells such as fibroblasts and osteoblasts, and can cause indirect tissue damage.
  • the concentration of protease may range from about 0.1 pg/mL to about 25 pg/mL, such as about 0.5 pg/mL to about 20 pg/mL, from about 1 pg/mL to about 15 pg/mL, about 0.5 pg/mL to about 5 pg/mL, about 1 pg/mL to about 2 pg/mL, about 2.5 pg/mL to about 5 pg/mL, about 3 pg/mL to about 8 pg/mL, about 5 pg/mL to about 15 pg/mL, about 10 pg/mL to about 18 pg/mL, about 15 pg/mL to about 22 pg/mL.
  • the hUC extract may be treated with the protease for a period of time ranging from about 5 minutes to about 18 hours, such as about 1 hour to about 16 hours, about 4 hours to about 12 hours, about 8 hours to about 16 hours, about 12 hours to about 16 hours, about 2 hours to about 8 hours, about 30 minutes to about 5 hours, or about 5 minutes to about 2 hours.
  • the hUC extract may be treated with about 0.5 pg/mL to about 5 pg/mL of protease for a period of time ranging from about 1 hour to about 16 hours.
  • the method of preparing the composition includes at least partially inactivating the protease.
  • an agent such as, e.g., ethylenediaminetetraacetic acid (EDTA), ilomastat (e.g., GM-6001 or Galardin®), or TIMP metallopeptidase inhibitor 1 (TIMP-1) may be added to passivate the enzyme.
  • EDTA ethylenediaminetetraacetic acid
  • ilomastat e.g., GM-6001 or Galardin®
  • TIMP metallopeptidase inhibitor 1 TIMP metallopeptidase inhibitor 1
  • Such agents may be selected to target the enzyme without damaging or minimizing damage to bioactive components in the hUC extract.
  • compositions e.g., modified extracts, herein may be advantageously combined with a buffer solution to maintain a target pH.
  • a buffer solution e.g., pH buffer or hydrogen ion buffer
  • pH buffer or hydrogen ion buffer is an aqueous solution of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very litle when a small amount of strong acid or base is added to it. Buffer solutions are used to keep pH at a nearly constant value in a wide variety of chemical applications.
  • the pH of a solution containing a buffering agent typically varies within a limited range, regardless of what else may be present in the solution. In biological systems, this allows enzymes to serve their intended functions. If the pH value of a solution rises or falls too much, the effectiveness of an enzyme decreases in a process, known as denaturation, which may be irreversible.
  • PBS phosphate buffered saline
  • Some exemplary buffering agents relevant to physiologic pH include citric acid and K ⁇ 2RO4.
  • citric acid By combining substances with p/C, values differing by only two or less and adjusting the pH, a wide range of buffers can be obtained.
  • Citric acid is a useful component of a buffer mixture because it has three p/C, values, separated by less than two. The buffer range can be extended by adding other buffering agents.
  • Mcllvaine's buffer solutions composed of Na2HP04 and citric acid, have a buffer range of pH 3 to 8.
  • buffers useful for biological systems suitable for the present disclosure include lactated Ringer’s solution (LRS), tris(hydroxymethyl)aminomethane (TRIS), Hank's Balanced Salt Solution (HBSS), Gey's Balanced Salt Solution (GBSS), TAPSO, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2- aminoethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine- N,N'-bis(2-ethanesulfonic acid) (PIPES), cacodylate, and 2-(N-morphobno)ethanesulfonic acid (MES).
  • LPS lactated Ringer’s solution
  • TAS tris(hydroxymethyl)aminomethane
  • HBSS Hank's Balanced Salt Solution
  • GBSS Gey's Balanced Salt Solution
  • TAPSO 4-(2-hydroxy
  • FIG. 12 shows an exemplary schematic for preparing a hUC gel composition in accordance with the discussion above and examples below.
  • the compositions may be prepared by mechanical processing of hUC tissue (e.g., bead-beating or other suitable technique) to obtain an extract, purifying the extract (which may include, e.g., removing cellular debris), treating the purified extract with a protease to reduce inflammatory components, and formulating the treated/purified hUC extract as a hydrogel suitable for injection.
  • tissue injuries including but not limited to, muscle, tendon, ligaments, etc.
  • the methods are designed to provide superior treatment of a site of pain, such as of peripheral neuropathy, compared with analgesic treatment or currently available amnion/birth-tissue-derived flowable products (e.g., OrthoFlo by Mimedx) and restore local tissues to a healthy state with normal function. This is intended to alleviate symptoms for patients who may undergo surgical intervention as well as patients who may not.
  • an injectable therapy the methods are designed to be minimally invasive.
  • the minimally invasive and versatile nature of this treatment as an injectable therapy intends to deliver accessibility to a treatment for a range of anatomical regions for painful neuropathy in the body.
  • the methods involve injection of hUC-derived material into a site of injury.
  • Medical professionals are capable of assessing the appropriate site based on symptoms and diagnosis, which may include feet, hands, and joints, such as shoulders, elbows, wrists and knees.
  • doctors can determine the appropriate surgical procedures for delivery of the agents, such as combining injection with image guided delivery or surgical resection of affected regions.
  • compositions herein may be formulated as a gel, e.g., a hydrogel, for injection at or proximate to the site of injury or otherwise in need of treatment.
  • a gel e.g., a hydrogel
  • Formulating the composition as a gel may provide for a longer duration of treatment, e.g., the gel may remain at the intended target site longer due to factors such as its viscosity, cohesion, etc. Accordingly, sustained release may be achieved by using gel compositions.
  • the gel may be formulated to provide desired properties, such as degradation rate, density, stiffness, and/or cargo load.
  • the methods may include multiple treatments over a period of time, such as on an on-going or permanent, chronic basis. For example, any number of treatments, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more treatments may be made. Such treatments may be spaced by days, weeks, months, or even years.
  • the methods may also include combination therapies employing administration of the hUC compositions described herein in conjunction with one or more recognized therapies for neuropathy, such as changes in diet and/or administration of NSAIDs, analgesics or anti-convulsants (discussed in greater detail above and incorporated herein by reference).
  • compositions were prepared from hUC extracts as follows. Samples of hUC membrane tissue were loaded in tubes and 700 pL of IX PBS was added (FIG. 1A). The samples were then homogenized for 3 cycles at a speed setting of 6 m/s for 60 seconds in a CoolPrep ® sample holder followed by centrifuging at a speed of 5000 x g for 10 min (FIG. IB).
  • hUC extract prepared according to Example 1 was loaded into a syringe, extruded through a 26G needle (FIG. 2), and polymerized into gel at 37°C with collagen as a gel forming agent, e.g., a formulation suitable for use as a minimally invasive injectable therapy.
  • FIG. 3 depicts collagen gel and hUC membrane extract polymerization after 30 minutes incubation at 37°C in circular molds.
  • FIG. 4 depicts collagen gel and hUC membrane extract polymerization, where a pre-polymerized gel extract mixture was expelled from a syringe with a 26G needle followed by 60 minutes incubation at 37°C.
  • FIG. 8A shows results for samples in which the hUC membrane tissue (umbilical cord membrane (UCM)) was homogenized for 1 cycle, 2 cycles, and 3 cycles at 4 m/s at 60 seconds per cycle, showing a slightly broader size distribution for the longer homogenization time.
  • UCM umbilical cord membrane
  • the extracts contained a variable particle disperity and size distribution profile depending on preparation conditions (FIG. 8B).
  • hUC extracts were prepared with 3 minutes homogenization of hUC membrane tissue per cycle at 4000 rpm, for a total of 4-5 cycles.
  • hUC extracts were prepared with 60 seconds homogenization of hUC membrane tissue per cycle at 6 m/s, for a total of 3 cycles.
  • U937 cell culture was treated with 20 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to generate macrophage- like cells, then given Ml differentiation stimulus (50 ng/mL LPS + 10 ng/mL IFN-g) (“Stim”) or no stimulus (“Unstim”). Ml cultures were treated with the respective hUC extracts (“UC”) for comparison. Results show that the hUC extracts modulated the immune response of human U937 cell-line derived macrophage-like cells in vitro during an inflammatory challenge by altering their secretion response of immune biomarkers IL-Ib and IL-10. FIG. 9 shows results at a 48-hour timepoint.
  • FIGS. 10A-10B shows results attimepoints of 24, 48, 72, and 96 hours.
  • Modified/treated hUC extracts were prepared to study treatment by MMP-7 protease.
  • hUC tissue was mechanically processed by 1 minute homogenization per cycle at 6 m/s, for a total of 3 cycles and then treated with MMP-7 (0.8 pg/mL) for 16 hours (“Treated UC”). This is shown schematically in the process of FIG. 12.
  • a separate control was prepared without MMP-7 treatment (“UC”).
  • UC MMP-7 treatment
  • FIG. 13 the extract treated with MMP-7 exhibited reduced expression of decorin (DCN) as compared to an untreated control. Decorin was measured with an ELISA kit (ThermoFisher Scientific).
  • FIG. 14A shows the total protein content (pg/mL) measured for the treated and control samples, indicating similar levels.
  • Total protein content was measured using a BCA (bicinchoninic acid) protein Assay Kit (ThrmoFisher Scientific) and tested 600 different proteins, of which 448 protein biomarkers were identified. This suggests that the MMP-7 treatment was successful in removing decorin content without significant damage to other proteins, including potentially beneficial components.
  • FIGS. 14A-14B prepared according to Example 6 were further investigated in an immunomodulation assay to determine their effect on immune biomarkers IL-Ib and IL-10.
  • U937 cell culture was treated with 20 ng/mL phorbol 12- myristate 13-acetate (PMA) for 48 hours to generate macrophage-like cells, then given Ml differentiation stimulus (50 ng/mL LPS + 10 ng/mL IFN-g) (“Stim”) or no stimulus (“Unstim”).
  • Ml cultures were also treated with the untreated hUC extract (“UC”) and the MMP-7 treated hUC extract (“Treated UC”).
  • the assay is illustrated schematically in FIG. 15. Results in FIGS. 16A and 16B shown that protease treatment led to reduced secretion response of IL-Ib and IL-10. Without being bound by theory, it is believed that lower amounts of decorin in the treated hUC extracts led to reduced inflammatory response.
  • hUC extracts were prepared and treated with MMP-7 as described in Example 6. Decorin levels of the untreated hUC extract control (“UC”) and the MMP-7 treated hUC extract (“Treated UC”) were measured as described in Example 6. The results shown in FIG. 17 confirm a reduction in decorin for the treated hUC extracts.
  • FIGS. 18A and 18B report the levels of IL-Ib and IL-10, respectively, for unstimulated culture without hUC extract (“Unstim”), simulated culture without hUC extract (“Stim”), simulated culture with untreated hUC extract (“UC”), simulated culture with MMP-7 treated hUC extract (“Treated UC”), and stimulated culture with MMP-7 (0.8 pg/mL), without hUC extract (“MMP-7”). These results are consistent with those of FIGS. 16A-16B, showing that protease treatment of hUC extracts led to significant reductions in IL-Ib and IL-10.
  • MMP-7 alone was not observed to affect IL-Ib as compared to the “Stim” control (FIG. 18A), and caused a slight (not statistically significant) reduction in IL-10 relative to control (FIG. 18B). This suggests that the reduction in inflammatory response is not due to residual protease, supporting the conclusion that removing decorin through protease treatment lowers immune response.

Abstract

La présente invention concerne des biomatériaux améliorés extraits d'un matériau de cordon ombilical humain (hUC). Les matériaux sont rompus mécaniquement pour produire des particules micronisées et sont en outre traités avec une protéase et éventuellement mélangés avec un agent de formation de gel. Les matériaux peuvent avoir des profils inflammatoires/anti-inflammatoires améliorés et peuvent fournir une utilité particulière dans le traitement de la neuropathie périphérique par administration locale des extraits de hUC.
PCT/US2022/015256 2021-03-03 2022-02-04 Compositions dérivées de cordon ombilical humain et leurs utilisations pour le traitement de la neuropathie WO2022186946A1 (fr)

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AU2022229090A AU2022229090A1 (en) 2021-03-03 2022-02-04 Human umbilical cord-derived compositions and uses thereof for treating neuropathy
KR1020237030006A KR20230166076A (ko) 2021-03-03 2022-02-04 신경병증 치료를 위한 인간 제대 유래 조성물 및 이의 용도
CA3210411A CA3210411A1 (fr) 2021-03-03 2022-02-04 Compositions derivees de cordon ombilical humain et leurs utilisations pour le traitement de la neuropathie
JP2023553116A JP2024508876A (ja) 2021-03-03 2022-02-04 ヒト臍帯由来組成物及び神経障害の治療のためのその使用
IL305497A IL305497A (en) 2021-03-03 2022-02-04 Preparations derived from the human umbilical cord and their uses for the treatment of neuropathy
CN202280032294.9A CN117320734A (zh) 2021-03-03 2022-02-04 人类脐带衍生组合物及其用于治疗神经病变的用途
EP22705956.5A EP4284399A1 (fr) 2021-03-03 2022-02-04 Compositions dérivées de cordon ombilical humain et leurs utilisations pour le traitement de la neuropathie

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US17/461,830 US20220280573A1 (en) 2021-03-03 2021-08-30 Human umbilical cord-derived compositions and uses thereof for treating neuropathy
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