Methods of treating chronic wounds using caspase inhibitors

Abstract

This disclosure relates to wound treatment. It discloses compositions and methods for treating wounds, particularly chronic wounds, using caspase-3 inhibitors. It also discloses methods for detecting wounds based on the level of cleaved caspase-3 and / or caspase-3 activity in wound samples.

Description

Technical Field

[0001] This invention relates generally to wound treatment, and more specifically to compositions and methods for treating wounds, involving the use of cysteine ​​inhibitors.

[0002] background The integrity of healthy skin plays a crucial role in maintaining physiological homeostasis and preventing infection. The wound healing process is a finely regulated sequence of events involving multiple cell types and signaling pathways. Upon injury, inflammatory cells are recruited to prevent infection. Fibrin clots form to seal the wound until a new skin barrier can be built. Fibroblasts migrate to the wound bed and form granulation tissue, while activated keratinocytes at the wound edge migrate across the wound bed until the epithelial tongue meets at the center of the wound. From here, keratinocytes differentiate to form a new skin barrier. During epidermal trauma, keratinocytes at the wound edge undergo an epithelial-to-mesenchymal transition (EMT) from a non-motile epithelial state to a mesenchymal-like state, in which they lose cell-cell contact and become motile. Migrating cells reorganize their actin cytoskeleton and secrete proteases to remodel the dermal extracellular matrix (ECM) and enable migration across the wound. Directly behind the migrating cells, keratinocytes rapidly proliferate to provide sufficient cells to cover the wound. These events are coordinated through transient regulation of multiple signaling pathways. Failure or delay in initiating these events can lead to chronic or non-healing wounds.

[0003] Older adults often suffer from underlying medical conditions such as vascular disease, venous insufficiency, and diabetes, making them prone to chronic wounds that either fail to heal or heal slowly. Chronic wounds, such as diabetic foot ulcers, frequently lead to lower limb amputations in older adults. With the increasing aging of the global population and the high prevalence of diabetes, chronic wounds have become a significant social and medical burden. The involvement of multiple signaling and regulatory pathways in wound healing means that current wound therapies (many of which target individual pathways or proteins) are largely ineffective in treating chronic wounds. Therefore, more effective treatments for chronic wounds are needed.

[0004] The goal is to improve at least one of the problems described above, or at least provide a useful alternative.

[0005] Overview This article discloses a method for treating wounds in subjects, which involves administering a therapeutically effective dose of a cysteine-3 inhibitor to the subjects.

[0006] This article discloses a caspase-3 inhibitor for treating wounds in subjects, wherein a therapeutically effective amount of the caspase-3 inhibitor is administered to the subjects.

[0007] This article discloses the use of caspase-3 inhibitors in the manufacture of drugs for treating wounds in subjects, wherein a therapeutically effective amount of caspase-3 inhibitor is administered to the subjects.

[0008] This article discloses a method for promoting wound re-epithelialization in subjects, which involves administering a therapeutically effective amount of a cysteine-3 inhibitor to the subjects.

[0009] This article discloses a method for detecting chronic wounds in subjects, which includes detecting the level and / or activity of cleaved caspase-3 in samples from subjects, wherein an increase in the level and / or activity of cleaved caspase-3 relative to a reference indicates that the subject has a chronic wound.

[0010] This article discloses a method for treating chronic wounds in subjects, comprising: (a) detecting the level and / or activity of cleaved caspase-3 in a sample from the subject, wherein an increase in the level and / or activity of cleaved caspase-3 relative to a reference indicates that the subject has a chronic wound; and (b) administering a therapeutically effective amount of a caspase-3 inhibitor to the subject with a chronic wound.

[0011] This article discloses pharmaceutical compositions comprising (a) a cystase-3 inhibitor and (b) spermine and / or spermidine.

[0012] This article discloses drug combinations comprising (a) a caspase-3 inhibitor and (b) spermine and / or spermidine.

[0013] This document discloses pharmaceutical compositions or combinations as defined herein, for use as medicines.

[0014] This article discloses a method for treating wounds in subjects, which includes administering a pharmaceutical composition or combination as defined herein to the subject.

[0015] This article discloses pharmaceutical compositions or combinations as defined herein for the treatment of wounds in subjects.

[0016] This document discloses the use of pharmaceutical compositions or combinations as defined herein in the manufacture of a medicament for treating wounds in a subject. Brief description of the attached diagram Embodiments of the invention will now be described by way of non-limiting examples with reference to the accompanying drawings, in which: Figure 1This is a schematic diagram of a proposed model of caspase-3-mediated wound healing control and the potential role of caspase-3 inhibitors in chronic wounds. (A) Caspase-3 is transiently inhibited in acute wound healing but persistently activated in chronic wounds. (B) In acute wounds, transiently inhibited caspase-3 promotes the expression of pro-healing mRNAs, leading to increased cell migration and efficient healing. (C) In chronic wounds, persistently activated caspase-3 prevents the expression of pro-healing mRNAs, resulting in defective healing.

[0018] Figure 2 Caspase-3 activity was shown in ex vivo injured human skin samples. (A) Representative ex vivo human wound (scale bar, 5 mm). (B) Caspase-3 activity in ex vivo human skin samples that were not injured and were injured. Error bars, n = mean ± SD of 3 experiments. P-values ​​were obtained from one-way ANOVA. *, p < 0.05, **, p < 0.01. Bottom * indicates p-value for statistical analysis between wound and non-wound samples from the corresponding time point. Top * indicates p-value for statistical analysis between different time points and 0 hours after injury.

[0019] Figure 3 The caspase-3 activity is shown in a monolayer keratinocyte scratch model. Error bars, n = mean ± SD of 3 experiments. P-values ​​are from one-way ANOVA. *, p < 0.05, **, p < 0.01.

[0020] Figure 4 Caspase-3 activity is shown in acute and disturbed rat wound models. (A) Representative acute and disturbed rat wounds (scale bar, 10 mm). (B) Caspase-3 activity in acute and disturbed rat wound models. Error bars, n = mean of 3 experiments ± SD. P-values ​​are from two-way ANOVA. *, p < 0.05, **, p < 0.01.

[0021] Figure 5 shows the addition of the caspase-3 inhibitor Z-DEVD-FMK and spermine surface to a disturbed rat wound model. (A) Representative disturbed rat wounds under treatment (scale bar, 10 mm). (B) Relative rates of wound closure in disturbed rat wounds under different treatments. (C) H&E staining of disturbed rat wounds treated with and without Z-DEVD-FMK. Arrows indicate reepithelialization of rat wounds. (D) Caspase-3 activity in disturbed rat wounds on day 3 with and without Z-DEVD-FMK treatment. (E) Expression of key factors for wound healing in disturbed rat wounds with and without Z-DEVD-FMK treatment (n = 6 animals per treatment). Data analysis was performed using the Mann-Whitney U test. *, p < 0.05; **, p < 0.01.

[0022] Detailed description Caspases are an evolutionarily conserved family of cysteine-dependent proteases essential for apoptosis. These enzymes are produced as inactive proenzymes, undergoing proteolytic processing at a conserved aspartic residue to produce two subunits. The two subunits dimerize, and the two dimers further assemble to form a functional heterotetrameric enzyme. Caspase-3, known as the "executioner" of apoptosis, has been found to have multiple non-apoptotic functions in development, cell proliferation and migration, cell fate determination, cytoskeleton remodeling, and signal transduction by cleaving various substrate factors.

[0023] The inventors have discovered that caspase-3 acts as a key regulator of wound healing by inhibiting the expression of pro-healing mRNAs. Caspase-3 activity undergoes a transient decrease in the early stages of wound healing, which promotes the expression of multiple key factors required for cell migration and wound closure. Higher levels of caspase-3 activity in chronic wounds can prevent the upregulation of these factors and lead to delayed healing. Conversely, inhibition of caspase-3 can improve wound healing, even for wounds that do not heal or heal slowly. Unlike conventional wound healing therapies that target single pathways or proteins, targeting key regulators of wound healing, such as caspase-3, is advantageous because it can trigger multiple pathways and processes to accelerate wound healing. The inventors have also discovered that the natural polyamines spermine and spermidine can promote cell migration and improve wound healing. Therefore, the combined use of caspase-3 inhibitors with spermine and / or spermidine can further promote the healing of chronic wounds.

[0024] Therefore, this disclosure provides methods for treating wounds (including chronic wounds) in subjects using caspase-3 inhibitors. Caspase-3 inhibitors can be used alone or in combination with spermine and / or spermidine. Methods for detecting wounds based on caspase-3 levels in samples from subjects are also provided.

[0025] This article discloses a method for treating wounds in subjects, which involves administering a therapeutically effective dose of a cysteine-3 inhibitor to the subjects.

[0026] This article also discloses a method for promoting wound re-epithelialization in subjects, which involves administering a therapeutically effective amount of a caspase-3 inhibitor to the subjects.

[0027] This article discloses a caspase-3 inhibitor for treating wounds in subjects and / or promoting reepithelialization of wounds in subjects, wherein a therapeutically effective amount of the caspase-3 inhibitor is administered to the subject.

[0028] This article discloses the use of caspase-3 inhibitors in the manufacture of medicines for treating wounds in subjects and / or for promoting wound re-epithelialization in subjects, wherein a therapeutically effective amount of caspase-3 inhibitor is administered to the subject.

[0029] As used herein, the term "wound" refers to damage to tissue. Wounds include both open wounds (where the underlying tissue is exposed to the external environment, such as, for example, lacerations, punctures, burns, or surgical incisions) and closed wounds (where the underlying tissue is not exposed to the external environment, such as, for example, pressure sores, wounds caused by blunt trauma, and wounds caused by surgical implants). Wounds can be acute or chronic. Wounds can exhibit a spectrum of healing rates, with acute wounds and non-healing wounds located at opposite ends of this spectrum. Technicians will be able to determine the expected timeframe for wound healing based on, for example, the severity of the wound, the location of the wound, the type of wound (e.g., open or closed), and the age and health status of the injured subject.

[0030] In this article, "acute wound" refers to a rapidly occurring injury (such as cuts, lacerations, contusions, burns, etc.) that typically heals quickly and is expected to move through the normal stages of the healing process at the anticipated rate, eventually leading to complete wound closure. Acute wounds can be expected to heal within three months. Acute wounds can occur anywhere on the body and range in severity from superficial scrapes to deep injuries that damage blood vessels, nerves, and muscle tissue.

[0031] In this article, “chronic wound” refers to a wound that does not heal or heals slowly, failing to progress through the usual healing stages in an orderly manner or at the expected rate. For example, a wound that does not heal within three months can be considered chronic. Chronic wounds can be characterized at least in part by one or more of the following: (1) a prolonged, self-sustaining state of wound inflammation; (2) a lack, defective, and / or slow-forming wound extracellular matrix; (3) poorly responsive (senescent) cells at the wound site, particularly fibroblasts, limiting extracellular matrix production; and (4) re-epithelialization or failure or reduced rate of wound closure. A chronic wound may be a wound that heals slowly, at a slower rate than expected, but still shows some healing over time. A chronic wound may also be a wound that does not heal, failing to show improvement or closure despite appropriate care.

[0032] As used herein, the terms “treatment”, “treating”, etc., refer to achieving the desired pharmacological and / or physiological effect. The effect can be therapeutic in terms of partially or completely curing a disease or condition and / or in relation to adverse effects attributable to that disease or condition. These terms also cover any treatment of a subject’s condition or disease and include: (a) suppressing the disease or condition, i.e., preventing its development; or (b) alleviating the disease or condition, i.e., causing the remission of the disease or condition.

[0033] In the context of treating a disease or condition, a "therapeutic effective amount" or "effective amount" refers to a quantity of active agent administered to a subject as a single dose or as part of a series or slow-release system that is effective in treating the disease or condition. The effective amount will vary depending on the subject's health and physical condition, as well as the subject's classification group, the severity of the disease or condition, the formulation of the active agent or pharmaceutical composition, the assessment of the medical condition, and other relevant factors. Technicians will be able to determine the effective amount of active agent by considering factors such as the subject's age, weight, and clinical condition.

[0034] In this document, "subject" refers to an organism to be treated using the methods of this disclosure. In some embodiments, the subject is a mammal, including both humans and non-human mammals, such as dogs, cats, pigs, bovines, equines, rodents, or primates. In one embodiment, the subject is a human. The subject may be a patient.

[0035] caspase-3 inhibitors As used herein, the term "caspase-3 inhibitor" refers to any molecule that partially, substantially, or completely prevents or blocks the production and / or activity of caspase-3. The term "caspase-3 inhibitor" encompasses inhibitors of caspase-3 production as well as inhibitors of caspase-3 activity. Caspase-3 inhibitors include small molecules, peptides (e.g., allosteric antagonists or substrate mimics), proteins (e.g., antibodies), nucleic acids (e.g., aptamers, antisense oligonucleotides, and small interfering RNA), glycans, and conjugates, or combinations of two or more of these. Inhibitors can be naturally isolated molecules or synthetic molecules (e.g., chemically or biosynthetically synthesized molecules). Inhibitors can be nucleic acids encoding inhibitory peptides or polypeptides. Inhibitors can inhibit caspase activity directly (e.g., by preventing interaction with a substrate or by altering the structure of the enzyme) or indirectly (e.g., by affecting the subcellular localization of the enzyme). Caspase-3 inhibitors can target, for example, epithelial cells or keratinocytes. One or more inhibitors may be used in the methods described herein.

[0036] The inhibitor produced can be any molecule that negatively affects the synthesis, processing, or maturation of caspase-3. Inhibitors can be, for example, inhibitors of caspase-3 gene expression; antisense oligonucleotides or double-stranded RNAs such as small interfering RNA or microRNAs that reduce or prevent the transcription of caspase-3 mRNA or degrade caspase-3 mRNA; proteins that impair the proper folding of caspase-3; proteases that degrade caspase-3 after synthesis; or compounds that inhibit the cleavage of pro-caspase-3 and thus prevent the production of active caspase-3 (cleaved caspase-3).

[0037] Inhibitors of caspase-3 activity can be caspase-3 antagonists. Antagonists can bind to or mask the caspase-3 molecule itself with sufficient affinity and specificity to partially or substantially neutralize caspase-3 activity. Inhibitors of caspase-3 activity can also be caspase-3-specific antibodies, such as polyclonal or monoclonal antibodies, or any other molecule that prevents caspase-3 from binding to its target, thereby reducing or preventing the triggering of caspase-3-mediated reactions.

[0038] In some embodiments, the caspase-3 inhibitor targets both caspase-3 and caspase-7, which are structurally similar and have similar allosteric sites. In other embodiments, the caspase-3 inhibitor is a selective caspase-3 inhibitor, i.e., it preferentially targets or binds to caspase-3 relative to other caspases.

[0039] In some embodiments, the caspase-3 inhibitor is a small molecule inhibitor. Small molecule inhibitors may have a molecular weight of about 1000 Da or less. Small molecule inhibitors include, but are not limited to, metabolites, metabolic analogs, amino acids, amino acid analogs, nucleotides, nucleotide analogs, heteroorganic compounds, and organometallic compounds. Non-limiting examples of small molecule caspase-3 inhibitors include flubendazole, L-tryptophan, fenprofen, diflunisal, pranoprofen, emricasan, VX-166, M826, M867, indigo-based sulfonamides, 5-fluoro-1H-indole-2-carboxylic acid (2-mercaptoethyl)amide (FICA), 2-(2,4-dichlorophenoxy-N-(2-mercaptoethyl)-acetamide) (DICA), and their pharmaceutically acceptable salts and solvates. The structures of some of these compounds are provided below. In one embodiment, the caspase-3 inhibitor is flubendazole, L-tryptophan, fenprofen, diflunisal, pranoprofen, or a pharmaceutically acceptable salt or solvation thereof.

[0040] In some embodiments, caspase-3 inhibitors are peptides or peptide mimics. Peptide and peptide mimic inhibitors can competitively bind to caspase-3 substrates. Caspase-3 recognizes a tetrapeptide motif Asp-XX-Asp, where X can be any amino acid. The C-terminal Asp is absolutely required, while variations at the other three positions are tolerable. This substrate specificity can be used to design caspase-3 inhibitors. For example, caspase-3 is known to cleave the peptide sequence DEVDG (Asp-Glu-Val-Asp-Gly) between D and G in vitro. Peptides WEHD, VDVAD, and DEVD are other examples of peptides that bind to caspase-3. Reversible or irreversible inhibitors of caspase activation can be generated by conjugating caspase-specific peptides to certain aldehyde, nitrite, or ketone compounds. Fluoromethyl ketone (FMK)-derived peptides or chloromethyl ketone (CMK)-derived peptides, such as Z-IETD-FMK, act as potent irreversible inhibitors. Inhibitors synthesized with benzyloxycarbonyl groups (also known as BOC or Z) at the N-terminus and O-methyl side chain exhibit enhanced cell permeability, thus facilitating their application in vivo. Non-limiting examples of peptide inhibitors of caspase-3 include Z-VAD-FMK, Z-DEVD-FMK, and Ac-DEVD-CMK.

[0041] In some implementations, the cysteine-3 inhibitor is a nucleic acid inhibitor. Non-limiting examples of nucleic acid inhibitors include antisense compounds (e.g., antisense oligonucleotides (ASO), gapmers, etc.); nucleic acid molecules that mediate RNA interference, including but not limited to short hairpin RNA (shRNA), small interfering RNA (siRNA) and their variants and precursors (e.g., small fragment siRNA, small interfering RNA neutrals, Dicer substrate siRNA, etc.), as well as microRNAs (miRNAs) and precursors; and guide RNAs (gRNAs) that mediate sequence-specific base editing, gene or RNA editing, or DNA or RNA cleavage associated with the Cas protein. The inhibitor may be a vector encoding the nucleic acid inhibitor (e.g., a plasmid vector, a viral vector, etc.).

[0042] In some implementations, the caspase-3 inhibitor reduces the production or activity of caspase-3 by at least about 10%. For example, the caspase-3 inhibitor may reduce the production or activity of caspase-3 by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0043] It should be understood that additional bioactive agents can be screened for caspase-3 inhibitory activity. The terms "candidate agent" or "candidate inhibitor" as used herein describe any molecule, such as small molecules, peptides, polypeptides, polysaccharides, sugars (including polysaccharides), polynucleotides, lipids, etc. Typically, more than one assay mixture is run in parallel with different agent concentrations to obtain differential responses to various concentrations. Typically, one of these concentrations is used as a negative control, i.e., zero concentration or below the detection level. Additionally, a positive control can be used, i.e., an agent known to inhibit caspase-3.

[0044] Candidates can be obtained from a variety of sources, including libraries of synthetic or natural compounds. For example, numerous methods are available for the random and directed synthesis of a wide range of organic compounds and biomolecules, including peptides, polypeptides, and oligonucleotides. Optionally, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily generated. In addition to screening using bioactivity assays, candidate agents can undergo computer simulation screening (such as by computationally docking the agent to a target molecule or target molecule site). The ZINC database provides a collection of commercially available chemical compounds for computer simulation screening.

[0045] Combination therapy This disclosure also provides combination therapies for treating wounds, including the administration of (a) a caspase-3 inhibitor and (b) one or more polyamines, such as spermine or spermidine. The inventors have discovered that intracellular levels of spermine and spermidine are upregulated at the wound site, and that these polyamines promote alterations in cellular behavior that enable reepithelialization of the wound. Therefore, combining spermine and / or spermidine with a caspase-3 inhibitor for wound treatment has the advantage of further accelerating wound closure, particularly in chronic wounds.

[0046] Therefore, in one embodiment of the disclosed method, a caspase-3 inhibitor is administered to a subject together with a therapeutically effective amount of spermine and / or spermidine to treat the subject's wound.

[0047] This document discloses pharmaceutical combinations comprising (a) a caspase-3 inhibitor and (b) spermine and / or spermidine. The caspase-3 inhibitor and spermine and / or spermidine may be contained in a single composition or in separate compositions. Additionally, the caspase-3 inhibitor and spermine and / or spermidine may be formulated for administration via the same route or via different routes. In one embodiment, the caspase-3 inhibitor and spermine and / or spermidine are contained in a single composition. In another embodiment, the caspase-3 inhibitor and spermine and / or spermidine are contained in separate compositions.

[0048] This article discloses pharmaceutical compositions comprising (a) a cystase-3 inhibitor and (b) spermine and / or spermidine.

[0049] This document discloses pharmaceutical compositions or combinations as defined herein, for use as medicines.

[0050] This article discloses a method for treating wounds in subjects, which includes administering a pharmaceutical composition or combination as defined herein to the subject.

[0051] This article discloses pharmaceutical compositions or combinations as defined herein for use in treating wounds or promoting wound re-epithelialization in subjects.

[0052] The use of pharmaceutical compositions or combinations as defined herein in the manufacture of medicaments for treating wounds or for promoting wound re-epithelialization in subjects is disclosed.

[0053] This article discloses a method for treating wounds in subjects or promoting reepithelialization of wounds in subjects, the method comprising administering to the subject a therapeutically effective amount of a combination of a caspase-3 inhibitor and a therapeutically effective amount of spermine and / or spermidine.

[0054] The terms “combination” and “in combination with” are not intended to imply that therapies or treatments must be administered simultaneously and / or formulated for delivery together, although such delivery methods are within the scope described herein. A treatment in a combination may be administered simultaneously, before, or after one or more other additional therapies or treatments. Treatments or treatment regimens may be administered in any order. Typically, each agent will be administered according to a dosage and / or timing regimen determined for that agent. It will be further understood that additional treatments used in a combination may be administered together or individually in different compositions. Generally, the treatments used in a combination are intended to be used at levels not exceeding those used individually. In some embodiments, the levels used in a combination are lower than those used individually.

[0055] Therapeutic agents can be administered simultaneously or sequentially, depending on factors such as the nature of the wound to be treated, the composition of the agent, or the desired route of administration. As used herein, “simultaneously” means that two or more therapeutic agents are administered at the same time or in a substantially simultaneous manner (e.g., immediately after each other). “Sequentially” means that two or more therapeutic agents are administered at different times. In sequential administration, caspase-3 inhibitors may be administered before or after spermine and / or spermidine. A time delay may exist between sequential administrations of the therapeutic agents. The time interval can be any predetermined time interval, but is preferably a time interval that provides synergistic effects between the caspase-3 inhibitor and the polyamine. Therapeutic agents can be administered simultaneously or sequentially via the same route or via different routes.

[0056] In one embodiment, the caspase-3 inhibitor and spermine and / or spermidine are administered simultaneously. In an alternative embodiment, the caspase-3 inhibitor and spermine and / or spermidine are administered sequentially.

[0057] The combination therapy described herein may also include other therapeutic agents, such as antimicrobial agents (e.g., antiseptics, antibiotics, or antifungals), analgesics (both narcotic and non-narcotic), anti-inflammatory agents (e.g., steroids), growth factors (e.g., PDGF), antihistamines, and / or vitamins (e.g., vitamins B, C, or E). These agents may be administered together with caspase-3 inhibitors and / or polyamines (e.g., simultaneously or in the same composition) or sequentially.

[0058] Pharmaceutical Composition Caspase-3 inhibitors and spermine or spermidine may be included in a suitable pharmaceutical composition for administration.

[0059] Depending on the location of the wound to be treated, the drug composition can be administered topically or systemically, such as via oral, intrafacial, intra-articular, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intravascular, intravitreal, liposome, local, mucosal, extra-gastric, subconjunctival, skin, subcutaneous, sublingual, surface, buccal, or percutaneous routes, or combinations thereof. The drug composition can be administered via catheter, via irrigation, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via implant, via dressing, or via any combination thereof.

[0060] Depending on the intended route of administration, the therapeutic agents, pharmaceutical compositions, combined articles, and medicaments of the present invention may take, for example, the form of tablets, caplets, capsules, hard capsules, soft capsules, gelatin capsules, flat capsules, lozenges, sugar lozenges, dispersants, suppositories, ointments, creams, gels, hydrogels, foams, mud dressings, pastes, powders, dressings, plasters, solutions, patches, aerosols, nasal sprays, inhalers, ointments, suspensions, aqueous liquid suspensions, non-aqueous liquid suspensions, oil-in-water emulsions, water-in-oil emulsions, solutions, sterile solids, crystalline solids, amorphous solids, solids for reconstitution, delayed-release formulations, sustained-release formulations, or combinations thereof. Other suitable formulations include liposome formulations, nanoparticle formulations, pluronic gel-based formulations, carboxymethyl cellulose (CMC)-based formulations, and hydroxypropyl methyl cellulose (HPMC)-based formulations. Those skilled in the art may select appropriate formulations and dosages based on, for example, the location, type, and severity of the wound, and the desired route of administration.

[0061] The pharmaceutical compositions described herein may also contain pharmaceutically acceptable carriers and / or pharmaceutically acceptable salts. Suitable carriers include isotonic saline solutions, such as phosphate-buffered saline. Suitable diluents and excipients also include, for example, water, saline, dextran, glycerol, and combinations thereof. Furthermore, if desired, substances such as wetting agents, solubilizers or emulsifiers, stabilizers or pH buffers, viscosity control agents, preservatives, antioxidants, emollients, odor control agents, or flavor compounds may be present.

[0062] The term "pharmaceutically acceptable delivery vehicle" refers to any drug delivery vehicle that does not induce antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable delivery vehicles can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, and amino acid copolymers.

[0063] Pharmaceutically acceptable salts may also be present, such as mineral salts, including hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, including acetate, propionate, malonate, benzoate, etc.

[0064] Surface composition In one embodiment, the pharmaceutical composition is a surface composition. The surface composition may be in the form of, for example, liquid, cream, ointment, gel, lotion, lotion, paste, jelly, hydrogel, soap, spray, foam, powder, liniment, patch, or impregnated dressing.

[0065] Suitable carrier materials for surface application include any carrier or medium commonly used as a matrix for creams, ointments, gels, emulsions, lotions, pastes, jelly, sprays, foams, powders, or coatings for surface application, including but not limited to emulsifiers, inert carriers, including hydrocarbon matrices, emulsified matrices, water-soluble matrices, or combinations thereof. Suitable solvents, emollients, and emulsifiers for hydrophobic surface formulations include lanolin, paraffin wax, beeswax, emulsified waxes, dimethyl silicone oil, mineral oil, silicone oil, vegetable oils, alkyl esters of fatty acids and fatty acids or dicarboxylic acids, triglycerides, fatty alcohols and fatty alcohol ethers, and sterols. Other suitable solvents, emollients, and emulsifiers include polyols and polyether derivatives such as glycerin, sorbitol, polyethylene glycol, polypropylene glycol, ionic and amphoteric surfactants, amphoteric surfactants, and nonionic surfactants.

[0066] Ointments and creams can be formulated, for example, with an aqueous or oil-based base, by adding suitable thickeners and / or gelling agents. Lotions can be formulated with an aqueous or oil-based base and will typically also contain one or more emulsifiers, stabilizers, dispersants, or thickeners. Liquid sprays can be delivered from pressurized packaging, for example, via specially shaped closures.

[0067] In some embodiments, the therapeutic agents described herein are formulated with an oily matrix or ointment to form a semi-solid composition having a desired shape. The composition can be shaped for easy application or insertion into wounds, ulcers, or surgical sites. In addition to the active ingredient, these semi-solid compositions may contain dissolved and / or suspended bactericides, preservatives, and / or buffering systems. The petrolatum component in these matrices can be any paraffin wax with viscosities ranging from mineral oils employing incorporated isobutylene, colloidal silica, or stearates to paraffin waxes. Such matrices can be prepared by melt-incorporating high-melting-point waxes into fluid mineral oils or by incorporating polyethylene into mineral oils at elevated temperatures. Polysiloxanes (also known as silicones) are suitable for these matrices.

[0068] In other embodiments, the compositions herein comprise a hydrogel carrier, such as a Pluronic gel (i.e., a nonionic polyethylene oxide-polyoxyethylene copolymer gel) or a gel based on HPMC, CMC, and other cellulose-based components. The gel may be moldable to conform to the size and shape of a wound and may be used to confine the release of a therapeutic agent to or immediately adjacent to the application site, and additionally provide delayed and / or sustained release of the therapeutic agent. Suitable hydrogel materials include natural polymers such as polysaccharides (e.g., starch, dextran, pectin, alginate, cellulose chitosan, hyaluronic acid, gellan gum, etc.) and peptides (e.g., collagen). Natural polymers may also be processed, for example, through chemical derivatization, such as forming esters and ethers or pharmaceutically acceptable salts. Alternatively, the hydrogel may also be based on a synthetic polymer, non-limiting examples of which include polymers based on polyalkylene oxides, poly(ethyl)acrylate and poly(meth)acrylate polymers, polyalkyl(meth)acrylate polymers, vinyl polymers, polylactide and polyglycolic acid polymers, polycaprolactam and polycaprolactone polymers, polyurethane polymers, and polyurea polymers.

[0069] Controlled release formulation Controlled or sustained release can be achieved by adding time-release additives, such as polymer structures or matrices known in the art. Suitable carriers include mixtures or coatings of polymers that provide release of the active agent at a constant rate over an extended time period.

[0070] The choice of matrix material is based on the desired release timeframe, typically ranging from at least one week to one month, although longer timeframes may be desired. In some cases, linear release can be most useful, although in others, pulsed release or "bulk release" may provide more efficient results.

[0071] In some embodiments, the carrier comprises one or more biodegradable polymers, and the therapeutic agent is delivered primarily through matrix degradation. Examples of biodegradable polymers include synthetic polymers such as polymers of hydroxy acids, such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(orthoesters), polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), natural polymers such as collagen, albumin, and other hydrophilic proteins, zein, and other alcohol-soluble glutenins, and combinations thereof. Typically, these materials degrade through enzymatic hydrolysis or in vivo exposure to water, via surface or bulk erosion.

[0072] The carrier may also include one or more non-degradable hydrophilic polymers, such as polyethylene glycol (PEG), polypropylene glycol, poloxamer, hydroxypropyl cellulose, polyvinyl alcohol, and other water-soluble excipients. The carrier may also include one or more hydrophobic polymers. Non-limiting examples of such polymers are ethyl cellulose, acrylic resins, copolymers of methacrylic acid and ethyl acrylate, vinyl acetate copolymers, polystyrene-butadiene copolymers, and silicone rubbers.

[0073] In some embodiments, the polymer matrix is ​​in the form of microparticles or nanoparticles. Microparticles may be in the form of microspheres, wherein the therapeutic agent is dispersed within a solid polymer matrix or microcapsule, wherein the core is a different material from the polymer shell, and the therapeutic agent is dispersed or suspended in the core, which may be liquid or solid in nature. Particles may also be in the form of liposomes (comprising a lipid bilayer separating an aqueous internal compartment from a bulk aqueous phase) or micelles (comprising a closed lipid monolayer having a hydrophobic core and polar surfaces or a polar core and hydrophobic surfaces). The therapeutic agent may be dispersed in an aqueous or hydrophobic phase in liposomes or micelles. Unless specifically defined herein, microparticles, microspheres, microcapsules, nanoparticles, nanospheres, and nanocapsules are used interchangeably.

[0074] Alternatively, the polymer matrix can be cast into sheets or films ranging from nanometers to centimeters; powders produced by grinding or other standard techniques; or gels such as hydrogels. Such matrices can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art.

[0075] In other embodiments, the therapeutic agent is incorporated into or encapsulated in a solid or semi-solid bulk matrix (such as a cross-linked hydrogel, patch, or dressing) for implantation at the wound site.

[0076] Wound treatment The compositions, combinations, and methods described herein can be used to treat wounds, particularly chronic wounds.

[0077] Non-limiting examples of chronic wounds include ulcers such as pressure ulcers (also known as bedsores), diabetic ulcers, diabetic foot ulcers, arterial ulcers, venous ulcers, venous stasis ulcers, vasculitis ulcers, burn ulcers, trauma-induced ulcers, infected ulcers, and pyoderma gangrene ulcers. Chronic wounds also include recurrent wounds caused by chronic skin or epithelial conditions such as acne, psoriasis, atopic dermatitis, and keratitis. Chronic wounds also include surgical wounds that do not heal or heal slowly, including dehiscences, which are wounds that have ruptured or split open, typically surgical incisions that are sutured or stapled.

[0078] In one implementation, the wound is an epithelial wound. An “epithelial wound” refers to any damage or disruption to epithelial tissue, such as the skin or the lining of various organs and body cavities.

[0079] In one implementation, the wound is a skin wound. A “skin wound” means any damage or destruction to the epidermis and / or dermis of the skin.

[0080] Wound treatment may include one or more of the following: increasing the rate of wound re-epithelialization; increasing the degree of wound re-epithelialization; increasing the rate of wound closure; and / or increasing the degree of wound closure.

[0081] In some implementations, the methods described herein promote wound re-epithelialization. As used herein, the term "re-epithelialization" refers to the process by which epithelial cells (e.g., fibroblasts, keratinocytes) at the wound margin differentiate, proliferate, and migrate to cover the wound. Methods can increase the rate and / or extent of wound re-epithelialization. Re-epithelialization can be determined using methods known in the art, such as by visual assessment, by histological analysis using cell or tissue staining of biopsy samples, or by imaging techniques such as optical coherence tomography.

[0082] In some implementations, the subject has impaired wound healing capacity. The subject may suffer from diseases or conditions that delay wound healing, such as diabetes, chronic skin or epithelial disorders, vascular diseases leading to chronic vascular insufficiency (e.g., atherosclerosis or peripheral artery disease), cancer, immunodeficiency disorders, autoimmune disorders, or malnutrition. Optionally or additionally, the subject may be undergoing treatments that impair wound healing. Subjects with impaired wound healing capacity may have chronic wounds that can be advantageously resolved using the methods of this disclosure.

[0083] The disclosed compositions can be applied to or near the wound site. For skin wounds, the compositions are preferably applied topically. Topical application can be in any suitable form, such as liquids, creams, ointments, gels, lotions, lotions, pastes, jelly, hydrogels, soaps, sprays, foams, powders, liniments, or patches, as described herein. The compositions can also be incorporated into inserts, wound dressings, hydrogels, or other materials that come into contact with the wound.

[0084] As described herein, caspase-3 inhibitors and / or polyamines can be formulated for sustained release, such as using polymer delivery systems, liposome delivery systems, and hydrogels. These can be loaded with therapeutic agents and injected or implanted at or near the wound site, where the caspase-3 inhibitors and / or polyamines are released for the duration of therapeutic efficacy.

[0085] Any of the cysteine-3 inhibitors and combination therapies can be administered in a single-dose, multiple-dose, continuous, or intermittent manner (e.g., at regular intervals), depending on, for example, the subject's clinical condition or the condition of the wound. Administration of the composition can be substantially continuous over an indeterminate period of time, e.g., until the wound closes. Optionally, the composition can be administered continuously over a pre-selected period of time or at a series of intervals.

[0086] Wound detection This article also provides a method for detecting wounds in subjects using caspase-3 as a biomarker. This method is beneficial for detecting wounds that are difficult to visually assess, such as closed wounds. It can be used to differentiate between acute and chronic wounds, as chronic wounds may exhibit persistently higher levels of caspase-3 or caspase-3 activity over a period of time than acute wounds. The method can also be used to identify wounds that may respond to treatment with caspase-3 inhibitors.

[0087] This article discloses a method for detecting wounds in subjects, which includes detecting the level and / or activity of cleaved caspase-3 in samples from subjects, wherein an increase in the level and / or activity of cleaved caspase-3 relative to a reference indicates that the subject has a wound.

[0088] In some implementations, the method differentiates chronic wounds from acute wounds based on an increase in lysed caspase-3 levels and / or caspase-3 activity. The method may differentiate chronic wounds from acute wounds based on the extent and / or duration of the increase in lysed caspase-3 levels and / or caspase-3 activity. For example, acute wounds typically result in a smaller increase in lysed caspase-3 levels and / or caspase-3 activity compared to chronic wounds, and the increase in acute wounds is generally more transient compared to chronic wounds.

[0089] This article discloses a method for detecting chronic wounds in subjects, which includes detecting the level and / or activity of cleaved caspase-3 in samples from subjects, wherein an increase in the level and / or activity of cleaved caspase-3 relative to a reference indicates that the subject has a chronic wound.

[0090] This article discloses a method for treating chronic wounds in subjects, comprising: (a) detecting the level and / or activity of cleaved caspase-3 in a sample from the subject, wherein an increase in the level and / or activity of cleaved caspase-3 relative to a reference indicates that the subject has a chronic wound; and (b) administering a therapeutically effective amount of a caspase-3 inhibitor to the subject with a chronic wound.

[0091] This article discloses a method for treating wounds in subjects, comprising: (a) detecting the level and / or activity of cleaved caspase-3 in a sample from the subject, wherein an increase in the level and / or activity of cleaved caspase-3 relative to a reference indicates that the wound may be responsive to caspase-3 inhibitor therapy; and (b) administering a therapeutically effective amount of a caspase-3 inhibitor to a subject with a wound that may be responsive to caspase-3 inhibitor therapy.

[0092] As used herein, "cleaved caspase-3" refers to one or both of the 17 kDa and 12 kDa polypeptide products resulting from the cleavage of caspase-3. Caspase-3 is naturally produced as a caspase zymogen and undergoes enzymatic cleavage to generate two protein cleavage products. The heterotetramerization of the cleavage products forms the active enzyme.

[0093] As used herein, “sample” includes any biological sample that can be extracted from a subject, whether untreated, processed, diluted, or concentrated. Within its scope, a sample includes collections of similar fluids, cells, or tissues isolated from a subject (e.g., surgically removed tissue, biopsies, including fine-needle aspiration), as well as fluids, cells, or tissues present within the subject. Any suitable method for obtaining a biological sample may be employed; exemplary methods include, for example, venipuncture, swabbing (e.g., oral swabbing), and surgical biopsy. Samples may be aggregated from multiple aliquots.

[0094] In one implementation, the sample is a wound sample. The wound sample may be taken from wound exudate, the wound edge, or the center of the wound.

[0095] In some implementations, samples are collected at least 24 hours after wound appearance. Samples can be collected at least 24 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after wound appearance. Samples can also be collected approximately 24 hours, approximately 48 hours, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, or approximately 14 days after wound appearance.

[0096] In some embodiments, the method includes detecting cleaved caspase-3 levels and / or caspase-3 activity over a period of time, wherein a sustained increase in cleaved caspase-3 levels and / or caspase-3 activity relative to a reference over the period of time indicates that the subject has a chronic wound. The time period for detection may depend on the expected healing time of the wound; for example, for wounds expected to heal rapidly, detection may be performed over a shorter time period.

[0097] In some implementations, the time period for detecting cleaved caspase-3 and / or caspase-3 activity is at least 5 days. The detection time period can be at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. The detection time period can be approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, approximately 14 days, or longer than 14 days.

[0098] Two or more samples may be collected during this time period to determine whether there is a sustained increase in lysed caspase-3 and / or caspase-3 activity. For example, samples may be collected at the beginning and end of the time period. Additional samples may also be collected at any point during the time period, such as daily, every other day, weekly, etc. Technicians can determine the appropriate sampling frequency to assess a sustained increase in lysed caspase-3 and / or caspase-3 activity based on factors such as the time period being tested, the condition and / or healing progress of the wound, and the subject's clinical status.

[0099] As used herein, “reference,” “control,” “reference sample,” or “control sample” refers to a sample, cell, tissue, standard, or level used for comparative purposes. In one embodiment, the reference is obtained from a healthy and / or uninjured body part (e.g., tissue or cell) of the same subject or individual. For example, healthy and / or uninjured cells or tissue adjacent to a wound site. In another embodiment, the reference is obtained from untreated tissue and / or cells of the same subject or individual. In yet another embodiment, the reference is obtained from a healthy and / or uninjured body part (e.g., tissue or cell) of an individual who is not a subject or individual. In yet another embodiment, the reference is obtained from untreated tissue and / or cells of an individual who is not a subject or individual. The reference may be a population average level of a biomarker (e.g., lysed caspase-3) in healthy cells or tissues. In yet another embodiment of the method for detecting chronic wounds, the reference may be obtained from an acute wound from the same or different subjects. The reference may also be a population average level of caspase-3 in acute wound samples.

[0100] As used herein, the term “increased” or “increased” in relation to biomarkers such as cystapatin-3 can refer to a statistically significant and measurable increase in the biomarker compared to a reference. An increase can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times or more.

[0101] Cleavage caspase-3 can be detected using any method known in the art for detecting proteins. Non-limiting examples of such methods include immunological methods (e.g., using Western blotting, immunofluorescence imaging, or immunohistochemical staining of the sample) using labeled antibodies or antibody fragments bound to one or more cleavage caspase-3 products; protein function or activity assays; protein binding assays using non-immunological means, such as using labeled aptamers, lectins, or other binding agents; and mass spectrometry.

[0102] Caspase-3 activity can be detected using any method known in the art for detecting the enzymatic activity of caspase-3, such as using a labeled substrate that generates a signal (e.g., a visible, luminescent, or fluorescent signal) when hydrolyzed by caspase-3 protein.

[0103] Reagent test kit This disclosure also extends to kits for detecting cleaved caspase-3 or caspase-3 activity in samples obtained from subjects. The kits may allow for the detection of wounds in subjects when the level of cleaved caspase-3 or caspase-3 activity in a sample is increased compared to a reference.

[0104] The kit may contain one or more reagents or materials for detecting cleaved caspase-3 or caspase-3 activity in samples from a subject. For example, the kit may contain a labeled antigen-binding molecule (e.g., an antibody or aptamer) for binding to and detecting cleaved caspase-3. Optionally or additionally, the kit may contain a labeled caspase-3 substrate for generating a signal upon hydrolysis by caspase-3 protein.

[0105] The kit may also include appropriate reagents for detecting the label, positive and negative controls, washing solutions, blot membranes, microtiter plates, dilution buffers, etc. For example, a protein assay kit may include (i) at least one naturally cleaved caspase-3 polypeptide (which may be used as a positive control), and (ii) one or more antigen-binding molecules that specifically bind to the cleaved caspase-3 polypeptide. The antigen-binding molecules are appropriately and detectably labeled. The kit may also have various devices (e.g., one or more) and reagents (e.g., one or more) for performing the assays described herein, and / or printed instruction material for quantifying the level of cleaved caspase-3 or caspase-3 activity using the kit. Reagents described herein, which may optionally associate with the detectable label, may be presented in the form of microfluidic cards, chips, chambers, or microarrays. The kit may also include instructions for selecting a suitable caspase-3 inhibitor based on the level of cleaved caspase-3 or caspase-3 activity.

[0106] This article also provides compositions comprising (a) a sample obtained from a subject; and (b) an antigen-binding molecule for detecting cleaved cysteine-3 in the sample. The sample may be a wound sample.

[0107] As used herein, “and / or” means and covers any and all possible combinations of one or more related listed items, as well as combinations that are missing when interpreted in terms of alternatives (or).

[0108] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used herein include plural referents. For example, the term “an agent” includes more than one agent, including mixtures thereof.

[0109] Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0110] Throughout this specification and the following claims, unless the context otherwise requires, the phrase "consisting essentially of" and variations such as "consistses essentially of" shall be understood as indicating that the listed elements are essential, i.e., necessary, elements of the invention. This phrase allows for the presence of other unlisted elements that do not substantially affect the features of the invention, but excludes additional unspecified elements that would affect the fundamental and novel features of the defined method.

[0111] References to any prior publications (or information derived therefrom) or to any known matter in this specification are not and should not be construed as an endorsement or acknowledgment or in any way imply that such prior publications (or information derived therefrom) or known matters constitute part of the general knowledge in the field covered by this specification.

[0112] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications beyond those specifically described. It should be understood that the invention includes all such variations and modifications falling within its spirit and scope. The invention also includes all steps, features, compositions, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0114] Some embodiments of the invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the general scope of the foregoing description.

[0115] Example method To generate the most clinically relevant data, profiles of caspase-3 activity during wound healing were determined using human ex vivo injured skin models, monolayer keratinocyte scraping models, and acute and disturbed rat wound models.

[0116] 1) Human Ex vivo Wound Model: Surgical waste skin tissue was collected from clinicians. To analyze polyamine levels in ex vivo injured human skin tissue, 6 mm puncture biopsies were created, and excisional wounds were formed within each biopsy. The excisional wounds created in ex vivo human skin were cultured at the air-liquid interface in William E medium with 10% FBS and harvested at 0, 2, 4, 8, 16, 24, 36, and 48 hours post-injury. Wound biopsies were rapidly frozen and lysed for measuring caspase-3 activity.

[0117] 2) Monolayer Keratinocyte Scraping Model: The immortalized keratinocyte line N / TERT was used in this model. Notches were created by scraping the confluent monolayer of N / TERT keratinocytes to simulate wounds. Keratinocytes were collected at different time points after scraping.

[0118] 3) Acute rat wound model: Six-week-old Sprague Dawley rats were anesthetized with 4% isoflurane, 20% oxygen, and 10% nitrous oxide, and maintained with 1.5% isoflurane. After subcutaneous injection of 0.03 mg / ml buprenorphine (Vetergesic), the rats' backs were shaved, and two full-thickness resection 6 mm biopsy puncture wounds were made on each side of the dorsal midline. Wounds were collected at designated time points after injury.

[0119] 4) Disturbed Rat Wound Model: The disturbed rat wound model was used as an animal model of chronic non-healing wounds. Disturbed rat wounds were created by placing an oversized scaffold containing 20% ​​collagen and aging-inducing FK866 into excised rat wounds, resulting in foreign body reactions within 10 days and inflammation that did not subside for at least 15 days after scaffold removal. The disturbed rat wounds were treated with 200 μM Z-DEVD-FMK (a caspase-3-specific inhibitor) delivered in a Pluronic gel, and the treated wounds were harvested at days 0, 1, 2, and 5 post-injury to determine the expression of migration and proliferation markers at the wound margins (n ​​= 6 animals at each time point). Photographs of rats were taken daily for recording, scoring, and analysis to measure multiple parameters, including wound size and depth. Images were evaluated using size and depth, with larger size and depth resulting in higher severity scores. If two wounds had similar size and depth, active inflammation (degree of redness) was assigned a higher severity score. Standard H&E histological analysis was performed on re-epithelialization, granulation tissue formation, and angiogenesis to score the wound.

[0120] As described above, wound edge tissue and scraped keratinocytes were collected at multiple time points after injury until wound closure. Skin samples and keratinocytes were lysed using lysis buffer (10 mM Tris HCl pH 7.5, 10% glycerol, 0.2 mM EDTA, 150 mM NaCl, and a mixture of protease inhibitors). Caspase-3 activity was measured using a caspase-3 assay kit (#39383, Abcam, UK). Cell migration efficiency was determined by staining for EMT markers and immunoblotting with anti-E-cadherin (1:200; #3195; CellSignaling, USA) and anti-SLUG (1:200; ab27568; Abcam, UK). Household proteins actin and tubulin were identified by immunoblotting using an anti-β actin antibody (#sc-47776, Santa Cruz, USA) and an anti-β tubulin antibody (#sc-5274, Santa Cruz, USA).

[0121] Example 1: Caspase-3 activity was transiently inhibited upon trauma. Human ex vivo injured skin Incision wounds created in ex vivo human skin were cultured at the gas-liquid interface using a rich culture medium, and samples were collected at different time points after injury. Figure 2 A). Caspase-3 activity in human ex vivo wounds exhibited a dynamic profile, reaching its lowest level 16 hours post-injury and then gradually increasing until it returned to levels similar to those immediately post-injury. Caspase-3 activity showed almost no change in uninjured skin samples. Figure 2 B).

[0122] Single-layer keratinocyte scraping model The profile of caspase-3 activity was determined using a monolayer keratinocyte scraping model. Caspase-3 activity in scraped keratinocytes was found to exhibit a dynamic pattern similar to that observed in ex vivo human wounds. Caspase-3 activity was gradually inhibited up to 4 hours, after which it recovered to levels observed immediately following scraping. Figure 3 These data also confirm that caspase-3 activity is dynamic and transiently inhibited during wound healing.

[0123] Acute rat wound and disturbed rat wound models The role of caspase-3 in wound healing was explored using a rat wound model. Figure 4 A). In an acute rat wound model, caspase-3 activity showed a dynamic profile, reaching its lowest level at 48 hours post-injury, while it was dysregulated in disturbed rat wounds and remained at significantly higher levels at all time points. Figure 4 B). These data demonstrate that transient inhibition of cystase-3 occurs upon injury and is misregulated in a rat model of perturbed wounds.

[0124] Example 2: Surface application of Z-DEVD-fmk, a specific inhibitor of caspase-3, promotes wound closure in disturbed rat wounds. To determine the effect of caspase-3 inhibition on the healing of disturbed wounds, the surface of the caspase-3 specific inhibitor Z-DEVD-fmk in Pluronic gel was applied to disturbed rat wounds. Z-DEVD-fmk treatment significantly promoted healing efficiency compared to the untreated control (Fig. 5A, Fig. 5B). Spermine has been reported to promote cell migration in acute wound models, and it was also shown to promote improved healing in disturbed wounds. Treatment with the caspase-3 inhibitor significantly promoted cell migration and re-epithelialization, as shown by H&E staining (Fig. 5C). Caspase-3 activity was significantly inhibited by Z-DEVD-fmk treatment (Fig. 5D). In disturbed wounds, the levels of cell migration factors Slug, Snail, uPA, uPAR, and TGF-β1 were low. However, their expression levels were significantly upregulated in disturbed rat wounds under Z-DEVD-fmk treatment (Fig. 5E). These data demonstrate that caspase-3 is a potential therapeutic target for chronic wounds.

[0125] In summary, transient inhibition of caspase-3 activity is essential for efficient cell migration during wound healing. In a perturbed rat wound model, caspase-3 inhibition resulted in improved wound closure and increased expression of cell migration factors. Therefore, targeting dysregulated caspase-3 is a potential therapeutic strategy for promoting chronic wound healing.

[0126] It should be understood that numerous further modifications and arrangements of the various aspects of the described embodiments are possible. Therefore, the described aspects are intended to encompass all such changes, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

1. A method for treating a wound in a subject, the method comprising administering to the subject a therapeutically effective amount of a cysteine-3 inhibitor.

2. The method according to claim 1, wherein the wound is a chronic wound.

3. The method according to claim 1 or 2, wherein the wound is an epithelial wound.

4. The method according to any one of claims 1 to 3, wherein the wound is a skin wound.

5. The method according to any one of claims 1 to 4, wherein the cystase-3 inhibitor is a small molecule, peptide, polypeptide, nucleic acid, or a combination thereof.

6. The method of claim 5, wherein the caspase-3 inhibitor is flubendazole, L-tryptophan, fenprofen, diflunisal, pranoprofen, or a pharmaceutically acceptable salt or solvation thereof.

7. The method according to any one of claims 1 to 6, wherein the cysteine-3 inhibitor is applied at or near the wound site.

8. The method according to any one of claims 1 to 7, wherein the subject is a mammal.

9. The method according to any one of claims 1 to 8, wherein the subject has impaired wound healing ability.

10. The method according to any one of claims 1 to 9, wherein the cysteine-3 inhibitor promotes reepithelialization of the wound.

11. The method according to any one of claims 1 to 10, wherein the cysteine-3 inhibitor is administered simultaneously or sequentially with a therapeutically effective amount of spermine and / or spermidine.

12. A caspase-3 inhibitor for treating a wound in a subject, wherein a therapeutically effective amount of the caspase-3 inhibitor is administered to the subject.

13. Use of a caspase-3 inhibitor in the manufacture of a medicament for treating a wound in a subject, wherein a therapeutically effective amount of the caspase-3 inhibitor is administered to the subject.

14. A method for promoting reepithelialization of a wound in a subject, the method comprising administering to the subject a therapeutically effective amount of a cysteine-3 inhibitor.

15. A method for detecting chronic wounds in a subject, the method comprising detecting cleaved caspase-3 levels and / or caspase-3 activity in a sample from the subject, wherein an increase in cleaved caspase-3 levels and / or caspase-3 activity relative to a reference indicates that the subject has chronic wounds.

16. A method for treating a subject's chronic wound, the method comprising: (a) Detecting the level and / or activity of cleaved caspase-3 in a sample from the subject, wherein an increase in the level and / or activity of cleaved caspase-3 relative to a reference indicates that the subject has a chronic wound; and (b) administering a therapeutically effective amount of a caspase-3 inhibitor to the subject with the chronic wound.

17. The method of claim 15 or 16, wherein the level of lysed caspase-3 and / or caspase-3 activity is detected over a period of time, and wherein a sustained increase in the level of lysed caspase-3 and / or caspase-3 activity relative to a reference over the period of time indicates that the subject has a chronic wound.

18. The method of claim 17, wherein the time period is at least 5 days.

19. The method according to any one of claims 15 to 18, wherein the sample is a wound sample.

20. A pharmaceutical composition comprising (a) a cystase-3 inhibitor and (b) spermine and / or spermidine.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is a surface composition.

22. A drug combination comprising (a) a cystase-3 inhibitor and (b) spermine and / or spermidine.

23. The pharmaceutical composition according to claim 20 or 21, or the pharmaceutical composition according to claim 22, for use as a medicine.

24. A method of treating a wound of a subject, the method comprising administering to the subject a pharmaceutical composition according to claim 20 or 21 or a pharmaceutical combination according to claim 22.

25. The pharmaceutical composition according to claim 20 or 21, or the pharmaceutical composition according to claim 22, for treating a wound in a subject.

26. Use of the pharmaceutical composition according to claim 20 or 21 or the pharmaceutical composition according to claim 22 in the manufacture of a medicament for treating a wound of a subject.

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