Formulation and process for modulating wound healing

Abstract

To provide methods and compounds for wound healing by modulating autophagy.SOLUTION: A formulation for modulating autophagy comprises a first modulating compound (FAM) selected from compounds having the general structure (I), where L represents a linker selected from -C≡C-, (a tolan), -CH=CH- (a stilbene, preferably trans); or-CRa=CRb-stilbene derivative; where Ra and Rb are independently H or phenyl that may be substituted with -(R3)p or -(R4)q; R1 to R4 are independently substituents at any available position of the phenyl rings, preferably at 3, 3', 4, 4', and / or 5, 5'; and m, n, p, and q are independently 0, 1, 2, or 3 representing the number of substituents of the rings, respectively, but at least one of m or n must be≥1.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to Provisional Application Serial No. 62 / 144,539, filed April 8, 2015, the contents of which are expressly incorporated herein by reference. [Background technology]

[0002] 2. Background of the Invention The present invention relates generally to the field of cell biology, and more particularly to compounds, formulations, and methods for modulating autophagy to treat wound or skin-related diseases or conditions. The name "autophagy", also known as autophagocytosis, is derived from the Greek words meaning "to eat" and "self". Autophagy is generally defined as self-digestion.

[0003] Autophagy is a lysosomal reuse or recycling pathway commonly used by cells to perform homeostatic functions by degrading naturally aged proteins and organelles and resorbing nucleotides, amino acids, and free fatty acids for new molecule synthesis and ATP production. As a cellular level self-preservation mechanism, autophagy can be upregulated in response to extracellular or intracellular stresses and signals, such as starvation, growth factor deprivation, ER stress, and pathogen infection. In some pathological conditions, it is desirable to stimulate the autophagy process, whereas in other pathological conditions, such as wound healing, it is desirable to inhibit or delay the autophagy process to reduce cell destruction. Thus, modulation of autophagy can result in either promotion / stimulation or inhibition / slowdown of cell proliferation and cell death processes.

[0004] The most common form of autophagy involves (1) the formation of an isolated membrane that extends, the terminus of which eventually fuses to (2) encompass the cellular contents within a double-membrane vesicle known as an autophagosome. The autophagosome then (3) fuses with a lysosome, which provides enzymes and (4) digests the autophagosome's contents, which then become available to the cell again as raw materials or component nutrients. Autophagy shows some similarities to the parallel proteasomal degradation of ubiquitin-tagged proteins, but differs in that the autophagosome not only degrades proteins, but also cytoplasm, mitochondria, organelles, and other cellular structures. In this sense, it is known as a bulk degradation system.

[0005] The autophagy process can be either beneficial or detrimental to the cell depending on external factors and conditions, so the process must be tightly controlled. Both yeast and mammalian systems have been studied, utilizing up to 36 proteins. Figure 1 illustrates the process in mammals.

[0006] The yeast autophagy-related gene product (Atg8) has three mammalian homologs: (1) LC3, (2) GABAA receptor-associated protein (GABARAP), and (3) Golgi-associated ATPase enhancer (GATE-16). Among these, LC3 is the most extensively studied and is frequently used as an autophagy marker in mammals. Immediately after translation (proLC3), LC3 is processed to LC3-I at the C-terminus by Atg4A or Atg4B. 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 during the process, resulting in the conversion of soluble LC3-I to autophagy vesicle-bound LC3-II. This lipid conjugation allows LC3-II to be used as a marker of autophagy activity.

[0007] Wound progression is driven by many mechanisms including local tissue hypoperfusion, prolonged inflammation, free radical damage, apoptosis, and necrosis. These are typically divided into three phases following injury including (i) the inflammatory phase, (ii) the cell proliferation phase, and (iii) the remodeling phase. Each of these phases is associated with a unique biological and histological fingerprint, and autophagy plays a special role in each phase. For example, during the inflammatory phase, autophagy is initially protective of the tissue in that it acts to prevent cells at the wound edge from dying. In the proliferation phase, which involves the proliferation and migration of cells of multiple cell types to close the wound, autophagy helps to control β1 integrin and other cell migration proteins to form a controlled column of cells at the leading edge of the wound. 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 lamins, helps to regenerate the basement membrane during the proliferation and migration phase.

[0008] During the wound healing process, collagen fibrils provide structural topology, rigidity, and organization that allow for proper cell migration. In chronic wounds and burns, this environment is subject to a variety of factors, including pH changes, chronic inflammation, and often the loss of the basement membrane, which contains the essential collagen ducts used by wound cells (e.g., keratinocytes, fibroblasts, myofibroblasts) to migrate and protect the body from invading microorganisms and environmental factors (e.g., thermoregulation) to fill the wound and restore the intact layer of skin. During the natural healing process, skin cells secrete a soluble form of collagen known as procollagen, which can enzymatically form higher order structures or undergo an entropy-driven self-organization process (Prockop, D. and D. Hulmes (1994)). Procollagen is secreted from cells as triple helices and can self-organize into fibrils due to its liquid crystalline nature. This process is highly dependent on pH, ionic strength, temperature, and concentration. The fibrils then form into individual collagen fibers and randomly organize into higher order three-dimensional networks and structures.

[0009] U.S. Patent Nos. 6,599,945 and 7,094,809 disclose several hydroxytrans compounds and their use in inhibiting the formation of infectious herpes virus particles or for treating gonorrhea caused by Neisseria gonorrhoeae. WO 2009 / 126700 discloses similar compounds for use in skin care and cosmetic applications such as UV light. And U.S. Patent Nos. 8,716,355 (WO 2011 / 0130468) and 8,680,142 (WO 2011 / 0160301) disclose similar hydroxytrans compounds for use as antitumor agents. However, the potential utility of these or other hydroxytrans compounds as autophagy modulating compounds was unknown until the present invention. U.S. Patent Nos. 6,008,260, 6,197,834, and 6,355,692 disclose certain hydroxylated stilbenes, specifically resveratrol, but none of these documents disclose the use of such modified tolane or stilbene compounds as autophagy regulators.

[0010] It would be advantageous if the process of autophagy could be regulated, stimulated or enhanced in some conditions and slowed down or inhibited in others. Summary of the Invention

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

[0012] These autophagy regulators are liquid crystal in nature and act in specific combinations to treat wound or skin-related diseases or conditions. The invention further describes using these liquid crystals to create bioactive formulations, such as hydrogels and alginates, which can also function as bioactive membranes to promote healing in wounds or skin-related conditions. Thus, in some embodiments, the invention comprises a formulation containing FAM and AAM. The formulation can be a hydrogel, such as an alginate. The formulation can include cationic salts of FAM, or complexes of FAM and AAM with cations. In other formulations, FAM and / or AAM can be combined with cyclodextrin.

[0013] A particular FAM may be a toran or a stilbene (including cis and trans stilbenes) and may have any of the various substituents described herein. In some embodiments, the substituents are hydroxyl or alkoxyl groups that increase the solubility and polarity of the molecule. Any FAM may be administered with any AAM. For example, the FAM may be a toran and the AAM may be a vitamin, an acidic sugar, an amino acid, or a quinolone. Similarly, the FAM may be a stilbene and the AAM may be a vitamin, an acidic sugar, an amino acid, or a quinolone.

[0014] In some embodiments, the methods promote wound healing in certain indications, wound or skin conditions include aging, autoimmune diseases with inflammation, avascular necrosis, bacterial infection, cancer, diabetic neuropathy, endometriosis, fungal infection, gout, hair loss, infectious arthritis, inflammation, inflammatory bowel disease, ischemia, Lyme disease, organ / tissue transplants, parasitic infections, psoriatic arthritis, psoriasis, pseudogout, rheumatoid arthritis, scleroderma, scurvy, sepsis, skin diseases, surgical scars, surgical adhesions, transfection procedures, ulcerative colitis, ulcers, viruses, The wounds are selected from one or more of the following: infection, warts, surgical wounds, incisions, lacerations, cuts and abrasions, skin graft donor wounds, traumatic wounds, infected wounds, ischemic wounds, burns, blistering wounds, non-infected wounds, contusions, cuts, lacerations, non-penetrating wounds, open wounds, penetrating wounds, perforating wounds, puncture wounds, abscessed wounds, subcutaneous wounds, chronic ulcers, gastric ulcers, skin ulcers, peptic ulcers, duodenal ulcers, gastric ulcers, gout, hypertensive ischemic ulcers, stasis ulcers, sublingual ulcers, submucosal ulcers, symptomatic ulcers, trophic ulcers, tropical ulcers, and venereal ulcers.

[0015] In some methods, the wound to be healed is dermal in nature, such as cuts, abrasions, ulcers of many types and degrees, aging, skin inelasticity, etc. In other methods, the wound may be an eye or ear wound, and in other methods, the wound may be oral in nature, such as cancer sores, herpes virus infections, tooth extraction wounds, gingivitis, etc. [Brief description of the drawings]

[0016] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate the invention in several aspects thereof 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.

[0017] [Figure 1] Schematic diagram showing the role of LC3 in autophagy.

[0018] [Diagram 2] 1 is a schematic diagram showing autophagy-related human diseases.

[0019] [Diagram 3] 1 is a bar graph of LC3-II fluorescent staining as described in Example 5.

[0020] [Figure 4] 1 is a bar graph of an LC3-II Western blot as described in Example 6.

[0021] [Diagram 5] 1 is a bar graph of the LC3-II / LC3-I ratio as described in Example 6.

[0022] [Figure 6] 13 is a graph of AKT signaling as described in Example 8.

[0023] [Figure 7] 13 is a graph of fibroblast growth factors as described in Example 9.

[0024] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate the invention in several aspects thereof 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description Numerical ranges, measurements, and parameters used to characterize the present invention, such as angles, amounts of components, polymer molecular weights, reaction conditions (pH, temperature, charge levels, etc.), physical dimensions, etc., are necessarily approximations and, while reported as precisely as possible, inherently contain imprecision resulting from each measurement. Accordingly, all numbers expressing ranges of size used in the specification and claims should be understood in all instances as modified by the term "about." All numerical ranges are understood to include all possible incremental subranges within the outer boundaries of that range. Thus, a range of 30-90 units, for example, discloses 35-50 units, 45-85 units, and 40-80 units, etc. 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 of density 1.0), and wound areas are expressed in cm as area / area (a / a). 2 It is represented as:

[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 regulating autophagy, comprising administering a first autophagy-modulating (FAM) compound and an optional supplemental 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 given sequentially or simultaneously. If given sequentially, the order may be FAM first, then AMM, or AMM first, then FAM. If given simultaneously, they may be given in separate individual formulations, or as a combination of components in a single formulation. The compounds may be administered once a day up to about six times a day, depending on the formulation excipients. 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 described in pharmaceutical treatises, e.g., Remington's Pharmaceutical Sciences, 18 th Edition (1990), and its successor, Remingtons: The Science and Practice of Pharmacy, 22 nd edition (2012), etc. Topical formulations may be combined with solvents, emulsifiers, emollients, solvents, etc., among others, to make solutions, suspensions, creams, ointments, and hydrogels.

[0029] In certain embodiments, the present invention relates to a formulation containing a FAM compound and an AAM compound. 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, can range from about 200:1 to 1:200. In a liquid formulation, the FAM can comprise about 0.01% to about 40% (w / v) of the formulation, and the AMM can comprise about 0.01% to about 99.9% (w / v). In certain embodiments, the FAM can comprise about 0.1% to about 40% (w / v) of the formulation, and the AMM can comprise about 0.1% to about 60% (w / v). The formulation concentration of FAM is optimally 0.5-15% (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 those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, 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 each incorporated by reference in their entirety, including all data, tables, figures, and texts set forth in the cited references.

[0031] As used throughout this application, the following terms have the following meanings.

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

[0033] [ka]

[0034] In the formula, L is -C=C- and -CR a =CR b -, R a and R b are independently H or -(R 3 ) p Or -(R 4 ) q phenyl optionally substituted with R 1 ~R 4 are independently a substituent at any available position on the phenyl ring; m, n, p, and q are each independently 0, 1, 2, or 3, which represent 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 is, independently, -R is selected from (C1-C6) alkyl, (C2-C6) alkenyl, or (C2-C6) alkynyl, and is optionally substituted with 1 to 3 substituents selected from -OH, -SH, -halo, -NH2, or NO2; 5 , Y is O, S, or NH; R 6 is H or R 5 Selected from -YR 6 , -ZR, Z is -N(C=O)- or -O(C=O)- 5 , -Halo, -NO2, -SO3Na, -azide, and -Glycosides - and their salts, is selected from However, the FAM is not resveratrol or 4,4'-(ethyne-1,2-diyl)diphenol (TOLCINE).

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

[0036] Autophagy regulation refers to either the upregulation or downregulation of the autophagy process in cells.Depending on a particular disease state or condition, it is desirable to achieve one or the other direction of autophagy regulation, as described below.And, referring to the discussion of hormesis, the dose of any particular FAM or AAM compound or combination or complex can achieve upregulation or downregulation, or both.

[0037] As used herein, the term "-(C1-C6) alkyl" refers to straight-chain and branched acyclic saturated hydrocarbons having 1 to 6 carbon atoms. Representative straight-chain -(C1-C6) alkyl groups include methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl. Representative branched-chain -(C1-C6) 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 commonly, the subscript refers to the number of carbon atoms in the chain. Thus, the term "-(C1-C4)alkyl" refers to straight-chain and branched acyclic saturated hydrocarbons having from 1 to 4 carbon atoms.

[0038] As used herein, the term "-(C2-C6)alkenyl" refers to straight-chain and branched acyclic hydrocarbons having 2-6 carbon atoms and containing one or more carbon-carbon double bonds. Representative straight-chain and branched -(C2-C6)alkenyl groups include -vinyl, allyl, 1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and the like.

[0039] As used herein, the term "-(C2-C6)alkynyl" refers to straight-chain and branched acyclic hydrocarbons having 2-6 carbon atoms and containing one or more carbon-carbon triple bonds. Representative straight-chain and branched -(C2-C6)alkynyl groups include -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, -4-pentynyl, and the like.

[0040] As used herein, "-(C1-C10 "Alkoxy" means a straight or branched acyclic hydrocarbon having one or more ether groups and 1 to 10 carbon atoms. Representative straight and branched (C1-C 10 ) Alkoxy includes -methoxy, -ethoxy, -propoxy, -butyloxy, -pentyloxy, -hexyloxy, -heptyloxy, -methoxymethyl, -2-methoxyethyl, -5-methoxypentyl, -3-ethoxybutyl, and the like.

[0041] As used herein, "-(C1-C6)alkoxy" means a straight or branched acyclic hydrocarbon having one or more ether groups and 1 to 6 carbon atoms. Representative straight and branched (C1-C5)alkoxy include -methoxy, -ethoxy, -propoxy, -butyloxy, -pentyloxy, -hexyloxy, -methoxymethyl, -2-methoxyethyl, -5-methoxypentyl, -3-ethoxybutyl, and the like.

[0042] As used herein, the term "-(C 12 "Cycloalkyl" refers to a cyclic saturated hydrocarbon having 3 to 12 carbon atoms. Representative (C3-C 12 ) Cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl and the like.

[0043] As used herein, the term "-(C 12 "Cycloalkenyl" refers to a cyclic hydrocarbon having 4 to 12 carbon atoms and containing one or more carbon-carbon double bonds. Representative -(C3-C 12)Cycloalkenyl includes -cyclobutenyl, -cyclopentenyl, -cyclopentadienyl, -cyclohexenyl, -cyclohexadienyl, -cycloheptenyl, -cycloheptadienyl, -cycloheptatrienyl, -cyclooctenyl, -cyclooctadienyl, -cyclocyclooctatrienyl, -cyclooctatetraenyl, -cyclononenyl, -cyclononadienyl, -cyclodecenyl, -cyclodecadienyl, -norbornenyl, and the like.

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

[0045] As used herein, "hydroxy(C1-C6)alkyl" refers to any of the above C1-C6 alkyl groups substituted with one or more hydroxy groups. 1~6 Representative hydroxy(C1-C6)alkyl groups include hydroxymethyl, hydroxyethyl, hydroxypropyl, and hydroxybutyl groups, 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 unsubstituted or substituted. When optional substituents are present, and unless otherwise indicated, the optional substituents include one, two, or three groups independently selected from the group consisting of -(C1-C6)alkyl, OH, halo, -C(halo)3, -CH(halo)2, -CH2(halo), NH2, -NH(C1-C6)alkyl, CN, SH, phenyl, benzyl, (=O), halo(C1-C6)alkyl-, hydroxy(C1-C6)alkyl-. Thus, specific substituted embodiments include the following:

[0047] As used herein, "dihydroxy(C1-C6)alkyl" refers to any of the above C1-C6 alkyl groups substituted with two hydroxy groups. 1~6 Representative dihydroxy (C1-C6) alkyl groups include dihydroxyethyl, dihydroxypropyl, and dihydroxybutyl groups, 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, "-CH2(halo)" means a methyl group where one of the hydrogens of the methyl group has been replaced with a halogen. Representative -CH2(halo) groups include -CH2F, -CH2Cl, -CH2Br, and -CH2I.

[0050] As used herein, "-CH(halo)2" refers to a methyl group in which two of the hydrogens of the methyl group have been replaced with halogens. Representative -CH(halo)2 groups include -CHF2, -CHCl2, -CHBr2, -CHBrCl, -CHClI, and -CHI2.

[0051] As used herein, "-C(halo)3" means a methyl group where each of the hydrogens of the methyl group has been replaced with a halogen. Representative -C(halo)3 groups include -CF3, -CCl3, -CBr3, and -CI3.

[0052] As used herein, "(halo) p "(C1-C6)alkyl-" means a (C1-C6)alkyl chain substituted at p positions with halo, where p is 1, 2, or 3. The halo substituents may be substituted on the same or different carbons in the (C1-C6)alkyl. Representative "(halo) p (C1-C6) alkyl-" groups include, for example, -CH2CHF2, -CH2CH2CH2Cl, -CHBr2, -CHBrCl, -CHClI, -CH2CHI2, -CH2CH2CHClCH2Br, and the like.

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

[0054] As used herein, "glycoside" refers to a 5- or 6-membered cyclic sugar attached to a compound of formula I. The linkage is generally a glycosidic bond from the anomeric carbon of the sugar, which may be formed (1) through an oxygen atom, thus forming an "O-glycoside", (2) through a nitrogen atom, thus forming an "N-glycoside", or (3) through a sulfur atom, thus forming an "S-glycoside". Glycosides may be monosaccharides or disaccharides with one or two ring structures. Representative glycosides formed from six-membered sugar glucosides, galactosides, mannosides, and altrosides and glycosides formed from five-membered sugars include ribosides, arabinosides, xylosides, and lyxosides. The sugars may contain any substituents, although many embodiments contain only the naturally occurring -H and -OH substituents that define the respective sugars.

[0055] There is an overlap in the literature between 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 "infarction", which are used interchangeably unless otherwise specified. Wounds are classified by many criteria, including size or area (large or small), depth or layer of injury, cause, difficulty in healing, etc. Wounds may be classified by i) minor tissue loss due to surgical incision, minor abrasion, and minor bite, or ii) severe tissue loss, such as ischemic ulcer, pressure ulcer, fistula, laceration, severe bite, burn, and donor site wound (in soft and hard tissue), and infarction. 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 dermal layer. Apart from the stratum corneum, the epidermal layer of the skin is the outer (epithelial) layer, while the deeper connective tissue layer of the skin is called the "dermis" which contains the nerves and end organs of sensation.

[0057] A method of "promoting wound healing" results in a wound healing that is more rapid as a result of the treatment than the healing of a similar wound in the absence of the treatment. "Promoting wound healing" can also mean that the method inter alia regulates the proliferation and / or growth of keratinocytes, or that the wound heals with less scarring, less wound contraction, less collagen deposition, and a larger surface area. In certain instances, "promoting wound healing" can also mean that a particular method of wound healing has an improved success rate (e.g., skin graft incorporation rate) when used with the methods of the present invention.

[0058] The phenomenon of hormesis is generally associated with compounds that induce biologically opposing effects in a dose-dependent manner and is well documented 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. In general, there is a stimulatory or beneficial effect at low doses and an inhibitory or toxic effect at high doses. Hormesis has also been characterized as, inter alia, self-protection, adaptive response, and preconditioning, and by the shape of its dose-response curve, including, inter alia, β-curve, biphasic, bell-shaped, U-shaped or inverted U-shaped, bimodal, functional antagonism, and dual response. Known hormetic substances (or "hormetins") include vitamin A, ferulic acid, chalcone, rapamycin, epigallocatechin-3-gallate, and many others.

[0059] Hormesis can also represent an adaptive response in which moderate stress is applied to provide the organism with adaptive resistance when faced with severe stressors. Environmental stressors such as oxidative metabolism and heat stress serve as hormetins used to induce specific responses or adaptive changes (Mattson MP. Hormesis defined, Ageing Res Rev. 2008;7: 1-7). Hormetins have 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, supra). These dose-response effects have been analyzed for a wide range of natural signaling molecules, including nitric oxide, adenosine, opioids, adrenergic agonists, 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 defend themselves against predatory species.However, many of these substances can have beneficial effects when utilized in lower concentrations.

[0060] Hydrogels are dilute cross-linked aqueous systems that comprise water and gelling agents, such as polymer plastics or polysaccharides, that can absorb and retain significant amounts of water to form a three-dimensional network. The hydrogel structure is generated by the interaction of water with hydrophilic groups or domains present in the polymer network upon hydration. Hydrogels are primarily classified as weak or strong, depending on their steady-state flow behavior.

[0061] As the polymer concentration increases, gelation occurs and the dispersion of the polymer initiates branching and crosslinking. When a critical concentration is reached, the sol becomes a gel and a sol-gel transition occurs. Gels may be considered to be chemically bonded or physically bonded. Physical gels can be subcategorized as strong or weak depending on the nature of the bonds, with chemical gels being strong and physically close together, and the bonds being permanent. Strong physical bonds include lamellar crystallites, glassy nodules, or double and triple helices, while weak physical gels include hydrogen bonds, block copolymers, micelles, and ionic associations.

[0062] Hydrogels, due to their significant water content, have a degree of flexibility similar to that of natural tissues and may exhibit viscoelastic or purely elastic behavior, and cohesiveness. The properties of hydrogels can be modified by controlling polarity, surface properties, mechanical properties, and swelling behavior. Gels can exhibit significant volume changes in response to small changes in pH, ionic strength, temperature, electric fields, 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 crosslinkers used to prepare the hydrogel. Labile bonds can be broken under physiological conditions, enzymatically or chemically, most often by hydrolysis ( 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 that have negatively charged groups at physiological pH can be crosslinked by the addition of multivalent cations and monovalent cations (e.g., K + , Na + ) or negatively charged groups (e.g. SO - 3) It is possible to form a stable gel by blocking the repulsion of Na + Alginate - , chitosan-polylysine, chitosan-glycerol phosphate (Syed KH 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, WE& Nostrum, CF(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 seaweed, that is favored due to its biocompatibility, low toxicity, relatively low cost, and Ca 2+Alginate hydrogels have been widely studied and used in many biomedical applications due to their ability to form hydrogels with the addition of divalent cations such as . Alginate hydrogels have been used in a variety of 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 the alginate gel in a controlled manner depending on the crosslinking agent and crosslinking method used. In addition, alginate gels can be administered orally or injected, making them 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 one to four glycosidic bonds. The ability of alginate to provide viscosity in sol-gels depends on its molecular weight (MM). The molecular weight (MM) of algal alginates has been found to range from 48 to 186 kDa (38), and some alginates isolated from Azotobacter vinelandii exhibit MMs ranging from 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 (M), guluronic acid (G) or alternating (MG) residues, depending on the species of origin (Smidsrod, O. & Draget, K. (1996). Chemistry and physical properties of alginates. Carbohydrates European, 14, 6-12). The G blocks of alginates are supported by divalent cations (e.g., Ca 2+) to form hydrogels. The composition (i.e., M / G ratio), sequence, G-block length, and molecular weight are important factors that alter the physical properties of alginates and alginate hydrogels (e.g., increasing G-block length increases ionic binding and mechanical stiffness of the gel). These same properties control the gel stability as well as the release rate of drug-containing alginates. Alginates with low M / G ratios form strong, brittle gels, whereas alginates with high M / G ratios form weak, soft, but more elastic gels. Finally, bacterial alginates are acetylated to varying degrees at the O-2 and / or O-3 positions of mannuronic acid residues (Skjak-Braek, G.; Grasdalen, H. & Larsen, B. (1986). Monomer sequence and acetylation pattern in some bacterial alginates. Carbohydrates Research, 154, 239-250). Variability in molecular weight, monomer block structure, and acetylation all affect the physicochemical and rheological properties of the gel polymer.

[0066] The majority of alginates contain divalent cations, either calcium or Na. + or K + On the other hand, Mg 2+ Other ions such as magnesium ions have been proposed, but the concentration of magnesium ions required to initiate the gelation process is 5-10 times higher than that of calcium ions (Topuz, F., Henke, A., Richtering, W. & Groll, J. (2012). Magnesium ions and alginate do form hydrogels: a rheological study. Soft Matter. 8:4877-4881). Alginates may be reduced in water solubility as alginic acid or calcium alginate, where the ions are protected from ionization resulting in insoluble alginate. Water soluble alginates are those containing Na + or K +These can be easily made by forming salts using monovalent anions such as NH4 or non-polar groups such as NH4 which are generally water soluble. The first autophagy regulator (FAM)

[0067] As described above, the first autophagy modulating agent comprises two phenyl rings joined by a linker L and one or more R 1 or R 2 In some embodiments, L is -C≡C-, and these FAM compounds are commonly known as "trans" which 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 about the double bond. In some embodiments, the FAM compound is a 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. They may also be stilbenes, in which case they are "phenylstilbene derivatives", and may be trans-stilbenes or cis-stilbenes.

[0068] The substituent R is shown in the structure, at least one of which must be present (i.e., at least one of m or n is ≧1). 1 and R 2 There are two "primary" phenyl rings containing a and R b There are also up to two "secondary" phenyl rings in the ring. On each phenyl ring (up to four possible, two primary and two secondary), there are 0 to 5 each of the substituents R 1~4 In certain embodiments, there may be 1 to 3 R on the primary phenyl ring. 1 and / or 1-3 R 2 In some embodiments, R on the primary phenyl ring is 1 and / or R 2The positions of R are mostly para and meta positions, i.e., 3, 4, or 5 on one phenyl ring and 3', 4', and 5' on the other phenyl ring, but it is also possible to have substituents at the ortho positions (2, 2', 6, and 6'). 1 Substituents are present, and correspondingly, 0 to 3 R on the second phenyl ring 2 Conversely, there may be one, two, or three R on the second phenyl ring. 2 Substituents are present, and correspondingly, 0 to 3 R 1 Substituents may be present. Additionally, in some embodiments, the secondary phenyl ring may be substituted with 1 to 3 substituents R 3 and R 4 All permutations of these are possible, for example, one R 1 and one R 2 , two R 1 and two R 2 , the three R's 1 and the three R's 2 , 1 R 1 and two R 2 , 1 R 1 and the three R's 2 , two R 1 and one R 2 , two R 1 and two R 2 , two R 1 and the three R's 2 , the three R's 1 and one R 2 , or the three R's 1 and two R 2 is possible. 3 and R 4 The same is true for each R 1~4 are independently selected, and if two or more are present, they may be the same or different. Examples of some specific first autophagy regulators are given in Table A, and because tolanes are analogs of stilbenes, detailed structures are 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 of the 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, MA, Kondo, K. and Tsuda, Y. (1992). Synthesis and Nematocidal activity of Hydroxy stilbenes. Chem. Pharm. Bull. 40(5): 1130- 1136; and Thakkar, K., Geahlen, RL 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., those having at least one R a or R b Phenylstilbene derivatives in which is a phenyl ring, as well as many other stilbenes, can be synthesized according to the method presented in the dissertation of Zhenlin Bai, Substituted Stilbenes and 1,2-Diaryl-l,2-diazidoethanes as Potential Anticancer Agents: Syntheses and Estrogenic / Antiestrogenic Properties in MCF-7-2a Cells, im Fachbereich Biologie, Chemie, Pharmazie der Freien Universitat Berlin (2006). Glucoside derivatives can be obtained according to the procedure described in WO 2007 / 020673 A1. Tolanes can be synthesized using the general procedure described in U.S. Patent No. 6,599,945 B2 to Docherty & Tsai.

[0080] It may be noted that the above FAM compounds are typically polar and have certain electronegative substituents at each end (e.g., -OH, -OCH3, -NO2, -halo, -O(C=O)R, etc.) Although this is not believed to be essential, it may be desirable to impart liquid crystal-like behavior in which the molecules adopt a lyotropic or partially ordered structure in solution.

[0081] FAM compounds also include salts of the compounds identified above. FAM compounds, particularly those mono- or polyhydroxylated compounds, readily release one or more protons to form anions depending on the pH. Such anions can combine with cations, such as monovalent, divalent, and trivalent cations, to form salts. In monovalent cations (M+), a single FAM is bound to form M + FAM - Similarly, divalent cations (M 2+ ), two FAM molecules are bound to form M 2+ (F.A.M. - ) di-salts and trivalent cations (M 3+ ), three FAM molecules are bound to form M +3 (F.A.M. - )3 salts. The salts are often readily soluble in aqueous media, which can facilitate formulation. Exemplary, but not limiting, cations for FAM salt formation include Na + or K + , Mg 2+ , Mn 2+ , Zn 2+ , Ca 2+ , Cu + , Cu 2+ Fe 2+ , and Fe 3+ Includes: Supplementary Autophagy Modulators (AAMs) and Formulations

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

[0083] Vitamins A, B, C, D, E, and K may all be useful as AAMs. By convention, the term "vitamin" does not include other essential nutrients such as nutrients minerals, essential fatty acids, or essential amino acids (which are required in greater amounts than vitamins), nor the numerous other nutrients that promote health. Currently, 13 vitamins are widely recognized. Vitamins are classified by their biological and chemical activity, not by their structure. Thus, each "vitamin" refers to several "vitamin" compounds that all exhibit the biological activity associated with the particular vitamin. Such sets of chemicals are grouped under alphabetical vitamin "umbrella descriptive" names, such as "vitamin A," which includes multiple compounds such as: Vitamers, by definition, are convertible to the active form of the vitamin in the body, and sometimes are also interconvertible. In certain embodiments, salt forms of vitamins that generally do not have long aliphatic side chains are good AAMs. In certain embodiments, oxygen-containing vitamins are preferred AAMs.

[0084] Most vitamins also form salts that are also within the scope of AAM. For example, vitamins may combine with cationic elements such as sodium, potassium, magnesium, manganese, calcium, copper, zinc, or iron to form salts. In addition, 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, stearates, lactates, succinates, maleates, citrates, etc.

[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, xanthophylls, 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 esters, 9-cis-β-carotene, retinyl palmitate, acitretin, vitamin A2 (3,4-dehydroretinol), A3 (3-hydroxyretinol), and salts thereof. All isomers and stereochemical forms of these isoprenoids are encompassed by the present invention.

[0086] B vitamins include, but are not limited to, the following compounds: thiamine (B1); riboflavin (B2); niacin or niacinamide (forms of B3); pantothenic acid, panthenol, pantothenol, 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 (forms of B9); and cobalamin, cyanocobalamin, hydroxycobalamin, methylcobalamin, adenoxylcobalamin (forms of B12); and salts thereof.

[0087] Vitamin C refers to ascorbic acid, its ascorbate anion, and ascorbate salts, as well as ascorbyl palmitate and its 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, but not limited to, calcidiol, calciferol (INN), ergocalciferol, and lumisterol (D1 forms); ergocalciferol, fromergergol, and 25-hydroxyvitamin D2 (D2 forms); cholecalciferol, 7-dehydrocholesterol, and 25-hydroxycholecalciferol (or 25-hydroxyvitamin D3, abbreviated 25(OH)D3, (D3 forms); 22-dihydroergocalciferol (D4); sitocalciferol, 7-dehydrositosterol (D5); 25-D-glucuronic acid, 25-D-hexuronic acid, 25-hydroxyvitamin D2-25-β-D-glucuronide, and salts thereof.

[0089] Vitamin E refers to a group of fat-soluble compounds that are either tocopherols or tocotrienols, of which the most active is α-tocopherol. Other tocopherols include beta, gamma, and delta. Similarly, tocotrienols also exist in alpha, beta, gamma, and delta forms. All isomeric 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) and 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, which include a group of compounds formed by esterifying the vitamin E molecule with carboxylic acids (e.g., alpha-tocopheryl succinate); d-α-tocopherol, which is often a mixture of two or more enantiomers of other tocopherols (β, γ, δ, ε, ζ, η) or exists as a tocotrienol, n-propionate, or linoleate, such as vitamin E acetate or alpha-tocopheryl acetate. Water-soluble forms of vitamin E include: R-(+)-alpha magnesium lipoate, 6-hydroxy-2,5,7,8-tramethylchroman-2-carboxylic acid (Trolox), or a salt of vitamin E.

[0090] Vitamin K refers to a group of compounds with a 2-methyl-1,4 naphthoquinone core and a side chain at the 3 position. The vitamers K1 (phylloquinone, phytomenadione, or phytonadione) and K2 (menaquinone) occur naturally. In fact, there is not one K2, but a series of compounds with isoprenoid side chains of various lengths, and the menaquinone family is sometimes referred to as MK-n, where n is the number of isoprenoid groups, with n=4 being the most common. In addition, several synthetic vitamin K analogs have been made, including K3 (menadione), K4, K5 (2-methyl-4-amino-1-naphthol hydrochloride), vitamin K6 (2-methyl-1,4-naphthalenediamine), and K7, which have no side chains. Many vitamin K compounds form salts, with the divalent salts being the most useful as AAMs. For example, salts of cations can take the following forms: M(Ki)2, where M is a divalent cation, or M(Ki)3, where M is a trivalent cation. In certain embodiments, useful AAMs include a dimer of vitamin K and a salt of a divalent cation, such as Ca or Mg.

[0091] Vitamin P (although a somewhat outdated term) refers to a group of flavonoids that have 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 three chemical classes: 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 acid

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

[0093] [ka]

[0094] where R is any of several well-recognized side chains. There are 20 amino acids encoded by the universal code, and humans synthesize 11 of these, making the other 9 "essential" amino acids and must be consumed 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 isomeric and stereochemical forms of these amino acids and their salts are encompassed by the present invention. In certain embodiments, useful amino acids include, but are not limited to, tyrosine, phenylalanine, cysteine, serine, threonine, and tryptophan. Additionally, certain amino acid derivatives are useful, such as lycopene and N-acyl cysteine ​​(NAC). acidic sugar

[0095] Acidic sugars include monosaccharides and disaccharides formed from 4-6 membered aldoses and ketoses. They are usually acidic because protons are easily released from many 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 isomeric and stereochemical forms of these sugars are encompassed by the present invention. Quinone Derivatives

[0096] Quinone derivatives include those having one, two, or three rings, and therefore:

[0097] [ka]

[0098] Based on 1,4-benzoquinone,

[0099] [ka]

[0100] Based on 1,4-naphthoquinone,

[0101] [ka]

[0102] Based on 9,10-anthraquinone,

[0103] [ka]

[0104] These quinone derivatives may contain substituents at any position except the ketone, which are generally selected from hydroxyl, methoxy, methyl, ethyl, halo, and amino. One to four 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: 2,6-dimethoxy-1,4-benzoquinone, 2,3,5,6-tetramethyl-1,4-benzoquinone, 1,4-benzoquinonetetracarboxylic acid, blatellaquinone, 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- These include 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 (spinochrome B). Other 1,4-naphthoquinone derivatives include menadione (sometimes called vitamin K3 or 2-methyl-1,4-naphthoquinone).

[0106] Examples of 9,10 anthraquinolones include the dihydroxy derivatives 1,2-dihydroxyanthraquinone (alizarin), 1,3-dihydroxyanthraquinone (purproxanthin, xanthopurpurin), 1,4-dihydroxyanthraquinone (quinizarin), 1,5-dihydroxyanthraquinone (anthramphin), 1,6-dihydroxyanthraquinone, 1,7-dihydroxyanthraquinone, 1,8-dihydroxyanthraquinone (danthrone, chrysazin), 2,3-dihydroxyanthraquinone, 2,6-dihydroxyanthraquinone, and 2,7-dihydroxyanthraquinone; the trihydroxy derivatives 1,2,3-trihydroxyanthraquinone (antragallol), 1,2,4-trihydroxyanthraquinone, These include purpurin, 1,2,5-trihydroxyanthraquinone (oxyanthraphin), 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 described above. In addition, complexes may be formed between FAM compounds and specific AAM compounds. Such FAM+AAM complexes include at least those that contain vitamins, such as ascorbate and ascorbyl palmitate; those that contain amino acids, such as alginate, lysinate, aspartate, glutamate; and those that contain acidic sugars, such as glucosides, ribosides, galactosides, mannosides, etc. Example 4 provides some specific examples of both salts and complexes of FAM and AAM compounds.

[0108] The nature of FAM compounds as liquid crystals may facilitate and / or mediate their role in wound healing. The polar nature of liquid crystals allows them to self-assemble into polymer-like structures, and thus they can (i) give rise to unique hydrogels, and / or (ii) enhance the sol-gel transition state to create a unique set of liquid crystal hydrogels by adding these molecules to conventional hydrogels. These gels may be modified to create any of the various aforementioned gel types, from strong to weak chemical bonds, or to create biodegradable gels. Without wishing to be bound by a particular theory, it is believed that these FAM molecules are useful for wound healing applications in part because the molecules themselves behave like collagen fibrils assembling into higher order structures, which can aid in scaffolding and cell migration during wound healing. Furthermore, the addition of these liquid crystal hydrogels may promote proper collagen alignment and orientation, which 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 includes cyclodextrin.Typical cyclodextrin formulations consist of FAM with a FAM:cyclodextrin ratio 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 sufficient dissolution of FAM in cyclodextrin. The AAM added 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. The utility of FAM and AAM in regulating autophagy

[0110] In recent years, scientists have been studying the effects of regulating the autophagy pathway as a way to treat various severe diseases. Indeed, dysregulation of autophagy has been linked to major diseases including heart disease, cancer, and diabetes (see Figure 2 from Klionsky DJ (2010). The Autophagy Connection. Developmental Cell. Jul 20; 19(1): 11-2.) This paper describes the link between the autophagy pathway and major human diseases. While not naming individual "myopathies," the ability to regulate this pathway is of major relevance to regulating disease outcomes. Depending on the disease state, it may be beneficial to upregulate or downregulate cellular levels of autophagy.

[0111] In several disease conditions, the pathology is associated with suppressed autophagy activity or activation of autophagy, the associated signaling pathways of which lead to the suppression of inflammation. Thus, diseases or conditions in which upregulating autophagy may be beneficial include wound healing, promoting hair regrowth, bacterial infection, inflammation, viral infection, Parkinson's disease, aging, Alzheimer's, neurodegenerative diseases, neuropathy, cardiovascular disease, burns, heart disease, Alzheimer's disease, atherosclerosis, arteriosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, inflammatory bowel disease, colitis, diabetes, types I and II diabetes, amyloidosis, bursitis, 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's congenital amaurosis, lysosomal storage diseases, arthritis, psoriasis, osteopenia, osteoporosis, surgical scars, surgical adhesions, space travel (bone density disorder), tendonitis, and ulcerative colitis.

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

[0113] In wound healing, increased levels of autophagy help protect tissues, reduce inflammation, and promote the synthesis of procollagen, hyaluronan, and elastin. As shown herein, FAM and AAM compounds have been used alone or in combination to promote wound healing, hair growth, and skin repair after damage from UV radiation exposure.

[0114] In bacterial infections, various types of bacteria attempt to interfere with the autophagy pathway, which prevents cellular uptake of bacteria and ultimately leads to autophagolysosomal degradation of the bacteria. Two clinically important skin pathogens, Streptococcus aureus, interfere with 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 with group A streptococci are characterized by the prevention of bacterial cellular uptake by encapsulation; once bacteria are taken up by keratinocytes, most streptococci are killed within hours (HM Schrager, JG Rheinwald and MR 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. No mechanistic studies of the action of autophagy against group A streptococci have been performed in keratinocytes, so further studies will be needed to understand the relevance and efficiency of this putative antimicrobial strategy in the skin.S. aureus induces autophagy through its alpha-toxin (Schnaith et al, above, and MB Mestre, CM Fader, C. Sola and MI 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 cause a decline in nutrient and energy levels that trigger 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)). It remains to be determined whether autophagy inhibits or enhances S. aureus infection of the skin in vivo. EXAMPLES

[0115] Example 1: Synthesis of specific FAMs of the present invention The following compounds were prepared and given the identification numbers shown in Table B:

[0116] [Table 2-1]

[0117] [Table 2-2]

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

[0119] Example 2: A simplified procedure for the synthesis of 4-hydroxy-4'-methoxytolane

[0120] [ka]

[0121] The synthesis of 4-hydroxy-4'-methoxytolane (4-((4'-methoxyphenyl)ethynyl)phenol) is described in (Pavia, MR; Cohen, MP; Dilley, GJ; Dubuc, GR; Durgin, TL; Forman, FW; Hediger, ME; Milot, G.; Powers, TS; Sucholeiki, I.; Zhou, S.; Hangauer, DG 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, JC Tetraalkylammonium salts in heck-type reactions using an alkali metal hydrogen carbonate or an alkali metal acetate as the baseThe synthesis of angular [4]- and [5]phenylene was a Heck-type reaction modified from Schmidt-Radde, RH; Vollhardt, K.; Peter C. The total synthesis of angular [4]- and [5]phenylene J Am Chem Soc, 1992, 114, 9713-9715). The product was a yellowish powder. 1 HNMR (CDCl 3, 300 MHz): 5 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, CHO). The product obtained was 98.2% pure and was used for all subsequent studies.

[0122] Example 3: Summary of the synthesis procedure for 2,4,4'-trimethoxytolane The synthesis was a Heck-type reaction similar to that of Example 2.

[0123] [ka]

[0124] The resulting product was an off-white powder. 1 HNMR (CDCl 3, 400MHz): 5ppm: 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, CH3O), 3.82(s, 6H, 2CH3O) and found to be 99.3% pure.

[0125] Example 4: Melting points of FAM salts and FAM and AAM salt complexes: All salts were prepared by combining magnesium hydroxide, zinc oxide, ascorbic acid, or ascorbyl palmitate with sufficient amounts of each stilbene, trene, or combination to make a salt solution. Each solution was then dried using a rotary evaporator (Centrifan, Harvard Biosciences) set at 40°C in a 20mL scintillation vial, and evaporated more slowly with a mixture of ethanol and ice. Once completely dried by evaporation, the salts were ground into a fine powder and their melting points 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 the calibration before 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, JN 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 dermal fibroblast neonatal, ThermoFisher) cells were seeded at 5,000 cells / well in black well clear bottom plates (Coning, Corning, NY). The cells were then treated with medium alone, FAM, AAM, or their combinations for 8 hours. After the treatment time, cells were fixed with 4% (w / v) PFA (paraformaldehyde) for 15 min at room temperature. Cells were then washed with PBS (phosphate buffered saline, pH 7.4) and blocked with 5% (w / v) BSA in PBS for 1 h. Cells were washed again with PBS and incubated with primary antibody LC3-IIB (rabbit monoclonal antibody, Thermo-Fisher) for 3 h. Cells were rinsed again with PBS and secondary fluorescent antibody (Alexafluor 488 rabbit anti-goat, Thermo-Fisher) was added for 30 min. Plates were then imaged using 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 cultured at 1 × 10 per T25 tissue culture treated flask. 6Cells were seeded at a density of 100x1000 cells / ml. Cells were then treated with FAM, AAM, or a combination for 8 hours. Cells were then trypsinized, washed with 1x PBS, and lysed in RIPA buffer + protease inhibitors on ice. Cells were sonicated and a BCA protein assay (Pierce Scientific) was performed to measure protein concentration. Concentrations were then normalized to 100 μg per sample and run on a 12% (w / v) polyacrylamide gel containing loading dye and appropriate molecular weight markers (all reagents were purchased from National Diagnostics). Gels were then transferred, blocked, and a primary monoclonal antibody against MAPLC3-2 (Thermo-Fisher) was added to the membrane and allowed to incubate overnight. After appropriate washing, Eu-labeled secondary antibody (Molecular Devices) was added and the membrane was imaged using a Spectramax i3x with Scan Later module. Gels were then stripped and reprobed for actin to confirm proper loading. Changes in average band intensity were measured using SoftMax Pro 6.5.1 software. Results are shown in Figures 4 and 5.

[0129] Example 7: Autophagy, Wound Healing, and Skin:Very low levels of autophagy exist in skin and function to degrade protein aggregates, damaged organelles, and change 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, preventing excessive inflammation and induction of cathelicidin (found in 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 was shown to promote autophagic vesicle formation, improve wound re-epithelialization time, and reduce IL-8 levels, methanedicarboxylic aldehyde levels (MDA, an indicator of oxidative stress), and myeloperoxidase levels (an indicator of hypochlorous acid (HOCl), hydrogen peroxide (H2O2), and chloride anion (Cl-) production) (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 results in 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 formulations described in this patent are hormetic agents that have been shown to induce autophagy in a dose-dependent manner for the treatment of a variety of diseases and conditions, including increased wound closure and re-epithelialization, as shown below.

[0131] Example 8: Protein kinase B (AKT) : HDFn cells were cultured in T-25 flasks at 1 × 10 per flask. 6 Cells were seeded at a density of 100x and allowed to attach and spread overnight. Cells were then treated with control media, FAM, AAM, or combination for 8 hours. Cells were removed using trypsin followed by trypsin neutralizer and spun down to collect cell pellets. Cells were washed with ice-cold 1x PBS and then lysed on ice with RIPA buffer + protease inhibitors. Samples were spun down, aliquoted, and frozen until they could be assayed using ELISA. Cells were sonicated and diluted 1:5 as recommended in the kit instructions. Samples were then assayed using the AKT ELISA (Thermo-Fisher) and concentrations were measured using the AKT standard curve. Results are shown in Figure 6.

[0132] Example 9: Fibroblast growth factor (FGF) :HDFn cells were seeded into 6-well tissue culture treated plates and allowed to reach a confluency of 70-80% (cells / area). A cell scraper was then used to create a "wound" in the center of the plate to simulate damage to the cell monolayer. The dishes were then washed with culture medium to remove cellular debris. The cells were then treated with control medium, FAM, AAM, or the combination, and then sampled at 3, 8, and 24 hours. Samples were spun down, divided into aliquots, and frozen until they could be assayed using ELISA. An EGF Streptavidin-HRP ELISA kit was used to measure the concentration of EGF in each sample compared to a known standard curve. The results are shown in Figure 7.

[0133] Example 10: In vivo evaluation of LCB on wound healing:Twenty 5-6 week old Balbc / J (stock no. 000651) male mice were transferred to Jackson Labs in vivo laboratory, Sacramento, CA. Mice were ear notched for identification and housed in polycarbonate cages that were individually and reliably ventilated with HEPA filtered air. Corn cob bedding, called Bed-o-cob, was used and cages were changed every 2 weeks. The animal room was illuminated throughout with artificial fluorescent lighting with a controlled 12-h light / dark cycle. Ambient temperature and relative humidity ranges in the animal room were 22 ± 4 °C and 50 ± 15%, respectively. The animal room was set to allow 15 air changes per hour. Filtered tap water was acidified to pH 2.8-3.1 and rodent chow was fed ad libitum. After 5-7 days of acclimation, mice were randomized by weight into two cohorts of 10 mice each. On study day 0, mice were anesthetized and two full-thickness excision wounds (approximately 6 mm) were made on the dorsal (back) side of the mouse. One of the wounds was covered with a semi-occlusive polyurethane dressing (Tegaderm™). The dressing was kept on the wound for five (5) consecutive days starting on the day of wounding (d0). The dose of 4-hydroxy-4'-methoxytran was based on the dose that promoted lesion healing in mice in previous herpes virus studies. Measurements of the wounds were taken on days 5, 7, 9, and 11. Digital images were taken of the wounds for each mouse. The test agent was a viscous paste, and the wounds were surrounded in place with a glued saddle to eliminate cross contamination and wound closure due to contraction. Thus, any changes in wound area are due to re-epithelialization. All wounds are covered with glycerin (control) or glycerin and test agent for five days. On day 5, test agents were removed and changes in wound area were measured (as area / area%) to assess compound persistence at the wound site. By day 5, glycerin caused 26.1% wound closure, while 4-hydroxy-4'-methoxytolane (2.5%) caused 92.8% wound closure and 5% 4-hydroxy-4'-methoxytolane caused 91.4% wound closure. Prior to statistical analysis, tests for normality were performed and Z scores were calculated to estimate the normal distribution area of ​​each wound on day 1 for all mice across groups. 2= 0.989. Statistics are calculated based on one-way ANOVA comparing changes in wound area on day 5. All mice tolerated the treatment well and all weekly clinical observations reported bright, alert, responsive and hydrated mice.

[0134] [Table 4]

[0135] Example 11: Excised skin samples treated with control or 5% (w / v) 4-hydroxy-4'-methoxytran. H&E staining of control-treated cells showed a 1-2 cell thick layer of keratinocytes over a loosely organized layer of myofibroblasts, with connective tissue containing a few deep sebaceous glands beginning to form. Skin treated with 4-hydroxy-4'-methoxytran showed extensive keratinocyte proliferation with a cell layer 7-8 cells thick. The dermis showed regular and proliferative myofibroblasts, as well as connective tissue surrounding highly proliferative, tendrils of sebaceous glands that extended to the surface to remodel hair follicles.

[0136] Example 12: Autophagy in Skin Appendages and Hair Regrowth (Alopecia) : Autophagosome-like structures have been detected by electron microscopy in hair and sebaceous glands. Although the physiological relevance of autophagy in skin appendages is currently poorly understood, current existing data suggest that induction of autophagy may prevent hair loss via a Wnt1-dependent cell rejuvenation process in which damaged cells undergo cell death and hair stem cells are stimulated to produce hair growth (Castilho RM, 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 Radiation Damage and Anti-Aging Skin is the largest organ in the human body and is in contact with the environment. Thus, the skin is constantly subject to damage, both external and internal, that threaten its balance and change its appearance. This damage is often manifested as a chronic low-level inflammation. For example, excessive exposure to UV is known to be reflected in various skin symptoms such as actinic erythema, solar elastosis, premature appearance of the effects of skin aging, in that the skin becomes sagging, deeply wrinkled, rough, dry, dotted with hypopigmented or pigmented spots, and the blood vessels become dilated. These manifestations of UV exposure, which reflect serious structural changes in the skin tissue, are unsightly and disfiguring, and many people tend to want to get rid of them. TEM and IF microscopic analysis of cultured dermal fibroblasts from women of various ages revealed a pathological outflow of autophagic fluid. Treatment of young dermal fibroblasts with lysosomal protease inhibitors to mimic the condition of aged dermal fibroblasts reduced autophagy activity, altered fibroblast content of type I procollagen, hyaluronan, and elastin, and caused collagen fibril disintegration. Together, these findings suggest that impaired autophagy induction leads to deterioration of dermal integrity and skin fragility (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 cellular organelles and protein aggregates are properly degraded and do not accumulate and cause cellular dysfunction, and enhanced autophagy activity is seen in response to caloric restriction (CR), which has been shown to increase life expectancy.

[0139] Example 14: Hydroxypropyl-β-cyclodextrin 40% (w / v) solution: Prepare a solution by adding 40.0 g of hydroxypropyl-β-cyclodextrin to 70 mL of water in a sterile graduated beaker and mixing thoroughly. When the solution is clear, bring to 100 mL quantity (QS). Weigh out 1.0 g of liquid crystalline compound (FAM) and transfer to a sterile glass bottle. Add 1.5 mL of ethanol to the bottle and dissolve completely. Slowly add 1 mL of cyclodextrin while stirring to ensure the drug remains in solution. Add 5 mL of water while stirring to ensure the drug remains in solution. Sonicate if necessary. The formulation should be a clear solution. Filter using a 0.2 μm filter. Freeze the suspension below -40°C and lyophilize. The lyophilized cake may be reconstituted with sterile water prior to use.

[0140] Example 15: Preparation of FAM cyclodextrin formulations as injectable liquid crystal preparations :Weigh out 100 mg of 4,4'-dihydroxytolane compound into a 5 ml scintillation tube. Add 1.5 ml of absolute ethanol to the tube and shake until the 4,4'-dihydroxytolane is completely dissolved. Weigh out 5 grams of pyrogen-free hydroxypropyl-β-cyclodextrin (Sigma) on an analytical balance and place into a graduated cylinder. Add water while shaking until the volume reaches 90 ml. Add the above ethanolic solution of FAM to the aqueous solution containing hydroxypropyl-β-cyclodextrin while stirring. Add water to the clear solution to make the total volume 100 ml. Sterile filter the solution through a 0.22 micron filter. Freeze the suspension below -40°C and lyophilize. The lyophilized cake may be reconstituted with sterile water for injection before use.

[0141] Example 16: Preparation of infusion solutions: A solution is prepared from the following components; FAM-cyclodextrin 0.625 parts; saccharin sodium 0.3 parts; sorbic acid 0.1 parts; ethanol 30.0 parts; flavoring agent 1.0 parts; distilled water qs ad 100.0 parts. FAM-cyclodextrin complex and flavoring agent are dissolved in ethanol, and sorbic acid and saccharin are dissolved in distilled water. The two solutions are mixed together uniformly, and the mixed solution is filtered until no suspended matter remains. 1 ml of the filtrate contains FAM and is an oral dosage unit composition with effective therapeutic action.

[0142] Example 17: Preparation of micronized drugs and drug suspensions : 16 grams of micronized FAM is milled using a 4 inch mill size and compressed nitrogen gas / compressed air (dew point >40°C) as the grinding gas. The material is manually fed into the hopper and placed on top of the feed stage. The material is drawn into a closed circular chamber by the pressurized grinding gas. The powder is suspended in a high velocity stream in the grinding chamber. The particle size distribution is measured with a particle size analyzer. The grinding conditions are then adjusted to obtain an acceptable micron size material.

[0143] Example 18: FAM or AAM Modulator-Cyclodextrin Complex Gel :Weigh out 100 mg of FAM and place in a sterile test tube. Dissolve FAM in 2-3 ml of purified absolute ethanol. Prepare 50 ml of a 10-50% (w / v) solution of hydroxypropyl-β-cyclodextrin (other cyclodextrins may also be used, based on the need for water absorption, such as α-cyclodextrin, γ-cyclodextrin, and β-cyclodextrin with specific modifications) in a 150 ml sterile beaker and heat the solution to 70-80 °C with stirring on a hot plate. Slowly add the ethanol solution of FAM to the beaker with stirring. At this stage, AAM may be added at 1-25% (w / v). The addition of FAM initiates the gel-sol transition and gelation can be further promoted by adding gelling molecules such as sodium pectinate dissolved in deionized water if necessary. 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 the solubility in water. To accelerate the gelation process, FAM or its magnesium dimer can be added to the alginate copolymer during mixing and the M / G ratio can be adjusted to obtain a stable FAM:Mg 2+ : To create alginate biodegradable sheets. FAM cations interact with the pockets formed by the G-type alginate copolymers via hydrogen bonds (see Figure 15). All ratios can be adjusted to optimize FAM concentration and polymerization. Mg 2+ In addition, Ca 2+ , K + , or Zn 2+ Other ions, such as may be added to further aid in 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 made in a sterile environment and are non-irritating, non-sensitizing, and biodegradable. This makes liquid crystalline FAM molecules ideal molecules to promote alginate gelation, polymerization, control copolymer block structures, vary 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 alter the gel viscosity.

[0146] A variety of polymeric sugar molecules, when combined with the liquid crystal FAM and AAM molecules described in this application, can create unique hydrogels, alginates, and drug delivery systems that can be used in creating novel wound care products, including, but 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 FAMs or AAMs may be added to any of the above formulations to improve solubility, adjust pH, balance cation or anion concentrations, improve adhesion to skin, increase or decrease aqueous solubility, create concentration gradients, improve gel-sol transitions, increase or decrease electrical conductivity, increase or decrease capacitance, or adjust overall resistance or impedance.

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

[0149] Example 19: FAM or AAM Modulator-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) with mixing, if desired. To this solution add a therapeutically effective amount of FAM, previously dissolved in ethanol to the desired concentration of 1-50% (w / v), and, if desired, add monovalent or divalent cationic salt additives until the gel reaches the desired consistency. For FAM's salts, the previous step is not necessary. The liquid crystalline nature of the FAM molecule results in unique co-block polymers that associate through hydrogen, electrostatic, and ionic bonds. This solution can then be used to coat cotton fabric or other fibers to create alginate dressings. This 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 of all embodiments or to limit the invention to the particular aspects disclosed. Obvious modifications or variations are possible in light of the above teachings, and such modifications and variations may be included within the scope of the invention, as determined by the appended claims, when interpreted in accordance with the breadth to which they are fairly, legally, and equitably entitled.

Claims

1. 1. A method for promoting wound healing in a patient having a chronic wound or skin condition, comprising: A first autophagy-modulating compound having the structure (I): 【Chemistry 1】 In the formula, L is -C=C- and -CR ａ =CR ｂ - is a linker selected from R ａ and R ｂ are independently H or -(R ３ ) ｐ Or -(R ４ ) ｑ phenyl optionally substituted with R １ ~R ４ are independently a substituent at any available position on the phenyl ring; m, n, p, and q are each independently 0, 1, 2, or 3, which represent 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 is, independently, (C １ -C ６ ) alkyl, (C ２ -C ６ ) alkenyl, or (C ２ -C ６ ) alkynyl; —OH, —SH, -halo, —NH ２ , or NO ２ -R may be substituted with 1 to 3 substituents selected from ５ , Y is O, S, or NH; R ６ is H or R ５ -YR ６ , Z is -N(C=O)- or -O(C=O)-; ５ , - Hello, -NO ２ 、 -SO ３ No, -azide, and -Glycoside - and their salts, is selected from The method comprises administering to the patient a therapeutically effective amount of a formulation comprising the first autophagy-modulating compound that is not resveratrol or 4,4'-(ethyne-1,2-diyl)diphenol (also known as TOLECINE, 4,4'-dihydroxytol).

2. 2. The method of claim 1, wherein the first autophagy-modulating compound is a tolane where L is -C≡C-.

3. The method of claim 2, wherein the tolane is a hydroxylated tolane having 1 to 4 hydroxyl substituents.

4. The method of claim 3, wherein the tolane is selected from 2,4,4'-trihydroxytolane, 4,3',5'-trihydroxytolane, 2,2',4,4'-tetrahydroxytolane, 3,3',5,5'-tetrahydroxytolane, and 4-hydroxy-4'-(trifluoro)methyltolane.

5. 4. The method of claim 3, wherein the tolan is selected from 4,4'-dihydroxy-3-methoxytol, 2,4,4'-trihydroxytol-beta-D-glucoside, and 4-hydroxy-4'-methoxytol.

6. 2. The method of claim 1, wherein the first autophagy-modulating compound is a trans-stilbene, wherein L is -CH=CH-.

7. The method of claim 6, wherein the stilbene is a hydroxylated stilbene having 1 to 4 hydroxyl substituents.

8. The method of any one of claims 1 to 7, further comprising co-administration of an auxiliary autophagy-modulating compound.

9. 10. The method of claim 8, wherein the supplemental autophagy-modulating compound is administered simultaneously with the first autophagy-modulating compound.

10. 10. The method of claim 8, wherein the supplemental autophagy-modulating compound is administered prior to administering the first autophagy-modulating compound.

11. 9. The method of claim 8, wherein the supplemental autophagy-modulating compound is selected from the group consisting of substituted or unsubstituted parabenzoquinones, substituted or unsubstituted orthobenzoquinones, and substituted or unsubstituted anthraquinones.

12. 9. The method of claim 8, wherein the supplemental autophagy-modulating compound is selected from the group consisting of an amino acid, an acidic monosaccharide, and a vitamin or a salt thereof.

13. 13. The method of claim 12, wherein the vitamin is an oxygen-containing vitamin.

14. 13. The method of claim 12, wherein the vitamin is an isoprenoid-containing vitamin.

15. 2. The method of claim 1, wherein the first autophagy-modulating compound upregulates autophagy activity.

16. The wound or skin condition is selected from the group consisting of aging, autoimmune diseases with inflammation, avascular necrosis, bacterial infection, cancer, diabetic neuropathy, endometriosis, fungal infection, gout, hair loss, infectious arthritis, inflammation, inflammatory bowel disease, ischemia, Lyme disease, organ / tissue transplants, parasitic infections, psoriatic arthritis, psoriasis, pseudogout, rheumatoid arthritis, scleroderma, scurvy, sepsis, skin diseases, surgical scars, surgical adhesions, transfection procedures, ulcerative colitis, ulcers, viral infections, warts, surgical wounds, incisions, lacerations, cuts and 16. The method of claim 1 or 15, wherein the wound is one or more selected from the group consisting of abrasions, skin graft donor wounds, traumatic wounds, infected wounds, ischemic wounds, burns, blistering wounds, non-infected wounds, contusions, incisions, lacerations, non-penetrating wounds, open wounds, penetrating wounds, perforating wounds, puncture wounds, abscessed wounds, subcutaneous wounds, chronic ulcers, gastric ulcers, skin ulcers, peptic ulcers, duodenal ulcers, gastric ulcers, gout, hypertensive ischemic ulcers, congestive ulcers, sublingual ulcers, submucosal ulcers, symptomatic ulcers, trophic ulcers, tropical ulcers, and venereal ulcers.

17. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease giving rise to a need for modulation of autophagy is one or more skin diseases, such as keratosis, photoaging, psoriasis, skin rashes, sunburn, and photoreactive processes.

18. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease creates a need for one or more of the autophagy modulators, which may be used orally in the form of a mouthwash or spray to protect and promote healing of damaged oral tissues such as oral sores and burns, post-tooth extraction wounds, endodontic wounds, especially those associated with the treatment of cysts and abscesses, ulcers and lesions of bacterial, viral, or autoimmune origin, mechanical, chemical, thermal, infectious, and lichenoid wounds, herpes ulcers, aphthous stomatitis, acute necrotizing ulcerative gingivitis, and caustic sores.

19. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease creates a need for one or more of the autophagy modulating agents, which may be used in an ophthalmic formulation to treat wounds, such as wounds resulting from corneal ulcers, radial keratotomy, corneal transplants, epikeratophakia, and other surgically induced intraocular wounds.

20. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease creates a need for one or more of the autophagy modulating agents, which may be used in anorectal creams and suppositories to treat conditions such as pruritus, proctitis, anal fissures, and hemorrhoids.

21. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease creates a need for one or more of the autophagy modulators that may be used for the following effects: moisturizing and protecting, healing dry cracked skin, healing severe dry skin due to other diseases (venous dermatitis), protecting the skin from UV photodamage, and treating seborrheic conditions.

22. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease creates a need for one or more of the autophagy-modulating agents that may be used to treat a wound or sore associated condition that can be successfully treated by the present invention, such as burns, anthrax, tetanus, gas gangrene, scarlet fever, erysipelas, sycosis barbae, folliculitis, impetigo contagiosa, or bullous impetigo.

23. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease gives rise to a need for one or more of the autophagy modulators that may be used to treat at least one sign of aging in the skin selected from the group consisting of improving the elasticity of the skin, improving the firmness of the skin, and reducing the appearance of wrinkles or cellulite in the skin, and / or (i) treat non-acnegenic inflammation in the skin.

24. 20. The method of claim 1 or 15, wherein the condition, disorder, or disease creates a need for one or more of the autophagy modulating agents, which may be used as a treatment for oral and paraoral ulcers, e.g., resulting from radiation and / or chemotherapy.

25. A formulation comprising the first autophagy-regulating compound according to any one of claims 1 to 7 dispersed in a hydrogel.

26. 26. The formulation of claim 25, wherein the hydrogel is a liquid crystalline hydrogel formed in part by the first autophagy-modulating compound.

27. 26. The formulation of claim 25, wherein the hydrogel is an alginate.

28. 8. A formulation comprising a first autophagy modulating compound according to any one of claims 1 to 7 in combination with a cyclodextrin in a ratio of about 1:1 to about 1:10 of first autophagy modulating compound:cyclodextrin.

29. A formulation comprising a first autophagy-modulating compound according to any one of claims 1 to 7 in a complex with ascorbic acid and a cation.

30. 1. A method for modulating autophagy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a formulation, the formulation comprising: comprising a first autophagy-modulating compound having structure (I): 【Chemistry 2】 In the formula, L is -C≡C- and -CR ａ =CR ｂ - is a linker selected from R ａ and R ｂ are independently H or -(R ３ ) ｐ Or -(R ４ ) ｑ phenyl optionally substituted with R １ ~R ４ are independently a substituent at any available position on the phenyl ring; m, n, p, and q are each independently 0, 1, 2, or 3, which represent 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 is, independently, (C １ ~C ６ ) alkyl, (C ２ ~C ６ ) alkenyl, or (C ２ ~C ６ ) alkynyl; —OH, —SH, -halo, —NH ２ , or NO ２ -R may be substituted with 1 to 3 substituents selected from ５ , Y is O, S, or NH; R ６ is H or R ５ -YR ６ , Z is -N(C=O)- or -O(C=O)-; ５ , - Hello, -NO ２ 、 -SO ３ No, -azide, and -glycosides, and salts thereof, The method is selected from the group consisting of

31. The patient is suffering from a condition requiring autophagy upregulation, and the condition is selected from the group consisting of wound healing, hair regrowth, bacterial infection, inflammation, viral infection, Parkinson's disease, aging, Alzheimer's, neurodegenerative disease, neurological disorders, cardiovascular disease, 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, amyotrophic lateral sclerosis, pulmonary arterial disease, pulmonary fibrosis ...

31. The method of claim 30, comprising one or more of the following conditions: inflammatory diseases, rheumatoid arthritis, bursitis, dermatitis, vasculitis, autoimmune diseases involving inflammation, blood disorders, aplastic anemia, endometriosis, hepatitis, herpes, HIV, multiple sclerosis, retinal detachment, age-related macular degeneration, retinitis pigmentosa, and Leber's congenital amaurosis, lysosomal storage diseases, arthritis, psoriasis, osteopenia, osteoporosis, surgical scars, surgical adhesions, space travel (bone density disorder), tendonitis, and ulcerative colitis.

32. 31. The method of claim 30, wherein the patient is suffering from a condition requiring autophagy downregulation, the condition comprising one or more of aging, cancer, polycystic kidney disease and liver disease, renal disease, liver disease, asthma, diabetic retinopathy, fibromyalgia, ankylosing spondylitis, celiac disease, Graves' disease, lupus, metabolic disease, nephritis, rheumatoid arthritis, osteolysis, ischemia-reperfusion (I / R) injury, organ and tissue transplantation, scleroderma, and sepsis.

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