Composition containing hyaluronic acid-crosslinked tropoelastin and method of use thereof

Abstract

For example, soft tissue conditions can be treated by administering compositions comprising tropoelastin, including compositions comprising tropoelastin cross-linked to hyaluronic acid, to increase water content within the soft tissue. Additionally, soft tissue conditions can be treated by administering compositions of tropoelastin cross-linked to hyaluronic acid in a ratio of 2:1 or greater, which increases water content within the soft tissue compared to otherwise identical compositions of tropoelastin cross-linked to hyaluronic acid in a ratio of less than 2:1.

Description

Technical Field

[0001] Background Disclosure Field The present disclosure relates to a composition comprising tropoelastin crosslinked to hyaluronic acid that increases the water content in soft tissues and methods of using the same.

Background Art

[0002] Description of Related Art The skin is an integumentary organ that interacts with the environment to protect the body from physical and biological damage. To perform this role, the skin needs to be elastic to respond to stretching and impact and recover from stretching and impact. Elastin is an essential component of the dermis and gives the skin elasticity and integrity. Since elastin and other skin components are gradually lost through aging, damage by sunlight, and after trauma, the need to restore these components to repair the skin is emphasized. Aging and tissue damage are associated with the denaturation of the extracellular matrix, leading to loss of tissue structure and / or function. Sagging skin, lax subcutaneous tissue, reduced density of the extracellular matrix, wrinkles, stretch marks, and fibrosis are physical signs of denaturation. The elastic profile of skin tissue results from a complex process known as elastic fiber formation involving multiple factors, the main of which is tropoelastin (TE), the monomeric subunit of elastin. Elastic fiber formation generally refers to a physiological process that occurs in the latter half of fetal development to early postnatal period, by which elastic fibers are newly created by cells including fibroblasts, smooth muscle cells, etc. from tropoelastin monomers and other related factors. During development, elastin is primarily incorporated into the dermis prenatally and within the first few years after birth. While some elastin deposits exist in adult tissue, elastic fiber formation is limited with age or injury. Consequently, elastin is gradually lost during aging as a result of gradual decline in elastin production associated with proteolysis over time. This has a substantial impact on skin tissue, and the combination of elastin reduction and loss of other macromolecular components such as collagen and hyaluronic acid (HA) leads to a decrease in the structural integrity, water content (hydration), and elasticity of the skin. Therefore, there is a strong demand for compositions and methods that restore the structural integrity, water content (or hydration), and elasticity of skin tissue. [Overview of the Initiative]

[0003] overview This disclosure provides compositions for treating soft tissue conditions in subjects requiring such treatment. In some embodiments, this disclosure provides methods for increasing water content in the soft tissue of subjects requiring such treatment. In further embodiments, this disclosure provides compositions and methods for repairing the soft tissue of subjects requiring such treatment. This disclosure provides a method for increasing the water content in the soft tissue of a subject in need, comprising administering a composition comprising tropoelastin and optionally hyaluronic acid to the subject's soft tissue. Furthermore, the present disclosure provides a method for increasing the water content in the soft tissue of a subject in need, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, the composition increasing the water content in the subject's soft tissue compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but is otherwise identical.

[0004] In each or any of the embodiments described above or below, the composition comprises tropoelastin crosslinked to hyaluronic acid without a crosslinking agent. In the embodiments, the tropoelastin is crosslinked to hyaluronic acid via at least one intermolecular crosslinking which includes an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. In each or any of the embodiments described above or below, the composition comprises human tropoelastin. In further embodiments, the tropoelastin comprises recombinant tropoelastin, for example, recombinant human tropoelastin. In some embodiments, the tropoelastin comprises tropoelastin monomer. In each or any of the embodiments described above or below, the composition contains tropoelastin in an amount of about 1 mg / ml to about 100 mg / mL. In certain embodiments, tropoelastin is present in an amount of about 10 to about 50 mg / mL. In further embodiments, tropoelastin is present in an amount of about 30 mg / mL. In each or any of the embodiments described above or below, the composition comprises derivatized hyaluronic acid. In further embodiments, tropoelastin is crosslinked with derivatized hyaluronic acid. In each or any of the embodiments described above or below, the composition contains tropoelastin crosslinked with about 0.1% to about 5% hyaluronic acid. In certain embodiments, the tropoelastin is crosslinked with about 0.5% hyaluronic acid. In further embodiments, hyaluronic acid is present in an amount of about 1 mg / mL to about 15 mg / mL. In even further embodiments, hyaluronic acid is present in an amount of about 10 mg / mL or less, about 5 mg / mL or less, about 4 mg / mL or less, about 3 mg / mL or less, about 2 mg / mL or less, or about 1 mg / mL or less. In each or any of the embodiments described above or below, the composition comprises a tropoelastin:hyaluronic acid ratio. In some embodiments, the tropoelastin:hyaluronic acid ratio is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0005] In each or any of the embodiments described above or below, the method comprises administering a composition containing tropoelastin crosslinked to hyaluronic acid to the soft tissue of a subject, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 2:1 or greater, and the second water content is increased compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. In each or any of the embodiments described above or below, the tropoelastin:hyaluronic acid ratio is approximately 2:1 or greater, and the composition increases the water content in the subject's soft tissues compared to a composition with a tropoelastin:hyaluronic acid ratio of less than 2:1 but otherwise identical. In further embodiments, the tropoelastin:hyaluronic acid ratio is approximately 2:1 or greater, and the amount of hyaluronic acid is approximately 5 mg / mL or less. In each or any of the embodiments described above or below, the tropoelastin:hyaluronic acid ratio is approximately 6:1 or higher, and the composition increases the water content in the subject's soft tissues compared to a composition with a tropoelastin:hyaluronic acid ratio of less than 6:1 but otherwise identical. In further embodiments, the tropoelastin:hyaluronic acid ratio is approximately 6:1 or higher, and the amount of hyaluronic acid is approximately 5 mg / mL or less.

[0006] In each or any of the embodiments described above or below, the soft tissue is skin. In further embodiments, the skin is very dry skin, dry skin, or hydrated skin prior to administration of the composition. In even further embodiments, the skin has a capacitance of less than about 90 a.u., less than about 85 a.u., less than about 80 a.u., less than about 75 a.u., less than about 70 a.u., less than about 65 a.u., less than about 60 a.u., less than about 55 a.u., less than about 50 a.u., less than about 45 a.u., less than about 40 a.u., less than about 35 a.u., less than about 30 a.u., less than about 25 a.u., less than about 20 a.u., less than about 15 a.u., less than about 10 a.u., or less than about 5 a.u. prior to administration of the composition. In each or any of the embodiments described above or below, the Disclosure provides methods and compositions for treating soft tissue conditions of the skin. In the embodiments, skin conditions include facial wrinkles, fine lines, thinning skin, aging skin, scar tissue, and skin furrows. In the embodiments, the methods of the Disclosure include administering the compositions into the skin by injection. In certain embodiments, the compositions are administered into the dermis, subcutaneous tissue, or subcutaneously. In each or any of the embodiments described above or below, the method increases glycosaminoglycan deposits in the soft tissues of a subject requiring it. In certain embodiments, the method increases endogenous glycosaminoglycan deposits, such as an increase in endogenous hyaluronic acid deposits in soft tissues. In further embodiments, glycosaminoglycan deposits are increased on the cell surfaces of the papillary or upper reticular dermis of the skin. In embodiments, glycosaminoglycan deposits are increased by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold. In each or any of the embodiments described above or below, the method increases the water content in the soft tissue by about 10% to about 80%, or about 20% to about 50%. In further embodiments, the method increases the water content in the soft tissue by about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.

[0007] In each or any of the embodiments described above or below, the composition comprising tropoelastin is administered to the soft tissue for a sufficient time to increase the expression of glycosaminoglycans and / or hyaluronan synthases (e.g., hyaluronan synthase 1 (HAS1), hyaluronan synthase 2 (HAS2), and / or hyaluronan synthase 3 (HAS3)) in the soft tissue (e.g., expression increases by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 30, 40, 50, 60, 70, 80, 90, 100%, or more compared to soft tissue that is otherwise identical but not administered with the composition). In each or any of the embodiments described above or below, the Disclosure provides a method for increasing the water content in the soft tissue of a subject in need thereof, comprising preparing a subject having soft tissue having a first water content and administering a composition comprising tropoelastin crosslinked to hyaluronic acid to the soft tissue in an amount effective to produce a second water content in the soft tissue, wherein the tropoelastin:hyaluronic acid ratio of the composition is about 2:1 or greater, and the second water content is increased compared to soft tissue administered with the same composition, but with a tropoelastin:hyaluronic acid ratio of less than 2:1. In further embodiments, the tropoelastin:hyaluronic acid ratio of the composition is about 6:1, and the second water content is increased compared to soft tissue administered with the same composition, but with a tropoelastin:hyaluronic acid ratio of less than 6:1. In each or any of the embodiments described above or below, the method of the Disclosure produces a second water content that remains higher than a first water content for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 24 weeks, or at least 48 weeks. In each or any of the embodiments described above or below, the method stimulates collagen synthesis in soft tissue. In certain embodiments, the method disclosed herein stimulates elastin synthesis in soft tissue. In other embodiments, the method of the Disclosure stimulates the synthesis of both collagen and elastin in soft tissue. In some embodiments, the method of the Disclosure increases tissue elasticity. In embodiments, the method of the Disclosure induces cell infiltration at the administration site.

[0008] This disclosure also provides a method for stimulating collagen synthesis in the soft tissue of the skin of a subject in need, comprising administering a composition comprising tropoelastin and optionally hyaluronic acid to the subject's soft tissue. In one embodiment, the composition has a tropoelastin:hyaluronic acid ratio of about 2:1 or higher, and increases collagen synthesis in the subject's soft tissue compared to a composition with a tropoelastin:hyaluronic acid ratio of less than about 2:1 but otherwise identical. The disclosure also provides a method for stimulating collagen synthesis in the soft tissue of the skin of a subject in need, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid (for example, a composition in which the ratio of tropoelastin to hyaluronic acid is about 2:1 or higher), wherein the composition increases collagen synthesis in the subject's soft tissue compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but is otherwise identical.

[0009] In each or any of the embodiments described above or below, the composition comprises tropoelastin crosslinked to hyaluronic acid without a crosslinking agent. In the embodiments, the tropoelastin is crosslinked to hyaluronic acid via at least one intermolecular crosslinking which includes an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. In each or any of the embodiments described above or below, the composition comprises human tropoelastin. In further embodiments, the tropoelastin comprises recombinant tropoelastin, for example, recombinant human tropoelastin. In some embodiments, the tropoelastin comprises tropoelastin monomer. In each or any of the embodiments described above or below, the composition contains tropoelastin in an amount of about 1 mg / ml to about 100 mg / mL. In certain embodiments, tropoelastin is present in an amount of about 10 to about 50 mg / mL. In further embodiments, tropoelastin is present in an amount of about 30 mg / mL. In each or any of the embodiments described above or below, the composition comprises derivatized hyaluronic acid. In each or any of the embodiments described above or below, the composition contains tropoelastin crosslinked with about 0.1% to about 5% hyaluronic acid. In certain embodiments, the tropoelastin is crosslinked with about 0.5% hyaluronic acid. In further embodiments, hyaluronic acid is present in an amount of about 1 mg / mL to about 15 mg / mL. In even further embodiments, hyaluronic acid is present in an amount of about 10 mg / mL or less, about 5 mg / mL or less, about 4 mg / mL or less, about 3 mg / mL or less, about 2 mg / mL or less, or about 1 mg / mL or less. In each or any of the embodiments described above or below, the composition comprises a tropoelastin:hyaluronic acid ratio. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 2:1, approximately 3:1, approximately 4:1, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or 10:1.

[0010] This disclosure also provides a method for increasing glycosaminoglycan deposition in the skin of a subject in need thereof, comprising administering a composition comprising tropoelastin and optionally hyaluronic acid to the skin. In one embodiment, the composition has a tropoelastin:hyaluronic acid ratio of about 2:1 or higher, and increases glycosaminoglycan deposition in the soft tissue of the subject compared to a composition with a tropoelastin:hyaluronic acid ratio of less than about 2:1 but otherwise identical. The disclosure also provides a method for increasing glycosaminoglycan deposition in the skin of a subject in need, comprising administering tropoelastin crosslinked to hyaluronic acid (for example, a composition having a tropoelastin:hyaluronic acid ratio of about 2:1 or greater) to the skin, wherein the glycosaminoglycan deposition is increased compared to soft tissue administered with a composition in which the tropoelastin is not crosslinked to hyaluronic acid but is otherwise identical. In each or any of the embodiments described above or below, the composition comprises tropoelastin crosslinked to hyaluronic acid without a crosslinking agent. In the embodiments, the tropoelastin is crosslinked to hyaluronic acid via at least one intermolecular crosslinking which includes an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid.

[0011] In each or any of the embodiments described above or below, the composition comprises human tropoelastin. In further embodiments, the tropoelastin comprises recombinant tropoelastin, for example, recombinant human tropoelastin. In some embodiments, the tropoelastin comprises tropoelastin monomer. In each or any of the embodiments described above or below, the composition contains tropoelastin in an amount of about 1 mg / ml to about 100 mg / mL. In certain embodiments, tropoelastin is present in an amount of about 10 to about 50 mg / mL. In further embodiments, tropoelastin is present in an amount of about 30 mg / mL. In each or any of the embodiments described above or below, the composition comprises derivatized hyaluronic acid. In each or any of the embodiments described above or below, the composition contains tropoelastin crosslinked with about 0.1% to about 5% hyaluronic acid. In certain embodiments, the tropoelastin is crosslinked with about 0.5% hyaluronic acid. In further embodiments, hyaluronic acid is present in an amount of about 1 mg / mL to about 15 mg / mL. In even further embodiments, hyaluronic acid is present in an amount of about 10 mg / mL or less, about 5 mg / mL or less, about 4 mg / mL or less, about 3 mg / mL or less, about 2 mg / mL or less, or about 1 mg / mL or less. In each or any of the embodiments described above or below, the composition has a tropoelastin:hyaluronic acid ratio. In some embodiments, the tropoelastin:hyaluronic acid ratio is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0012] This disclosure also provides a method for increasing hyaluronan synthase expression (e.g., hyaluronan synthase 1 (HAS1), hyaluronan synthase 2 (HAS2), and / or hyaluronan synthase 3 (HAS3)) in the skin of a subject who requires it, the method comprising administering a composition comprising tropoelastin and optionally hyaluronic acid to the skin. In one embodiment, the composition comprises a tropoelastin:hyaluronic acid ratio of about 2:1 or higher, and increases hyaluronan synthase expression in the soft tissue of the subject compared to a composition with a tropoelastin:hyaluronic acid ratio of less than about 2:1 but otherwise identical. In some embodiments, hyaluronan synthase expression increases by about 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to hyaluronan synthase expression in untreated skin. This disclosure also provides a method for increasing hyaluronan synthase expression (e.g., hyaluronan synthase 1 (HAS1), hyaluronan synthase 2 (HAS2), and / or hyaluronan synthase 3 (HAS3)) in the skin of a subject requiring such effect, comprising administering to the skin a composition comprising tropoelastin crosslinked to hyaluronic acid (e.g., a composition in which the tropoelastin:hyaluronic acid ratio is about 2:1 or higher), wherein the hyaluronan synthase is increased compared to soft tissue administered with a composition in which tropoelastin is in monomeric form but otherwise identical (e.g., a composition in which the tropoelastin:hyaluronic acid ratio is about 2:1). In some embodiments, hyaluronan synthase expression is increased by about 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to hyaluronan synthase expression in untreated skin.

[0013] In each or any of the embodiments described above or below, the composition comprises tropoelastin crosslinked to hyaluronic acid without a crosslinking agent. In the embodiments, the tropoelastin is crosslinked to hyaluronic acid via at least one intermolecular crosslinking which includes an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. In each or any part of the above or below embodiments, the composition contains human tropoelastin. In a further embodiment, tropoelastin includes recombinant tropoelastin, such as recombinant human tropoelastin. In some embodiments, tropoelastin includes tropoelastin monomers. In each or any part of the above or below embodiments, the composition contains tropoelastin in an amount of about 1 mg / ml to about 100 mg / mL. In certain embodiments, tropoelastin is present in an amount of about 10 to about 50 mg / mL. In a further embodiment, tropoelastin is present in an amount of about 30 mg / mL. In each or any part of the above or below embodiments, the composition contains derivatized hyaluronic acid. In each or any part of the above or below embodiments, the composition contains tropoelastin crosslinked with about 0.1% to about 5% hyaluronic acid. In certain embodiments, tropoelastin is crosslinked with about 0.5% hyaluronic acid. In a further embodiment, hyaluronic acid is present in an amount of about 1 mg / mL to about 15 mg / mL. In even further embodiments, hyaluronic acid is present in an amount of about 10 mg / mL or less, about 5 mg / mL or less, about 4 mg / mL or less, about 3 mg / mL or less, about 2 mg / mL or less, or about 1 mg / mL or less. In each or any part of the above or below embodiments, the composition has a tropoelastin:hyaluronic acid ratio. In some embodiments, the tropoelastin:hyaluronic acid ratio is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0014] The present disclosure also provides a method for improving the skin composition (e.g., skin brightness, skin elasticity, and / or increased extracellular matrix protein) of a subject in need thereof, the method comprising administering to the skin a composition comprising tropoelastin and optionally hyaluronic acid. In one embodiment, the composition has a tropoelastin:hyaluronic acid ratio of about 2:1 or greater, and the tropoelastin:hyaluronic acid ratio improves the skin composition of the subject compared to a composition that is otherwise identical but has a tropoelastin:hyaluronic acid ratio of less than about 2:1. The present disclosure also provides a method for improving the skin composition (e.g., skin brightness, skin elasticity, and / or increased extracellular matrix protein) of a subject in need thereof, the method comprising administering a composition comprising tropoelastin crosslinked to hyaluronic acid (e.g., a composition having a tropoelastin:hyaluronic acid ratio of about 2:1 or greater), wherein the skin composition is improved compared to soft tissue administered the composition that is otherwise identical but in which tropoelastin is not crosslinked to hyaluronic acid.

[0015] In some embodiments of each or any part of the above or below embodiments, the composition comprises tropoelastin crosslinked to hyaluronic acid without a crosslinking agent. In an embodiment, tropoelastin is crosslinked to hyaluronic acid via at least one intermolecular crosslink comprising an amide bond between an amine of tropoelastin and a carboxyl group of hyaluronic acid. In some embodiments of each or any part of the above or below embodiments, the composition comprises human tropoelastin. In a further embodiment, tropoelastin comprises recombinant tropoelastin, such as recombinant human tropoelastin. In some embodiments, tropoelastin comprises tropoelastin monomers. In some embodiments of each or any part of the above or below embodiments, the composition comprises tropoelastin in an amount from about 1 mg / ml to about 100 mg / mL. In certain embodiments, tropoelastin is present in an amount from about 10 to about 50 mg / mL. In a further embodiment, tropoelastin is present in an amount of about 30 mg / mL. In each or any of the embodiments described above or below, the composition comprises derivatized hyaluronic acid. In each or any of the embodiments described above or below, the composition contains tropoelastin crosslinked with about 0.1% to about 5% hyaluronic acid. In certain embodiments, the tropoelastin is crosslinked with about 0.5% hyaluronic acid. In further embodiments, hyaluronic acid is present in an amount of about 1 mg / mL to about 15 mg / mL. In even further embodiments, hyaluronic acid is present in an amount of about 10 mg / mL or less, about 5 mg / mL or less, about 4 mg / mL or less, about 3 mg / mL or less, about 2 mg / mL or less, or about 1 mg / mL or less. In each or any of the embodiments described above or below, the composition has a tropoelastin:hyaluronic acid ratio. In some embodiments, the tropoelastin:hyaluronic acid ratio is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0016] Further features and advantages of the subject art will be described below. Some of these will be obvious from this description, while others will become apparent through the implementation of the subject art. The advantages of the subject art will be realized and achieved, in particular, through the written specification and the embodiments relating thereto, as well as the structures indicated in the accompanying drawings. It should be understood that both the general explanation above and the detailed explanation below are illustrative and explanatory, and are intended to provide further explanation of the subject technology.

[0017] Various features of embodiments useful in explaining the present invention are described below with reference to the drawings. These illustrated embodiments are intended to illustrate the present invention and are not intended to limit it. The drawings include the following figures. [Brief explanation of the drawing]

[0018] [Figure 1A]The water content of skin explants 1, 2, 5, and 7 days after intradermal injection of the following: control; 12 mg / mL hyaluronic acid (HA) alone; 10 mg / mL recombinant human tropoelastin (rhTE) monomer (i.e., uncrosslinked) mixed with 12 mg / mL HA (F7); 30 mg / mL rhTE monomer (i.e., uncrosslinked) mixed with 12 mg / mL HA (F6); 10 mg / mL rhTE crosslinked with 0.5% dHA in phosphate-buffered saline (PBS) (F9); and 30 mg / mL rhTE crosslinked with 0.5% dHA in PBS (F8). [Figure 1B] Immunofluorescence staining of acid glycosaminoglycans (GAGs) in papillary dermis 5 and 7 days after injection is shown. [Figure 1C] Immunofluorescence staining of acidic GAG in the upper reticular dermis 5 and 7 days after injection is shown. [Figure 2] Figure 2A shows representative hematoxylin and eosin (H&E) stained cross-sectional images from rat skin biopsies taken two weeks after intradermal injection. Figure 2A shows the results after intradermal injection of 10 mg / mL rhTE crosslinked with 0.5% derivatized hyaluronic acid (dHA) in PBS. Figure 2B shows the results after intradermal injection of 30 mg / mL rhTE crosslinked with 0.5% dHA in PBS. Figure 2C shows the results after intradermal injection of 10 mg / mL rhTE monomer (i.e., uncrosslinked) mixed with 12 mg / mL HYC-24L+. Figure 2D shows the results after intradermal injection of 30 mg / mL rhTE monomer (i.e., uncrosslinked) mixed with 12 mg / mL HYC-24L+. Figure 2E shows the results after intradermal injection of 12 mg / mL HYC-24L+. Figure 2F shows the results after intradermal injection of a saline control. Collagen tissue appears as pink fibers, HA appears as light purple to dark purple regions, and cell nuclei appear as punctate dark purple staining. [Figure 3]Representative images (100×) of immunofluorescence-duplex labeled recombinant human elastin (red) and rat elastin (green) from cross-sections of skin biopsies taken 8 weeks after intradermal injection are shown. Figure 3A shows the results after intradermal injection of 10 mg / mL rhTE crosslinked with 0.5% dHA in PBS. Figure 3B shows the results after intradermal injection of 30 mg / mL rhTE crosslinked with 0.5% dHA in PBS. Figure 3C shows the results after intradermal injection of 10 mg / mL rhTE monomer (i.e., uncrosslinked) mixed with 12 mg / mL HYC-24L+. Figure 3D shows the results after intradermal injection of 30 mg / mL rhTE monomer (i.e., uncrosslinked) mixed with 12 mg / mL HYC-24L+. Figure 3E shows the results after intradermal injection of 12 mg / mL HYC-24L+. Figure 3F shows the results after intradermal injection of a saline control. The nuclei are counterstained with DAPI (blue). [Figure 4] Representative confocal microscopy images of fibroblasts incubated in the absence or presence of L-ascorbic acid 2-phosphate sesquimagnesium salt (APM) with and without low (×1) and high (×2) doses of tropoelastin are shown. Z-stack images of the fibroblast culture from the upper layer (layer 1) to the lower layer (layer 4) allowed for the detection of elastin (red), type I collagen (green), and nuclei (blue) within different layers. [Figure 5A] The qPCR analysis of hyaluronan synthase 1 (HAS1) mRNA is shown, indicating HAS1 levels 24 hours after injection of skin patches containing the test formulations (F1-F5, see Table 1). All values ​​are mean (SD), and *P<0.05 compared to the control. [Figure 5B] The qPCR analysis of hyaluronan synthase 1 (HAS1) mRNA is shown, indicating HAS1 levels 120 hours after injection of skin patches containing the test formulations (F1-F5, see Table 1). All values ​​are mean (SD), and *P<0.05 compared to the control. [Figure 6A] The images show glycosaminoglycan (GAG) deposits in Alcian blue-stained neonatal dermal fibroblasts 7 days after the addition of hyaluronic acid (HA), tropoelastin (TE), or both. The control without HA or TE is labeled "cells." [Figure 6B] The images show glycosaminoglycan (GAG) deposits in Alcian blue-stained adult dermal fibroblasts 7 days after the addition of hyaluronic acid (HA), tropoelastin (TE), or both. The control without HA or TE is labeled "cells." [Figure 6C] Figure 6A shows the quantification of the experiment (neonatal) exemplified by the example, and the control without HA or TE is labeled as "cells". [Figure 6D] Figure 6B shows the quantification of the experiment (adults) exemplified by the example, and the control without HA or TE is labeled as "cells". [Modes for carrying out the invention]

[0019] Detailed explanation This disclosure, which describes various configurations of the subject art by illustrating them with examples, will make the various configurations of the subject art readily apparent to those skilled in the art. As you know, the subject art can be configured in many other ways, and some of its details can be modified in various other ways without departing from the scope of the subject art. Therefore, the outline, drawings and detailed description should be considered examples rather than limitations. The detailed description below is intended to describe various configurations of the subject art and not to represent the only configurations in which the subject art can be implemented. The accompanying drawings are incorporated herein and constitute part of the detailed description. The detailed description includes specific details for the purpose of providing an understanding of the subject art. However, it will be clear that those skilled in the art can implement the subject art even without these specific details. In some cases, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject art. For ease of understanding, similar components are shown with the same element number.

[0020] This disclosure provides methods and compositions for treating or restoring soft tissue conditions in subjects requiring such treatment. As used herein, “soft tissue” means tissues that connect, support, or surround bones and internal organs. For example, soft tissues include skin, muscle, fat, tendons, and fascia. Soft tissue “condition” includes disease, disorder, pathological condition, non-pathological condition, or deviation from homeostasis. In embodiments, this disclosure provides a method for increasing the water content in the soft tissue of a subject who requires it. As used herein, “water content” means the hydration level in soft tissue, including the amount of water present in the cells and / or extracellular space of the tissue. Methods for measuring the water content of soft tissue are technically well known. For example, in the context of this disclosure, the water content, or hydration level, of skin can be measured based on the change in dielectric constant due to hydration of the skin surface. Preferably, skin hydration is measured with the CORNEOMETER®, a handheld probe from Courage+Khazaka Electronics GmbH (Cologne, Germany) (see Examples).

[0021] As used herein, “increase water content” or “increase water content” means increasing the water content or hydration level present in the cells and / or extracellular space of a tissue, including, for example, increasing it relative to a comparison tissue. In some embodiments, the comparison tissue may include soft tissue before treatment with the composition of the Disclosure. In some embodiments, the comparison tissue may include soft tissue that is not treated or has been administered a composition other than those disclosed herein. In some embodiments, the method of the Disclosure increases the water content in the soft tissue by about 10% to about 80%, or about 20% to about 50%. In further embodiments, the method of the Disclosure increases the water content in the soft tissue by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the compositions of the Disclosure increase the water content by about 30% compared to soft tissue that is not treated or is treated with compositions other than those disclosed herein. This disclosure provides a method for increasing the water content in soft tissue, comprising administering a composition comprising tropoelastin and optionally hyaluronic acid to the soft tissue of a subject. In one embodiment, the tropoelastin is crosslinked with hyaluronic acid. In a further embodiment, the tropoelastin is crosslinked with derivatized hyaluronic acid. In another embodiment, the tropoelastin is human tropoelastin. In yet another embodiment, the tropoelastin comprises recombinant tropoelastin, for example recombinant human tropoelastin. In yet another embodiment, the tropoelastin comprises a tropoelastin monomer.

[0022] In some embodiments, the method of the present disclosure includes a composition comprising tropoelastin present in an amount of about 1 mg / ml to about 100 mg / ml. For example, in the embodiments, tropoelastin is present in an amount of about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 15 mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, about 35 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, or about 100 mg / ml. In certain embodiments, tropoelastin is present in an amount of approximately 30 mg / mL. The amount and concentration of tropoelastin to be administered are determined by both the area and volume of the tissue to be treated, usually the tissue contents, and the required level of water content increase. In embodiments, tropoelastin is administered to 1 cm of tissue. 3 It is administered to tissues in amounts of approximately 1 μg to 1 mg per unit. For skin, 1 cm 2 This can be calculated using approximately 1 μg to 1 mg. Other possible doses include 1 cm³ of tissue. 3 Approximately 0.1 μg to 10 mg per 1 cm of tissue 3 Approximately 1 mg to 20 mg per unit area, or 1 cm² of tissue. 3The dosage ranges from approximately 1 mg to 100 mg per unit area. In certain embodiments, the dosage is approximately 1 cm³ of tissue. 3 The amount may be less than approximately 0.1 μg or more than approximately 100 mg per dose. The concentration of tropoelastin in the administered composition may be varied to ensure that the required amount of tropoelastin is administered.

[0023] In embodiments, the tropoelastin of this disclosure is substantially equivalent to the isoform of tropoelastin naturally present in the tissue to be treated. Furthermore, the tropoelastin should be provided in a form substantially free of impurities. Fragments of tropoelastin, i.e., cleaved forms of the isoform of tropoelastin unintentionally produced by tropoelastin manufacturing, may be considered impurities in this context. In certain embodiments, the tropoelastin incorporated into the therapeutic formulation is at least 65% of the length of a suitable full-length isoform of tropoelastin, more preferably 80% of the length of a suitable full-length isoform of tropoelastin. In other embodiments, the tropoelastin is more than 85%, more than 90%, or more than 95% of the full length. In embodiments, tropoelastin of the present disclosure is modified to reduce protease degradation. For example, as described in WO 2000 / 04043, the protein species may be selected to some extent to remain substantially the full-length tropoelastin species naturally found in the tissues to be treated. Alternatively, the therapeutic formulation may incorporate a protease inhibitor or molecule that blocks a signaling pathway known to increase protease expression. Such molecules include serine protease inhibitors, matrix metalloproteinase inhibitors, galactosides, elastin signaling inhibitory antibodies, and small molecule inhibitors.

[0024] In embodiments involving the treatment of human subjects, tropoelastin comprises a sequence of an isotype of tropoelastin expressed in humans. In some embodiments, the isotype may be selected from the group consisting of SHEL (see WO 1994 / 14958) and SHELδ26A (see WO 1999 / 03886) and protease-resistant derivatives of these isotypes (see WO 2000 / 0403). In certain embodiments, the tropoelastin isotype is SHELδ26A. In certain embodiments, tropoelastin has a defined degree of purity with respect to the amount of tropoelastin in the administration composition compared to the amount of other proteins or molecules in the composition. In one embodiment, tropoelastin is in a composition having a purity of at least about 75%, preferably about 85%, more preferably about 90%, or about 95%. In certain embodiments, it will be found that tropoelastin may be provided in the form of tropoelastin, preferably the full-length isoform of tropoelastin, or in the form of a composition essentially consisting of tropoelastin, preferably the full-length isoform of tropoelastin. Finally, if tropoelastin is already substantially intramolecularly crosslinked, cells cannot utilize tropoelastin to form elastic fibers.

[0025] Typically, administration compositions containing tropoelastin do not contain exogenous factors for elastic fiber formation, particularly lysyl oxidase. In certain embodiments, tropoelastin is provided in substantially monomeric form according to the treatment plan. In certain embodiments, tropoelastin is provided in a form substantially lacking intramolecular crosslinking according to the treatment plan. In certain embodiments, tropoelastin is provided in a composition comprising tropoelastin and a solvent for tropoelastin, such as an aqueous solution, according to the treatment plan. In embodiments, the compositions of the present disclosure include one or more compounds that enhance the utilization of tropoelastin. Examples of compounds that enhance the utilization of tropoelastin include diclofenac, lys'lastine, amino acids (e.g., Gly, Val, Ala), vitamins (e.g., C, E), sunscreens, and chemical enhancers.

[0026] In some embodiments, the methods of the present disclosure include tropoelastin crosslinked to hyaluronic acid. In embodiments, the present disclosure provides a method comprising administering a composition comprising tropoelastin crosslinked to hyaluronic acid to the soft tissue of a subject, wherein the composition increases the water content in the subject's soft tissue compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but is otherwise identical. In some embodiments, the methods of the present disclosure include compositions comprising tropoelastin crosslinked to hyaluronic acid without a crosslinking agent. For example, in embodiments, tropoelastin is crosslinked to hyaluronic acid without a crosslinking agent such as an enzymatic crosslinking agent. In certain embodiments, the compositions of the present disclosure include tropoelastin crosslinked to hyaluronic acid without treatment under alkaline conditions. In some embodiments, tropoelastin is crosslinked to hyaluronic acid via at least one intermolecular crosslinking comprising an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid.

[0027] In certain embodiments, the composition containing tropoelastin is crosslinked with about 0.1% (w / v) to about 5% (w / v) of hyaluronic acid. In some embodiments, the hyaluronic acid is crosslinked with about 0.1% (w / v), about 0.2% (w / v), about 0.3% (w / v), about 0.4% (w / v), about 0.5% (w / v), about 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), about 1.0% (w / v), about 1.1% (w / v), and about 1.2% (w / v). %(w / v), approx. 1.3%(w / v), approx. 1.4%(w / v), approx. 1.5%(w / v), approx. 1.6%(w / v), approx. 1.7%(w / v), approx. 1.8%(w / v) , about 1.9% (w / v), about 2.0% (w / v), about 2.1% (w / v), about 2.2% (w / v), about 2.3% (w / v), about 2.4% (w / v), about 2.5% (w / v), approximately 2.6% (w / v), approximately 2.7% (w / v), approximately 2.8% (w / v), approximately 2.9% (w / v), approximately 3.0% (w / v), approximately 3.1% (w / v) , about 3.2% (w / v), about 3.3% (w / v), about 3.4% (w / v), about 3.5% (w / v), about 3.6% (w / v), about 3.7% (w / v), about 3.8% It accounts for approximately 3.9%(w / v), 4.0%(w / v), 4.1%(w / v), 4.2%(w / v), 4.3%(w / v), 4.4%(w / v), 4.5%(w / v), 4.6%(w / v), 4.7%(w / v), 4.8%(w / v), 4.9%(w / v), or 5.0%(w / v). In certain embodiments, tropoelastin is cross-linked with approximately 0.5%(w / v) of hyaluronic acid.

[0028] In further embodiments, tropoelastin is cross-linked with hyaluronic acid present in amounts of approximately 1 mg / mL to approximately 15 mg / mL. In yet another embodiment, tropoelastin is cross-linked with hyaluronic acid present in amounts of approximately 10 mg / mL or less, approximately 5 mg / mL or less, approximately 4 mg / mL or less, approximately 3 mg / mL or less, approximately 2 mg / mL or less, or approximately 1 mg / mL or less. In a specific embodiment, tropoelastin is cross-linked with hyaluronic acid present in amounts of approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL, approximately 7 mg / mL, approximately 8 mg / mL, approximately 9 mg / mL, approximately 10 mg / mL, approximately 11 mg / mL, approximately 12 mg / mL, approximately 13 mg / mL, approximately 14 mg / mL, or approximately 15 mg / mL. In certain embodiments, tropoelastin in the composition may be crosslinked with derivatized hyaluronic acid. In some embodiments, the methods of the present disclosure include a composition having a tropoelastin to hyaluronic acid ratio ("tropoelastin:hyaluronic acid"). As used herein, the "tropoelastin:hyaluronic acid ratio" includes the ratio of the mass / volume concentrations of each component in the composition. For example, a tropoelastin:hyaluronic acid ratio of about 6:1 includes a composition having about 30 mg / mL (or about 3% (w / v)) of tropoelastin and about 5 mg / mL (or about 0.5% (w / v)) of hyaluronic acid. In some embodiments, the tropoelastin:hyaluronic acid ratio is about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0029] In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 2:1 or higher, and the composition increases the water content in the subject's soft tissues compared to a composition that is otherwise identical but has a tropoelastin:hyaluronic acid ratio of less than approximately 2:1. Accordingly, embodiments of the method disclosed herein utilize a composition that, despite having a smaller amount of hyaluronic acid, will increase the water content in the soft tissues more significantly than a comparative composition having a larger amount of hyaluronic acid. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 2:1 or higher, and the amount of hyaluronic acid is approximately 5 mg / mL or less, and this composition will increase the water content in the soft tissues more significantly than a composition having more than 5 mg / mL of hyaluronic acid. In embodiments, the tropoelastin:hyaluronic acid ratio is approximately 2:1, the amount of tropoelastin is approximately 10 mg / mL, and the amount of hyaluronic acid is approximately 5 mg / mL. In certain embodiments, the tropoelastin:hyaluronic acid ratio is approximately 3:1 or higher, and the composition increases the water content in the subject's soft tissues compared to compositions with a tropoelastin:hyaluronic acid ratio of less than 3:1 but otherwise identical. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 3:1 or higher, and the amount of hyaluronic acid is approximately 5 mg / mL or less, and this composition significantly increases the water content in the soft tissues compared to compositions with more than 5 mg / mL of hyaluronic acid. In embodiments, the tropoelastin:hyaluronic acid ratio is approximately 3:1, the amount of tropoelastin is approximately 15 mg / mL, and the amount of hyaluronic acid is approximately 5 mg / mL.

[0030] In certain embodiments, the tropoelastin:hyaluronic acid ratio is approximately 4:1 or higher, and the composition increases the water content in the subject's soft tissues compared to compositions with a tropoelastin:hyaluronic acid ratio of less than 4:1 but otherwise identical. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 4:1 or higher, and the amount of hyaluronic acid is approximately 5 mg / mL or less, and this composition significantly increases the water content in the soft tissues compared to compositions with more than 5 mg / mL of hyaluronic acid. In embodiments, the tropoelastin:hyaluronic acid ratio is approximately 4:1, the amount of tropoelastin is approximately 20 mg / mL, and the amount of hyaluronic acid is approximately 5 mg / mL. In certain embodiments, the tropoelastin:hyaluronic acid ratio is approximately 5:1 or higher, and the composition increases the water content in the subject's soft tissues compared to compositions with a tropoelastin:hyaluronic acid ratio of less than 5:1 but otherwise identical. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 5:1 or higher, and the amount of hyaluronic acid is approximately 5 mg / mL or less, and this composition significantly increases the water content in the soft tissues compared to compositions with more than 5 mg / mL of hyaluronic acid. In embodiments, the tropoelastin:hyaluronic acid ratio is approximately 5:1, the amount of tropoelastin is approximately 25 mg / mL, and the amount of hyaluronic acid is approximately 5 mg / mL.

[0031] In certain embodiments, the tropoelastin:hyaluronic acid ratio is approximately 6:1 or higher, and the composition increases the water content in the subject's soft tissues compared to compositions with a tropoelastin:hyaluronic acid ratio of less than 6:1 but otherwise identical. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 6:1 or higher, and the amount of hyaluronic acid is 1 to approximately 5 mg / mL or less, and this composition significantly increases the water content in the soft tissues compared to compositions with more than 5 mg / mL of hyaluronic acid. In embodiments, the tropoelastin:hyaluronic acid ratio is approximately 6:1, the amount of tropoelastin is approximately 30 mg / mL, and the amount of hyaluronic acid is approximately 5 mg / mL. In certain embodiments, the tropoelastin:hyaluronic acid ratio is approximately 7:1 or higher, and the composition increases the water content in the subject's soft tissues compared to compositions with a tropoelastin:hyaluronic acid ratio of less than 7:1 but otherwise identical. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 7:1 or higher, and the amount of hyaluronic acid is approximately 5 mg / mL or less, and this composition significantly increases the water content in the soft tissues compared to compositions with more than 5 mg / mL of hyaluronic acid. In embodiments, the tropoelastin:hyaluronic acid ratio is approximately 7:1, the amount of tropoelastin is approximately 35 mg / mL, and the amount of hyaluronic acid is approximately 5 mg / mL.

[0032] In certain embodiments, the tropoelastin:hyaluronic acid ratio is approximately 8:1 or higher, and the composition increases the water content in the subject's soft tissues compared to compositions with a tropoelastin:hyaluronic acid ratio of less than 8:1 but otherwise identical. In some embodiments, the tropoelastin:hyaluronic acid ratio is approximately 8:1 or higher, and the amount of hyaluronic acid is approximately 5 mg / mL or less, and this composition significantly increases the water content in the soft tissues compared to compositions with more than 5 mg / mL of hyaluronic acid. In embodiments, the tropoelastin:hyaluronic acid ratio is approximately 8:1, the amount of tropoelastin is approximately 40 mg / mL, and the amount of hyaluronic acid is approximately 5 mg / mL. In some embodiments, the soft tissue is skin. In further embodiments, the skin is very dry skin, dry skin, or hydrated skin prior to administration of the composition. In even further embodiments, the skin has a capacitance of less than about 90 a.u., less than about 85 a.u., less than about 80 a.u., less than about 75 a.u., less than about 70 a.u., less than about 65 a.u., less than about 60 a.u., less than about 55 a.u., less than about 50 a.u., less than about 45 a.u., less than about 40 a.u., less than about 35 a.u., less than about 30 a.u., less than about 25 a.u., less than about 20 a.u., less than about 15 a.u., less than about 10 a.u., or less than about 5 a.u. prior to administration of the composition.

[0033] In other embodiments, the skin has a capacitance of 30–60 a.u. (e.g., very dry skin), 60–70 a.u. (e.g., dry skin), or 70–90 a.u. (e.g., hydrated skin) before administration of the composition, including when measured with a Corneometer (see Zuang et al. (1997) J. Appl. Cosmetol. 15, 95–102). In further embodiments, the skin has an increased capacitance after administration of the composition, including an increase in capacitance of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more. In some embodiments, the skin is determined to be very dry before administration of the composition and to be dry, hydrated, or very wet after administration of the composition. In other embodiments, the skin is determined to be dry before administration of the composition and to be hydrated or very wet after administration of the composition. In some embodiments, the skin is determined to be hydrated before administration of the composition and to be very wet after administration of the composition.

[0034] In some embodiments, the Disclosure provides methods and compositions for treating soft tissue conditions of the skin. In embodiments, skin conditions include facial wrinkles, fine lines, thinning skin, aging skin, scar tissue, and skin furrows. In embodiments, the methods of the Disclosure include administering the compositions into the skin by injection. In certain embodiments, the compositions are administered into the dermis, subcutaneous tissue, or subcutaneously. In some embodiments, the tissue is skin tissue, for example, skin tissue from an individual who is at least about 20 years old, about 20 to about 50 years old, or about 30 to about 60 years old. In some embodiments, the skin tissue treated according to this disclosure may be characterized by damage or rupture of elastic fibers at the junction of the dermis and epidermis, and a lower water content compared to healthy skin tissue. The skin tissue may be photoaged tissue. The skin tissue may exhibit one or more of the following features: loose skin, flaccid subcutaneous tissue, reduced extracellular matrix density, wrinkles, and stretch marks. The skin tissue is preferably located on the face, neck, or upper or lower limbs.

[0035] In some embodiments, the compositions of this disclosure are administered by injection. When the tissue is skin, the compositions are preferably administered into the dermis. In certain embodiments, the compositions are administered by fine-needle injection into the mid- or deep dermis. The injection may be performed using a subcutaneous needle having a gauge of 25G, preferably 27G or less, and more preferably 30G or 31G. The injection may be performed by manual application of the treatment to the skin using a single syringe and needle. In certain embodiments, a single treatment may include multiple injections into the treatment area. If each treatment requires multiple injections, they may be spaced 1 mm to 3 cm apart. In certain embodiments, injections may be performed using a device that enables automatic injection into the dermis of the skin, such as a mesotherapy gun, or an auxiliary injection device, such as an artiste injection device or an anteis injection device. In certain embodiments, the syringe or automatic injection device may be used with an adapter that allows for the attachment of multiple needles so that multiple injections can be applied at once. In certain embodiments, treatment may be performed using a solid needle system, such as a dermal roller, or a dermapen needling system (as described, for example, by Kalluri, H. et al 2011, AAPS Journal 13:473-4841).

[0036] There may be a period of approximately 1 day to 6 months between each treatment. Typical intervals between treatments include approximately 1 to 7 days, 7 to 14 days, 21 to 28 days, 28 to 49 days, and 49 to 100 days. There may be a total of approximately 1 to 24 treatments, or approximately 3 to 6 treatments. Generally, the treatment period is approximately 1 year or less, preferably approximately 3 weeks to 6 months, and preferably approximately 1 to 3 months. In this embodiment, there are intervals between each treatment of approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, or 100 days.

[0037] In some embodiments, treatment sites include the cheeks, eyes, neck, decolletage, hands, scar tissue, and areas near, around, within, or adjacent to stretch marks. In some embodiments, the composition includes additional components to assist in the treatment or repair of soft tissue conditions and / or the increase of water content within the soft tissue. For example, for the treatment of skin, additional components that assist in the recruitment or amplification of fibroblasts at the treatment site may be incorporated into the formulation. Examples of such components include the epidermal growth factor family, the transforming growth factor β family, the fibroblast growth factor family, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, platelet-derived growth factor, connective tissue growth factor, the interleukin family, and the tumor necrosis factor α family. In certain embodiments, the treatment plan may further include topical application of a substance capable of enhancing, treating, or repairing the soft tissue condition or increasing the water content of the soft tissue. Such substances will be well known to those skilled in the art, and may include, but are not limited to, dill extract for stimulating lysyl oxidase expression (Cenizo et al 2006 Exp. Dermatol. 15:574-81) and copper and / or zinc-based creams for reducing elastic fiber breakage (Mahoney et al 2009 Exp. Dermatol. 18:205-211).

[0038] In certain embodiments, the treatment may include the delivery of cells to the treatment site by tropoelastin. As an example of skin treatment, fibroblasts may be included in the therapeutic formulation or procedure to aid in the synthesis of elastic fibers at the treatment site. Fibroblasts may be supplied from an allogeneic source such as neonatal foreskin or by biopsy of an invisible skin site (e.g., behind the ear) and used as autotherapy. In some embodiments, the present disclosure provides a method for treating a subject's skin condition, comprising: preparing the subject with the skin condition; defining a treatment area of ​​the subject's skin, which is an area of ​​skin to be increased in water content; injecting a composition comprising cross-linked tropoelastin and hyaluronic acid into the treatment area to establish an increased amount of cross-linked tropoelastin and hyaluronic acid within the treatment area compared to the skin outside the treatment area; and maintaining the amount of cross-linked tropoelastin and hyaluronic acid within the treatment area for a predetermined period of time, thereby increasing the water content of the subject's skin. In this embodiment, the cross-linked tropoelastin and hyaluronic acid are maintained in the treatment area for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days.

[0039] In certain embodiments, the treatment site is repeatedly treated to ensure that tropoelastin is delivered in a form that cells can utilize as a substrate for constructing elastic fibers, and that it remains present at the treatment site for a sufficient period of time. In certain embodiments, each tissue site to be treated receives three treatments of the product spaced about 1 to 24 weeks apart, or about 2 to 12 weeks apart, or about 3 to 6 weeks apart. The treatment may consist of frequent injections across the area to be treated, each spaced about 10 mm apart in the lattice structure. Treatment may be performed using a fine-gauge needle, e.g., 27G, 29G, 30G, or 31G. The needle may be inserted into the tissue considering the angle and direction of the bevel, the injection depth, and the amount of material to be administered. The therapeutic agent may be injected into the tissue as a bolus, for example, by implanting about 10 μl to 100 μl, about 10 μl to 50 μl, or about 20 μl to 30 μl of the product into each injection site. After each injection is complete, the needle may be slowly withdrawn. Once all transplants are complete, the treatment site may be gently massaged if necessary to ensure the transplant material conforms to the contours of the surrounding tissue. The number of treatments, the interval between treatments, and the amount of tropoelastin delivered to each treatment site will be adjusted based on the tissue area to be treated and the level of elasticity to be restored.

[0040] In some embodiments, the methods of the present disclosure increase glycosaminoglycan deposits in the soft tissues of subjects requiring such deposits. In some embodiments, the increase in glycosaminoglycan deposits includes an increase in hyaluronic acid deposits. In some embodiments, the increase in glycosaminoglycan deposition in the subject's skin includes endogenous glycosaminoglycans, i.e., glycosaminoglycans produced by cells at or near the administration site and not induced by the composition itself. In certain embodiments, the method increases endogenous glycosaminoglycan deposition, for example, increasing endogenous hyaluronic acid deposition in soft tissue. In further embodiments, glycosaminoglycan deposition is increased on the cell surface of the papillary or upper reticular dermis of the skin. In the embodiment, glycosaminoglycan deposits increase by approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Methods for detecting glycosaminoglycan deposits are well known in the art. For example, in this embodiment, glycosaminoglycan deposits are detected by histological evaluation of a tissue biopsy.

[0041] In some embodiments, the Disclosure provides a method for increasing the expression of hyaluronan synthases (e.g., hyaluronan synthase 1 (HAS1), hyaluronan synthase 2 (HAS2), and / or hyaluronan synthase 3 (HAS3)) in the skin of a subject in need thereof, comprising administering to the skin a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 2:1 or greater, and the hyaluronan synthase expression is increased compared to soft tissue administered with the same composition, but with a tropoelastin:hyaluronic acid ratio of less than 2:1. In some embodiments, the tropoelastin:hyaluronic acid ratio of the composition is approximately 6:1 or greater, and the hyaluronan synthase expression is increased compared to soft tissue administered with the same composition, but with a tropoelastin:hyaluronic acid ratio of less than 6:1. In some embodiments, hyaluronan synthase expression increases by approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to hyaluronan synthase expression in untreated skin. Methods for evaluating hyaluronan synthase (e.g., hyaluronan synthase 1 (HAS1), hyaluronan synthase 2 (HAS2), and / or hyaluronan synthase 3 (HAS3)) expression in soft tissues such as skin are well known. In one embodiment, hyaluronan synthase expression is measured at the level of hyaluronan synthase gene expression, for example, by evaluating the mRNA level. In another embodiment, hyaluronan synthase expression is measured at the level of protein production. [Examples]

[0042] Examples Example 1: Tropoelastin increases the water content, elastin content, and glycosaminoglycan deposition in human skin explants and rat biopsy specimens. Tropoelastin and Hyaluronic Acid: Preparation and Formulation. Recombinant human tropoelastin (rhTE) was produced in Escherichia coli (E. coli) as previously described and purified as a 60 kDa monomer (Martin SL, Vrhovski B, Weiss AS. Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin. Gene. 1995;154(2):159-166). rhTE matched amino acid residues 27-724 of GenBank entry AAC98394 isotype SHELδ26A (Vrhovski B, Jensen S, Weiss AS. Coacervation characteristics of recombinant human tropoelastin. Eur. J. Biochem. 1997;250(1):92-98). rhTE is produced according to the current Good Manufacturing Practices (cGMP) code (Elastagen, Sydney, Australia). The hyaluronic acid was manufactured in accordance with cGMP and supplied by HTL SAS (Javene, France). Table 1 shows the nine formulations prepared for the research described herein. For the formulations of rhTE crosslinked with dHA, dHA was prepared as described in U.S. Patent No. 9,611,312.

[0043] [Table 1]

[0044] Ex vivo human skin explant preparation. Skin explants from a 63-year-old Caucasian woman who underwent abdominoplasty were cut into 15 × 20 mm rectangles and maintained in Explants Medium (BIO-EC; Longjumeau, France) at 37°C and 5% CO2. On day zero (0), the explants were injected with 50 μL of the test formulation (n=3 per group per time point for glycosaminoglycan [GAG] analysis) in a single injection or in three injections of 50 μL of the test formulation separated by a distance of 5 mm (n=3 for corneometry analysis). The test formulations were untreated control (NT), 12 mg / mL HA only, and formulations F6, F7, F8, and F9. On days 5 and 7, the explants were collected for histological examination. Histological evaluation of glycosaminoglycans (GAGs) in human explants. Explants were fixed in formal solution for 24 hours, dehydrated, embedded in paraffin, and cut into 5 μm thin sections. To detect acidic GAGs, the slides were stained with Alcian blue and periodate Schiff (Mowry Method). Images were obtained using an Olympus BX43 microscope equipped with a DP72 camera, and the area percentage of positive staining was analyzed using Cell^D software. Evaluation of skin water content. To measure skin water content, a CM825 Corneometer® (Courage+Khazaka electronic; Cologne, Germany) was applied to the surface of the explant centered on three injection sites. Ten measurements were taken for each explant on days 0, 1, 2, 5, and 7. Data are presented as the average (SD) of 10 measurements from three explants for each condition.

[0045] Rat Protocol. All animal protocols were approved by the Allergan Animal Care and Use Committee. Six-week-old male CD rats were obtained from Charles River Labs (Wilmington, MA, USA) and reared for five days before the start of the experiment. Prior to injection, the rats were anesthetized with 4% isoflurane and bilateral abdominal bristles were trimmed. 20 μL of the test formulation was injected into the dermis using a 27G 1 / 2-inch needle. The test formulations were saline (control) and formulations F6, F7, F8, and F9 (n=8 per group at each time point). Histological evaluation of rat skin biopsies. Punch biopsies (8 mm) were collected at 2 and 8 weeks, fixed in 10% neutral buffered formalin, embedded in paraffin, and prepared as thin sections. Immunohistochemical staining was performed using rat elastin-specific primary antibodies (Ab23748; Abcam, Cambridge, UK) and human elastin-specific primary antibodies (MAB2503; Millipore, Burlington, MA, USA), as well as anti-mouse secondary antibodies (760-4814) and anti-rat secondary antibodies (760-4815) (Ventana Medical Systems, Tucson, AZ, USA). Immunohistochemical staining was performed using the Ventana Discovery Benchmark Ultra automated staining system (Ventana Medical Systems), and imaging was performed using NanoZoomer (Hamamatsu Photonics, Hamamatsu City, Japan). Thin sections were stained with hematoxylin and eosin (H&E), and qualified veterinary pathologists, blinded to treatment, scored the sections on a scale of 0 to 5 based on the severity of material diffusion, inflammation, and injection site fibrosis.

[0046] result Compared to untreated controls, injection of F8 (30 mg of rhTE cross-linked with 0.5% dHA, in a 6:1 ratio of tropoelastin to hyaluronic acid) significantly increased skin water content by 30.3% (P<0.01) at 7 days post-injection (Figure 1A). This formulation tended to increase water content on days 2 and 5. This increase in water content was greater than the increase achieved with a 2:1 ratio of tropoelastin to hyaluronic acid, and formulations containing uncross-linked rhTE monomer and HA (F6 and F7) had no effect on skin water content (Figure 1A). Immunohistochemical analysis of acidic GAGs revealed that intradermal injection of formulations derived from rhTE crosslinked with dHA (F8 and F9) resulted in increased GAG staining in the papillary and upper reticular dermis on days 5 and 7, while formulations of rhTE mixed with HYC-24L+ (F6 and F7) did not (Figures 1B and 1C). Two weeks after intradermal injection, both non-crosslinked formulations (F6 and F7) and crosslinked formulations (F8 and F9) were detectable throughout the skin layer in skin biopsies (Table 2). All test formulations induced minimal injection tract fibrosis and inflammation within two weeks; by eight weeks, no inflammation was observed in any group except formulation F6 (30 mg / mL rhTE mixed with HYC-24L+; Table 2), and there was little evidence of injection tract fibrosis.

[0047] [Table 2]

[0048] Histological analysis of rat skin biopsies at 2 weeks revealed that the cross-linked formulations (F8 and F9) induced substantial cell infiltration and material diffusion (Figures 2A and 2B). However, by 8 weeks, the hydrogel was less visible with the cross-linked formulations compared to the non-cross-linked formulations (Table 2). In contrast, injection of the non-cross-linked formulations (F6 and F7) resulted in significant material diffusion at both 2 and 3 weeks (Table 2) and exhibited minimal cell infiltration (Figures 2C and 2D). For comparison, injections with 12 mg / mL of HYC-24L+(HA) alone (Figure 2E) or with physiological saline (Figure 2F) are shown. Further analysis of the elastin composition revealed that injection of the rhTE+HA crosslinked formulation produced not only a stronger human elastin-positive signal from the mixture but also co-localization of rat and human elastin-positive signals (Figures 3A and 3B). This suggests the integration of the added novel elastin. In contrast, injection of the non-crosslinked formulation showed strong, specific rat elastin staining around the hydrogel and, similar to injection of HA alone, did not show co-localization of rat and human elastin staining in the tissue (Figures 3C, 3D, and 3E). The results for injection with physiological saline are shown in Figure 3F.

[0049] Example 2: Tropoelastin supports elastin organization and collagen production by fibroblasts. Cell culture and microscopy. Human dermal fibroblasts (C0045C; Thermo Fisher Scientific, Waltham, MA, USA) were seeded on glass coverslips and grown in Dulbecco's modified Eagle medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured for 24 days at 37°C and 5% CO2, with the medium changed every 2-3 days. To evaluate and promote collagen production, fibroblasts were incubated in or without 50 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (APM; Sigma-Aldrich). Cultures were treated with one dose of filter-sterilized rhTE (formulation F1; final medium concentration 0.25 mg / mL) on day 10, or with one dose each on days 10 and 17. Elastin and collagen fibers were stained with primary rabbit polyclonal elastin antibody (a-hTE-17-F; a gift from Dr. Dieter Reinhart, McGill University) and primary mouse monoclonal collagen antibody (COL-I; Sigma-Aldrich). Secondary antibodies (Alexa Fluor 568 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG; Life Technologies) were used for visualization. Nuclei were visualized by mounting coverslips with Prolong Glass antifade mountant containing Nuc Blue dye (Thermo Fisher Scientific). Samples were visualized using a Nikon C2 confocal microscope (Tokyo, Japan), and images were generated by converting the Z-stack into a maximum projection image of four equal continuous layers of the cell matrix using ImageJ software.

[0050] result Figure 4 shows four equal continuous layers from the top to the bottom of the cell matrix culture. In the absence of rhTE and APM, fibroblasts showed low levels of collagen and elastin synthesis. In the presence of APM, cells produced sufficient collagen, which was mainly limited to the top 75% of the layer. When incubated with one dose of rhTE in the absence of APM, an extensive network of elastin fibers was observed in the bottom layer; two doses of rhTE extended the branched elastin network throughout the culture. In the presence of both APM and rhTE, both fibrous collagen and branched elastin fibers were present; again, in this case, the elastin network developed at the bottom of the culture after one dose of rhTE or throughout the culture after two doses of rhTE separated by time (Figure 4).

[0051] Example 3: Tropoelastin stimulates the expression of hyaluronan synthase 1. A 3D in vitro skin patch model was used. The Phenion® Full Thickness Skin Model (Henkel, Dusseldorf, Germany) was used according to the manufacturer's instructions. Skin patches (n=3 for each test formulation) were cultured in a 37°C incubator supplemented with 5% CO2 in an Air-Liquid Interface (ALI) culture medium. The skin patches were injected with a total volume of 50 μL of the test formulation at four points across the surface of the skin patch. The test items were as follows: control (positive: physiological saline or 1% Triton X-100; negative: untreated), and formulations F1, F2, F3, F4, and F5. Intradermal injections of both free rhTE and rhTE+HA increased HAS1 mRNA at 120 hours (Figure 5B). The effect of rhTE injection on HAS1 expression was delayed: HAS1 mRNA levels were barely detectable at 24 hours but rose to detectable levels after 120 hours. Furthermore, HAS1 mRNA expression was proportional to the concentration of available rhTE implanted, with F1 (containing the highest concentration of rhTE) showing a greater effect, followed by F2 and F3; F4 (rhTE cross-linked with dHA at a relatively high 2:1 dHA-to-rhTE ratio) and F5 (HA only) showing the least effect.

[0052] Example 4: Tropoelastin stimulates GAG deposition by skin fibroblasts. Materials and Methods. Human dermal fibroblasts were supplied from a neonatal male (Thermo Fisher, C0045C, foreskin) and a 51-year-old male (Sigma, 142BR Lot 05 / H / 014). Dermal fibroblasts (1 × 10⁶) 4 Cells were seeded directly into the wells of a 12-well tissue culture plate in 4 ml of fresh medium (DMEM (Life Tech)) containing 10% (v / v) fetal bovine serum (FBS; Life Tech) and 1% (v / v) Pen / Strep (Sigma)). The cells were cultured at 37°C in 5% CO2, changing the medium four times every 2-3 days. On day 10 of culture, rhTE (15 mg / ml in phosphate-buffered saline (PBS); filtration sterile; 1.5 mg / well), HA (0.5% w / v in PBS; filtration sterile; 50 μl / well), or both were added to the wells, and the cells were cultured for a further 7 days. GAG deposition by dermal fibroblasts was evaluated 17 days after culture and 7 days after the addition of HA and / or rhTE using Alcian blue staining (Lopez-Gonzalez, et al. (2017) J. Dent. Res. 96, 832). Cultured cells and deposited ECM were fixed with 4% paraformaldehyde in PBS at room temperature for 1 hour, rinsed three times with PBS, and stained overnight at room temperature with Alcian blue solution (1% in 3% acetic acid, pH 2.5; Sigma B8438). Next, the samples were rinsed twice with 3% acetic acid and twice with H2O, and the stained cultures were photographed. The Alcian blue dye was extracted from the cell / ECM matrix with 6M guanidine hydrochloride (Sigma G4505) over 6 hours, and the absorbance of the resulting solution was measured at 630 nm using a Tecan plate reader.

[0053] Results: Tropoelastin supplementation of dermal fibroblast cultures resulted in a significant increase in GAG ECM accumulation in neonatal fibroblasts compared to unsupplemented neonatal fibroblasts (Figures 6A and 6C), and a significant increase in GAG ECM accumulation in adult fibroblasts compared to both unsupplemented and HA-supplemented adult fibroblasts (Figures 6B and 6D). The addition of tropoelastin restored GAG accumulation in adult fibroblast cultures to levels similar to those observed in neonatal fibroblast cultures.

[0054] Explanation as a clause on subject technology Various examples of the embodiments of this disclosure are described for convenience as numbered clauses (1, 2, 3, etc.). These are provided as examples and do not limit the subject art. Clause 1. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering a composition comprising tropoelastin and optionally hyaluronic acid to the subject's soft tissue, wherein the composition increases the water content within the subject's soft tissue. Clause 2. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering a composition comprising tropoelastin crosslinked to hyaluronic acid to the subject's soft tissue, wherein the composition increases the water content in the subject's soft tissue compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but is otherwise identical. Clause 3. The method according to Clause 2, wherein tropoelastin is cross-linked to hyaluronic acid without a cross-linking agent. Clause 4. The method according to Clause 2, wherein tropoelastin is cross-linked to hyaluronic acid via at least one intermolecular cross-linking comprising an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. Clause 5. The method according to Clause 1 or 2, wherein the tropoelastin comprises human tropoelastin. Clause 6. The method according to Clause 1 or 2, wherein the tropoelastin comprises recombinant tropoelastin. Clause 7. The method according to Clause 1 or 2, wherein the tropoelastin comprises recombinant human tropoelastin. Clause 8. The method according to Clause 1 or 2, wherein the tropoelastin comprises a tropoelastin monomer. Clause 9. The method according to any one of Clauses 1 to 8, wherein the hyaluronic acid is a derivatized hyaluronic acid.

[0055] Clause 10. The method according to any one of Clauses 1 to 9, wherein tropoelastin is present in an amount of approximately 1 mg / ml to approximately 100 mg / mL. Clause 11. The method according to any one of Clauses 1 to 10, wherein tropoelastin is present in an amount of approximately 10 to approximately 50 mg / mL. Clause 12. The method according to Clause 11, wherein tropoelastin is present in an amount of approximately 30 mg / mL. Clause 13. The method according to any one of Clauses 1 to 12, wherein tropoelastin is cross-linked with approximately 0.1% to approximately 5% hyaluronic acid. Clause 14. The method according to Clause 13, wherein tropoelastin is cross-linked with approximately 0.5% hyaluronic acid. Clause 15. The method described in any one of Clauses 1 to 14, wherein hyaluronic acid is present in an amount of approximately 1 mg / mL to approximately 15 mg / mL. Clause 16. The method according to Clause 2, wherein the tropoelastin:hyaluronic acid ratio is approximately 2:1, approximately 3:1, approximately 4:1, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1. Clause 17. The method according to Clause 2, wherein the tropoelastin:hyaluronic acid ratio is approximately 2:1 or greater, and the composition increases the water content in the soft tissue of a subject compared to soft tissue administered with a composition in which the tropoelastin:hyaluronic acid ratio is less than that of the composition, but which is otherwise identical. Clause 18. The method according to Clause 17, wherein the amount of hyaluronic acid is approximately 5 mg / mL or less, and the composition increases the water content in the soft tissue of a subject compared to a composition having more than approximately 5 mg / mL of hyaluronic acid but otherwise identical. Clause 19. The method according to Clause 2, wherein the tropoelastin:hyaluronic acid ratio is approximately 6:1 or greater, and the composition increases the water content in the soft tissues of a subject compared to a composition in which the tropoelastin:hyaluronic acid ratio is less than 6:1 but which is otherwise identical. Clause 20. The method according to Clause 19, wherein the amount of hyaluronic acid is approximately 5 mg / mL or less, and the composition increases the water content in the subject's soft tissues compared to a composition having more than approximately 5 mg / mL of hyaluronic acid but otherwise identical.

[0056] Clause 21. The method described in any one of Clauses 1 to 20, wherein the soft tissue is skin. Clause 22. The method according to Clause 21, wherein the step of administering the composition includes injecting the composition into the skin. Clause 23. The method according to Clause 21, wherein the step of administering the composition includes intradermal injection of the composition into the dermis. Clause 24. The method according to Clause 21, wherein the step of administering the composition includes injecting the composition into subcutaneous tissue. Clause 25. The method according to Clause 21, wherein the step of administering the composition includes subcutaneous injection of the composition. Clause 26. The method according to any one of Clauses 1 to 25, wherein the composition is an injectable composition. Clause 27. The method according to any one of Clauses 1 to 26, wherein the step of administering the composition increases glycosaminoglycan deposition in the tissue. Clause 28. The method according to Clause 27, wherein the step of administering the composition increases the accumulation of endogenous glycosaminoglycans in the tissue. Clause 29. The method according to Clause 27, wherein the step of administering the composition increases hyaluronic acid deposition in the tissue. Clause 30. The method according to Clause 29, wherein the hyaluronic acid is endogenous. Clause 31. The method according to Clause 27, wherein glycosaminoglycan deposits are increased on the surface of papillary or upper reticular dermal cells within the tissue. Clause 32. The method according to Clause 27, wherein the glycosaminoglycan deposit increases by only about 1x and about 15x. Clause 33. The method according to Clause 27, wherein glycosaminoglycan deposits increase by only about two to about ten times. Clause 34. The method according to Clause 27, wherein the glycosaminoglycan deposit increases by approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0057] Clause 35. The method described in any one of Clauses 1 to 34, wherein the water content of the soft tissue increases by approximately 10% to approximately 80%. Clause 36. The method described in any one of Clauses 1 to 35, wherein the water content of the soft tissue increases by approximately 20% to approximately 50%. Clause 37. The method described in any one of Clauses 1 to 36, wherein the water content of the soft tissue increases by approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, or approximately 50%. Clause 38. The method according to any one of Clauses 1 to 37, wherein the step of administering the composition stimulates collagen synthesis in the soft tissue. Clause 39. The method according to any one of Clauses 1 to 38, wherein the step of administering the composition stimulates elastin synthesis in the soft tissue. Clause 41 The method according to any one of Clauses 1 to 40, wherein the step of administering the composition causes cell infiltration at the administration site. Clause 42. The method according to any one of Clauses 1 to 41, wherein the step of administering the composition increases the elasticity of the tissue.

[0058] Clause 43. The method described in any one of Clauses 1 to 42, wherein the soft tissue is skin. Clause 44. The method according to Clause 43, wherein the skin is very dry, dry, or hydrated prior to administration of the composition. Clause 45. The method according to Clause 44, wherein the skin has a capacitance of less than about 90 a.u., less than about 85 a.u., less than about 80 a.u., less than about 75 a.u., less than about 70 a.u., less than about 65 a.u., less than about 60 a.u., less than about 55 a.u., less than about 50 a.u., less than about 45 a.u., less than about 40 a.u., less than about 35 a.u., less than about 30 a.u., less than about 25 a.u., less than about 20 a.u., less than about 15 a.u., less than about 10 a.u., or less than about 5 a.u. before administration of the composition.

[0059] Clause 46. A method for increasing the water content in the soft tissue of a subject in need thereof, comprising: preparing a subject having soft tissue having a first water content; and administering to the soft tissue an amount effective in causing a second water content in the soft tissue, wherein the tropoelastin:hyaluronic acid ratio of the composition is about 2:1 or greater, and the second water content is increased compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. Clause 47. The method according to Clause 46, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 6:1, and the second water content is increased compared to soft tissue administered with the same composition, but with a tropoelastin:hyaluronic acid ratio of less than approximately 6:1. Clause 48. The method described in any one of Clauses 46 to 47, wherein the second water content is approximately 10% to approximately 80% greater than the first water content. Clause 49. The method described in any one of Clauses 46 to 48, wherein the second water content is approximately 20% to approximately 50% greater than the first water content. Clause 50. The method described in any one of Clauses 46-49, wherein the second water content is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% more than the first water content. Clause 51. The method according to any one of Clauses 46-50, wherein the second moisture content remains greater than the first moisture content for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 24 weeks, or at least 48 weeks.

[0060] Clause 52. The method according to any one of Clauses 46 to 51, wherein tropoelastin is cross-linked to hyaluronic acid without a cross-linking agent. Clause 53. The method according to Clause 52, wherein tropoelastin is cross-linked to hyaluronic acid via at least one intermolecular cross-linking comprising an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. Clause 54. The method according to any one of Clauses 46-53, wherein the tropoelastin is human tropoelastin. Clause 55. The method according to any one of Clauses 46 to 53, wherein tropoelastin is recombinant tropoelastin. Clause 56. The method according to any one of Clauses 46 to 53, wherein tropoelastin is recombinant human tropoelastin. Clause 57. The method according to any one of Clauses 46 to 56, wherein the tropoelastin comprises a tropoelastin monomer. Clause 58. The method according to any one of Clauses 46 to 57, wherein the hyaluronic acid is a derivatized hyaluronic acid.

[0061] Clause 59. The method according to any one of Clauses 46 to 58, wherein tropoelastin is present in an amount of approximately 1 mg / ml to approximately 100 mg / mL. Clause 60. The method according to any one of Clauses 46 to 59, wherein tropoelastin is cross-linked with approximately 0.1% to approximately 5% hyaluronic acid. Clause 61. The method described in any one of Clauses 46 to 60, wherein hyaluronic acid is present in an amount of approximately 1 mg / mL to approximately 15 mg / mL. Clause 62. The method according to Clause 61, wherein hyaluronic acid is present in amounts of approximately 10 mg / mL or less, approximately 5 mg / mL or less, approximately 4 mg / mL or less, approximately 3 mg / mL or less, approximately 2 mg / mL or less, and approximately 1 mg / mL or less. Clause 63. The method according to any one of Clauses 46 to 62, wherein the tropoelastin:hyaluronic acid ratio is approximately 2:1, approximately 3:1, approximately 4:1, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1. Clause 64. The method according to Clause 63, wherein the tropoelastin:hyaluronic acid ratio is approximately 2:1 or greater. Clause 65. The method described in any one of Clauses 46-64, wherein the soft tissue is skin. Clause 66. The method of Clause 65, wherein the step of administering the composition includes injecting the composition into the skin. Clause 67. The method of Clause 65, wherein the step of administering the composition includes intradermal injection into the dermis. Clause 68. The method of Clause 65, wherein the step of administering the composition includes injection into subcutaneous tissue. Clause 69. The method according to Clause 65, wherein the step of administering the composition includes subcutaneous injection. Clause 70. The method according to Clause 46, wherein the subject has a soft tissue condition selected from the group consisting of facial wrinkles, fine lines, thinned skin, aging skin, scar tissue, and skin furrows. Clause 71. The method according to Clause 46, wherein the composition is an injectable composition.

[0062] Clause 72. A method for repairing soft tissue, comprising preparing soft tissue in need of repair and contacting the soft tissue with a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the method increases the water content within the tissue. Clause 73. The method according to Clause 72, wherein the soft tissue contains fibroblasts. Clause 74. The method according to any one of Clauses 72 to 73, wherein tropoelastin is cross-linked to hyaluronic acid without a cross-linking agent. Clause 75. The method according to Clause 74, wherein tropoelastin is cross-linked to hyaluronic acid via at least one intermolecular cross-linking comprising an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. Clause 76. The method according to any one of Clauses 72 to 75, wherein tropoelastin is recombinant tropoelastin. Clause 77. The method according to any one of Clauses 72 to 75, wherein the tropoelastin is recombinant human tropoelastin. Clause 78. The method according to any one of Clauses 72 to 77, wherein the tropoelastin comprises a tropoelastin monomer. Clause 79. The method according to any one of Clauses 72 to 78, wherein the hyaluronic acid is a derivatized hyaluronic acid. Clause 80. The method according to any one of Clauses 72 to 79, wherein tropoelastin is present in an amount of approximately 1 mg / ml to approximately 100 mg / mL. Clause 81. The method according to any one of Clauses 72 to 81, wherein tropoelastin is present in an amount of approximately 10 to approximately 50 mg / mL. Clause 82. The method according to Clause 81, wherein tropoelastin is present in an amount of approximately 30 mg / mL. Clause 83. The method according to any one of Clauses 72 to 82, wherein tropoelastin is cross-linked with approximately 0.1% to approximately 5% hyaluronic acid. Clause 84. The method according to any one of Clauses 72 to 83, wherein tropoelastin is cross-linked with approximately 0.5% hyaluronic acid. Clause 85. The method according to any one of Clauses 72 to 84, wherein hyaluronic acid is present in an amount of approximately 1 mg / mL to approximately 15 mg / mL. Clause 86. The method according to any one of Clauses 72 to 85, wherein hyaluronic acid is present in an amount of approximately 10 mg / mL or less, approximately 5 mg / mL or less, approximately 4 mg / mL or less, approximately 3 mg / mL or less, approximately 2 mg / mL or less, or approximately 1 mg / mL or less. Clause 87. The method according to any one of Clauses 72 to 86, wherein the tropoelastin:hyaluronic acid ratio is approximately 2:1 or greater.

[0063] Clause 88. The method described in any one of Clauses 72 to 87, wherein the water content increases by approximately 10% to approximately 80%. Clause 89. The method according to any one of Clauses 72 to 88, wherein the step of bringing soft tissue into contact with the composition increases the deposition of glycosaminoglycans. Clause 90. A method for increasing glycosaminoglycan deposition in the skin of a subject in need thereof, comprising administering to the skin a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is about 2:1 or greater, and the glycosaminoglycan deposition is increased compared to soft tissue administered with the same composition, but with a tropoelastin:hyaluronic acid ratio of less than 2:1. Clause 91. The method according to Clause 90, wherein the step of administering the composition increases glycosaminoglycan deposition on the cell surface of the papillary or upper reticular dermis of the skin. Clause 92. The method according to Clause 91, wherein the glycosaminoglycan deposit increases by only about 1x and about 15x. Clause 93. The method according to Clause 91 or 92, wherein the glycosaminoglycan deposit increases by only about two to about ten times. Clause 94. The method described in any one of Clauses 91 to 93, wherein the glycosaminoglycan deposit increases by approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0064] Clause 95. The method according to any one of Clauses 90 to 94, wherein tropoelastin is cross-linked to hyaluronic acid without a cross-linking agent. Clause 96. The method according to Clause 95, wherein tropoelastin is cross-linked to hyaluronic acid via at least one intermolecular cross-linking comprising an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. Clause 97. The method according to any one of Clauses 90 to 96, wherein the tropoelastin is recombinant human tropoelastin. Clause 98. The method according to any one of Clauses 90 to 97, wherein tropoelastin is present in an amount of approximately 1 mg / ml to approximately 100 mg / mL. Clause 99. The method according to any one of Clauses 90 to 98, wherein tropoelastin is cross-linked with approximately 0.1% to approximately 5% hyaluronic acid. Clause 100. The method according to any one of Clauses 90 to 99, wherein hyaluronic acid is present in an amount of approximately 1 mg / mL to approximately 15 mg / mL.

[0065] Clause 101. A method for increasing hyaluronan synthase expression in the skin of a subject, comprising administering to the skin a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 2:1 or greater, and the hyaluronan synthase expression is increased compared to soft tissue administered with the same composition, but with a tropoelastin:hyaluronic acid ratio of less than 2:1. Clause 102. The method according to Clause 101, wherein tropoelastin is cross-linked to hyaluronic acid without a cross-linking agent. Clause 103. The method according to Clause 101 or 102, wherein tropoelastin is crosslinked to hyaluronic acid via at least one intermolecular crosslinking comprising an amide bond between the amine of tropoelastin and the carboxyl group of hyaluronic acid. Clause 104. The method according to any one of Clauses 101 to 103, wherein hyaluronan synthase expression is increased by approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to hyaluronan synthase expression in untreated skin. Clause 105. The method described in any one of Clauses 101 to 104, wherein hyaluronan synthase mRNA expression is increased. Clause 106. The method according to any one of Clauses 101 to 104, wherein hyaluronan synthase protein expression is increased.

[0066] Clause 107. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the composition increases the water content in the subject's soft tissue compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but is otherwise identical, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 2:1 or greater, and the increase in water content is greater compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition but otherwise identical. Clause 108. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 2:1 or greater, and the composition increases the water content in the subject's soft tissue compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. Clause 109. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 3:1 or greater, and the composition increases the water content in the subject's soft tissue compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. Clause 110. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 4:1 or greater, and the composition increases the water content in the subject's soft tissue compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. Clause 111. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 5:1 or greater, and the composition increases the water content in the subject's soft tissue compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. Clause 112. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 6:1 or greater, and the composition increases the water content in the subject's soft tissue compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. Clause 113. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 7:1 or greater, and the composition increases the water content in the subject's soft tissue compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. Clause 114. A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering to the subject's soft tissue a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the tropoelastin:hyaluronic acid ratio of the composition is approximately 8:1 or greater, and the composition increases the water content in the subject's soft tissue compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical.

[0067] Further considerations In some embodiments, any provision of this specification may be dependent on any one independent provision or any one dependent provision. In one embodiment, any provision (e.g., dependent or independent provision) may be combined with any one or more other provisions (e.g., dependent or independent provisions). In one embodiment, a claim may include some or all of the words (e.g., steps, operations, means, or components) listed in a provision, sentence, phrase, or paragraph. In one embodiment, a claim may include some or all of the words listed in one or more provisions, sentences, phrases, or paragraphs. In one embodiment, some of the words in each provision, sentence, phrase, or paragraph may be removed. In one embodiment, additional words or elements may be added to a provision, sentence, phrase, or paragraph. In one embodiment, the subject art may be carried out without using some of the components, elements, functions, or operations described herein. In one embodiment, the subject art may be carried out using additional components, elements, functions, or operations.

[0068] The preceding description is provided so that those skilled in the art can implement the various configurations described herein. In particular, various figures and configurations have been described in relation to the subject art, but it should be understood that these are for illustrative purposes only and should not be interpreted as limiting the scope of the subject art. The specific order or hierarchy of the disclosed process steps is understood to be an example of a typical approach. It is understood that the specific order or hierarchy of the process steps may be rearranged based on the adoption of the design. Some steps may be performed simultaneously. The attached method claims present elements of various steps in an exemplary order and are not intended to limit the user to the specific order or hierarchy presented.

[0069] As used herein, the expression “at least one of ~” preceding a set of items and accompanied by the terms “and” or “or” to distinguish any of the items, qualifies the list as a whole, rather than each member of the list (i.e., each item). The expression “at least one of ~” does not require the selection of at least one of each listed item; rather, it allows the meaning to encompass at least one of any one of the items, and / or at least one of any combination of items, and / or at least one of each item. For example, the expression “at least one of A, B, and C” or “at least one of A, B, or C” refers to A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0070] Furthermore, to the extent that the terms “include,” “have,” etc. are used in the specification and claims, such terms are intended to be comprehensive in the same manner as the term “comprise” is interpreted when the term “comprise” is used as a transitional term in the claims. As used herein, the term “about” refers to the actual value stated and, as those skilled in the art will understand, allows for approximations, inaccuracies, and limitations of measurements under appropriate conditions. In one or more embodiments, the terms “about,” “substantially,” and “approximately” may give industry-accepted tolerances for the relatives between their corresponding terms and / or items, e.g., tolerances from less than 1 percent to 10 percent of the actual value stated, and other appropriate tolerances. As used herein, the term "comprising" indicates the existence of a specified integer, but acknowledges the possibility of other integers not specified. The term does not imply any particular ratio of the specified integer. Variants of the word "comprising," such as "comprise" and "comprises," have corresponding similar meanings. In this specification, the word “typical” means “useful as an example, illustration, or illustration.” No embodiment described as “typical” in this specification should necessarily be construed as preferable or advantageous to any other embodiment.

[0071] Unless otherwise specified, a singular reference to an element is not intended to mean "one or just one," but rather "one or more." Masculine pronouns (e.g., his) include feminine and neutral genders (e.g., her and its), and vice versa. The term "several" refers to one or more. Underlined and / or italicized headings and subheadings are for convenience only and do not limit the subject art or refer to the interpretation of the description of the subject art. All structural and functional equivalents to the elements of the various configurations described throughout this disclosure, which are known to those skilled in the art or will become known thereafter, are expressly incorporated herein by reference and intended to be encompassed by the subject art. Furthermore, nothing is disclosed herein intended to be dedicated to the public, whether such disclosure is expressly stated in the above description or not. While the detailed description contains many features, these should not be interpreted as limiting the scope of the subject art, but rather as merely describing various examples and aspects of the subject art. It should be understood that the scope of the subject art includes other embodiments not detailed above. Furthermore, for a method to be included within the scope of this disclosure, it is not necessary to address (or achieve) every problem that can be solved by the various embodiments of this disclosure. The use of “can” and its derivatives herein should be understood as meaning “probably” or “optionally” as opposed to “positive possibility.” Another aspect of the present invention may be as follows: [1] A method for treating a soft tissue condition of a subject requiring the treatment thereof, comprising administering a composition comprising tropoelastin crosslinked with hyaluronic acid to the soft tissue of the subject, wherein the composition increases the water content in the soft tissue of the subject compared to a composition in which the tropoelastin is not crosslinked with hyaluronic acid but is otherwise identical. [2] The method according to [1], wherein the tropoelastin is crosslinked with the hyaluronic acid without a crosslinking agent. [3] The method according to [2], wherein the tropoelastin is crosslinked to the hyaluronic acid via at least one intermolecular crosslink including an amide bond between the amine of the tropoelastin and the carboxyl group of the hyaluronic acid. [4] The method according to [1], wherein the tropoelastin comprises human tropoelastin. [5] The method according to [1], wherein the tropoelastin comprises recombinant tropoelastin. [6] The method according to [1], wherein the tropoelastin comprises recombinant human tropoelastin. [7] The method according to [1], wherein the tropoelastin comprises a tropoelastin monomer. [8] The method according to [1], wherein the tropoelastin is present in an amount of about 1 mg / ml to about 100 mg / mL. [9] The method according to [1], wherein the tropoelastin is present in an amount of about 10 to about 50 mg / mL.

[10] The method according to [9], wherein the tropoelastin is present in an amount of about 30 mg / mL.

[11] The method according to [1], wherein the hyaluronic acid is a derivatized hyaluronic acid.

[12] The method according to [1], wherein the tropoelastin is cross-linked with approximately 0.1% to approximately 5% hyaluronic acid.

[13] The method according to

[12] , wherein the tropoelastin is crosslinked with approximately 0.5% hyaluronic acid.

[14] The method according to [1], wherein the hyaluronic acid is present in an amount of about 1 mg / mL to about 15 mg / mL.

[15] The method according to [1], wherein the hyaluronic acid is present in an amount of approximately 10 mg / mL or less, approximately 5 mg / mL or less, approximately 4 mg / mL or less, approximately 3 mg / mL or less, approximately 2 mg / mL or less, or approximately 1 mg / mL or less.

[16] The method according to [1], wherein the tropoelastin:hyaluronic acid ratio is approximately 2:1, approximately 3:1, approximately 4:1, approximately 5:1, approximately 6:1, approximately 7:1, approximately 8:1, approximately 9:1, or approximately 10:1.

[17] The method according to

[16] , wherein the tropoelastin:hyaluronic acid ratio of the composition is about 2:1 or more, and the composition increases the water content in the soft tissue of the subject compared to soft tissue administered with a composition having a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical.

[18] The method according to

[17] , wherein the amount of hyaluronic acid is about 5 mg / mL or less, and the composition increases the water content in the soft tissue of the subject compared to a composition having more than 5 mg / mL of hyaluronic acid but otherwise being the same.

[19] The method according to

[17] , wherein the ratio of tropoelastin to hyaluronic acid is about 6:1 or more, and the composition increases the water content in the soft tissue of the subject compared to a composition in which the ratio of tropoelastin to hyaluronic acid is less than 6:1 but is otherwise identical.

[20] The method according to

[19] , wherein the amount of hyaluronic acid is about 5 mg / mL or less, and the composition increases the water content in the soft tissue of the subject compared to a composition having more than 5 mg / mL of hyaluronic acid but otherwise being the same.

[21] The method according to [1], wherein the soft tissue is skin.

[22] The method according to

[21] , wherein the skin is very dry, dry, or hydrated before administration of the composition.

[23] The method according to

[22] , wherein the skin has a capacitance of less than about 90 a.u., less than about 85 a.u., less than about 80 a.u., less than about 75 a.u., less than about 70 a.u., less than about 65 a.u., less than about 60 a.u., less than about 55 a.u., less than about 50 a.u., less than about 45 a.u., less than about 40 a.u., less than about 35 a.u., less than about 30 a.u., less than about 25 a.u., less than about 20 a.u., less than about 15 a.u., less than 10 a.u., or less than about 5 a.u. before administration of the composition.

[24] The method according to

[21] , wherein the soft tissue condition is selected from the group consisting of facial wrinkles, fine wrinkles, thinned skin, aged skin, scar tissue, and skin furrows.

[25] The method according to

[21] , wherein the step of administering the composition comprises injecting the composition into the skin.

[26] The method according to

[21] , wherein the step of administering the composition includes intradermal injection of the composition into the dermis.

[27] The method according to

[21] , wherein the step of administering the composition comprises injecting the composition into subcutaneous tissue.

[28] The method according to

[21] , wherein the step of administering the composition includes subcutaneous injection of the composition.

[29] The method according to [1], wherein the composition is an injectable composition.

[30] The method according to [1], wherein the step of administering the composition increases glycosaminoglycan deposition in the tissue.

[31] The method according to [1], wherein the water content of the soft tissue increases by about 10% to about 80%.

[32] The method according to [1], wherein the water content of the soft tissue increases by approximately 20% to approximately 50%.

[33] The method according to [1], wherein the water content of the soft tissue increases by approximately 20%, approximately 25%, approximately 30%, approximately 35%, approximately 40%, approximately 45%, or approximately 50%.

[34] A method for increasing the water content in the soft tissues of a subject who requires it, the following: Prepare a subject having soft tissue with a first water content; and A composition containing tropoelastin crosslinked with hyaluronic acid is administered to the soft tissue in an amount effective in generating a second water content in the soft tissue. Includes, The tropoelastin:hyaluronic acid ratio of the aforementioned composition is approximately 2:1 or higher, and The method wherein the second water content is increased compared to soft tissue administered with the composition, having a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical.

[35] The method according to

[34] , wherein the tropoelastin:hyaluronic acid ratio of the composition is about 6:1, and the second water content is increased compared to soft tissue administered with the same composition in all other respects, but with a tropoelastin:hyaluronic acid ratio of less than about 6:1.

[36] The method according to

[34] , wherein the second water content is about 10% to about 80% more than the first water content.

[37] The method according to

[34] , wherein the second water content remains greater than the first water content for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 24 weeks, or at least 48 weeks.

[38] The method according to

[34] , wherein the tropoelastin is present in an amount of about 1 mg / ml to about 100 mg / mL.

[39] The method according to

[34] , wherein the tropoelastin is cross-linked with approximately 0.1% to approximately 5% hyaluronic acid.

[40] The method according to

[34] , wherein the soft tissue is skin.

[41] A method for increasing cell infiltration into the soft tissue of a subject in need thereof, comprising administering to the soft tissue of the subject a composition comprising tropoelastin crosslinked to hyaluronic acid, wherein the composition increases cell infiltration into the soft tissue of the subject compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but otherwise identical.

[42] The method according to

[41] , wherein the method increases the formation of elastin fibers.

[43] The method according to any one of the above

[41] to

[42] , wherein the method increases the formation of new collagen fibers.

[44] A method for replenishing the skin of a subject in need thereof, comprising administering a composition comprising tropoelastin crosslinked to hyaluronic acid to the soft tissue or skin of the subject, wherein the composition increases the formation of elastin fibers in the soft tissue or skin of the subject compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but otherwise identical.

[45] The method according to

[44] , wherein the method increases the formation of new collagen fibers.

[46] A method for improving the skin composition of a subject in need thereof, comprising administering a composition comprising tropoelastin crosslinked to hyaluronic acid to the soft tissue or skin of the subject, wherein the composition increases the formation of elastin fibers in the soft tissue or skin of the subject compared to a composition in which the tropoelastin is not crosslinked to hyaluronic acid but is otherwise identical.

[47] The method according to

[46] , wherein the method causes the formation of a novel matrix at the administration site.

[48] ​​The method according to

[46] , wherein the method increases the formation of elastin fibers and collagen fibers.

[49] The method according to

[46] , wherein the improvement of skin composition is an improvement in skin brightness.

[50] The method according to

[46] , wherein the improvement of skin composition is to improve skin elasticity.

[51] The method according to

[46] , wherein the improvement of skin composition is an increase in extracellular matrix proteins at the administration site.

Claims

1. A composition comprising hyaluronic acid-crosslinked tropoelastin for treating the soft tissue condition of a subject requiring treatment, The composition, when administered to the soft tissue of the subject, increases the water content in the subject's soft tissue compared to a composition that is otherwise identical but in which the tropoelastin is not cross-linked with hyaluronic acid. The tropoelastin is crosslinked to the hyaluronic acid via at least one intermolecular crosslinking that includes an amide bond between the amine of the tropoelastin and the carboxyl group of the hyaluronic acid. The aforementioned tropoelastin is present in an amount of 30 mg / mL, The tropoelastin is cross-linked with 0.5% hyaluronic acid, and The water content in the soft tissue increases by 20% to 50%. The above composition.

2. The tropoelastin includes human tropoelastin, or The tropoelastin includes recombinant tropoelastin, or The tropoelastin includes recombinant human tropoelastin, or The tropoelastin comprises a tropoelastin monomer. The composition according to claim 1.

3. The hyaluronic acid is a derivatized hyaluronic acid. The composition according to claim 1.

4. The composition according to claim 1, wherein the hyaluronic acid is present in an amount of 5 mg / mL.

5. The ratio of tropoelastin to hyaluronic acid is 6:

1. and / or The tropoelastin:hyaluronic acid ratio of the composition is 6:1, and the composition increases the water content in the soft tissue of the subject compared to soft tissue administered with a tropoelastin:hyaluronic acid ratio less than that of the composition, but otherwise identical. and / or, The amount of hyaluronic acid is 5 mg / mL, and the composition increases the water content in the soft tissue of the subject compared to a composition that has more than 5 mg / mL of hyaluronic acid but is otherwise identical. The composition according to claim 1.

6. The aforementioned soft tissue is skin, The skin is very dry, dry, or hydrated before administration of the composition, or The skin has a capacitance of less than 90 a.u., less than 85 a.u., less than 80 a.u., less than 75 a.u., less than 70 a.u., less than 65 a.u., less than 60 a.u., less than 55 a.u., less than 50 a.u., less than 45 a.u., less than 40 a.u., less than 35 a.u., less than 30 a.u., less than 25 a.u., less than 20 a.u., less than 15 a.u., less than 10 a.u., or less than 5 a.u. before administration of the composition. and / or, The aforementioned soft tissue condition is selected from the group consisting of facial wrinkles, fine lines, thinned skin, aged skin, scar tissue, and skin furrows. The composition according to claim 1.

7. The step of administering the composition includes injecting the composition into the skin, or The step of administering the composition includes intradermal injection of the composition into the dermis, or The step of administering the composition includes injecting the composition into subcutaneous tissue, or The step of administering the composition includes subcutaneous injection of the composition. The composition according to claim 6.

8. The aforementioned composition is an injectable composition. and / or The step of administering the composition increases glycosaminoglycan deposition in the tissue. The composition according to claim 1.

9. The water content of the soft tissue increases by 20%, 25%, 30%, 35%, 40%, 45%, or 50%. The composition according to claim 1.

10. A composition for use in a non-therapeutic method to increase the water content in the soft tissues of a subject who requires it, The composition comprises tropoelastin crosslinked with hyaluronic acid, The above method is as follows: To prepare a subject having soft tissue with a first water content; and The composition is administered to the soft tissue in an amount effective in causing a second water content in the soft tissue. Includes, The aforementioned tropoelastin is present in an amount of 30 mg / mL, The tropoelastin is cross-linked with 0.5% hyaluronic acid, and The amount of water in the soft tissue is increased by 20% to 50%. The aforementioned composition.

11. The aforementioned soft tissue is skin. The composition according to claim 10.

12. The composition according to claim 1, wherein the tropoelastin is crosslinked with the hyaluronic acid without a crosslinking agent.

13. The second water content has a tropoelastin:hyaluronic acid ratio less than that of the composition, but is increased in all other respects compared to soft tissue administered with the same composition; The tropoelastin:hyaluronic acid ratio of the composition is 6:1, and the second water content is increased compared to soft tissue administered with the same composition in all other respects, although the tropoelastin:hyaluronic acid ratio is less than 6:1; Or, The second moisture content remains greater than the first moisture content for at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 24 weeks, or at least 48 weeks. The composition according to claim 10.

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