CN117320734A - Human umbilical cord derived compositions and their use for treating neuropathy - Google Patents

Abstract

The present disclosure provides improved biomaterials extracted from human umbilical cord (hUC) material. The material is mechanically broken to produce micronized particles and further treated with a protease and optionally mixed with a gel forming agent. The material may have improved inflammatory/anti-inflammatory properties and may provide particular utility in the treatment of peripheral neuropathy by topical application of an hUC extract.

Description

Human umbilical cord derived compositions and their use for treating neuropathy

Technical Field

The present patent application claims priority from U.S. provisional patent application Ser. No. 63/156123, filed on 3/2021, and U.S. provisional patent application Ser. No. 63/237602, filed on 8/27/2021, each of which is incorporated herein by reference in its entirety.

Background

The present disclosure relates generally to the field of neurobiology, medicine, and medical procedures. More particularly, it relates to improved biological materials extracted from human umbilical cord (hUC) materials having improved inflammatory/anti-inflammatory properties, and the use of these materials in the treatment of neuropathy.

Painful neuropathy is mostly non-surgically treated, usually by systemic administration of analgesics or NSAIDs (non-steroidal anti-inflammatory drugs). As a first line of defense, analgesics and NSAIDs can address neuropathic pain depending on the type of injury; however, if the analgesic or NSAID therapy is not capable of alleviating pain, the secondary treatment options may become more invasive or risky, for example, administration of a steroid anti-inflammatory drug (e.g., a corticosteroid), an anticonvulsant (anti-convulsant) drug (e.g., pregabalin), or surgery may be the option.

Unfortunately, the limitations of current first-line therapies such as analgesics and NSAIDs are that they are not effective in addressing painful neuropathy in many patients. Many patients will receive secondary treatment options such as surgery or drugs (i.e., steroids or anticonvulsants) with a greater risk of side effects. Side effects that most patients receiving these more risky therapies typically experience include fluid retention (fluid retention), weight gain, headache, stomach pain and swelling. There is therefore a need for improved compositions and methods for treating neuropathy.

Disclosure of Invention

In at least one aspect, the present disclosure provides a physiologically buffered human umbilical cord (hoc) extract composition comprising micronized particles of hoc treated with ECM degrading protease. The hoc tissue may comprise, consist of, or consist essentially of a hoc membrane, a hoc matrix, or a combination of hoc membranes and hoc matrices. The composition may further comprise one or more gel forming agents, cross-linking agents, biomolecules, enzymes and/or buffers. For example, the composition may comprise an in situ polymerized gel forming agent. The in situ polymerized gel former can be present at about 0.1 to 8 mg/ml.

The composition may further comprise a cross-linking agent, such as genipin (genipin) or transglutaminase, as well as other suitable cross-linking agents/cross-linking agents. The gel forming agent, e.g., a polymeric gel forming agent, e.g., a thermal polymeric gel forming agent, may be or include one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, collagen XXVII. In some examples, the ECM degrading protease treated hoc tissue includes a hoc membrane and the thermal polymeric gel former is not fibrin. The composition may be a physiological saline-based suspension buffered at about pH 7.2 to 7.4.

The composition may further comprise one or more biomolecules, such as hyaluronic acid, chondroitin sulfate (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 XXII, collagen XXIII, collagen XXVI, and/or collagen XXVIII. In at least one example, the composition does not comprise chondroitin sulfate. The composition may comprise micronized particles. For example, the diameter of most micronized particles may be between about 140nm and about 160 nm.

A method of making such a composition is also provided. For example, the method may produce a human umbilical cord (hUC) extract, the method comprising: (a) providing a fruc membrane and/or fruc matrix; (b) Mechanically striking the hic membrane and/or the hic matrix to produce micronized particles; and (c) treating with ECM degrading protease: (i) the composition of step (a) prior to mechanical impact; (ii) The composition of step (b) during a mechanical impact; and (iii) one or more of the micronized particles resulting from step (b).

The methods herein may further comprise inactivating the protease. After inactivation of the ECM degrading protease, an in situ gel former, such as a polymeric gel former, may be added. The method may further comprise polymerizing the gel forming agent. Polymerization may occur in the presence of a cross-linking agent such as genipin or transglutaminase.

The gel forming agent may be or may include one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, collagen XXVII. The in situ gel forming agent, such as a polymeric gel forming agent, can be present at about 0.1mg/ml to 8 mg/ml. About 0.5 to 1.0cm may be provided in step (a) ² Includes a hUC membrane and/or a hUC tissue of a matrix. The hic membrane and/or matrix provided in step (a) may be dispersed in a physiological saline-based suspension buffered between about pH 6.0 and 8.0.

The mechanical impact is performed between 1 and about 5 cycles. Further, for example, the mechanical impact can be performed at a speed ranging from, for example, about 3400RPM to about 3700RPM for a duration of about 60 seconds per cycle. The methods herein may further comprise centrifuging the micronized particles at a rate of, for example, about 5000xg prior to ECM degrading protease treatment.

Proteases, such as ECM degrading proteases, may be collagenases or Matrix Metalloproteinases (MMPs), such as collagenase I, collagenase III, MMP-2, MMP-3 or MMP-7. For example, after inactivation of the ECM degrading protease, 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 XX, collagen XXI, collagen XXII, collagen XXIII, collagen XXVI, or collagen XXVIII may be added to the composition. The mechanical impact may provide most micronized particles having a particle size distribution between about 140nm and about 160nm in diameter, for example, having an average diameter ranging from about 140nm to about 150 nm.

The present disclosure also includes methods of treating peripheral neuropathy comprising administering (e.g., injecting) a composition described herein to a subject at or near a peripheral neuropathy site. For example, a method of treating peripheral neuropathy may comprise injecting a composition prepared by a method defined herein at or near a peripheral neuropathy site in a subject. The subject may be a human, primate, non-human mammal or another vertebrate or animal. The method may further comprise treating the subject with a second therapy, such as analgesic therapy, NSAID therapy, and/or anticonvulsant drug. The methods herein may also include administering, e.g., injecting, the composition to the subject at least a second, third, fourth, or fifth time or on a sustained or permanent, chronic basis.

As used herein, the specification, "a" and "an" mean one or more. As used herein, the terms "a" and "an" when used in conjunction with the word "comprising" mean one or more than one.

The use of the term "or" is used to mean "and/or" unless explicitly indicated to merely refer to alternatives or to the mutual exclusion of alternatives, although the disclosure supports definitions of alternatives and "and/or". As used herein, "another" means at least a second or two or more.

Throughout this application, the term "about" is used to indicate a value that includes the inherent error variation of the device, the method used to determine the value, or the variation that exists between study subjects. The term "about" is understood to encompass a particular amount or value of + -5% unless otherwise indicated.

Other objects, features and advantages of the present disclosure will become more apparent in the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Note that the mere fact that a particular compound belongs to one particular formula does not mean that it cannot belong to another formula either.

Drawings

The following drawings form a part of the present application and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of exemplary embodiments presented herein.

The patent or application document contains at least one drawing in color. The patent office will provide a copy of the color drawings of the patent or patent application publication upon request and at the expense of the necessary fee.

Figures 1A-1B illustrate the preparation of a fruc membrane extract as discussed in example 1. (FIG. 1A) about 0.7cm containing 700. Mu.L of 1 XPBS ² Is a pre-treated hUC in the tube of debrided (debrided) hUC. (FIG. 1B) when inThe sample holder was set at 6m/s for 60 seconds for 3 cycles of homogenization and then centrifuged at 5000XG for 10 minutes to homogenize the hUC.

Figure 2 shows the hic membrane extract expelled from the syringe with a 26G needle as discussed in example 2.

Figure 3 shows collagen gel and hic membrane extract polymerization as discussed in example 2, including incubation in a circular mold at 37 ℃ for 30 minutes.

Figure 4 shows collagen gel and hic membrane extract polymerization as discussed in example 2. The pre-polymerized gel extract mixture was discharged from the syringe with a 26G needle and incubated at 37 ℃ for 60 minutes.

Fig. 5 to 6 show aggregation curves as discussed in example 3. The gel loaded with the hoc extract polymerized in 10 minutes at 37 ℃ without the addition of the gel cross-linker and 8 minutes with the addition of the cross-linker, indicating a rapidly reacting in situ gel polymerization.

Fig. 7 shows gel degradation curves as discussed in example 2. The polymeric gel maintains a mass of 50% or more for a period of 1 day under physiological conditions without the addition of a cross-linking agent, and can last up to 36 days with the addition of a gel cross-linking agent.

Fig. 8A to 8B show particle size distributions as discussed in example 3. (FIG. 8A) hUC extract containing a suspension of monodisperse particles, wherein the major portion of the particles have a diameter between 160 and 180 nm. (FIG. 8B) extracts containing variable particle dispersion and particle size distribution curves depending on the preparation conditions.

Fig. 9 shows the results of an immunomodulatory assay as discussed in example 4. The hUC extract was observed by altering its immunobiomarkers: the secretory responses of IL-1b, IL-10 and MMP-9 regulate the in vitro immune response of human peripheral blood mononuclear cells (human peripheral blood mononuclear cells) during inflammatory attacks.

FIGS. 10A to 10B show modulation of immune responses in human U937 cell line-derived megaphaga-like cells (macrophaga-like cells) as discussed in example 4. The hUC extract was purified by altering its immune biomarker: the secretory response of IL-1 beta and IL-10 regulates the immune response of human U937 cell lines-derived megaphagous cells in vitro during inflammatory attacks.

Fig. 11 shows collagenase disruption of collagen polymer formation as discussed in example 5. The concentration of disrupted collagen polymer fragments is reduced by treatment with collagenase.

Fig. 12 shows an exemplary schematic for preparing the hoc gel composition. The houc composition may be prepared by machining the houc tissue to obtain an extract, purifying the extract (e.g., to remove cellular debris), treating the purified extract with a protease to reduce inflammatory components, and formulating the treated/purified houc extract into a hydrogel suitable for injection.

Figure 13 shows reduced expression of decorin as discussed in example 6. The MMP-7 treated hUC composition showed reduced expression of Decorin (DCN) compared to the untreated control group.

FIG. 14A shows modulation of immune responses in human U937 cell line-derived megaphages cells as described in example 7. Fig. 14B reports total protein content of treated and untreated hic extracts as described in example 7.

Fig. 15 shows an exemplary schematic for performing an immunomodulatory assay, as described in example 7.

Fig. 16A-16B show the results of an immunomodulatory assay of protease-treated hoc extracts, as described in example 7.

Figure 17 shows reduced expression of decorin in protease-treated hiuc extracts, as described in example 7.

Fig. 18A-18B show the results of another immunomodulatory assay of protease-treated hoc extracts, as described in example 7.

Detailed Description

As mentioned above, the use of amniotic membrane/childbirth materials is a promising approach in the area of tissue regeneration treatment, including neuropathy. The present disclosure provides methods for producing improved biomaterials and methods of use thereof. In general, the present disclosure is directed to physiological saline-based suspensions derived from human umbilical cord (hoc) material produced by mechanical impact (e.g., bead-beating) homogenization of a hoc membrane and a hoc matrix slice. This manufacturing technique aims to release a large amount of soluble bioactive components associated with inflammatory regulation and healthy tissue recovery into a physiological saline-based suspension while reducing the soluble content of the inflammatory components of the hic membrane and matrix, such as DNA, cytosolic damage-related molecular patterns (damage associated molecular patterns, DAMPs) and ECM protein fragments, in the same suspension. The suspension may be further treated to reduce inflammatory protein fragments by incubation with ECM degrading proteases (e.g., collagenase or Matrix Metalloproteinases (MMPs) such as MMP-7). The suspension may also be supplemented with a concentration of collagen gel to enhance sustained delivery of the therapeutic agent to the local tissue. Such hUC membrane and matrix derived physiological saline-based suspensions may be delivered to a tissue injury (e.g., neuropathy) site by, for example, injection. These and other features of the present disclosure are described in detail below.

I. Neuropathy of nerve

Neuropathy generally refers to nerve damage. Peripheral neuropathy describes damage to nerves other than the central nervous system, i.e., damage to nerves other than the brain and spinal cord. Peripheral neuropathy, for example, includes damage to sensory and motor nerves connecting the brain and spinal cord to other parts of the body. Peripheral nerve injury can impair sensation, movement, and function, depending on the extent of the injury and the peripheral nerve affected. Peripheral neuropathy includes reversible or permanent lesions that affect chronic symptoms that may be sudden onset of an acute, rapid progression or unnoticeable onset and progression over time. The cause of peripheral neuropathy may be genetic or idiopathic (unknown cause), and may be accompanied by other medical conditions or prescribed medical treatments.

Umbilical cord material and extraction method thereof

The compositions herein are derived from umbilical cord tissue, including membranes and/or matrices. Umbilical cord extracts may have benefits in treating neuropathy as compared to materials derived from other types of tissue. Without being bound by theory, it is believed that the compositions derived from umbilical cord tissue herein may result in reduced immune response, for example, because the umbilical cord tissue lacks human leukocyte antigens (human leukocyte antigen, HLA). In addition, umbilical cord tissue includes higher levels of bioactive growth factors, stem cells, free proteins, and glucosamine that can help promote nerve repair.

The compositions described herein may be prepared from the human umbilical cord materials described herein. These materials may be obtained from any suitable source. For example, at least one component may be obtained from human tissue. These ingredients may also be obtained from commercial sources. The components may be purified, substantially purified, partially purified or unpurified.

Human umbilical cord tissue may be obtained, for example, from commercial sources or from a hospital or surgical/obstetrical center. Tissue is typically obtained in a fresh or frozen state. The tissue may be washed to remove excess storage buffer, blood, or contaminants. For example, a brief centrifugation step may be used or excess liquid may be removed by other means. The tissue may be frozen using, for example, liquid nitrogen or other cooling means to facilitate subsequent homogenization. The source of umbilical cord tissue may be human umbilical cord (hUC). The entire hic material may be debrided, for example, by using a surgical cutting tool, a manual cutter, or an automatic cutter, to remove material unrelated to the membrane and/or substrate.

The hUC can also be processed to make extracts by using mechanical impact such as "bead impact" techniques. This treatment removes cell debris. Bead beating is a laboratory-scale mechanical method using, for example, glass, ceramic or steel beads, mixed with a sample suspended in an aqueous medium to process biological samples. The mixture of samples and beads is stirred, for example, by stirring or shaking. The beads collide with the tissue material, mechanically destroying the tissue to release the therapeutically bioactive molecules. The advantage of this over other mechanical treatment methods is the ability to produce micronized extracts with unique distributions of bioactive molecules and small particles, process many samples at a time without cross-contamination problems, and without liberating potentially harmful aerosols during the process.

In one example of the method, a quantity of beads, for example an equal volume of beads compared to the quantity of tissue, is added to the tissue suspension in the vessel and the sample is vigorously mixed in a laboratory vortex mixer. Although the treatment time may be relatively slow, 3 to 10 times longer than a professional shaker, it can effectively treat tissue and is inexpensive. A magnification procedure with a larger volume and faster processing time is conceivable.

Successful bead beating depends not only on the design features of the rocker (taking into account shaking oscillations per minute, shaking throw or distance, shaking direction and vial direction) but also on the choice of the correct bead size, the composition of the beads (glass, ceramic, steel) and the bead load in the vial.

High energy bead blasters typically warm the sample due to frictional collisions of beads during homogenization. Cooling of the sample may be required during or after the bead impact to prevent damage to heat sensitive proteins such as enzymes. Sample warming can be controlled by bead beating for short time intervals and/or cooling on ice/dry ice between each interval, by handling the sample in a pre-chilled aluminum vial rack, by circulating a gaseous coolant through the rocker during bead beating. In some examples herein, the sample in the processing chamber is cooled with dry ice, for example using a device from MP Biomedicals.

Different bead breaker configurations suitable for larger sample volumes use a rotating fluorocarbon rotor in a 15-, 50-, or 200-ml chamber to agitate the beads. In this configuration, the chamber may be surrounded by a static cooling jacket. Using the same rotor/chamber configuration, large commercial machines are capable of handling several liters of cell suspension. Currently, these machines are limited to processing single cell organisms such as yeast, algae and bacteria.

This initial treatment may produce micronized particles of the hoc tissue. For example, the hoc extract may include particles having a unimodal or bimodal particle size distribution, depending on the processing conditions. In some examples herein, the average diameter of the composition (before and/or after treatment with the protease as further discussed below) may be about 50nm to about 500nm, for example about 70nm to about 250nm, 80nm to about 180nm, about 120nm to about 350nm, about 150nm to about 300nm, about 165nm to about 200nm, about 140nm to about 160nm, about 200nm to about 275nm, about 175nm to about 325nm, or about 250nm to about 450nm. In some examples, particles above a given threshold (e.g., removal of proteoglycans and/or large tissue fragments released during homogenization, etc.) may be removed, resulting in a desired unimodal or bimodal particle size distribution. Particle size can be measured, for example, by nanoparticle tracking analysis (nanoparticle tracking analysis, NTA) or multi-angle kinetic light scattering (Multi Angle Dynamics Light Scattering, MADLS).

The tissue may optionally be frozen prior to the striking process. The freezing step may be performed by any suitable cooling process. For example, the tissue may be flash frozen using liquid nitrogen. Alternatively, the material may be placed in an isopropanol/dry ice bath, or may be flash frozen in other coolants. Commercially available flash freezing processes may be used. In addition, the material may be placed in a refrigerator to allow it to equilibrate more slowly to storage temperature rather than flash freezing. The tissue may be stored at any desired temperature. For example, -20 ℃ or-80 ℃ or other temperatures useful for storage. Destroying tissue at the time of freezing, rather than prior to freezing, is an alternative method of preparing tissue.

The hUC preparation may be in liquid, suspension, or dry (including but not limited to freeze-dried) form. Antimicrobial agents, such as antibiotics or antifungals, may be added. The material may be packaged and stored prior to use, for example at room temperature or, for example, at-20 ℃ or-80 ℃.

In some embodiments, the hoc material used to prepare the composition, e.g., a gel composition, is present herein as a dry formulation (dry formulation). The dry formulation can be stored in a smaller volume and may not require the same low temperature storage requirements to prevent degradation of the formulation over time. The dry formulation may be stored and reconstituted (reconstitute) prior to use. For example, a dry formulation may be prepared by preparing a freeze-minced hic as described herein, and then removing at least a portion of the water in the composition. The water may be removed from the formulation by any suitable means. An exemplary method of removing water is freeze drying using a commercially available freeze dryer or freeze dryer. For example, via Virtis, gardiner, n.y.; FTS Systems, stone Ridge, N.Y; and SpeedVac (Savant Instruments inc., farm, n.y.) find suitable equipment. In certain embodiments, the water content of the dry formulation will be less than about 20%, as low as about 10%, as low as about 5% or as low as about 1% by weight of the formulation. In some embodiments, substantially all of the water is removed. The freeze-dried composition may then be stored. The storage temperature may vary from less than about-196 ℃, -80 ℃, -50 ℃ or-20 ℃ to greater than about 23 ℃. If desired, the composition may be characterized (weight, protein content, etc.) prior to storage.

The lyophilized composition may be reconstituted in a suitable solution or buffer prior to use. Exemplary solutions include, but are not limited to, phosphate buffered saline (phosphate buffered saline, PBS), darbert's Modified Eagle's Medium (DMEM), and balanced salt solutions (balanced salt solution, BSS). The pH value of the solution can be adjusted according to the requirement. The concentration of hUC can be varied as desired. In some examples herein, a more concentrated solution of hUC is useful, while in other examples, a solution with a low concentration of hUC is useful. Other compounds may be added to the solution. Exemplary compounds that may be added to the reconstituted formulation include, but are not limited to, pH adjusters, buffers, collagen, hyaluronic Acid (HA), antibiotics, surfactants, stabilizers, proteins, and the like (described further below).

Umbilical cord extract composition

According to the present disclosure, there is provided a hoc composition as described above. These compositions can be further processed or supplemented with other materials described herein, including but not limited to one or more of gel forming agents, cross-linking agents, biomolecules, enzymes, and/or buffers. The compositions herein may be formulated, for example, in solution or gel form, for administration to a subject.

A. Gel forming agent

The compositions herein may include one or more gel forming agents. Suitable gel formers may be thermally polymerized at a temperature in the human body of about 37 ℃ (98 to 99°f). Exemplary gel formers include, but are not limited to, collagen I, II, III, IV, V, VIII, X, XI, XXIV and XXVII; polyethylene glycol (PEG); polylactic acid-glycolic acid copolymer (PLGA); poly (ethylene glycol) diacrylate (poly (ethylene glycol) diacrylate, PEGDA); methacrylated gelatin (gelatin methacryloyl, gelMA); methacrylic acid hyaluronic acid (methacrylated hyaluronic acid, meHA); and (3) fibrin. Fibrin, also known as factor Ia, is a fibrous, non-globular protein involved in blood clotting.

The amount of gel forming agent may generally be from 0.1g/mL to 8mg/mL, such as from 0.5g/mL to about 5mg/mL, from 1mg/mL to 4mg/mL, or from about 3.5mg/mL to about 4.5mg/mL.

Collagen protein

In at least one aspect, a composition comprising a hic extract can comprise one or more collagen types. Fibril forming (Fibril-forming) or network-forming (network-forming) collagen, including types I, II, III, IV, V, VIII, X, XI, XXIV or XXVII, may be used as the in situ polymerized gel former (discussed below). Other collagens may also be included in the composition.

In another aspect, a composition comprising an hUC extract may comprise one or more collagen types, none of which are fibril forming or reticulate forming collagen.

Collagen is the major structural protein in the extracellular matrix of various connective tissues in the body. As a major component of connective tissue, it is the protein most abundant in mammals, accounting for 25% to 35% of the total protein content. Collagen is composed of amino acids that bind together to form a triple helix of elongated fibrils called the collagen helix. It is mainly found in fibrous tissues such as tendons, ligaments, and skin.

The composition of the hUC extract may comprise any one or more of the following:

fibrous (I, II, III, V, XI, XXIV, XXVII type)

Non-fibrous form

FACIT (Fibril Associated Collagens with Interrupted Triple Helices, fiber-related collagen with interrupted triple helix) (IX, XII, XIV, XVI, XIX, XX, XXI, XXII type)

Reticulation collagen (VIII, IV, X type)

Multiplexin (Multiple Triple Helix domains with Interruptions, with interrupted multiple triple-helical domains) (XV, XVIII type)

MACIT (Membrane Associated Collagens with Interrupted Triple Helices, membrane-associated collagen with interrupted triple helix) (XIII, XVII type)

Transmembrane related collagen (XXIII)

Others (VI, VII, XXVI, XXVIII type)

The five most common types are type I (skin, tendons, vasculature, organs, bones (main component of organic part of bone), type II (cartilage; main collagen component of cartilage), type III (reticulation; main component of reticulation fibers; commonly found beside type I), type IV (epithelial secretory layer forming basal layer, basal membrane) and type V (cell surface, hair and placenta).

In four phases of wound healing, collagen is understood to perform some or all of the following functions in wound healing:

guide function: collagen fibers are used to guide fibroblasts. Fibroblasts migrate along connective tissue matrix.

Chemotactic properties: the large surface area available on collagen fibers can attract fibroblasts, which aid in healing.

Nucleation: in the presence of certain neutral salt molecules, collagen can act as a nucleating agent, leading to the formation of fibrous structures. The collagen wound dressing can be used as a guide for directing new collagen deposition and microvascular growth.

Hemostatic properties: platelets interact with collagen to form a hemostatic plug.

B. Crosslinking agent

The compositions herein may additionally or alternatively include a crosslinker, also referred to herein as a crosslinker. The cross-linking agent typically provides a bond linking one polymer chain to another. These linkages may take the form of covalent or ionic bonds, and the polymer may be a synthetic polymer or a natural polymer (such as a protein). In polymer chemistry, "crosslinking" generally refers to the use of crosslinking to promote a change in the physical properties of a polymer. When "cross-linking" is used in biology, it is generally meant that reagents are used to link proteins together to examine protein-protein interactions or to enhance the overall biological material.

Exemplary cross-linking agents useful in the present disclosure include, but are not limited to, genipin, transglutaminase, iminoester cross-linking agent Xin Erya dimethyl amino acid (imidoester crosslinker dimethyl suberimidate), N-hydroxysuccinimide cross-linking agent BS3 (N-hydroxysuccinimide-ester crosslinker BS 3), and formaldehyde. Further, for example, the compositions herein may include one or more photocrosslinkable ingredients, for example, wherein UV light may be used to initiate crosslinking.

Crosslinking agent Xin Erya dimethyl amino acid, N-hydroxysuccinimide ester crosslinking agent BS3 and formaldehyde typically form bonds by inducing nucleophilic attack of the amine groups of the lysine and subsequent covalent bonding via the crosslinking agent. Zero length carbodiimide (EDC) cross-linker acts by converting the carboxyl groups to amine reactive isourea (isourea) intermediates that bind to lysine residues or other available primary amines. SMCC or its water-soluble analogue, sulfo-SMCC, can be used in the preparation of antibody-hapten conjugates for antibody development.

Genipin is a compound found in extracts of gardenia fruits. It is an aglycone derived from an iridoid glycoside (iridoid glycoside) known as geniposide in the fruit of gardenia (Gardenia jasminoides). Genipin is a natural cross-linking agent for cross-linking proteins, collagen, gelatin and chitosan. It has a low acute toxicity (acute toxicity) of LD50 i.v.382mg/kg in mice, and therefore toxicity is much lower than glutaraldehyde with many other commonly used synthetic crosslinkers. In addition, genipin is useful as a modulator of drug delivery and as an intermediate in alkaloid synthesis. In vitro experiments show that genipin blocks the action of enzymolysis coupled protein 2.

Another class of cross-linker/cross-linker transglutaminases is gamma-amido (gamma-carboxamide groups) (- (C=O) NH) which essentially catalyzes predominantly the side chain of the glutamate residue ₂ ) And subsequent release of ammonia (NH) ₃ ) Epsilon-amino (-NH) group of the side chain of the amino acid residue ₃ ) An enzyme that forms an isopeptide bond therebetween. The lysine and the glutamic acid residues must bind to the peptide or protein so that this crosslinking (between different molecules) or intramolecular (in the same molecule) reaction occurs. The bonds formed by transglutaminase are highly resistant to proteolytic degradation (proteolysis). These enzymes may also deamidate the glutamic acid residues to glutamic acid residues in the presence of water. For example, transglutaminase isolated from Streptomyces mobaraensis (Streptomyces mobaraensis) is a calcium independent enzyme. Mammalian transglutaminase among other transglutaminases requires Ca ²⁺ Ions act as cofactors.

Transglutaminases form widely cross-linked, generally insoluble protein polymers. These biopolymers are essential for the organism to create a barrier and a stable structure. Examples are thrombosis (factor XIII), and skin and hair. Catalytic reactions are generally considered irreversible and must be closely monitored via a control mechanism.

The amount of crosslinker/crosslinker can range from about 0.1mM to about 5mM, such as from about 0.5mM to about 3mM, from about 3mM to about 4mM, from about 1.5mM to about 2.5mM, or from about 1mM to about 2mM.

C. Other biological molecules

In addition to the agents already discussed, the hoc composition may also include one or more ingredients, such as hyaluronic acid, chondroitin sulfate, chitosan, and/or polyethylene glycol (PEG).

Hyaluronic acid, also known as hyaluronic acid, is an anionic, non-sulfated glycosaminoglycan, widely distributed in connective, epithelial and nervous tissues. It is unique in glycosaminoglycans in that it is non-sulfated, forms in the plasma membrane rather than the Gao Jishi body, and can be very large, such as human synovial membrane HA (human synovial HA) averaging about 7 million Da, or about two tens of thousands of disaccharide monomers per molecule, with other mentioned sources being 3 to 4 million Da. Hyaluronic acid, one of the major components of the extracellular matrix, contributes significantly to cell proliferation and migration, and may also be involved in the progression of certain malignant tumors.

Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) that includes alternating sugars (N-acetylgalactosamine and aldonic acid). It is generally found to attach to proteins as part of proteoglycans. The chondroitin chain can have more than 100 individual saccharides, each capable of being sulfated in different positions and amounts.

In some embodiments herein, the compositions comprise reduced amounts of one or more types of proteoglycans (e.g., biglycan, decorin, versican (versican), etc.) and/or sulfated GAGs associated with natural hUC tissue. In some examples, the composition does not include one or more types of proteoglycans and/or sulfated GAGs associated with the native hUC tissue. For example, the compositions herein may have reduced amounts of biglycan, decorin, and/or multifunctional proteoglycans compared to native hUC tissue. In some examples, the compositions herein are free (e.g., have a lower grade than detected) of one or more proteoglycans, such as biglycan, decorin, and/or multifunctional proteoglycans, present in the native hic tissue.

Chitosan is a linear polysaccharide consisting of randomly distributed β - (1→4) -linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units) obtained from chitin. It is prepared by treating shrimp and other crustacean shells with an alkaline material such as sodium hydroxide.

Polyethylene glycol (PEG) is a polyether compound, also known as polyethylene oxide (PEO) or Polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is generally denoted as H- (O-CH) ₂ -CH ₂ ) _n -OH. Researchers studying peripheral nerve and spinal cord injury are exploring the possibility that PEG could be used to fuse axons.

D. Enzyme treatment

After one or more treatment steps of the hoc tissue (e.g., mechanical impact with bead impact), the resulting hoc extract may include disrupted protein fragments, such as protein monomers and ECM fragments. Such ingredients may cause or lead to adverse reactions when administered to a patient. For example, certain components of the hoc extract may elicit or be associated with an inflammatory and/or immune response when injected at the site of nerve injury. The compositions herein may be prepared by treating with a protease to selectively remove, reduce or inactivate certain components while retaining biologically active components useful in promoting nerve repair. The components that may be at least partially, substantially or completely removed may include, for example, natural proteoglycans, such as decorin, biglycan and/or multifunctional proteoglycans.

Without being bound by theory, it is believed that protease treatment may result in degradation or reduced expression of proteoglycans, such as decorin, present in the ECM associated with inflammation via the TLR4 pathway. For example, decorin is believed to affect inflammatory signaling events (inflammatory signaling events), recognized as DAMP by inflammatory cells. Removal or reduction of the amount of proteoglycans (e.g., decorin) when the compositions herein are administered can reduce the risk of the subject developing an inflammatory response. Embodiments of the present disclosure may effectively guide the immune system to promote healing while reducing or preventing potentially damaging aspects of the immune response.

For example, the preparation of the hoc composition herein may comprise 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 enzymatic treatment may be performed prior to the introduction of the gel forming agent (as described above) and/or may be performed prior to the further introduction of one or more other ingredients comprising the collagen type and/or other gel forming agents as described above.

Collagenase is an enzyme that breaks peptide bonds in collagen. They contribute to the destruction of extracellular structures in the pathogenesis of bacteria such as Clostridium (Clostridium). They are thought to be a virulence factor (viral factor) that promotes the transmission of gas gangrene. They are usually targeted to connective tissue in muscle cells and other body organs. Once collagenase is secreted from cells, collagen, a key component of the extracellular matrix of animals, is produced by cleavage of procollagen by collagenase. This prevents the formation of large structures within the cell itself. In addition to being produced by certain bacteria, collagenases can also be produced by the body as part of its normal immune response. This production is induced by cytokines which stimulate fibroblasts and osteoblasts and other cells and can cause indirect tissue damage.

The concentration of protease may range from about 0.1 μg/mL to about 25 μg/mL, such as from about 0.5 μg/mL to about 20 μg/mL, from about 1 μg/mL to about 15 μg/mL, from about 0.5 μg/mL to about 5 μg/mL, from about 1 μg/mL to about 2 μg/mL, from about 2.5 μg/mL to about 5 μg/mL, from about 3 μg/mL to about 8 μg/mL, from about 5 μg/mL to about 15 μg/mL, from about 10 μg/mL to about 18 μg/mL, from about 15 μg/mL to about 22 μg/mL. Further, for example, the hoc extract can be treated with the protease for a period of time ranging from about 5 minutes to about 18 hours, such as from about 1 hour to about 16 hours, from about 4 hours to about 12 hours, from about 8 hours to about 16 hours, from about 12 hours to about 16 hours, from about 2 hours to about 8 hours, from about 30 minutes to about 5 hours, or from about 5 minutes to about 2 hours. In a non-limiting example, the hUC extract can be treated with from about 0.5 μg/mL to about 5 μg/mL protease for a period of from about 1 hour to about 16 hours.

According to some examples herein, a method of preparing a composition comprises at least partially inactivating a protease. For example, reagents such as ethylenediamine tetraacetic acid (EDTA), ilomastat (e.g., GM-6001 or) Or TIMP metallopeptidase inhibitor 1 (TIMP-1) to inactivate the enzyme. Such agents may be selected to target enzymes without damaging or minimizing damage to the bioactive components in the hoc extract.

E. Buffering agents

The compositions herein, e.g., modified extracts, may advantageously be combined with a buffer solution to maintain a target pH. Typically, the buffer solution (e.g., a pH buffer or a hydrogen ion buffer) is an aqueous solution of a mixture of a weak acid and its conjugate base, and vice versa. The pH change is small after adding a small amount of strong acid or alkali. Buffer solutions are used to maintain pH at nearly constant values in a variety of chemical applications.

The pH of a solution containing a buffer agent typically varies within a limited range, regardless of what may be present in the solution. In biological systems, this allows the enzyme to perform its intended function. If the pH of the solution rises or falls too much, the enzyme effectiveness may decrease in a process known as denaturation, which may be irreversible. Most biological samples for research are stored in a buffer solution, typically Phosphate Buffered Saline (PBS), at a pH of about 7.4.

Some exemplary buffers associated with physiological pH include citric acid and KH2PO4. By combining substances whose pKa values differ by only 2 or less and adjusting the pH, a wide range of buffers can be obtained. Citric acid is a useful ingredient of the buffer mixture because it has three pKa values, which are separated by a value less than 2. The buffer range can be extended by adding other buffers. From Na ₂ HPO ₄ And citric acid, the buffer ranges of the various McIlvaine buffer solutions are pH3 to 8. Other buffers suitable for use in the biological systems of the present disclosure include Lactated Ringer's Solution (LRS),TRIS (hydroxymethyl) aminomethane (TRIS), hank's Balanced Salt Solution, HBSS), grignard equilibrium salt solution (Gey's Balanced Salt Solution, GBSS), TAPSO, 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (4- (2-hydroxyyethyl) -1-piperazineethanesulfonic acid, HEPES), N- [ TRIS (hydroxymethyl) methyl]2-aminoethanesulfonic acid (N- [ tris (hydroxymethyl) methyl)]-2-aminoethanesulfonic acid, TES), 3- (N-morpholino) propanesulfonic acid (3- (N-morpholino) propanesulfonic acid, MOPS), piperazine-N, N '-bis (2-ethanesulfonic acid) (piperazine-N, N' -bis (2-ethanesulfonic acid, PIPES), dimethylarsinate (cacodinate) and 2- (N-morpholino) ethanesulfonic acid (2- (N-morpholino) ethanesulfonic acid, MES).

Fig. 12 shows an exemplary schematic of preparing a hoc gel composition according to the discussion above and the following examples. As shown, the composition may be prepared by mechanical processing of the hoc tissue (e.g., bead beating or other suitable technique) to obtain an extract, purifying the extract (which may include, for example, removal of cell debris), treating with a protease to reduce inflammatory components, and formulating the treated/purified hoc extract into a hydrogel suitable for injection.

IV. treatment method

In accordance with the present disclosure, there is also provided a method for treating tissue damage (including but not limited to muscle, tendon, ligament, etc.) in a subject, in particular for treating neuropathy. These methods aim to provide better treatment of pain such as peripheral neuropathy sites and to restore normal functional health to local tissues, compared to analgesic treatment or currently available amniotic membrane/childbirth tissue derived flowable products (e.g., orthoFlo of Mimedx). This aims to alleviate symptoms in patients who may and may not receive surgical intervention. As an injectable therapy, these methods are designed to be minimally invasive. The treatment is an injectable therapy with minimally invasive and diverse properties aimed at providing therapeutic accessibility to a range of anatomical regions of painful neuropathy in the body.

Thus, in at least one aspect, the method is directed to injecting a hoc derived material into a lesion site. The medical professional is able to evaluate the appropriate sites based on symptoms and diagnosis, which may include feet, hands, and joints such as shoulders, elbows, wrists, and knees. Similarly, the physician may determine the appropriate surgical procedure to deliver the agent, such as a combination of injection and image-guided delivery or surgical excision of the affected area.

As described above, the compositions herein may be formulated as a gel, such as a hydrogel, for injection at or near the site of injury or at a site in need of treatment. Formulating the composition as a gel may provide a longer treatment duration, e.g., the gel may stay longer at the intended target site due to factors such as its viscosity, cohesion, etc. Thus, sustained release can be achieved by using a gel composition. The gel may be formulated to provide desired characteristics such as degradation rate, density, rigidity, and/or cargo loading.

The method may comprise multiple treatments over a period of time, such as on a continuous or permanent, long-term basis. For example, any number of processes may be performed, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more processes. Such treatments may be spaced days, weeks, months or even years apart.

The method may further comprise a combination therapy combining administration using the hoc compositions described herein with one or more recognized neuropathy therapies, such as altering the diet and/or administering NSAIDs, analgesics, or anticonvulsants (discussed in more detail above and incorporated herein by reference).

V. examples

The following examples are included to illustrate exemplary embodiments of the present disclosure, however, not to be limiting in nature. It is to be understood that the present disclosure includes additional embodiments consistent with the foregoing description and the following examples. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute exemplary modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1

Preparation of hUC extract

Compositions were prepared from the hUC extracts as follows. The hUC membrane tissue samples were loaded into tubes and 700. Mu.L 1 XPBS (FIG. 1A) was added. Then the sample is arranged inThe sample holder was homogenized for 3 cycles at a speed of 6m/s for 60 seconds and then centrifuged at 5000Xg for 10 minutes (FIG. 1B).

Example 2

Preparation of hUC gel

Studies were performed to assess gel formation using the hoc extract and gel former. The hoc extract prepared according to example 1 was loaded into a syringe, extruded through a 26G needle (fig. 2), and polymerized into a gel at 37 ℃ using collagen as a gel former, for example, a formulation suitable for use as a minimally invasive injection therapy. Figure 3 depicts polymerization of collagen gel with the hoc membrane extract after incubation in a circular mold at 37 ℃ for 30 minutes. Fig. 4 depicts the polymerization of collagen gel with a hic membrane extract, wherein the pre-polymerized gel extract mixture was discharged from a syringe with a 26G needle, followed by incubation at 37 ℃ for 60 minutes.

Samples were prepared with varying amounts of collagen (1 mg/mL, 2mg/mL and 4 mg/mL) as gel former and incubated with and without genipin (2 mM) as cross-linker for 30 minutes to determine the effect on polymerization time. Absorbance at 410nm was used as an indication of the degree of polymerization. The results are shown in FIGS. 5 and 6. The gel loaded with the hoc extract was observed to polymerize in 8 minutes at 37 ℃ without the addition of the gel cross-linking agent and in 10 minutes with the addition of the cross-linking agent, indicating rapid in situ gel polymerization. In the absence of cross-linking agent, faster polymerization is observed with higher amounts of gel former. Samples prepared with 4mg/mL collagen and without genipin exhibited the fastest polymerization time, followed by samples prepared with 2mg/mL collagen and without genipin. Samples prepared using 4mg/mL and 2mg/mL collagen and using 2mM genipin exhibited similar polymerization times. Samples prepared with 1mg/mL collagen and 2mM genipin showed the slowest polymerization.

Further studies were performed on samples prepared with the hUC extract in combination with 4mg/mL collagen as a gel former and varying amounts of genipin (0, 0.5mM, 1mM, 2mM, and 5 mM) as a cross-linker to investigate the degradation of the polymeric gel over time (total 64 days). The samples were incubated in 1mL of digested collagenase solution at 37℃for 30 minutes over a period of 6 weeks. After polymerization and removal of excess liquid, studies were performed and then weighed weekly to determine initial mass and evaluate resistance to degradation. Fresh enzyme solution was used after each weighing. It was observed that the polymerized gel retained more than 50% of its mass under physiological conditions for 1 day without the addition of a cross-linking agent, and for up to 36 days with the addition of a gel cross-linking agent (fig. 7).

Example 3

Characterization of particle size of hUC extract

The hoc extract prepared according to example 1 was analyzed to determine the particle size distribution. Malvern Zetasizer Ultra analyzer was used to measure the average diameter by multi-angle dynamic light scattering (Multi Angle Dynamics Light Scattering, MADLS). FIG. 8A shows the results in which hUC membrane tissue (umbilical cord membrane (umbilical cord membrane, UCM)) was homogenized at 4m/s for 1 cycle, 2 cycles, and 3 cycles of samples at 60 seconds per cycle, showing that the size distribution was slightly broader with longer homogenization time. These data support consistency of sample preparation, e.g., consistent mixtures of components from homogenized hic membrane tissue. The sample contained a suspension of monodisperse particles, with a major portion of the particles ranging in diameter between 160 and 180nm (fig. 8A). The speed (4 m/s, 5m/s or 6 m/s) and the number of cycles (1, 3 or 5 cycles, 60 seconds each) were further investigated to investigate the effect on particle size. Depending on the preparation conditions, the extract contained variable particle dispersion and size distribution curves (fig. 8B).

Example 4

Immunomodulation of hUC extracts

For the ability of the hoc extract to modulate the immune response of human peripheral blood mononuclear cells derived from the human U937 cell line, in vitro studies were performed during two inflammatory attacks by altering their secretory response to the following immune biomarkers: IL-1β, IL-10 and MMP-9 (FIGS. 9 and 10A-10B). In the first study (fig. 9), the hoc membrane tissue was homogenized at 4000rpm per cycle for 3 minutes for a total of 4 to 5 cycles to prepare a hoc extract. In a second study (FIGS. 10A-10B), hUC membrane tissue was homogenized at 6m/s per cycle for 60 seconds for a total of 3 cycles to prepare hUC extract. For immunomodulation experiments, U937 cell cultures were treated with 20ng/mL phorbol 12-myristate13-acetate (PMA) for 48 hours to generate megaphagous cells, followed by administration of M1 differentiation stimulation (50 ng/mL LPS+10ng/mL IFN-. Gamma.) ("Stim") or no stimulation ("uninstim"). The M1 cultures were treated with the respective hUC extracts ("UC") for comparison. The results show that the hUC extract regulates the immune response of human U937 cell line-derived megaphagous cells in vitro during inflammatory attack by altering the secretory response of its immune biomarkers IL-1 beta and IL-10. Fig. 9 shows the results at the 48 hour time point. Fig. 10A to 10B show the results at 24, 48, 72 and 96 hour time points.

Example 5

Protease (collagenase) treatment of hUC extracts

The results of the study with collagenase treatment (25. Mu.g/mL) are shown in FIG. 11 for comparison with the control group. As shown, the concentration of the destroyed collagen polymer fragments treated with collagenase was reduced compared to the untreated control group. Samples treated with collagenase and control samples were homogenized at a rate setting of 4m/s (1 cycle of 60 minutes) and 6m/s (3 cycles of 60 minutes), and collagenase groups decreased in collagen polymer fragments relative to control groups at both rate settings.

Example 6

Protease treatment of hUC extracts (MMP-7)

Modified/treated hUC extracts were prepared to investigate MMP-7 protease treatment. The hUC tissue was mechanically treated by homogenization for 1 minute per cycle of 6m/s for a total of 3 cycles, followed by MMP-7 (0.8. Mu.g/mL) for 16 hours ("treated UC"). This is schematically shown in the process of fig. 12. Another control group without MMP-7 treatment ("UC") was prepared. As shown in fig. 13, the MMP-7 treated extract exhibited reduced expression of Decorin (DCN) compared to the untreated control group. Decorin was measured in an ELISA kit (ThermoFisher Scientific).

Additional hUC extracts were prepared and treated with MMP-7 under the same conditions as FIG. 13; the decorin performance scale shown in fig. 14A demonstrates a reduction in decorin of about 45% via protease treatment. Differences in the relative amounts of decorin may be related to differences between tissues, e.g. obtained from different donors or from the same tissue source. Fig. 14B shows the total protein content (μg/mL) measured for the treated samples and the control samples, showing similar grades. The total protein content was measured using BCA (bicinchoninic acid ) protein detection kit (ThrmoFisher Scientific) and 600 different proteins were tested, of which 448 protein biomarkers were identified. This indicates that MMP-7 treatment successfully removed the decorin component without significant damage to other proteins including potentially beneficial components.

Example 7

Immunomodulation of protease-treated hUC extracts

The samples of figures 14A to 14B prepared according to example 6 were further investigated in an immunomodulatory assay to determine their effect on the immune biomarkers IL-1 beta and IL-10. U937 cell cultures were treated with 20ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 hours to generate megaphagous cells, followed by M1 differentiation stimulation (50 ng/mL LPS+10ng/mL IFN-. Gamma.) ("Stim") or no stimulation ("Unstim"). The M1 culture was also treated with untreated hUC extract ("UC") and MMP-7 treated hUC extract ("treated UC"). This measurement is schematically illustrated in fig. 15. The results in FIGS. 16A and 16B show that protease treatment resulted in a decrease in the secretion response of IL-1. Beta. And IL-10. Without being bound by theory, it is believed that the lower amount of decorin in the treated hoc extract results in a reduced inflammatory response.

Additional studies were performed to investigate the effect of residual proteases from the treated hUC extract on IL-1β and IL-10. The hUC extract was prepared and treated with MMP-7 as described in example 6. The decorin grade of untreated hUC extract control ("UC") and MMP-7 treated hUC extract ("treated UC") was measured as described in example 6. The results shown in fig. 17 demonstrate the reduction of decorin in the treated hic extract.

Immunomodulatory assays as described above were performed on treated and untreated hUC extract samples. FIGS. 18A and 18B report the grades of IL-1β and IL-10 for unstimulated cultures without hUC extracts ("Unstim"), stimulated cultures without hUC extracts ("Stim"), stimulated cultures with untreated hUC extracts ("UC"), stimulated cultures with MMP-7 treated hUC extracts ("treated UC"), and stimulated cultures with MMP-7 (0.8 μg/mL) and without hUC extracts ("MMP-7"), respectively. These results are consistent with those of figures 16A-16B, showing that protease treatment of the hic extract resulted in significant reduction of IL-1 β and IL-10. No effect on IL-1β was observed for MMP-7 alone compared to the "Stim" control group (fig. 18A), and a slight (no statistical significance) decrease in IL-10 was caused relative to the control group (fig. 18B). This indicates that the reduction of inflammatory response is not due to residual protease supporting the conclusion that removal of decorin via protease treatment would reduce immune response.

All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure and the knowledge of those skilled in the art. Although the compositions and methods of this disclosure have been described in terms of exemplary embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. It will be apparent to those skilled in the art that all such similar substitutes and modifications are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims (36)

1. A physiologically buffered human umbilical cord (hoc) extract composition, comprising micronized particles of an ECM-degrading protease-treated hoc.

2. The composition of claim 1, wherein the hoc comprises a hoc membrane, a hoc matrix, or a combination of a hoc membrane and a hoc matrix.

3. The composition of claim 1, further comprising a gel former, such as an in situ polymerized gel former.

4. A composition according to claim 3 wherein the gel forming agent is present at about 0.1 to 8 mg/ml.

5. The composition of claim 1, further comprising a cross-linking agent, such as genipin or transglutaminase.

6. The composition of claim 3, wherein the gel-forming agent comprises one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, collagen XXVII, polyethylene glycol, polylactic-co-glycolic acid, poly (ethylene glycol) diacrylate, methacrylated gelatin, or methacrylated hyaluronic acid.

7. The composition of claim 1, wherein the ECM-degrading protease treated fruc comprises a fruc membrane and the gel-forming agent is not fibrin.

8. The composition of claim 1, wherein the composition is a physiological saline-based suspension buffered at about pH 7.2 to 7.4, or wherein the composition is formulated as a gel such as a hydrogel.

9. The composition of claim 1, wherein the composition further comprises 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 XIX, collagen XX, collagen XXI, collagen XXII, collagen XXIII, collagen XXV, collagen XXVI, and/or collagen XXVIII.

10. The composition of claim 1, wherein a majority of the micronized particles have a diameter between about 80nm and about 180nm, such as between about 140nm and about 160 nm.

11. A method of producing a human umbilical cord (hic) extract comprising:

(a) Providing a hUC membrane and/or a hUC matrix;

(b) Mechanically striking the hoc membrane and/or the hoc matrix to produce micronized particles; and

(c) Treatment with ECM degrading protease: (i) the composition of step (a) prior to mechanical impact; (ii) The composition of step (b) during a mechanical impact; or (iii) one or more of the micronized particles resulting from step (b).

12. The method of claim 11, further comprising inactivating the protease.

13. A method according to claim 12, wherein a gel former, such as an in situ polymeric gel former, is added after inactivation of the ECM degrading protease.

14. The method of claim 13, further comprising polymerizing the in situ gel forming agent.

15. The method of claim 14, wherein polymerization occurs in the presence of a crosslinking agent.

16. The method of claim 15, wherein the cross-linking agent is genipin or transglutaminase.

17. The method of claim 13, wherein the gel-forming agent comprises one or more of fibrin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VIII, collagen X, collagen XI, collagen XXIV, collagen XXVII, polyethylene glycol, polylactic-co-glycolic acid, poly (ethylene glycol) diacrylate, methacrylated gelatin, or methacrylated hyaluronic acid.

18. The method of claim 13, wherein the gel-forming agent is present at about 0.1 to 8 mg/ml.

19. The method of claim 11, wherein 0.5 to 1.0cm is provided in step (a) ² And/or a hUC matrix.

20. The method of claim 11, wherein the hoc membrane and/or the hoc matrix provided in step (a) is dispersed in a physiological saline-based suspension buffered between about pH 6.0 and 8.0.

21. The method of claim 11, wherein the mechanical impact is performed for between 1 and about 5 cycles, each cycle lasting about 60 seconds, at a speed ranging from about 3400RPM to about 3700RPM.

22. The method of claim 21, further comprising centrifuging the micronized particles prior to ECM degrading protease treatment.

23. The method of claim 11, wherein the ECM degrading protease is a collagenase or a Matrix Metalloproteinase (MMP).

24. The method of claim 23, wherein the protease is one or more of collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V, collagenase VI, collagenase VII, MMP-2, MMP-3, or MMP-7.

25. The method of claim 23, wherein the collagenase is one or more of collagenase I or collagenase III.

26. The method of claim 11, wherein after inactivation of the ECM degrading protease, one or more of hyaluronic acid, chondroitin sulfate, chitosan, PEG, collagen I, collagen II, collagen III, collagen IV, collagen V, 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 XXII, collagen XXIII, collagen XXVI, or collagen XXVIII is added to the composition.

27. The method of claim 11, wherein the mechanical impact provides a majority of micronized particles having a diameter between about 80nm and about 180nm, such as between about 140nm and about 160 nm.

28. A method of treating peripheral neuropathy comprising injecting the composition of claim 1 into a peripheral neuropathy site in a subject.

29. A method of treating peripheral neuropathy comprising injecting the composition prepared by the method of claim 11 into a peripheral neuropathy site in a subject.

30. The method of claim 28, wherein the subject is a human.

31. The method of claim 28, further comprising treating the subject with a second therapy such as analgesic therapy, NSAID therapy, or anticonvulsant drug.

32. The method of claim 28, further comprising injecting said composition a second time into said site.

33. A composition comprising micronized particles of human umbilical cord (hoc) tissue and a buffer, wherein the composition is formulated for injection into a subject.

34. The composition of claim 33, wherein the composition is formulated as a gel.

35. The composition of claim 33, wherein the hoc tissue has been protease treated.

36. The composition of claim 33, wherein the composition comprises less than 1.0 μg/mL of decorin.