Use of cell membrane-binding signaling factor

Abstract

To provide a method for promoting regeneration of a tissue.SOLUTION: A method for promoting regeneration of a tissue of a subject includes a) a step of administering a composition to the subject, where the composition includes a heterogeneous mixture of composites including: i. a first composite containing first cyclodextrin molecules and Wingless (Wnt) protein covalently bonded to first cell membrane lipid; and ii. a second composite containing second cyclodextrin molecules and Hedgehog (Hh) protein covalently bonded to second cell membrane lipid.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 422900, filed November 16, 2016, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Membrane bound signaling factors, including proteins from the Wingless (Wnt) and Hedgehog (Hh) families, have potential use in a variety of diseases, but current methods for obtaining these proteins do not yield stable and effective molecules. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein are compositions comprising a complex of a lipid-modified protein as disclosed herein and a cyclodextrin.

[0004] Also disclosed herein are topical compositions comprising a complex of a lipid-modified protein and a cyclodextrin as disclosed herein.

[0005] In some embodiments, the cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or methyl-β-cyclodextrin. In some embodiments, the cyclodextrin is a chemically modified cyclodextrin that has been modified by hydrogenation, hydroformylation, methylation, oxidation, reduction, or carbon-carbon coupling. In some embodiments, the cyclodextrin is methyl-β-cyclodextrin.

[0006] In some embodiments, the lipid-modified protein comprises one or more of a Wingless (Wnt) protein or a Hedgehog (Hh) protein associated with a cell membrane lipid. In some embodiments, the Hh protein is one or more of a Sonic Hedgehog (Shh) protein, a Desert Hedgehog (Dhh) protein, or an Indian Hedgehog (Ihh) protein. In some embodiments, the Wnt protein is one or more of Wnt3a, Wnt7b, or Wnt10b. In some embodiments, the lipid-modified protein comprises other proteins that do not belong to the Wingless (Wnt) family or the Hedgehog (Hh) family.

[0007] In some embodiments, the lipid-modified protein is obtained from a stem cell population. In some embodiments, the stem cells are embryonic stem cells, parthenogenetic stem cells, adult stem cells, fetal stem cells, or induced pluripotent stem cells. In some embodiments, the stem cells are mammalian stem cells. In some embodiments, the stem cells are human stem cells. In some embodiments, the stem cells are genetically engineered to overexpress a Wnt protein or an Hh protein. In some embodiments, the stem cells are genetically immortalized. In some embodiments, the stem cells are genetically engineered to express telomere reverse transcriptase (hTERT).

[0008] In some embodiments, the composition further comprises at least one kosmotrope, hi some embodiments, the at least one kosmotrope is propylene glycol, proline, trehalose, ectoine, or trimethylamine-N-oxide.

[0009] In some embodiments, the topical composition is an aqueous formulation. In some embodiments, the topical composition further comprises at least one kosmotrope and an antimicrobial agent. In some embodiments, the at least one kosmotrope is trehalose. In some embodiments, the antimicrobial agent comprises silver particles. In some embodiments, the silver particles are silver nanoparticles or silver microparticles. In some embodiments, the pH of the topical composition is between about 4.5 and about 8.0.

[0010] Also disclosed herein are methods of promoting tissue regeneration in tissue in need thereof comprising exposing the tissue to a composition or topical composition disclosed herein.

[0011] Also disclosed herein are methods of promoting tissue rejuvenation in tissue in need thereof comprising exposing the tissue to a composition or topical composition disclosed herein.

[0012] Also disclosed herein is a method of restoring sensory nerve function to a tissue in need thereof comprising exposing the tissue to a composition or topical composition disclosed herein.

[0013] In some embodiments, the tissue is skin, hi some embodiments, the tissue is scar tissue.

[0014] Also disclosed herein is a method of promoting hair growth, comprising exposing hair follicles to a composition or topical composition disclosed herein. Also disclosed herein is a method of improving hair appearance, comprising exposing hair follicles to a composition or topical composition disclosed herein. In some embodiments, hair growth is promoted in a subject with age-related alopecia, alopecia totalis, telogen and anagen effluvium, or alopecia areata. In some embodiments, the hair follicle is on the scalp of the subject. In some embodiments, the hair follicle is in the eyelashes of the subject. In some embodiments, the hair follicle is in the eyebrows of the subject. In some embodiments, the hair follicle is on the face of the subject. In some embodiments, the hair follicle is on the chest of the subject. In some embodiments, the hair follicle is on the arms of the subject. In some embodiments, the hair follicle is on the legs of the subject.

[0015] Also disclosed herein are methods of improving the appearance of skin comprising exposing the skin to a composition or topical composition disclosed herein, in some embodiments, the improved appearance is one or more of skin texture, wrinkles, age spots, and age spots.

[0016] The present specification also discloses a method for producing a composition or topical composition disclosed herein, comprising culturing stem cells capable of producing Wnt and Hh proteins in a medium, incubating the stem cells in a harvest solution containing cyclodextrin to obtain a cyclodextrin complex of a lipid-modified protein, and mixing the cyclodextrin / lipid-modified protein complex with one or more pharma- ceutically acceptable excipients to form a topical formulation.

[0017] In some embodiments, the harvest solution further comprises at least one kosmotrope. In some embodiments, the harvest solution comprises a stabilizing agent. In some embodiments, the stabilizing agent is a kosmotrope. In some embodiments, the kosmotrope is trehalose. In some embodiments, the concentration of the kosmotrope in the harvest solution is about 5% to about 30%. In some embodiments, the concentration of the kosmotrope is about 20%.

[0018] In some embodiments, the harvest solution comprises an aqueous solution of cyclodextrin. In some embodiments, the cyclodextrin is methyl-β-cyclodextrin. In some embodiments, the concentration of cyclodextrin in the harvest solution is about 1 mM to about 20 mM. In some embodiments, the concentration of cyclodextrin in the harvest solution is about 10 mM.

[0019] In some embodiments, the solution of the cyclodextrin / lipid-modified protein complex is stored at 4° C. or below. In some embodiments, the solution of the cyclodextrin / lipid-modified protein complex is lyophilized. In some embodiments, the one or more pharma- ceutically acceptable additives include one or more preservatives. In some embodiments, the one or more pharma-ceutically acceptable additives include one or more antimicrobial agents. [Brief description of the drawings]

[0020] [Figure 1] The chemical and physical structure of cyclodextrin is shown. [Diagram 2] Schematic showing the effect of trehalose on stabilizing lipid films during dehydration. [Diagram 3] 1 shows the interplay between local and regional factors in morphogenesis and tissue growth. [Figure 4A] Schematic mechanism of membrane-bound lipid-modified protein capture using cyclodextrins. This figure shows that cyclodextrins in solution can capture lipid-modified proteins attached to cell membranes. [Figure 4B]Schematic mechanism of entrapment of membrane-bound lipid-modified proteins with cyclodextrins. The figure shows that cyclodextrin entrapment of the complexes can be further enhanced by the addition of trehalose, which has a kosmotropic effect by replacing the water surrounding the protein molecules, thus enabling the use of freeze-drying preservation methods. [Figure 5A] A smooth, compact, multi-layered cell culture is shown prior to exposure. [Figure 5B] Cultures are shown after incubation with cyclodextrin, where most of the cells have lost adhesion and the culture has broken apart. [Figure 6A] 1 shows that mice in treatment groups 1-4 demonstrate an early transition to anagen in the presence of new anagen spots, "anagen waves." This figure shows mice in treatment group 1. [Figure 6B] 1 shows that mice in treatment groups 1-4 demonstrate an early transition to anagen in the presence of new anagen spots, "anagen waves." This figure shows mice in treatment group 2. [Figure 6C] 1 shows that mice in treatment groups 1-4 demonstrate an early transition to anagen in the presence of new anagen spots, "anagen waves." This figure shows mice in treatment group 3. [Figure 6D] FIG. 1 shows that mice in treatment groups 1-4 demonstrate an early transition to anagen in the presence of new anagen spots, "anagen waves." This figure shows mice in treatment group 4. [Figure 7A] It is shown that treated animals showed a hair growth response regardless of the active ingredient concentration. [Figure 7B] 4 shows that treated animals exhibited an increased number of anagen patches. [Figure 7C] 4 shows that response to treatment was confirmed by increased skin darkness in the treatment group. [Figure 8A]The telogen state of hair follicles in treated mice is shown. Samples were taken on day 1 of the study to ensure that the mice were in the first telogen phase. Mice in the control group (Group 1, FIG. 8A) maintained a telogen (T) state of hair follicles on the final day of the study (Day 20) (FIGS. 8B and 8C). [Figure 8B] The telogen state of hair follicles in treated mice is shown. Samples were taken on day 1 of the study to ensure that the mice were in the first telogen phase. Mice in the control group (Group 1, FIG. 8A) maintained a telogen (T) state of hair follicles on the final day of the study (Day 20) (FIGS. 8B and 8C). [Figure 8C] The telogen state of hair follicles in treated mice is shown. Samples were taken on day 1 of the study to ensure that the mice were in the first telogen phase. Mice in the control group (Group 1, FIG. 8A) maintained a telogen (T) state of hair follicles on the final day of the study (Day 20) (FIGS. 8B and 8C). [Figure 9A] Figure 2 shows the telogen status of hair follicles in treated mice. Mice in the treatment group (Group 2) showed a mixture of telogen and early anagen (EA) hair follicles in areas that did not contain neoanagen patches. [Figure 9B] Figure 2 shows the telogen status of hair follicles in treated mice. Mice in the treatment group (Group 3) showed a mixture of telogen and early anagen (EA) hair follicles in areas that did not contain neoanagen patches. [Figure 9C] Figure 2 shows the telogen status of hair follicles in treated mice. Mice in the treatment group (Group 4) showed a mixture of telogen and early anagen (EA) hair follicles in areas that did not contain neoanagen patches. [Figure 10A] Hair growth in treated mice is shown. Mice in the treatment group (Group 2) exhibited neoanagen patches with typical anagen hair follicle morphology. [Figure 10B] Hair growth in treated mice is shown. Mice in the treatment group (Group 3) showed neoanagen patches with typical anagen hair follicle morphology. [Figure 10C] Hair growth in treated mice is shown. Mice in the treatment group (Group 4) showed neoanagen patches with typical anagen hair follicle morphology. [Figure 11A]Hair growth in two untreated mice is shown. In control animals, telogen persisted with minimal or no Wnt activity, as evidenced by very few LRG5 positive cells. [Figure 11B] Hair growth in two untreated mice is shown. In control animals, telogen persisted with minimal or no Wnt activity, as evidenced by very few LRG5 positive cells. [Figure 12A] Hair follicles in treated mice. This figure shows hair follicles in treatment group 2. This is a micrograph of a new hair follicle. Proliferating LGR5-positive bulge cells (B) migrate downward and gather in the new hair bulb matrix (MX). [Figure 12B] Hair follicles in treated mice. This figure shows hair follicles from treatment group 3. Photomicrographs of new anagen follicles. Proliferating LGR5-positive bulge cells (B) migrate downward and congregate into the early anagen hair bulb (EA) adjacent to the telogen (T) follicle. [Figure 12C] Hair follicles in treated mice. This figure shows hair follicles from treatment group 4. Photomicrograph of a new anagen follicle. The new hair follicle (EA) is underneath the old telogen blanket (T). LGR5 positive cells expand the bulge area (B) and congregate downwards into the new hair bulb matrix (MX). [Figure 13A] Hair growth in the treatment group is shown. Treated animals show hair stem cell mobilization by Sox9 positivity. This figure shows the early anagen phase. [Figure 13B] Hair growth in the treatment group is shown. Treated animals show hair stem cell mobilization by Sox9 positivity. This figure shows the anagen phase. [Figure 14A] FIG. 1 shows the effect of methyl-β-cyclodextrin (MBCD) at various concentrations in the absence of other growth factors on the in vitro growth of hair follicles after 2 days of culture. [Figure 14B] FIG. 1 shows the effect of methyl-β-cyclodextrin (MBCD) at various concentrations in the absence of other growth factors on the in vitro growth of hair follicles after 2 days of culture. [Figure 14C]FIG. 1 shows the effect of methyl-β-cyclodextrin (MBCD) at various concentrations in the absence of other growth factors on the in vitro growth of hair follicles after 2 days of culture. [Figure 14D] FIG. 1 shows the effect of methyl-β-cyclodextrin (MBCD) at various concentrations in the absence of other growth factors on the in vitro growth of hair follicles after 5 days of culture. [Figure 14E] FIG. 1 shows the effect of methyl-β-cyclodextrin (MBCD) at various concentrations in the absence of other growth factors on the in vitro growth of hair follicles after 5 days of culture. [Figure 14F] FIG. 1 shows the effect of methyl-β-cyclodextrin (MBCD) at various concentrations in the absence of other growth factors on the in vitro growth of hair follicles after 5 days of culture. [Figure 14G] FIG. 1 shows the effect of methyl-β-cyclodextrin (MBCD) at various concentrations in the absence of other growth factors on the in vitro growth of hair follicles after 5 days of culture. [Figure 15A] 1 shows that hair follicles grown with 0.25 mM MBCD complex exhibit enhanced length growth. [Figure 15B] 1 shows that hair follicles grown with 0.25 mM and 0.5 mM MBCD complexes exhibited enhanced thickness growth with statistical significance (p<0.01 for 0.25 mM group, p<0.05 for 0.5 mM group). [Figure 16A] 1 shows the testing of a composition containing embryonic stem cell membrane components and MBCD encapsulated with trehalose on human skin. The figure shows an untreated area. [Figure 16B] 1 shows a test of a composition containing MBCD encapsulated with embryonic stem cell membrane components and trehalose in human skin, showing a treated contralateral area with visibly restored vellus hair, long vellus hair, and reduced age spots. [Figure 16C] 1 shows the testing of a composition containing embryonic stem cell membrane components and MBCD encapsulated with trehalose on human skin. The figure shows an untreated area. [Figure 16D]1 shows a test of a composition containing MBCD encapsulated with embryonic stem cell membrane components and trehalose in human skin, showing a treated contralateral area with visibly restored vellus hair, long vellus hair, and reduced age spots. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Although Wingless (Wnt) and Hedgehog (Hh) proteins have been isolated and characterized, the applications of these factors are quite limited: the sources of these proteins are commonly cells engineered to overexpress a single specific protein, which is removed from the cell surface by mild detergents.

[0022] This specification discloses a complex of lipid-modified proteins derived from stem cells with cyclodextrin. As used herein, the term "lipid-modified" refers to a protein having a lipid covalently attached thereto. For Wnt and Hh, these proteins are modified with the fatty acid palmitic acid, although modifications with other lipids, including cholesterol, are also within the scope of the disclosed compositions and methods.

[0023] Stem cells undergoing the transition from pluripotency to the germ layer stage express significant amounts of Wnd and Hh proteins. However, Wnd and Hh proteins cannot be found in cell culture supernatants, although the culture supernatants contain various soluble growth factors. Physiological expression of Wnt and Hh leads to lipid modification, so that they associate with the surface membrane of expressing cells rather than being secreted into the extracellular fluid. Exposure of stem cells to cyclodextrin solutions successfully allows extraction of lipid-modified Wnt and Hh proteins bound to cholesterol-containing cell membranes, thus forming soluble complexes of lipid-modified proteins bound to cyclodextrin. Addition of trehalose to the soluble lipid-modified protein / cyclodextrin complexes stabilizes the complexes, allowing long-term storage of the lyophilized complexes.

[0024] Wnt and Hh proteins are effective in cell proliferation, hair growth, and tissue regeneration in vitro and in vivo. Because a combination of various factors is required for proper stem cell function and therapeutic effect, the use of heterogeneous Wnt (e.g., Wnt3a, Wnt7b, Wnt10b, etc.) and Hh mixtures derived from defined normal stem cell cultures is advantageous over single factors derived from modified cells engineered to express specific proteins.

[0025] Thus, the present specification discloses a method for the capture of membrane-associated lipid-modified proteins from stem cells, including embryonic stem cells, induced pluripotent stem cells, and adult stem cells. Examples of lipid-modified protein structures include, but are not limited to, proteins (ligands) of the Wnt and Hh families. The cells are engineered to maximize the expression of these proteins and then exposed to cyclodextrin, which is known for its ability to capture hydrophobic molecules. The cyclodextrin complex is further coated with trehalose to provide protection against drying and protein denaturation.

[0026] Prior to this disclosure, these lipid-modified proteins were extracted from cells with organic solvents or detergents. These methods have the drawback of providing limited stability and functionality to the extracted proteins. Organic solvents can denature protein components and remove lipid modifications essential for protein activity. Detergent extraction produces micelles of lipoproteins, which can further be encapsulated within liposomes. Although detergent extraction is superior to solvent extraction, the aforementioned micelles and liposomes are unstable structures with limited shelf life.

[0027] The method described herein ensures capture of lipid-modified proteins and opens the possibility of long-term storage by lyophilization. The cyclodextrin / lipid-modified protein complexes are further preserved using trehalose, a kosmotropic agent that replaces the water surrounding the protein and lipids and ensures structural preservation.

[0028] Lipid-modified protein / cyclodextrin complexes isolated from stem cell cultures are useful in tissue repair, wound repair and regeneration, skin revitalization, hair growth, and cosmetics.

[0029] (Cyclodextrin) There are three naturally occurring cyclodextrins: cyclodextrin-α, cyclodextrin-β, and cyclodextrin-γ. Cyclodextrins form stable aqueous complexes with many other chemicals. A typical cyclodextrin contains 6-8 glucopyranoside units and can be topologically represented as a ring with a large opening exposing the secondary hydroxyl groups to the solvent and a smaller opening exposing the primary hydroxyl groups to the solvent. This arrangement makes the interior of the ring, while not hydrophobic, significantly less hydrophilic than the aqueous environment and therefore capable of accommodating other hydrophobic molecules. Meanwhile, its exterior is sufficiently hydrophilic to confer water solubility to the cyclodextrin (or their complexes) (Figure 1).

[0030] Since the formation of the inclusion complex significantly modifies the physical and chemical properties (mainly water solubility) of the guest molecule, inclusion complexes of hydrophobic molecules with cyclodextrins have the ability to penetrate through biological tissues and release the bioactive hydrophobic compounds under certain conditions (including but not limited to pH change, heat, enzymes capable of cleaving the α-1,4 bond between glucose monomers, or substitution with other hydrophobic molecules (e.g., cholesterol)).

[0031] Depending on the number of glucose rings in the molecule, cyclodextrins are classified as α (alpha)-cyclodextrin (a 6-saccharide ring molecule), β (beta)-cyclodextrin (a 7-saccharide ring molecule), or γ (gamma)-cyclodextrin (an 8-saccharide ring molecule). Cyclodextrins are hydrophobic on the inside and hydrophilic on the outside, allowing them to form complexes with hydrophobic compounds. Thus, cyclodextrins can increase the solubility and bioavailability of these compounds. This is of great interest in pharmaceutical applications as well as food supplement applications where hydrophobic compounds are delivered. α-, β-, and γ-cyclodextrins are all generally rated as safe by the FDA.

[0032] Chemical modifications of naturally occurring cyclodextrins can be designed to enhance solubility, accommodate certain hydrophobic molecules, provide termini available for conjugation to other molecules, and provide specific functions (binding to specific cellular components, self-assembly into macromolecular structures, etc.) Common modifications include random methylation and hydroxypropylation.

[0033] Both β-cyclodextrin and methyl-β-cyclodextrin (MBCD) remove cholesterol from cultured cells. The methylated form (MBCD) is more efficient than β-cyclodextrin in removing cholesterol from cultured cells. Water-soluble MBCD forms soluble inclusion complexes with cholesterol, which increases the solubility of cholesterol in aqueous solutions. MBCD is employed to formulate cholesterol-free products, as the bulky and hydrophobic cholesterol molecules easily fit inside the cyclodextrin rings, which are then removed. MBCD is also employed in research to disrupt lipid rafts by removing cholesterol from membranes.

[0034] (Cosmotrope) Kosmotropes favor the interaction of water molecules, thereby (in effect) stabilizing the intramolecular interactions of macromolecules such as proteins. Examples of kosmotropes include, but are not limited to, propylene glycol, proline, trehalose, ectoine, and trimethylamine-N-oxide. Trehalose (mycose, tremalose) is a disaccharide consisting of two glucose molecules.

[0035] In fungi and bacteria, the main biological purpose of trehalose is water regulation, since it forms a gel phase during intracellular dehydration, protecting intracellular organelles during this period and allowing rapid rehydration upon return to the appropriate environment thereafter. In humans, trehalose performs a hydration function in addition to general antioxidant properties, but its main role is as a cellular chaperone regulating intracellular functions such as protein folding and unfolding.

[0036] [ka]

[0037] Trehalose is classified as a kosmotrope or water structure marker, i.e., trehalose / water interactions are much stronger than water / water interactions and may be involved in the bioprotective effects of trehalose.

[0038] Trehalose can prevent protein aggregation, thus acting as a stabilizer and improving the shelf life of therapeutic proteins. Studies with model proteins have shown that trehalose has the ability to stop moisture-induced aggregation of bovine serum albumin by interfering with the formation of intramolecular disulfide bonds. Trehalose is effective in stabilizing lipid membranes and protecting them against dehydration. Without trehalose, lipid bilayers would undergo a liquid crystalline to gel transition during dehydration, leading to premature damage to the bilayer structure. By displacing water, trehalose occupies the space between the lipids and maintains an organized liquid crystalline structure upon rehydration (Figure 2).

[0039] (Hedgehog (Hh) family and Wingless (Wnt) family) Mammals have three Hedgehog homologues, Desert (Dhh), Indian (Ihh), and Sonic (Shh) Hedgehog, of which Sonic is the best studied. This signaling pathway has been studied in knockout mice, showing cell specificity in the brain, skeletal, and muscle tissues, gastrointestinal tract, lung, and heart. Recent studies point to a role for Hedgehog signaling in regulating adult stem cells involved in the maintenance and regeneration of adult tissues. This pathway is also involved in the development of several cancers. Drugs that specifically target Hedgehog signaling to combat cancer are actively being developed.

[0040] The addition of lipophilic groups is a widespread modification that occurs on approximately 1000 proteins with diverse structures and functions (Table 1). At least five different types of lipids can be covalently attached to proteins. These include, but are not limited to, fatty acids, isoprenoids, sterols, phospholipids, and glycosylphosphatidylinositol (GPI) anchors. Proteins may contain more than one type of lipid, for example, myristate + palmitate, palmitate + cholesterol, or farnesyl + palmitate. The most common effect of lipid modification is an increase in affinity for membranes.

[0041] [Table 1]

[0042] The Hh protein is produced as a precursor molecule, with a C-terminal protease domain and an N-terminal signaling unit, and undergoes a number of unique modifications during its synthesis. The N-terminus of Hh is modified with the fatty acid palmitate on a conserved cysteine ​​residue that is exposed at the very N-terminus of the protein after its signal sequence is removed. The palmitoyl group is attached to the NH 2 The bond is to the group via an amide.

[0043] Wnt molecules are palmitoylated and therefore much more hydrophobic than would be predicted from their primary amino acid sequence. The amino acid in Wnt proteins that appears to be modified is the first conserved cysteine ​​(C77), a residue that is present in all Wnts and that, according to mutational analysis, is essential for Wnt function.

[0044] Due to lipid modifications that confer hydrophobicity, Hh and Wnt cannot be distributed throughout the body: these proteins are membrane bound and can only be transmitted from cell to cell between cells in direct contact, whereas soluble factors (FGF, EGF, etc.) are distributed throughout the body and can act on both local and distant cells.

[0045] The cells of origin (stem cells) expressing the Engrailed (En) transcription factor secrete Hh, and only cells adjacent to En-expressing cells can respond to Hedgehog following interaction of Hh with the receptor protein Patched (Ptc).

[0046] Cells in which Ptc is activated by Hh synthesize Wnt proteins. Wnt lipid-modified proteins act as intercellular signals to pattern adjacent rows of cells by activating their cell surface receptor, Frizzled. Thus, the actions of Wnt and Hh on adjacent cells define a positional code that describes distinct anatomical features, whereas soluble factors define a temporal code for cell proliferation and tissue growth (Figure 3).

[0047] Hair follicles are thought to be organelles formed by the interaction of neuroectoderm and mesoderm. Hair follicle neogenesis occurs in the embryo by invagination of the epithelial placode into the surrounding dermis. Postnatal hair follicles undergo a three-phase renewal cycle: anagen, catagen, and telogen. The first complete postnatal hair follicle cycle (first anagen, first catagen, first telogen) is completed within the first 3.5 weeks after birth, followed by the next hair cycle (second anagen, second catagen, second telogen).

[0048] In skin, the formation of hair follicles from the developing epidermis requires signals from fibroblasts in the underlying dermis. Hair follicle morphogenesis occurs during the late embryonic and early neonatal periods. In adult skin, new hair follicles do not normally arise.

[0049] Hair follicle neogenesis can be induced in adult mouse skin upon gene transfer of Wnt / β-catenin or upon activation of Wnt / β-catenin in the epidermis by wounding. Inhibition of Wnt signaling by DKK1 (Dickkopf-related protein 1) indicates the functional importance of Wnt signaling in hair follicle development. Several Wnt molecules are expressed in hair follicles and may be involved in this function. Wnt3a and Wnt7a are expressed in hair follicle matrix cells and are responsible for the maintenance of dermal papilla cells in the anagen phase. These cells may be capable of responding to Wnt, as they express components of the Wnt signaling cascade, including frizzled7, disheveled2, GSK3β, β-catenin, and Lef1. Thus, the Wnt pathway is thought to be a key regulator during hair follicle morphogenesis. Wnt signaling proceeds via EDA / EDAR / NF-κB (ectodysplasin A / ectodysplasin A receptor / nuclear factor kappa light-chain enhancer of activated B cells) signaling. NF-κB acts as a signal mediator that regulates the Wnt pathway and upregulates Shh expression. Dermal Shh and platelet-derived growth factor (PDGF) signaling upregulate dermal noggin expression. Noggin is a potent inhibitor of bone morphogenetic protein (BMP) signaling and helps to counteract BMP-mediated β-catenin inhibition. This interplay of signaling between epidermal and dermal lineages helps in epidermal Shh signal amplification.

[0050] The involvement of Shh in hair development was suggested by manipulation of Shh expression during and across embryonic development. During normal hair follicle development, Shh is expressed within the hair follicle within the epithelial placode, and its receptor Ptc is detected in the underlying mesenchymal condensations during early embryonic stages.

[0051] In vivo experiments suggest that Shh stimulates the telogen-to-anagen transition, possibly in concert with other local factors. Transient expression of Shh could reactivate the hair growth cycle in disease conditions.

[0052] In mammals, despite a considerable tissue regeneration capacity, after large wounds tissue morphology and function are not fully restored, leading to the formation of scar tissue. This limited regeneration capacity is due in part to the rapid intervention of fibrous tissue, something that impedes subsequent tissue regeneration, but may have a defensive advantage in inhibiting harmful microorganisms. Upon injury, only bones, liver, and neonatal fingertips are capable of regenerating. Age is another determinant of tissue repair, with animals gradually losing their regeneration capacity as they age.

[0053] Repaired skin usually heals as a scar, but is weaker than intact skin and has a more disorganized extracellular matrix (ECM) than unwounded skin. Skin wounds do not normally regenerate hair follicles. As a result, postnatal mammalian skin repair is not the same as the regenerative process in early gestational fetal wounds, where the regenerated tissue is nearly indistinguishable from unwounded tissue.

[0054] Although Wnt signaling is not essential for maintaining elevated β-catenin levels during the proliferative phase of skin healing, Wnt proteins may participate in stimulating epidermal β-catenin during repair.Similar to their function in skin development, Wnt and / or β-catenin signaling play important roles in various aspects of cutaneous wound repair and are involved in the organization of epithelial architecture and remodeling of the epidermal compartment.

[0055] The relative levels and activity of β-catenin contribute to the epidermal wound phenotype, with high β-catenin levels and activity resulting in an expanded hyperplastic epidermal compartment and therefore contributing to hypertrophic scar formation. β-catenin levels are Wnt-dependent and may also be influenced by transforming growth factor β (TGFβ) levels.

[0056] Wnt signaling regulates cell proliferation in the epidermis, which directly affects the speed and extent of skin wound healing.Wnt also serves as a niche signal for at least two types of skin stem cells, the skin stem cells in the bulge region of hair follicles and the skin stem cells in the basal layer of interfollicular epidermis, which contribute to skin wound repair.Local application of liposomal Wnt3a to non-healing wounds supplements endogenous Wnt signaling, leading to improved skin wound healing.

[0057] Hedgehog signaling has been shown to directly contribute to normal and accelerated wound healing in mice. When Hh signaling is inhibited, wound healing is severely impaired in all respects (wound closure, epithelialization, granulation, vascularity, and proliferation).

[0058] In skin, the antennal disc develops in parallel with early hair follicles and contains sensory Merkel cells. Epidermal Wnt signaling and subsequent epidermal Eda / Edar (ectodysplasin / ectodysplasin receptor) signaling promote Merkel cell morphogenesis by inducing Shh expression in early hair follicles. Although developmentally associated with hair follicles, fate mapping has shown that Merkel cells have a predominant origin outside the hair follicle lineage. These findings suggest that antennal disc development requires Wnt-dependent mesenchymal signals to establish reciprocal signaling within the developing ectodermal Shh signaling from early hair follicles to extrafollicular Merkel cell precursors. Locally upregulated Shh acting as a morphogen is essential for lineage specification during development and postnatal antennal disc stem cell maintenance.

[0059] In amphibians, Hh signaling and its hierarchical correlation with Wnt signaling control limb regeneration. Wnt signaling has been shown to promote self-renewal in both intestinal epithelium and hematopoietic stem cells (HSCs). Stem cells of many tissues are responsive to Wnt (Table 2).

[0060] [Table 2]

[0061] Along with lipid-modified proteins bound to cell membranes, lipids are also involved in cell signaling. These lipids include, but are not limited to, sphingolipid-based lipids (e.g., ceramide, sphingosine, sphingosine-1-phosphate, glucosylceramide, ceramide-1-phosphate, phosphatidylinositol bisphosphate (PIP 2 )), lipid agonists, phosphatidylinositol-based lipids (e.g., phosphatidylinositol bisphosphate (PIP 2 )), activators of G protein-coupled receptors (e.g., lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), platelet activating factor (PAF), endocannabinoids, prostaglandins, FAHFAs, retinol derivatives), and activators of nuclear receptors (e.g., steroid hormones, retinoic acid, prostaglandins).

[0062] (composition) Thus, the present specification discloses a composition comprising a lipid-modified Hedgehog (Hh) and / or Wingless (Wnt) protein and at least one cyclodextrin. The lipid-modified Hh and / or Wnt protein is isolated from a human stem cell as described herein. In certain embodiments, the stem cell is a pluripotent stem cell, a multipotent stem cell, a monolineage dividing progenitor cell, or an immortalized cell lineage.

[0063] In certain embodiments, the source cells for Wnt and Hh proteins are human embryonic stem cells, parthenogenetic stem cells, or induced pluripotent stem cells. Other cells of interest include any in vitro grown cells that have been identified as fetal or adult stem cells or harvested from fetal appendages. Other cells can also be modified by genetic engineering to confer immortality through cell cycle deregulation (e.g., telomerase expression). In certain embodiments, stem cells are immortalized by genetic engineering expression of telomere reverse transcriptase (hTERT). Source cells can be cultured using established methods and cell culture media known to those skilled in the art.

[0064] In some embodiments, the Hh or Wnt protein is harvested by first discarding the medium, rinsing the culture with an isotonic buffer (e.g., saline, Hank's balanced salt solution, etc.), and then mixing the cells with the harvest solution with slow continuous or intermittent agitation for about 1 hour to about 5 hours. Some of the cells may lose their attachment to the substrate. The harvest solution containing the soluble lipid-modified protein / cyclodextrin complexes is further filtered (e.g., 0.1-0.5 μm pass) to remove debris and stored at 4° C.

[0065] A suitable harvest solution for obtaining lipid-modified Wnt and Hh proteins from stem cells comprises an isotonic solution containing 1-20 mM cyclodextrin. In some embodiments, the concentration of cyclodextrin is about 1-5 mM, about 5-10 mM, about 10-15 mM, about 15-20 mM, about 2-10 mM, about 5-20 mM, about 8-20 mM, about 12-20 mM, about 8-12 mM, about 5 mM, about 7 mM, about 9 mM, about 10 mM, about 11 mM, about 13 mM, or about 15 mM. The volume of the harvest solution is about 0.1-1.0 mL / cm per dish or flask. 2 In some embodiments, the volume of the harvest solution is about 0.25 mL / cm per dish or flask. 2 It is.

[0066] The harvest solution is incubated with the source cells with slow continuous or intermittent mixing for about 1 hour to about 5 hours, hi some embodiments, the harvest solution is incubated with the cells for about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours.

[0067] In some embodiments, the cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin. In some embodiments, natural cyclodextrin is chemically modified by hydrogenation, hydroformylation, oxidation, reduction, and carbon-carbon coupling reactions. Such well-known modifications include 2-hydroxypropyl β-cyclodextrin and methyl-β-cyclodextrin. In some embodiments, the cyclodextrin is methyl-β-cyclodextrin (MBCD).

[0068] In certain embodiments, the harvest solution further contains a kosmotrope. One method for preparing the harvest solution includes adding a kosmotropic agent to replace the water surrounding the protein molecules. An example of a kosmotropic agent includes trehalose, which is added to the cyclodextrin complex solution at a final concentration between about 5% and about 30%. In another embodiment, the kosmotrope concentration is about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 15% to about 25%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%, or any concentration between these values. In one embodiment, the trehalose concentration is 20% w / v added immediately after harvest.

[0069] In some embodiments, the harvest solution comprises an aqueous solution of 10 mM cyclodextrin and 20% trehalose.

[0070] The cyclodextrin complexes may further be stored lyophilized using low temperature methods (eg, freeze-drying).

[0071] Detection of Wnt and Hh proteins in the compositions of the present disclosure can be performed using commercially available quantitative ELISA detection kits.

[0072] One embodiment of a lipid-modified protein / cyclodextrin complex disclosed herein is illustrated in Figures 4A and 4B.

[0073] Use of cyclodextrin Wnt / Hh complexes can be formulated for in vivo applications with attention to the following principles: (1) Preparation and storage temperatures should be kept below 40°C. (2) The final product should not contain hydrophobic molecules that may displace the cyclodextrin payload (i.e., lipid-modified proteins). (3) The final product may contain antimicrobial stabilizers or preservatives (such as, but not limited to, silver (Ag) nano- or microparticles, phenoxyethanol, caprylyl glycol, pentylene glycol, etc.) or other agents used for topical application.

[0074] In an exemplary embodiment, the composition containing the Wnt / Hh-cyclodextrin complex is an aqueous solution, and optionally further contains silver particles. The composition may further include one or more of amino acids, peptides, proteins, water-soluble vitamins, and trace elements. Examples of the components of the composition include, but are not limited to, water-soluble growth factors, steroid hormones, and analogs thereof. Examples of water-soluble growth factors include, but are not limited to, fibroblast growth factor (FGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), stem cell growth factor (HGF), and the like.

[0075] The silver particles (nanoparticles or microparticles) may be present in a concentration of 0.01-1.0% w / v, hi some embodiments, the silver particles are present in a concentration of 0.1-0.55 w / v.

[0076] According to some embodiments, topical compositions or formulations prepared according to the present disclosure may take the form of a liquid, paste, cream, lotion, powder, ointment, or gel composition.

[0077] According to some embodiments, the composition form is a paste, which refers to a semi-solid dosage form containing one or more substances intended for topical application.

[0078] According to some embodiments, the composition form is a cream. As used herein, the term "cream" refers to a semi-solid emulsion or viscous liquid, either oil-in-water or water-in-oil. As used herein, the term "emulsion" refers to a colloidal system in which both the dispersed phase and the dispersion medium are immiscible liquids, and the dispersed liquid is distributed throughout the dispersion medium liquid in the form of small granules. A stable elementary emulsion contains at least two liquids and an emulsifier. Common types of emulsions are oil-in-water, where oil is the dispersed liquid and an aqueous solution (such as water) is the dispersion medium, and conversely, water-in-oil, where an aqueous solution is the dispersed phase. It is also possible to prepare non-aqueous emulsions. Oil-in-water creams include hand creams and foundation creams. Water-in-oil creams include cold creams and emollient creams.

[0079] According to some embodiments, the composition form is a lotion, which refers to a liquid or semi-liquid preparation containing one or more active ingredients in a suitable vehicle. A lotion may be a suspension of solids in an aqueous medium, an emulsion, or a solution.

[0080] A "solution" is generally considered to be a homogeneous mixture of two or more substances. A solution is often, but not necessarily, a liquid. In a solution, the molecules of the solute (or the dissolved substance) are evenly distributed among the molecules of the solvent. Solvents that may be useful in the compositions of the present disclosure include water, as well as organic solvents such as alcohols.

[0081] According to some embodiments, the composition form is an ointment. An ointment is a semi-solid preparation, often intended for external application to the skin. In general, ointment bases are divided into carbohydrate-based (oil-based), adsorption-based (anhydrous), emulsion-based (water-oil type), and water-soluble-based. Due to their anhydrous nature, ointments generally do not require preservatives. Ointments have higher water retention and occlusion properties than creams, forming a protective film on the skin. The occlusive effect tends to enhance penetration and prolong its duration.

[0082] According to some embodiments, the composition form of the composition is a gel. As used herein, the term "gel" refers to a viscous, jelly-like semi-solid or solid prepared from high molecular weight polymers in an aqueous or alcoholic base.

[0083] Additional composition forms can be prepared using techniques well known in the pharmaceutical art.For example, such techniques are described in "Remington: The Science and Practice of Pharmacy", 20th edition, edited by Gennaro AR et al., published by Lippincott Williams & Wilkins, Philadelphia, 2000.The contents of said publications are incorporated by reference.

[0084] Numerous additional ingredients can be added to the compositions disclosed herein for functional, aesthetic, and marketing purposes, including emulsifiers, preservatives, humectants, thickeners, fragrances, dyes, herbal extracts, and vitamins, provided that the additional ingredients selected are chemically and physically compatible. As used herein, the term "compatible" means that the ingredients of the composition are capable of being mixed with one another under normal conditions of use without any interactions that would substantially diminish the effectiveness of the composition.

[0085] According to some embodiments, the composition comprises a polysorbate, such as polysorbate-20, polysorbate-40, polysorbate-80, and mixtures thereof.

[0086] As used herein, the term "carrier" refers to a pharma- ceutically acceptable inactive agent or vehicle for delivering one or more active agents to a subject, and is often referred to as an "additive." The carrier must have a high enough purity and low enough toxicity to be suitable for administration to a subject to be treated. The carrier should also maintain the stability and bioavailability of the lipid-modified protein / cyclodextrin complex disclosed herein. The carrier may be liquid or solid, and is selected so that when combined with the active agent and other components of a composition, it provides the desired bulk, firmness, etc., in the planned manner of the intended administration.

[0087] According to some embodiments, the described compositions include an aqueous carrier. The level and type of carrier is selected depending on compatibility with other ingredients and other desired properties of the product. The aqueous carrier is present in the composition at a level of, for example, about 30 to about 98%, about 50 to about 95%, or about 70 to about 95% by weight.

[0088] Examples of carriers include water and aqueous solutions of lower alkyl alcohols. Examples of lower alkyl alcohols include monohydric alcohols having 1 to 6 carbons, such as ethanol. According to some embodiments, the aqueous carrier is substantially water.

[0089] The pH of the composition of the present disclosure is, for example, about 4 to about 8. When a skin benefit agent is included in the composition, the pH may be adjusted to a pH that provides optimal effect. To obtain a desired pH, buffering agents and pH adjusting agents may be included in the composition. Examples of pH adjusting agents herein include acetate, phosphate, citrate, triethanolamine, and carbonate.

[0090] The viscosity (resistance to flow) of the compositions of the present disclosure may vary over a wide range and may depend on the thickener. For example, according to some embodiments, the compositions of the present disclosure may include a thickener that provides the composition with a viscosity of about 500 mPas to about 1,000,000 Pas. According to some embodiments, the thickener provides the composition with a viscosity of about 1,000 mPas to about 100,000 mPas.

[0091] Non-limiting examples of thickeners that result in microemulsions include carboxylic acid / carboxylate copolymers. These copolymers can maintain the composition at the appropriate viscosity during use without being sticky or oily, and can also disperse and stabilize water-insoluble ingredients when included in the composition. Examples of commercially available carboxylic acid / carboxylate copolymers include Acrylate / C 10-30 Alkyl acrylate copolymers include, for example, PEMULEN® TR-I, PEMULEN® TR-2, CARBOPOL® 1342, CARBOPOL® 1382, and CARBOPOL® ETD 2020 (all available from BF Goodrich Company).

[0092] To neutralize the carboxylic acid / carboxylate copolymer, a neutralizing agent such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, monoethanolamine, diethanolamine, triethanolamine, diisopropanolamine, aminomethylpropanol, tromethamine, tetrahydroxypropylethylenediamine, and mixtures thereof may be included in the composition.

[0093] Examples of cellulosic polymers include, but are not limited to, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxyethylethylcellulose, hydroxypropylmethylcellulose, nitrocellulose, sodium cellulose sulfate, sodium carboxymethylcellulose, microcrystalline cellulose, cellulose powder, and mixtures thereof. According to some embodiments, the cellulosic polymer is hydroxyethylcellulose, carboxymethylcellulose, and mixtures thereof. Commercially available compounds that are particularly useful herein include hydroxyethylcellulose under the brand name Natrosol Hydroxyethylcellulose and carboxymethylcellulose under the brand name Aqualon Cellulose Gum (both available from Aqualon).

[0094] Other examples of thickening agents include pullulan, mannan, scleroglucan, polyvinylpyrrolidone, polyvinyl alcohol, guar gum, hydroxypropyl guar gum, xanthan gum, acacia gum, gum arabic, tragacanth, galactan, carob gum, karaya gum, locust bean gum, carrageenan, pectin, amylopectin, agar, quince seed (Cydonia oblonga Mill), starch (rice, corn, potato, wheat), algae colloids (algae extract), etc. Examples of microbiological polymers include, but are not limited to, dextran, succinoglucan, starch-based polymers (such as carboxymethyl starch and methylhydroxypropyl starch), etc. Examples of alginic acid-based polymers include, but are not limited to, sodium alginate and propylene glycol alginate. Examples of acrylate polymers include, but are not limited to, sodium polyacrylate, polyacrylamide, and polyethyleneimine. Examples of inorganic water-soluble materials include, but are not limited to, bentonite, magnesium aluminum silicate, laponite, hectonite, and silicic anhydride.

[0095] Exemplary thickening agents also include polyalkylene glycols having a molecular weight greater than about 1000. Exemplary compounds include polyethylene oxide, polyoxyethylene, and polyethylene glycol, polypropylene oxide, polyoxypropylene, and polypropylene glycol; and polypropylene glycol and mixed polyethylene-polypropylene glycol or polyoxyethylene-polyoxypropylene copolymers. Examples of polyethylene glycol polymers include, but are not limited to, PEG-2M (also known as POLYOX WSR® N-10, available from Union Carbide, also available as PEG-2,000), PEG-5M (also known as POLYOX WSR® N-35 and POLYOX WSR® N-80, both available from Union Carbide, also available as PEG-5,000 and Polyethylene Glycol 300,000), PEG-7M (also known as POLYOX WSR® N-750, available from Union Carbide), PEG-9M (also known as POLYOX WSR® N-3333, available from Union Carbide), and PEG-14M (also known as POLYOX WSR® N-3000, available from Union Carbide).

[0096] Examples of commercially available additional water soluble polymers include, but are not limited to, xanthan gum (KELTROL™, available from Kelco), carbomers (CARBOPOL™ 934, CARBOPOL™ 940, CARBOPOL™ 950, CARBOPOL™ 980, and CARBOPOL™ 981, all available from BF Goodrich Co.), (acrylates / steareth-20 methacrylate) copolymers, and the like. Examples of suitable polyacrylamides include glyceryl polymethacrylate (ACRYSOL™ 22, available from Rohm and Haas), polyacrylamide (SEPIGEL™ 305, available from Seppic), glyceryl polymethacrylate (LUBRAGEL™ NP) and a mixture of glyceryl polymethacrylate, propylene glycol and PVM / MA copolymer (LUBRAGEL™ OIL, available from ISP), scleroglucan (CLEAROGEL™ SCI I, available from Michel Mercier Products Inc., New Jersey, USA), ethylene oxide and / or propylene oxide based polymers (CARBOWAX™ PEG, POLYOX™ WASR, and UCON™ FLUIDS, all available from Amerchol).

[0097] Other exemplary agents include commercially available amphoteric polymers such as Polyquaternium 22 (MERQUAT® 280, MERQUAT® 295), Polyquaternium 39 (MERQUAT® PLUS 3330, MERQUAT® PLUS 3331), and Polyquaternium 47 (MERQUAT® 2001, MERQUAT® 200IN), all available from Calgon Corporation.

[0098] As used herein, the term "humectant" refers to a substance that promotes moisture retention through its hygroscopic properties. Humectants function by being absorbed into the skin, attracting moisture from the atmosphere, which then serves as a reservoir for the stratum corneum.

[0099] Examples of water-soluble humectants include, but are not limited to, polyhydric alcohols (such as butylene glycol (1,3-butanediol), pentylene glycol (1,2-pentanediol), glycerin, sorbitol, propylene glycol, hexylene glycol, ethoxylated glucose, 1,2-hexanediol, 1,2-pentanediol, hexanetriol, dipropylene glycol, erythritol, trehalose, diglycerin, xylitol, maltitol, maltose, glucose, fructose, and the like), other water-soluble compounds (such as urea, sodium chondroitin sulfate, sodium hyaluronate, sodium adenosine phosphate, sodium lactate, pyrrolidone carboxylic acid, glucosamine, cyclodextrin, and the like), and mixtures thereof. Additional examples include water soluble alkoxylated nonionic polymers such as polyethylene glycols and polypropylene glycols of molecular weight up to about 1000 (e.g., PEG-200, PEG-400, PEG-600, PEG-1000) and mixtures thereof.

[0100] Examples of commercially available humectants include, but are not limited to, butylene glycol (1,3-butylene glycol available from Celanese), pentylene glycol (HYDROLITE®-5 available from Dragoco), glycerin (STAR® and SUPEROL® available from Procter & Gamble, CRODEROL® GA7000 available from Croda Universal Ltd., PRECERIN® series available from Unichema, and NOF products sold under the same trade name as the chemical name), propylene glycol (LEXOL® PG-865 / 855 available from Inolex, 1,2-Propylene glycol USP), sorbitol (LIPONIC series available from Lipo, SORBO, ALEX, A-625, and A-641 available from ICI, and UNISWEET 70, UNISWEET CONC available from UPI), dipropylene glycol (available under the same trade name as the chemical from BASF), diglycerin (DIGLYCEROL available from Solvay GmbH), xylitol (available under the same trade name as the chemical from Kyowa and Eisai), maltitol (MALBIT available from Hayashibara), sodium chondroitin sulfate (available under the same trade name as the chemical from Freeman and Bioiberica, and ATOMERGIC SODIUM CHONDROITIN SULFATE (available from Atomergic Chemetals), sodium hyaluronate (available under the same trade name as the chemical name from Chisso, ACTIMOIST (available under the trade name as the chemical name from Active Organics, AVIAN SODIUM HYALURONATE series available from Intergen, HYALURONIC ACID Na available from Ichimaru Pharcos), sodium adenosine phosphate (available under the trade name as the chemical name from Asahi Kasei, Kyowa, and Daiichi Pharmaceutical), sodium lactate (available under the trade name as the chemical name from Merck, Wako, and Showa Kako), cyclodextrin (American Mize), Examples of suitable oils include CAVITRON™ available from Maize, RHODOCAP™ series available from Rhone-Poulenc, and DEXPEARL™ available from Tomen, polyethylene glycols (CARBOWAX™ series available from Union Carbide), and mixtures of glyceryl polymethacrylate, propylene glycol, and PVM / MA copolymers (LUBRAJEL™ Oil available from Guardian Labs).

[0101] As used herein, the term "preservative" refers to a substance that prevents or inhibits the growth of undesirable microorganisms in a product containing moisture. Preservatives approved for use in pharmaceutical preparations, such as topical preparations, can be found in the current Federal Regulations published in Title 21 of the Code of Federal Regulations, the contents of which are incorporated herein by reference. Examples of preservatives include, but are not limited to, ascorbic acid, ascorbyl palmitate, biopein, BHT (butylated hydroxytoluene), butylated hydroxyanisole, butylated hydroxytoluene, b-butylparaben, calcium ascorbate, calcium sorbate, citric acid, cinnamon cassia, chlorocresol, diazolidinyl urea, dilauryl thiodipropionate, EDTA (ethylenediaminetetraacetic acid tetrasodium salt), erythorbic acid, grapefruit seed extract, hydroxyphenyl trimethicone, benzoic acid ... hydroxyhenzoate, methylparaben, Neopein, phenonip, phenoxyethanol, potassium bisulfite, potassium metabisulfite, potassium sorbate, propylparaben, rosemary oil extract, sodium ascorbate, sodium benzoate, sodium bisulfite, sodium metabisulfite, sodium sorbate, sodium sulfite, sorbic acid, sulfur dioxide, Suprarein, thiodipropionic acid, silver particles, and / or tocopherol. Additionally, preservation may be accomplished by storage at reduced temperatures (e.g., below 4°C or frozen).

[0102] In some embodiments, the compositions are applied topically once a day, twice a day, three times a day, every other day, weekly, or any period necessary to achieve the desired results. Typically, the compositions are applied topically to the desired treatment area and allowed to be absorbed by the skin.

[0103] (therapeutic use) Therapeutic applications of cyclodextrin / lipid-modified protein complex-containing compositions include, but are not limited to, hair growth or regrowth purposes, in vitro expansion of hair follicle stem cells for the generation of hair follicles that can be transplanted into an autologous recipient, wound healing, restoring touch, improving the appearance of skin, and tissue reactivation.

[0104] The tissue targeted by the cyclodextrin complex should contain receptors for the payload protein (eg, Frizzled, Patched, etc.) and exchange hydrophobic molecules (eg, cholesterol from cell membranes or lipid secretions such as sebum).

[0105] The compositions and formulations disclosed herein may be administered topically. The cyclodextrin complex-containing composition may be administered topically in a dosage that contains about 0.1% to about 100% of the collected solution. In certain embodiments, the composition is administered topically in a dosage that is about 5% to about 25% (v / w, v / v, or w / v), about 10% to about 25%, about 15% to about 25%, about 20% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%, about 17.5%, about 20%, about 22.5%, or about 25%, or any range between these values.

[0106] Doses and desired drug concentrations of the compositions of the present disclosure may vary depending on the particular application envisioned. Determination of appropriate doses is well within the skill of a physician of ordinary skill in the art. Animal studies provide a reliable basis for determining effective doses for human treatment. Interspecies scaling of effective doses can be performed based on the principles described in The use of interspecies scaling in toxicokinetics, by Mardenti, J. and Chappell, W. (Toxicokinetics and New Drug Development, Yacobi et al., eds., Pergamon Press, New York, 1989, pp. 42-96). As used herein, the term "therapeutically effective" amount refers to the amount required to perform a specific treatment, such as hair growth. "Treatment" refers to both therapeutic treatments and prophylactic measures aimed at preventing or slowing (reducing) the target symptoms or disease. Those in need of treatment include those already suffering from the disease, as well as those susceptible to the disease or those in need of preventing the disease. In some embodiments, the disease is present.

[0107] For hair growth or regrowth applications, the compositions disclosed herein may be applied to healthy scalp to maintain an extended anagen phase, or to various forms of alopecia to convert telogen to anagen or induce new hair growth.In certain embodiments, the compositions disclosed herein are applied topically to a portion of the scalp to induce hair growth or hair regrowth.In other embodiments, the compositions are applied to other areas of the body where hair growth or hair regrowth is desired, such as, but not limited to, eyebrows, eyelashes, face, chest, arms, legs, or genital area.

[0108] In certain embodiments, the compositions disclosed herein are applied topically for wound healing, such as for accelerated healing of atonic wounds or scar-free wound healing.

[0109] In another embodiment, the compositions disclosed herein are applied topically to a wound to aid in wound healing.

[0110] In yet another embodiment, the compositions disclosed herein are applied topically to existing scars to minimize the appearance of scars.

[0111] In yet another embodiment, the compositions disclosed herein are applied topically to areas of compromised tactile sensation to improve tactile sensation.

[0112] In some embodiments, the compositions disclosed herein are applied topically to improve a youthful appearance, reduce wrinkles, improve skin elasticity, reduce the visible effects of skin aging and environmental stress, reduce hyper- or hypo-pigmentation, improve the appearance of age spots, improve skin texture, and / or improve the uniformity of skin pigmentation.

[0113] (Example) Example 1: Capture and detection of Hh / Wnt from cells derived from partially differentiated embryonic stem cell cultures Embryonic stem cells were grown on an adhesive substrate consisting of a thin layer of MATRIGEL® in serum-free medium supplemented with bFGF (10 ng / mL) and activin A (5 ng / mL) according to currently published methods. After reaching confluence, half of the cultures received the same medium but without the growth factors bFGF and activin A. Cell culture supernatant samples were analyzed for follistatin concentration.

[0114] Individual cultures, either undifferentiated or partially differentiated, were exposed to 10 mM solutions of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or methyl-β-cyclodextrin (MBCD) for 60 to 180 min. Before exposure, the cell cultures were smooth, compact, and multilayered (Figure 5A). After incubation with cyclodextrin, the cultures became fragmented, with most cells losing their adhesion (Figure 5B).

[0115] The MBCD solutions incubated with the cells were then analyzed for concentrations of Shh, Wnt3a, and Wnt7a, and the results are shown in Table 3.

[0116] [Table 3]

[0117] The addition of α- or β-cyclodextrin had a proportional, albeit diminished, effect.

[0118] Example 2: Comparative study of hair growth in animal models Partially differentiated embryonic stem cell cultures were cultured at 1mL / 10 6 The cyclodextrin / lipid-modified protein complex (hereafter referred to as the "active ingredient") was obtained by exposure to a harvested aqueous solution containing 10 mM methyl-β-cyclodextrin and 20% trehalose injected at the cell volume for 3 h at room temperature.

[0119] The formulated active ingredient (containing 0.75% phenoxyethanol and caprylyl glycol as preservatives) was tested to target hair growth (or regrowth). A mouse model of hair growth was used to test prototype formation. Six-week-old male and female mice in the first telogen phase were randomly assigned to three different concentration treatment groups and a negative control.

[0120] On day 1, all animals were anesthetized with isoflurane and the entire back (from shoulder to rump) was clipped free of hair with scissors. Test articles were applied continuously for 14 days starting on day 0. Test articles were gently rubbed onto the dorsal skin of each mouse every day. New gloves were worn for each treatment type. Animals were kept alone during the treatment period to avoid mice in the same cage licking the test article.

[0121] Body weights and clinical observations were performed weekly. Macroscopic digital photographs were taken on days 2, 7, 10, 14, and 21. On day 21, skin samples were taken from all mice and fixed in formalin for histological analysis.

[0122] All treatment groups, regardless of the concentration of active ingredient, showed a response with neo-anagen spots, as evidenced by increased skin darkness and neo-anagen spot counts in shaved areas (FIGS. 6, 7A-C).

[0123] Hematoxylin-eosin staining shows that in the control group, telogen phase started on day 1 and continued until the end of the experiment (Figure 8). The treatment group showed a telogen to anagen transition in areas not yet showing visible hair growth (Figure 9) and typical anagen follicles in the new hair patches (Figure 10).

[0124] In telogen, hair follicle stem cells are quiescently localized in the bulge area of ​​old hair follicles. In the absence of Wnt, LGR5 expression is minimal or absent. CK14 is a skin basal cell marker found throughout the outer root sheath of the hair follicle (Figure 11). During the anagen transition, Wnt signaling stabilizes β-catenin and induces Lgr5, a putative hair follicle stem cell marker. Lgr5+ cells prime the actual hair follicle shaft for cell migration to the dermal papilla (Figure 12).

[0125] SOX9 is a precursor factor that governs hair follicle stem cell fate and plasticity and is essential for outer root sheath (ORS) differentiation and formation of the hair stem cell compartment at the bulge. Sox9 expression is dependent on sonic hedgehog (Shh) signaling. Treated animals are Sox9 positive and show hair stem cell mobilization (Figure 13).

[0126] Example 3: Ex vivo human hair follicle cultures Plucked human hairs with a clearly present hair bulb area were immersed in cell growth medium immediately after harvest. Some hair samples were exposed to lipid-modified protein / MBCD complexes (encapsulated MBCD) derived from embryonic stem cell cultures described in Example 2 (referred to as active ingredient) or to unencapsulated MBCD as a control, at the same concentration of 0.25 mM of the cyclodextrin component. No other growth factors (e.g., EGF, KGF, etc.) were used in the hair follicle cultures. After 2 days of culture (Figure 14A-C), cell attachment and slight proliferation was observed in both MBCD-containing and control hair follicle cultures, although proliferation was greater in hair follicle cultures containing MBCD-encapsulated factors. After 5 days of exposure (Figure 14D-G), control hair follicles were senescent and detached from the matrix, whereas hair follicle cultures exposed to MBCD continued to increase in diameter and to proliferate adherent cells.

[0127] Human hair follicles from non-balding men were dissociated from scalp samples. A total of 15 hair follicles per group were transferred to standard DMEM:F12 medium (5% fetal bovine serum) and exposed to either control or active ingredient at concentrations of 0.1 mM, 0.25 mM, or 0.5 mM of the cyclodextrin component. Hair length and follicle thickness were measured on days 0 and 7.

[0128] Within 7 days, the majority of the hair follicles had grown in terms of length, with an increase in length ranging from 0 to 63%. Hair follicles treated with 0.25 mM of the active ingredient grew by 42% ± 13%, while follicles receiving the other treatments grew by 33 to 34%. Under the same test conditions, the growth in the thickness of the hair follicles reached statistical significance (p<0.01) (Figures 15A-B).

[0129] Example 4: Clinical trial of a formulation containing a cell membrane-bound lipid-modifying signal transduction factor The active ingredient, a cyclodextrin / lipid-modified protein complex, was added to 1 mL / 10 of a partially differentiated human embryonic stem cell culture. 6The cells were produced by exposure to a harvesting aqueous solution containing 10 mM methyl-β-cyclodextrin and 20% trehalose injected in a volume of 100 cells at room temperature for 3 hours. The composition was applied topically onto the dorsal area of ​​the wrist of one hand, while the other hand was left untreated. This area of ​​the hand is covered with peripheral arm hair, which is the same in pattern and density on the left and right, but shows clear signs of telogen upon mechanical abrasion. Application was by spreading approximately one drop (30 μL) onto the skin, allowing it to dry until tacky, and then rubbing until no longer tacky. The area was treated daily for 5 days and evaluated after 2 weeks. Peripheral arm hair growth was clearly noticeable in the treated area (FIGS. 16C-D). In addition, age-related wrinkles and spotty pigmentation were clearly reduced, and skin texture was improved. Subjects reported improved tactile sensation based on increased ability to discriminate between two points. The observations suggest a reinvigorating effect in the skin (FIGS. 16A-B).

[0130] Unless otherwise indicated, all numbers used in the specification and claims expressing amounts of ingredients, properties (such as molecular weight), reaction conditions, and the like, should be understood to be modified in any place by the word "about". As used herein, the terms "about" and "approximately" mean within 10 to 15%, preferably within 5 to 10%. Thus, unless otherwise indicated, the numerical parameters specified in the following specification and claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. While there is no intention to limit the application of the doctrine of equivalents to the scope of the claims, at the very least, each numerical parameter should be considered in light of the number of significant digits reported and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in the corresponding testing measurements.

[0131] In the context of the present invention (particularly in the context of the claims), the singular includes both the singular and the plural, unless otherwise specified or clearly contradicted by the context. The recitation of numerical ranges herein is merely intended to serve as a shorthand method of individually referring to each individual numerical value within the range. Unless otherwise specified herein, each of these individual numerical values ​​is incorporated herein as if each were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise specified or clearly contradicted by the context. Any examples or illustrative language (e.g., "for example") provided herein are intended merely to highlight the present invention and are not intended to limit the scope of the present invention other than as described in the claims. The description herein should not be construed as indicating any non-claimed element essential to the practice of the present invention.

[0132] Groupings of optional elements or embodiments of the invention described herein are not to be construed as limitations. Each group member may be referenced and claimed individually or in combination with other group members or other elements herein. One or more members of a group may be added to or deleted from a group for reasons of convenience and / or patentability. When such additions or deletions are made, the specification shall be deemed to include the modified group and the specification shall satisfy the recitation requirement of all Markush groups recited in the claims.

[0133] Certain embodiments of the present invention have been described herein, including the best mode of the invention for carrying out the invention. Of course, variations on these preferred embodiments will be apparent to those of ordinary skill in the art in light of the foregoing description. The inventors expect that such variations will be adopted by those of ordinary skill in the art as appropriate, and intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all variations thereof is encompassed by the present invention unless otherwise specified or clearly contradicted by context.

[0134] Specific embodiments described herein may be further limited in the claims by the phrases "consisting of" or "consisting essentially of." When used in the claims, whether originally recited or added by amendment, the transitional phrase "consisting of" excludes all elements, steps, or ingredients not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to those materials and steps specified and that do not materially affect the basic and novel characteristic(s). The embodiments of the invention described in such claims are inherently or explicitly recited and enabled by this specification.

[0135] Additionally, throughout the specification, there are numerous references to patents and publications. Each of the above references and publications is incorporated herein by reference in their entirety.

[0136] Finally, it is to be understood that the embodiments of the invention described herein are illustrative of the principles of the invention. Other variations that can be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the invention can be used in accordance with the teachings herein. Accordingly, the invention is not limited to that precisely as described and illustrated.

[0137] [Note] [Appendix 1] A composition comprising a complex of a lipid-modified protein and a cyclodextrin.

[0138] [Appendix 2] The cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or methyl-β-cyclodextrin; 2. The composition of claim 1.

[0139] [Appendix 3] The cyclodextrin is a chemically modified cyclodextrin that has been modified by hydrogenation, hydroformylation, methylation, oxidation, reduction, or carbon-carbon coupling. 3. The composition of claim 1 or 2.

[0140] [Appendix 4] The cyclodextrin is methyl-β-cyclodextrin. 4. The composition of any one of claims 1 to 3.

[0141] [Appendix 5] The lipid-modified protein includes one or more of Wingless (Wnt) protein or Hedgehog (Hh) protein bound to a cell membrane lipid; 5. The composition of any one of claims 1 to 4.

[0142] [Appendix 6] The Hh protein is one or more of Sonic Hedgehog (Shh) protein, Desert Hedgehog (Dhh) protein, and Indian Hedgehog (Ihh) protein. 6. The composition of claim 5.

[0143] [Appendix 7] The Wnt protein is one or more of Wnt3a, Wnt7b, or Wnt10b; 6. The composition of claim 5.

[0144] [Appendix 8] The lipid-modified protein includes other proteins that do not belong to the Wingless (Wnt) family or the Hedgehog (Hh) family. 5. The composition of any one of claims 1 to 4.

[0145] [Appendix 9] The lipid-modified protein is obtained from a stem cell population. 9. The composition of any one of claims 1 to 8.

[0146] [Appendix 10] The stem cells are embryonic stem cells, parthenogenetic stem cells, adult stem cells, fetal stem cells, or induced pluripotent stem cells; 10. The composition of claim 9.

[0147] [Appendix 11] The stem cells are mammalian stem cells. 11. The composition of claim 9 or 10.

[0148] [Appendix 12] The stem cells are human stem cells. 12. The composition of claim 9 or 11.

[0149] [Appendix 13] The stem cells are genetically engineered to overexpress a Wnt protein or an Hh protein. 13. The composition of any one of claims 9 to 12.

[0150] [Appendix 14] The stem cells are immortalized by genetic engineering. 14. The composition of any one of claims 9 to 13.

[0151] [Appendix 15] The stem cells are genetically engineered to express telomere reverse transcriptase (hTERT). 15. The composition of claim 14.

[0152] [Appendix 16] Further comprising at least one kosmotrope, 16. The composition of any one of claims 1 to 15.

[0153] [Appendix 17] The at least one cosmotrope is propylene glycol, proline, trehalose, ectoine, or trimethylamine-N-oxide; 17. The composition of claim 16.

[0154] [Appendix 18] The at least one kosmotrope is trehalose; 17. The composition of claim 16.

[0155] [Appendix 19] A topical composition comprising a complex of a lipid-modified protein and a cyclodextrin according to any one of claims 1 to 18.

[0156] [Appendix 20] The composition is an aqueous formulation. 21. The topical composition of claim 19 or 20.

[0157] [Appendix 21] Further comprising at least one cosmotrope and an antimicrobial agent; 20. The topical composition of claim 19.

[0158] [Appendix 22] The at least one kosmotrope is trehalose; 22. The topical composition of claim 21.

[0159] [Appendix 23] The antimicrobial agent comprises silver particles. 22. The topical composition of claim 21.

[0160] [Appendix 24] The silver particles are silver nanoparticles or silver microparticles. 22. The topical composition of claim 21.

[0161] [Appendix 25] the pH is between about 4.5 and about 8.0; 25. The topical composition of any one of claims 19 to 24.

[0162] [Appendix 26] Exposing tissue to a composition according to any one of claims 1 to 25. A method for promoting tissue regeneration in a tissue in need thereof.

[0163] [Appendix 27] Exposing tissue to a composition according to any one of claims 1 to 25. A method for promoting tissue rejuvenation in tissues in need thereof.

[0164] [Appendix 28] Exposing tissue to a composition according to any one of claims 1 to 25. A method for restoring sensory nerve function in tissue.

[0165] [Appendix 29] The tissue is skin. 29. The method of any one of claims 26 to 28.

[0166] [Appendix 30] The tissue is scar tissue. 29. The method of any one of claims 26 to 28.

[0167] [Appendix 31] comprising exposing a hair follicle to a composition according to any one of claims 1 to 25; How to promote hair growth.

[0168] [Appendix 32] The hair growth is promoted in subjects with age-related alopecia, alopecia totalis, telogen and anagen effluvium, or alopecia areata; 32. The method according to claim 31 .

[0169] [Appendix 33] The hair follicle is on the scalp of the subject. 33. The method according to claim 31 or 32.

[0170] [Appendix 34] The hair follicle is an eyelash of a subject. 33. The method according to claim 31 or 32.

[0171] [Appendix 35] The hair follicle is the eyebrow of a subject. 33. The method according to claim 31 or 32.

[0172] [Appendix 36] The hair follicle is on the face of a subject. 33. The method according to claim 31 or 32.

[0173] [Appendix 37] The hair follicle is on the subject's chest. 33. The method according to claim 31 or 32.

[0174] [Appendix 38] The hair follicle is on the arm of a subject. 33. The method according to claim 31 or 32.

[0175] [Appendix 39] The hair follicle is on the leg of the subject. 33. The method according to claim 31 or 32.

[0176] [Appendix 40] comprising exposing the skin to a composition according to any one of claims 1 to 24; A method for improving the appearance of skin.

[0177] [Appendix 41] The appearance that is improved is one or more of skin texture, wrinkles, age spots, and age spots. 41. The method according to claim 40.

[0178] [Appendix 42] Exposing a hair follicle to a composition according to any one of claims 1 to 24, How to improve the appearance of hair.

[0179] [Appendix 43] Cultivating stem cells capable of producing Wnt protein and Hh protein in a medium; incubating said stem cells in a harvest solution containing cyclodextrin to obtain a cyclodextrin complex of the lipid-modified protein; and mixing said cyclodextrin / lipid-modified protein complex with one or more pharma- ceutically acceptable excipients to form a topical formulation; Equipped with 23. A method for producing a composition according to any one of claims 1 to 22.

[0180] [Appendix 44] The harvest solution further comprises at least one kosmotrope; 44. The method according to claim 43.

[0181] [Appendix 45] The harvest solution includes a stabilizing agent. 44. The method according to claim 43.

[0182] [Note 46] The stabilizer is a kosmotrope. 46. ​​The method according to claim 45.

[0183] [Appendix 47] The cosmotrope is trehalose. 47. The method according to claim 44 or 46.

[0184] [Appendix 48] The concentration of kosmotrope in the harvest solution is about 5% to about 30%. 48. The method of claim 44, 46, or 47.

[0185] [Appendix 49] The concentration of the kosmotrope is about 20%. 49. The method of claim 44, 46, 47, or 48.

[0186] [Appendix 50] The harvest solution comprises an aqueous solution of cyclodextrin; 50. The method of any one of claims 43 to 49.

[0187] [Appendix 51] The cyclodextrin is methyl-β-cyclodextrin. 51. The method of any one of claims 43 to 50.

[0188] [Appendix 52] the concentration of the cyclodextrin in the harvest solution is from about 1 mM to about 20 mM; 52. The method of any one of claims 43 to 51.

[0189] [Appendix 53] The concentration of the cyclodextrin in the harvest solution is about 10 mM. 53. The method of any one of claims 43 to 52.

[0190] [Appendix 54] The solution of the cyclodextrin / lipid-modified protein complex is stored at 4° C. or below. 54. The method of any one of claims 43 to 53.

[0191] [Appendix 55] The solution of cyclodextrin / lipid-modified protein complex is lyophilized. 54. The method of any one of claims 43 to 53.

[0192] [Appendix 56] The one or more pharma- ceutically acceptable excipients include one or more preservatives. 44. The method according to claim 43.

[0193] [Appendix 57] The one or more pharma- ceutically acceptable excipients include one or more antibacterial agents. 44. The method according to claim 43.

Claims

1. a. A cyclodextrin molecule having a hydrophobic inner surface and a hydrophilic outer surface, and capable of forming complexes with hydrophobic compounds, and b. Lipid-modified Hedgehog (Hh) protein and lipid-modified Wingless (Wnt) protein A pharmaceutical composition comprising, c. The lipid-modified Hh protein or the lipid-modified Wnt protein binds to the cyclodextrin molecule and is collected from the stem cell population using the collected solution. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the cyclodextrin molecule comprises an α-cyclodextrin molecule, a β-cyclodextrin molecule, a γ-cyclodextrin molecule, or a methyl-β-cyclodextrin molecule (MβCD).

3. The pharmaceutical composition according to claim 1, wherein the cyclodextrin molecule is a chemically modified cyclodextrin.

4. The pharmaceutical composition according to claim 1, wherein the lipid-modified Hh protein comprises Sonic Hedgehog (Shh) protein, Desert Hedgehog (Dhh) protein, or Indian Hedgehog (Ihh) protein.

5. The pharmaceutical composition according to claim 1, wherein the lipid-modified Wnt protein comprises Wnt3a protein, Wnt7b protein, or Wnt10b protein.

6. The pharmaceutical composition according to claim 1, wherein the lipid-modified Wnt protein or the lipid-modified Hh protein is collected from a stem cell population.

7. The pharmaceutical composition according to claim 6, wherein the stem cell population is genetically engineered to overexpress the Wnt protein or the Hh protein.

8. The pharmaceutical composition according to claim 1, wherein the lipid that can covalently bind to the protein comprises myristate, palmitate, palmitreate, oleate, farnesyl, geranylgeranyl, phophatidyl-ethanolamine, cholesterol, or a glycophosphatidylinositol (GPI) anchor.

9. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is in the form of a liquid, paste, cream, powder, ointment, lotion, emulsion, or gel.

10. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is approximately 5% to approximately 25% (v / w, v / v, or w / v) of the collected solution.

11. The pharmaceutical composition according to claim 1, wherein the tissue is a hair follicle, skin, or scar tissue.

12. The pharmaceutical composition according to claim 11, wherein the tissue is a hair follicle, and the hair follicle is from the scalp of the subject.

13. The pharmaceutical composition according to claim 2, wherein the cyclodextrin molecule is methyl-β-cyclodextrin.

14. The pharmaceutical composition according to claim 6, wherein the stem cell population is a population of mammalian stem cells.

15. The pharmaceutical composition according to claim 6, wherein the stem cell population is a population of human stem cells.

16. The pharmaceutical composition according to claim 6, wherein the stem cell population is immortalized by genetic manipulation.

17. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is mixed with one or more pharmaceutically acceptable additives to form a topical formulation.

18. The pharmaceutical composition according to claim 1, wherein the Wnt protein and / or the Hh protein are not treated with a detergent or organic solvent.

19. The pharmaceutical composition according to claim 1, wherein the cyclodextrin molecule is present in the collected solution at a concentration of about 1 mM to about 20 mM.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the pharmaceutical composition is a topical composition.

21. The pharmaceutical composition according to any one of claims 1 to 19, wherein the pharmaceutical composition is an aqueous formulation.

22. The pharmaceutical composition according to any one of claims 1 to 19, wherein the pH value of the pharmaceutical composition is between 4.5 and 8.

0.

23. A pharmaceutical composition according to any one of claims 1 to 19, further comprising at least one antibacterial agent.

24. The pharmaceutical composition according to claim 23, wherein the antibacterial agent comprises silver particles, silver nanoparticles, or silver microparticles.

25. The pharmaceutical composition according to any one of claims 1 to 19, wherein the lipid-modified Hh protein or the lipid-modified Wnt protein is further coated with trehalose.

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