Human Umbilical Cord Compositions and Methods for Intra-Articular Therapy

Abstract

Described are aqueous and non-immunogenic injectable compositions derived from human umbilical cord and methods for their production. The non-immunogenic compositions are used for the treatment of joints in human subjects in need of joint treatment. The non-immunogenic compositions may comprise an aqueous human umbilical cord filtrate prepared without using exogenous enzymes that cause exogenous enzymatic degradation / digestion.

Description

Technical Field

[0001] The present invention generally relates to the field of umbilical cord-derived compositions, and more particularly to non-immunogenic compositions obtained from fresh human umbilical cords for intra-articular injection and intra-articular treatment for a subject in need thereof.

Background Art

[0002] In the medical field, the uses of compositions derived from human umbilical cords are diverse. For example, these advantageous uses may include harvesting stem cells therefrom for treatments applied to various blood diseases, cancers, and immune system disorders. During the preparation of human umbilical cord-derived compositions, umbilical cord tissue is often placed in harsh mechanical and enzymatic treatment conditions, and under such conditions, certain cells (e.g., stem cells) may be separated from the umbilical cord tissue, expanded / cultured, and cryopreserved, resulting in a dramatic change in the initial endogenous cell profile and extracellular profile of the umbilical cord tissue. Furthermore, exogenous additives, such as various growth factors / cytokines, including interferon alpha (INF-α), may be included in these isolates either before, during, or after the treatment of these umbilical cord isolates, thereby further changing the isolates compared to the original umbilical cord tissue. These changes can result in the loss of the original endogenous cell profile and / or extracellular profile of the umbilical cord tissue, which may also reduce the effectiveness of the desired treatment.

Summary of the Invention

[0003] From the above perspective, an object of the present invention is to provide a human umbilical cord-derived composition that mimics, contains, and / or retains an extracellular profile similar to the (e.g., in vivo) endogenous profile of the human umbilical cord, particularly as compared to the various umbilical cord isolates described above. These compositions described herein are prepared using fresh human umbilical cords (collected and processed within 48 to 72 hours after extraction from a human subject) and, unlike the compositions of the prior art, advantageously do not undergo biochemical digestion and / or enzymatic digestion, so that the compositions contain and / or retain most of the extracellular profile (of the in vivo human umbilical cord).

[0004] In certain embodiments, an aqueous, non-immunogenic, injectable composition for intra-articular treatment of a human subject in need of joint treatment is disclosed. In certain embodiments, the aqueous and non-immunogenic composition comprises an aqueous human umbilical cord filtrate obtained from a human umbilical cord. In certain embodiments, the aqueous and non-immunogenic composition consists of an aqueous human umbilical cord filtrate obtained from a human umbilical cord. In certain embodiments, the aqueous and non-immunogenic composition consists essentially of an aqueous human umbilical cord filtrate obtained from a human umbilical cord.

[0005] In certain embodiments, the aqueous human umbilical cord filtrate is a solution that shows no sedimentation, separation, and / or precipitation after 12 months, 24 months, up to 60 months, or more when stored at -20 °C or -80 °C. In certain embodiments, the aqueous human umbilical cord filtrate is a solution that shows no sedimentation, separation, and / or precipitation after 10 days, 20 days, 30 days, up to 60 days, or more when stored at 4 °C to 8 °C. In certain embodiments, since no exogenous enzymes are introduced into the aqueous human umbilical cord filtrate during its preparation, exogenous enzyme degradation / digestion is avoided. In certain embodiments, the aqueous human umbilical cord filtrate is obtained by filtering the crushed human umbilical cord. In certain embodiments, the aqueous and non-immunogenic composition has microparticles less than 100 μm. In certain embodiments, the human umbilical cord is double-filtered to obtain the aqueous human umbilical cord filtrate. In certain embodiments, the human umbilical cord is triple-filtered to obtain the human umbilical cord filtrate. In certain embodiments, the aqueous and non-immunogenic injectable composition has microparticles less than 50 μm. In certain embodiments, the aqueous and non-immunogenic injectable composition has microparticles less than 35 μm. In certain embodiments, the aqueous and non-immunogenic injectable composition has microparticles less than 10 μm. In certain embodiments, the aqueous and non-immunogenic injectable composition is sterile. In certain embodiments, the aqueous and non-immunogenic injectable composition is acellular.

[0006] In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×10 2 pg / mL to 2.5×10 3 pg / mL of vascular endothelial growth factor receptor (VEGFR1), 2.5×10 2 pg / mL to 1.42×10 4 pg / mL of hepatocyte growth factor (HGF), 8.13×10 2 pg / mL to 5.15×10 4 pg / mL of interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)), 2.0×10 1 pg / mL to 1.58×10 3 pg / mL of platelet-derived growth factor-BB (PDGF-BB), 4.73×101 pg / ml to 2.07×10 3 basic fibroblast growth factor (bFGF) at a concentration of pg / ml, 1.51×10 7 pg / mL to 3.5×10 8 contains at least one of endogenous hyaluronic acid (HA) at a concentration of pg / mL, and any combination thereof.

[0007] In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×10 2 pg / mL to 2.5×10 3 vascular endothelial growth factor receptor-1 (VEGFR1) at a concentration of pg / mL, 2.5×10 2 pg / mL to 1.42×10 4 hepatocyte growth factor (HGF) at a concentration of pg / mL, 8.13×10 2 pg / mL to 5.15×10 4 interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) at a concentration of pg / mL, 2.0×10 1 pg / mL to 1.58×10 3 platelet-derived growth factor-BB (PDGF-BB) at a concentration of pg / mL, 4.73×10 1 pg / ml to 2.07×10 3 basic fibroblast growth factor (bFGF) at a concentration of pg / ml, 1.51×10 7 pg / mL to 3.5×10 8 contains at least two of endogenous hyaluronic acid (HA) at a concentration of pg / mL, and any combination thereof.

[0008] In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×10 2 pg / mL to 2.5×10 3 vascular endothelial growth factor receptor-1 (VEGFR1) at a concentration of pg / mL, 2.5×10 2 pg / mL to 1.42×10 4 hepatocyte growth factor (HGF) at a concentration of pg / mL, 8.13×10 2pg / mL to 5.15×10 4 An interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) at a concentration of 2.0×10 1 pg / mL to 1.58×10 3 Platelet-derived growth factor-BB (PDGF-BB) at a concentration of 4.73×10 1 pg / ml to 2.07×10 3 Basic fibroblast growth factor (bFGF) at a concentration of 1.51×10 7 pg / mL to 3.5×10 8 Endogenous hyaluronic acid (HA) at a concentration of pg / mL, and at least three of any combination thereof.

[0009] In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×10 2 pg / mL to 2.5×10 3 Vascular endothelial growth factor receptor-1 (VEGFR1) at a concentration of 2.5×10 2 pg / mL to 1.42×10 4 Hepatocyte growth factor (HGF) at a concentration of 8.13×10 2 pg / mL to 5.15×10 4 An interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) at a concentration of 2.0×10 1 pg / mL to 1.58×10 3 Platelet-derived growth factor-BB (PDGF-BB) at a concentration of 4.73×10 1 pg / ml to 2.07×10 3 Basic fibroblast growth factor (bFGF) at a concentration of 1.51×10 7 pg / mL to 3.5×10 8 Endogenous hyaluronic acid (HA) at a concentration of pg / mL, and at least four of any combination thereof.

[0010] In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×102 pg / mL to 2.5×10 3 pg / mL concentration of vascular endothelial growth factor receptor-1 (VEGFR1), 2.5×10 2 pg / mL to 1.42×10 4 pg / mL concentration of hepatocyte growth factor (HGF), 8.13×10 2 pg / mL to 5.15×10 4 pg / mL concentration of interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)), 2.0×10 1 pg / mL to 1.58×10 3 pg / mL concentration of platelet-derived growth factor-BB (PDGF-BB), 4.73×10 1 pg / ml to 2.07×10 3 pg / ml concentration of basic fibroblast growth factor (bFGF), 1.51×10 7 pg / mL to 3.5×10 8 pg / mL concentration of endogenous hyaluronic acid (HA), and at least five of any combination thereof.

[0011] In certain embodiments, the aqueous human umbilical filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×10 2 pg / mL to 2.5×10 3 pg / mL concentration of vascular endothelial growth factor receptor-1 (VEGFR1), 2.5×10 2 pg / mL to 1.42×10 4 pg / mL concentration of hepatocyte growth factor (HGF), 8.13×10 2 pg / mL to 5.15×10 4 pg / mL concentration of interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)), 2.0×10 1 pg / mL to 1.58×10 3 pg / mL concentration of platelet-derived growth factor-BB (PDGF-BB), 4.73×10 1 pg / ml to 2.07×10 3 pg / ml concentration of basic fibroblast growth factor (bFGF), 1.51×10 7 pg / mL to 3.5×10 8It contains at least six of endogenous hyaluronic acid (HA) at a concentration of pg / mL, and any combination thereof.

[0012] In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×10 2 pg / mL to 2.5×10 3 vascular endothelial growth factor receptor-1 (VEGFR1) at a concentration of pg / mL, 2.5×10 2 pg / mL to 1.42×10 4 hepatocyte growth factor (HGF) at a concentration of pg / mL, 8.13×10 2 pg / mL to 5.15×10 4 interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) at a concentration of pg / mL, 2.0×10 1 pg / mL to 1.58×10 3 platelet-derived growth factor-BB (PDGF-BB) at a concentration of pg / mL, 4.73×10 1 pg / ml to 2.07×10 3 basic fibroblast growth factor (bFGF) at a concentration of pg / ml, 1.51×10 7 pg / mL to 3.5×10 8 It contains at least seven of endogenous hyaluronic acid (HA) at a concentration of pg / mL, and any combination thereof.

[0013] In certain embodiments, the aqueous human umbilical cord filtrate is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.0×10 2 pg / mL to 2.5×10 3 vascular endothelial growth factor receptor-1 (VEGFR1) at a concentration of pg / mL, 2.5×10 2 pg / mL to 1.42×10 4 hepatocyte growth factor (HGF) at a concentration of pg / mL, 8.13×10 2 pg / mL to 5.15×10 4 interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) at a concentration of pg / mL, 2.0×10 1 pg / mL to 1.58×10 3Platelet-derived growth factor-BB (PDGF-BB) at a concentration of pg / mL, 4.73×10 1 pg / ml to 2.07×10 3 Basic fibroblast growth factor (bFGF) at a concentration of pg / ml, 1.51×10 7 pg / mL to 3.5×10 8 Endogenous hyaluronic acid (HA) at a concentration of pg / mL, and at least eight of any combination thereof.

[0014] In certain embodiments, the aqueous human umbilical cord filtrate further comprises an isotonic solution. In certain embodiments, the isotonic solution is phosphate buffered saline (1×PBS), lactated Ringer's (sodium chloride 6 g / L, sodium lactate 3.1 g / L, potassium chloride 0.3 g / L, calcium chloride 0.2 g / L, pH 6.5), isotonic saline (0.9 wt% sodium chloride), plasmalyte® (sodium chloride 5.26 g / L, potassium chloride 0.37 g / L, magnesium chloride hexahydrate 0.30 g / L, sodium acetate trihydrate 3.68 g / L, sodium gluconate 5.02 g / L, pH 7.4), or Normosol® (sodium chloride 5.26 g / L, KCl 0.37 g / L, magnesium chloride 0.30 g / L, sodium acetate anhydrous 2.22 g / L, sodium gluconate 5.02 g / L, pH 7.4). In certain embodiments, the aqueous human umbilical cord filtrate further comprises amniotic fluid.

[0015] In certain embodiments, the filtrate described immediately above may be further combined with an isotonic solution (diluent) capable of further diluting the concentration of the growth factor to a desired range. In certain embodiments, when an isotonic solution is included, the composition is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.23×10 2 pg / mL to 1.9×10 3 Vascular endothelial growth factor receptor (VEGFR1) in the concentration range of pg / mL, 3.47×10 2 pg / mL to 1.0×10 3 Hepatocyte growth factor (HGF) in the concentration range of pg / mL, 1.35×10 3 pg / mL to 3.43×10 3Interleukin antagonists (interleukin-1 receptor antagonist (IL-1ra)) in the concentration range of pg / mL, 2.0×10 1 pg / mL to 1.05×10 3 Platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, 7.95×10 1 pg / mL to 1.83×10 3 Basic fibroblast growth factor (bFGF) in the concentration range of pg / mL, 1.51×10 7 pg / mL to 1.0×10 8 Endogenous hyaluronic acid (HA) in the concentration range of pg / mL, and at least one of any combination thereof.

[0016] In certain embodiments, when an isotonic solution is included, the composition is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.23×10 2 pg / mL to 1.9×10 3 Vascular endothelial growth factor receptor (VEGFR1) in the concentration range of pg / mL, 3.47×10 2 pg / mL to 1.0×10 3 Hepatocyte growth factor (HGF) in the concentration range of pg / mL, 1.35×10 3 pg / mL to 3.43×10 3 Interleukin antagonists (interleukin-1 receptor antagonist (IL-1ra)) in the concentration range of pg / mL, 2.0×10 1 pg / mL to 1.05×10 3 Platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, 7.95×10 1 pg / mL to 1.83×10 3 Basic fibroblast growth factor (bFGF) in the concentration range of pg / mL, 1.51×10 7 pg / mL to 1.0×10 8 Endogenous hyaluronic acid (HA) in the concentration range of pg / mL, and at least two of any combination thereof.

[0017] In certain embodiments, when an isotonic solution is included, the composition comprises acellular Wharton's jelly, exosomes, endogenous growth factors, vascular endothelial growth factor receptor (VEGFR1) in a concentration range of 1.23×10 2 pg / mL to 1.9×10 3 pg / mL, hepatocyte growth factor (HGF) in a concentration range of 3.47×10 2 pg / mL to 1.0×10 3 pg / mL, interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) in a concentration range of 1.35×10 3 pg / mL to 3.43×10 3 pg / mL, platelet-derived growth factor-BB (PDGF-BB) in a concentration range of 2.0×10 1 pg / mL to 1.05×10 3 pg / mL, basic fibroblast growth factor (bFGF) in a concentration range of 7.95×10 1 pg / mL to 1.83×10 3 pg / mL, endogenous hyaluronic acid (HA) in a concentration range of 1.51×10 7 pg / mL to 1.0×10 8 pg / mL, and at least three of any combination thereof.

[0018] In certain embodiments, when an isotonic solution is included, the composition comprises acellular Wharton's jelly, exosomes, endogenous growth factors, vascular endothelial growth factor receptor (VEGFR1) in a concentration range of 1.23×10 2 pg / mL to 1.9×10 3 pg / mL, hepatocyte growth factor (HGF) in a concentration range of 3.47×10 2 pg / mL to 1.0×10 3 pg / mL, interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) in a concentration range of 1.35×10 3 pg / mL to 3.43×10 3 pg / mL, platelet-derived growth factor-BB (PDGF-BB) in a concentration range of 2.0×10 1 pg / mL to 1.05×10 3 pg / mL, and 7.95×10 1 pg / mL to 1.83×103 basic fibroblast growth factor (bFGF) in the concentration range of pg / mL, 1.51×10 7 pg / mL to 1.0×10 8 endogenous hyaluronic acid (HA) in the concentration range of pg / mL, and at least four of any combination thereof.

[0019] In certain embodiments, when an isotonic solution is included, the composition is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.23×10 2 pg / mL to 1.9×10 3 vascular endothelial growth factor receptor (VEGFR1) in the concentration range of pg / mL, 3.47×10 2 pg / mL to 1.0×10 3 hepatocyte growth factor (HGF) in the concentration range of pg / mL, 1.35×10 3 pg / mL to 3.43×10 3 interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) in the concentration range of pg / mL, 2.0×10 1 pg / mL to 1.05×10 3 platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, 7.95×10 1 pg / mL to 1.83×10 3 basic fibroblast growth factor (bFGF) in the concentration range of pg / mL, 1.51×10 7 pg / mL to 1.0×10 8 endogenous hyaluronic acid (HA) in the concentration range of pg / mL, and at least five of any combination thereof.

[0020] In certain embodiments, when an isotonic solution is included, the composition is acellular Wharton's jelly, exosomes, endogenous growth factors, 1.23×10 2 pg / mL to 1.9×10 3 vascular endothelial growth factor receptor (VEGFR1) in the concentration range of pg / mL, 3.47×10 2 pg / mL to 1.0×10 3 hepatocyte growth factor (HGF) in the concentration range of pg / mL, 1.35×10 3pg / mL to 3.43×10 3 An interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) in the concentration range of pg / mL, 2.0×10 1 pg / mL to 1.05×10 3 Platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, 7.95×10 1 pg / mL to 1.83×10 3 Basic fibroblast growth factor (bFGF) in the concentration range of pg / mL, 1.51×10 7 pg / mL to 1.0×10 8 Endogenous hyaluronic acid (HA) in the concentration range of pg / mL, and at least six of any combination thereof.

[0021] In certain embodiments, when an isotonic solution is included, the composition comprises acellular Wharton's jelly, exosomes, endogenous growth factors, 1.23×10 2 pg / mL to 1.9×10 3 Vascular endothelial growth factor receptor (VEGFR1) in the concentration range of pg / mL, 3.47×10 2 pg / mL to 1.0×10 3 Hepatocyte growth factor (HGF) in the concentration range of pg / mL, 1.35×10 3 pg / mL to 3.43×10 3 An interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) in the concentration range of pg / mL, 2.0×10 1 pg / mL to 1.05×10 3 Platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, 7.95×10 1 pg / mL to 1.83×10 3 Basic fibroblast growth factor (bFGF) in the concentration range of pg / mL, 1.51×10 7 pg / mL to 1.0×10 8 Endogenous hyaluronic acid (HA) in the concentration range of pg / mL, and at least seven of any combination thereof.

[0022] In certain embodiments, when an isotonic solution is included, the composition comprises acellular Wharton's jelly, exosomes, endogenous growth factors, vascular endothelial growth factor receptor (VEGFR1) in a concentration range of 1.23×10 2 pg / mL to 1.9×10 3 pg / mL, hepatocyte growth factor (HGF) in a concentration range of 3.47×10 2 pg / mL to 1.0×10 3 pg / mL, interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) in a concentration range of 1.35×10 3 pg / mL to 3.43×10 3 pg / mL, platelet-derived growth factor-BB (PDGF-BB) in a concentration range of 2.0×10 1 pg / mL to 1.05×10 3 pg / mL, basic fibroblast growth factor (bFGF) in a concentration range of 7.95×10 1 pg / mL to 1.83×10 3 pg / mL, endogenous hyaluronic acid (HA) in a concentration range of 1.51×10 7 pg / mL to 1.0×10 8 pg / mL, and at least eight of any combination thereof.

[0023] In certain embodiments, the composition further comprises an effective amount of exogenous hyaluronic acid to restore endogenous extracellular matrix function, to restore endogenous collagen function, and / or to treat and / or alleviate the symptoms of an inflammatory disease in the subject in the joint cavity. In certain embodiments, the exogenous hyaluronic acid is present in the composition at a concentration of 0.5 wt% to 5.0 wt%. In other embodiments, the composition has an exogenous hyaluronic acid at a concentration of about 0.75 wt% to about 4.0 wt%. In other embodiments, the composition has an exogenous hyaluronic acid at a concentration of about 1.5 wt% to about 3.5 wt%. In other embodiments, the composition has an exogenous hyaluronic acid at a concentration of about 2 wt% to about 3.0 wt%.

[0024] In certain embodiments, the composition is configured as a sterile injectable composition for intra-articular treatment of a human subject in need of joint treatment. In this embodiment, the composition is preferably a viscous aqueous composition, which has a viscosity sufficient to be injected into the joint cavity while minimizing pain and / or discomfort of the subject, and has a thickness and consistency sufficient to improve and / or repair the extracellular matrix, and / or provide additional support to the extracellular matrix, promote water absorption into the extracellular matrix, and provide collagen to the joint cavity, thereby promoting the cushioning and lubricity of the joint and providing shock absorption to the joint cavity of the subject.

[0025] In certain embodiments, a method of injecting an aqueous and non-immunogenic injectable composition for joint treatment into a human subject in need of joint treatment is also disclosed. The method includes step (a) of injecting an effective amount of the composition into the joint cavity of the affected joint of the human subject to improve and / or restore the endogenous extracellular matrix function in the joint cavity, to improve and / or restore the endogenous collagen function in the joint cavity, and / or to treat and / or alleviate the symptoms of an inflammatory disease of the subject, or any combination thereof.

[0026] In certain embodiments, the composition is sterile. In certain embodiments, the composition further comprises an effective amount of exogenous hyaluronic acid to improve and / or restore the endogenous extracellular matrix function in the joint cavity, to improve and / or restore the endogenous collagen function in the joint cavity, and to treat and / or alleviate the symptoms of an inflammatory disease of a human subject. In certain embodiments, the exogenous hyaluronic acid is present in the composition at a concentration of 0.5 wt% to 5.0 wt%. In other embodiments, the composition has an exogenous hyaluronic acid at a concentration of about 0.75 wt% to about 4.0 wt%. In other embodiments, the composition has an exogenous hyaluronic acid at a concentration of about 1.5 wt% to about 3.5 wt%. In other embodiments, the composition has an exogenous hyaluronic acid at a concentration of about 2 wt% to about 3.0 wt%.

[0027] In certain embodiments, the joint treatment is provided by intra-articular injection. This method involves aseptically injecting a composition into and / or near the joint cavity of the affected joint of a human subject in need of joint treatment.

[0028] In certain embodiments, injecting an effective amount of the composition into the affected joint of a human subject to restore the endogenous extracellular matrix function in the joint cavity, to restore the endogenous collagen function in the joint cavity, and / or to treat and / or alleviate the symptoms of an inflammatory disease of the subject is repeated at predetermined time intervals. In certain embodiments, step (a) is repeated daily. In certain embodiments, step (a) is repeated weekly. In certain embodiments, step (a) is repeated monthly. In certain embodiments, step (a) is repeated bi-weekly. In certain embodiments, step (a) is repeated semi-weekly. In certain embodiments, step (a) is repeated bi-monthly. In certain embodiments, step (a) is repeated semi-monthly.

[0029] In certain embodiments, an effective amount of the composition for restoring endogenous extracellular matrix function in the joint cavity, for restoring endogenous collagen function in the joint cavity, and / or for treating and / or alleviating the symptoms of an inflammatory disease in a human subject is 0.5 mL. In certain embodiments, an effective amount of the composition for restoring endogenous extracellular matrix function in the joint cavity, for restoring endogenous collagen function in the joint cavity, and / or for treating and / or alleviating the symptoms of an inflammatory disease in a human subject is 1 mL. In certain embodiments, an effective amount of the composition for restoring endogenous extracellular matrix function in the joint cavity, for restoring endogenous collagen function in the joint cavity, and / or for treating and / or alleviating the symptoms of an inflammatory disease in a human subject is 2 mL. In certain embodiments, an effective amount of the composition for restoring endogenous extracellular matrix function in the joint cavity, for restoring endogenous collagen function in the joint cavity, and / or for treating and / or alleviating the symptoms of an inflammatory disease in a human subject is 3 mL. In certain embodiments, an effective amount of the composition for restoring endogenous extracellular matrix function in the joint cavity, for restoring endogenous collagen function in the joint cavity, and / or for treating and / or alleviating the symptoms of an inflammatory disease in a human subject is 4 mL. In certain embodiments, an effective amount of the composition for restoring endogenous extracellular matrix function in the joint cavity, for restoring endogenous collagen function in the joint cavity, and / or for treating and / or alleviating the symptoms of an inflammatory disease in a human subject is 5 mL.

[0030] In certain embodiments, the human subject in need of joint treatment has osteoarthritis, rheumatoid arthritis, psoriatic arthritis, lupus, gout, or any combination thereof.

[0031] Embodiments of the present invention may include one or more or any combination of the above features and configurations.

[0032] Additional features, aspects, and advantages of the present invention are described in the following detailed description, some of which will be readily apparent to those skilled in the art from the description or will be recognized by practicing the invention as described herein. It is to be understood that both the foregoing summary and the following detailed description present various embodiments of the present invention and are intended to provide an overview or framework for understanding the nature and characteristics of the claimed invention. The accompanying drawings are included to provide a further understanding of the present invention and are incorporated herein and constitute a part of this specification.

Brief Description of the Drawings

[0033] These and other features, aspects, and advantages of the present invention will be better understood by reading the following detailed description of the invention with reference to the accompanying drawings.

[0034]

Figure 1

[0035]

Figure 2

Mode for Carrying Out the Invention

[0036] The present invention will now be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. However, the invention may be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. The exemplary embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the various figures, like reference numerals refer to like elements. Further, in this specification and the following claims, a number of terms are used which have the following meanings as defined.

[0037] Note that the singular forms "a", "an", and "the" as used in this specification and the appended claims include the plural unless the context clearly dictates otherwise.

[0038] Concentrations, amounts, and other numerical data may be presented or expressed herein in a range format. Such a range format is used for merely convenience and brevity and should, therefore, be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also all the individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly recited. By way of example, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values of "about 1 to about 5" but also the individual values and sub-ranges within the specified range. Thus, this numerical range includes not only the individual values of 2, 3, 4, etc. and sub-ranges such as 1-3, 2-4, 3-5, etc., but also the individual values of 1, 2, 3, 4, 5. The same principle applies to ranges that indicate only one numerical value as the minimum or maximum value. Further, such an interpretation should apply regardless of the breadth of the range or the nature of the property being described.

[0039] The compositions and methods described herein may comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, or limitations described herein. (Composition)

[0040] Disclosed herein are human umbilical cord-derived compositions that, among other things, retain an extracellular profile similar to the endogenous profile of an in vivo human umbilical cord, as compared to the various umbilical cord isolates described above. These compositions are prepared using fresh human umbilical cords (collected and processed within 48 to 72 hours after extraction from a human subject) and, unlike prior art compositions, have advantageously not been subjected to biochemical and / or enzymatic digestion and thus contain and / or retain a majority of the extracellular profile (as compared to the endogenous profile of an in vivo human umbilical cord). Further, since these compositions are easy and convenient to administer (e.g., preparation at the point of use and use in a dental office, clinic, or emergency room) and have non-immunogenic properties, they can be used in a number of different medical purposes and treatments. These medical purposes and treatments include, but are not limited to, intra-articular treatment of a human subject in need of joint treatment to improve and / or restore the endogenous extracellular matrix function in the joint cavity, to improve and / or restore the endogenous collagen function in the joint cavity, to treat and / or alleviate the symptoms of an inflammatory disease of the subject, or for any combination thereof.

[0041] Disclosed herein are compositions (e.g., compositions) that may be configured for intra-articular treatment and that contain an aqueous human umbilical cord filtrate. In certain embodiments, exogenous enzymatic degradation / digestion is avoided because no exogenous enzymes are introduced thereto during the preparation of the composition, and in particular, the composition will have a better endogenous extracellular profile (similar to that of an in vivo human umbilical cord) as compared to conventional compositions using umbilical cord tissue and / or umbilical cord-derived cells.

[0042] Hyaluronic acid is a major component of the extracellular matrix and other human connective tissues and plays many structural roles in the body. The endogenous hyaluronic acid and sulfated glycosaminoglycans (sGAGs) found in the compositions described herein increase the tensile strength of the extracellular matrix within the joint cavity. Additionally, hyaluronic acid can trigger intracellular events that lead to the promotion of cell movement and proliferation. It has been found that injecting hyaluronic acid alone restores the viscoelasticity of the joint. Another property of hyaluronic acid is its ability to absorb water, i.e., hygroscopicity. This property is desirable for joint treatment as it draws water into the joint, providing additional cushioning, lubrication, and shock absorption.

[0043] The aqueous human umbilical cord filtrate of the components is preferably prepared from human umbilical cords through one or more separation steps (e.g., filtration steps). The human umbilical cord filtrate preferably contains acellular Wharton's jelly, exosomes, endogenous growth factors, vascular endothelial growth factor receptor 1 (VEGFR1), hepatocyte growth factor (HGF), interleukin antagonist (IL-1ra), platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), endogenous hyaluronic acid (HA), or combinations thereof, whereby when the disclosed composition is used for its desired purpose, joint lubrication and healing are advantageously promoted in the subject. As shown in Figure 2, in the filtrate, the concentration of VEGFR1 is in the range of 1.0×10 2 pg / mL to 2.5×10 3 pg / mL, the concentration of HGF is in the range of 2.5×10 2 pg / mL to 1.42×10 4 pg / mL, the concentration of IL-1ra is in the range of 8.13×10 2 pg / mL to 5.15×10 4 pg / mL, the concentration of PDGF-BB is in the range of 2.0×10 1 pg / mL to 1.58×10 3 pg / mL, the concentration of bFGF is in the range of 4.73×10 1 pg / ml to 2.07×10 3 pg / ml, and the concentration of HA is 1.51×107 pg / mL to 3.5×10 8 in the range of pg / mL and any combination thereof. In certain embodiments, any endpoint of the above ranges may function as an endpoint of an additional range within that range.

[0044] In certain embodiments, the aqueous human umbilical cord filtrate may contain particles remaining from the human umbilical cord tissue, and these particles have a diameter of less than 100 μm, preferably less than 50 μm, more preferably less than 35 μm, and even more preferably less than 10 μm. Further, the aqueous human umbilical cord filtrate is a solution in which no sedimentation, separation, and / or precipitation is observed even after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more in the stored state. In certain embodiments, for the preparation steps of these compositions disclosed in FIG. 1 and immediately below, the aqueous human umbilical cord filtrate is phosphate buffered saline (or lactated Ringer's (sodium chloride 6 g / L, sodium lactate 3.1 g / L, potassium chloride 0.3 g / L, CaCl 0.2 g / L, pH 6.5), isotonic saline (0.9 wt% sodium chloride), plasmalyte® (sodium chloride 5.26 g / L, potassium chloride 0.37 g / L, magnesium chloride hexahydrate 0.30 g / L, sodium acetate trihydrate 3.68 g / L, sodium gluconate 5.02 g / L, pH 7.4), Normosol® (sodium chloride 5.26 g / L, potassium chloride 0.37 g / L, magnesium chloride 0.30 g / L, sodium acetate anhydrous 2.22 g / L, sodium gluconate 5.02 g / L, pH 7.4)), and the like, and the isotonic solution only aids in the preparation of each component of the compositions disclosed herein. For example, it has no degrading effect on acellular Wharton's jelly, exosomes, endogenous growth factors, VEGFR1, HGF, interleukin antagonists (e.g., IL-1ra), bFGF, PDGF-BB, endogenous hyaluronic acid, or combinations thereof in the aqueous human umbilical cord filtrate, and / or has only a minimal degrading effect. In certain embodiments, in addition to the aqueous human umbilical cord filtrate disclosed herein, the use of amniotic fluid is also contemplated. Amniotic fluid may be used as a diluent instead of the isotonic solution. Amniotic fluid has a high concentration of human growth factor (HGF), which may be desirable when using the disclosed compositions. When adding any of the above-described isotonic solutions to the above-disclosed filtrate, the isotonic solution may act as a diluent to further dilute the growth factor concentration in the filtrate to a desired range.When isotonic solution is added to the filtrate disclosed above, the concentration of VEGFR1 is 1.23×10. 2 pg / mL to 1.9×10 3 pg / mL, and the concentration of HGF is 3.47×10 2 pg / mL to 1.0×10 3 pg / mL, and the concentration of IL-1ra is 1.35×10 3 pg / ml to 3.43×10 3 pg / mL, and the concentration of PDGF-BB is 2.00×10 1 pg / mL to 1.05×10 2 pg / mL, and the concentration of bFGF is 7.95×10 1 pg / mL to 1.38×10 2 pg / mL, and the concentration of HA is 1.51×10 7 pg / mL to 1.0×10 8 pg / mL, and any combination thereof. In certain embodiments, any endpoint of the above ranges may function as an endpoint of an additional range within that range.

[0045] In certain embodiments, exogenous hyaluronic acid may be added to the aqueous human umbilical cord filtrate. In certain embodiments, exogenous hyaluronic acid is present in the composition at a concentration of 0.5 wt% to 5.0 wt%. In other embodiments, the composition has exogenous hyaluronic acid at a concentration of about 0.75 wt% to about 4.0 wt%. In other embodiments, the composition has exogenous hyaluronic acid at a concentration of about 1.5 wt% to about 3.5 wt%. In other embodiments, the composition has exogenous hyaluronic acid at a concentration of about 2.0 wt% to about 3.0 wt%.

[0046] Hyaluronic acid, also known as hyaluronan or hyaluronate, is used interchangeably with these terms in this specification. Hyaluronic acid is a glycosaminoglycan composed of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine. The hyaluronic acid used in this specification may be in the form of a salt or may not be in the form of a salt. Examples of hyaluronic acid in the form of a salt include sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, and magnesium hyaluronate. In some embodiments, the hyaluronic acid used in this specification may be obtained from bacterial biomanufacturing. These bacteria include, but are not limited to, Streptococcus faecalis, Streptococcus zooepidemicus, Escherichia coli, Agrobacterium, Streptococcus lactis, and Bacillus subtilis. In other embodiments, recombinant hyaluronic acid production is the source of exogenous hyaluronic acid used in this specification. An example of recombinant hyaluronic acid production is the expression of hyaluronic acid synthase and UDP-glucose dehydrogenase in a host bacterium to produce large amounts of hyaluronic acid in a fed-batch culture process. In other embodiments, the hyaluronic acid used in this specification may be obtained by extraction from animal tissues, including, but not limited to, rooster combs, bovine synovial fluid, and bovine vitreous humor. In some embodiments, hyaluronic acid may be purchased from commercial suppliers, including, but not limited to, Kewpie, Awa Biopharm, Dongchen Group, Fufeng Group, Focus Chem, and Bloomage Biotech.

[0047] The hyaluronic acid used in this specification may have various molecular weights. The term "molecular weight" may refer to both the weight-average molecular weight and the number-average molecular weight. In some embodiments, the hyaluronic acid used in this specification may have a molecular weight of from about 0.25 MDa to about 8.0 MDa.

[0048] In some embodiments, exogenous hyaluronic acid in aqueous human umbilical cord filtrate is crosslinked. Hyaluronic acid may be crosslinked using various crosslinking agents, including but not limited to 1,4-butanediol diglycidyl ether (BDDE), poly(ethylene glycol) diglycidyl ether (PEGDE), pentaerythritol tetraglycidyl ether (PETGE), divinyl sulfone, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), (phenylenebis-(ethyl)-carbodiimide, 1,6-hexamethylenebis(ethylcarbodiimide), adipic acid dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane. The degree of crosslinking referred to herein is defined as the proportion of free hyaluronic acid (non-crosslinked hyaluronic acid). In some embodiments, the exogenous hyaluronic acid is highly crosslinked and the proportion of free hyaluronic acid is low, for example 5% - 25%. In some embodiments, the exogenous hyaluronic acid is moderately crosslinked and the proportion of free hyaluronic acid is about 26% - 74%. In some embodiments, the exogenous hyaluronic acid is lightly crosslinked and the proportion of free hyaluronic acid is high, 75% - 95%. In some embodiments, the hyaluronic acid is not crosslinked, i.e., 100% free hyaluronic acid. Due to the degree of crosslinking of hyaluronic acid, the half-life of hyaluronic acid in the body is prolonged, and therapeutic effects such as joint cushioning and shock absorption can be observed over a long period of time.

[0049] As further mentioned above, the composition may be used as an allograft within the human body in a number of different purposes and procedures, including but not limited to intra-articular treatment. In this aspect, it is important to maintain the sterility of the aqueous human umbilical filtrate. It should be noted that since the aqueous human umbilical filtrate is non-immunogenic, it causes little immune response within the subject even when used for the desired purpose. However, sterility should be maintained so that contaminants that may cause an immune response and / or may cause infection (e.g., viral contaminants, bacterial contaminants, chemical contaminants, etc.) do not contaminate the composition when the composition is placed within or on the subject.

[0050] In certain aspects, the composition is configured for intra-articular treatment. In this aspect, the resulting composition preferably has a viscosity sufficient to provide lubricity, cushioning, and / or shock absorption to the joint cavity of the subject by replenishing the structural proteins in the extracellular matrix, supplying various growth factors and other nutrients from the composition, and in some aspects supplying exogenous hygroscopic hyaluronic acid. For example, this composition may be injected into the knee, ankle, hip, shoulder, or other joint of the subject. Also, it is contemplated that this composition may be injected into other joints or joint cavities for substantially similar purposes. In another aspect, the composition is configured to be injected into the heel or foot of the subject for the treatment of plantar fasciitis. (Method for producing the composition)

[0051] Figure 1 shows a schematic diagram of the steps involved in generating an aqueous human umbilical cord filtrate of the composition described herein. As further shown in Figure 1, none of the steps involve the introduction of exogenous enzymes that would result in exogenous enzymatic degradation / digestion. The method for generating an aqueous human umbilical cord filtrate configured for joint treatment includes steps (a) to (h) described immediately below. Before step (a), the umbilical cord / umbilical cord tissue is examined for infectious diseases in order to confirm that it is healthy / non-afflicted and to further minimize risks during the preparation of the composition and during final use after adjustment. Before beginning the treatment of the umbilical cord in steps (a) to (h), the umbilical cord is maintained in the temperature range of 4°C to 8°C.

[0052] As shown in Figure 1, step (a) involves preparing the human umbilical cord (i.e., the tissue and the cells that make up that tissue) preferably within 24 to 96 hours, more preferably within 24 to 72 hours after extraction from a human subject, in order to ensure the freshness of the human umbilical cord and minimize degradation due to necrosis, necroptosis, and / or apoptosis. In this step, it is preferable to process less than 80 grams at a time in order to perform appropriate grinding / micronization (in subsequent step (c)).

[0053] After completion of step (a), step (b) is performed. Step (b) preferably uses phosphate buffered saline (PBS) (i.e., 1×PBS) (alternatively, lactated Ringer's (sodium chloride 6 g / L, sodium lactate 3.1 g / L, potassium chloride 0.3 g / L, CaCl 0.2 g / L, pH 6.5), isotonic saline (0.9 wt% sodium chloride), plasmalyte® (sodium chloride 5.26 g / L, KCl 0.37 g / L, magnesium 0.37 g / L, magnesium chloride hexahydrate 0.30 g / L, sodium acetate trihydrate 3.68 g / L, sodium gluconate 5.02 g / L, pH 7.4), Normosol® (sodium chloride 5.26 g / L, KCl 0.37 g / L, magnesium chloride 0.30 g / L, anhydrous sodium acetate 2.22 g / L, sodium gluconate 5.02 g / L, pH 7.4)), and involves placing less than 80 grams of umbilical cord into a container containing a predetermined amount (e.g., 300 mL to 1000 mL, preferably 500 mL) of the isotonic solution. The container is placed on a stirring plate, a stir bar is placed into the container (containing PBS and the umbilical cord), and the umbilical cord is stirred in the isotonic solution for 5 to 15 minutes (at medium to high speed) to wash the umbilical cord portion. Next, the "used" isotonic solution is transferred, and a predetermined amount (e.g., 300 mL to 1000 mL, preferably 500 mL) of fresh isotonic solution is poured into the container, and the washing step (b) is repeated 1 to 5 times by washing the umbilical cord again. At any time before step (a), during the implementation of step (a), after step (b), or during the implementation of step (b), further, it is examined whether there are blood clots and / or blood pools / blood stagnations in the human umbilical cord and / or umbilical cord portion. If present, the blood clots are removed using suction or other mechanical removal means (e.g., scalpel, gauze, and forceps) so that the immunogenic components (e.g., hemoglobin and / or heme-related components derived from the umbilical cord donor) in the finally obtained composition are minimized. During the implementation of these washing steps, it is essential to maintain a sterile working environment and / or a aseptic working environment to prevent and / or reduce the contamination of contaminants during the production of the composition.

[0054] Upon completion of step (b), in step (c), the washed umbilical cord is transferred to a grinding device and / or a mincing device as disclosed in "Tissue Mincing Tool" of US D716,601 and / or "Systems And Methods For Processing Cells" of US Patent No. 8,967,512, the entire disclosures of which are incorporated herein by reference, and a predetermined amount (e.g., 75 mL to 125 mL, preferably 100 mL) of isotonic solution is placed into the device. The washed umbilical cord is then ground and / or minced by the grinding tool / mincing tool while the head of the grinding tool / mincing tool rotates in the range of 40 to 200 revolutions per minute (RPM) until the umbilical cord is completely ground (or as close as possible to a completely ground state), thereby obtaining ground human umbilical cord tissue. During the implementation of this grinding / mincing step, it is essential to maintain a sterile working environment to prevent and / or reduce the contamination of contaminants during the generation of the composition. In certain embodiments, to perform steps (d) and / or (e) described below, further maintain the sterility during the generation of the composition, and / or minimize the contamination of contaminants, the grinding tool / mincing tool may be directly connected to the device (i.e., a closed-system environment as disclosed, for example, in US Patent No. 8,967,512). Alternatively, steps (d) and / or (e) may be performed in an open-system environment / laboratory environment.

[0055] When step (c) is completed, in step (d), the ground / chopped human umbilical cord tissue of step (c) is separated into solid residues and an aqueous human umbilical cord supernatant. This first separation step may be carried out by a filtration process (either positive pressure or negative pressure). For example, the chopped / ground human umbilical cord tissue (of step (c) contained in a predetermined amount (e.g., 75 mL to 125 mL, preferably 100 mL) of isotonic solution) is placed directly on a filter having a desired porosity (e.g., 200 μm or 150 μm or 100 μm such as a qualitative grade mesh or a quantitative grade mesh or a net filter), and then a force (either positive pressure or negative pressure) may or may not be applied such that solid residues (solids having a size greater than 200 μm or 150 μm or 100 μm) remain on the filter and an aqueous human umbilical cord supernatant (containing solids less than 200 μm or 150 μm or 100 μm) passes through the filter. The filtration step takes approximately 15 seconds to 2 minutes. Here too, in order to prevent and / or reduce contamination in step (d), it is essential to maintain a sterile working environment and / or an aseptic working environment.

[0056] Upon completion of step (d), optionally, step (e) may be performed on the aqueous human umbilical cord supernatant. Step (e) preferably includes a plurality of filtration steps including: (i) filtering the aqueous human umbilical cord supernatant with a first filter having a porosity of 30 μm to 40 μm to obtain a second human umbilical cord supernatant; (ii) filtering the second human umbilical cord supernatant with a second filter having a porosity of 10 μm to 25 μm to obtain a third human umbilical cord supernatant; and (iii) filtering the third human umbilical cord supernatant with a third filter having a porosity of 4 μm to 10 μm to obtain an aqueous human umbilical cord filtrate. In certain embodiments, the applied force is negative pressure (vacuum), which is preferred because such negative pressure is less likely to damage the filter and less likely to lead to subsequent quality control issues for the final compositions disclosed herein. The aqueous human umbilical cord filtrate preferably contains acellular Wharton's jelly, exosomes, endogenous growth factors, VEGFR1, HGF, interleukin antagonist (IL-1ra), bFGF, PDGF-BB, endogenous hyaluronic acid, or any combination thereof. Each filtration step is completed at a vacuum of 1 - 5 psi in approximately 15 seconds to 2 minutes. Further, the aqueous human umbilical cord filtrate obtained in the above filtration steps is a solution that shows no sedimentation, separation, and / or precipitation even after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 24 months, 60 months, or more in storage. Filtering the human umbilical cord supernatant with a filter having a porosity of 10 μm or less results in a cell-free supernatant as the solution contains no cells. Thus, in some embodiments, the compositions described herein are acellular.

[0057] After completion of step (d) and / or (e), optionally, step (f) may be performed. In this step, the human umbilical cord filtrate is diluted with an isotonic solution or amniotic fluid to obtain a composition standardized to known factors (e.g., original cord weight, average growth factor content, etc.).

[0058] After completion of step (d) and / or (e), optionally, step (g) is carried out simultaneously with (and / or before or during step (e) and / or (f)). In this step, the solid residue of step (d) is further processed into a micronized human umbilical cord composition by carrying out a process of dehydration (lyophilization), freeze-drying, pulverization, and / or (cryogenic grinding) configured to obtain particles (polydisperse particles) having a size of more than 1 μm to 300 μm, preferably more than 1 μm to 100 μm, more preferably more than 1 μm to 50 μm, and even more preferably more than 1 μm to 35 μm. In a particular embodiment, step (e) is a cryogenic grinding process (e.g., as described in US20160287749, US20170203004, and US Patent No. 10105398, each of which is incorporated herein by reference in its entirety), in which the solid residue of step (d) is dehydrated, placed in a liquid nitrogen-cooled cryogenic grinding chamber, and ground therein to obtain a micronized human umbilical cord composition having a particle size of more than 1 μm to less than 300 μm, preferably more than 1 μm to 100 μm, more preferably more than 1 μm to 50 μm, and even more preferably more than 1 μm to 35 μm. The micronized human umbilical cord composition contains collagen, fibronectin, endogenous hyaluronic acid, elastin, or any combination thereof. The micronized human umbilical cord may be combined with the aqueous human umbilical cord filtrate of step (d), (e), or (f) and stored for other uses, such as the preparation of a two-part composition for other contemplated therapeutic uses.

[0059] In certain embodiments, after performing steps (d), (e), and / or (f) and before the following step (h), exogenous hyaluronic acid is added to the aqueous human umbilical cord filtrate. In some embodiments, highly cross-linked hyaluronic acid is added to the aqueous human umbilical cord filtrate. In some embodiments, moderately cross-linked hyaluronic acid is added to the aqueous human umbilical cord filtrate. In other embodiments, lightly cross-linked hyaluronic acid is added to the aqueous human umbilical cord filtrate. In some embodiments, non-cross-linked hyaluronic acid is added to the aqueous human umbilical cord filtrate. In certain embodiments, the exogenous hyaluronic acid is present in the composition at a concentration of 0.5 wt% to 5.0 wt%. In other embodiments, the composition has exogenous hyaluronic acid at a concentration of about 0.75 wt% to about 4.0 wt%. In other embodiments, the composition has exogenous hyaluronic acid at a concentration of about 1.5 wt% to about 3.5 wt%. In other embodiments, the composition has exogenous hyaluronic acid at a concentration of about 2 wt% to about 3.0 wt%.

[0060] In some embodiments, the exogenous hyaluronic acid is added to the aqueous human umbilical cord filtrate as a solid. In other embodiments, the exogenous hyaluronic acid is added to the aqueous human umbilical cord filtrate as an aqueous solution.

[0061] In certain embodiments, the aqueous human umbilical cord filtrate is sterile and the aqueous human umbilical cord filtrate is non-immunogenic.

[0062] After the completion of steps (d), (e) and / or (f), in step (h), the aqueous human umbilical cord filtrate (from step (d), (e) or (f)) may be placed in a sterile container and sealed for subsequent use, and the aqueous human umbilical cord filtrate is sterile. (Method of Use)

[0063] Without wishing to be bound by theory, the compositions disclosed herein are particularly useful for intra-articular treatment and, advantageously, are thought to cause little to no immunogenic response due to the non-immunogenic properties / nature of the composition.

[0064] Intra-articular treatment may be administered to patients with injuries or degenerations of soft tissues and connective tissues, and / or patients with various chronic diseases. These chronic diseases include, but are not limited to, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, lupus, gout, and other arthritis symptoms. In many cases, intra-articular treatment includes the injection of corticosteroids, analgesics, NSAIDs, and / or hyaluronic acid. Intra-articular treatment is basically provided by injection into any joint cavity in the body. Most commonly, intra-articular treatment is performed on the knee, hip, shoulder, and ankle, but other joint cavities such as finger joints and toe joints, wrist, elbow, jaw, spine, and neck (although not limited to these) may be the subject of intra-articular treatment.

[0065] Hyaluronic acid, which is found endogenously in the compositions described herein and in some embodiments is added exogenously to the compositions, has been found to be extremely effective as an intra-articular treatment in various arthritides. Hyaluronic acid naturally exists in intra-articular synovial fluid and cartilage and functions as a shock absorber, protective film, and lubricant on the surface of articular cartilage. It has been found that in many patients suffering from various arthritides such as osteoarthritis, the concentration of hyaluronic acid in the joint cavity is decreased. Hyaluronic acid has been found to not only play a role as a protective agent in the joint cavity but also have an anti-inflammatory effect.

[0066] VEGFR1, HGF, interleukin antagonist (IL-1ra), bFGF, and PDGF-BB, which are endogenously present in aqueous human umbilical cord filtrate, all provide various therapeutic effects for the alleviation of various joint diseases by providing cell proliferation signaling and anti-inflammatory effects.

[0067] For example, it is contemplated that the compositions disclosed herein are used to treat various arthropathies by intra-articular therapy, more particularly, restoration of endogenous extracellular matrix function in the joint cavity, restoration of endogenous collagen function in the joint cavity, and / or treatment and / or alleviation of symptoms of an inflammatory disease in a subject. In this particular use, the compositions disclosed herein are injected into the joints (knee, shoulder, ankle, wrist, elbow) of a subject.

[0068] In certain embodiments, the composition may be injected into the desired joint cavity at desired time intervals to achieve the desired result. In certain embodiments, the composition is injected daily. In certain embodiments, the composition is administered weekly. In certain embodiments, the composition is injected monthly. In certain embodiments, the composition is injected bi-weekly. In certain embodiments, the composition is injected semi-weekly. In certain embodiments, the composition is injected bi-monthly. In certain embodiments, the composition is injected semi-monthly.

[0069] The composition is delivered in an effective amount to restore endogenous extracellular matrix function in the joint cavity, to restore endogenous collagen function in the joint cavity, and / or to treat and / or alleviate symptoms of an inflammatory disease in the subject. This effective amount may be from 0.5 mL to 5 mL, and any amount within this range may function as an additional range endpoint.

[0070] In certain embodiments, methods for treating orthopedic and / or podiatric symptoms / diseases are also disclosed. For example, in certain embodiments, by directly injecting the compositions disclosed herein into the foot of a subject (subcutaneously in the area between the ball of the foot and the heel), and / or injecting immediately adjacent to the bone portion forming the heel of the subject, plantar fasciitis and / or heel diseases may be treated. This method includes treating the symptoms / diseases by aseptically injecting the mixed composition into and / or adjacent to the target area of a subject suffering from orthopedic and / or podiatric symptoms / diseases. In this embodiment, the aqueous human umbilical cord filtrate is sterile and non-immunogenic. For example, when treating plantar fasciitis with the above methods and compositions, the composition has a thickness and viscosity sufficient to provide cushioning (subcutaneous cushion) for treating and alleviating the pain associated with plantar fasciitis. In particular, the Wharton's jelly (mucopolysaccharides and proteoglycans) in the filtrate promotes the cushioning and protective purposes of the above treatment.

[0071] In another example, it is further envisioned that the disclosed compositions have general uses in the medical field such as general wound packing (resulting from acute trauma that causes surgical procedures and / or open external and / or internal wounds) and / or wound healing. In this aspect, the aqueous human umbilical cord filtrate may be used alone or in combination with micronized human umbilical cord. In some aspects, the micronized human umbilical cord may be combined with amniotic fluid instead of the aqueous human umbilical cord filtrate. In these aspects, it is envisioned that first the wound is evaluated and, for example, the overall viscosity and thickness of the (mixed) two-component composition necessary to pack and / or treat the subject's wound are roughly determined. Next, after the composition is aseptically mixed to an effective viscosity for blood coagulation, the subject's wound is aseptically packed with the aseptically mixed composition to coagulate blood within the aseptically packed wound. In certain aspects, both the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are aseptic and non-immunogenic, and in this method, the micronized human umbilical cord composition and the aqueous human umbilical cord filtrate are mixed in a ratio of 2:1 to 1:2. If a very highly viscous mixed composition is desired, the ratio of the micronized human umbilical cord composition is increased and the ratio of the aqueous human umbilical cord filtrate is decreased for mixing, and conversely, if a low-viscosity mixed composition is desired, the ratio of the aqueous human umbilical cord filtrate is increased and the ratio of the micronized human umbilical cord composition is decreased for mixing. The above packing may be repeated as necessary.

[0072] In certain embodiments, each of the individual components of the two-component compositions disclosed herein may be used individually (alone) for a particular purpose. For example, when using the disclosed filtrates individually, the purpose of using all of the filtrates (filtrates only) would be to provide not only soluble scaffolds and stromal components, but also growth factors and exosomes in the filtrates. For example, when using the filtrates to provide cushioning material in a denatured heel pad or joint cavity. As another example, when using the disclosed micronized compositions individually (alone), the purpose of using all of the microparticles would be to pack a wet wound bed or dental cavity when the target area is too wet to add filtrates, or when another filtrate such as platelet-rich plasma (PRP) is desired.

[0073] The foregoing description provides embodiments of the invention by way of example only. It is contemplated that other embodiments may perform similar functions and / or achieve similar results. All such equivalent embodiments and examples are within the scope of the invention and are intended to be encompassed by the appended claims.

Claims

**Claim 1** An aqueous, non-immunogenic, injectable composition for treating joints in a human subject in need of joint treatment, wherein the composition comprises an aqueous human umbilical cord filtrate that contains no exogenous enzymes and has microparticles less than 100 μm in the aqueous and non-immunogenic composition, the aqueous, non-immunogenic, injectable composition. **Claim 2** The aqueous human umbilical cord filtrate (a) acellular Wharton's jelly, (b) exosomes, (c) endogenous growth factors, (d) 1.51 × 10 ７ pg / mL to 3.5 × 10 ８ pg / mL concentration range of endogenous hyaluronic acid (HA), (e) 1.0 × 10 ２ pg / mL to 2.5 × 10 ３ pg / mL concentration range of vascular endothelial growth factor receptor (VEGFR1), (f) 2.5 × 10 ２ pg / mL to 1.42 × 10 ４ pg / mL concentration range of hepatocyte growth factor (HGF), (g) 8.13 × 10 ２ pg / mL to 5.15 × 10 ４ An interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)) in the concentration range of pg / mL, (h) 2.0 × 10 １ pg / mL to 1.58 × 10 ３ platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, (i) 4.73×10 １ pg / mL to 2.07×10 ３ pg / mL concentration range of basic fibroblast growth factor (bFGF), and (j) any combination thereof, and contains at least four of them, an aqueous, non-immunogenic, injectable composition for treating joints in a human subject in need of joint treatment according to claim 1. **Claim 3** An aqueous, non-immunogenic, injectable composition for treating joints in a human subject in need of joint treatment according to claim 1 or 2, further comprising an isotonic solution. **Claim 4** The isotonic solution is at least one of phosphate buffered saline, Ringer's lactate, a solution consisting essentially of sodium chloride, sodium acetate anhydrous, sodium gluconate, potassium chloride, and magnesium chloride, and a solution consisting essentially of sodium chloride, potassium chloride, magnesium chloride hexahydrate, sodium acetate trihydrate, and sodium gluconate, an aqueous, non-immunogenic, injectable composition for treating joints in a human subject in need of joint treatment according to any one of claims 1 to 3. **Claim 5** The aqueous and non-immunogenic composition (a) acellular Wharton's jelly, (b) exosomes, (c) endogenous growth factors, (d) 1.51×10 ７ pg / mL to 1.0×10 ８ pg / mL concentration range of endogenous hyaluronic acid (HA), (e) 1.23 × 10 ２ pg / mL to 1.9 × 10 ３ pg / mL concentration range of vascular endothelial growth factor receptor (VEGFR1), (f) 3.47 × 10 ２ pg / mL to 1.0 × 10 ３ pg / mL in the concentration range of hepatocyte growth factor (HGF), (g) 1.35 × 10 ３ pg / mL to 3.43 × 10 ３ pg / mL concentration range of interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)), (h) 2.0 × 10 １ pg / mL to 1.05 × 10 ３ platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, (i) 7.95 × 10 １ pg / mL to 1.83 × 10 ３ pg / mL of basic fibroblast growth factor (bFGF), and (j) any combination thereof, and contains at least four of them, an aqueous, non-immunogenic, injectable composition for treating joints in a human subject in need of joint treatment according to any one of claims 1 to 4. **Claim 6** The aqueous and non-immunogenic composition is acellular, an aqueous, non-immunogenic, injectable composition for treating joints in a human subject in need of joint treatment according to any one of claims 1 to 5. **Claim 7** The aqueous and non-immunogenic composition further comprises an effective amount of exogenous hyaluronic acid for restoring the endogenous extracellular matrix function in the joint cavity, for restoring the endogenous collagen function in the joint cavity, and / or for treating and / or alleviating the symptoms of an inflammatory disease in the subject, the composition for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, according to any one of claims 1 to 6.

8. The aqueous and non-immunogenic composition according to any one of claims 1 to 7, for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, comprises exogenous hyaluronic acid at a concentration of 0.5% to about 5.0% by weight based on the total composition.

9. The aqueous human umbilical cord filtrate is sterile, the composition for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, according to any one of claims 1 to 8.

10. The composition for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, according to any one of claims 1 to 9, further comprises amniotic fluid.

11. The aqueous and non-immunogenic composition according to any one of claims 1 to 10, for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, comprises fine particles less than 50 μm.

12. The aqueous and non-immunogenic composition according to any one of claims 1 to 11, for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, comprises fine particles less than 35 μm.

13. The aqueous and non-immunogenic composition according to any one of claims 1 to 12, for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, comprises fine particles less than 10 μm.

14. The aqueous and non-immunogenic composition according to claim 13, for treating a joint of a human subject in need of joint treatment, which is aqueous, non-immunogenic, and injectable, is acellular.

15. A method of injecting the composition according to claim 1 for treating a joint into a human subject in need of joint treatment, A method comprising: (a) injecting an effective amount of said composition into the joint cavity of the affected joint of the human subject to improve and / or restore the endogenous extracellular matrix function in the joint cavity, to improve and / or restore the endogenous collagen function in the joint cavity, to treat and / or alleviate the symptoms of the inflammatory disease of the subject, or for any combination of these.

16. The method according to claim 15, wherein the composition is sterile.

17. The method according to claim 15 or 16, wherein the composition further comprises an effective amount of exogenous hyaluronic acid to improve and / or restore the endogenous extracellular matrix function in the joint cavity, to improve and / or restore the endogenous collagen function in the joint cavity, to treat and / or alleviate the symptoms of the inflammatory disease of the subject, or for any combination of these.

18. The method according to any one of claims 15 to 17, wherein step (a) is repeated at predetermined time intervals.

19. The method according to claim 18, wherein step (a) is repeated daily, weekly, bi-weekly, semi-monthly, monthly, bi-monthly, or every half month.

20. The composition is (a) acellular Wharton's jelly, (b) exosomes, (c) endogenous growth factors, (d) 1.51×10 ７ pg / mL to 3.5×10 ８ pg / mL concentration range of endogenous hyaluronic acid (HA), (e) 1.0 × 10 ２ pg / mL to 2.5 × 10 ３ pg / mL concentration range of vascular endothelial growth factor receptor (VEGFR1), (f) 2.5 × 10 ２ pg / mL to 1.42 × 10 ４ pg / mL concentration range of hepatocyte growth factor (HGF), (g) 8.13 × 10 ２ pg / mL to 5.15 × 10 ４ pg / mL concentration range of interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)), (h) 2.0 × 10 １ pg / mL to 1.58 × 10 ３ platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, (i) 4.73 × 10 １ pg / mL to 2.07 × 10 ３ pg / mL of basic fibroblast growth factor (bFGF), and (j) any combination of these, and comprises at least four of them. The method according to claim 15.

21. The method according to claim 15, wherein the composition further comprises an isotonic solution.

22. The composition is (a) acellular Wharton's jelly, (b) exosomes, (c) endogenous growth factors, (d) 1.51 × 10 ７ pg / mL to 1.0 × 10 ８ pg / mL concentration range of endogenous hyaluronic acid (HA), (e) 1.23 × 10 ２ pg / mL to 1.9 × 10 ３ pg / mL concentration range of vascular endothelial growth factor receptor (VEGFR1), (f) 3.47 × 10 ２ pg / mL to 1.0 × 10 ３ pg / mL in the concentration range of hepatocyte growth factor (HGF), (g) 1.35 × 10 ３ pg / mL to 3.43 × 10 ３ pg / mL concentration range of interleukin antagonist (interleukin-1 receptor antagonist (IL-1ra)), (h) 2.0 × 10 １ pg / mL to 1.05 × 10 ３ platelet-derived growth factor-BB (PDGF-BB) in the concentration range of pg / mL, (i) 7.95 × 10 １ pg / mL to 1.83 × 10 ３ pg / mL of basic fibroblast growth factor (bFGF), and (j) any combination of these, and comprises at least four of them. The method according to claim 21.

23. The method according to any one of claims 15 to 22, wherein the effective amount of the composition is selected from the group consisting of 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL.

24. The human subject in need of joint treatment has at least one of osteoarthritis, rheumatoid arthritis, psoriatic arthritis, lupus, gout, plantar fasciitis, and any combination of these. The method according to one of claims 15 to 23.

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