WO2021226060A1 - Formulations of hyaluronic acid and amniotic or gestational fluid, and uses of the same - Google Patents

Abstract

Methods of preparing a mixture of human amniotic fluid (and/or tissue or fluid of the placental/gestational area) and high molecular weight hyaluronic acid are described. The mixtures are amenable for long-term storage (e.g., at room temperature or with cryopreservation) with minimal loss of biological function. The mixtures are stable at room temperature. The formulations are useful for treating, for example, joint injuries, osteoarthritis, eye injuries, and cosmetic conditions.

Description

FORMUUATIONS OF HYAUURONIC ACID AND AMNIOTIC OR GESTATIONAU

FUUID, AND USES OF THE SAME

This application claims the benefit of priority of U.S. Provisional Application 63/019,732, filed May 4, 2020, which is incorporated herein by reference in its entirety.

FIEUD OF THE INVENTION

Described herein are methods of preparing a composition comprising sterile human amniotic fluid (and/or tissue or fluid of the placental/gestational area) and derivatives of gestational fluids (e.g., Wharton’s jelly, coelomic fluid, and cord blood) and high molecular weight hyaluronic acid that is stable during long-term storage (e.g., at room temperature or with cryopreservation).

BACKGROUND OF THE INVENTION

Amniotic fluid (AF) contains nutrients and growth factors that facilitate fetal growth. AF surrounds the fetus during pregnancy and provides protection and nourishment for the fetus. AF provides mechanical cushioning and antimicrobial effectors that protect the fetus. AF comprises proteins, growth factors, hyaluronic acid (HA), cytokines, chemokines and carbohydrates. Some of the growth factors found in AF include epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), transforming growth factor beta-1 (TGF- b 1), insulin-like growth factor I (IGF-I), and erythropoietin (EPO).

Current technologies that utilize amniotic base materials are lacking because they fail to adequately preserve the growth factor protein arrays and cellular content that make it a potential regenerative source. Furthermore, existing amnio-based technologies fail to preserve the natural and intrinsic architecture of the native tissues making them less suitable for application by utilizing outdated cryopreservation techniques or damaging lyophilization (freeze-drying) techniques. Furthermore, to date, there is little to no data of utilizing amniotic base materials for aesthetic application in a clinical setting.

Thus, there is a need for compositions comprising AF that have improved properties.

SUMMARY OF THE INVENTION

In one aspect of the invention is provided a composition comprising a) tissue and/or fluid of the placental/gestational area, such as, amniotic fluid (or other placental tissue such as amniotic membrane tissue, umbilical cord tissue, tissue of the placental disc, and/or tissue of the amniotic sac, or other components; and/or tissue grafts and osteochondral pastes) and b) hyaluronic acid (HA) having a molecular weight of at least 20,000 Daltons. In another aspect is provided a composition comprising a) a derivative of a gestational fluid (e.g., Wharton’s jelly, coelomic fluid, and cord blood) and b) hyaluronic acid (HA) having a molecular weight of at least 20,000 Daltons. In some embodiments of either aspect, the molecular weight of the HA is at least 50,000 Daltons. In some embodiments, the molecular weight of the HA is at least 100,000 Daltons. In some embodiments, the molecular weight of the HA is at least 200,000 Daltons. In some embodiments, the molecular weight of the HA is at least 300,000 Daltons. In some embodiments, the molecular weight of the HA is at least 400,000 Daltons. In some embodiments, the molecular weight of the HA is between 400,000 Daltons and 1,300,000 Daltons. In certain embodiments, the molecular weight of the HA is between 20,000 Daltons and 1,000,000 Daltons. In various embodiments, the molecular weight of the HA is 20,000 Daltons, 30,000 Daltons, 40,000 Daltons, 50,000 Daltons, 60,000 Daltons, 70,000 Daltons, 80,000 Daltons, 90,000 Daltons, 100,000 Daltons, 110,000 Daltons, 120,000 Daltons, 130,000 Daltons, 140,000 Daltons, 150,000 Daltons, 160,000 Daltons, 170,000 Daltons, 180,000 Daltons, 190,000 Daltons, 200,000 Daltons, 210,000 Daltons, 220,000 Daltons, 230,000 Daltons, 240,000 Daltons, 250,000 Daltons, 260,000 Daltons, 270,000 Daltons, 280,000 Daltons, 290,000 Daltons, 300,000 Daltons, 310,000 Daltons, 320,000 Daltons, 330,000 Daltons, 340,000 Daltons, 350,000 Daltons, 360,000 Daltons, 370,000 Daltons, 380,000 Daltons, 390,000 Daltons, 400,000 Daltons, 410,000 Daltons, 420,000 Daltons, 430,000 Daltons, 440,000 Daltons, 450,000 Daltons, 460,000 Daltons, 470,000 Daltons, 480,000 Daltons, 490,000 Daltons, 500,000 Daltons, 510,000 Daltons, 520,000 Daltons, 530,000 Daltons, 540,000 Daltons, 550,000 Daltons, 560,000 Daltons, 570,000 Daltons, 580,000 Daltons, 590,000 Daltons, 600,000 Daltons, 610,000 Daltons, 620,000 Daltons, 630,000 Daltons, 640,000 Daltons, 650,000 Daltons, 660,000 Daltons, 670,000 Daltons, 680,000 Daltons, 690,000 Daltons, 700,000 Daltons, 710,000 Daltons, 720,000 Daltons, 730,000 Daltons, 740,000 Daltons, 750,000 Daltons, 760,000 Daltons, 770,000 Daltons, 780,000 Daltons, 790,000 Daltons, 800,000 Daltons, 810,000 Daltons, 820,000 Daltons, 830,000 Daltons, 840,000 Daltons, 850,000 Daltons, 860,000 Daltons, 870,000 Daltons, 880,000 Daltons, 890,000 Daltons, 900,000 Daltons, 910,000 Daltons, 920,000 Daltons, 930,000 Daltons, 940,000 Daltons, 950,000 Daltons, 960,000 Daltons, 970,000 Daltons, 980,000 Daltons, 990,000 Daltons, or 1,000,000 Daltons.

In some embodiments, the therapeutic composition is an injectable composition having a viscosity of no more than about 50 Pa sec. In some embodiments, the therapeutic composition is formulated for injection through a 20-30 gauge needle. In some embodiments, the therapeutic composition is formulated for injection through a 25-27 gauge needle.

In some embodiments, the composition further comprises a carrier fluid. The carrier fluid may comprise one or more of plasma, serum, water for injection, hyaluronan, saline, phosphate buffered saline, chondroitin sulfate, glucosamine, mannosamine, proteoglycan, proteoglycan fragments, chitin, and chitosan.

In some embodiments, the composition further comprises stem cells.

In some embodiments, the composition further comprises exosomes or secretive-type or secretome cells, e.g., stems cells, epithelial cells, and other cells harvested from the placenta and umbilical cord.

In another aspect is provided a method of treating an ophthalmic condition comprising administering to the eye any of the above compositions. In certain embodiments, the concentration of the HA having a molecular weight of at least 50,000 Daltons in the composition is from 0.5 to 1.5%.

In one aspect of the invention, the tissue and/or fluid of the placental/gestational area comprises amniotic fluid and/or other placental tissue such as amniotic membrane tissue, umbilical cord tissue, tissue of the placental disc, and/or tissue of the amniotic sac, or other components.

In another aspect of the invention, the tissue of the placental/gestational area (e.g., amniotic fluid) includes and/or is replaced by tissue grafts or osteochondral pastes.

In another aspect is provided a method of treating a joint injury comprising administering to the joint any of the above compositions. In some embodiments, the amount of the composition administered is effective to improve joint function. In some embodiments, the joint is a knee joint, a hip joint, a shoulder joint, an elbow joint, an ankle joint, a wrist joint, a spine joint, a tarsal joint, a metatarsal joint, a carpal joint, a metacarpal joint, or a temporal mandibular joint. In some embodiments, the joint injury is a meniscus injury. In certain embodiments, the meniscus injury is a meniscus tear. In some embodiments, the joint injury has osteoarthritis.

In another aspect is provided a method of treating a soft tissue injury comprising administering to or near an injured soft tissue any of the above compositions.

In another aspect is provided a method of treating a tendon injury comprising administering to or near the injured tendon any of the above compositions.

In another aspect is provided a method of treating a tendinopathy. In another aspect is provided a method of treating osteochondral injuries.

In another aspect is provided a method for treating a cosmetic condition comprising administering to a subject any of the above compositions. In some embodiments, the cosmetic condition is a skin condition. In some embodiments, the skin condition is selected from the group consisting of skin wrinkles, skin aging, loss of skin elasticity, cellulite, skin lines, and scarring. In some embodiments, the composition is administered to the site of the cosmetic condition. In some embodiments, the composition is administered by injection, e.g., with a 20-gauge needle, a 21 -gauge needle, a 22-gauge needle, or a 23 -gauge needle. In some embodiments, the composition is administered to the skin surface, e.g., by micro- needling.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts a flowchart describing a manufacturing process of a composition of the invention as described in Example 1.

FIG. 2 depicts a flowchart describing a manufacturing process of a composition of the invention as described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

An “active agent” refers to a physiologically or pharmacologically active substance that acts locally and/or systemically in the body. An active agent is a substance that is administered to an individual for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), or diagnosis (e.g., diagnostic agent) of a disease or disorder. The term “ophthalmic active agent”, or “ophthalmic drug” refers to an agent that is administered to a patient to alleviate, delay onset of, or prevent one or more symptoms of a disease or disorder of the eye or a component thereof. Active agents may also include materials that alleviate joint pain, promote healing of tissue in the joint, or treat osteoarthritis, for example.

The term “effective amount” or “therapeutically effective amount” refers to an amount effective to alleviate, delay onset of, or prevent one or more symptoms of a disease or disorder. The “effective amount” of the formulations described herein are sufficient, when administered to a patient in need thereof, to effect treatment for disease-states, conditions, or disorders for which the compounds have utility. The amount of the formulation that constitutes a therapeutically effective amount will vary depending on such factors as the compound and its biological activity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of treatment, the type of disease-state or disorder being treated and its severity, the drugs used in combination with or coincidentally with the compounds of the invention, and the age, body weight, general health, sex, and diet of the patient. Such a therapeutically effective amount can be determined by one of ordinary skill in the art.

The term “growth factors” refers to a group of proteins or hormones that stimulate the cellular growth. Growth factors play an important role in promoting cellular differentiation and cell division, and they occur in a wide range of organisms. Growth factors can stimulate cell migration (e.g., mitogenic cytokines), function as chemotactic agents, inhibit cell migration or invasion of tumor cells, modulate differentiated functions of cells, be involved in apoptosis and promote survival of cells. Growth factors can be secreted as diffusible factors and can also exist in membrane- anchored forms.

“Amniotic factors” are molecules naturally present in the amniotic fluid. Amniotic factors include carbohydrates, proteins and peptides such as enzymes and hormones, lipids, metabolic substrates and products such as lactate and pyruvate, and electrolytes.

As used herein, the tissue and/or fluid of the placental/gestational area includes amniotic fluid or other placental tissue such as amniotic membrane tissue, umbilical cord tissue, tissue of the placental disc, and/or tissue of the amniotic sac, or other components.

The term “amniotic fluid,” unless specified otherwise, refers to the entire fluid and non-fluid components present in the amniotic cavity, such as the liquid, solid, semi-solid, or cellular constituents contained therein, whether in suspension or not. "Modified amniotic fluid" refers to amniotic fluid that differs from amniotic fluid that is found in its natural state. In some embodiments, the modified amniotic fluid has some components (solvent, growth factors, amino acids, salts, proteins, and the like) removed from the amniotic fluid. In other embodiments, the modified amniotic fluid has some components (solvent, growth factors, amino acids, salts, proteins, and the like) added to the amniotic fluid. In yet other embodiments, the modified amniotic fluid has some components (solvent, growth factors, amino acids, salts, proteins, and the like) removed from the amniotic fluid and has some components (solvent, growth factors, amino acids, salts, proteins, and the like) added to the modified amniotic fluid.

The term "hyaluronic acid" (HA) refers to the glycosaminoglycan composed of disaccharide repeats of N-acetylglucos amine and glucuronic acid found in nature, also known as hyaluronan, as well as derivatives of HA having chemical modifications such as, but not limited to, esters of HA, amide derivatives, alkyl-amine derivatives, low molecular weight and high molecular weight forms of HA, and cross-linked forms and the like. Thus, the disaccharide chain may be linear or non-linear. HA can be cross-linked by attaching cross linkers such as thiols, methacrylates, hexadecylamides, and tyramines. HA can also be cross- linked directly with formaldehyde and divinylsulfone. In one preferred example, HA chains can be cross-linked using 1,4-butanediol diglycidyl ether (BDDE) as the cross-linking agent. The term "hyaluronic acid" or HA includes HA itself and pharmaceutically acceptable salts or derivatives thereof.

The term “biocompatible” or “biologically compatible” generally refers to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient. Biocompatible materials generally do not elicit a significant inflammatory or immune response when administered to a patient.

The term “pharmaceutically acceptable” refers to compounds, carriers, excipients, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit/risk ratio. A pharmaceutically acceptable carrier or compound will not abrogate the biological activity or properties of the amniotic fluid, derivative of a gestational fluid, or HA.

The term “molecular weight” generally refers to the relative average chain length of the bulk polymer, e.g., HA molecule, unless otherwise specified. In practice, molecular weight can be estimated or characterized using various methods including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

Therapeutic Compositions

Disclosed herein are compositions comprising hyaluronic acid with tissue and/or fluid of the placental/gestational area, such as, amniotic fluid (or other placental tissue such as amniotic membrane tissue, umbilical cord tissue, tissue of the placental disc, and/or tissue of the amniotic sac or other components; or tissue grafts and osteochondral pastes), and/or gestational fluid derivatives (e.g., Wharton’s jelly, coelomic fluid, and cord blood). The hyaluronic acid may be added to tissue of the placental/gestational area (e.g., amniotic fluid). The hyaluronic acid may be added to gestational fluid. The compositions comprising amniotic fluid (or other placental tissue) and hyaluronic acid can be formulated into pharmaceutical compositions. The compositions comprising gestational fluid derivatives and hyaluronic acid can be formulated into pharmaceutical compositions. These compositions may be sufficiently stable so as to retain most amniotic factors after short-term or long-term storage under temperature-controlled conditions either as a liquid or as lyophilized powder.

Amniotic fluid isolated from a mother, e.g., a full term mother having a C-section, may contain nutrients and growth factors that facilitate fetal growth, provides mechanical cushioning and antimicrobial effectors that protect the fetus, and allows assessment of fetal maturity and disease. Amniotic fluid may also comprise one or more of the following: growth factors, pro-inflammatory cytokines and anti-inflammatory cytokines, as well as a variety of macromolecules including carbohydrates, proteins and peptides, lipids, lactate, pyruvate, electrolytes, enzymes, and hormones.

Growth factors and their receptors may control a wide range of biological functions, such as but not limited to, regulating cellular proliferation, survival, migration and differentiation. Growth factors in AF may be useful to treat a variety of conditions, including joint injuries, osteoarthritis and ophthalmic injuries. Growth factors that may be present in the AF include one or more of EGF, IGF-I, vascular endothelial growth factor A (VEGF-a), TNF-a, hepatocyte growth factor (HGF), fibroblast growth factor 7 (FGF7), matrix metallopeptidase (MMP-9), granulocyte-colony stimulating factor (GCSF), matrix metalloproteinase-7 (MMP-7), matrix metalloproteinase-7 (MMP-13), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), fibroblast growth factor 4 (FGF-4), endocrine gland-derived vascular endothelial growth factor (EG-VEGF), interleukin 8 (IL-8), fibroblast growth factor 21 (FGF-21), angiopoietin-2 (ANG2), Glial cell-derived neurotrophic factor (GDNF), fibroblast growth factor 19 (FGF-19), TIMP metallopeptidase inhibitor 2 (TIMP-2), angiopoietin-1 (ANG-1), TGF-bI, macrophage colony-stimulating factor (M-CSF), angiotensinogen, platelet derived growth factor- AA (PDGF-AA), and stem cell factor (SCF).

EGF is a small polypeptide hormone with mitogenic properties in vivo and in vitro. EGF elicits biologic responses by binding to a cell surface receptor that is a transmembrane glycoprotein containing a cytoplasmic protein tyrosine kinase. EGF responses are mediated by ligand binding and activation of this intrinsic protein kinase. TGF-a has a structure similar to EGF and can bind to the same receptor as does EGF. Vascular endothelial growth factor (VEGF) was originally described as an endothelial cell-specific mitogen. VEGF is produced by many cell types including tumor cells, macrophages, platelets, keratinocytes, and renal mesangial cells. VEGF can promote bone formation, hematopoiesis, wound healing, and development. VEGF-A is a signal protein that stimulates vasculogenesis and angiogenesis (Hoeben Am, et al., Pharmacol. Rev 2004, 56:549-580). These activities of VEGF may be particularly useful in treatment of joint injuries.

TGF-b is a multifunctional peptide that can control proliferation, differentiation, and other functions in many cell types. TGF-bI, for example, may induce terminal differentiation of intestinal epithelial cells and to accelerate the rate of healing of intestinal wounds by stimulating cell migration. Various cells may synthesize TGF- b and essentially all of them have specific receptors for this peptide. HGF, the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and has major roles in development, tissue regeneration and cancer. Met proto-oncogene may improve or enhance the immunomodulating activity of the HA- comprising formulations described herein.

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs), and may regulate a broad spectrum of biological functions, including cellular proliferation, survival, migration, and differentiation. FGF signal pathways include the RAS/MAP kinase pathway, PI3 kinase/AKT pathway, and PLCy pathway. Without wishing to be bound by theory, FGFs may improve or enhance the tissue regeneration activity of the HA-comprising formulations described herein.

Such tissue regeneration may occur in any one or more of skin, blood vessel, muscle, adipose, tendon/ligament, cartilage, bone, tooth, and nerve tissues. Matrix metalloproteinases (MMPs), also called matrixins, function in the extracellular environment of cells and degrade both matrix and non-matrix proteins. MMPs may play central roles in morphogenesis, wound healing, tissue repair and remodeling in response to injury. MMPs may improve or enhance the tissue regeneration activity of the HA-comprising formulations described herein.

Amniotic fluid and/or derivatives of gestational fluids in the compositions described herein may contain many pro- and anti-inflammatory cytokines. Without wishing to be bound by theory, pro- and anti-inflammatory cytokines can play important immunoregulatory roles, with inflammation involving an interplay between pro- and anti-inflammatory cytokines. The amniotic fluid or the derivative of gestational fluid may comprise one or more pro-inflammatory cytokines, such as one or more of interleukin- 1 (IF- 1), tumor necrosis factor (TNF), gamma- interferon (IFN-gamma), IF- 12, IF- 18, granulocyte-macrophage colony stimulating factor, Eotaxin-2 (CCF24), interleukin 6 (IF-6), pulmonary and activation-regulated chemokine PARC or chemokine (C-C motif) ligand 18 (CCL18), total GRO which consisted of three subunits GROa/CXCLl, GROp/CXCL2, and GROy/CXCL3, expression of the neutrophil-activating CXC chemokine (ENA-78/CXCL-5), chemokine (C- C motif) ligand 21 (CCL21 or 6Ckine), macrophage inflammatory protein 3 alpha (MIP-3a or CCL20), monokine induced by gamma (MIG or CXCL-9), MIP-la, chemokine (C-C motif) ligand 5 (CCL-5), also known as RANTES (regulated on activation, normal T cell expressed and secreted), Interleukin-1 alpha (IL-la), macrophage inflammatory protein-ΐb (MIP-Ib or CCL4), tumor necrosis factor (TNFa) and monocyte chemotactic protein 2 (MCP-2 or CCL8).

The amniotic fluid or the derivative of gestational fluid may comprise one or more anti-inflammatory cytokines, such as one or more of IL4, IL-10, IL-13, IL-27, IFN-alpha and transforming growth factor-beta (e.g., TGF-bI), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), vascular endothelial growth factor D (VEGF-D), interleukin- 1 receptor antagonist (IL-IRa), interleukin 5 (IL-5) and interleukin 21 (IL-21).

Hyaluronic acid (HA) is a main component of the extracellular matrix. HA is a glycosaminoglycan polymer that is composed of disaccharide units. Each of the disaccharide units is connected to another by a b(1 3^^oo8kϋo linkage. The size range of HA is from 5,000 to over 20,000,000 Daltons. HA can be found in the eye, synovial fluid, skin, joints and gestational tissues. HA can be extracted from rooster combs, or produced by bacteria.

The inventors have found that combining HA of certain sizes with amniotic fluid (or other tissue and/or fluid of the placental/gestational area), or a derivative of a gestational fluid, provides for advantageous protection and treatment of injured joints, as well as ophthalmic injuries. Without wishing to be bound by theory, HA is generally not a toxic or immunogenic material. HA is found within joint fluid, and can provide for viscoelastic effects and anti-inflammatory effects. The HA in the compositions and formulations described herein may provide for protection to the joint against degradation and pathological changes of synovial fluid HA. The HA in the compositions and formulations described herein can provide for shock absorption and lubrication to promote healing, and to enhance the effects of amniotic fluid. The HA in the compositions and formulations described herein may work synergistically with amniotic fluid so as to prolong the presence of amniotic fluid in the joint so that the various amniotic fluid factors described herein can remain present in, and carry out an effect in, the injured joint or eye tissue.

In various embodiments, HA may be in the form of a derivative. Derivatives of HA include, but are not limited to, biodegradable polymers grafted to individual polysaccharide moieties in HA. Such polymers include, but are not limited to, polyglycolic acid, polylactic acid, and copolymers of lactic acid and glycolic acid.

In some embodiments, additives are added to the HA to enhance and/or prolong the effects of HA. HA additives can have the effect of optimizing the flow and viscoelastic properties of HA, and/or alter chemical stability or biological activity of HA in a manner not found in pure HA. Hence, additives can be used for different applications compared to pure HA. Example HA additives can include, but are not limited to, materials and minerals having a particle size of from 10 pm to 60 pm or from 25 pm to 45 pm, or less than 25 pm. Such materials can include, for example, calcium phosphate materials that can be comprised of, for example, calcium hydroxyapatite, calcium fluoroapatite, calcium chioroapatite, calcium carbonate apatite, tetracalcium phosphate, calcium pyrophosphate, tricalcium phosphate or octacalcium phosphate particles, and mixtures thereof. HA additives such as antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), and the like may also be used.

Method of Preparation

The compositions comprising amniotic fluid (or other tissue and/or fluid of the placental/gestational area) and hyaluronic acid may comprise amniotic fluid obtained from a pregnant woman. The compositions can be formulated such that the formulation may have no less than 0.5%, less than 1%, less than 1.5%, less than 2%, less than 2.5% or less than 3% of amniotic membrane particulate matter, for example, cells, large particles and other undissolvables are removed, preferably by high speed centrifugation to obtain clarified amniotic fluid.

Those of skill in the art are well-acquainted with methods of safely and humanely obtaining samples of AF (or other tissue and/or fluid of the placental/gestational area), and of the need to maintain sterility of the AF tissue during such procedures. Suitable sources, e.g. of human AF tissue, include AF that is obtained from patients who are undergoing amniocentesis, patients who are undergoing a Caesarean section delivery, and patients undergoing normal delivery using a specially designed receptacle to collect the fluid after rupture of membranes. In certain embodiments, the amniotic fluid collection procedure is performed in a sterile operating room environment during an elective C-section. The woman may be undergoing a pre-Caesarian surgical method. During the procedure of obtaining the amniotic fluid, an ultrasound device may be used to provide guidance for the process of obtaining human fluid from the woman. The procedure may comprise one or more of the following steps: inserting a blunt tip needle into the amniotic sac of the woman, attaching the blunt tip needle to a three-way stopcock, connecting a Luer lock syringe to the three-way stopcock, connecting a first end of a length of sterile tubing with the three-way stopcock, and collecting sterilely the amniotic fluid through the blunt tip needle and sterile tubing into a collection container. The sterile collection container may include a pump with a suction device.

Amniotic fluid (or other tissue and/or fluid of the placental/gestational area), or a derivative of a gestational fluid, may be further processed to remove cells, large particles and other solids. Such processing may comprise one or more of centrifugation and filtration. In certain embodiments, a morcellation tissue extraction technique is used (e.g., cutting tissue into small pieces). The amniotic fluid may be sterilized, such as by addition of an antibacterial, antiviral, and/or antifungal. The derivative of a gestational fluid may be sterilized, such as by addition of an antibacterial, antiviral, and/or antifungal.

In various methods, centrifugation filtration may be conducted with 5pm to 10pm filters (low protein binding filter) to complete the removal of cells and large particles, followed by submicron filtration (e.g., 0.2 pm, 0.45 pm, or 1.0 pm filters) to remove gross contaminates. Alternatively or in addition, membrane filters such as those made with polyethersulphone (PES) may be used.

Additional HA may then be added to the amniotic fluid. In various embodiments, to each 1 mL of amniotic fluid, an additional 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL,

1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL,

2.9 mL, 3.0 mL, 3.1 mL, 3.2 mL, 3.3 mL, 3.4 mL, 3.5 mL, 3.6 mL, 3.7 mL, 3.8 mL, 3.9 mL,

4.0 mL, 4.1 mL, 4.2 mL, 4.3 mL, 4.4 mL, 4.5 mL, 4.6 mL, 4.7 mL, 4.8 mL, 4.9 mL, 5.0 mL,

5.1 mL, 5.2 mL, 5.3 mL, 5.4 mL, 5.5 mL, 5.6 mL, 5.7 mL, 5.8 mL, 5.9 mL, 6.0 mL, 6.1 mL,

6.2 mL, 6.3 mL, 6.4 mL, 6.5 mL, 6.6 mL, 6.7 mL, 6.8 mL, 6.9 mL, 7.0 mL, 7.1 mL, 7.2 mL,

7.3 mL, 7.4 mL, 7.5 mL, 7.6 mL, 7.7 mL, 7.8 mL, 7.9 mL, 8.0 mL, 8.1 mL, 8.2 mL, 8.3 mL,

8.4 mL, 8.5 mL, 8.6 mL, 8.7 mL, 8.8 mL, 8.9 mL, 9.0 mL, 9.1 mL, 9.2 mL, 9.3 mL, 9.4 mL,

9.5 mL, 9.6 mL, 9.7 mL, 9.8 mL, 9.8 mL, or 10.0 mL of HA may be added. In various embodiments, to each 1 mL of amniotic fluid, an additional 0.1 mL to 0.5 mL, 0.3 mL to 0.8 mL, 0.5 mL to 1.0 mL, 0.7 mL to 1.2 mL, 0.9 mL to 1.4 mL, 1.1 mL to 1.6 mL, 1.3 mL to 1.8 mL, 1.5 mL to 2.0 mL, 1.7 mL to 2.2 mL, 1.9 mL to 2.4 mL, 2.1 mL to 2.6 mL, 2.3 mL to 2.8 mL, 2.5 mL to 3.0 mL, 2.7 mL to 3.2 mL, 2.9 mL to 3.4 mL, 3.1 mL to 3.6 mL, 3.2 mL to 3.7 mL, 3.4 mL to 3.9 mL, 3.6 mL to 4.1 mL, 3.8 mL to 4.3 mL, 4.0 mL to 4.5 mL, 4.2 mL to 4.7 mL, 4.4 mL to 4.9 mL, 4.6 mL to 5.1 mL, 4.8 mL to 5.3 mL, 5.0 mL to 5.5 mL, 5.2 mL to 5.7 mL, 5.4 mL to 5.9 mL, 5.6 mL to 6.1 mL, 5.8 mL to 6.3 mL, 6.0 mL to 6.5 mL, 6.2 mL to 6.7 mL, 6.4 mL to 6.9 mL, 6.6 mL to 7.1 mL, 6.8 mL to 7.3 mL, 7.0 mL to 7.5 mL, 7.2 mL to 7.7 mL, 7.4 mL to 7.9 mL, 7.6 mL to 8.1 mL, 7.8 mL to 8.3 mL, 8.0 mL to 8.5 mL, 8.2 mL to 8.7 mL, 8.4 mL to 8.9 mL, 8.6 mL to 9.1 mL, 8.8 mL to 9.3 mL, 9.0 mL to 9.5 mL, 9.2 mL to 9.7 mL, or 9.5 mL to 10.0 mL of HA may be added.

Additional HA may then be added to the derivative of a gestational fluid. In various embodiments, to each 1 mL of the fluid, an additional 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.1 mL, 3.2 mL, 3.3 mL, 3.4 mL, 3.5 mL, 3.6 mL, 3.7 mL, 3.8 mL, 3.9 mL, 4.0 mL, 4.1 mL, 4.2 mL, 4.3 mL, 4.4 mL, 4.5 mL, 4.6 mL, 4.7 mL, 4.8 mL, 4.9 mL, 5.0 mL, 5.1 mL, 5.2 mL, 5.3 mL, 5.4 mL, 5.5 mL, 5.6 mL, 5.7 mL, 5.8 mL, 5.9 mL, 6.0 mL, 6.1 mL, 6.2 mL, 6.3 mL, 6.4 mL, 6.5 mL, 6.6 mL, 6.7 mL, 6.8 mL, 6.9 mL, 7.0 mL, 7.1 mL, 7.2 mL, 7.3 mL, 7.4 mL, 7.5 mL, 7.6 mL, 7.7 mL, 7.8 mL, 7.9 mL, 8.0 mL, 8.1 mL, 8.2 mL, 8.3 mL, 8.4 mL, 8.5 mL, 8.6 mL, 8.7 mL, 8.8 mL, 8.9 mL, 9.0 mL, 9.1 mL, 9.2 mL, 9.3 mL, 9.4 mL, 9.5 mL, 9.6 mL, 9.7 mL, 9.8 mL, 9.8 mL, or 10.0 mL of HA may be added. In various embodiments, to each 1 mL of the fluid, an additional 0.1 mL to 0.5 mL, 0.3 mL to 0.8 mL, 0.5 mL to 1.0 mL, 0.7 mL to 1.2 mL, 0.9 mL to 1.4 mL, 1.1 mL to 1.6 mL, 1.3 mL to

1.8 mL, 1.5 mL to 2.0 mL, 1.7 mL to 2.2 mL, 1.9 mL to 2.4 mL, 2.1 mL to 2.6 mL, 2.3 mL to

2.8 mL, 2.5 mL to 3.0 mL, 2.7 mL to 3.2 mL, 2.9 mL to 3.4 mL, 3.1 mL to 3.6 mL, 3.2 mL to

3.7 mL, 3.4 mL to 3.9 mL, 3.6 mL to 4.1 mL, 3.8 mL to 4.3 mL, 4.0 mL to 4.5 mL, 4.2 mL to

4.7 mL, 4.4 mL to 4.9 mL, 4.6 mL to 5.1 mL, 4.8 mL to 5.3 mL, 5.0 mL to 5.5 mL, 5.2 mL to

5.7 mL, 5.4 mL to 5.9 mL, 5.6 mL to 6.1 mL, 5.8 mL to 6.3 mL, 6.0 mL to 6.5 mL, 6.2 mL to

6.7 mL, 6.4 mL to 6.9 mL, 6.6 mL to 7.1 mL, 6.8 mL to 7.3 mL, 7.0 mL to 7.5 mL, 7.2 mL to

7.7 mL, 7.4 mL to 7.9 mL, 7.6 mL to 8.1 mL, 7.8 mL to 8.3 mL, 8.0 mL to 8.5 mL, 8.2 mL to

8.7 mL, 8.4 mL to 8.9 mL, 8.6 mL to 9.1 mL, 8.8 mL to 9.3 mL, 9.0 mL to 9.5 mL, 9.2 mL to

9.7 mL, or 9.5 mL to 10.0 mL of HA may be added.

HA in the compositions and formulations described herein may be in the form of a pharmaceutically acceptable salt, such as alkali metal salts (e.g., sodium salts, potassium salts, or lithium salts). The HA may be extracted from cockscombs. The HA may be extracted from porcine subcutaneous tissue. The HA may be prepared from biological fermentation methods. In some embodiments, the molecular weight of the HA is at least 100,000 Daltons. In some embodiments, the molecular weight of the HA is at least 150,000 Daltons. In some embodiments, the molecular weight of the HA is at least 200,000 Daltons. In some embodiments, the molecular weight of the HA is at least 250,000 Daltons. In some embodiments, the molecular weight of the HA is at least 300,000 Daltons. In some embodiments, the molecular weight of the HA is at least 400,000 Daltons. In some embodiments, the molecular weight of the HA is between 400,000 Daltons and 1,300,000 Daltons. In certain embodiments, the molecular weight of the HA is between 500,000 Daltons and 1,000,000 Daltons. In some embodiments, the molecular weight of the HA is between 500,000 Daltons and 700,000 Daltons. In some embodiments, the molecular weight of the HA is between 600,000 Daltons and 800,000 Daltons. In some embodiments, the molecular weight of the HA is between 700,000 Daltons and 900,000 Daltons. In some embodiments, the molecular weight of the HA is between 800,000 Daltons and 1,000,000 Daltons. In some embodiments, the molecular weight of the HA is between 900,000 Daltons and 1,100,000 Daltons. In some embodiments, the molecular weight of the HA is between 1,000,000 Daltons and 1,200,000 Daltons. In some embodiments, the molecular weight of the HA is between 1,100,000 Daltons and 1,300,000 Daltons.

Methods of Storage

The compositions comprising amniotic fluid (or other tissue and/or fluid of the placental/gestational area) and hyaluronic acid described herein may be stable over extended periods of time at room temperature (e.g., from 15 to 25 °C). Also, the compositions comprising a derivative of a gestational fluid and hyaluronic acid described herein may be stable over extended periods of time at room temperature (e.g., from 15 to 25 °C). Molecules contained within the fluid may be stabilized against degradation so as to avoid the need for chemical or physical modification to maintain the biological activity of the molecules over extended periods of time. The compositions prepared according to the methods described herein can be stored for long periods of time, allowing for a broad range of application methods, including distribution and storage as aerosols, gels, solutions, powders, etc. It can be advantageous to keep the compositions at room temperature so as to avoid degradation from freeze/thawing.

Assays may be undertaken to determine the extent of degradation from storage at room temperature. Exemplary assays for total protein content include, but are not limited to, a bicinchoninic acid (BCA) assay, Bradford assay, Lowry assay, and a UV absorption assay (e.g., at 280 nm). Individual proteins may be assayed using high throughput methods such as high density screening arrays. The biological activity of one or more amniotic growth factors can also be assessed in vitro. The activity of any one or more of the amniotic growth factors of the stored product can be assessed as a percentage of the fresh (raw) product, or as a percentage of the product prior to storage. Therefore, in some embodiments, little or no statistically significant changes in the biological activity of the amniotic factors are observed when using the AF-HA composition stored at room temperature for up to 2 weeks, up to 3 weeks, up to 4 weeks, up to one month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months or more than 6 months. In other embodiments, the activity of any one of the proteins in the amniotic fluid are reduced by no more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 17%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, or less than 5% relative to the activity of the same proteins in the product prior to storage.

Formulations

In various embodiments, any of the compositions described herein are formulated into, and optionally packaged into, sterile dosage units which can be stored and distributed for use by attending physicians. These formulations can be in the form of sterile packaged syringes for injection, dropper bottles (typically a 30 day supply for application once or twice daily to the eye), or aerosols. In certain embodiments, the dosages for the injectables are selected from 0.25 cc, 0.5 cc, and 1.0 cc. The injectables can be formulated for subcutaneous administration, intramuscular administration, ophthalmological administration, or administration into a joint. The efficacy of the formulation can be determined by one or more of Physician evaluations, patient self-evaluations, imaging studies and Quality of life evaluations.

The formulation can be administered in concentrated form, diluted with sterile water or buffer, or formulated as a gel, ointment, solution, suspension or aerosol. The formulation can include one or more additional therapeutic, prophylactic or diagnostic agents, either in the solution, gel, ointment or suspension, or as particles (nanoparticles, liposomes, microparticles) or implants. The formulation may comprise one or more excipients. Exemplary excipients include, but are not limited to, solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients may be materials which are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. Compositions can be formulated for storage as a fluid or solid (i.e., powder). In preferred embodiments, the composition is formulated for storage as a liquid (i.e., above freezing temperatures).

For ophthalmic application, the pH of the formulations can be similar to that of tear fluid, which is 7.4. In certain embodiments, the pH is 7.00 to 7.10, 7.05 to 7.15, 7.10 to 7.20, 7.15 to 7.25, 7.20 to 7.30, 7.25 to 7.35, 7.30 to 7.40, 7.35 to 7.45, 7.40 to 7.50, 7.45 to 7.55, 7.50 to 7.60, 7.55 to 7.65, 7.60 to 7.70, 7.65 to 7.75, 7.70 to 7.80, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8.

Buffers or pH adjusting agents or vehicles can be added to adjust and stabilize the pH at a desired level. Suitable buffers are well known by those skilled in the art, and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.

A formulation for ophthalmic application may be buffered at the pH of maximum stability of the drug(s) they contain. The buffers may be included to minimize any change in pH during the storage life of the drug; this can result from absorbed carbon dioxide from the air or from hydroxyl ions from a glass container. Changes in pH can affect the solubility and stability of drugs; consequently, it is important to minimize fluctuations in pH. The buffer system may be sufficient to maintain the pH throughout the expected shelf-life of the product, but with a low buffer capacity so that when the ophthalmic solution is instilled into the eye, the buffer system of the tears will rapidly bring the pH of the solution back to that of the tears. Low concentrations of buffer salts may be used to prepare buffers of low buffer capacity.

The preparation of aqueous ophthalmic drops requires careful consideration of the need for isotonicity, a certain buffering capacity, the desired pH, the addition of antimicrobial agents and/or antioxidants, the use of viscosity-increasing agents, and the choice of appropriate packaging. Ophthalmic drops are considered isotonic when the tonicity is equal to that of a 0.9% solution of NaCl.

The formulation may comprise additional salts so as to be isotonic with tears. For example, the solution may comprise 0.9% NaCl. Alternatively, the solution may comprise tonicity agents having an isotonicity equivalent to 0.9% NaCl so as to minimize discomfort. Suitable tonicity agents include, but are not limited to, glycerin, mannitol, sorbitol, NaCl, and other electrolytes.

In some embodiments, the formulation is distributed or packaged in a liquid form. Alternatively, formulations for administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration.

The formulations may also contain one or more preservatives to prevent bacterial contamination. Suitable preservatives are known in the art, and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as PURITE®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.

Solutions, suspensions, or emulsions for administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.

In the certain embodiments, the formulations comprise amniotic fluid and hyaluronic acid, but do not contain any additives, and are packaged in sterile form. In the certain embodiments, the formulations comprise hyaluronic acid and a derivative of a gestational fluid, but do not contain any additives, and are packaged in sterile form. In certain embodiments, the formulations comprise amniotic fluid (or other tissue and/or fluid of the placental/gestational area) and hyaluronic acid, and are packaged in a sterile form.

Methods of Treatment and Administration

Compositions and formulations comprising tissue and/or fluid of the placental/gestational area, such as amniotic fluid (or other placental tissue such as amniotic membrane tissue, umbilical cord tissue, tissue of the placental disc, and/or tissue of the amniotic sac, or other components; and/or tissue grafts and osteochondral pastes) and hyaluronic acid, or a derivative of a gestational fluid and hyaluronic acid, have a variety of uses. The uses may vary based on the concentrations of growth factors and low toxicity and inflammation. In various embodiments, the formulation is effective to alleviate pain. In various embodiments, the formulation is effective to reduce inflammation. In various embodiments, the formulation is effective to treat a tissue injury or tissue degeneration. In certain embodiments, the formulation is administered directly to the eye to treat dry eye due to aging or long term contact use. In other embodiments, the formulation is administered into a joint to alleviate pain or enhance healing.

The compositions and formulations described herein that comprise amniotic fluid (or other tissue of the placental/gestational area) and hyaluronic acid may be used to treat an ophthalmological injury, disease or condition, e.g., an eye disorder. The compositions and formulations described herein that comprise hyaluronic acid and a derivative of a gestational fluid may be used to treat an ophthalmological injury, disease or condition, e.g., an eye disorder. Examples of eye disorders that may be treated with the formulations described herein, and according to the methods described herein, include but are not limited to, disorders due to age, damage from trauma or infection, and autoimmune disease. The compositions and formulations described herein may be used to promote recovery of the eye after surgery, such as cataract surgery. These include keratitis, conjunctivitis, corneal dystrophic diseases, Fuchs’ endothelial dystrophy, dry eye due to Sjogren's syndrome, Stevens-Johnson syndrome, and other autoimmune dry eye diseases, and environmental dry eye diseases, corneal neovascularization diseases, post-corneal transplant rejection prophylaxis and treatment, some types of uveitis, edema, degeneration, and retinopathies. Other conditions which may be treated include injury, bum, abrasion of the cornea, cataracts, and conditions arising from long-time contact lens use.

In various embodiments, the concentration of HA is 0.5 to 1.5% in the composition or formulation. In various embodiments, the concentration of HA is about 1% in the composition or formulation. In various embodiments, the concentration of HA is 1% in the composition or formulation. In some embodiments, the compositions and formulations may be used to treat an eye injury. In some embodiments, the compositions and formulations may be used to treat a cataract. In some embodiments, the compositions and formulations may be used to treat an eye in which a cataract was removed. In some embodiments, the compositions and formulations may be used to treat an eye into which a cornea was transplanted. In some embodiments, the compositions and formulations may be used to treat a detached retina. In some embodiments, the compositions and formulations may be used to treat dry eye. The dry eye may arise from aging or long term contact use.

The compositions and formulations described herein that comprise amniotic fluid and hyaluronic acid, or hyaluronic acid and a derivative of a gestational fluid, may be used in plastic surgery. In one embodiment, the composition or formulation is injected into areas with wrinkles, thin skin, or poor healing. In certain embodiments, the formulation is injected with a 20 gauge needle. In certain embodiments, the formulation is injected with a 21 gauge needle. In certain embodiments, the formulation is injected with a 22 gauge needle. In certain embodiments, the formulation is injected with a 23 gauge needle. The compositions and formulations may be administered periodically (e.g., monthly, bimonthly, semiannually, or annually) to reduce aging of the skin. The compositions and formulations may be administered periodically (e.g., monthly, bimonthly, semiannually, or annually) to improve appearance of the skin.

In some embodiments, any of the compositions or formulations described herein that comprise amniotic fluid and hyaluronic acid are administered into a joint to alleviate pain or enhance healing. In certain embodiments, the formulation is injected with a 25 gauge needle. In certain embodiments, the formulation is injected with a 26 gauge needle. In certain embodiments, the formulation is injected with a 27 gauge needle. In some embodiments, any of the compositions or formulations described herein that comprise amniotic fluid and hyaluronic acid are administered into a tendon to alleviate pain or enhance healing. The injection of formulation may be performed under guidance, e.g., by ultrasound, endoscopy, or endo-ultrasound. In certain embodiments, the formulation is injected with a 25 gauge needle. In certain embodiments, the formulation is injected with a 26 gauge needle. In certain embodiments, the formulation is injected with a 27 gauge needle. The injection of formulation may be performed under guidance, e.g., by ultrasound, endoscopy, or endo- ultrasound.

In some embodiments, any of the compositions or formulations described herein that comprise a derivative of a gestational fluid and hyaluronic acid are administered into a joint to alleviate pain or enhance healing. In certain embodiments, the formulation is injected with a 25 gauge needle. In certain embodiments, the formulation is injected with a 26 gauge needle. In certain embodiments, the formulation is injected with a 27 gauge needle. In some embodiments, any of the compositions or formulations described herein that comprise a derivative of a gestational fluid and hyaluronic acid are administered into a tendon to alleviate pain or enhance healing. The injection of formulation may be performed under guidance, e.g., by ultrasound, endoscopy, or endo-ultrasound. In certain embodiments, the formulation is injected with a 25 gauge needle. In certain embodiments, the formulation is injected with a 26 gauge needle. In certain embodiments, the formulation is injected with a 27 gauge needle. The injection of formulation may be performed under guidance, e.g., by ultrasound, endoscopy, or endo-ultrasound.

In some embodiments, any of the compositions or formulations described herein that comprise amniotic fluid and hyaluronic acid are used to treat a urinary tract infection. In some embodiments, any of the compositions or formulations described herein that comprise amniotic fluid and hyaluronic acid are used to treat a skin wound. In some embodiments, any of the compositions or formulations described herein that comprise amniotic fluid and hyaluronic acid are used to treat a bum injury. In some embodiments, any of the compositions or formulations described herein that comprise amniotic fluid and hyaluronic acid are used to treat a mouth sore.

In some embodiments, any of the compositions or formulations described herein that comprise a derivative of a gestational fluid and hyaluronic acid are used to treat a urinary tract infection. In some embodiments, any of the compositions or formulations described herein that comprise a derivative of a gestational fluid and hyaluronic acid are used to treat a skin wound. In some embodiments, any of the compositions or formulations described herein that comprise a derivative of a gestational fluid and hyaluronic acid are used to treat a bum injury. In some embodiments, any of the compositions or formulations described herein that comprise a derivative of a gestational fluid and hyaluronic acid are used to treat a mouth sore.

Many joint conditions may be treated, including pain, arthritis, degeneration, cartilage wear or tear, and damaged joints (such as fracture). The formulation may also be administered to bones that have had prosthetic implants, pins, screws or plates attached or implanted into them, to promote healing and repair, and to reduce inflammation. Injections may be administered to assist in resurfacing and repair or regeneration of cartilage. The formulation may also be administered to assist in soft tissue repair, such as repair of torn or strained ligaments or tendons.

In one embodiment, the formulation is administered at the site of injury. In another embodiment, the formulation is sprayed onto, soaked into, or powder dispersed onto the implant or prosthetic. This can include matrices, implants and sutures.

Dosage and dosing regimens are dependent on the intended use of the formulation, and is known to those skilled in the art. The concentration and dosage (number of times per day of amount of formulation for period of time) will vary depending on the condition to be treated, the severity of the condition, and the inclusion of other therapeutic, prophylactic or diagnostic agents. The appropriate amounts are determined on an individual basis, measuring response to treatment over time, as demonstrated in the examples.

For joint injuries, variables include the size of the joint, the severity of the injury to be treated, the purpose, for example, to enhance healing following surgery may require a shorter term higher dose as compared to more long term, lower dosages for treatment age related damage. The formulation may be administered over one to three injections that can be done daily, every other day, once a week, biweekly, once a month, or at longer intervals, depending on the indication and on the severity of the injury or inflammation. Dosing frequency or dosage may be decreased over time.

The formulations and compositions described herein are sufficiently non-toxic so as to be injected as often as the physician chooses, unlike steroids that can only be injected infrequently, typically two to three times a year.

EXAMPLES

The following examples are for illustrative purposes only and do not limit the scope of the invention in any way.

Example 1: Production of Composition of the Invention

This example provides an overview of the steps to produce a composition of the invention.

Amniotic fluid is retrieved from eligible donors who have met all of the requirements of 21 C.F.R. § 1271 (FDA, 2015). Donor amniotic fluid is aseptically procured, contained, and transported per the Standard Operating Procedures (SOP) of Genesis Biologies, Inc. for amniotic fluid.

This process entails the centrifugation and filtering of amniotic fluid. The primary objective is:

1. Preserve growth factor and cellular content: epithelial cell, amniotic fluid cells with mesenchymal stromal characteristics.

2. Removal of contaminate and unwanted by byproducts.

3. Ratio of amniotic fluid supernatant and amniotic fluid cellular pellet.

Step 1:

Raw amniotic fluid is placed in a centrifugation unit and processed at: 450-800 x g for 10 minutes at 4° Celsius.

Step 2:

Amniotic fluid supernatant is drawn off and sterile filtered in accordance with the existing SOP protocol with X micron screen.

Step 3:

Purified amniotic fluid weight is recorded. Ratio is established; Purified amniotic fluid is added to mixing container/chamber;

Total weight of purified amniotic fluids supernatant are weighed in grams (g).

Step 4:

Fixed ratio of HA is added to purified amniotic fluid supernatant (AF Base)

HA Specifications:

• Non-animal stabilized HA (NASHA)

• Crosslinked with BDDE (1.4-butanediol diglycidyl ether) - 23 mg/mL

• Biphasic - 200,000 particles per ml (175-200pm)

• Concentration of 25 mg/g

• Phosphate buffered to 7.0ph

Formulation is mixed in collection chamber at Y x g for X mins at Z° Celsius.

Step 5:

Fixed ratio of CryoStor5 is added to AF Base and HA formulation.

Step 6:

Separate final formulation in 1ml volumes into 2ml cryovials per SOP standard protocol.

Step 7:

Cryovials are labeled per SOP standard protocol.

Step 8:

Cryovials are cryopreserved according to SOP standard protocol.

Step 9:

Packaging and Storage per SOP protocols.

A flowchart describing the manufacturing process described in this example is depicted in FIG. 1.

Example 2: Production of Composition of the Invention

The process described in this example entails the filtering of amniotic fluid. The primary objective is:

1. Preserve growth factor and cellular content: epithelial cell, amniotic fluid cells with mesenchymal stromal characteristics.

2. Removal of contaminate and unwanted by byproducts.

3. Ratio of amniotic fluid, HA and cryopreservation agent (CPA).

Step 1: Raw amniotic fluid is filtered of gross contaminates such as:

• Red Blood Cells

• Fetal Meconium

• Other contaminating factors

Step 2:

Filtered amniotic fluid is weighed in grams (g).

Step 3:

Fixed ratio of HA is added to purified amniotic fluid (AF Base)

HA Specifications:

• Non-animal stabilized HA (NASHA)

• Crosslinked with BDDE (1.4-butanediol diglycidyl ether) - 23 mg/mL

• Biphasic - 200,000 particles per ml (175-200pm)

• Concentration of 25 mg/g

• Phosphate buffered to 7.0ph

Formulation is mixed in collection chamber at Y x g for X mins at Z° Celsius.

Step 4:

Fixed ratio of CryoStor5 (CS5) is added to AF Base and HA formulation.

Step 5:

Separate final formulation in 1ml volumes into 2ml cryovials per SOP standard protocol.

Step 6:

Cryovials are labeled per SOP standard protocol.

Step 7:

Cryovials are cryopreserved according to SOP standard protocol.

Step 8:

Packaging and Storage per SOP protocols.

A flowchart describing the manufacturing process described in this example is depicted in

FIG. 2.

Example 3: Composition of the Invention

This example presents an amniotic fluid composition of the invention. The composition is described below in Table 1. All components of the composition are free of bioburdens, pathogens, communicable diseases or inflammatory-causing agents which could cause tissue damage. The composition is hypo-allergenic and aseptically processed as set forth above.

Table 1: Composition Specification.

The samples were prefilled into 1 mL or 5 mL syringes and sterilized. The samples can be stored at room temperature or in a clinical freezer (-40 to -80 degrees Celsius). Frozen samples are stable beyond three to five years. The samples can be delivered as an injectable through a 25-27 gauge needle. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Each reference cited in the present application is incorporated by reference in its entirety.

Claims

1. A composition comprising: a) amniotic fluid or a derivative of a gestational fluid; and b) hyaluronic acid having a molecular weight of at least 20,000 Daltons.

2. The composition of claim 1, wherein the composition comprises a) amniotic fluid.

3. The composition of claim 1, wherein the composition comprises a) a derivative of a gestational fluid.

4. The composition of claim 1, wherein the molecular weight of the hyaluronic acid is at least 100,000 Daltons.

5. The composition of claim 4, wherein the molecular weight of the hyaluronic acid is at least 200,000 Daltons.

6. The composition of claim 5, wherein the molecular weight of the hyaluronic acid is at least 400,000 Daltons.

7. The composition of claim 5, wherein the molecular weight of the hyaluronic acid is between 400,000 Daltons and 1,300,000 Daltons.

8. The composition of claim 7, wherein the molecular weight of the hyaluronic acid is between 500,000 Daltons and 1,000,000 Daltons.

9. The composition of any one of claims 1 to 8, wherein said therapeutic composition is an injectable composition having a viscosity of no more than about 50 Pa sec.

10. The composition of any one of claims 1 to 9, wherein the therapeutic composition is formulated for injection through a 20-30 gauge needle.

11. The composition of claim 10, wherein the therapeutic composition is formulated for injection through a 25-27 gauge needle.

12. The composition of any one of any one of claims 1 to 11, further comprising a carrier fluid.

13. The composition of any one of claims 1 to 12, wherein the carrier fluid comprises one or more of plasma, serum, water for injection, hyaluronan, saline, phosphate buffered saline, chondroitin sulfate, glucosamine, mannosamine, proteoglycan, proteoglycan fragments, chitin, and chitosan.

14. The composition of any one of claims 1 to 13, further comprising stem cells.

15. The composition of any one of claims 1 to 14, further comprising a cellular component selected from epithelial cells and/or cells harvested from the placenta or umbilical cord.

16. A method of treating an ophthalmic condition comprising administering to the eye the composition of any one of claims 1 to 15.

17. The method of claim 5, wherein the concentration of the hyaluronic acid having a molecular weight of at least 50,000 Daltons in the composition is from 0.5 to 1.5%.

18. A method of treating a joint injury comprising administering to the joint the composition of any one of claims 1 to 15.

19. The method of claim 18, wherein the amount of the composition administered is effective to improve joint function.

20. The method of claim 18 or claim 19, wherein the joint is a knee joint, a hip joint, a shoulder joint, an elbow joint, an ankle joint, a wrist joint, a spine joint, a tarsal joint, a metatarsal joint, a carpal joint, a metacarpal joint, or a temporal mandibular joint.

21. The method of any one of claims 18 to 20, wherein the joint injury is a meniscus injury.

22. The method of claim 21, wherein the meniscus injury is a meniscus tear.

23. The method of any one of claims 18 to 20, wherein the joint injury is osteoarthritis.

24. A method of treating a soft tissue injury comprising administering at or near an injured soft tissue the composition of any one of claims 1 to 15.

25. A method of treating a tendon injury comprising administering at or near the tendon the composition of any one of claims 1 to 15.

26. A method for treating a cosmetic condition comprising administering to a subject the composition of any one of claims 1 to 15.

27. The method of claim 26, wherein the cosmetic condition is a skin condition.

28. The method of claim 27, wherein the skin condition is selected from the group consisting of skin wrinkles, skin aging, loss of skin elasticity, cellulite, skin lines, and scarring.

29. The method of any one of claims 26 to 28, wherein the composition is administered to the site of the cosmetic condition.

30. The method of any one of claims 26 to 29, wherein the composition is administered by injection.

31. The method of any one of claims 27 or 28, wherein the composition is administered to the skin surface.

32. The method of claim 31, wherein the composition is administered by micro-needling.

33. A composition comprising: a) tissue and/or fluid of the placental/gestational area; and b) hyaluronic acid having a molecular weight of at least 20,000 Daltons.

34. The composition of claim 33, wherein the placental tissue is selected from the group consisting of: amniotic membrane tissue, umbilical cord tissue, tissue of the placental disc, and tissue of the amniotic sac.

35. A composition comprising: a) a tissue graft and/or osteochondral paste; and b) hyaluronic acid having a molecular weight of at least 20,000 Daltons.

36. The composition of claim 35, wherein the tissue graft is derived from the placenta.