Compositions and methods for stimulating hair growth

Abstract

Methods and compositions for stimulating hair growth are disclosed. The compositions for stimulating hair growth include two or more of hyaluronic acid, osteopontin and another CD44 binding ligand. The methods for stimulating hair growth include administering such compositions into the skin of a patient or injecting such compositions into the skin of a patient.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 236,999, filed August 25, 2021, the contents of which are incorporated by reference.

[0002] Array List

[0003] This application contains a Sequence Listing that has been submitted electronically in XML file format, which is hereby incorporated by reference in its entirety. Said XML copy, created on August 23, 2022, is named AMP-22-1033WO2_SL.xml and is 9KB in size. [Background technology]

[0003]

[0004] The present invention relates generally to compositions and methods for stimulating hair growth.

[0004]

[0005] Hair loss often has negative social and psychological effects on individuals who suffer from it. Many factors are believed to contribute to hair loss, including genetics, hormones, environmental exposure, drugs, psychological stress, and nutrition. One known treatment is hair transplantation, which requires anesthesia, is expensive, time-consuming, and sometimes painful. Other approaches include massage and acupuncture, but these have not been shown to be effective. Hormones and other drugs have been used to treat hair loss, but these treatments often cause undesirable side effects, such as hair growth in unwanted areas. Thus, there is a need for an effective therapy to stimulate hair growth. Summary of the Invention

[0005]

[0006] In one embodiment, the composition for stimulating hair growth comprises osteopontin and hyaluronic acid. The hyaluronic acid may have an average molecular weight ranging from about 20 KDa to 1350 KDa. The hyaluronic acid may be cross-linked, and in some embodiments may have a cross-link density of about 20% or more. In some embodiments, the hyaluronic acid is present in a concentration of 25 mcg / mL or more, or from about 25 mcg / mL to about 100 mcg / mL. In some embodiments, the composition may also comprise one or more of serglycin, chondroitin sulfate, fibrin, IGFBP4, and GFP10, and in particular one or more of serglycin, chondroitin sulfate, and fibrin. In some embodiments, the compositions may include a hyaluronidase inhibitor, such as a hyaluronidase inhibitor selected from high molecular weight poly(styrene-4-sulfonate) (PSS), gossypol, sodium aurothiomalate, fenoprofen, glycerrhizic acid, fatty acids, plant-derived compounds, heparin, and O-sulfated HA (sHA), or combinations thereof.

[0006]

[0007] In another embodiment, the composition for stimulating hair growth comprises hyaluronic acid and one or more of serglycin, chondroitin sulfate, fibrin, IGFBP4 and GFP10. In particular, the composition can comprise one or more of serglycin, chondroitin sulfate and fibrin. The hyaluronic acid can have an average molecular weight ranging from about 20 KDa to 1350 KDa. The hyaluronic acid can be crosslinked and, in some embodiments, can have a crosslink density of about 20% or more. In some embodiments, the hyaluronic acid is present at a concentration of 25 mcg / mL or more, or from about 25 mcg / mL to about 100 mcg / mL. In some embodiments, the composition can also include osteopontin. In some embodiments, the compositions may also include a hyaluronidase inhibitor selected from, for example, high molecular weight poly(styrene-4-sulfonate) (PSS), gossypol, sodium aurothiomalate, fenoprofen, glycerrhizic acid, fatty acids, plant-derived compounds, heparin, and O-sulfated HA (sHA), or combinations thereof.

[0007]

[0008] In one embodiment, a method of stimulating hair growth in the skin of a patient in need thereof comprises administering to the patient's skin a composition comprising hyaluronic acid and a CD44 binding ligand. In some embodiments, administering comprises applying the composition to the surface of the skin, while in other embodiments, administering comprises injecting the composition into the dermis layer of the skin. The composition can be injected into the skin at a depth of about 400 microns to about 2 mm. In some embodiments, the composition is administered in multiple injections in an amount of about 400 injections / cm' skin to about 650 injections / cm' skin. In some embodiments, the needle is a microneedle. In some embodiments, the composition can be encapsulated in a liposome, such as a liposome comprising a hydrogenated phospholipid.

[0008]

[0009] In some embodiments, the method of stimulating hair growth further comprises applying iontophoresis to the skin.

[0009]

[0010] In some embodiments, the method of stimulating hair growth further comprises applying electroporation to the skin.

[0010]

[0011] In some embodiments, the method of stimulating hair growth further comprises applying laser ablation to the skin.

[0011]

[0012] In some embodiments, the method of stimulating hair growth further comprises applying radio frequency thermal ablation to the skin.

[0012]

[0013] In some embodiments, the method of stimulating hair growth further comprises applying a microneedle device to the skin.

[0013]

[0014] In one embodiment, a method of administering a composition for hair growth to a patient in need of treatment for hair loss includes injecting the composition into the patient's skin, where the composition comprises hyaluronic acid and a CD44 binding ligand. The composition can be injected through a needle, for example, where the needle is inserted 400 microns to about 2 mm into the skin prior to injection. In some embodiments, the needle is a microneedle. In some embodiments, the composition can be encapsulated in a liposome, for example a liposome comprising a hydrogenated phospholipid.

[0014]

[0015] In one embodiment, the composition for stimulating hair growth comprises two or more of osteopontin, hyaluronic acid, serglycin, chondroitin sulfate, fibrin, IGFBP4 and GFP10.

[0015]

[0016] In one embodiment, a composition for stimulating hair growth comprising hyaluronic acid at a concentration of about 1 mcg / mL to about 250 mcg / mL. The hyaluronic acid can have an average molecular weight, for example, in the range of about 4,000 Da to about 10,000 Da, in the range of about 10,000 Da to about 100,000 Da, in the range of about 15 kDa to about 50 kDa, in the range of about 75 kDa to about 350 kDa, or in the range of about 20 kDa to about 1350 kDa. In one embodiment, the hyaluronic acid has an average molecular weight greater than about 950 kDa.

[0016]

[0017] In some embodiments of the composition, the hyaluronic acid is cross-linked. For example, the hyaluronic acid can have a cross-link density of about 20% or greater.

[0017]

[0018] In certain embodiments of the composition, hyaluronic acid is from about 200mcg / mL to about 950mcg / mL, from about 200mcg / mL to about 900mcg / mL, from about 200mcg / mL to about 850mcg / mL, from about 200mcg / mL to about 800mcg / mL, from about 200mcg / mL to about 750mcg / mL, from about 200mcg / mL to about 700mcg / mL, from about 200mcg / mL to about 650mcg / mL , about 200mcg / mL to about 600mcg / mL, about 200mcg / mL to about 550mcg / mL, about 200mcg / mL to about 500mcg / mL, about 200mcg / mL to about 450mcg / m L, about 200mcg / mL to about 400mcg / mL, about 200mcg / mL to about 350mcg / mL, about 200mcg / mL to about 300mcg / mL, about 200mcg / mL to about 250mcg / m L, about 100mcg / mL to about 750mcg / mL, about 100mcg / mL to about 700mcg / mL, about 100mcg / mL to about 600mcg / mL, about 100mcg / mL to about 500mcg / mL, about 100mcg / mL to about 400mcg / mL, about 100mcg / mL to about 300mcg / mL, about 100mcg / mL to about 200mcg / mL, about 50mcg / mL to about 500mcg / mL, about 50mcg / mL to about 400mcg / mL, about 50mcg / mL to about 300mcg / mL, about 50mcg / mL to about 200mcg / mL, about 50mcg / mL to about 100mcg / mL, about 25mcg / mL to about 250mcg / mL, about 25mcg / mL to about 100mcg / mL, about 100mcg / mL to about 250mcg / mL, or less than about 100mcg / mL.

[0018]

[0019] In some embodiments, the composition for stimulating hair growth comprising hyaluronic acid at a concentration of about 250 mcg / mL or less further comprises one or more of osteopontin, hyaluronic acid, serglycin, chondroitin sulfate, fibrin, IGFBP4 and GFP10.

[0019]

[0020] In certain embodiments of the invention, a method of stimulating hair growth in the skin of a patient in need thereof comprises administering to the patient's skin a composition comprising hyaluronic acid at a concentration of about 1 mcg / mL to about 250 mcg / mL. In such methods, administering can include injecting the composition into the dermis layer of the skin, for example, to a depth of about 400 microns to about 2 mm into the skin. In other ... 2 Skin ~ approx. 650 injections / cm 2 It may include injecting multiple injections into the amount of skin.In some embodiments, the method further includes further steps such as applying iontophoresis to the skin, applying electroporation to the skin, applying laser ablation to the skin, applying radiofrequency thermal ablation to the skin, and / or applying a microneedle device to the skin.In some embodiments, the composition further includes a CD44 binding ligand, such as osteopontin.

[0020]

[0021] In some embodiments of the present invention, a method of administering a composition for hair growth to a patient in need of treatment for hair loss comprises injecting the composition into the patient's skin, wherein the composition comprises hyaluronic acid at a concentration of about 1 mcg / mL to about 250 mcg / mL. In some embodiments, the composition is injected through a needle, for example, wherein the needle is inserted 400 microns to about 2 mm into the skin prior to injection. In some embodiments, the needle is a microneedle. In some embodiments, the composition further comprises a CD44 binding ligand, for example osteopontin.

[0021]

[0022] The following detailed description of embodiments of compositions and methods for stimulating hair growth will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the precise configurations shown.

[0022]

[0023] In the drawings: [Brief description of the drawings]

[0023] [Figure 1]

[0024] Figure 1 illustrates the sequences and domains of osteopontin. SEQ ID NO: 1 represents the amino acids of exon 4 of osteopontin; SEQ ID NO: 2 represents the amino acid residues of exon 5 of osteopontin; SEQ ID NO: 3 represents integrin binding domain #2 (amino acid residues 162-168) of osteopontin. [Diagram 2]

[0025] FIG. 2 illustrates the domains and structure of osteopontin; SEQ ID NO:4 represents the integrin binding site of osteopontin; SEQ ID NO:5 represents the integrin binding site of osteopontin. [Diagram 3]

[0026] Figure 3 illustrates the domains and structure of osteopontin. SEQ ID NO:6 represents amino acid residues 17-314 of osteopontin. [Figure 4]

[0027] Figure 4 illustrates the sequences and domains of osteopontin. SEQ ID NO:3 represents the integrin binding domain #2 of osteopontin (amino acid residues 162-168); SEQ ID NO:7 represents the calcium binding domain of osteopontin (amino acid residues 216-228); and SEQ ID NO:8 represents the sequence of osteopontin including the signal peptide (amino acid residues 1-16). [Diagram 5]

[0028] FIG. 5 shows mice 18 days after injection with a control solution and solutions of 25, 50 and 100 mcg / mL low molecular weight hyaluronic acid. [Figure 6]

[0029] FIG. 6 shows mice 18 days after injection with a control solution and solutions of 25, 50 and 100 mcg / mL low molecular weight hyaluronic acid. [Figure 7]

[0030] FIG. 7 shows mice 18 days after injection with a control solution and solutions of 25, 50 and 100 mcg / mL high molecular weight hyaluronic acid. [Figure 8]

[0031] FIG. 8 shows mice 18 days after injection with a control solution and solutions of 25, 50 and 100 mcg / mL high molecular weight hyaluronic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024]

[0032] The present invention provides methods and compositions useful for stimulating hair growth.

[0025]

[0033] I. Composition

[0034] In certain embodiments, the hair growth stimulating composition comprises a naturally occurring ligand for CD44 or a fragment or derivative thereof.

[0026]

[0035] In certain embodiments, the naturally occurring ligand for CD44 is one or more of osteopontin (OPN, SPP1), serglycin (SRGN), chondroitin sulfate, collagen, fibronectin, fibrin, or a CD44-binding fragment, isoform, or derivative thereof.

[0027]

[0036] CD44-binding fragments, isoforms or derivatives of naturally occurring ligands for CD44 include synthetic peptides that bind to CD44 and are generated based on the CD44-binding domains of osteopontin, serglycin, chondroitin sulfate, collagen, fibronectin and / or fibrin.

[0028]

[0037] In some embodiments, the hair growth stimulating composition comprises hyaluronic acid.

[0029]

[0038] In certain embodiments, the composition for stimulating hair growth comprises one or more naturally occurring ligands for CD44, or fragments or derivatives thereof, and hyaluronic acid.

[0030]

[0039] In some embodiments, the hair growth stimulating composition comprises a combination of two or more of hyaluronic acid, osteopontin, serglycin, chondroitin sulfate, collagen, fibronectin, fibrin, and their proteolytically or synthetically produced CD44 binding fragments, isoforms, or derivatives. In some embodiments, the hair growth composition comprises three or more of hyaluronic acid, osteopontin, serglycin, chondroitin sulfate, collagen, fibronectin, fibrin, and their proteolytically or synthetically produced fragments, isoforms, or derivatives that bind to CD44. Any of the above may also include a hyaluronidase inhibitor and / or one or more other components.

[0031]

[0040] Various non-limiting embodiments include the compositions set forth in the table below, where an "X" indicates that the element is included in an embodiment of the invention:

[0032] [Table 1-1]

[0033] [Table 1-2]

[0034] [Table 1-3]

[0035] [Table 1-4]

[0036]

[0041] In some embodiments, the composition for stimulating hair growth can include one or more ligands of CD44. Such ligands can include osteopontin. Other ligands within the scope of the present invention include serglycin, chondroitin sulfate (e.g., chondroitin 4-sulfate), collagen, fibronectin, fibrin, insulin growth factor binding protein (IGFBP) (e.g., IGFBP-1, IGFBP-2, IGFBP-3, IGFBP4, IGFBP-5 and IGFBP-6), green fluorescent protein and hyaluronic acid.

[0037]

[0042] In certain embodiments, a composition can comprise a fragment, isoform, or derivative of a ligand of CD44, where such fragment, isoform, or derivative has at least 95% identity, at least 90% identity, at least 85% identity, at least 80% identity, at least 75% identity, at least 70% identity, at least 65% identity, or at least 60% identity to the corresponding ligand of CD44.

[0038]

[0043] In certain embodiments, the composition comprises about 0.01% or more CD44 binding ligand by weight, about 0.025% or more CD44 binding ligand by weight, about 0.050% or more CD44 binding ligand by weight, about 0.075% or more CD44 binding ligand by weight, about 0.1% or more CD44 binding ligand by weight, about 0.25% or more CD44 binding ligand by weight, about 0.5% or more CD44 binding ligand by weight, about 0.75% or more CD44 binding ligand by weight, about 1% or more CD44 binding ligand by weight, about 2.5% or more CD44 binding ligand by weight, about 5% or more CD44 binding ligand, about 7.5% or more CD44 binding ligand by weight, or about 10% or more CD44 binding ligand by weight. In certain embodiments, the composition comprises about 0.01% to about 0.025% by weight of CD44 binding ligand, about 0.025% to about 0.05% by weight of CD44 binding ligand, about 0.025% to about 0.075% by weight of CD44 binding ligand, about 0.025% to about 0.1% by weight of CD44 binding ligand, about 0.01% to about 0.1% by weight of CD44 binding ligand, about 0.05% to about 0.075% by weight of CD44 binding ligand, about 0.05% to about 0.1% by weight of CD44 binding ligand, about 0.075% to about 0.1% by weight of CD44 binding ligand, about 0.1% to about 0.2% by weight of CD44 binding ligand, about 0.1% to about 0.5% by weight of CD44 binding ligand. ligand, about 0.2% to about 0.4% by weight of CD44 binding ligand, about 0.5% to about 1% by weight of CD44 binding ligand, about 0.4% to about 0.6% by weight of CD44 binding ligand, about 0.6 to about 0.8% by weight of CD44 binding ligand, about 0.8% to about 1% by weight of CD44 binding ligand, about 1% to about 2% by weight of CD44 binding ligand, about 1% to about 5% by weight of CD44 binding ligand, about 2% to about 4% by weight of CD44 binding ligand, about 5% to about 10% by weight of CD44 binding ligand, about 4% to about 6% by weight of CD44 binding ligand, about 6 to about 8% by weight of CD44 binding ligand, or about 8% to about 10% by weight of CD44 binding ligand.

[0039]

[0044] 1. Osteopontin

[0045] Osteopontin is an extracellular signaling protein that is a natural ligand for CD44. Figures 1-4 illustrate the sequence and domain structure of osteopontin. Without being bound by theory, it is believed that osteopontin can induce activation of CD44 by proteolytic cleavage of CD44, resulting in intracellular release of the intracellular domain (ICD) of CD44. The CD44-ICD is then free to migrate into the nucleus and regulate gene expression. Osteopontin is discussed in International Patent Publication No. 2018175630, which is incorporated herein by reference in its entirety.

[0040]

[0046] In some embodiments, a composition useful for stimulating hair growth comprises osteopontin or a CD44-binding fragment, isoform, or derivative thereof. In some embodiments, the CD44-binding fragment, isoform, or derivative of osteopontin comprises a peptide generated by proteolytic cleavage of a naturally occurring (e.g., full-length) osteopontin protein. In some embodiments, the CD44-binding fragment, isoform, or derivative of osteopontin comprises residues 290-305 of osteopontin, residues 291-304 of osteopontin, comprises residues 292-303 of osteopontin, comprises residues 293-302 of osteopontin, comprises residues 294-301 of osteopontin, or comprises residues 295-300 of osteopontin. In some embodiments, the CD44-binding fragment, isoform, or derivative of osteopontin comprises SEQ ID NO:2. In some embodiments, the CD44-binding fragment, isoform, or derivative of osteopontin comprises SEQ ID NO:3.

[0041]

[0047] In certain embodiments, the composition comprises about 0.01% by weight or more of osteopontin, about 0.025% by weight or more of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.050% by weight or more of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.075% by weight or more of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.1% by weight or more of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.25% by weight or more of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.5% by weight or more of osteopontin or a CD44-binding fragment thereof. The osteopontin or CD44-binding fragment thereof, isoform or derivative may comprise about 0.75% by weight or more of osteopontin or its CD44-binding fragment, isoform or derivative, about 1% by weight or more of osteopontin or its CD44-binding fragment, isoform or derivative, about 2.5% by weight or more of osteopontin or its CD44-binding fragment, isoform or derivative, about 5% by weight or more of osteopontin or its CD44-binding fragment, isoform or derivative, about 7.5% by weight or more of osteopontin or its CD44-binding fragment, isoform or derivative, or about 10% by weight or more of osteopontin or its CD44-binding fragment, isoform or derivative. In one embodiment, the composition comprises about 0.01% to about 0.025% by weight of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.025% to about 0.05% by weight of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.025% to about 0.075% by weight of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.025% to about 0.1% by weight of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.01% to about 0.1% by weight of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.05% to about 0.075% by weight of osteopontin or a CD44-binding fragment thereof, isoform or derivative, about 0.05% to about 0.1% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 0.075% by weight to about 0.1% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 0.1% by weight to about 0.2% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 0.1% by weight to about 0.5% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 0.2% by weight to about 0.4% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 0.5% by weight to about 1% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 0.4% by weight to about 0.6% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 0.6% by weight to about 0.8% by weight of osteopontin or its CD44-binding fragment, isoform or derivative or derivatives, about 0.8% to about 1% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 1% to about 2% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 1% to about 5% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 2% to about 4% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 5% to about 10% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 4% to about 6% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, about 6% to about 8% by weight of osteopontin or its CD44-binding fragment, isoform or derivative, or about 8% to about 10% by weight of osteopontin or its CD44-binding fragment, isoform or derivative.

[0042]

[0048] 2. Other ligands for CD44

[0049] In some embodiments, the composition useful for stimulating hair growth comprises one or more ligands for CD44 other than or in addition to osteopontin. Such ligands include serglycin, chondroitin sulfate (e.g., chondroitin 4-sulfate), collagen, fibronectin, fibrin, insulin growth factor binding protein (IGFBP) (e.g., IGFBP-1, IGFBP-2, IGFBP-3, IGFBP4, IGFBP-5, and IGFBP-6), and green fluorescent protein (e.g., GFP10). Another ligand for CD44 is hyaluronic acid, which is discussed further below. In some embodiments, the composition comprises IGFBP4 and GFP10.

[0043]

[0050] Without being bound by theory, it is believed that each of these components may function as a ligand that binds to CD44. In some embodiments, a composition useful for stimulating hair growth comprises a ligand that binds to CD44. In some embodiments, a composition useful for stimulating hair growth comprises a combination of two or more (e.g., three or four) ligands that bind to CD44.

[0044]

[0051] Serglycin

[0052] In some embodiments, the composition comprises about 0.01% or more serglycin by weight, about 0.025% or more serglycin by weight, about 0.050% or more serglycin by weight, about 0.075% or more serglycin by weight, about 0.1% or more serglycin by weight, about 0.25% or more serglycin by weight, about 0.5% or more serglycin by weight, about 0.75% or more serglycin by weight, about 1% or more serglycin by weight, about 2.5% or more serglycin by weight, about 5% or more serglycin by weight, about 7.5% or more serglycin by weight, or about 10% or more serglycin by weight. In certain embodiments, the composition comprises about 0.01% to about 0.025% by weight of serglycine, about 0.025% to about 0.05% by weight of serglycine, about 0.025% to about 0.075% by weight of serglycine, about 0.025% to about 0.1% by weight of serglycine, about 0.01% to about 0.1% by weight of serglycine, about 0.05% to about 0.075% by weight of serglycine, about 0.05% to about 0.1% by weight of serglycine, about 0.075% to about 0.1% by weight of serglycine, about 0.1% to about 0.2% by weight of serglycine, about 0.1% to about 0.5% by weight of serglycine, % serglycine, about 0.2% to about 0.4% by weight serglycine, about 0.5% to about 1% by weight serglycine, about 0.4% to about 0.6% by weight serglycine, about 0.6 to about 0.8% by weight serglycine, about 0.8% to about 1% by weight serglycine, about 1% to about 2% by weight serglycine, about 1% to about 5% by weight serglycine, about 2% to about 4% by weight serglycine, about 5% to about 10% by weight serglycine, about 4% to about 6% by weight serglycine, about 6 to about 8% by weight serglycine, or about 8% to about 10% by weight serglycine.

[0045]

[0053] B. Chondroitin

[0054] In some embodiments, the composition comprises chondroitin or a salt thereof, such as chondroitin sulfate. In some embodiments, the composition comprises about 0.01% by weight or more of chondroitin or a salt thereof, about 0.025% by weight or more of chondroitin or a salt thereof, about 0.050% by weight or more of chondroitin or a salt thereof, about 0.075% by weight or more of chondroitin or a salt thereof, about 0.1% by weight or more of chondroitin or a salt thereof, about 0.25% by weight or more of chondroitin or a salt thereof, about 0.5% by weight or more of chondroitin or a salt thereof, about 0.75% by weight or more of chondroitin or a salt thereof, about 1% by weight or more of chondroitin or a salt thereof, about 2.5% by weight or more of chondroitin or a salt thereof, about 5% by weight or more of chondroitin or a salt thereof, about 7.5% by weight or more of chondroitin or a salt thereof, or about 10% by weight or more of chondroitin or a salt thereof.In one embodiment, the composition comprises about 0.01% by weight to about 0.025% by weight of chondroitin or a salt thereof, about 0.025% by weight to about 0.05% by weight of chondroitin or a salt thereof, about 0.025% by weight to about 0.075% by weight of chondroitin or a salt thereof, about 0.025% by weight to about 0.1% by weight of chondroitin or a salt thereof, about 0.01% by weight to about 0.1% by weight of chondroitin or a salt thereof, about 0.05% by weight to about 0.075% by weight of chondroitin or a salt thereof, about 0.05% by weight to about 0.1% by weight of chondroitin or a salt thereof, about 0.075% by weight to about 0.1% by weight of chondroitin or a salt thereof, about 0.1% by weight to about 0.2% by weight of chondroitin or a salt thereof, or about 0.1% by weight to about 0.5% by weight of chondroitin or a salt thereof. , about 0.2% to about 0.4% by weight of chondroitin or a salt thereof, about 0.5% to about 1% by weight of chondroitin or a salt thereof, about 0.4% to about 0.6% by weight of chondroitin or a salt thereof, about 0.6 to about 0.8% by weight of chondroitin or a salt thereof, about 0.8% to about 1% by weight of chondroitin or a salt thereof, about 1% to about 2% by weight of chondroitin or a salt thereof, about 1% to about 5% by weight of chondroitin or a salt thereof, about 2% to about 4% by weight of chondroitin or a salt thereof, about 5% to about 10% by weight of chondroitin or a salt thereof, about 4% to about 6% by weight of chondroitin or a salt thereof, about 6 to about 8% by weight of chondroitin or a salt thereof, or about 8% to about 10% by weight of chondroitin or a salt thereof.

[0046]

[0055] C fibrin

[0056] In certain embodiments, the composition comprises about 0.01% or more by weight fibrin, about 0.025% or more by weight fibrin, about 0.050% or more by weight fibrin, about 0.075% or more by weight fibrin, about 0.1% or more by weight fibrin, about 0.25% or more by weight fibrin, about 0.5% or more by weight fibrin, about 0.75% or more by weight fibrin, about 1% or more by weight fibrin, about 2.5% or more by weight fibrin, about 5% or more by weight fibrin, about 7.5% or more by weight fibrin, or about 10% or more by weight fibrin. In one embodiment, the composition comprises about 0.01% by weight to about 0.025% by weight of fibrin, about 0.025% by weight to about 0.05% by weight of fibrin, about 0.025% by weight to about 0.075% by weight of fibrin, about 0.025% by weight to about 0.1% by weight of fibrin, about 0.01% by weight to about 0.1% by weight of fibrin, about 0.05% by weight to about 0.075% by weight of fibrin, about 0.05% by weight to about 0.1% by weight of fibrin, about 0.075% by weight to about 0.1% by weight of fibrin, about 0.1% by weight to about 0.2% by weight of fibrin, about 0.1% by weight to about 0.5 % by weight of fibrin, about 0.2% by weight to about 0.4% by weight of fibrin, about 0.5% by weight to about 1% by weight of fibrin, about 0.4% by weight to about 0.6% by weight of fibrin, about 0.6% by weight to about 0.8% by weight of fibrin, about 0.8% by weight to about 1% by weight of fibrin, about 1% by weight to about 2% by weight of fibrin, about 1% by weight to about 5% by weight of fibrin, about 2% by weight to about 4% by weight of fibrin, about 5% by weight to about 10% by weight of fibrin, about 4% by weight to about 6% by weight of fibrin, about 6% by weight to about 8% by weight of fibrin, or about 8% by weight to about 10% by weight of fibrin.

[0047]

[0057] D. Collagen

[0058] In certain embodiments, the composition comprises about 0.01% or more collagen by weight, about 0.025% or more collagen by weight, about 0.050% or more collagen by weight, about 0.075% or more collagen by weight, about 0.1% or more collagen by weight, about 0.25% or more collagen by weight, about 0.5% or more collagen by weight, about 0.75% or more collagen by weight, about 1% or more collagen by weight, about 2.5% or more collagen by weight, about 5% or more collagen, about 7.5% or more collagen, or about 10% or more collagen by weight. In certain embodiments, the composition comprises about 0.01% to about 0.025% by weight collagen, about 0.025% to about 0.05% by weight collagen, about 0.025% to about 0.075% by weight collagen, about 0.025% to about 0.1% by weight collagen, about 0.01% to about 0.1% by weight collagen, about 0.05% to about 0.075% by weight collagen, about 0.05% to about 0.1% by weight collagen, about 0.075% to about 0.1% by weight collagen, about 0.1% to about 0.2% by weight collagen, about 0.1% to about 0.5% by weight collagen, % by weight of collagen, about 0.2% by weight to about 0.4% by weight of collagen, about 0.5% by weight to about 1% by weight of collagen, about 0.4% by weight to about 0.6% by weight of collagen, about 0.6% by weight to about 0.8% by weight of collagen, about 0.8% by weight to about 1% by weight of collagen, about 1% by weight to about 2% by weight of collagen, about 1% by weight to about 5% by weight of collagen, about 2% by weight to about 4% by weight of collagen, about 5% by weight to about 10% by weight of collagen, about 4% by weight to about 6% by weight of collagen, about 6% by weight to about 8% by weight of collagen, or about 8% by weight to about 10% by weight of collagen.

[0048]

[0059] Fibronectin

[0060] In some embodiments, the composition comprises greater than about 0.01% fibronectin by weight, greater than about 0.025% fibronectin by weight, greater than about 0.050% fibronectin by weight, greater than about 0.075% fibronectin by weight, greater than about 0.1% fibronectin by weight, greater than about 0.25% fibronectin by weight, greater than about 0.5% fibronectin by weight, greater than about 0.75% fibronectin by weight, greater than about 1% fibronectin by weight, greater than about 2.5% fibronectin by weight, greater than about 5% fibronectin by weight, greater than about 7.5% fibronectin by weight, or greater than about 10% fibronectin by weight. In one embodiment, the composition comprises about 0.01% to about 0.025% by weight of fibronectin, about 0.025% to about 0.05% by weight of fibronectin, about 0.025% to about 0.075% by weight of fibronectin, about 0.025% to about 0.1% by weight of fibronectin, about 0.01% to about 0.1% by weight of fibronectin, about 0.05% to about 0.075% by weight of fibronectin, about 0.05% to about 0.1% by weight of fibronectin, about 0.075% to about 0.1% by weight of fibronectin, about 0.1% to about 0.2% by weight of fibronectin, about 0.1% to about 0.5% by weight of fibronectin. fibronectin, about 0.2% to about 0.4% by weight of fibronectin, about 0.5% to about 1% by weight of fibronectin, about 0.4% to about 0.6% by weight of fibronectin, about 0.6 to about 0.8% by weight of fibronectin, about 0.8% to about 1% by weight of fibronectin, about 1% to about 2% by weight of fibronectin, about 1% to about 5% by weight of fibronectin, about 2% to about 4% by weight of fibronectin, about 5% to about 10% by weight of fibronectin, about 4% to about 6% by weight of fibronectin, about 6 to about 8% by weight of fibronectin, or about 8% to about 10% by weight of fibronectin.

[0049]

[0061] f insulin-like growth factor binding protein

[0062] In certain embodiments, the composition comprises about 0.01% or more by weight of insulin-like growth factor binding protein, about 0.025% or more by weight of insulin-like growth factor binding protein, about 0.050% or more by weight of insulin-like growth factor binding protein, about 0.075% or more by weight of insulin-like growth factor binding protein, about 0.1% or more by weight of insulin-like growth factor binding protein, about 0.25% or more by weight of insulin-like growth factor binding protein, about 0.5% or more by weight of insulin-like growth factor binding protein, about 0.75% or more by weight of insulin-like growth factor binding protein, about 1% or more by weight of insulin-like growth factor binding protein, about 2.5% or more by weight of insulin-like growth factor binding protein, about 5% or more by weight of insulin-like growth factor binding protein, about 7.5% or more by weight of insulin-like growth factor binding protein, or about 10% or more by weight of insulin-like growth factor binding protein.In certain embodiments, the composition comprises about 0.01% to about 0.025% by weight of insulin-like growth factor binding protein, about 0.025% to about 0.05% by weight of insulin-like growth factor binding protein, about 0.025% to about 0.075% by weight of insulin-like growth factor binding protein, about 0.025% to about 0.1% by weight of insulin-like growth factor binding protein, about 0.01% to about 0.1% by weight of insulin-like growth factor binding protein, about 0.05% to about 0.075% by weight of insulin-like growth factor binding protein, about 0.05% to about 0.1% by weight of insulin-like growth factor binding protein, about 0.075% to about 0.1% by weight of insulin-like growth factor binding protein, about 0.1% to about 0.2% by weight of insulin-like growth factor binding protein, about 0.1% to about 0.5% by weight of insulin-like growth factor binding protein, The insulin-like growth factor binding protein may comprise 0.2% by weight to about 0.4% by weight of insulin-like growth factor binding protein, about 0.5% by weight to about 1% by weight of insulin-like growth factor binding protein, about 0.4% by weight to about 0.6% by weight of insulin-like growth factor binding protein, about 0.6% by weight to about 0.8% by weight of insulin-like growth factor binding protein, about 0.8% by weight to about 1% by weight of insulin-like growth factor binding protein, about 1% by weight to about 2% by weight of insulin-like growth factor binding protein, about 1% by weight to about 5% by weight of insulin-like growth factor binding protein, about 2% by weight to about 4% by weight of insulin-like growth factor binding protein, about 5% by weight to about 10% by weight of insulin-like growth factor binding protein, about 4% by weight to about 6% by weight of insulin-like growth factor binding protein, about 6% by weight to about 8% by weight of insulin-like growth factor binding protein, or about 8% by weight to about 10% by weight of insulin-like growth factor binding protein.

[0050]

[0063] G green fluorescent protein

[0064] In certain embodiments, the composition comprises about 0.01% or more by weight of green fluorescent protein, about 0.025% or more by weight of green fluorescent protein, about 0.050% or more by weight of green fluorescent protein, about 0.075% or more by weight of green fluorescent protein, about 0.1% or more by weight of green fluorescent protein, about 0.25% or more by weight of green fluorescent protein, about 0.5% or more by weight of green fluorescent protein, about 0.75% or more by weight of green fluorescent protein, about 1% or more by weight of green fluorescent protein, about 2.5% or more by weight of green fluorescent protein, about 5% or more by weight of green fluorescent protein, about 7.5% or more by weight of green fluorescent protein, or about 10% or more by weight of green fluorescent protein. In one embodiment, the composition comprises about 0.01% by weight to about 0.025% by weight of green fluorescent protein, about 0.025% by weight to about 0.05% by weight of green fluorescent protein, about 0.025% by weight to about 0.075% by weight of green fluorescent protein, about 0.025% by weight to about 0.1% by weight of green fluorescent protein, about 0.01% by weight to about 0.1% by weight of green fluorescent protein, about 0.05% by weight to about 0.075% by weight of green fluorescent protein, about 0.05% by weight to about 0.1% by weight of green fluorescent protein, about 0.075% by weight to about 0.1% by weight of green fluorescent protein, about 0.1% by weight to about 0.2% by weight of green fluorescent protein, about 0.1% by weight to about 0.5% by weight of green fluorescent protein. protein, about 0.2% to about 0.4% by weight of green fluorescent protein, about 0.5% to about 1% by weight of green fluorescent protein, about 0.4% to about 0.6% by weight of green fluorescent protein, about 0.6 to about 0.8% by weight of green fluorescent protein, about 0.8% to about 1% by weight of green fluorescent protein, about 1% to about 2% by weight of green fluorescent protein, about 1% to about 5% by weight of green fluorescent protein, about 2% to about 4% by weight of green fluorescent protein, about 5% to about 10% by weight of green fluorescent protein, about 4% to about 6% by weight of green fluorescent protein, about 6 to about 8% by weight of green fluorescent protein, or about 8% to about 10% by weight of green fluorescent protein.

[0051]

[0065] 3. Hyaluronic Acid

[0066] In some embodiments, the composition useful for stimulating hair growth comprises hyaluronic acid. Hyaluronic acid is the natural ligand for CD44, which is now known to be proinflammatory and stimulate hair growth. Hyaluronic acid is a natural linear polymer containing repeating disaccharide units of β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine, and is represented by the following formula I:

[0067] Formula I:

[0052] [ka]

[0053]

[0068] In some embodiments, hyaluronic acid has a low average molecular weight, which as used herein refers to a range of about 15,000 Da to about 40,000 Da. In some embodiments, hyaluronic acid has a medium average molecular weight, which as used herein refers to a range of about 75,000 Da to about 350,000 Da. In some embodiments, hyaluronic acid has a high average molecular weight, which as used herein refers to about 950,000 Da or more.

[0054]

[0069] In one embodiment, the hyaluronic acid has an average molecular weight in the range of about 4,000 Da or less to about 10,000 Da. In one embodiment, the hyaluronic acid has an average molecular weight in the range of about 10,000 Da to about 100,000 Da. In one embodiment, the hyaluronic acid has an average molecular weight in the range of about 100,000 Da to about 1,500,000 Da or more.

[0055]

[0070] In one embodiment, the hyaluronic acid has an affinity for about 1 kDa to about 10 kDa, about 10 kDa to about 50 kDa, about 50 kDa to about 100 kDa, about 100 kDa to about 150 kDa, about 200 kDa to about 250 kDa, about 300 kDa to about 350 kDa, about 400 kDa to about 450 kDa, about 500 kDa to about 5 50kDa, about 600kDa to about 650kDa, about 700kDa to about 750kDa, about 800kDa to about 850kDa, about 900kDa to about 1000kDa, about 1000kDa to about 1100kDa, about 1100kDa to about 1200kDa, about 1200kDa to about 1300kDa, about 1300kDa ~about 1400kDa, about 1400kDa to about 1500kDa, about 1kDa to about 100kDa, about 100kDa to about 250kDa, about 250kDa to about 500kDa, about 500kDa to about 750kDa, about 750kDa to about 1000kDa, about 1000kDa to about 1250kDa, about 1250kDa about 1500 kDa, about 1 kDa to about 250 kDa, about 1 kDa to about 500 kDa, about 100 kDa to about 500 kDa, about 250 kDa to about 750 kDa, about 500 kDa to about 1000 kDa, about 750 kDa to about 1250 kDa, or about 1000 kDa to about 1500 kDa.

[0056]

[0071] In some embodiments, the hyaluronic acid is cross-linked. Cross-linking may improve the longevity of hyaluronic acid, and in some embodiments, some degree of cross-linking is desirable. In some embodiments, the hyaluronic acid has sufficient cross-linking to last about one week. However, without being bound by mechanism of action, it is believed that hyaluronic acid is effective in stimulating hair growth by interacting with CD44 receptor. Therefore, it is desirable that the hyaluronic acid is not cross-linked so extensively that the cross-linking interferes with the ability of hyaluronic acid to interact with CD44 receptor.

[0057]

[0072] The hydroxyl (--OH), carboxyl (--COOH), and / or amide (--NHCOCH 3The functional groups can be crosslinked via ether bonds (R--O--R), ester bonds (R--COO--R), or carbodiimides, respectively. In some embodiments, hyaluronic acid is crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), glutaraldehyde (GTA), poly(ethylene glycol) diglycidyl ether (PEGDE), ethylene glycol diglycidyl ether (EGDE), divinyl sulfonate (DVS), or pentaerythritol tetraacrylate (PT).

[0058]

[0073] Hyaluronic acid is approximately 1 x 10 -7 mol / cm 3 That's about 2 x 10 -7 mol / cm 3 That's about 3 x 10 -7 mol / cm 3 That's about 4 x 10 -7 mol / cm 3 That's about 5 x 10 -7 mol / cm 3 That's about 6 x 10 -7 mol / cm 3 That's about 7 x 10 -7 mol / cm 3 That's about 8 x 10 -7 mol / cm 3 That's about 9 x 10 -7 mol / cm 3 That's about 1 x 10 -6 mol / cm 3 That's about 2 x 10 -6 mol / cm 3 That's about 3 x 10 -6 mol / cm 3 That's about 4 x 10 -6 mol / cm 3 That's about 5 x 10 -6 mol / cm 3 That's about 6 x 10 -6 mol / cm 3 That's about 7 x 10 -6 mol / cm 3 That's about 8 x 10 -6 mol / cm 3 That's about 9 x 10 -6mol / cm 3 or more or about 1×10 -5 mol / cm 3 In one embodiment, the hyaluronic acid can have a crosslink density of about 1×10 -7 mol / cm 3 ~Approx. 1×10 -5 mol / cm 3 , about 1×10 -7 mol / cm 3 ~Approx. 1×10 -6 mol / cm 3 , about 1×10 -7 mol / cm 3 ~Approx. 5×10 -7 mol / cm 3 , about 5×10 -7 mol / cm 3 ~Approx. 1×10 -6 mol / cm 3 , about 1×10 -7 mol / cm 3 ~about 2×10 -7 mol / cm 3 , about 2×10 -7 mol / cm 3 ~Approx. 4×10 -7 mol / cm 3 , about 4×10 -7 mol / cm 3 ~about 6×10 -7 mol / cm 3 , about 6×10 -7 mol / cm 3 ~Approx. 8×10 -7 mol / cm 3 or about 8 x 10 -7 mol / cm 3 ~Approx. 1×10 -6 mol / cm 3 , about 1×10 -6 mol / cm 3 ~Approx. 1×10 -5 mol / cm 3 , about 1×10 -6 mol / cm 3 ~Approx. 5×10 -6 mol / cm 3 , about 5×10 -6 mol / cm 3 ~Approx. 1×10 -5 mol / cm3 , about 1×10 -6 mol / cm 3 ~about 2×10 -6 mol / cm 3 , about 2×10 -7 mol / cm 3 ~Approx. 4×10 -7 mol / cm 3 , about 4×10 -7 mol / cm 3 ~about 6×10 -7 mol / cm 3 , about 6×10 -7 mol / cm 3 ~Approx. 8×10 -7 mol / cm 3 , about 8×10 -7 mol / cm 3 ~Approx. 1×10 -5 mol / cm 3 or about 5 x 10 -7 mol / cm 3 ~Approx. 5×10 -6 mol / cm 3 The crosslink density may be

[0059]

[0074] The composition of the present invention may contain hyaluronic acid in an amount sufficient to provide therapeutic effect, for example, stimulating hair growth in a patient in need thereof.However, higher concentrations of hyaluronic acid may result in undesirable inflammation.In some embodiments, the composition of the present invention contains hyaluronic acid in an amount sufficient to provide therapeutic effect, for example, stimulating hair growth in a patient in need thereof, but not sufficient to provide unacceptable inflammatory reaction.As used throughout this description, mcg / mL and ug / mL both refer to micrograms per milliliter. In some embodiments, the hyaluronic acid is at least about 10mcg per mL of the composition, at least about 15mcg per mL of the composition, at least about 20mcg per mL of the composition, at least about 25mcg per mL of the composition, at least about 30mcg per mL of the composition, at least about 35mcg per mL of the composition, at least about 40mcg per mL of the composition, at least about 45mcg per mL of the composition, at least about 50mcg per mL of the composition, at least about 55mcg per mL of the composition, at least about 60mcg per mL of the composition, at least about 65mcg per mL of the composition, at least about 70mcg per mL of the composition, at least about 75mcg per mL of the composition, at least about 80mcg per mL of the composition, at least about 85mcg per mL of the composition, at least about 90mcg per mL of the composition, at least about 95mcg per mL of the composition, at least about 100mcg per mL of the composition, about 110mcg or more per mL of the composition, about 120mcg or more per mL of the composition, about 130mcg or more per mL of the composition, about 140mcg or more per mL of the composition, about 150mcg or more per mL of the composition, about 160mcg or more per mL of the composition, about 170mcg or more per mL of the composition, about 180mcg or more per mL of the composition, about 190mcg or more per mL of the composition, about 200mcg or more, about 210mcg or more per mL of the composition, about 220mcg or more per mL of the composition, about 230mcg or more per mL of the composition, about 240mcg or more per mL of the composition, about 250mcg or more per mL of the composition, about 260mcg or more per mL of the composition, about 270mcg or more per mL of the composition, about 280mcg or more per mL of the composition, about 290mcg or more per mL of the composition,about 300mcg or more per mL of the composition, about 310mcg or more per mL of the composition, about 320mcg or more per mL of the composition, about 330mcg or more per mL of the composition, about 340mcg or more per mL of the composition, about 350mcg or more per mL of the composition, about 360mcg or more per mL of the composition, about 370mcg or more per mL of the composition, about 380mcg or more per mL of the composition, about 390mcg or more per mL of the composition, about 400mcg or more per mL of the composition, about 410mcg or more per mL of the composition, about 420mcg or more per mL of the composition, about 430mcg or more per mL of the composition, about 440mcg or more per mL of the composition, about 450mcg or more per mL of the composition, about 460mcg or more per mL of the composition, about 470mcg or more per mL of the composition, about 480mcg or more per mL of the composition, about 490mcg or more per mL of the composition, about 500mcg or more per mL of the composition, about 510mcg or more per mL of the composition, about 520mcg or more per mL of the composition, about 530mcg or more per mL of the composition, about 540mcg or more per mL of the composition, about 550mcg or more per mL of the composition, about 560mcg or more per mL of the composition, about 570mcg or more per mL of the composition, about 580mcg or more per mL of the composition, about 590mcg or more per mL of the composition, about 600mcg or more per mL of the composition, about 610mcg or more per mL of the composition, about 620mcg or more per mL of the composition, about 630mcg or more per mL of the composition, about 640mcg or more per mL of the composition, about 650mcg or more per mL of the composition, about 660mcg or more per mL of the composition, about 670mcg or more per mL of the composition, about 680mcg or more per mL of the composition, about 700mcg or more per mL of the composition, about 710mcg or more per mL of the composition, about 720mcg or more per mL of the composition, about 730mcg or more per mL of the composition, about 740mcg or more per mL of the composition, about 750mcg or more per mL of the composition, about 760mcg or more per mL of the composition, about 770mcg or more per mL of the composition, about 780mcg or more per mL of the composition, about 790mcg or more per mL of the composition, about 800mcg or more per mL of the composition, about 810mcg or more per mL of the composition, about 820mcg or more per mL of the composition,is present in an amount of about 830mcg or more per mL of the composition, about 840mcg or more per mL of the composition, about 850mcg or more per mL of the composition, about 860mcg or more per mL of the composition, about 870mcg or more per mL of the composition, about 880mcg or more per mL of the composition, about 890mcg or more per mL of the composition, about 900mcg or more per mL of the composition, about 910mcg or more per mL of the composition, about 920mcg or more per mL of the composition, about 930mcg or more per mL of the composition, about 940mcg or more per mL of the composition, about 950mcg or more per mL of the composition, about 960mcg or more per mL of the composition, about 970mcg or more per mL of the composition, about 980mcg or more per mL of the composition.

[0060]

[0075] In some embodiments, hyaluronic acid is from about 1 mcg per mL of the composition to about 250 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 250 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 200 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 150 mcg per mL of the composition, from 10 mcg per mL of the composition to about 100 mcg per mL of the composition, from about 25 mcg per mL of the composition to about 250 mcg per mL of the composition, from about 25 mcg per mL of the composition to about 250 mcg per mL of the composition. About 200mcg per mL of composition, about 25mcg per mL to about 150mcg per mL of composition, about 25mcg per mL to about 100mcg per mL of composition, about 50mcg per mL to about 250mcg per mL of composition, about 50mcg per mL to about 200mcg per mL of composition, about 50mcg per mL to about 150mcg per mL of composition, about 50mcg per mL to about 100mcg per mL of composition, about 75mcg per mL of composition ~ about 250mcg per mL of the composition, about 75mcg per mL to about 200mcg per mL of the composition, about 75mcg per mL to about 150mcg per mL of the composition, about 75mcg per mL to about 100mcg per mL of the composition, about 100mcg per mL to about 250mcg per mL of the composition, about 100mcg per mL to about 200mcg per mL of the composition, about 100mcg per mL to about 150mcg per mL of the composition, about Present in an amount ranging from 150mcg to about 250mcg per mL of the composition, from about 200mcg per mL of the composition to about 250mcg per mL of the composition, from about 60mcg per mL of the composition to about 80mcg per mL of the composition, from about 50mcg per mL of the composition to about 75mcg per mL of the composition, from about 25mcg per mL of the composition to about 75mcg per mL of the composition, from about 10mcg per mL of the composition to about 50mcg per mL of the composition or from about 10mcg per mL of the composition to about 25mcg per mL of the composition.

[0061]

[0076] In some embodiments, hyaluronic acid is from about 1 mcg per mL of the composition to about 500 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 500 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 450 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 400 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 350 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 300 mcg per mL of the composition, from about 25 mcg per mL of the composition to about 500 mcg per mL of the composition. g, about 25mcg per mL to about 450mcg per mL of the composition, about 25mcg per mL to about 400mcg per mL of the composition, about 25mcg per mL to about 350mcg per mL of the composition, about 25mcg per mL to about 300mcg per mL of the composition, about 50mcg per mL to about 500mcg per mL of the composition, about 50mcg per mL to about 450mcg per mL of the composition, about 50mcg per mL to about 400mcg per mL of the composition, about 50 mcg to about 350mcg per mL of the composition, about 50mcg per mL to about 300mcg per mL of the composition, about 75mcg per mL to about 500mcg per mL of the composition, about 75mcg per mL to about 450mcg per mL of the composition, about 75mcg per mL to about 400mcg per mL of the composition, about 75mcg per mL to about 350mcg per mL of the composition, about 75mcg per mL to about 300mcg per mL of the composition, about 100mcg per mL to about 1 mL of the composition about 500mcg, about 100mcg per mL to about 450mcg per mL of the composition, about 100mcg per mL to about 400mcg per mL of the composition, about 100mcg per mL to about 350mcg per mL of the composition, about 100mcg per mL to about 300mcg per mL of the composition, about 150mcg per mL to about 500mcg per mL of the composition, about 150mcg per mL to about 450mcg per mL of the composition, about 150mcg per mL to about 400mcg per mL of the composition,is present in an amount ranging from about 150mcg per mL of the composition to about 350mcg per mL of the composition, from about 150mcg per mL of the composition to about 300mcg per mL of the composition, from about 200mcg per mL of the composition to about 500mcg per mL of the composition, from about 200mcg per mL of the composition to about 450mcg per mL of the composition, from about 200mcg per mL of the composition to about 400mcg per mL of the composition, from about 200mcg per mL of the composition to about 350mcg per mL of the composition, from about 200mcg per mL of the composition to about 300mcg per mL of the composition, or from about 200mcg per mL of the composition to about 250mcg per mL of the composition.

[0062]

[0077] In some embodiments, hyaluronic acid is from about 1 mcg per mL of the composition to about 750 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 750 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 700 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 650 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 600 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 550 mcg per mL of the composition, from about 25 mcg per mL of the composition to about 750 mcg per mL of the composition. g, about 25mcg per mL to about 700mcg per mL of the composition, about 25mcg per mL to about 650mcg per mL of the composition, about 25mcg per mL to about 600mcg per mL of the composition, about 25mcg per mL to about 550mcg per mL of the composition, about 50mcg per mL to about 750mcg per mL of the composition, about 50mcg per mL to about 700mcg per mL of the composition, about 50mcg per mL to about 650mcg per mL of the composition, about 50 mcg to about 600mcg per mL of the composition, about 50mcg per mL to about 550mcg per mL of the composition, about 75mcg per mL to about 750mcg per mL of the composition, about 75mcg per mL to about 700mcg per mL of the composition, about 75mcg per mL to about 650mcg per mL of the composition, about 75mcg per mL to about 600mcg per mL of the composition, about 75mcg per mL to about 550mcg per mL of the composition, about 100mcg per mL to about 1 mL of the composition about 750mcg, about 100mcg per mL to about 700mcg per mL of the composition, about 100mcg per mL to about 650mcg per mL of the composition, about 100mcg per mL to about 600mcg per mL of the composition, about 100mcg per mL to about 550mcg per mL of the composition, about 150mcg per mL to about 750mcg per mL of the composition, about 150mcg per mL to about 700mcg per mL of the composition, about 150mcg per mL to about 650mcg per mL of the composition,About 150mcg per mL of the composition to about 600mcg per mL of the composition, about 150mcg per mL of the composition to about 550mcg per mL of the composition, about 200mcg per mL of the composition to about 750mcg per mL of the composition, about 200mcg per mL of the composition to about 700mcg per mL of the composition, about 200mcg per mL of the composition to about 650mcg per mL of the composition, about 200mcg per mL of the composition to about 600mcg per mL of the composition, about 200mcg per mL of the composition to about 650mcg per mL of the composition About 550mcg per mL, about 300mcg per mL to about 750mcg per mL of the composition, about 300mcg per mL to about 700mcg per mL of the composition, about 300mcg per mL to about 650mcg per mL of the composition, about 300mcg per mL to about 600mcg per mL of the composition, about 300mcg per mL to about 550mcg per mL of the composition, about 400mcg per mL to about 750mcg per mL of the composition, about about 400mcg per mL to about 700mcg per mL of the composition, about 400mcg per mL to about 650mcg per mL of the composition, about 400mcg per mL to about 600mcg per mL of the composition, about 400mcg per mL to about 550mcg per mL of the composition, about 500mcg per mL to about 750mcg per mL of the composition, about 500mcg per mL to about 700mcg per mL of the composition, about 500mcg per mL to about 1 mL of the composition about 650mcg, about 500mcg per mL of the composition to about 600mcg per mL of the composition, about 500mcg per mL of the composition to about 550mcg per mL of the composition, about 600mcg per mL of the composition to about 750mcg per mL of the composition, about 600mcg per mL of the composition to about 700mcg per mL of the composition, about 600mcg per mL of the composition to about 650mcg per mL of the composition, or about 700mcg per mL of the composition to about 750mcg per mL of the composition.

[0063]

[0078] In some embodiments, the hyaluronic acid is from about 1 mcg per mL of the composition to about 950 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 950 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 900 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 850 mcg per mL of the composition, from about 10 mcg per mL of the composition to about 800 mcg per mL of the composition, from about 25 mcg per mL of the composition to about 950 mcg per mL of the composition, from about 25 mcg per mL of the composition to about 900 mcg per mL of the composition , about 25mcg per mL to about 850mcg per mL of the composition, about 25mcg per mL to about 800mcg per mL of the composition, about 50mcg per mL to about 950mcg per mL of the composition, about 50mcg per mL to about 900mcg per mL of the composition, about 50mcg per mL to about 850mcg per mL of the composition, about 50mcg per mL to about 800mcg per mL of the composition, about 75mcg per mL to about 950mcg per mL of the composition, about 75mcg per mL to about 750mcg per mL of the composition g to about 900mcg per mL of the composition, about 75mcg per mL to about 850mcg per mL of the composition, about 75mcg per mL to about 800mcg per mL of the composition, about 100mcg per mL to about 950mcg per mL of the composition, about 100mcg per mL to about 900mcg per mL of the composition, about 100mcg per mL to about 850mcg per mL of the composition, about 100mcg per mL to about 800mcg per mL of the composition, about 150mcg per mL to about 1 mL of the composition about 950mcg per mL of the composition, about 150mcg per mL to about 900mcg per mL of the composition, about 150mcg per mL to about 850mcg per mL of the composition, about 150mcg per mL to about 800mcg per mL of the composition, about 200mcg per mL to about 950mcg per mL of the composition, about 200mcg per mL to about 900mcg per mL of the composition, about 200mcg per mL to about 850mcg per mL of the composition, about 200mcg per mL to about 800mcg per mL of the composition,About 300mcg per mL of composition to about 950mcg per mL of composition, about 300mcg per mL of composition to about 900mcg per mL of composition, about 300mcg per mL of composition to about 850mcg per mL of composition, about 300mcg per mL of composition to about 800mcg per mL of composition, about 400mcg per mL of composition to about 950mcg per mL of composition, about 400mcg per mL of composition to about 900mcg per mL of composition, about 400mcg per mL of composition to about 1 mL of composition About 950mcg per mL, about 400mcg per mL to about 900mcg per mL of the composition, about 400mcg per mL to about 850mcg per mL of the composition, about 400mcg per mL to about 800mcg per mL of the composition, about 500mcg per mL to about 950mcg per mL of the composition, about 500mcg per mL to about 900mcg per mL of the composition, about 500mcg per mL to about 850mcg per mL of the composition, about about 500mcg per mL to about 800mcg per mL of the composition, about 600mcg per mL to about 950mcg per mL of the composition, about 600mcg per mL to about 900mcg per mL of the composition, about 600mcg per mL to about 850mcg per mL of the composition, about 600mcg per mL to about 600mcg per mL of the composition, about 700mcg per mL to about 950mcg per mL of the composition, about 700mcg per mL to about 1000mcg per mL of the composition about 900mcg, about 700mcg per mL of the composition to about 850mcg per mL of the composition, about 700mcg per mL of the composition to about 800mcg per mL of the composition, about 800mcg per mL of the composition to about 950mcg per mL of the composition, about 800mcg per mL of the composition to about 900mcg per mL of the composition, about 800mcg per mL of the composition to about 850mcg per mL of the composition, or about 900mcg per mL of the composition to about 950mcg per mL of the composition.

[0064]

[0079] In certain embodiments, the composition comprises about 0.001% or more by weight of hyaluronic acid, about 0.0025% or more by weight of hyaluronic acid, about 0.0050% or more by weight of hyaluronic acid, about 0.0075% or more by weight of hyaluronic acid, about 0.01% or more by weight of hyaluronic acid, about 0.025% or more by weight of hyaluronic acid, about 0.05% or more by weight of hyaluronic acid, about 0.075% or more by weight of hyaluronic acid, about 0.1% or more by weight of hyaluronic acid, about 0.25% or more by weight of hyaluronic acid, about 0.5% or more by weight of hyaluronic acid, about 0.75% or more by weight of hyaluronic acid, or about 1% or more by weight of hyaluronic acid. In one embodiment, the composition comprises about 0.001% by weight to about 0.0025% by weight of hyaluronic acid, about 0.0025% by weight to about 0.005% by weight of hyaluronic acid, about 0.0025% by weight to about 0.0075% by weight of hyaluronic acid, about 0.0025% by weight to about 0.01% by weight of hyaluronic acid, about 0.001% by weight to about 0.01% by weight of hyaluronic acid, about 0.005% by weight to about 0.0075% by weight of hyaluronic acid, about 0.005% by weight to about 0.01% by weight of hyaluronic acid, about 0.0075% by weight to about 0.01% by weight of hyaluronic acid, about 0.01% by weight to about 0.02% by weight of hyaluronic acid, about 0.01% by weight to about 0.05% by weight of hyaluronic acid. % hyaluronic acid, about 0.02% to about 0.04% by weight of hyaluronic acid, about 0.05% to about 0.1% by weight of hyaluronic acid, about 0.04% to about 0.06% by weight of hyaluronic acid, about 0.06 to about 0.08% by weight of hyaluronic acid, about 0.08% to about 0.1% by weight of hyaluronic acid, about 0.1% to about 0.2% by weight of hyaluronic acid, about 0.1% to about 0.5% by weight of hyaluronic acid, about 0.2% to about 0.4% by weight of hyaluronic acid, about 0.5% to about 1% by weight of hyaluronic acid, about 0.4% to about 0.6% by weight of hyaluronic acid, about 0.6 to about 0.8% by weight of hyaluronic acid, or about 0.8% to about 1% by weight of hyaluronic acid.

[0065]

[0080] In some embodiments, the composition according to an embodiment of the present invention comprises commercially available hyaluronic acid composition.For example, suitable commercially available hyaluronic acid composition includes, but is not limited to, hyaluronic acid sold under the trademarks JUVEDERM (trademark), RESTYLANE-L (trademark), CAPTIQUE (trademark), BELOTERO BALANCE (trademark), PREVELLE SILK (trademark), ELEVESS (trademark), HYLAFORM (trademark), EUFLEXXA (trademark), GEL-ONE (trademark), HYALGAN (trademark), ORTHOVISC (trademark), MONOVISC (trademark), SUPARTZ (trademark), SYNVISC (trademark) and SYNVISC-ONE (trademark).

[0066]

[0081] C. Hyaluronidase inhibitors

[0082] In some embodiments according to the present invention, the composition comprises a hyaluronidase inhibitor. Examples of hyaluronidase inhibitors include, but are not limited to, high molecular weight poly(styrene-4-sulfonate) (PSS), gossypol, sodium aurothiomalate, fenoprofen, glycerrhizic acid, heparin, and O-sulfated hyaluronic acid (sHA) and dextran sulfate or combinations thereof.

[0067]

[0083] In some embodiments, the composition comprises about 0.01% by weight or more of hyaluronidase inhibitor, about 0.025% by weight or more of hyaluronidase inhibitor, about 0.050% by weight or more of hyaluronidase inhibitor, about 0.075% by weight or more of hyaluronidase inhibitor, about 0.1% by weight or more of hyaluronidase inhibitor, about 0.25% by weight or more of hyaluronidase inhibitor, about 0.5% by weight or more of hyaluronidase inhibitor, about 0.75% by weight or more of hyaluronidase inhibitor, about 1% by weight or more of hyaluronidase inhibitor, about 2.5% by weight or more of hyaluronidase inhibitor, about 5% by weight or more of hyaluronidase inhibitor, about 7.5% by weight or more of hyaluronidase inhibitor, or about 10% by weight or more of hyaluronidase inhibitor. In one embodiment, the composition comprises about 0.01% to about 0.025% by weight of hyaluronidase inhibitor, about 0.025% to about 0.05% by weight of hyaluronidase inhibitor, about 0.025% to about 0.075% by weight of hyaluronidase inhibitor, about 0.025% to about 0.1% by weight of hyaluronidase inhibitor, about 0.01% to about 0.1% by weight of hyaluronidase inhibitor, about 0.05% to about 0.075% by weight of hyaluronidase inhibitor, about 0.05% to about 0.1% by weight of hyaluronidase inhibitor, about 0.075% to about 0.1% by weight of hyaluronidase inhibitor, about 0.1% to about 0.2% by weight of hyaluronidase inhibitor, about 0.1% to about 0.5% by weight of hyaluronidase inhibitor. inhibitor, about 0.2% to about 0.4% by weight of hyaluronidase inhibitor, about 0.5% to about 1% by weight of hyaluronidase inhibitor, about 0.4% to about 0.6% by weight of hyaluronidase inhibitor, about 0.6 to about 0.8% by weight of hyaluronidase inhibitor, about 0.8% to about 1% by weight of hyaluronidase inhibitor, about 1% to about 2% by weight of hyaluronidase inhibitor, about 1% to about 5% by weight of hyaluronidase inhibitor, about 2% to about 4% by weight of hyaluronidase inhibitor, about 5% to about 10% by weight of hyaluronidase inhibitor, about 4% to about 6% by weight of hyaluronidase inhibitor, about 6 to about 8% by weight of hyaluronidase inhibitor, or about 8% to about 10% by weight of hyaluronidase inhibitor.

[0068]

[0084] D. Carriers and Other Additives

[0085] In some embodiments, the composition includes a carrier medium. Such a carrier medium can be a biocompatible fluid suitable for injection into the skin of a mammal. In some embodiments, the carrier medium includes a saline solution. In some embodiments, hyaluronic acid functions as the carrier medium as well as the active ingredient.

[0069]

[0086] In some embodiments, the composition includes one or more additives. Such additives may include preservatives or biocides.

[0070]

[0087] In some embodiments, the composition comprises a microemulsifier, a nanoemulsifier, a solid lipid nanoparticle, a nanostructured lipid carrier, a liposome, or a vesicle.

[0071]

[0088] In some embodiments, the composition can include a fatty acid (e.g., oleic acid), an ester of a fatty acid and an alcohol (e.g., isopropyl myristate, isopropyl palmitate, ethyl oleate), a medium chain triglyceride, triacetin, or a terpene (e.g., limonene, methol, cinoele). In some embodiments, the composition can include a surfactant. For example, suitable surfactants include, but are not limited to, TWEEN™ (polysorbate), CREMOPHOR™ (mixture of macrogol glycerol hydroxystearate, PEG-40 castor oil, polyoxyl 40 hydrogenated castor oil), TRANSCUTOL™ P (diethylene glycol monoethyl ether), PLUROL OLEIQUE™ (polyglyceryl-3-oleate), PLUROL ISOSTEARIQUE™ (isostearic acid esters and higher oligomers of polyglycerols) and LABRASOL™ (mixture of mono-, di-, and triglycerides of C8 and C10 fatty acids and mono- and diesters of PEG) and lecithin. In some embodiments, the composition can include a co-surfactant. For example, suitable co-surfactants include, but are not limited to, short- and medium-chain alcohols and polyglyceryl derivatives, including ethanol, isopropanol, isopropyl myristate, and propylene glycol.

[0072]

[0089] In certain embodiments, the composition comprises one or more of soybean oil, jojoba oil, aloe vera oil, soy phosphatidylcholine, water, polysorbate 80, ethanol, benzyl alcohol, isopropyl alcohol, glycerin, glyceryl monostearate, and propylene glycol.

[0073]

[0090] II. Method

[0091] Manufacturing method

[0092] A process for the manufacture of a therapeutic composition according to one embodiment of the present invention includes mixing an effective amount of an active agent. In some embodiments, an effective amount of hyaluronic acid is mixed with an effective amount of osteopontin. In some embodiments, the hyaluronic acid has an average molecular weight in the range of about 4,000 Da to 10,000 Da, about 10,000 Da to about 100,000 Da, about 15 kDa to about 50 kDa, about 75 kDa to about 350 kDa, or about 20 kDa to 1350 kDa. In some embodiments, the hyaluronic acid has an average molecular weight greater than about 950 kDa. In some embodiments, an effective amount of hyaluronic acid is mixed with an effective amount of a CD44 binding ligand. In some embodiments, an effective amount of osteopontin is mixed with an effective amount of a CD44 binding ligand. In some embodiments, an effective amount of each of hyaluronic acid and osteopontin is mixed with an effective amount of a CD44 binding ligand. In one embodiment of the composition according to the present invention, an effective amount of a hyaluronidase inhibitor is mixed with one or more of hyaluronic acid, osteopontin and CD44 binding ligand. The process can also include a step of preparing a physiologically acceptable carrier medium to which the active agent is added. Preferably, the physiologically acceptable carrier medium is injectable.

[0074]

[0093] In some embodiments, the method includes forming a microemulsion or nanoemulsion. The microemulsion or nanoemulsion can include oil, water, a surfactant, and a co-surfactant to form a colloidal dispersion with droplet sizes ranging from about 10 nm to about 100 nm. In some embodiments, the microemulsion or nanoemulsion can include a fatty acid (e.g., oleic acid), an ester of a fatty acid and an alcohol (e.g., isopropyl myristate, isopropyl palmitate, ethyl oleate), a medium chain triglyceride, triacetin, or a terpene (e.g., limonene, methol, cinoele). In some embodiments, the microemulsion or nanoemulsion can include a surfactant. For example, suitable surfactants include, but are not limited to, TWEEN™ (polysorbate), CREMOPHOR™ (mixture of macrogol glycerol hydroxystearate, PEG-40 castor oil, polyoxyl 40 hydrogenated castor oil), TRANSCUTOL™ P (diethylene glycol monoethyl ether), PLUROL OLEIQUE™ (polyglyceryl-3-oleate), PLUROL ISOSTEARIQUE™ (isostearic acid esters of polyglycerol and higher oligomers) and LABRASOL™ (mixture of mono-, di- and triglycerides of C8 and C10 fatty acids and mono- and diesters of PEG) and lecithin. In some embodiments, the microemulsion or nanoemulsion can include a co-surfactant. For example, suitable co-surfactants include, but are not limited to, short- and medium-chain alcohols and polyglyceryl derivatives, including ethanol, isopropanol, isopropyl myristate and propylene glycol. In certain embodiments, the formation of a microemulsion or nanoemulsion involves the use of a high pressure homogenizer, a microfluidizer and / or an ultrasonicator.

[0075]

[0094] In an embodiment, the method of manufacture comprises mixing a composition according to an embodiment of the present invention with solid nanoparticles. The solid nanoparticles may comprise inorganic materials, such as metal oxides (e.g., zinc oxide, titanium dioxide) or polymers that are solid at room temperature.

[0076]

[0095] In some embodiments, the method of manufacture includes mixing a composition according to an embodiment of the present invention with solid lipid nanoparticles, which include lipids that are solid at room temperature and have a surfactant surface coating to stabilize them as droplets having a size of less than about 100 nm when dispersed in water.

[0077]

[0096] In an embodiment, the method of manufacture comprises mixing a composition according to an embodiment of the present invention with a nanostructured lipid carrier, which may comprise a fluid lipid phase and a surfactant layer embedded in a solid lipid matrix or localized on the surface of solid platelets.

[0078]

[0097] In one embodiment, the method of manufacture comprises mixing a composition according to an embodiment of the present invention with liposomes, which may comprise spherical vesicles composed of amphiphilic phospholipids and cholesterol, which self-assemble into multilamellar, large unilamellar and small unilamellar vesicles.

[0079]

[0098] In some embodiments, the method of manufacture includes mixing a composition according to an embodiment of the present invention with flexible vesicles. Flexible vesicles can include materials that will assemble into bilayer structures as well as components that provide flexibility. In some embodiments, flexible vesicles include ethosomes (i.e., phospholipids with a high percentage of ethanol), niosomes (i.e., non-ionic surfactants), invasomes (i.e., a mixture of phospholipids, ethanol, and terpene penetration enhancers), SECosomes (i.e., surfactants, ethanol, and cholesterol), or PEVs (i.e., penetration enhancer vesicles). In some embodiments, PEVs can include oleic acid, limonene, or propylene glycol.

[0080]

[0099] In an embodiment, the method of manufacture comprises mixing a composition according to an embodiment of the present invention with a polymeric micelle or a polymeric dendrimer. The polymeric micelle can be a colloidal carrier having a hydrophilic outer shell and a hydrophobic inner core. The polymeric micelle can be nano-sized. The polymeric dendrimer can include a branched polymer structure.

[0081] [000100] In certain embodiments, any of the above CD44-binding fragments, isoforms or derivatives of naturally occurring ligands for CD44 are proteolytically produced, while in other embodiments, such are synthetically produced.

[0082] [000101] Method of administration [000102] A method of administering a composition according to an embodiment of the present invention includes delivery of a composition according to an embodiment of the present invention to a hair follicle. In some embodiments, delivery is achieved by topical administration, i.e., application of the composition to the surface of the skin and allowing the composition to penetrate the skin. In some embodiments, the administration method includes a step of enhancing penetration prior to topical administration of the composition. In some embodiments, delivery is achieved by injection into the skin.

[0083] [000103] In some embodiments, topical delivery is performed following or in conjunction with the application of iontophoresis. For example, iontophoresis is the application of a weak electrical current (e.g., 0.1-1.0 mA / cm) to enhance skin penetration of the composition. 2 Without being bound by theory, it is believed that iontophoresis may enhance skin penetration by electromigration, electroosmosis and / or enhanced passive diffusion.

[0084] [000104] In some embodiments, local delivery is performed following or in conjunction with the application of electroporation. For example, electroporation can include the application of high intensity, high voltage (e.g., 50-1500 V), brief duration (10 microseconds to 10 milliseconds) electrical pulses to form aqueous pores in the lipid bilayer of the stratum corneum of the skin.

[0085] [000105] In some embodiments, localized delivery is performed after or in conjunction with the application of sonophoresis. For example, sonophoresis can include the application of high frequency (e.g., about 500 kHz to 1250 kHz) or low frequency (e.g., about 20 to about 100 kHz) or low frequency (starting with one of the high frequency or low frequency and progressing to the other of the high frequency or low frequency).

[0086] [000106] In some embodiments, localized delivery is performed after or in conjunction with the application of laser ablation, which can include the generation of photomechanical waves by laser ablation of a target material (e.g., a polymer) disposed on the surface of the skin.

[0087] [000107] In some embodiments, localized delivery is performed after or in conjunction with magnetophoresis, which can involve the application of a magnetic field, such as a pulsed electromagnetic field, to the skin.

[0088] [000108] In some embodiments, local delivery is performed after application of radiofrequency thermal ablation. Thermal ablation can include application of extreme heat (e.g., about 300°C for microseconds) at the skin surface. Without being bound by theory, it is believed that thermal ablation can vaporize a portion of the stratum corneum to create microscale channels. Thermal ablation can be accomplished using commercially available devices, including VIADOR™ (Syneron Medical Ltd, Israel) and PASSPORT' (Nitto-Denko, Japan). In some embodiments, thermal ablation can be accomplished using an Erbium:Yttrium-Gallium-Garnet (Er:YAG) laser emitting at 2,790 nm or an Yttrium-Scandium-Gallium-Garnet (YSGG) laser emitting at 2,940 nm. In some embodiments, fractional laser ablation is applied to sub-mm regions, generating spots that mimic microneedle array-type patterns (e.g., 40-300 µm at a density of 50-600 cm).

[0089] [000109] In some embodiments, localized delivery is performed following application of a microneedle device.

[0090] [000110] A method for administering a composition according to an embodiment of the present invention comprises injecting a therapeutic amount of the composition into the skin of a patient in need of treatment. In some embodiments, a composition according to an embodiment of the present invention is administered as a bolus, which as used herein refers to a dose delivered over a period of less than 10 minutes. In some embodiments, a composition according to an embodiment of the present invention is administered as an infusion, which as used herein refers to a dose delivered over a period of 10 minutes or more.

[0091] [000111] Such injection can be via a single needle, microneedle or similar device, or an array of needles, microneedles or similar devices. In some embodiments, the composition according to an embodiment of the present invention is delivered via a conventional syringe. In some embodiments, subcutaneous delivery is performed via a hollow microneedle injector. In some embodiments, subcutaneous delivery is performed with a microneedle patch, which is coated with a composition according to an embodiment of the present invention, for example, which is coated with a composition according to an embodiment of the present invention by 3D printing. In some embodiments, the composition is delivered via a jet injector. The term "needle" as used herein refers to any such device for piercing the skin and injecting a composition according to an embodiment of the present invention.

[0092] [000112] Preferably, the composition is administered to the patient near a hair follicle. Thus, in some embodiments, the composition is administered by injecting a therapeutic amount of the composition into the dermis of the patient. In some embodiments, the composition is administered by injecting a therapeutic amount of the composition into the subcutaneous tissue of the patient. In some embodiments, the composition is administered to the patient about 0.4 mm to about 2 mm into the skin (i.e., about 0.4 mm to about 3 mm from the surface of the skin). In certain embodiments, the composition is administered to a patient at a depth of about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, about 2 mm, about 2.1 mm, about 2.2 mm, about 2.3 mm, about 2.4 mm, about 2.5 mm, about 2.6 mm, about 2.7 mm, about 2.8 mm, about 2.9 mm, or about 3 mm into the skin. In some embodiments, the composition has a diameter of about 0.5 mm to about 1 mm, about 1 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, about 2.5 mm to about 3 mm, about 1 mm to about 3 mm, about 1.5 mm to about 3 mm, about 0.4 mm to about 0.6 mm, about 0.4 mm to about 0.8 mm, about 0.4 mm to about 1 mm, about 0.4 mm to about 1.2 mm, Approximately 0.4mm to approximately 1.4mm, approximately 0.4mm to approximately 1.6mm, approximately 0.4mm to approximately 1.8mm, approximately 0.4mm to approximately 2mm, approximately 0.4mm to approximately 2.2mm, approximately 0.4mm to Approximately 2.4mm, approximately 0.4mm to approximately 2.6mm, approximately 0.4mm to approximately 2.8mm, approximately 0.4mm to approximately 3mm, approximately 0.6mm to approximately 0.8mm, approximately 0.6mm to approximately 1mm, approximately 0. 6mm to 1.2mm, 0.6mm to 1.4mm, 0.6mm to 1.6mm, 0.6mm to 1.8mm, 0.6mm to 2mm, 0.6mm to 2.2mm, 0.6mm to 2.4mm, 0.6mm to 2.6mm, 0.6mm to 2.8mm, 0.6mm to 3mm, 0.8mm to 1mm, 0.8mm m to about 1.2 mm, about 0.8 mm to about 1.4 mm, about 0.8 mm to about 1.6 mm, about 0.8 mm to about 1.8 mm, about 0.8 mm to about 2 mm, about 0.8 mm to about 2.2 mm, about 0.8 mm to about 2.4 mm, about 0.8 mm to about 2.6 mm, about 0.8 mm to about 2.8 mm, about 0.8 mm to about 3 mm, about 1 mm to about 1.2 mm, about 1 mm to about 1.4mm, about 1mm to about 1.6mm, about 1mm to about 1.8mm, about 1mm to about 2mm, about 1mm to about 2.2mm, about 1mm to about 2.4mm, about 1mm to about 2.6mm, about 1mm to about 2.8mm, about 1mm to about 3mm, about 1.2mm to about 1.4mm, about 1.2mm to about 1.6mm, about 1.2mm to about 1.8mm, about 1.2mm to about 2mm, about 1.2mm to about 2.2mm, about 1.2mm to about 2.4mm, Approximately 1.2mm to approximately 2.6mm, approximately 1.2mm to approximately 2.8mm, approximately 1.2mm to approximately 3mm, approximately 1.4mm to approximately 1.6mm, approximately 1.4mm to approximately 1.8mm, approximately 1.4mm to approximately 2mm, approximately 1.4mm to approximately 2 .2mm, about 1.4mm to about 2.4mm, about 1.4mm to about 2.6mm, about 1.4mm to about 2.8mm, about 1.4mm to about 3mm, about 1.6mm to about 1.8mm, about 1.6mm to about 2mm, about 1.6 mm to about 2.2 mm, about 1.6 mm to about 2.4 mm, about 1.6 mm to about 2.6 mm, about 1.6 mm to about 2.8 mm, about 1.6 mm to about 3 mm, about 1.8 mm to about 2 mm, about 1.8 mm to about 2.2 mm, about 1.8 mm to about 2.4 mm, about 1.8 mm to about 2.6 mm, about 1.8 mm to about 2.8 mm, about 1.8 mm to about 3 mm, about 2.0 mm to about 2.2 mm, about 2.0 mm to about 2.4 mm, about 2.0 mm to about 3 mm About 2.6 mm, about 2.0 mm to about 2.8 mm, about 2.0 mm to about 3 mm, about 2.2 mm to about 2.4 mm, about 2.2 mm to about 2.6 mm, about 2.2 mm to about 2.8 mm, about 2.2 mm to about 3 mm, about 2.4 mm to about 2.6 mm, about 2.4 mm to about 2.8 mm, about 2.4 mm to about 3 mm, about 2.6 mm to about 2.8 mm, about 2.6 mm to about 3 mm, or about 2.8 mm to about 3 mm is administered into the patient's skin.

[0093] [000113] In some embodiments, the composition according to an embodiment of the present invention can be administered in multiple injections. In some embodiments, the composition according to an embodiment of the present invention can be administered in about 1 injection per cm of skin. 2 ~Approximately 1000 injections / cm of skin 2 , about 200 injections / cm of skin 2 ~Approximately 800 injections / cm of skin 2 or approximately 400 injections / cm of skin 2 ~Approximately 650 injections / cm of skin 2In one embodiment, the composition is administered via about 200 injections per cm of skin. 2 , approximately 250 injections / cm of skin 2 , about 300 injections / cm of skin 2 , approximately 350 injections / cm of skin 2 , about 400 injections / cm of skin 2 , approximately 450 injections / cm of skin 2 , about 500 injections / cm of skin 2 , approximately 550 injections / cm of skin 2 , approximately 600 injections / cm of skin 2 or approximately 650 injections / cm of skin 2 The contents of U.S. Patent No. 11,337,993 are incorporated herein by reference.

[0094] [000114] Methods of Treatment [000115] A method of stimulating hair growth in a patient in need thereof comprises administering a composition according to an embodiment of the present invention to or into the patient's skin. In some embodiments, the composition is administered topically by applying the composition to the surface of the patient's skin. In some embodiments, the composition is administered into the dermis or subcutaneous tissue of the patient's skin, for example, by injection as described herein.

[0095] [000116] In some embodiments, a composition according to an embodiment of the invention is applied to the skin of a patient once a day for one day, once a day for one week, once a day for one month, once a day for one year, twice a day for one day, twice a day for one week, twice a day for one month, twice a day for one year, once a week for one week, once a week for one month, once a week for one year, twice a week for one week, twice a week for one month, twice a week for one year, once a month for one month, once a month for two months, once a month for six months, once a month for one month. once per month for one year, twice per month for one month, twice per month for two months, twice per month for six months, twice per month for one year, once per 2 months for two months, once per 2 months for four months, once per 2 months for six months, once per 2 months for one year, once per 3 months for three months, once per 3 months for six months, once per 3 months for nine months, once per 3 months for one year, once per 4 months for four months, once per 4 months for eight months, once per 4 months for one year, once per 6 months for six months, once per 6 months for one year, or as needed.

[0096] [000117] EXAMPLES

[0097] [000118] Example 1. Injection of Hyaluronic Acid ("HA") at Concentrations of 100, 250 and 1000 μg / Ml [000119] Three different concentrations (100, 250, and 1000 μg / mL) of hyaluronic acid of "high" (molecular weight distribution >950 kDa), "medium" (molecular weight distribution 75-350 kDa), and "low" molecular weight (molecular weight distribution 15-40 kDa) were injected into mice, respectively. High molecular weight concentrations above 250 μg / mL were found to induce a strong inflammatory response, with 1000 μg / mL being the worst. At the low molecular weight concentrations, the response was milder at 250 μg / mL compared to high molecular weight HA, but there was still strong inflammation at 1000 μg / mL. Adverse side effects were observed with hyaluronic acid of all molecular weights at concentrations above 250 μg / mL.

[0098] [000120] Example 2. Injection of HA at concentrations of 25, 50, and 100 μg / mL [000121] Follow-up experiments included lower HA concentrations (less than 250 μg / mL). A small incision was made using a fine needle (insulin syringe) to facilitate injection. 3 μL of either control, high (molecular weight distribution ≥ 950 kDa), medium (molecular weight distribution 75-350 kDa) or low molecular weight (molecular weight distribution 15-40 kDa) hyaluronic acid was injected into the dorsal skin of P53 mice on three (3) consecutive days. In Figures 5-8, looking at the back of the mouse with the head facing up, the control (1% BSA) injection is at the top left, the 25 μg / ml injection is at the top right, the 100 μg / ml injection is at the bottom right, and the 50 μg / ml injection is at the bottom left.

[0099] [000122] Figures 5 and 6 show representative images of two mice, each of which received an injection of low molecular weight HA. Figures 7 and 8 show representative images of two mice, each of which received an injection of high molecular weight HA. Figure 5 shows growth on 25 μg / ml low molecular weight HA and 100 μg / ml low molecular weight HA spots. Figure 6 shows growth on 25 μg / ml low molecular weight HA and 100 μg / ml low molecular weight HA spots. Figure 7 shows growth on 50 μg / ml high molecular weight HA and 100 μg / ml high molecular weight HA spots. Figure 8 shows growth on 25 μg / ml high molecular weight HA and 50 μg / ml high molecular weight HA spots.

[0100] [000123] For high molecular weight, injection sites were monitored for 18 days for complete anagen. Pigmentation was evident at P14 for both high and low molecular weight HA. Two mice had good induction with both low and high molecular weight HA, but no induction with medium dose HA. Without being bound by theory, it is possible that the depth of injection of the medium dose HA influenced the strength of induction.

[0101] [000124] Figures 7 and 8 show that high molecular weight HA induces at 50 μg / mL and 100 μg / mL without the massive inflammatory response found in Example 1. In the case of low molecular weight HA, only 100 μg / mL induced hair growth. As shown in Figure 6, low molecular weight HA mouse 2 has a good anagen spot at 25 μg / mL (the control injection site is visible below), but only in one mouse.

[0102] [000125] It will be appreciated by those skilled in the art that changes may be made to the exemplary embodiments shown and described above without departing from the broad inventive concept thereof. Accordingly, the present invention is not limited to the exemplary embodiments shown and described, but is intended to cover modifications within the spirit and scope of the present invention as defined by the claims. For example, certain features of the exemplary embodiments may or may not be part of the claimed invention, and various features of the disclosed embodiments may be combined. Unless specifically stated herein, the terms "a," "an," and "the" should be read as meaning "at least one" rather than limited to one element.

[0103] [000126] Furthermore, to the extent that the method of the present invention does not rely on a particular order of steps described herein, the particular order of steps should not be construed as a limitation on the claims. Any claims directed to the method of the present invention should not be limited to performing those steps in the written order, and one of ordinary skill in the art can readily appreciate that steps can be varied and still remain within the spirit and scope of the present invention.

Claims

1. A composition for use in a method for stimulating hair growth comprising hyaluronic acid at a concentration of greater than 250 mcg / mL.

2. 10. The composition of claim 1, wherein the hyaluronic acid has an average molecular weight in the range of about 4,000 Da to 10,000 Da.

3. 2. The composition of claim 1, wherein the hyaluronic acid has an average molecular weight in the range of about 20 KDa to 1350 KDa.

4. 10. The composition of claim 1, wherein the hyaluronic acid is crosslinked.

5. 10. The composition of claim 1, wherein the hyaluronic acid has a crosslink density of about 20% or greater.

6. 10. The composition of claim 1, wherein the hyaluronic acid is present in a concentration of from 250 mcg / mL to about 1000 mcg / mL.

7. The composition of claim 1, wherein the method comprises administering a composition containing hyaluronic acid at a concentration greater than 250 mcg / mL to the skin of a patient in need of stimulating hair growth.

8. 8. The composition of claim 7, wherein administering comprises injecting the composition into the dermis layer of the skin.

9. 8. The composition of claim 7, wherein the composition is injected into the skin to a depth of about 400 microns to about 2 mm.

10. 8. The composition of claim 7, wherein the composition is administered in multiple injections in an amount of about 400 injections / cm2 of skin to about 650 injections / cm2 of skin.

11. The composition of claim 7, further comprising a CD44-binding ligand.

12. 12. The composition of claim 11, wherein the CD44 binding ligand is osteopontin.

13. A composition comprising hyaluronic acid at a concentration greater than 250 mcg / mL for use in a method of administering a composition for hair growth to a patient in need of treatment for hair loss, the method comprising injecting the composition into the skin of the patient.

14. 14. The composition of claim 13, wherein the composition is injected through a needle, the needle being inserted about 400 microns to about 2 mm into the skin prior to injection.

15. 15. The composition of claim 14, wherein the needle is a microneedle.

16. 15. The composition of claim 14, further comprising a CD44 binding ligand.

17. 17. The composition of claim 16, wherein the CD44 binding ligand is osteopontin.

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