Formulations and methods for wound healing modulation

Abstract

To provide a method and a compound for wound healing by modulating autophagy.SOLUTION: A compound is provided that has structure (I), and is not resveratrol or 4,4'-(ethyne-1,2-diyl)diphenol [L: -C≡C- or the like; R1-R2: are independently substituents at any available position of the phenyl rings and selected from (C1-C6) alkyl, (C2-C6) alkenyl, and (C2-C6) alkynyl; m, n: are, independently, 0, 1, 2, or 3, representing the number of substituents on the rings, and at least one of m and n is ≥1].SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of Provisional Application Serial No. 62 / 144,539, filed Apr. 8, 2015, the contents of which are hereby expressly incorporated herein by reference.

Background Art

[0002] Background of the Invention The present invention generally relates to the field of cell biology, and more particularly to compounds, formulations, and methods for modulating autophagy to treat wounds or diseases or conditions associated with the skin. The term “autophagy,” also known as autophagocytosis, is derived from the Greek words meaning “eat” and “self.” Autophagy is generally defined as self-digestion.

[0003] Autophagy is a recycling or reuse pathway of lysosomes commonly used by cells to perform a homeostatic function by breaking down aged proteins and organelles and reabsorbing nucleotides, amino acids, and free fatty acids for new molecular synthesis and ATP production. Autophagy can be upregulated in response to extracellular or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection as a cell-level self-preservation mechanism. In some pathological conditions, it is desirable to stimulate the autophagy process, but in other pathological conditions such as wound healing, it is desirable to suppress or delay the autophagy process to reduce cell destruction. Thus, modulation of autophagy can cause either promotion / stimulation or suppression / slowing of cell proliferation and cell death processes.

[0004] The most common form of autophagy involves (1) the formation of an elongating isolation membrane, the ends of which ultimately fuse to enclose cellular contents within a double-membrane vesicle known as the autophagosome. Next, the autophagosome fuses with (3) a lysosome that provides enzymes, (4) digesting the contents of the autophagosome, and the contents then become available to the cell again as nutrients for raw materials or building blocks. Autophagy shows some similarities to the parallel proteasomal degradation of ubiquitinated tagged proteins, but autophagosomes differ in that they contain not only proteins but also cytoplasm, mitochondria, organelles, and other cellular structures. In this sense, it is known as a bulk degradation system.

[0005] Since the autophagy process can be either beneficial or harmful to the cell depending on external factors and conditions, the process must be tightly regulated. Both yeast and mammalian systems have been studied and utilize up to 36 proteins. Figure 1 shows the process in mammals.

[0006] The yeast autophagy-related gene product (Atg8) has three mammalian homologs: (1) LC3, (2) GABA A receptor-associated protein (GABARAP), and (3) Golgi-associated ATPase enhancer (GATE-16). Among these, LC3 has been the most intensively studied and is frequently used as a mammalian autophagy marker. Immediately after translation (proLC3), LC3 is processed at the C-terminus by Atg4A or Atg4B to LC3-I. Upon induction or enhancement of autophagy, LC3-I is conjugated to the substrate phosphatidylethanolamine (PE) via E1 (yeast Atg7) and E2 (yeast Atg3). The PE-LC3-I conjugate is called LC3-II. This conjugation occurs during autophagosome formation in the process, resulting in the conversion of soluble LC3-I to autophagosome-bound LC3-II. This lipid conjugation enables LC3-II to be used as a marker of autophagy activity.

[0007] The progression of a wound is caused by many mechanisms including local tissue hypoperfusion, prolonged inflammation, free radical damage, apoptosis, and necrosis. These are typically divided into three post-injury stages, including (i) the inflammatory stage, (ii) the cell proliferation stage, and (iii) the remodeling stage. Each of these stages is associated with a biologically and histologically distinct fingerprint, and autophagy plays a special role at each stage. For example, during the inflammatory stage, autophagy initially protects tissue in that it acts to prevent cells at the wound edge from dying. During the proliferation stage, which involves the proliferation and migration of cells of multiple cell types to close the wound, autophagy helps to form an ordered row of cells at the leading edge of the wound by controlling β1 integrin and other cell migration proteins. In remodeling, the direction of these migrating cells is often controlled by the directionality and deposition of collagen secreted by these transformed fibroblasts as procollagen. Collagen, along with other components such as fibronectin and laminin, helps to regenerate the basement membrane during the proliferation and migration stages.

[0008] During the wound healing process, collagen fibrils impart a structural topology, stiffness, and composition that enable proper cell migration. In chronic wounds and burns, this environment is subject to various factors, including loss of the basement membrane, which contains essential collagen tubes used to fill the wound and restore the layer of intact skin to protect the body from invading microorganisms and environmental factors (e.g., temperature regulation) by moving and invading wound cells (e.g., keratinocytes, fibroblasts, myofibroblasts). During the natural healing process, skin cells secrete collagen in a soluble form known as procollagen, which can enzymatically form higher-order structures or undergo an entropy-driven self-assembly process (Prockop, D. and D. Hulmes (1994)). Procollagen is secreted from cells as a triple helix and can self-assemble into fibrils due to its liquid crystalline properties. This process is highly dependent on pH, ionic strength, temperature, and concentration. The fibrils then form individual collagen fibers and are randomly organized into higher-order three-dimensional networks and structures.

[0009] U.S. Patent Nos. 6,599,945 and 7,094,809 disclose several hydroxytran compounds and their use in inhibiting the formation of infectious herpes virus particles or their use in treating gonorrhea caused by Neisseria gonorrhoeae. International Publication No. WO 2009 / 126700 discloses the use of similar compounds for skin care, such as ultraviolet light, and for cosmetic purposes. And U.S. Patent Nos. 8,716,355 (International Publication No. WO 2011 / 0130468) and 8,680,142 (International Publication No. WO 2011 / 0160301) disclose similar hydroxytrans for use as anti-tumor agents. However, the potential usefulness of these, or other hydroxytrans, as autophagy regulatory compounds was not known until the present invention was made. U.S. Patent Nos. 6,008,260, 6,197,834, and 6,355,692 disclose certain hydroxylated stilbenes, specifically resveratrol. None of these documents disclose the use of such modified trans or stilbene compounds as autophagy regulators.

[0010] It would be advantageous if the process of autophagy could be regulated and, under some conditions, stimulated or enhanced, and under other conditions, slowed or inhibited. SUMMARY OF THE INVENTION

[0011] The present invention relates to compounds, formulations, and methods for modulating autophagy, particularly for applications or purposes of promoting wound healing in patients having chronic wounds or skin conditions. In one aspect, the present invention comprises a method of promoting wound healing, comprising administering at least one first autophagy modulating (FAM) compound described herein. In most embodiments for promoting wound healing, the autophagy modulation is a directional upregulation of autophagic activity. In certain embodiments, the FAM is administered with an auxiliary autophagy modulating (AAM) compound. The FAM and AAM may be administered simultaneously, or one may be administered before the other. When administered simultaneously, they may be in the same dosage form, or prepared as two different dosages or formulations. Since the AAM is often a hermetic compound, it can modulate the effect of the FAM by stimulating or increasing its effect, or by suppressing or inhibiting its effect.

[0012] These autophagy modulators are essentially liquid crystalline and act in specific combinations to treat wounds or diseases or conditions related to the skin. The present invention further describes using these liquid crystals to create bioactive formulations such as hydrogels and alginates that can also function as bioactive membranes to promote healing in conditions related to wounds or the skin. Thus, in some aspects, the present invention comprises a formulation containing FAM and AAM. The formulation can be a hydrogel, such as an alginate. The formulation can include a cationic salt of the FAM, or a complex of the FAM and AAM with a cation. In other formulations, the FAM and / or AAM can be formulated with cyclodextrin.

[0013] Certain FAMs can be trans or stilbenes (including cis and trans stilbenes) and can have any of the various substituents described herein. In some embodiments, the substituent is a hydroxyl or alkoxyl group that increases the solubility and polarity of the molecule. Any FAM can be administered with any AAM. For example, the FAM can be trans and the AAM can be a vitamin, acidic sugar, amino acid, or quinolone. Similarly, the FAM can be a stilbene and the AAM can be a vitamin, acidic sugar, amino acid, or quinolone.

[0014] In some embodiments, the method promotes wound healing in certain symptoms, and the wound or skin condition is aging, autoimmune disease with inflammation, avascular necrosis, bacterial infection, cancer, diabetic neuropathy, endometriosis, fungal infection, gout, alopecia, infectious arthritis, inflammation, inflammatory bowel disease, ischemia, Lyme disease, organ / tissue transplantation, parasitic infection, psoriatic arthritis, psoriasis, pseudogout, rheumatoid arthritis, scleroderma, scurvy, sepsis, skin disease, surgical scar, surgical adhesion, transfection method, ulcerative colitis, ulcer, viral infection, wart, surgical wound, incision, laceration, cut and abrasion, donor site wound by skin graft, traumatic wound, infectious wound, ischemic wound, burn, blistering wound, non-infectious wound, contusion, incised wound, laceration, non-penetrating wound, open wound, penetrating wound, perforating wound, stab wound, abscess, subcutaneous wound, chronic ulcer, gastric ulcer, skin ulcer, peptic ulcer, duodenal ulcer, gastric ulcer, gout, hypertensive ischemic ulcer, congestive ulcer, sublingual ulcer, submucosal ulcer, symptomatic ulcer, nutritional ulcer, tropical ulcer, and venereal ulcer.

[0015] In some methods, the wound to be healed is a wound of epithelial skin nature, such as cuts, abrasions, ulcers of many types and degrees, aging, non-elasticity of the skin, etc. In other methods, the wound can be a wound of the eye or ear, and in other methods, the wound can be a wound of the oral cavity nature, such as oral leukoplakia, herpes virus infection, extraction wound, gingivitis, etc.

Brief Description of the Drawings

[0016] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate several aspects of the invention and together with the description serve to explain the principles of the invention. In the drawings, the thickness of lines, layers, and regions may be exaggerated for clarity.

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[0024] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate several aspects of the invention and together with the description serve to explain the principles of the invention. In the drawings, the thickness of lines, layers, and regions may be exaggerated for clarity.

Modes for Carrying Out the Invention

[0025] Detailed Description For example, numerical ranges, measured values, and parameters used to characterize the present invention, such as angles, amounts of components, polymer molecular weights, reaction conditions (pH, temperature, charge level, etc.), physical dimensions, etc., are necessarily approximate values and, although reported as accurately as possible, inherently include inaccuracies derived from each measurement. Accordingly, all numbers representing ranges of magnitudes used in the specification and claims should be understood to be modified in all cases by the term "about." All numerical ranges are understood to include all possible incremental sub-ranges within the outer boundaries of that range. Thus, a range of 30 to 90 units discloses, for example, sub-ranges of 35 to 50 units, 45 to 85 units, and 40 to 80 units. Unless otherwise defined, percentages are weight / weight (wt / wt), formulations are generally weight / volume (w / v), grams per 100 mL (equivalent to wt / wt in an aqueous solution with a density of 1.0), and wound area is expressed as area / area (a / a) in cm 2 as represented.

[0026] All patents, published patent applications, and non-patent literature cited herein are hereby incorporated by reference in their entirety.

[0027] In some embodiments, the present invention comprises a method of modulating autophagy comprising administration of a first autophagy modulating (FAM) compound and any auxiliary autophagy modulating (AAM) compound. The FAM and AAM compounds are described in more detail in the following sections. The FAM and AAM compounds may be administered sequentially or simultaneously. When administered sequentially, the order may be first FAM and then AAM, or first AAM and then FAM. When administered simultaneously, they may be administered in separate individual formulations or as a single formulation in combination of the components. The compounds may be administered from once a day up to about six times a day depending on the excipients of the formulation. Routes of administration include topical, transdermal, oral, nasal, ocular, otic, IV, IM, subcutaneous, rectal, and vaginal.

[0028] The use of pharmaceutical excipients in the preparation of formulations is generally well understood from pharmaceutical treatises such as Remington's Pharmaceutical Sciences, 18 th Edition (1990), and subsequent editions such as Remingtons: The Science and Practice of Pharmacy, 22 nd edition (2012). Topical formulations can be made into solutions, suspensions, creams, ointments, and hydrogels, among other things, by combining them with solvents, emulsifiers, emollients, solvents, and the like.

[0029] In certain embodiments, the present invention relates to formulations containing FAM compounds and AAM compounds. The relative amount of FAM to AAM in the formulation, expressed as a molar ratio, can range from about 500:1 to about 1:500 (FAM:AAM), or in certain embodiments, from about 200:1 to 1:200. In liquid formulations, FAM can comprise from about 0.01% to about 40% (w / v) of the formulation, and AMM can comprise from about 0.01% to about 99.9% (w / v) of the formulation. In certain embodiments, FAM can comprise from about 0.1% to about 40% (w / v) of the formulation, and AMM can comprise from about 0.1% to about 60% (w / v) of the formulation. The formulation concentration of FAM is optimally from 0.5 to 15% (w / v). Chemical and Biological Definitions

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described herein. All references cited herein, including books, journal articles, published U.S. or foreign patent applications, issued U.S. or foreign patents, and other references, are hereby incorporated by reference in their entirety, including all data, tables, figures, and text shown in the cited references.

[0031] The following terms used throughout this application have the meanings set forth below.

[0032] "The first autophagy regulator" or "FAM compound" or "FAM" means a compound of formula I,

[0033] [Chemical formula]

[0034] wherein L is a linker selected from -C=C- and -CR a =CR b -, and R a and R b are independently H, or phenyl optionally substituted with -(R 3 ) p or -(R 4 ) q ; R 1 ~R 4 are independently substituents at any available position on the phenyl ring, m, n, p, and q are each independently 0, 1, 2, or 3 representing the number of substituents on the ring, and at least one of m or n must be ≥1, each R 1 , R 2 , R 3 , and R 4 are independently (C 1 ~C 6 )alkyl, (C 2 ~C 6 )alkenyl, or (C 2 ~C 6 )alkynyl, and may be substituted with 1 - 3 substituents selected from -OH, -SH, -halo, -NH 2 , or NO 2 ; 5 , Y is O, S, or NH, and R 6 is H or R 5-YR selected from 6 , -ZR where Z is -N(C=O)- or -O(C=O)- 5 , -halo, -NO 2 , -SO 3 Na, -azide, and -glycoside -and salts thereof, selected from provided that FAM is not resveratrol or 4,4'-(ethyne-1,2-diyl)diphenol (TOLCINE).

[0035] "Auxiliary autophagy regulator" or "AAM compound" or "AAM" means a compound described herein that also has an autophagy regulatory effect. The effect may be stimulatory or inhibitory depending on the compound. Without wishing to be bound by any particular theory, AAM compounds may have an inhibitory effect by competing at rate-limiting steps such as the cellular uptake mechanism. More specific AAM compounds will be described in a later section.

[0036] Autophagy regulation refers to either upregulation or downregulation of the autophagy process in cells. Depending on the particular medical condition or state, as will be described later, it may be desirable to achieve one direction or the other of autophagy regulation. And referring to the considerations of hormesis, the dose of any particular FAM or AAM compound or formulation or complex may achieve upregulation or downregulation, or both.

[0037] As used herein, the term "-(C 1 ~C 6 )alkyl" refers to straight-chain and branched acyclic saturated hydrocarbons having 1 to 6 carbon atoms. Representative straight-chain -(C 1 ~C 6) The alkyl groups include methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl. Representative branched-chain -(C 1 ~C 6 ) alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, neopentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl and 1,2-dimethylpropyl, methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 3-ethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, and the like. More generally, the subscript numbers refer to the number of carbon atoms in the chain. Thus, the term "-(C 1 ~C 4 ) alkyl" refers to straight-chain and branched acyclic saturated hydrocarbons having 1 to 4 carbon atoms.

[0038] As used herein, the term "-(C 2 ~C 6 ) alkenyl" refers to straight-chain and branched acyclic hydrocarbons having 2 to 6 carbon atoms and containing one or more carbon-carbon double bonds. Representative straight-chain and branched -(C 2 ~C 6 ) alkenyl groups include -vinyl, allyl, 1-butenyl, -2-butenyl, -isobutenylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and the like.

[0039] As used herein, the term "-(C 2 ~C 6 ) alkynyl" refers to straight-chain and branched acyclic hydrocarbons having 2 to 6 carbon atoms and containing one or more carbon-carbon triple bonds. Representative straight-chain and branched -(C 2 ~C 6) The alkynyl group includes -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, -4-pentynyl, etc.

[0040] As used herein, "-(C 1 ~C 10 ) alkoxy" means a straight-chain or branched acyclic hydrocarbon having one or more ether groups and 1 to 10 carbon atoms. Representative straight-chain and branched (C 1 ~C 10 ) alkoxy includes -methoxy, -ethoxy, -propoxy, -butyloxy, -pentyloxy, -hexyloxy, -heptyloxy, -methoxymethyl, -2-methoxyethyl, -5-methoxypentyl, -3-ethoxybutyl, etc.

[0041] As used herein, "-(C 1 ~C 6 ) alkoxy" means a straight-chain or branched acyclic hydrocarbon having one or more ether groups and 1 to 6 carbon atoms. Representative straight-chain and branched (C 1 ~C 5 ) alkoxy includes -methoxy, -ethoxy, -propoxy, -butyloxy, -pentyloxy, -hexyloxy, -methoxymethyl, -2-methoxyethyl, -5-methoxypentyl, -3-ethoxybutyl, etc.

[0042] As used herein, the term "-(C 3 ~C 12 ) cycloalkyl" refers to a cyclic saturated hydrocarbon having 3 to 12 carbon atoms. Representative (C 3 ~C 12 ) cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, etc.

[0043] As used herein, the term "-(C 4 ~C12 ) "Cycloalkenyl" refers to a cyclic hydrocarbon having 4 to 12 carbon atoms and containing one or more carbon-carbon double bonds. A typical -(C 3 ~C 12 ) Cycloalkenyl includes -cyclobutenyl, -cyclopentenyl, -cyclopentadienyl, -cyclohexenyl, -cyclohexadienyl, -cycloheptenyl, -cycloheptadienyl, -cycloheptatrieneyl, -cyclooctenyl, -cyclooctadienyl, -cyclocyclooctatrieneyl, -cyclooctatetraeneyl, -cyclononenyl, -cyclononadienyl, -cyclodecenyl, -cyclodecadienyl, -norbornenyl, and the like.

[0044] As used herein, "-(7- to 12-membered) bicyclic aryl" means a bicyclic aromatic carbocyclic ring containing 7 to 12 carbon atoms. Representative -(7- to 12-membered) bicyclic aryl groups include -indenyl, -naphthyl, and the like.

[0045] As used herein, "hydroxy(C 1 ~C 6 ) alkyl" means any of the above C 1~6 alkyl groups substituted with one or more hydroxy groups. Representative hydroxy(C 1 ~C 6 ) alkyl groups include hydroxymethyl group, hydroxyethyl group, hydroxypropyl group, and hydroxybutyl group, particularly hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1,2-dihydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, 2-hydroxy-1-methylpropyl, and 1,3-dihydroxyprop-2-yl.

[0046] Any of the groups defined above may be substituted. As used herein, the term "optionally substituted" refers to a group that is either unsubstituted or substituted. In the presence of any substituent and unless otherwise indicated, any substituent includes -(C 1 ~C6 ) alkyl, OH, halo, -C(halo) 3 , -CH(halo) 2 , -CH 2 (halo), NH 2 , -NH(C 1 -C 6 ) alkyl, CN, SH, phenyl, benzyl, (=O), halo(C 1 -C 6 ) alkyl-, hydroxy(C 1 ~C 6 ) alkyl- are each independently selected from the group consisting of 1, 2, or 3 groups. Thus, certain substituted embodiments include the following.

[0047] As used herein, "dihydroxy(C 1 ~C 6 ) alkyl" means any of the above C 1~6 alkyl groups substituted with two hydroxy groups. Representative dihydroxy(C 1 ~C 6 ) alkyl groups include dihydroxyethyl group, dihydroxypropyl group, and dihydroxybutyl group, particularly 1,2-dihydroxyethyl, 1,3-dihydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxybutyl, 1,4-dihydroxybutyl, and 1,3-dihydroxyprop-2-yl.

[0048] As used herein, the terms "halo" and "halogen" refer to fluoro, chloro, bromo, or iodo.

[0049] As used herein, "-CH 2 (halo)" means a methyl group in which one of the hydrogens of the methyl group is replaced by a halogen. Representative -CH 2 (halo) groups include -CH 2 F, -CH 2 Cl, -CH 2 Br, and -CH 2 I.

[0050] As used herein, “-CH(halo) 2 ” means a methyl group in which two hydrogens of the methyl group are replaced by halogens. Representative -CH(halo) 2 groups include -CHF 2 , -CHCl 2 , -CHBr 2 , -CHBrCl, -CHClI, and -CHI 2 .

[0051] As used herein, “-C(halo) 3 ” means a methyl group in which each of the hydrogens of the methyl group is replaced by a halogen. Representative -C(halo) 3 groups include -CF 3 , -CCl 3 , -CBr 3 , and -CI 3 .

[0052] As used herein, “(halo) p (C 1 ~C 6 )alkyl-” means a (C 1 ~C 6 )alkyl chain in which p positions are substituted with halo, where p is 1, 2, or 3. The halo substituents may be substituted on the same or different carbons in the (C 1 ~C 6 )alkyl. Representative “(halo) p (C 1 ~C 6 )alkyl-” groups include, for example, -CH 2 CHF 2 , -CH 2 CH 2 CH 2 Cl, -CHBr 2 , -CHBrCl, -CHClI, -CH 2 CHI 2 , -CH 2 CH 2 CHClCH 2 Br, etc.

[0053] As used herein, "azide" means a substituent of the formula -N=N=N.

[0054] As used herein, "glycoside" means a 5- or 6-membered cyclic sugar attached to a compound of formula I. The attachment is generally a glycosidic bond from the anomeric carbon of the sugar and can be formed (1) via an oxygen atom, thus an "O-glycoside" can be formed, (2) via a nitrogen atom, thus an "N-glycoside" can be formed, or (3) via a sulfur atom, thus an "S-glycoside" can be formed. The glycoside can be a monosaccharide or disaccharide having one or two ring structures. Representative glycosides formed from 6-membered sugar glucosides, galactosides, mannoses, and altrosides and glycosides formed from 5-membered sugars include ribosides, arabinosides, xylosides, and lyxosides. The sugar may contain any substituent, but many embodiments contain only the natural -H and -OH substituents that define each sugar.

[0055] There is overlap in the literature in the use of the terms "wound," "ulcer," and "erosion," and these terms are often used randomly. Thus, in this context, the term "wound" encompasses the terms "ulcer," "lesion," "erosion," and "infarct," and these terms are used interchangeably unless otherwise specified. Wounds are classified by many criteria including size or area (large or small), depth or layer of the injury, cause, difficulty of healing, etc. Wounds can be classified as (i) small tissue loss due to surgical incisions, minor abrasions, and minor bites, or (ii) severe tissue loss such as ischemic ulcers, pressure sores, fistulas, lacerations, severe bites, burns, and donor site wounds (in soft and hard tissues), and infarcts. All types and classifications of wounds are encompassed by the present invention.

[0056] The term "skin" is used in a very broad sense to include the epidermal layer of the integument and, if the skin surface is more or less damaged, also the underlying dermis layer. Except for the stratum corneum, the epidermal layer of the skin is the outer (epithelial) layer, and the deeper connective tissue layer of the skin is called the "dermis" and includes sensory nerves and end organs.

[0057] A method that "promotes wound healing" results in wound healing that is more rapid than the healing of a similar wound in the absence of treatment. Also, "promotion of wound healing" can mean that the method particularly modulates the proliferation and / or growth of keratinocytes, or that the wound heals with less scarring, less wound contraction, less collagen deposition, and on a larger surface area. In certain examples, "promotion of wound healing" can also mean that, when used with the methods of the present invention, a particular method of wound healing has an improved success rate (e.g., the uptake rate of a skin graft).

[0058] The phenomenon of hormesis is generally associated with compounds that elicit biologically opposing effects in a dose-dependent manner and is well described in the literature, for example, Calabrese EJ, Bachmann KA, Bailer AJ, Bolger PM, Borak J, et al. (2007), Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hermetic dose-response framework. Toxicol Appl Pharmacol. 222: 122-128; Penniston KL, Tanumihardjo SA. The acute and chronic toxic effects of vitamin A. Am J Clin Nutr. 2006;83: 191-201; and Cook R, Calabrese EJ. The importance of hormesis to public health. Cien saude Colet. 2007;12:955-963. Generally, at low doses there are stimulatory or beneficial effects and at high doses there are inhibitory or toxic effects. Hormesis is also characterized, among other things, as self-protection, adaptive response, and preconditioning, and is characterized by the shape of its dose-response curve, which particularly includes a beta-curve, biphasicity, bell-shaped, U-shaped or inverted U-shaped, bimodality, functional antagonism, and dual response. Known hormetic substances (or "hormetins") include vitamin A, ferulic acid, chalcones, rapamycin, epigallocatechin-3-gallate, and many others.

[0059] Hormesis can also represent an adaptive response in which moderate stress is applied to confer adaptive resistance to organisms when faced with severe stress factors. Environmental stresses such as oxidative metabolism and heat stress serve as hormetin, which is used to induce specific responses or adaptive changes (Mattson MP. Hormesis defined, Ageing Res Rev. 2008;7: 1-7). Hormetin has been shown to activate various stress and detoxification pathways, including heat shock proteins, antioxidants, protein chaperones, metabolism, calcium homeostasis, and growth factors (Mattson MP, Cheng A. Neurohormetic phytochemicals: Low-dose toxins that induce adaptive neuronal stress responses.Trends Neurosc.2006;29:632-639; and Mattson, 2008 mentioned above). These dose-response effects have been analyzed for a wide range of natural signaling molecules, including nitric oxide, adenosine, opioids, adrenergic agents, prostaglandins, estrogens, androgens, 5-hydroxytryptamine, and dopamine (Calabrese EJ, Bachmann KA, Bailer AJ, Bolger PM, Borak J, et al.(2007), Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework.Toxicol Appl Pharmacol.222: 122-128 and Hayes DP.Nutritional hormesis.Eur J Clin Nutr.2007;61: 147-159). Many of these hormetic substances are concentrated by bacteria, fungi, viruses, and plants to protect themselves from predatory species.However, many of these substances can have beneficial effects when utilized at lower concentrations.

[0060] A hydrogel is a dilute cross-linked aqueous system comprising water and a gelling agent such as a polymeric plastic or a polysaccharide that can absorb and retain a significant amount of water to form a three-dimensional network structure. The hydrogel structure is generated by the interaction of hydrophilic groups or domains present in the polymer network with water upon hydration. Hydrogels are mainly classified as weak or strong depending on their flow behavior in the steady state.

[0061] Gelation occurs as the polymer concentration increases, and branching and cross-link formation begin due to the dispersion of the polymer. When the critical concentration is reached, the sol becomes a gel and a sol-gel transition occurs. The gel can be considered to be either chemically or physically bonded. Physical gels can be subcategorized as strong or weak depending on the nature of the bonds, and chemically gels with strong physical proximity have bonds that are permanent. Strong physical bonds include lamellar microcrystals, glassy nodules, or double and triple helices, while weak physical gels include hydrogen bonds, block copolymers, micelles, and ionic associations.

[0062] Due to its substantial water content, a hydrogel has a degree of flexibility similar to natural tissues and may exhibit viscoelastic or purely elastic behavior, as well as adhesiveness. The properties of a hydrogel can be modified by controlling its polarity, surface properties, mechanical properties, and swelling behavior. The gel may exhibit significant volume changes in response to small changes in pH, ionic strength, temperature, electric field, and light. Biodegradable hydrogels containing labile bonds are advantageous in applications such as tissue engineering, wound healing, and drug delivery. These labile bonds can be present in the polymer backbone or in the cross-linking agent used to prepare the hydrogel. Labile bonds can be broken enzymatically or chemically, most often by hydrolysis, under physiological conditions ( Bajpai, A.K., Shukla, S.K., Bhanu, S. & Kankane, S. (2008). Responsive polymers in controlled drug delivery. Progress in Polymer Science. 33: 1088 - 1118).

[0063] Ionic polymers having negatively charged groups at physiological pH can be crosslinked by the addition of polyvalent cations, and monovalent cations (e.g., K + Na + etc.) can also shield the repulsion of negatively charged groups (e.g., SO - 3 ) to form a stable gel. Examples include Na + alginate - , chitosan - polylysine, chitosan - glycerophosphate (Syed K. H. Gulrez 1 and Saphwan Al - Assaf. Progress in molecular and environmental bioengineering from analysis and modeling to technology applications Chapter 5: Hydrogels: Methods of preparation, characterization and applications. Publisher InTech. August, 01, 2011(34)) and chitosan - dextran hydrogels (Hennink, W.E. & Nostrum, C.F. (2002). Novel crosslinking methods to design hydrogels. Advanced drug delivery reviews. 54: 13).

[0064] Alginate is a naturally occurring anionic polysaccharide usually obtained from brown seaweeds, and its biocompatibility, low toxicity, relatively low cost, and Ca 2+Due to its ability to form hydrogels upon the addition of divalent cations such as those mentioned above, it has been widely studied and used in many biomedical applications. Alginate hydrogels have been used in various applications including wound healing and drug delivery. Alginate wound dressings maintain a moist wound environment, minimize bacterial infection at the wound site, and promote wound healing. Drug molecules can be released from alginate gels in a controlled manner depending on the crosslinking agent and method used. Furthermore, alginate gels can be administered orally or by injection, and are therefore widely useful in the pharmaceutical field.

[0065] Alginate is a polysaccharide composed of variable amounts of β-D-mannuronic acid and its C5-epimer, α-L-guluronic acid, linked by 1-4 glycosidic bonds. The ability of alginate to impart viscosity in a sol-gel depends on its molecular weight (MM). The molecular weight (MM) of alginate from algae has been found to range from 48 to 186 kDa (38), and some alginates isolated from Azotobacter vinelandii exhibit an MM in the range of 80 to 4,000 kDa (Galindo, E.; Pena, C.; Nunez, C.; Segura, D. & Espin, G. (2007). Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoate production by Azotobacter vinelandii. Microbial Cell Factories, 6, 1-1). The saccharide monomers are distributed in blocks of consecutive mannuronic acid residues (M), guluronic acid residues (G), or alternating residues (MG) depending on the source species (Smidsrod, O. & Draget, K. (1996). Chemistry and physical properties of alginates. Carbohydrates European, 14, 6-12). The G-block of alginate binds divalent cations (e.g., Ca 2+) participates in intermolecular cross-linking with [substance not specified] to form a hydrogel. Composition (i.e., M / G ratio), sequence, G-block length, and molecular weight are important factors that change the physical properties of alginate and alginate hydrogels (e.g., an increase in G-block length increases ionic bonds and the mechanical rigidity of the gel). These same properties control the stability of the gel, along with the release rate of the drug-containing alginate. Alginate with a low M / G ratio forms a strong and brittle gel, while alginate with a high M / G ratio forms a weak and soft but more elastic gel. Finally, bacterial alginate is acetylated at the O-2 and / or O-3 positions of mannuronic acid residues, to a variable extent (Skjak-Braek, G.; Grasdalen, H. & Larsen, B. (1986). Monomer sequence and acetylation pattern in some bacterial alginates. Carbohydrates Research, 154, 239-250). Variations in molecular weight, monomer block structure, and acetylation all affect the physicochemical and rheological properties of the gel polymer.

[0066] Most of the alginate uses divalent cations such as calcium or monovalent ions such as Na + or K + while other ions such as Mg 2+ have been proposed, but the concentration of magnesium ions required to initiate the gelation process is 5 to 10 times higher than that of calcium (Topuz, F., Henke, A., Richtering, W. & Groll, J. (2012). Magnesium ions and alginate do form hydrogels: a rheological study. Soft Matter. 8:4877-4881). Alginate may have reduced water solubility, such as in the form of alginic acid or calcium alginate where the ions are protected from ionization to yield an insoluble alginate. Water-soluble alginate is Na + or K +A monovalent anion such as or NH which is generally water-soluble 4 It can be easily prepared by forming a salt using a non-polar group such as. The first autophagy regulator (FAM)

[0067] As described above, the first autophagy regulator contains two phenyl rings linked by a linker L, and one or more R attached to the phenyl ring 1 or R 2 is a compound of formula I having. In some embodiments, L is -C≡C-, and these FAM compounds are generally known as "trans" in which the phenyl rings are linear from phenyl ring to phenyl ring. In other embodiments, L is -CH=CH-, and these FAM compounds are known as "stilbenes" which are cis- and trans-isomers with respect to the double bond. In some embodiments, the FAM compound is trans-stilbene. In still other embodiments, L is -CR a =CR b -, where R a and / or R b may be a phenyl ring or H. These are also stilbenes, in which case they are "phenylstilbene derivatives", and may also be trans-stilbene or cis-stilbene.

[0068] Substituents R 1 and R 2 which are shown in the structure and at least one of which must be present (i.e., at least one of m or n is ≧1) are contained in two "primary" phenyl rings. Optionally, there are also up to two "secondary" phenyl rings within any R a and R b . On each phenyl ring (up to 4 possible, two primary and two secondary), 0 to 5 of each substituent R 1~4 can be present. In certain embodiments, there are 1 to 3 R 1 and / or 1 to 3 R 2 substituents on the primary phenyl ring. In some embodiments, R 1 and / or R on the primary phenyl ring2 The position is mostly para and meta positions, that is, the 3, 4, or 5 positions on one phenyl ring and the 3’, 4’ and 5’ positions on the other phenyl ring, but it is also possible to have substituents at the ortho positions (2, 2’, 6, and 6’). There is 1, 2, or 3 R 1 substituents on the first phenyl ring, and correspondingly 0 to 3 R 2 substituents may exist on the second phenyl ring. Conversely, there is 1, 2, or 3 R 2 substituents on the second phenyl ring, and correspondingly 0 to 3 R 1 substituents may exist on the first phenyl ring. Furthermore, in some embodiments, the secondary phenyl ring may contain 1 to 3 substituents R 3 and R 4 . All permutations among these are possible. For example, 1 R 1 and 1 R 2 , 2 R 1 and 2 R 2 , 3 R 1 and 3 R 2 , 1 R 1 and 2 R 2 , 1 R 1 and 3 R 2 , 2 R 1 and 1 R 2 , 2 R 1 and 2 R 2 , 2 R 1 and 3 R 2 , 3 R 1 and 1 R 2 , or 3 R 1 and 2 R 2 are possible. The same is true for R 3 and R 4 . Each R 1~4 is independently selected, and when there are two or more, they may be the same or different. Examples of some specific first autophagy regulators are given in Table A. Since tran is an analog of stilbene, the detailed structure is not necessary for each individual compound.

[0069]

Table 1-1

[0070]

Table 1-2

[0071]

Table 1-3

[0072]

Table 1-4

[0073]

Table 1-5

[0074]

Table 1-6

[0075]

Table 1-7

[0076]

Table 1-8

[0077]

Table 1-9

[0078]

Table 1-10

[0079] Many stilbene compounds are well-studied, naturally occurring molecules, and some are readily available on the market. Others can be synthesized by conventional methods such as those described in Ali, M.A., Kondo, K. and Tsuda, Y. (1992). Synthesis and Nematocidal activity of Hydroxy stilbenes. Chem. Pharm. Bull. 40(5): 1130 - 1136; and Thakkar, K., Geahlen, R.L. and Cushman, M. (1993). Synthesis and Protein-tyrosine kinase inhibitory activity of polyhydroxylated stilbene analogues of piceatannol. J. Med. Chem. 36: 2950 - 2955). Phenylstilbene derivatives, i.e., phenylstilbene derivatives in which at least one R a or R b is a phenyl ring, and many other stilbenes can be synthesized according to the methods shown in the dissertation of Zhenlin Bai, Substituted Stilbenes and 1,2-Diaryl-1,2-diazidoethanes as Potential Anticancer Agents: Syntheses and Estrogenic / Antiestrogenic Properties in MCF-7-2a Cells, in the Department of Biology, Chemistry, Pharmacy of the Free University of Berlin (2006). Glucoside derivatives can be obtained according to the procedures described in International Publication No. WO 2007 / 020673 A1. Tran can be synthesized using the general procedures described in U.S. Patent No. 6,599,945 B2 to Docherty & Tsai.

[0080] The above FAM compounds are usually polar and have specific electronegative substituents (e.g., -OH, -OCH 3 , -NO2 It can be noted that it has, for example, - halo, - O(C = O)R, etc. Although this is not considered essential, there may be cases where it is desirable for the molecule to give liquid crystal-like behavior where it is lyotropic or has a partially regular structure in the solution state.

[0081] FAM compounds also include salts of the compounds identified above. FAM compounds, especially their mono- or polyhydroxylated compounds, readily release one or more protons depending on the pH to form anions. Such anions can combine with cations such as monovalent, divalent, and trivalent cations to form salts. For monovalent cations (M+), a single FAM binds to form M + FAM - salts. Similarly, for divalent cations (M 2+ ), two FAM molecules bind to form M 2+ (FAM - ) 2 salts, and for trivalent cations (M 3+ ), three FAM molecules bind to form M +3 (FAM - ) 3 salts. Salts can often be readily dissolved in aqueous media, which can facilitate formulation. Examples of cations for FAM salt formation, but not limited to, include Na + or K + , Mg 2+ , Mn 2+ , Zn 2+ , Ca 2+ , Cu + , Cu 2+ Fe 2+ , and Fe 3+ . Auxiliary autophagy modulators (AAM) and formulations

[0082] In certain embodiments, FAM is used in combination with an auxiliary autophagy modulator (AAM). When used, AMM can take any of several forms described herein and can fall into the following classifications: vitamins, amino acids, acidic sugars, and quinene derivatives. Vitamins

[0083] Vitamins A, B, C, D, E, and K can all be useful as AAMs. By convention, the term "vitamin" does not include other essential nutrients such as minerals, essential fatty acids, or essential amino acids (which require more than the amount of vitamins) nor many other nutrients that promote health. Currently, 13 vitamins are widely recognized. Vitamins are classified by their biological and chemical activities, not by their structures. Thus, each "vitamin" refers to several "vitamer" compounds that all exhibit biological activities associated with a particular vitamin. Such a set of chemical substances is grouped under the alphabetical "collective descriptor" name of a vitamin such as "vitamin A" that includes multiple compounds as follows. Vitamers are, by definition, convertible to the active form of the vitamin in the body and are sometimes interconvertible as well. In certain embodiments, salt forms of vitamins that generally do not have long-chain aliphatic side chains are excellent AAMs. In certain embodiments, oxygen-containing vitamins are suitable AAMs.

[0084] Also, most vitamins form salts that are also within the scope of AAMs. For example, vitamins can form salts by binding to cationic elements such as sodium, potassium, magnesium, manganese, calcium, copper, zinc, or iron. Further, vitamins can form diethanolamine salts, 2-amino-2-ethyl-1,3-propanediol salts, triethanolamine salts, morpholine salts, piperazine salts, piperidine salts, arginine salts, lysine salts, and histidine salts. Some vitamins form acetate salts, palmitate salts, oleate salts, linoleate salts, stearate salts, lactate salts, succinate salts, maleate salts, citrate salts, and the like.

[0085] Vitamin A refers to a group of lipid-soluble unsaturated isoprenoid compounds including, but not limited to, retinol, retinal, retinoic acid, carotenoids, retinyl acetate, retinyl palmitate, α-carotene, β-carotene, γ-carotene, β-cryptoxanthin, xanthophyll, cryptoxanthin, 13-cis retinoic acid, 13-trans retinoic acid, tretinoin, ATRA (all-trans retinoic acid), rutin, 11-cis-retinal, 11-cis-retinol, 9-cis-retinal, lecithin, retinyl ester, 9-cis-β-carotene, retinyl palmitate, acitretin, vitamin A 2 (3,4-dehydroretinol), A 3 (3-hydroxyretinol), and salts thereof. All isomers and stereochemical forms of these isoprenoids are encompassed by the present invention.

[0086] Vitamin B includes, but is not limited to, the following compounds: thiamine (B1); riboflavin (B2); niacin or niacinamide (forms of B3); pantothenic acid, panthenol, panthenol, and calcium pantothenate (forms of B5); pyridoxine, pyridoxine 5'-phosphate, pyridoxal, pyridoxal phosphate, pyridoxal 5'-phosphate, pyridoxamine, pyridoxamine 5'-phosphate, 4-pyridoxic acid (forms of B6); biotin, vitamin H, or coenzyme R (forms of B7); folic acid, folate, vitamin M, vitamin Bc, pteroyl-L-glutamic acid, and pteroyl-L-glutamate salts (forms of B9); and cobalamin, cyanocobalamin, hydroxycobalamin, methylcobalamin, adenosylcobalamin (forms of B12); and salts thereof.

[0087] Vitamin C refers to ascorbic acid, its ascorbic acid anion, and ascorbates, as well as ascorbyl palmitate and their salts (e.g., ascorbyl palmitate, magnesium ascorbyl palmitate, manganese ascorbyl palmitate, calcium ascorbyl palmitate, zinc ascorbyl palmitate, iron ascorbyl palmitate), benzyl ascorbate, and 2-ascorbyl phosphate.

[0088] Vitamin D refers to a group of fat-soluble secosteroid molecules, including calcidiol, calciferol (INN), ergocalciferol and lumisterol (form of D1); ergocalciferol, formergergol, and 25-hydroxyvitamin D 2 (form of D2); cholecalciferol, 7-dehydrocholesterol, and 25-hydroxy cholecalciferol (or 25-hydroxyvitamin D 3 , abbreviated as 25(OH)D 3 , (form of D3); 22-dihydroergocalciferol (D4); sitocalciferol, 7-dehydrositosterol (D5); 25-D-glucuronic acid, 25-D-hexuronic acid, 25-hydroxyvitamin D 2 -25-β-D-glucuronide, and their salts, including but not limited to these.

[0089] Vitamin E refers to a group of fat-soluble compounds that are either tocopherols or tocotrienols, and the most active among them is α-tocopherol. Other tocopherols include beta, gamma, and delta. Similarly, tocotrienols also exist in the forms of alpha, beta, gamma, and delta. All isomers and stereochemical forms of these tocopherols and tocotrienols and their salts are encompassed by the present invention. For example, synthetic vitamin E is a mixture of eight isomers and is usually labeled as "all-rac" or "dl". Tocopherol and tocotrienol derivatives include all R and all S stereoisomers of tocopherol (RRR, RRS, RSR, SRR, RSS, SRS, and SSS) as well as two stereoisomers of tocotrienol (e.g., R or S-α-tocotrienol). Other examples include: conjugated vitamin E molecules; vitamin E or tocopherol or tocotrienol esters; alpha-tocopheryl acetate; vitamin E esters containing a group of compounds formed by esterifying vitamin E molecules with carboxylic acids (e.g., alpha-tocopheryl succinate); in many cases, a mixture of two or more enantiomers of other tocopherols (β, γ, δ, ε, ζ, η), or tocotrienol, n-propionate, or linoleate, such as d-α-tocopherol present as vitamin E acetate or alpha-tocopheryl acetate. Water-soluble forms of vitamin E include: R-(+)-alpha lipoic acid magnesium, 6-hydroxy-2,5,7,8-trimethylchroman-2-carboxylic acid (trolox), or salts of vitamin E.

[0090] Vitamin K refers to a group of compounds having a 2-methyl-1,4-naphthoquinone core and a side chain at the 3-position. Vitamer K 1 (phylloquinone, phytomenadione, or phytondione) and K 2 (menaquinone) occur naturally. Indeed, K 2is not a single compound but a series of compounds with isoprenoid side chains of various lengths. The menaquinone family is sometimes called MK-n, where n is the number of isoprenoid groups, and n = 4 is the most common. Additionally, there is K without a side chain 3 (menadione), K 4 , K 5 (2-methyl-4-amino-1-naphthol hydrochloride), vitamin K 6 (2-methyl-1,4-naphthalenediamine), and several synthetic vitamin K analogs including K 7 have been prepared. Many vitamin K compounds form salts, and the divalent salts are the most useful as AAMs. For example, the salts of cations can take the following forms: M(Ki) where M is a divalent cation 2 , or M(Ki) where M is a trivalent cation 3 . In certain embodiments, useful AAMs include salts of dimers of vitamin K and divalent cations such as Ca or Mg.

[0091] Vitamin P (although an older term) refers to a group of flavonoids having a general structure of a 15-carbon skeleton consisting of two phenyl rings (A and B) and a heterocyclic ring (C). This carbon structure can be abbreviated as C6-C3-C6. Based on the nature and position of the substituents on the skeleton, flavonoids fall into one of the following three chemical classifications: 1) flavonoids or bioflavonoids based on a flavone core (2-phenyl-1,4-benzopyrone); (2) isoflavonoids based on a 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) core structure; and (3) neoflavonoids based on a 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) core structure. Amino acids

[0092] Certain amino acids and their derivatives are also useful as AAMs. As is well known, amino acids have the general formula

[0093]

Chemical formula

[0094] and wherein R is any of several well-known side chains. There are 20 amino acids that are coded by a universal code, and humans synthesize 11 of these, so the other 9 are "essential" amino acids and must be obtained in the diet. The 20 are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. All isomers and stereochemical forms of these amino acids and salts of these amino acids are included in the present invention. In certain embodiments, useful amino acids include, but are not limited to, tyrosine, phenylalanine, cysteine, serine, threonine, and tryptophan. Further, certain amino acid derivatives, such as lycopene and N-acetylcysteine (NAC), are useful. Acidic sugars

[0095] Acidic sugars include monosaccharides and disaccharides formed from 4- to 6-membered aldoses and ketoses. They are usually acidic because protons are readily released from many of their hydroxyl groups. Useful monosaccharides include, but are not limited to, erythrose, erythulose, threose, ribose, ribulose, arabinose, xylose, xylulose, glucose, dextrose (or D-glucose), mannose, galactose, fructose, and sorbose. Useful disaccharides include, but are not limited to, maltose, sucrose, lactose, cellobiose, and trehalose. All isomers and stereochemical forms of these saccharides are included in the present invention. Quinone derivatives

[0096] Quinone derivatives include those having 1, 2, or 3 rings and thus,

[0097]

Chemical formula

[0098] 1,4 - benzoquinone based on

[0099] [Chemical formula]

[0100] 1,4 - naphthoquinone based on

[0101] [Chemical formula]

[0102] 9,10 - anthraquinone based on

[0103] [Chemical formula]

[0104] and 1,3 - indandione based on are included. These quinone derivatives may contain substituents at any position other than the ketone, and the substituents are generally selected from hydroxyl, methoxy, methyl, ethyl, halo, and amino. 1 to 4 hydroxyl substituents are particularly useful. For example, other examples of hydroxy - 1,4 - benzoquinone derivatives include 2 - hydroxy - 1,4 - benzoquinone, 2,3 - dihydroxy - 1,4 - benzoquinone, 2,5 - dihydroxy - 1,4 - benzoquinone, 2,6 - dihydroxy - 1,4 - benzoquinone, 2,3,5 - trihydroxy - 1,4 - benzoquinone, and 2,3,5,6 - tetrahydroxy - 1,4 - benzoquinone. Other 1,4 - benzoquinone derivatives include the following: 2,6 - dimethoxy - 1,4 - benzoquinone, 2,3,5,6 - tetramethyl - 1,4 - benzoquinone, 1,4 - benzoquinone tetracarboxylic acid, brateraquone, 2,5 - dichloro - 3,6 - dihydroxybenzoquinone (chloranilic acid), and 2 - isopropyl - 5 - methylbenzo - 1,4 - quinone (thymoquinone).

[0105] Examples of mono-, di-, and tetra-hydroxy-1,4-naphthoquinones include 2-hydroxy-1,4-naphthoquinone (lawsone), 5-hydroxy-1,4-naphthoquinone (juglone), 6-hydroxy-1,4-naphthoquinone, 2,3-dihydroxy-1,4-naphthoquinone, 2,5-dihydroxy-1,4-naphthoquinone, 2,6-dihydroxy-1,4-naphthoquinone, 2,7-dihydroxy-1,4-naphthoquinone, 2,8-dihydroxy-1,4-naphthoquinone, 5,6-dihydroxy-1,4-naphthoquinone, 5,7-dihydroxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone (naphthazarin), 6,7-dihydroxy-1,4-naphthoquinone, and 2,3,5,7-tetrahydroxynaphthoquinone (spinachrome B). Other 1,4-naphthoquinone derivatives include menadione (also known as vitamin K3 or 2-methyl-1,4-naphthoquinone).

[0106] Examples of 9,10 anthraquinones include the dihydroxy derivatives 1,2-dihydroxyanthraquinone (alizarin), 1,3-dihydroxyanthraquinone (purproxanthin, xanthopurpurin), 1,4-dihydroxyanthraquinone (quinizarin), 1,5-dihydroxyanthraquinone (anthralfin), 1,6-dihydroxyanthraquinone, 1,7-dihydroxyanthraquinone, 1,8-dihydroxyanthraquinone (dantron, chrysazin), 2,3-dihydroxyanthraquinone, 2,6-dihydroxyanthraquinone, and 2,7-dihydroxyanthraquinone; the trihydroxy derivatives 1,2,3-trihydroxyanthraquinone (anthragallol), 1,2,4-trihydroxyanthraquinone (purpurin), 1,2,5-trihydroxyanthraquinone (oxyanthralfin), 1,2,6-trihydroxyanthraquinone (flavopurpurin), 1,2,7-trihydroxyanthraquinone (isopurpurin, anthrapurpurin), 1,2,8-trihydroxyanthraquinone (oxychrysazin), 1,3,5-trihydroxyanthraquinone, 1,3,6-trihydroxyanthraquinone, 1,3,7-trihydroxyanthraquinone, 1,3,8-trihydroxyanthraquinone, 1,4,5-trihydroxyanthraquinone, 1,4,6-trihydroxyanthraquinone, 1,6,7-trihydroxyanthraquinone, and 2,3,6-trihydroxyanthraquinone.

[0107] Salt complexes may be formed from FAM and / or AAM compounds as already described. Further, the complex may be formed between a FAM compound and a specific AAM compound. Such FAM+AAM complexes include those containing vitamins, such as ascorbate and ascorbyl palmitate; those containing amino acids, such as alginate, lysinate, aspartate, glutamate; and those containing acidic sugars, such as glucoside, riboside, galactoside, mannoside, etc. at least. Example 4 gives some specific examples of both salts and complexes of FAM and AAM compounds.

[0108] The properties of FAM compounds as liquid crystals can promote and / or mediate their roles in wound healing. The polar nature of liquid crystals allows them to self-organize into polymer-like structures, and thus, they can create unique sets of liquid crystal hydrogels by (i) generating their own hydrogels and / or (ii) adding these molecules to conventional hydrogels to enhance the sol-gel transition state. These gels may be modified to create any of the various aforementioned gel types, from strong chemical bonds to weak chemical bonds, or to create biodegradable gels. Without wishing to be bound by a particular theory, these FAM molecules are thought to be useful for applications in wound healing, one reason being that the molecules themselves behave like collagen fibrils that assemble higher-order structures and can serve as scaffolds and aid cell migration during wound healing. Furthermore, the addition of these liquid crystal hydrogels can promote proper collagen alignment and orientation that reduces the risk of scar or keloid formation during the wound healing process.

[0109] In addition to hydrogel formulations, another useful formulation of FAM compounds is one that contains cyclodextrin. Common cyclodextrin formulations consist of FAM with a ratio of FAM:cyclodextrin of about 1:1, 1.5:1, 1.5:2, 1.5:3, 1:3, 1.5:4, 1:4, 1.5:5, 1:5, 1.5:6, 1:6, 1.5:7, 1:7, 1.5:8, 1:8, 1.5:9, 1:9, 1.5:10, or 1:10. These ratios allow for sufficient dissolution of FAM in cyclodextrin. The added AAM can be from about 0.1% to about 99% (w / v), for example, 0.1 - 10%, 10 - 20%, 20 - 30%, 30 - 40%, 50 - 60%, 60 - 70%, and 80 - 90% (w / v) or more. Usefulness of FAM and AAM in Autophagy Regulation

[0110] In recent years, scientists have studied the effects of modulating the autophagy pathway as a way to treat various severe diseases. In fact, dysregulation of autophagy has been associated with major diseases including heart disease, cancer, and diabetes (see Figure 2 from Klionsky D.J. (2010). The Autophagy Connection. Developmental Cell. Jul 20; 19(1): 11-2.). This paper describes the relevance between the autophagy pathway and major human diseases. Although not naming individual "myopathies", the ability to regulate this pathway has a major relevance to the regulation of disease outcome. Depending on the disease state, it may be beneficial to upregulate or downregulate the cellular level of autophagy.

[0111] In some disease states, the pathological cause is related to suppressed autophagy activity or activation of autophagy, and the associated signaling pathways result in suppression of inflammation. Therefore, diseases or conditions in which it may be beneficial to upregulate autophagy include wound healing, promotion of hair regrowth, bacterial infection, inflammation, viral infection, Parkinson's disease, aging, Alzheimer's, neurodegenerative diseases, neuropathy, cardiovascular diseases, burns, heart disease, Alzheimer's disease, atherosclerosis, arteriosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, inflammatory bowel disease, colitis, diabetes, type I and type II diabetes, amyloidosis, synovitis, dermatitis, vasculitis, autoimmune diseases with inflammation, blood diseases, aplastic anemia, endometriosis, hepatitis, herpes, HIV, multiple sclerosis, retinal detachment, age-related macular degeneration, retinitis pigmentosa, and Leber congenital amaurosis, lysosomal storage diseases, arthritis, psoriasis, osteopenia, osteoporosis, surgical scars, surgical adhesions, space travel (bone density impairment), tendinitis, and ulcerative colitis.

[0112] In other pathological conditions, the pathological causes are related to the overexpression of autophagy activity and the activation of autophagy and its related signaling pathways. Accordingly, diseases or conditions in which it is beneficial to downregulate autophagy include aging, cancer, polycystic kidney and liver diseases, kidney diseases, liver diseases, asthma, diabetic retinopathy, fibromyalgia, ankylosing spondylitis, celiac disease, Graves' disease, lupus, metabolic diseases, nephritis, rheumatoid arthritis, osteolysis, ischemia-reperfusion (I / R) injury, organ and tissue transplantation, scleroderma, and sepsis.

[0113] In wound healing, an increase in autophagy levels helps protect tissues, reduce inflammation, and promote the synthesis of procollagen, hyaluronan, and elastin. As shown herein, FAM and AAM compounds are used alone or in combination to promote wound healing, hair growth, and skin repair after damage by UV irradiation exposure.

[0114] In bacterial infections, various types of bacteria attempt to prevent bacterial cell uptake and disrupt the autophagy pathway, ultimately leading to the degradation of bacteria in autophagolysosomes. Two clinically important skin pathogens, Streptococcus and Staphylococcus aureus, disrupt the autophagy pathway (see I. Nakagawa, et al, Autophagy defends cells against invading group A Streptococcus, Science 306, 1037-1040 (2004); and Schnaith, et al, Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death, J BiolChem 282, 2695-2706 (2007)). Invasive skin infections caused by group A Streptococcus are characterized by the prevention of bacterial cell uptake by encapsulation, and when bacteria are taken up by keratinocytes, most streptococci die within a few hours (H. M. Schrager, J. G. Rheinwald and M. R. Wessels: Hyaluronic acid capsule and the role of streptococcal entry into keratinocytes in invasive skin infection, J Clin Invest 98, 1954-1958 (1996)). Nakagawa et al (cited above) showed that autophagy is involved in the killing activity. Although some bacteria survive, the reduction in the number of extracellular streptococci may have a partial protective effect. Since the mechanism of action of autophagy against group A Streptococcus in keratinocytes has not been studied, further research will be needed to understand the validity and efficiency of this putative antibacterial strategy in the skin.Staphylococcus aureus induces autophagy via its alpha-toxin (Schnaith et al, above, and M. B. Mestre, C. M. Fader, C. Sola and M. I. Colombo: Alpha-hemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells, Autophagy 6, 110-125 (2010)). Pore-forming toxins induce a decrease in nutrient and energy levels that triggers autophagy as a rescue mechanism to re-establish cellular homeostasis (N. Kloft, et al: Pro-autophagic signal induction by bacterial pore-forming toxins, Med Microbiollmmunol 199, 299-309 (2010)). Whether autophagy suppresses or enhances Staphylococcus aureus infection of the skin in vivo has not been determined.

Example

[0115] Example 1: Synthesis of Specific FAMs of the Invention : The following compounds were prepared and given the identification numbers shown in Table B.

[0116]

Table 2-1

[0117]

Table 2-2

[0118] The stilbene compounds (BM2xxx series) were synthesized / obtained according to the procedures described in the above-mentioned Ali, M et al 1992 and Thakkar, K. et al 1993. The trane compounds (BM3xxx series) were synthesized according to the procedures described in U.S. Patent No. 6,599,945B2 of Docherty & Tsai.

[0119] Example 2: Simplified Synthetic Procedure for 4-Hydroxy-4’-Methoxytolan

[0120]

Chemical formula

[0121] The synthetic procedure of 4-hydroxy-4'-methoxytlane (4-((4'-methoxyphenyl)ethynyl)phenol) is as follows: (Pavia, M. R.; Cohen, M. P.; Dilley, G. J.; Dubuc, G.R.; Durgin, T. L.; Forman, F. W.; Hediger, M. E.; Milot, G.; Powers, T. S.; Sucholeiki, I.; Zhou, S.; Hangauer, D. G. The design and synthesis of substituted biphenyl libraries. Bioorg. Med. Chem. 1996,4, 659-666., Jeffery, T. Heck-type reactions in water. Tetrahedron Lett, 1994,35, 3051-3054, Jeffery, T.; Galland, J. C. Tetraalkylammonium salts in heck-type reactions using an alkali metal hydrogen carbonate or an alkali metal acetate as the base. It was a Heck-type reaction modified from Tetrahedron Lett, 1994, 35, 4103-4106, and Schmidt-Radde, R. H.; Vollhardt, K.; Peter C. The total synthesis of angular [4]- and [5]phenylene J Am Chem Soc, 1992, 114, 9713-9715). The obtained product was a yellowish powder, 1 HNMR (CDCl 3, 300 MHz): δ ppm: 7.44 (d, 4H, J = 8.7, Ar-H), 6.89 (d, 2H, J = 8.7, Ar-H), 6.82 (d, 2H, J = 8.7, Ar-H), 4.89 (s, 1H, OH), 3.85 (s, 3H, CH 3 O) was used for confirmation. The obtained product had a purity of 98.2% and was used in all subsequent tests.

[0122] Example 3: Summary of the Synthetic Procedure for 2,4,4’-Trimethoxytolan : The synthesis was a Heck-type reaction similar to that in Example 2.

[0123]

Chemical Structure

[0124] The obtained product was an off-white powder, 1 HNMR (CDCl 3, 400 MHz): δ ppm: 7.47 (d, 2H, J = 6.4, Ar-H), 7.40 (s, 1H, Ar-H), 6.85 (d, 2H, J = 6.8, Ar-H), 6.47 (dd, 2H, J = 2.4, Ar-H), 3.89 (s, 3H, CH 3 O), 3.82 (s, 6H, 2CH 3 O) was used for confirmation and it was found to have a purity of 99.3%.

[0125] Example 4: Melting Points of FAM Salts and FAM and AAM Salt Complexes:All salts were manufactured by formulating magnesium hydroxide, zinc oxide, ascorbic acid, or ascorbyl palmitate with each stilbene, tren, or combination in sufficient amounts to produce a salt solution. Next, each solution was dried using a rotary evaporator (Centrifan, Harvard Biosciences) set at 40 °C in a 20 mL scintillation vial, evaporated with a mixture of ethanol and ice to evaporate more slowly. Once evaporated to complete dryness, the salts were pulverized into a fine powder and their melting points were used to confirm salt formation. The melting points (Table C below) were measured using a Meltemp II apparatus equipped with a temperature probe and thermocouple that gave a digital readout. The apparatus was calibrated and compounds with known melting points were tested to confirm calibration prior to analysis of unknown compounds.

[0126]

Table 3

[0127] Example 5: LC3-II (Microtubule-Associated Protein 1 Light Chain 3) in Human Dermal FibroblastsStaining: The staining method was modified from the procedures described in Furuta, S, (2000) Ras is involved in the negative control of autophagy through the class I PI3 -kinase, Oncogene.23: 3898-3904; Ge, J.N. et al, (2008) Effect of starvation-induced autophagy on cell cycle of tumor cells, Chinese Journal of Cancer27:8 102-108; and Settembre, C. et al (2011) TFEB Links autophagy to lysosomal biogenesis, Science 332: 17 1429-1433. HDFn (human neonatal dermal fibroblasts, ThermoFisher) cells were seeded at 5,000 cells / well on clear bottom black wells (Coning, Corning, NY). The cells were then treated with medium alone, FAM, AAM, or combinations thereof for 8 hours. After the treatment time, the cells were fixed with 4% (w / v) PFA (paraformaldehyde) for 15 minutes at room temperature. The cells were then washed with PBS (phosphate buffered saline, pH 7.4) and blocked with 5% (w / v) BSA in PBS for 1 hour. The cells were washed again with PBS and incubated with a primary antibody against LC3-IIB (rabbit monoclonal antibody, Thermo-Fisher) for 3 hours. The cells were rinsed again with PBS and a secondary fluorescent antibody (Alexafluor 488 rabbit anti-goat, Thermo-Fisher) was added for 30 minutes. The plates were then imaged using a SpecraMax i3X (Molecular Devices, Sunnyvale, CA) and the number of LC3-II positive cells was measured using SoftMax Pro 6.5.1 software. The results are shown in Figure 3.

[0128] Example 6: Autophagy Regulation and Hormesis : LC3-II Western blot: Human dermal fibroblasts were seeded at 1×10 per T25 tissue culture treated flask 6Cells were seeded at a density of. The cells were then treated with FAM, AAM, or a combination for 8 hours. The cells were then trypsinized, washed with 1×PBS, and lysed on ice with RIPA buffer + protease inhibitor. The cells were sonicated and the protein concentration was measured by performing a BCA protein assay (Pierce Scientific). The concentration was then normalized to 100 μg per sample and run on a 12% (w / v) polyacrylamide gel containing a loading dye and appropriate molecular weight markers (all reagents were purchased from National Diagnostics). The gel was then transferred, blocked, and a primary monoclonal antibody against MAPLC3-2 (Thermo-Fisher) was added to the membrane and incubated overnight. After appropriate washing, a Eu-labeled secondary antibody (Molecular Devices) was added and the membrane was imaged using a Spectramax i3x equipped with a Scan Later module. The gel was then stripped and reprobed for actin to confirm appropriate loading. The change in average band intensity was measured using SoftMax Pro 6.5.1 software. The results are shown in Figures 4 and 5.

[0129] Example 7: Autophagy, Wound Healing, and Skin: Very low levels of autophagy exist in the skin, which functions to degrade protein aggregates and damaged organelles and change the skin color via the FGF-PI3K-AKT-MTOR signaling pathway in melanosomes (Belleudi, et al. The receptor tyrosine kinase FGFR2WKGFR controls early differentiation of human keratinocytes, PLoS One 2011; 6:e24194; PMID:21957444; http: / / dx.doi.org / 10.1371 / journal.pone.0024194; and Belleudi, et al, Expression and signaling of the tyrosine kinase FGFR2b / KGFR regulates phagocytosis and melanosome uptake in human keratinocytes. FASEB J 2011; 25: 170-81; PMID:20844240; http: / / dx.doi.org / 10.1096 / fj.10-162156.). Induction of autophagy in human keratinocytes negatively regulates p62 and prevents the induction of excessive inflammation and cathelicidin (found in the lysosomes of macrophages and PMNs) (Lee, et al Autophagy Negatively Regulates Keratinocyte Inflammatory Responses via Scaffolding Protein p62 / SQSTMl. J Immunol, published online 15 December 2010). In a deep wound second-degree burn model, the autophagy inducer rapamycin promotes autophagosome formation, improves wound re-epithelialization time, and reduces IL-8 levels, methanedicarboxylic aldehyde levels (an indicator of MDA oxidative stress), and myeloperoxidase levels (hypochlorous acid (HOCl), hydrogen peroxide (H 2 O 2) and an indicator of the production of chloride anions (Cl-) were shown to be decreased (Xiao et al, (2013) Rapamycin reduces burn wound progression by enhancing autophagy in deep second-degree burn in rats. Wound Rep.Reg. 21: 852-859). This indicates that induction of autophagy in the skin brings about an anti-inflammatory effect. Rapamycin is a known hormetic chemical that can prevent or treat various diseases when used in a dose-dependent manner.

[0130] The formulation described in this patent is a hormetic substance that has been shown to induce autophagy in a dose-dependent manner for the treatment of various diseases and conditions, including increased wound closure and re-epithelialization, as shown below.

[0131] Example 8: Protein Kinase B (AKT) : HDFn cells were seeded into T-25 flasks at a density of 1×10 6 cells per flask and allowed to adhere and spread overnight. The cells were then treated with control medium, FAM, AAM, or combinations for 8 hours. The cells were removed using trypsin, followed by a trypsin neutralizer, and centrifuged to recover the cell pellet. The cells were washed with ice-cold 1×PBS and then lysed on ice with RIPA buffer + protease inhibitor. The samples were centrifuged and aliquoted, and frozen until they could be assayed using ELISA. The cells were sonicated and diluted 1:5 as recommended in the kit instructions. The samples were then assayed using an AKT ELISA (Thermo-Fisher), and the concentration was measured using an AKT standard curve. The results are shown in Figure 6.

[0132] Example 9: Fibroblast Growth Factor (FGF): HDFn cells were seeded in 6-well tissue culture-treated plates and allowed to reach 70 - 80% (cells / area) confluence. Next, a "wound" was made in the center of the plate using a cell scraper to simulate damage to the cell monolayer. The dish was then washed with culture medium to remove cell debris. The cells were then treated with control medium, FAM, AAM, or combinations, and samples were taken at 3, 8, and 24 hours. The samples were centrifuged, aliquoted, and frozen until they could be assayed using ELISA. The concentration of EGF in each sample was measured using an EGF streptavidin-HRP ELISA kit and compared to a known standard curve. The results are shown in Figure 7.

[0133] Example 10: In Vivo Evaluation of LCB for Wound Healing:Twenty 5- to 6-week-old Balbc / J (stock number 000651) male mice were transferred to Jackson Labs, an in vivo research facility in Sacramento, California. The mice were ear-notched for identification and housed individually and securely ventilated in polycarbonate cages using HEPA-filtered air. A corn cob bedding called Bed-o-cob was used, and the cages were changed every two weeks. The animal room was illuminated throughout with artificial fluorescent lights having a controlled 12-hour light / dark cycle. The ambient temperature and relative humidity ranges in the animal room were 22 ± 4°C and 50 ± 15%, respectively. The animal room was set to have 15 air exchanges per hour. Filtered tap water was acidified to pH 2.8 - 3.1, and rodent solid feed was provided ad libitum. After 5 - 7 days of acclimation, the mice were randomized by weight into two cohorts of 10 each. On study day 0, the mice were anesthetized and two full-thickness excisional wounds (approximately 6 mm) were created on the dorsal (back) side of the mice. One of the wounds was covered using a semi-occlusive polyurethane dressing (Tegaderm™). The dressing covered the wound for 5 (five) consecutive days starting on the day of the wound (d0). The dose of 4-hydroxy-4'-methoxythran was based on the dose that had promoted lesion healing in mice in previous herpes virus studies. Wound measurements were taken on days 5, 7, 9, and 11. Digital images of the wounds of each mouse were taken. The test agent was a viscous paste and was surrounded in situ with a saddled dressing of the wound to eliminate cross-contamination and wound closure due to shrinkage. Thus, the change in wound area was due to re-epithelialization. For 5 days, all wounds were covered with glycerin (control) or glycerin and the test agent. On day 5, the test agent was removed and the change in wound area was measured (as area / area %) to evaluate the persistence of the compound at the wound site. By day 5, glycerin had produced 26.1% wound closure, while 4-hydroxy-4'-methoxythran (2.5%) had produced 92.8% wound closure and 5% 4-hydroxy-4'-methoxythran had produced 91.4% wound closure. Prior to statistical analysis, a normality test was performed and Z-scores were calculated to determine the normal distribution region of each wound on day 1 for all mice across the groups as r 2It was confirmed that = 0.989. The statistic is calculated based on a one-way analysis of variance comparing the changes in wound area on the 5th day. All mice tolerated the treatment, and all weekly clinical observations reported bright, sensitive, highly responsive, and hydrated mice.

[0134]

Table 4

[0135] Example 11: Excised Skin Samples Treated with Control or 5% (w / v) 4-Hydroxy-4’-Methoxytolan In the H&E staining of cells treated with the control, a keratinocyte layer 1 - 2 cells thick was seen on top of a gently organized layer of myofibroblasts, and the connective tissue had a few deep sebaceous glands that had begun to form. In the skin treated with 4-hydroxy-4'-methoxytiane, extensive proliferation of keratinocytes with a cell layer 7 - 8 cells thick was seen. The dermis showed regular and proliferative myofibroblasts, as well as connective tissue that extended to the surface and surrounded highly proliferative and ramified sebaceous glands that reconstructed hair follicles.

[0136] Example 12: Autophagy and Hair Regrowth (Hair Loss) in Skin Appendages : Autophagosome-like structures have been detected by electron microscopy in hair and sebaceous glands. The physiological relevance of autophagy in skin appendages is not well understood currently, but current existing data suggest that induction of autophagy may prevent hair loss through a Wnt1-dependent cellular rejuvenation process in which damaged cells undergo cell death and hair follicle stem cells are stimulated to cause hair growth (Castilho R.M., et al. (2009) mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging, Cell Stem Cell 5, 279 - 289; and Vishnyakova, et al. (2013) Possible Role of Autophagy Activation in Stimulation of Regeneration, Molecular Biology. 47(5): 692 - 700).

[0137] Example 13: UV Irradiation Damage and Anti-Aging The skin is the largest organ in the human body and is in contact with the environment. Thus, the skin is constantly subjected to damage from both external and internal sources that threaten its balance and alter its appearance. This damage often manifests as chronic low-level inflammation. For example, excessive exposure to UV is reflected in various skin symptoms such as photodermatitis, solar elastosis, premature appearance of the effects of skin aging, skin sagging, deep wrinkling, roughness, dryness, the appearance of depigmented or hyperpigmented spots, and blood vessel dilation. The manifestation of these UV exposures, which reflect profound structural changes in the skin tissue, is unsightly and many people tend to want to remove them. TEM and IF microscopic analyses of cultured dermal fibroblasts from women of various ages revealed a disorder in the pathological efflux of autophagic body fluids. Treating young dermal fibroblasts with a lysosomal protease inhibitor to mimic the state of aged dermal fibroblasts resulted in a decrease in autophagy activity, a change in the fibroblast content of type I procollagen, hyaluronan, and elastin, and caused the disintegration of collagen fibrils. These findings together suggest that impaired autophagy induction leads to deterioration of dermal integrity and skin vulnerability (Tashiro K, et al. (2014) Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components, Biochem Biophys Res Commun. 443(1): 167-172).

[0138] Autophagy ensures that damaged cell organelles and protein aggregates are properly degraded and do not accumulate to cause cell dysfunction. Also, an enhancement of autophagy activity is seen in response to calorie restriction (CR), which has been shown to extend lifespan.

[0139] Example 14: 40% (w / v) Solution of Hydroxypropyl-β-Cyclodextrin: Prepare a solution by adding 40.0 g of hydroxypropyl-β-cyclodextrin to 70 mL of water in a sterilized graduated beaker and mixing well. Make up to 100 mL with a suitable quantity (QS) when the solution becomes clear. Weigh 1.0 g of the liquid crystal compound (FAM) and transfer it to a sterilized glass bottle. Add 1.5 mL of ethanol to the bottle and dissolve completely. Slowly add 1 mL of cyclodextrin with stirring to ensure that the drug remains in the solution. Add 5 mL of water with stirring to ensure that the drug remains in the solution. Sonicate if necessary. The formulation should be a clear solution. Filter using a 0.2 μm filter. Freeze the suspension at -40 °C or below and lyophilize. The lyophilized cake may be reconstituted with sterile water before use.

[0140] Example 15: Preparation of FAM Cyclodextrin Formulation, an Injectable Liquid Crystal Formulation : Weigh 100 mg of 4,4'-dihydroxytran compound and place it in a 5 ml scintillation vial. Add 1.5 ml of absolute ethanol to the vial and shake until 4,4'-dihydroxytran is completely dissolved. Weigh hydroxypropyl-β-cyclodextrin (Sigma) free of pyrogenic substances on an analytical balance and place it in a graduated cylinder. Add water with shaking until the volume reaches 90 ml. Add the above ethanol solution of FAM to the aqueous solution containing hydroxypropyl-β-cyclodextrin with stirring. Add water to the clear solution to make the total volume 100 ml. Sterilize and filter this solution through a 0.22 micron filter. Freeze the suspension at -40 °C or below and lyophilize. The lyophilized cake may be reconstituted with sterile water for injection before use.

[0141] Example 16: Preparation of Infusion Solution: The solution is formulated from the following components; 0.625 parts of FAM-cyclodextrin; 0.3 parts of sodium saccharin; 0.1 parts of sorbic acid; 30.0 parts of ethanol; 1.0 part of flavoring agent; distilled water q.s. ad 100.0 parts. Dissolve the FAM-cyclodextrin complex and the flavoring agent in ethanol, and dissolve the sorbic acid and saccharin in distilled water. Mix the two solutions uniformly with each other, and filter the mixed solution until there is no suspension. 1 ml of the filtrate contains FAM and is an oral dosage unit composition having an effective therapeutic effect.

[0142] Example 17: Preparation of Micronized Drug and Drug Suspension : 16 grams of micronized FAM is pulverized using a 4-inch mill size and compressed nitrogen gas / compressed air (dew point > 40 °C) as the pulverizing gas. Manually supply the material to the hopper and place it at the upper part of the supply stage. The material is drawn into the sealed circular chamber by the pressurized pulverizing gas. The powder is suspended in a high-speed flow in the pulverizing chamber. Measure the particle size distribution with a particle size analyzer. Next, adjust the pulverizing conditions to obtain a material with an acceptable micron size.

[0143] Example 18: FAM or AAM Regulator-Cyclodextrin Complex Gel : Weigh 100 mg of FAM and place it in a sterilized test tube. Dissolve the FAM in 2 - 3 ml of purified anhydrous ethanol. Prepare a 10 - 50% (w / v) solution of 50 ml of hydroxypropyl-β-cyclodextrin (other cyclodextrins can also be used, such as α-cyclodextrin, γ-cyclodextrin, and specifically modified β-cyclodextrin, based on the need for water absorption) in a 150 ml sterilized beaker, and heat the solution to 70 - 80 °C while stirring on a hot plate. Slowly add the ethanol solution of FAM to the beaker while stirring. At this stage, AAM can be added at 1 - 25% (w / v). The addition of FAM initiates the gel-sol transition, and a gelling molecule such as sodium pectate dissolved in deionized water can be added as needed to further promote gelation. Other gel enhancers include monovalent or divalent cation FAM or AAM salts that form ionic crosslinks to promote gel formation.

[0144] The use of cations can be selected based on the desire to increase or decrease solubility in water. To facilitate the gelation process, FAM or its magnesium dimer is added to the alginate copolymer during mixing, and the M / G ratio is adjusted to obtain a stable FAM:Mg 2+ : alginate biodegradable sheet. The FAM cation interacts via hydrogen bonding with the pockets formed by the G-type alginate copolymer (see Figure 15). All ratios can be adjusted to optimize the FAM concentration and polymerization. Mg 2+ In addition to Mg, Ca 2+ , K + , or Zn 2+ and other ions such as can be added to further facilitate the gelation process.

[0145] Alginate G, M, or G / M copolymers form calcium alginate sheets upon the addition of divalent cations such as calcium. These sheets can be prepared in a sterile environment and are non-irritating, non-sensitizing, and biodegradable. Thereby, the liquid crystal FAM molecules are ideal molecules that promote the gelation, polymerization, and controlled copolymer block structure of alginate, change acetylation, and affect the physicochemical and rheological properties of the polymer. In addition to adjusting the molecular weight of individual alginate monomers, the selected FAM’s can also change the gel viscosity.

[0146] When various polymeric sugar molecules are combined with the liquid crystal FAM and AAM molecules described in this application, unique hydrogels, alginates, and drug delivery systems can be created for use in making novel wound care products. Examples include, but are not limited to, chitosan, hyaluronic acid, pectin, heparin, alginate, chondroitin sulfate A, D & E, PEG (polyethylene glycol), PLA (polylactic acid), and their polymers, and polyphosphazenes.

[0147] In some cases, additional FAM or AAM may be added to any of the above formulations to improve solubility, adjust the pH, balance the cation or anion concentration, improve adhesion to the skin, increase or decrease solubility in water, create a concentration gradient, improve gel-sol transitions, increase or decrease electrical conductivity, increase or decrease capacitance, or adjust the overall resistance or impedance.

[0148] The formulations described in this patent are liquid crystal hormesis substances that have been shown to induce autophagy in a dose-dependent manner for the treatment of various diseases and conditions, including increased wound closure and re-epithelialization.

[0149] Example 19: FAM or AAM Regulator-Alginate Complex Dissolve 1 gram of sodium alginate in deionized water in a 500 mL beaker and add the selected AAM (1 - 25% w / v) while mixing as needed. To this solution, add a therapeutically effective amount of FAM previously dissolved in ethanol to the desired concentration of 1 - 50% (w / v), and add a monovalent or divalent cation salt additive as needed until the gel reaches the desired consistency. For salts of FAM’s, the previous step is not necessary. Due to the liquid crystalline properties of the FAM molecules, unique co-block polymers that associate via hydrogen bonding, electrostatic bonding, and ionic bonding are formed. Next, this solution can be used to coat cotton fabric or other fibers to create alginate dressings. The same solution can also be used to create biodegradable sheets, films, beads, or gels.

[0150] The foregoing description of various aspects and embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the specific forms disclosed. Modifications or variations apparent from the above teachings are possible, and such modifications and variations are within the scope of the invention as determined by the appended claims when fairly, legally, and equitably construed within the scope of the rights to which they are entitled.

Claims

1. A formulation for promoting wound healing in a patient having a chronic wound or a skin disease, comprising a first autophagy regulatory compound having structure (I), wherein the first autophagy regulatory compound upregulates autophagy activity: 【Chemical 1】 The first autophagy regulatory compound is tran, wherein L is -C≡C-; R １ and R ２ are, independently, substituents at any available position of the phenyl ring; m and n are each independently 0, 1, 2, or 3 representing the number of substituents of the ring, and at least one of m or n must be ≧1; Each R １ and R ２ are, independently: (C １ -C ６ ) alkyl, (C ２ -C ６ ) alkenyl, or (C ２ -C ６ ) alkynyl, selected from -OH, -SH, -halo, -NH ２ , or NO ２ ; -R ５ which may be substituted with 1 to 3 substituents selected from; Y is O, S, or NH, and R ６ is H or R ５ -YR selected from ６ ; -ZR where Z is -N(C=O)- or -O(C=O)- ５ ; - halo; -NO ２ ; -SO ３ Na; - azide; and - glycoside; - and salts thereof; selected from; not 4,4'-(ethyne-1,2-diyl)diphenol (TOLECINE, also known as 4,4'-dihydroxytolan); at least one R １ is methoxy at the 4-position, or at least one R ２ is methoxy at the 4'-position; formulation.

2. The formulation according to claim 1, wherein the tran is hydroxylated tran having 1 to 4 hydroxyl substituents.

3. The formulation according to claim 1 or 2, for co-administration with an auxiliary autophagy regulatory compound.

4. The formulation according to claim 3, which is administered simultaneously with the auxiliary autophagy regulatory compound.

5. The formulation according to claim 4, which is administered after administration of the auxiliary autophagy regulatory compound.

6. The formulation according to claim 4, wherein the auxiliary autophagy regulatory compound is selected from the group consisting of substituted or unsubstituted para-benzoquinone; substituted or unsubstituted ortho-benzoquinone; and substituted or unsubstituted anthraquinone.

7. The formulation according to claim 4, wherein the auxiliary autophagy regulatory compound is selected from the group consisting of amino acids, acidic monosaccharides, and vitamins or salts thereof.

8. The formulation according to claim 7, wherein the vitamin is an oxygen-containing vitamin.

9. The formulation according to claim 7, wherein the vitamin is an isoprenoid-containing vitamin.

10. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more selected from aging, autoimmune diseases with inflammation, avascular necrosis, bacterial infection, cancer, diabetic neuropathy, endometriosis, fungal infection, infectious arthritis, inflammation, inflammatory bowel disease, ischemia, Lyme disease, organ / tissue transplantation, parasitic infection, psoriatic arthritis, psoriasis, pseudogout, rheumatoid arthritis, scleroderma, scurvy, sepsis, skin disease, surgical scar, surgical adhesion, transfection method, ulcerative colitis, ulcer, viral infection, wart, surgical wound, incision, laceration, cut and abrasion, donor site wound by skin graft, traumatic wound, infectious wound, ischemic wound, burn, blistering wound, non-infectious wound, contusion, incised wound, laceration, non-penetrating wound, open wound, penetrating wound, perforating wound, stab wound, pustule, subcutaneous wound, chronic ulcer, gastric ulcer, skin ulcer, peptic ulcer, duodenal ulcer, gastric ulcer, gout, hypertensive ischemic ulcer, congestive ulcer, sublingual ulcer, submucosal ulcer, symptomatic ulcer, trophic ulcer, tropical ulcer, and venereal ulcer.

11. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more skin diseases selected from keratosis, photoaging, psoriasis, skin rash, sunburn, and photoreaction process.

12. The preparation according to claim 1, wherein the chronic wound or skin disease is oral erosion and burn, post-extraction wound, intra-dental wound related to the treatment of cysts and abscesses, ulcers and lesions of bacterial, viral or autoimmune origin, mechanical, chemical, thermal, infectious, and lichenoid wounds; and one or more damaged oral tissues selected from herpes ulcer, aphthous stomatitis, acute necrotizing ulcerative gingivitis and oral burning sensation.

13. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more of corneal ulcer, radial keratotomy, corneal transplantation, epikeratophakia, and intraocular wounds induced by other surgeries.

14. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more of pruritus, proctitis, anal fissure, and hemorrhoid.

15. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more of dry cracked skin; severely dry skin due to other diseases (venous dermatitis); skin damaged by UV light; and seborrheic conditions.

16. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more wounds or erosions selected from burns, anthrax, tetanus, gas gangrene, scarlet fever (scalatina), erysipelas, sycosis barbae, folliculitis, impetigo contagiosa, or bullous impetigo.

17. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more of skin aging and / or non-acne-forming inflammation of the skin.

18. The preparation according to claim 1, wherein the chronic wound or skin disease is one or more of oral and perioral ulcers resulting from radiation and / or chemotherapy.

19. A preparation for upregulating autophagy activity in a patient in need of autophagy regulation, comprising a first autophagy regulatory compound having structure (I), wherein the patient is suffering from a condition in need of autophagy upregulation, the condition being wound healing, and the first autophagy regulatory compound upregulates autophagy activity: 【Chemical Formula 3】 The first autophagy regulatory compound is tran, wherein L is -C≡C-; R １ and R ２ are each, independently, a substituent at any available position of the phenyl ring; m and n each independently represent 0, 1, 2, or 3, which is the number of substituents on the ring, and at least one of m or n must be ≧1; Each R １ and R ２ are, independently: (C １ -C ６ ) alkyl, (C ２ -C ６ ) alkenyl, or (C ２ -C ６ ) alkynyl, and is optionally substituted with 1 to 3 substituents selected from -OH, -SH, -halo, -NH ２ , or NO ２ -R ５ ; Y is O, S, or NH, and R ６ is H or R ５ -YR selected from ６ ; -ZR where Z is -N(C=O)- or -O(C=O)- ５ ; - halo; -NO ２ ; -SO ３ Na; - azide; and - glycoside; - as well as salts thereof; is selected from; not 4,4'-(ethyne-1,2-diyl)diphenol (TOLECINE, also known as 4,4'-dihydroxytolan); at least one R １ is methoxy at the 4-position, or at least one R ２ is methoxy at the 4'-position; preparation.

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