BOOSTING AN IMMUNE SYSTEM

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional application No. 63/040,677, filed June 18, 2020; U.S. Provisional application No. 63/051,876, filed July 14, 2020; and U.S. Provisional application No. 63/057,256, filed July 27, 2020. The contents of each of which is incorporated by reference in its entirety.

BACKGROUND

Of the approximately 150,000 human deaths per day, approximately two-thirds are due to age- related causes. Aging leads to functional deterioration and progressive decline across multiple tissues, organs, and systems, including the immune system, that arise from the progressive accumulation of cellular and tissue damage. This damage may be attributed, in part, to dysfunction or disruption in one or more signaling pathways. Accordingly, there remains an unmet need for compositions and methods that stop, slow, or reverse these dysfunctions or disruptions and are capable of thereby treating aging-related disorders and/or reducing symptoms of aging.

SUMMARY

The present invention addresses this need. Accordingly, the present disclosure relates to compositions and methods for increasing lifespan, for preventing or treating a disease including an aging-related disorder, for reducing a symptom of aging, and/or boosting an immune system in a mammal. The present disclosure additionally relates to compositions and methods for improving effectiveness of a vaccine in a mammal.

An aspect of the present disclosure is a method for preventing or treating a disease including an aging-related disorder in a mammal, for increasing lifespan in a mammal, for reducing a symptom of aging in a mammal, and/or boosting an immune system in a mammal comprising. The method comprising a step of administering to the mammal a composition comprising a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator.

In embodiments, the ALDH modulator is an ALDH inhibitor or an ALDH agonist.

In some embodiments, the ALDH inhibitor inhibits ALDHl, ALDH1 Al, ALDH1 A2, ALDHl A3, ALDH2, and/or ALDH3A1. In various embodiments, the ALDH inhibitor is disulfiram, NCT-501, 4-

Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3- Hydroxybenzaldehyde, or ANS-66372. In some cases, the disulfiram is a pharmaceutically acceptable salt, solvate, or prodrug. The disulfiram may be a metabolite selected from diethyldithiocarbamate, methyl diethyldithiocarbamate, dithiomethylcarbamate, S-Methyl N,N- Diethylthiocarbamate, diethyl-amine, and carbon disulfide. In various cases, the disulfiram is variant having the structure of Formula (II), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

Formula (II), wherein,

Xi and X2 are each independently O or S; p is 1 or 2;

Ri, R2, R3, and R4 are each independently H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 4-10 membered heterocycloalkyl, C(0)OR ^la , or C(0)NR ^la R ^lb ; wherein each is optionally substituted with 1, 2, or 3 R ⁵ ; or R ¹ and R ² together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁶ . or R ³ and R ⁴ together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁷ ; each R ⁵ , R ⁶ , and R ⁷ is independently selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, CN, NO2, OR ^la , C(0)R ^lb , C(0)NR ^la R ¹ b, C(0)OR ^la , NR ^la R ^lb , S(0) ₂ R ^lb and S(0) ₂ NR ^la R ^lb ; each R ^la and R ^lb is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

In embodiments, the disease is acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI). The ALDH modulator may limit the progression of ARDS in patients with or without ALI. In some embodiments, the ALDH modulator is formulated in a single composition. In some cases, the single composition is a liquid, a suspension, a gel, a gel tab, a semisolid, a tablet, a sachet, a lozenge or a capsule. The single composition may be formulated for intravenous injection or infusion, intraarterial injection or infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intra-arterial injection or infusion. In various cases, the ALDH modulator is disulfiram and the single composition comprises between about 5 mg to about 500 mg of disulfiram.

In various embodiments, the ALDH modulator inhibits and/or reduces pyroptotic cell death. In some cases, the ALDH modulator is disulfiram and the single composition comprises between about 5 mg to about 500 mg of disulfiram.

In embodiments, the mammal is near or has reached maturity or the mammal is nearing or has reached halfway to its expected lifespan for the mammal’s species, size, sex, age, and/or health status. The mammal may have reached an age that is at least 60%, 70%, 80%, 90%, or 100% of its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

In some embodiments, the mammal is a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.

In various embodiments, the aging-related disorder or symptom of aging selected from one or more of actinic keratosis, age-related macular degeneration (AMD), Alzheimer’s disease, arthritis, atherosclerosis and cardiovascular disease, benign prostatic hyperplasia (BPH), bone atrophy, cachexia, cancer, cardiomyopathy, cataracts, chronic obstructive pulmonary disease (COPD), constipation, decrease in overall energy, decrease in visual acuity, delirium, dementia, depression, dermal atrophy (thinning of the skin), diminished peripheral vision, greater risk of heat stroke or hypothermia, hearing loss, hypertension, increased susceptibility to infection (including influenza and pneumonia), lentigines (aging spots), liver conditions ( e.g ., nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and cirrhosis), memory loss, metabolic syndrome, muscle atrophy (e.g., Sarcopenia and myopenia), frailty, muscle repair or rejuvenation deficiency, muscular dystrophy, osteoarthritis, osteoporosis, periodontitis, photoaging, reduced metabolism (including increased risk for obesity), reduced reflexes and coordination including difficulty with balance, respiratory disease (including acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI) and pulmonary fibrosis (e.g, idiopathic pulmonary fibrosis)), rheumatoid arthritis, sarcopenic obesity, sexual dysfunction, shingles, type 2 diabetes, urologic changes (including incontinence), vaginal atrophy, whitening or graying of hair, prolonged/inefficient wound healing, wrinkling/sagging skin (including loss of skin elasticity), and xerosis cutis (skin dryness); or the disease includes asthma, deafness, or a viral infections and/or a symptom of the disease comprises sepsis. In some cases, the aging-related disorder or symptom of aging is actinic keratosis, dermal atrophy (thinning of the skin), lentigines (aging spots), photoaging, vaginal atrophy, prolonged/inefficient wound healing, wrinkles, and/or xerosis cutis (skin dryness) and wherein the administering is oral or topical. The mammal may have at least one aging-related disorder or symptom of aging, as disclosed herein.

In embodiments, increasing lifespan comprises an at least 5% increase in lifespan relative to the expected or median lifespan of a mammal of similar species, sex, age, and/or health status. In some cases, increasing lifespan comprises an at least 10%, at least 15%, at least 20%, or at least 25% increase in lifespan.

In some embodiments, the therapeutically effective amount of the ALDH modulator boosts the immune system in the mammal. In various cases, boosting the immune system increases an effective immune response against an infectious agent. The infectious agent may be a virus, a bacterium, a fungus, a protozoan, a helminth, a prion, or a parasite. In some cases, the bacterium is Bordatella pertussis or Streptococcus pneumoniae or the virus is a Chickenpox virus, Coronavirus, Hepatitis A virus, Hepatitis B virus, Human papillomavirus, Human immunodeficiency virus (HIV), influenza, Japanese encephalitis virus, Measles, mumps, or rubella virus, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rotavirus, Shingles virus, Smallpox, Varicella virus, or Yellow fever virus. In various cases, the Coronavirus is Sars-CoV-2. The infectious agent may affect the mammal’s respiratory system or is transmitted via the mammal’s respiratory system and wherein the administering is by inhalation.

In various embodiments, the mammal has a healthy immune system.

In embodiments, the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

In some embodiments, the method further comprises a step of administering a potentiating agent that enhances, increases, and/or improves the effectiveness and/or desirable activity of the ALDH modulator. In some cases, the potentiating agent is tert-Butylhydroquinone (TBHQ). The combination of the ALDH modulator and TBHQ may inhibit and/or reduce pyroptotic cell death. In various cases, the ALDH modulator is disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is from about 0.02% to about 56% by weight of disulfiram. The TBHQ may further potentiate disulfiram’ s ability to treat acute lung injury (ALI), acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI), pulmonary fibrosis ( e.g idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, dry eye, actinic keratosis, alopecia, and/or skin cancer. In various cases, the TBHQ further potentiates disulfiram’ s ability to inhibit and/or reduce a pathological inflammatory response, alter a T-celfs age, and/or alter mitochondrial function in the cell. The combination of disulfiram and TBHQ may limit the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

In embodiments, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg and the amounts of disulfiram and TBHQ limit the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI). In some cases, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg and the amounts of disulfiram and TBHQ limit the progression of ARDS in patients with ALI.

Another aspect of the present disclosure is a method for improving effectiveness of a vaccine in a mammal in need thereof. The method comprising a step of administering a composition comprising a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator, wherein the mammal contemporaneously and/or subsequently will be administered a vaccine.

In embodiments, the ALDH modulator and the vaccine are administered contemporaneously.

In some embodiments, the vaccine is administered subsequent to the ALDH modulator’s administration.

In various embodiments, the therapeutically effective amount of the ALDH modulator boosts the immune system in the mammal. In some cases, boosting the immune system increases an immune response against a component contained in the vaccine. The increased immune response may promote future immunity against the component contained in the vaccine. In various cases, the component contained in the vaccine is an antigen obtained from, related to, homologous to, or expressed by an infectious agent.

In embodiments, the vaccine is a Chickenpox vaccine, Coronavirus vaccine, Diphtheria vaccine, Hepatitis A vaccine, Hepatitis B vaccine, Haemophilus influenzae type b vaccine, Human Immunovirus (HIV) vaccine, Human papillomavirus vaccine, influenza vaccine, Japanese encephalitis vaccine, Measles, mumps, or rubella (including MMR combined vaccine) vaccine, Meningococcal disease vaccine, Pneumococcal disease vaccine, Polio vaccine, Rabies vaccine, Respiratory syncytial virus (RSV) vaccine, Rotavirus vaccine, Shingles vaccine, Smallpox vaccine, Tetanus vaccine, Varicella virus vaccine, Whooping cough (part of the DTaP combined vaccine) vaccine, or Yellow fever vaccine. In some cases, the vaccine is a coronavirus vaccine, e.g., the coronavirus vaccine is directed against Sars-CoV-2.

In some embodiments, the mammal has a healthy immune system.

In various embodiments, the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

In embodiments, the ALDH modulator is an ALDH inhibitor or an ALDH agonist. In some cases, the ALDH inhibitor inhibits ALDHl, ALDHIAI, ALDH1A2, ALDH 1 A3, ALDH2, and/or ALDH3A1. The ALDH inhibitor may be disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, or ANS-66372. In various cases, the disulfiram is a pharmaceutically acceptable salt, solvate, or prodrug. The disulfiram may be a metabolite selected from diethyldithiocarbamate, methyl diethyldithiocarbamate, dithiomethylcarbamate, S-Methyl N,N-Diethylthiocarbamate, diethyl- amine, and carbon disulfide. In some cases, the disulfiram is variant having the structure of Formula (II), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

Formula (II), wherein,

Xi and X2 are each independently O or S; p is 1 or 2;

Ri, R2, R3, and R4 are each independently H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 4-10 membered heterocycloalkyl, C(0)OR ^la , or C(0)NR ^la R ^lb ; wherein each is optionally substituted with 1, 2, or 3 R ⁵ ; or R ¹ and R ² together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁶ . or R ³ and R ⁴ together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁷ ; each R ⁵ , R ⁶ , and R ⁷ is independently selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, CN, NO2, OR ^la , C(0)R ^lb , C(0)NR ^la R ¹ b, C(0)OR ^la , NR ^la R ^lb , S(0) ₂ R ^lb and S(0) ₂ NR ^la R ^lb ; each R ^la and R ^lb is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

In some embodiments, the administering of the ALDH modulator is oral, by injection, inhalation, or topical.

In various embodiments, the administering of the vaccine is oral, by injection, inhalation, or topical. In some cases, the injection is intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

In embodiments, the method further comprises administering a potentiating agent that enhances, increases, and/or improves the effectiveness and/or desirable activity of the ALDH modulator. In some cases, the potentiating agent is tert-Butylhydroquinone (TBHQ). The ALDH modulator may be disulfiram. In various cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is from about 0.02% to about 56% by weight of disulfiram.

Yet another aspect of the present disclosure is a method for reducing a predicted biological age of a cell. The method comprising a step of contacting the cell with a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator.

In embodiments, the ALDH modulator is an ALDH inhibitor selected from disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3-

Hydroxybenzaldehyde, and ANS-66372. In some cases, the disulfiram is a pharmaceutically acceptable salt, solvate, or prodrug. The disulfiram may be a metabolite selected from diethyldithiocarbamate, methyl diethyldithiocarbamate, dithiomethylcarbamate, S-Methyl N,N- Diethylthiocarbamate, diethyl-amine, and carbon disulfide. In various cases, the disulfiram is variant having the structure of Formula (II), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

Formula (II), wherein,

Xi and X2 are each independently O or S; p is 1 or 2; Ri, R2, R3, and R4 are each independently H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 4-10 membered heterocycloalkyl, C(0)OR ^la , or C(0)NR ^la R ^lb ; wherein each is optionally substituted with 1, 2, or 3 R ⁵ ; or R ¹ and R ² together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁶ . or R ³ and R ⁴ together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁷ ; each R ⁵ , R ⁶ , and R ⁷ is independently selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, CN, NO2, OR ^la , C(0)R ^lb , C(0)NR ^la R ¹ b, C(0)OR ^la , NR ^la R ^lb , S(0) ₂ R ^lb and S(0) ₂ NR ^la R ^lb ; each R ^la and R ^lb is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

In embodiments, the cell is in vitro, ex vivo, or in vivo.

In an aspect, the present disclosure provides a composition comprising an ALDH modulator for use in the method of any of the herein disclosed aspects or embodiments.

In another aspect, the present disclosure provides a method for preventing or treating a disease including an aging-related disorder in a mammal, for increasing lifespan in a mammal, for reducing a symptom of aging in a mammal, and/or boosting an immune system in a mammal. The method comprising a step of administering to the mammal a composition comprising a therapeutically effective amount of a gasdermin D inhibitor.

In yet another aspect, the present disclosure provides a method for improving effectiveness of a vaccine in a mammal in need thereof. The method comprising a step of administering a composition comprising a therapeutically effective amount of a gasdermin D inhibitor, wherein the mammal contemporaneously and/or subsequently will be administered a vaccine.

An aspect of the present disclosure is a method for reducing a predicted biological age of a cell. The method comprising a step of contacting the cell with a therapeutically effective amount of a gasdermin D inhibitor.

Another aspect of the present disclosure is a method for limiting the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI) . The method comprising a step of administering to the patient a therapeutically effective amount of disulfiram. Yet another aspect of the present disclosure is a method for treating acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI) . The method comprising a step of administering to the patient a therapeutically effective amount of disulfiram.

In embodiments, the therapeutically effective amount of disulfiram is from about 5 mg to about 500 mg. In some cases, the therapeutically effective amount of disulfiram is about 500 mg.

In some embodiments, the disulfiram is a pharmaceutically acceptable salt, solvate, or prodrug.

In various embodiments, the disulfiram is a metabolite selected from diethyldithiocarbamate, methyl diethyldithiocarbamate, dithiomethylcarbamate, S-Methyl N,N-Diethylthiocarbamate, diethyl-amine, and carbon disulfide.

In embodiments, the disulfiram is variant having the structure of Formula (II), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

Formula (II), wherein,

Xi and X2 are each independently O or S; p is 1 or 2;

Ri, R2, R3, and R4 are each independently H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 4-10 membered heterocycloalkyl, C(0)OR ^la , or C(0)NR ^la R ^lb ; wherein each is optionally substituted with 1, 2, or 3 R ⁵ ; or R ¹ and R ² together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁶ . or R ³ and R ⁴ together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁷ ; each R ⁵ , R ⁶ , and R ⁷ is independently selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, CN, NO2, OR ^la , C(0)R ^lb , C(0)NR ^la R ¹ b, C(0)OR ^la , NR ^la R ^lb , S(0) ₂ R ^lb and S(0) ₂ NR ^la R ^lb ; each R ^la and R ^lb is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl. In embodiments, the method further comprises administering to the patient a potentiating agent that enhances, increases, and/or improves the effectiveness and/or desirable activity of disulfiram. In some cases, the potentiating agent is tert-Butylhydroquinone (TBHQ). The amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is from about 0.02% to about 56% by weight of disulfiram.

In an aspect, the present disclosure provides a composition comprising from about 5 mg to about 500 mg of disulfiram. In this aspect, the amount of disulfiram limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

In another aspect, the present disclosure provides a composition comprising from about 5 mg to about 500 mg of disulfiram and up to about 37 mg tert-Butylhydroquinone (TBHQ). In this aspect, the amounts of disulfiram and TBHQ limit the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI). In embodiments, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg tert-Butylhydroquinone (TBHQ) and the amounts of disulfiram and TBHQ limit the progression of ARDS in patients with ALI.

In yet another aspect, the present disclosure provides a composition comprising from about 5 mg to about 500 mg of disulfiram and an amount of tert-Butylhydroquinone (TBHQ) from about 0.02% to about 56% by weight of disulfiram. In this aspect, the amounts of disulfiram and TBHQ limit the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

It shall be understood that different aspects and/or embodiments of the invention can be appreciated individually, collectively, or in combination with each other. Various aspects and/or embodiments of the invention described herein may be applied to any of the uses set forth below and in other methods for increasing lifespan in a mammal. Any description herein concerning a specific composition and/or method apply to and may be used for any other specific composition and/or method as disclosed herein. Additionally, any composition disclosed herein is applicable to any herein-disclosed method. In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A to FIG. ID show data obtained when virally-infected cells were treated with an active ingredient of the present disclosure. FIG. 1A shows cytokine levels from the supernatant of vesicular stomatitis virus encoding a red fluorescent protein (rVSV-AG-mCherry)-infected cells (infected at lOx MO I); asterisks indicate statistical significance relative to untreated control cells. Data are normalized by total cell count per well. In FIG. IB and FIG. 1C, respectively, show reticularity measurements of mitochondria and aging scores in T cells. In FIG. IB and FIG. 1C, horizontal bars represent the distribution of untreated controls from younger and older donors; the solid line represents the median and the lower and upper dashed lines represent the 25 ^th and 75 ^th quartiles, respectively. The line graph represents the median and 95% Cl for treated cells, with statistical significance relative to untreated control cells indicated by asterisks. In FIG. ID, “on- age” and “off-age” scores for T cells treated with either disulfiram or dimethyl fumarate. Distributions for younger and older control (untreated) cells are plotted using a Gaussian kernel density estimation. Statistical significance relative to untreated control cells is indicated by the following: *, P<0.5; **, P< 001; ***, P< 0001. IL, interleukin; MCP1, monocyte chemoattractant protein 1; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor.

FIG. 2 shows the effect of cells treated with disulfiram alone, tert-Butylhydroquinone (TBHQ) alone, or disulfiram and TBHQ in combination at different concentrations.

FIG. 3 shows the effect of cells treated with TBHQ and different concentrations of disulfiram.

FIG. 4A to FIG. 4C show synergistic effects of TBHQ on disulfiram activity. FIG. 4A is a table of synergy ratio of actual/expected effect at each concentration of TBHQ and disulfiram; cells in grey indicate positive synergy. FIG. 4B and FIG. 4C are graphs showing average synergy at different concentrations of TBHQ and disulfiram.

FIG. 5 is a table showing the percent difference in IC50 between disulfiram alone vs. disulfiram and TBHQ at various concentrations or constant ratio (last column). TBHQ combined with disulfiram shifts the IC50 to the left relative to disulfiram alone in a dose-dependent manner. TBHQ has no effect on pyroptosis inhibition or a clear IC50, indicating effects on early inhibition and shifted IC50 is synergistic.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to active ingredients, compositions, and methods for increasing lifespan, for preventing or treating a disease including an aging-related disorder, for reducing a symptom of aging, and/or boosting an immune system in a mammal. The present disclosure additionally relates to active ingredients, compositions, and methods for improving effectiveness of a vaccine in a mammal. Aldehyde dehydrogenase (ALDH) Modulators

Aldehyde dehydrogenases are a group of enzymes that catalyze the oxidation of aldehydes. They convert aldehydes to carboxylic acids. The produced carboxylic acids leave the liver (where the highest concentration of the enzymes are located) and are metabolized by the body’s muscle and heart. There are three different classes of these enzymes in mammals: class 1 (ALDH1, low Km, cytosolic), class 2 (ALDH2, low Km, mitochondrial), and class 3 (ALDH3, high Km, such as those expressed in tumors, stomach, and cornea). To date, nineteen ALDH genes have been identified within the human genome. These enzymes participate in a wide variety of biological processes including the detoxification of exogenously and endogenously generated aldehydes, e.g., from alcohol oxidation.

Aspects of the present disclosure relate to active ingredients, compositions, and methods for increasing lifespan, for preventing or treating an aging-related disorder, for reducing a symptom of aging, boosting an immune system in a mammal, and improving effectiveness of a vaccine in a mammal. The methods comprise administering to the mammal a composition comprising a therapeutically effective amount of an active ingredient that is an aldehyde dehydrogenase (ALDH) modulator.

In embodiments, the ALDH modulator is an ALDH modulator or an ALDH agonist.

In embodiments, the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, or ANS-66372.

Disulfiram is a specific inhibitor of an aldehyde-dehydrogenase (ALDHl). Disulfiram was approved by the US Food and Drug Administration in 1951 for alcohol aversion therapy after researchers observed that it induced the effects of a hangover after alcohol consumption. Disulfiram blocks the major metabolic reaction that converts alcohol into acetaldehyde. Disulfiram (also known as Tetraethylthiuram disulfide, Antabuse, Antabus, diethylcarbamothioylsulfanyl N,N-diethylcarbamodithioate, and 1-

(diethylthiocarbamoyldisulfanyl)-N,N-diethyl-methanethioa mide; CAS Reference Number: 97- 77-8) has a structure of Formula (I):

Formula (I). As used herein, the term disulfiram includes disulfiram itself and any pharmaceutically acceptable salt, solvate, or prodrug thereof.

As used herein, the term disulfiram includes disulfiram itself and any of its metabolites and/or their derivatives and variants thereof. Examples of metabolites include di ethyl dithiocarbamate, methyl diethyldithiocarbamate, dithiomethylcarbamate, S-Methyl N,N-Diethylthiocarbamate, diethyl- amine, and carbon disulfide.

NCT-501 is a potent and selective theophylline-based inhibitor of aldehyde dehydrogenase 1A1 (ALDHl Al). It inhibits hALDHl A1 with IC50 of 40 nM. It typically shows better selectivity over other ALDH isozymes and other dehydrogenases (hALDHlBl, hALDEBAl, and hALDH2, IC50 >57 mM)., IC50 value: 40 nM, Target: hALDHl Al . NCT-501 is well absorbed and distributed but rapidly metabolized and/or excreted. NCT-501 does penetrate the blood-, brain barrier. NCT-501 inhibits functional properties, such as self-renewal property, migratory potential and induced sell sensitivity in cisplatin resistant cells.

4-Diethylaminobenzaldehyde is a reversible aldehyde dehydrogenases (ALDHs) inhibitor, with a Ki of 4 nM for ALDHl. 4-Diethylaminobenzaldehyde displays potent anti -androgenic effect (IC50= 1.71mM).

Daidzin is an isoflavone that has anti-oxidant, anti-carcinogenic, and anti-atherosclerotic activities. It directly inhibits mitochondrial aldehyde dehydrogenase 2 (IC50 = 80 nM) and is an effective anti-dipsotropic isoflavone.

CM10 is a potent and selective aldehyde dehydrogenase 1A family inhibitor (ALDHlAi), with IC50s of 1700, 740, and 640 nM for ALDHl Al, ALDHl A2, and ALDHl A3, respectively. CM10 does not inhibit any of the other ALDH family members. CM10 can regulate metabolism and has anti-cancer activity.

EN40 is a potent, selective aldehyde dehydrogenase 3A1 (ALDH3A1) inhibitor as a covalent ligand, exhibits an IC50 value of 2 mM.

4-Hydroxynonenal (4-HNE) is an a,b unsaturated hydroxyalkenal and an oxidative/nitrosative stress biomarker. 4-Hydroxynonenal is a substrate and an inhibitor of acetaldehyde dehydrogenase 2 (ALDH2). 4-Hydroxynonenal can modulate a number of signaling processes mainly through forming covalent adducts with nucleophilic functional groups in proteins, nucleic acids, and membrane lipids. 4-Hydroxynonenal plays an important role in cancer through mitochondria. RV01 is an analogue of resveratrol. It inhibits DNA damage, reduces acetaldehyde dehydrogenase 2 (ALDH2) mRNA expression induced by ethanol, and exhibits hydroxyl radical scavenging activity. RV01 decreases iNOS expression, with anti-neuroinflammatory activity.

CVT-10216 is a highly selective, reversible aldehyde dehydrogenase-2 (ALDH-2) inhibitor with an IC50 of 29 nM. CVT-10216 also has inhibitory effect of ALDH-1 with an IC50 of 1.3 mM. CVT-10216 can reduce excessive alcohol drinking in alcohol-preferring rats and exhibit anxiolytic effects.

3-Hydroxybenzaldehyde is a precursor compound for phenolic compounds, such as Protocatechualdehyde (HY-N0295). 3-Hydroxybenzaldehyde is a substrate of aldehyde dehydrogenase (ALDH) in rats and humans (ALDH2). 3-Hydroxybenzaldehyde has vasculoprotective effects in vitro and in vivo.

ANS-6637 is a selective and reversible ALDH2 inhibitor. Its mechanism of action in the brain, to prevent pathophysiologic dopamine surge without changes to basal dopamine, suggests that ANS- 6637 has the potential to prevent drug seeking behavior, craving and relapse.

In embodiments, the ALDH modulator is an ALDH agonist.

In embodiments, the ALDH agonist is Alda-1. Alda-1 is an activator of aldehyde dehydrogenase 2 (ALDH2). Alda-1 increases activity of wild-type ALDH2*1 and variant ALDH2*2 (by ~2-fold and 11 -fold respectively). It is capable of partly restoring mutant ALDH2*2 activity, and protective against cardiac ischemia.

Gasdermin D Inhibitors

The pore-forming protein gasdermin (such as gasdermin D) is the final pyroptosis executioner downstream of inflammasome activation. Gasdermin D inhibitor compounds of the present disclosure inhibit gasdermin pore formation and subsequent secretion of inflammatory mediators such as IE- lb.

Aspects of the present disclosure relate to active ingredients, compositions, and methods for increasing lifespan, for preventing or treating a disease including an aging-related disorder, for reducing a symptom of aging, boosting an immune system in a mammal, and improving effectiveness of a vaccine in a mammal. The methods comprise administering to the mammal a composition comprising a therapeutically effective amount of an active ingredient that is a gasdermin D inhibitor. Gasdermin D (GSDMD or DFNA5L) is a recently-identified pore-forming protein. In embodiments, the gasdermin D inhibitor is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082.

Disulfiram is a specific inhibitor of an aldehyde-dehydrogenase (ALDHl). Disulfiram was approved by the US Food and Drug Administration in 1951 for alcohol aversion therapy after researchers observed that it induced the effects of a hangover after alcohol consumption. Disulfiram blocks the major metabolic reaction that converts alcohol into acetaldehyde.

Disulfiram (also known as Tetraethylthiuram disulfide, Antabuse, Antabus, diethylcarbamothioylsulfanyl N,N-diethylcarbamodithioate, and 1-

(diethylthiocarbamoyldisulfanyl)-N,N-diethyl-methanethioa mide; CAS Reference Number: 97- 77-8) has a structure of Formula (I):

Formula (I).

As used herein, the term disulfiram includes disulfiram itself and any pharmaceutically acceptable salt, solvate, or prodrug thereof.

As used herein, the term disulfiram includes disulfiram itself and any of its metabolites and/or their derivatives and variants thereof. Examples of metabolites include di ethyl dithiocarbamate, methyl diethyldithiocarbamate, dithiomethylcarbamate, S-Methyl N,N-Diethylthiocarbamate, diethyl- amine, and carbon disulfide.

Aloe emodin is a hydroxyanthraquinone present in aloe vera leaves. In vitro, aloe-emodin treatments led to the dissociation of heat shock protein 90 (HSP90) and ER a and increased ER a ubiquitination. Protein fractionation results suggest that aloe-emodin induces cytosolic ER a degradation. Aloe-emodin inhibits both proliferation and anchorage-independent growth of PC3 cells. Protein content analysis suggests that activation of the downstream substrates of mTORC2, Akt and PKCa, is inhibited by aloe-emodin treatment. Pull-down assay and in vitro kinase assay results indicate that aloe-emodin binds with mTORC2 in cells and inhibits its kinase activity. Aloe- emodin downregulates mRNA expression and promoter/gelatinolytic activity of Matrix Metalloproteinase (MMP)-2/9, as well as the RhoB expression at gene and protein level. Aloe- emodin suppresses the nuclear translocation and DNA binding of NF-KB. It has been shown to have specific in vitro antitumor activity. In vivo, aloe-emodin exhibits tumor suppression effects in an athymic nude mouse model. 2-iodomelatonin is a potent agonist of melatonin receptor 1 (MT1) with a Ki value of 28 pM. It is more 5-fold selectivity for MT1 over MT2. Due to its selective binding, 2-iodomelatonin can be used to identify, characterize and localize melatonin binding sites in the brain and peripheral tissues.

LDC7559 operates by blocking neutrophil extracellular trap (NET) in its late stages.

Necrosulfonamide is a necroptosis inhibitor which acts by selectively targeting the mixed lineage kinase domain-like protein (MLKL) to block the necrosome formation.

Tasimelteon is a melatonin receptor 1 (MT1) and MT2 agonist; it has 2 - 4 times greater affinity for the MT2 receptor than the MT1 receptor. Tasimelteon, as a novel circadian regulator, is the first product for the treatment of Non-24-hour Sleep-Wake Disorder (Non-24) that has been approved by either the FDA or the European Medicines Agency.

Ramelteon is a highly potent and selective melatonin receptor agonist with Ki values of 14 and 112 pM for human melatonin receptor 1 (MT1) and MT2.

Melatonin is a hormone made by the pineal gland that can activates the melatonin receptors. Melatonin plays a role in sleep and may possess antioxidative and anti-inflammatory properties.

Emodin is a broad-spectrum anticancer agent. Emodin inhibits casein kinase II (CKII) activity with IC50 of 2 mM.

BAY 11-7082 is a NF-KB inhibitor which decreases NF-KB by inhibiting TNF-a-induced phosphorylation of IkB-a. BAY 11-7082 inhibits ubiquitin-specific protease USP7 and USP21 with IC50s of 0.19 mM and 0.96 pM, respectively.

Variants of Disulfiram

As used herein, the term disulfiram includes disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

The term disulfiram includes variants of disulfiram, which, as used herein is, a compound having the structure of Formula (II), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

Formula (II), wherein,

Xi and X2 are each independently O or S; p is 1 or 2;

Ri, R2, R3, and R4 are each independently H, C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 4-10 membered heterocycloalkyl, C(0)0R ^la , or C(0)NR ^la R ^lb ; wherein each is optionally substituted with 1, 2, or 3 R ⁵ ; or R ¹ and R ² together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁶ . or R ³ and R ⁴ together with the N atom to which they are attached from a 4-10 membered ring, which is optionally substituted with 1, 2, 3, or 4 R ⁷ ; each R ⁵ , R ⁶ , and R ⁷ is independently selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, CN, NO2, OR ^la , C(0)R ^lb , C(0)NR ^la R ¹ b, C(0)OR ^la , NR ^la R ^lb , S(0) ₂ R ^lb and S(0) ₂ NR ^la R ^lb ; each R ^la and R ^lb is independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl.

In some embodiments, the compound of Formula (II) has the structure of Formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof:

Formula (I).

The compound of Formula (I) is l-(diethylthiocarbamoyldisulfanyl)-N,N-diethyl- methanethioamide, also known as Disulfiram.

Methods, Compositions, and Administrations

An aspect of the present disclosure is a method for increasing lifespan in a mammal, for preventing or treating an aging-related disorder in a mammal, for reducing a symptom of aging in a mammal, and/or boosting an immune system in a mammal. The method comprising: administering to the mammal a composition comprising a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram). In embodiments, the ALDH modulator is an ALDH modulator or an ALDH agonist. In embodiments, the ALDH modulator inhibits ALDH1, ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, and/or ALDH3A1. In embodiments, the ALDH agonist activates ALDH2.

In embodiments, the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, or ANS-66372. In embodiments, the ALDH modulator is Disulfiram, NCT-501, 4-

Diethylaminobenzaldehyde, or Daidzin. In embodiments, the ALDH modulator is Disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

In embodiments, the ALDH agonist is Alda-1.

In embodiments, the gasdermin D inhibitor is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082. In embodiments, the gasdermin D inhibitor is Disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

In embodiments, the administering is oral, by injection, inhalation, or topical. In embodiments, administering is more than one of oral, by injection, inhalation, or topical. In embodiments, the injection is intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

In embodiments, the mammal is near or has reached maturity.

In embodiments, the mammal is nearing or has reached halfway to its expected lifespan for the mammal’s species, size, sex, age, and/or health status. In embodiments, the mammal has reached an age that is at least 60%, 70%, 80%, 90%, or 100% of its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

In embodiments, increasing lifespan comprises an at least 5% increase in lifespan relative to the expected or median lifespan of a mammal of similar species, sex, age, and/or health status. In embodiments, increasing lifespan comprises an at least 10%, at least 15%, at least 20%, or at least 25% increase in lifespan.

In embodiments, the mammal is a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the mammal is a human.

In embodiments, the aging-related disorder or symptom of aging selected from one or more of actinic keratosis, age-related macular degeneration (AMD), Alzheimer’s disease, arthritis, atherosclerosis and cardiovascular disease, benign prostatic hyperplasia (BPH), bone atrophy, cachexia, cancer, cardiomyopathy, cataracts, chronic obstructive pulmonary disease (COPD), constipation, decrease in overall energy, decrease in visual acuity, delirium, dementia, depression, dermal atrophy (thinning of the skin), diminished peripheral vision, greater risk of heat stroke or hypothermia, hearing loss, hypertension, increased susceptibility to infection (including influenza and pneumonia), lentigines (aging spots), liver conditions ( e.g ., nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and cirrhosis), memory loss, metabolic syndrome, muscle atrophy (e.g., Sarcopenia and myopenia), frailty, muscle repair or rejuvenation deficiency, muscular dystrophy, osteoarthritis, osteoporosis, periodontitis, photoaging, reduced metabolism (including increased risk for obesity), reduced reflexes and coordination including difficulty with balance, respiratory disease (including acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI) and pulmonary fibrosis (e.g, idiopathic pulmonary fibrosis)), rheumatoid arthritis, sarcopenic obesity, sexual dysfunction, shingles, type 2 diabetes, urologic changes (including incontinence), vaginal atrophy, whitening or graying of hair, prolonged/inefficient wound healing, wrinkling/sagging skin (including loss of skin elasticity), and xerosis cutis (skin dryness). In embodiments, the aging-related disorder or symptom of aging is actinic keratosis, dermal atrophy (thinning of the skin), lentigines (aging spots), photoaging, prolonged/inefficient wound healing, wrinkles, and/or xerosis cutis (skin dryness) and wherein the administering is oral or topical. In embodiments, the mammal has at least one aging-related disorder or symptom of aging. A non-human mammal may have an aging-related disorder or symptom of aging that is homologous to the aging-related disorder or symptom of aging listed above.

In embodiments, the therapeutically effective amount of the active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) boosts the immune system in the mammal.

In embodiments, boosting the immune system increases an effective immune response against an infectious agent. In embodiments, the infectious agent is a virus, a bacterium, a fungus, a protozoan, a helminth, a prion, or a parasite. In embodiments, the bacterium is Bordatella pertussis or Streptococcus pneumoniae or the virus is a Chickenpox virus, Coronavirus, Hepatitis A virus, Hepatitis B virus, Human papillomavirus, Human immunodeficiency virus (HIV), influenza, Japanese encephalitis virus, Measles, mumps, or rubella virus, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rotavirus, Shingles virus, Smallpox, Varicella virus, or Yellow fever virus. In embodiments, the Coronavirus is Sars-CoV-2. In embodiments, when the infectious agent affects the mammal’s respiratory system or is transmitted via the mammal’s respiratory system and wherein the administering is by inhalation.

In embodiments, the administering is oral or by inhalation.

In embodiments, the mammal has a healthy immune system. In embodiments, the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

Compositions of the present disclosure are formulated to be suitable for in vivo administration to a mammal. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration. Pharmaceutical excipients can be aqueous liquids, such as water or saline. Pharmaceutical excipients can be lipid based, e.g., comprising a liquid or solid oil. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. The pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any composition described herein is administered parentally or in some oral formulations. In embodiments, the compositions described herein are suspended in a saline buffer (including, without limitation Ringer’s, TBS, PBS, HEPES, HBSS, and the like). Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, glycerol monostearate, mannitol, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any composition described herein, if desired, can also comprise pH buffering agents.

In embodiments, the compositions of the present disclosure are formulated for oral administration, for injection, or for topical administration. The administering the composition may comprise intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

The compositions suitable for parenteral administration (e.g, intravenous injection or infusion, intraarterial injection or infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intra-arterial injection or infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like, or in another acceptable format used in methods well known in the art. For patients who have difficulty swallowing a capsule, pill or the like, e.g, the elderly, the young, the infirm, or persons with assisted breathing, a composition for administration by injection or infusion may be preferred.

Compositions suitable for enteral administration (e.g, oral administration) may be formulated as a liquid, a suspension, a gel, a gel tab, a semisolid, a tablet, a sachet, a lozenge or a capsule, or in another acceptable format used in methods well known in the art. For patients who have difficulty swallowing a capsule, pill or the like, e.g., the elderly, the young, the infirm, or persons with assisted breathing, a liquid or suspension for oral administration may be preferred.

Compositions suitable for topical administration can be formulated in a solution, gel, lotion, ointment, cream, suspension, paste, liniment, powder, tincture, aerosol, patch, or the like in a pharmaceutically or cosmetically acceptable format used in methods well known in the art.

Compositions suitable for administration via inhalation. Such formulation will likely be in liquid form and will be delivered in a spray bottle, in an inhaler, or in a nebulizer. Inhaled compositions are particularly suited for diseases and disorder, including infections, that affect the mammal’s respiratory system and/or are transmitted via the mammal’s respiratory system.

The dosage of any herein-disclosed composition can depend on several factors including the characteristics of the mammal to be administered. Examples of characteristics include species, strain, breed, sex, age, weight, size, health, and/or disease status. Moreover, the dosage may depend on whether the administration is the first time the subject received a composition of the present disclosure or if the subject has previously received a composition of the present disclosure. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a composition) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific composition being administered, the time of administration, the route of administration, the nature of the formulation, and the rate of excretion. Some variations in the dosage can be expected.

Moreover, the dosage depends on the specific active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) administered.

In embodiments, the active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) is encapsulated in a microcapsule. The microcapsule may be a liposome, an albumin microsphere, a microemulsion, a nanoparticle (e.g, a lipid nanoparticle), and a nanocapsule. In embodiments, microcapsules, e.g, lipid nanoparticles and liposomes, include lipids selected from one or more of the following categories: cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids. In some cases, a cationic lipid and/or cationic polymer may be used to facilitate a charge-charge interaction with active ingredient. The microcapsule may comprise a PEGylated lipid. Examples of microcapsules and methods for manufacturing the same are described in the art. See, e.g, Prui el al, Crit Rev Ther Drug Carrier Syst, 2009; 26(6): 523-580; Wakasar, J Drug Target, 2018, 26(4):311-318, Langer, 1990, Science 249:1527-1533; Treat etal. , in “Liposomes in the Therapy of Infectious Disease and Cancer”, Lopez -Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Pelaz et al. “Diverse applications of nanomedicine.” (2017): 2313-2381; the contents of each of which is incorporated herein by reference in its entirety.

In embodiments, a composition may comprise one or more of capralactone, polylactide (PLA), polylactic-co-glycolic (PLGA), polyethylene glycol (PEG), polylactic-co-hydroxymethylglycolic acid (PLHMGA), carboxymethylcellulose, hydroxylmethylcellulose, gelatin-microcapsules, a poloxamer, or polymethylmethacrylate.

When the composition is for oral administration and is in solid form ( e.g ., a pill or capsule), the composition may comprise delay -release components. For example, a pill or capsule may comprise a coating that slows release of the agents and/or prevents release of the active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g., disulfiram) until the pill or capsule has arrived at a desired location of the mammal’s digestive system.

Yet another aspect of the present disclosure is composition comprising an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) for use in any herein disclosed method.

In certain embodiments, the active ingredient is disulfiram. The disulfiram is administered at a daily dosage of about 5 mg to about 500 mg per day. For example, disulfiram is administered at a total daily dosage of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg per day, and any total dosage therebetween. As examples, the daily dosage may be 5-10 mg, 10- 15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50-55 mg, 55- 60 mg, 60-65 mg, 65-70 mg, 70-75 mg, 75-80 mg, 80-85 mg, 85-90 mg, 90-95 mg, 95-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275- 300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 400-425 mg, 425-450 mg, 450-475 mg, or 475-500 mg. The daily dosage may be greater than 500 mg per day, e.g, 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,

640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg,

750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg,

860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,

970 mg, 980 mg, 990 mg, or 1000 mg. The daily dosage may be 0.5-1.0 g, 1.0-1.5 g, 1.5-2.0 g, 2.0-2.5 g, 2.5-3.0 g, 3.0-3.5 g, 3.5-4.0 g, 4.0-4.5 g, or 4.5-5.0 g. The disulfiram may be administered lx, 2x, or 3x per day to achieve the daily dosage. Thus, for a daily dose of 5 mg with a once per day administration, only a single administration of 5 mg will be given; for a daily dose of 5 mg with a twice per day administration, two administrations of about 2.5 mg will be given; and for a daily dose of 5 mg with a thrice per day administration, three administrations of about 1.7 mg will be given. Similarly, for a daily dose of 500 mg with a once per day administration, only a single administration of 500 mg will be given; for a daily dose of 500 mg with a twice per day administration, two administrations of about 250 mg will be given; and for a daily dose of 500 mg with a thrice per day administration, three administrations of about 170 mg will be given.

In some embodiments, 500 mg of Disulfiram is administered per dose or per day. In certain embodiments, the active ingredient Disulfiram limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

In some embodiments, the active ingredient Disulfiram is administered in one or more oral compositions. The oral compositions may be formulated as a liquid, a suspension, a gel, a gel tab, a semisolid, a tablet, a sachet, a lozenge or a capsule, or in another acceptable format used in methods well known in the art. In various embodiments, 500 mg of Disulfiram (in a single oral composition or in a plurality of distinct oral compositions) limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

In some embodiments, the active ingredient Disulfiram is administered in one or more compositions suitable for parenteral administration ( e.g ., intravenous injection or infusion, intraarterial injection or infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intra-arterial injection or infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like, or in another acceptable format used in methods well known in the art. In various embodiments, 500 mg of Disulfiram (in a single intravenous injection or as a plurality of intravenous injections) limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

Combinations with a Potentiating Agent

An active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g., disulfiram) may be combined (in a composition and/or in a method) with a potentiating ingredient. The potentiating ingredient, as used in the present disclosure enhances, increases, and/or improves the effectiveness and/or desirable activity of the active ingredient. Thus, a composition comprising the active ingredient alone will be less effective, for example, than a composition comprising the active ingredient and its potentiating ingredient. Similarly, a method in which the active ingredient is administered alone is less effective than a method in which the active ingredient is administered with a potentiating agent (either as a single composition or as distinct compositions that are administered contemporaneously or sequentially).

An increased and/or improved effectiveness may be determined by any assay, phenotype, marker, or indicator demonstrating a desired outcome from a treatment or administration of a composition. As examples, effectiveness may be shown as a reduction in markers/indicators of an aging cell, an increase in markers/indicators of a healthy cell, an extension of the active life of a cell, a reduction in apoptosis, increased longevity of a mammal, a reduction in the predicted biological age of a cell, increased titer of antibodies in response to a vaccine, inhibition of pyroptosis in an in vitro assay, an immune profile, and phenotypic changes in a cell that report health and/or activity. Increased and/or improved effectiveness may be objective (e.g, quantifiable) or subjective (e.g, qualifiable).

The potentiating ingredient, as used herein, is a compound that is generally recognized as safe (GRAS; e.g. , by the FDA), is listed in the FDA inactive ingredient database (IID (see, the World Wide Web at accessdata.fda.gov/ scripts/cder/iig/index.cfm), and/or is included in the FDA’s Substances Added to Food list (see, the World Wide Web at cfsanappsexternal.fda.gov/scripts/fdcc/?set=FoodSubstances and the regulations set forth in 21 CFR 73, 74, 172, 173, 181, 182, and 184, the contents of each of which is incorporated by reference in its entirety). The potentiating ingredient may be found in a plurality of the above-mentioned lists/databases. In some instances, the potentiating ingredient is considered an “inactive ingredient”. An inactive ingredient is any component of a drug product other than the active ingredient (see, the World Wide Web at fda.gov/drugs/drug-approvals-and-databases/ inactive- ingredients-approved-drug-products-search-frequently-asked-q uestions, the contents of which is incorporated by reference in its entirety). In the present disclosure, the active ingredient is an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram). In various embodiments of the present disclosure, the potentiating ingredient is any substance that can intentionally be added to a food as a food additive and which is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use. In particular, the potentiating ingredients are exempted from the usual Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tolerance requirements. The potentiating ingredient, as used herein, includes both the specific potentiating ingredient and any of its metabolites and/or their derivatives. Non-limiting examples of a metabolite includes a salt and ester of the potentiating ingredient. Other variations may include changes in Chirality, Isomers, Hydration states relative to the specific potentiating ingredient. The usefulness of a metabolite or a derivative of a potentiating ingredient that is useful in the present disclosure, is well-within the ability of a skilled artisan; such experiments needed to verify that the metabolite or derivative is useful would be similar to the experiments used to verify that the specific potentiating ingredient is useful, e.g ., in the compositions and methods of the present disclosure.

In various embodiments, the potentiating ingredient, as disclosed herein, enhances the active ingredient’s ability to inhibit pyroptosis.

In some embodiments, the enhanced, increased, and/or improved effectiveness and/or activity of the active ingredient, i.e ., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram), may be any amount between about 1.1 -fold and about 3 -fold, e.g, about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3-fold, and any fold therebetween. In embodiments, the enhanced, increased, and/or improved effectiveness and/or activity of the active ingredient, may be any amount between about 3-fold and about 5-fold, e.g, about 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5-fold, and any fold therebetween. The enhanced, increased, and/or improved effectiveness and/or activity of the active ingredient, may be any amount between about 5-fold and about 15-fold. As examples, the effectiveness may be increased about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15-fold, and any fold therebetween. In some embodiments, the enhanced, increased and/or improved effectiveness and/or activity of the active ingredient, is greater than about 15-fold. The increased and/or improved effectiveness may be greater than about 1%, e.g, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%, and any percentage therebetween.

An enhanced, increased, and/or improved effectiveness and/or activity of the active ingredient, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram), may be determined by any assay, phenotype, marker, or indicator demonstrating a desired outcome from a treatment or administration of a composition. As examples, effectiveness and/or activity may be shown as a reduction in markers/indicators of an aging cell, an increase in markers/indicators of a healthy cell, an extension of the active life of a cell, a reduction in apoptosis, increased longevity of a mammal, a reduction in the predicted biological age of a cell, increased titer of antibodies in response to a vaccine, inhibition of pyroptosis in an in vitro assay, an immune profile, and phenotypic changes in a cell that report health and/or activity. Increased and/or improved effectiveness may be objective (e.g, quantifiable) or subjective (e.g, qualifiable). Because the potentiating ingredient, enhances, increases, and/or improve the active ingredient’s effectiveness and/or activity ( e.g ., in pyroptosis inhibition), lower doses of the active ingredient may be administered to a mammal while still providing a desired outcome. This lower dose may minimize adverse effects resulting from the active ingredient administration, if any.

In certain embodiments, the active ingredient is disulfiram. The disulfiram is administered at a daily dosage of about 5 mg to about 500 mg per day. For example, disulfiram is administered at a total daily dosage of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg per day, and any total dosage therebetween. As examples, the daily dosage may be 5-10 mg, 10- 15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50-55 mg, 55- 60 mg, 60-65 mg, 65-70 mg, 70-75 mg, 75-80 mg, 80-85 mg, 85-90 mg, 90-95 mg, 95-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275- 300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 400-425 mg, 425-450 mg, 450-475 mg, or 475-500 mg. The daily dosage may be greater than 500 mg per day, e.g., 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,

640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg,

750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg,

860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,

970 mg, 980 mg, 990 mg, or 1000 mg. The daily dosage may be 0.5-1.0 g, 1.0-1.5 g, 1.5-2.0 g, 2.0-2.5 g, 2.5-3.0 g, 3.0-3.5 g, 3.5-4.0 g, 4.0-4.5 g, or 4.5-5.0 g. The disulfiram may be administered lx, 2x, or 3x per day to achieve the daily dosage. Thus, for a daily dose of 5 mg with a once per day administration, only a single administration of 5 mg will be given; for a daily dose of 5 mg with a twice per day administration, two administrations of about 2.5 mg will be given; and for a daily dose of 5 mg with a thrice per day administration, three administrations of about 1.7 mg will be given. Similarly, for a daily dose of 500 mg with a once per day administration, only a single administration of 500 mg will be given; for a daily dose of 500 mg with a twice per day administration, two administrations of about 250 mg will be given; and for a daily dose of 500 mg with a thrice per day administration, three administrations of about 170 mg will be given. The potentiating ingredient may be administered lx, 2x, or 3x per day.

In certain embodiments, the active ingredient is disulfiram. The disulfiram is administered at an individual dose of about 5 mg to about 500 mg per day. For example, disulfiram is administered at an individual dose of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg per day, and any total dosage therebetween. As examples, the individual dose may be 5-10 mg, 10-15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50-55 mg, 55-60 mg, 60-65 mg, 65-70 mg, 70-75 mg, 75-80 mg, 80-85 mg, 85-90 mg, 90-95 mg, 95-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275- 300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 400-425 mg, 425-450 mg, 450-475 mg, or 475-500 mg. The daily dosage may be greater than 500 mg per day, e.g., 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,

640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg,

750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg,

860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,

970 mg, 980 mg, 990 mg, or 1000 mg. The daily dosage may be 0.5-1.0 g, 1.0-1.5 g, 1.5-2.0 g, 2.0-2.5 g, 2.5-3.0 g, 3.0-3.5 g, 3.5-4.0 g, 4.0-4.5 g, or 4.5-5.0 g. The disulfiram may be administered lx, 2x, 3x, 4x, 5x, or more times per day at any of the herein-disclosed individual doses. The potentiating ingredient may be administered lx, 2x, 3x, 4x, 5x, or more times per day. By individual dose is meant the amount of the disulfiram that is administered to a patient at a specific time. The individual dose may be in a single composition (with or without the potentiating ingredient, e.g, TBHQ). The individual dose may be split between two or more separate compositions (each with or without the potentiating ingredient, e.g, TBHQ).

In certain embodiments, the potentiating ingredient, and the active ingredient, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) are administered in two separate formulations. In certain embodiments, the potentiating ingredient and the active ingredient are administered in a single formulation.

In some embodiments, the potentiating ingredient is ingredient is tert-Butylhydroquinone (TBHQ). TBHQ has the following chemical identifiers: CAS 1948-33-0, DrugBank DB07726, MedChem HY-100489, and SelleckChem S4990. TBHQ protects against Doxorubicin (DOX)-induced cardiotoxicity through activation of nuclear factor erythroid 2-related factor 2 (NrF2). TBHQ is also an ERK activator, and rescues Dehydrocorydaline (DHC)-induced cell proliferation inhibition in melanoma.

In certain embodiments, the potentiating ingredient, i.e., TBHQ, is administered at a ratio of about 1:1 to about 1:3000 moles of TBHQ to the active ingredient, e.g. , disulfiram. The TBHQ may be administered at a ratio of about 1:1, 1:1.01, 1:1.02, 1:1.03, 1:1.04, 1:1.05, 1:1.06, 1:1.07, 1:1.08, 1:1.09, 1:1.1, 1:1.11, 1:1.12, 1:1.13, 1:1.14, 1:1.15, 1:1.16, 1:1.17, 1:1.18, 1:1.19, 1:1.2, 1:1.21, 1 : 1.22, 1 : 1.23, 1 : 1.24, 1 : 1.25, 1 : 1.26, 1 : 1.27, 1 : 1.28, 1 : 1.29, or 1 : 1.3 moles TBHQ per mole of the active ingredient, e.g, disulfiram. The TBHQ may be administered at a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20 moles TBHQ per mole of the active ingredient, e.g. , disulfiram. The TBHQ may be administered at a ratio of about 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, or 1:200 moles TBHQ per mole of the active ingredient, e.g., disulfiram, and any ratio therebetween. As examples, the ratio may be about 1 : 10-1 : 15, 1:15-1 :20, 1:20-1:25, 1:25-1:30, 1:30-1:35, 1:35-1:40, 1:40-1:45, 1:45-1:50, 1:50-1:55, 1:55-1:60, 1:60-1:65, 1:65-1:70, 1:70-1:75, 1:75-1:80, 1:80-1:85, 1:85-1:90, 1:90-1:95, 1:95-1:100, 1:100-1:125, 1:125- 1:150, 1:150-1:175, 1:175-1:200 moles TBHQ per mole of the active ingredient, e.g., disulfiram. The TBHQ may be administered at a ratio of about 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, or 1:1000 moles TBHQ per mole of the active ingredient, e.g., disulfiram, and any ratio therebetween. As examples, the ratio may be about 1:100-1:125, 1:125-1:150, 1:150-1:175, 1:175-1:200, 1:200-1:225, 1:225-1:250, 1:250-1:275, 1:275-1:300, 1:300-1:325, 1:325-1:350, 1:350-1:375, 1:375-1:400, 1:400-1:425, 1:425-1:450, 1:450-1:475, 1:475-1:500, 1:500-1:525, 1:525-1:550, 1:550-1:575, 1:575-1:600, 1:600-1:625, 1:625-1:650, 1:650-1:675, 1:675-1:700, 1:700-1:725, 1:725-1:750, 1:750-1:775, 1:775-1:800, 1:800-1:825, 1:825-1:850, 1:850-1:875, 1:875-1:900, 1:900-1:925, 1:925-1:950, 1:950-1:975, or 1:975-1:1000 moles TBHQ per mole of the active ingredient, e.g, disulfiram. The TBHQ may be administered at a ratio of about 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900, or 1:3000 moles TBHQ per mole of the active ingredient, e.g, disulfiram, and any ratio therebetween. As examples, the ratio may be about 1:1000-1:1100, 1:1100-1:1200, 1:1200-1:1300, 1:1300-1:1400, 1:1400-1:1500, 1:1500-1:1600, 1:1600-1:1700, 1:1700-1:1800, 1:1800-1:1900, 1:1900-1:2000, 1:2000-1:2100, 1:2100-1:2200, 1:2200-1:2300, 1:2300-1:2400, 1:2400-1:2500, 1:2500-1:2600, 1:2600-1:2700, 1:2700-1:2800, 1:2800-1:2900, or 1:2900-1:3000 moles TBHQ per mole of the active ingredient, e.g., disulfiram.

In embodiments where the potentiating ingredient, i.e., TBHQ, and the active ingredient, e.g, disulfiram, are administered in a single formulation, the amount of TBHQ is about 0.02% to about 56% of the amount of the active ingredient, e.g, disulfiram (by weight). The amount of TBHQ may be about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the weight of the active ingredient, e.g, disulfiram. The amount of TBHQ may be about 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%, 7.2%, 7.4%, 7.6%, 7.8%, 8%, 8.2%, 8.4%, 8.6%, 8.8%, 9%, 9.2%, 9.4%, 9.6%, 9.8%, 10%, of the weight of the active ingredient, e.g., disulfiram, and any percentage therebetween. As examples, the percentage may be about 1%-1.5%, 1.5%-2%, 2%-2.5%, 2.5%-3%, 3%-3.5%, 3.5%-4%, 4%- 4.5%, 4.5%-5%, 5%-5.5%, 5.5%-6%, 6%-6.5%, 6.5%-7%, 7%-7.5%, 7.5%-8%, 8%-8.5%, 8.5%- 9%, 9%-9.5%, or 9.5%-10% of the weight of the active ingredient, e.g, disulfiram. The amount of TBHQ may be about 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, or 56% of the weight of the active ingredient, e.g, disulfiram, and any percentage therebetween. As examples, the percentage may be about 10%- 15%, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, or 50%-56% of the weight of the active ingredient, e.g, disulfiram. In embodiments where the potentiating ingredient, i.e., TBHQ, and the active ingredient, e.g, disulfiram, are administered in two formulations, the amount of TBHQ is about 0.02% to about 56% of the weight of the active ingredient, e.g, disulfiram. The amount of TBHQ may be about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the weight of the active ingredient, e.g, disulfiram. The amount of TBHQ may be about 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%, 7.2%, 7.4%, 7.6%, 7.8%, 8%, 8.2%, 8.4%, 8.6%, 8.8%, 9%, 9.2%, 9.4%, 9.6%, 9.8%, 10%, of the weight of the active ingredient, e.g., disulfiram, and any percentage therebetween. As examples, the percentage may be about 1%-1.5%, 1.5%-2%, 2%-2.5%, 2.5%-3%, 3%-3.5%, 3.5%-4%, 4%-4.5%, 4.5%-5%, 5%-5.5%, 5.5%-6%, 6%-6.5%, 6.5%-7%, 7%-7.5%, 7.5%-8%, 8%-8.5%, 8.5%-9%, 9%-9.5%, or 9.5%-10% of the weight of the active ingredient, e.g, disulfiram. The amount of TBHQ may be about 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, or 56% of the weight of the active ingredient, e.g, disulfiram, and any percentage therebetween. As examples, the percentage may be about 10%-15%, 15%-20%, 20%- 25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, or 50%-56% of the weight of the active ingredient, e.g, disulfiram.

In some embodiments, TBHQ is administered at a daily dosage from about 0.001 mg to about 280 mg. As examples, TBHQ is administered at a total daily dosage of about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, or 0.01 mg, and any daily dosage therebetween. As examples, the daily dosage of TBHQ may be about 0.001-0.002 mg, 0.002-0.003 mg, 0.003-0.004 mg, 0.004-0.005 mg, 0.005-0.006 mg, 0.006-0.007 mg, 0.007- 0.008 mg, 0.008-0.009 mg, or 0.009-0.01 mg. The TBHQ may be administered at a total daily dosage of about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, or 0.1 mg, and any daily dosage therebetween. As examples, the daily dosage of TBHQ may be about 0.01-0.02 mg, 0.02-0.03 mg, 0.03-0.04 mg, 0.04-0.05 mg, 0.05-0.06 mg, 0.06-0.07 mg, 0.07-0.08 mg, 0.08-0.09 mg, or 0.09-0.1 mg. The TBHQ may be administered at a total daily dosage of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg, and any daily dosage therebetween. As examples, the daily dosage of TBHQ may be about 0.1-0.2 mg, 0.2-0.3 mg, 0.3-0.4 mg, 0.4-0.5 mg, 0.5-0.6 mg, 0.6-0.7 mg, 0.7-0.8 mg, 0.8-0.9 mg, or 0.9-1 mg. The TBHQ may be administered at a total daily dosage of about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg, and any daily dosage therebetween. As examples, the daily dosage of TBHQ may be about 1-2 mg, 2-3 mg, 3-4 mg, 4-5 mg, 5-6 mg, 6-7 mg, 7-8 mg, 8-9 mg, or 9-10 mg. The TBHQ may be administered at a total daily dosage of about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, and any daily dosage therebetween. The TBHQ may be administered at a total daily dosage of about 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, and any daily dosage therebetween. The TBHQ may be administered at a total daily dosage of about 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, or 30 mg, and any daily dosage therebetween. The TBHQ may be administered at a total daily dosage of about 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, or 40 mg, and any daily dosage therebetween. The TBHQ may be administered at a total daily dosage of about 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, or 50 mg, and any daily dosage therebetween. As examples, the daily dosage of TBHQ may be about 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg. The TBHQ may be administered at a total daily dosage of about 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, or 280 mg and any daily dosage therebetween. As examples, the daily dosage of TBHQ may be about 100-110 mg, 110-120 mg, 120-130 mg, 130-140 mg, 140-150 mg, 150- 160 mg, 160-170 mg, 170-180 mg, 180-190 mg, 190-200 mg, 200-210 mg, 210-220 mg, 220-230 mg, 230-240 mg, 240-250 mg, 250-260 mg, 260-270 mg, or 270-280 mg. The potentiating ingredient, i.e., TBHQ, may be administered lx, 2x, or 3x per day to achieve the daily dosage. Thus, for a daily dose of 5 mg with a once per day administration, only a single administration of 5 mg will be given; for a daily dose of 5 mg with a twice per day administration, two administrations of about 2.5 mg will be given; and for a daily dose of 5 mg with a thrice per day administration, three administrations of about 1.7 mg will be given. Similarly, for a daily dose of 280 mg with a once per day administration, only a single administration of 280 mg will be given; for a daily dose of 280 mg with a twice per day administration, two administrations of about 140 mg will be given; and for a daily dose of 280 mg with a thrice per day administration, three administrations of about 93 mg will be given.

In some embodiments, TBHQ is administered at an individual dose from about 0.001 mg to about 280 mg. As examples, TBHQ is administered at individual doses of about 0.001 mg, 0.002 mg, 0.003 mg, 0.004 mg, 0.005 mg, 0.006 mg, 0.007 mg, 0.008 mg, 0.009 mg, or 0.01 mg, and any individual dose therebetween. As examples, the individual dose of TBHQ may be about 0.001- 0.002 mg, 0.002-0.003 mg, 0.003-0.004 mg, 0.004-0.005 mg, 0.005-0.006 mg, 0.006-0.007 mg, 0.007-0.008 mg, 0.008-0.009 mg, or 0.009-0.01 mg. The TBHQ may be administered at individual doses of about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, or 0.1 mg, and any individual dose therebetween. As examples, the individual dose of TBHQ may be about 0.01-0.02 mg, 0.02-0.03 mg, 0.03-0.04 mg, 0.04-0.05 mg, 0.05-0.06 mg, 0.06-0.07 mg, 0.07-0.08 mg, 0.08-0.09 mg, or 0.09-0.1 mg. The TBHQ may be administered at individual doses of about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg, and any individual dose therebetween. As examples, the individual dose of TBHQ may be about 0.1-0.2 mg, 0.2-0.3 mg, 0.3-0.4 mg, 0.4-0.5 mg, 0.5-0.6 mg, 0.6-0.7 mg, 0.7-0.8 mg, 0.8-0.9 mg, or 0.9-1 mg. The TBHQ may be administered at individual doses of about 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg, and any individual dose therebetween. As examples, the individual dose of TBHQ may be about 1-2 mg, 2-3 mg, 3-4 mg, 4-5 mg, 5-6 mg, 6-7 mg, 7-8 mg, 8-9 mg, or 9-10 mg. The TBHQ may be administered at individual doses of about 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, or 100 mg, and any individual dose therebetween. The TBHQ may be administered at individual doses of about 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg, and any individual dose therebetween. The TBHQ may be administered at individual doses of about 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, or 30 mg, and any individual dose therebetween. The TBHQ may be administered at individual doses of about 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, or 40 mg, and any individual dose therebetween. The TBHQ may be administered at individual doses of about 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, or 50 mg, and any individual dose therebetween. As examples, the individual dose of TBHQ may be about 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 50-60 mg, 60-70 mg, 70- 80 mg, 80-90 mg, or 90-100 mg. The TBHQ may be administered at individual doses of about 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, or 280 mg and any individual dose therebetween. As examples, the individual dose of TBHQ may be about 100-110 mg, 110-120 mg, 120-130 mg, 130-140 mg, 140-150 mg, 150-160 mg, 160-170 mg, 170-180 mg, 180-190 mg, 190- 200 mg, 200-210 mg, 210-220 mg, 220-230 mg, 230-240 mg, 240-250 mg, 250-260 mg, 260-270 mg, or 270-280 mg. The potentiating ingredient, i.e., TBHQ, may be administered lx, 2x, 3x, 4x, 5x, or more times per day at the above-mentioned the individual doses. By individual dose is meant the amount of the TBHQ that is administered to a patient at a specific time. The individual dose may be in a single composition (with or without disulfiram). The individual dose may be split between two or more separate compositions (each with or without disulfiram).

In some embodiments, the active ingredient is Disulfiram and the potentiating ingredient is tertiary butylhydroquinone (TBHQ). In some case, about 500 mg Disulfiram and up to about 37 mg TBHQ, e.g., 500 mg Disulfiram and 37 mg TBHQ, are combined (in a single dosage form or as distinct dosage forms). In certain embodiments, the combination of the active ingredient Disulfiram and is potentiating ingredient TBHQ limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI). In some embodiments, the combination of 500 mg Disulfiram and 37 mg TBHQ, e.g., 500 mg Disulfiram and 37 mg TBHQ (in a single dosage form or as distinct dosage forms) limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

In some embodiments, the active ingredient as Disulfiram and the potentiating ingredient as tertiary butylhydroquinone (TBHQ) are administered in one or more oral compositions. The oral compositions may be formulated as a liquid, a suspension, a gel, a gel tab, a semisolid, a tablet, a sachet, a lozenge or a capsule, or in another acceptable format used in methods well known in the art. In various embodiments, the combination of about 500 mg Disulfiram and up to about 37 mg TBHQ, e.g, 500 mg Disulfiram and 37 mg TBHQ, (in a single oral composition or as distinct dosage oral compositions) limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

In some embodiments, the active ingredient as Disulfiram and the potentiating ingredient as tertiary butylhydroquinone (TBHQ) are administered in one or more compositions suitable for parenteral administration (e.g, intravenous injection or infusion, intraarterial injection or infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intra-arterial injection or infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like, or in another acceptable format used in methods well known in the art. In various embodiments, the combination of about 500 mg Disulfiram and up to about 37 mg TBHQ, e.g., 500 mg Disulfiram and 37 mg TBHQ, (in a single intravenous injection or as distinct dosage intravenous injections) limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

In some embodiments, the active ingredient as Disulfiram and the potentiating ingredient as tertiary butylhydroquinone (TBHQ) are administered in either a composition suitable for parenteral administration (e.g, intravenous injection or infusion, intraarterial injection or infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intra-arterial injection or infusion) or an oral composition (e.g, formulated as a liquid, a suspension, a gel, a gel tab, a semisolid, a tablet, a sachet, a lozenge or a capsule, or in another acceptable format used in methods well known in the art). Thus, in a first embodiment, the Disulfiram is formulated as an oral composition and the TBHQ is formulated in a composition suitable for parenteral administration and, in a second embodiment, the TBHQ is formulated as an oral composition and the Disulfiram is formulated in a composition suitable for parenteral administration. In either embodiment, the combination of about 500 mg Disulfiram and up to about 37 mg TBHQ (e.g, 500 mg Disulfiram and 37 mg TBHQ) limits the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI).

Other potentiating ingredients may be combined with an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram).

One such other potentiating ingredient is cinnamaldehyde. Cinnamaldehyde has the following chemical identifiers: CAS: 14371-10-9, 104-55-2; DrugBank: DB14184; MedChem: HY-N0609; and SelleckChem: S3763. Cinnamaldehyde is the aldehyde that gives cinnamon its flavor and odor. Cinnamaldehyde occurs naturally in the bark of cinnamon trees and other species of the genus Cinnamomum like camphor and cassia. These trees are the natural source of cinnamon, and the essential oil of cinnamon bark is about 90% cinnamaldehyde. Cinnamaldehyde is also used as a fungicide. Proven effective on over 40 different crops, cinnamaldehyde is typically applied to the root systems of plants. Its low toxicity and well-known properties make it ideal for agriculture. To a lesser extent, cinnamaldehyde is an effective insecticide, and its scent is also known to repel animals like cats and dogs. Cinnamaldehyde is also known as a corrosion inhibitor for steel and other ferrous alloys in corrosive fluids. It can be used in combination with additional components such as dispersing agents, solvents and other surfactants. Concentrated cinnamaldehyde is a skin irritant, and the chemical is toxic in large doses, but no agencies suspect the compound is a carcinogen or poses a long-term health hazard. Most cinnamaldehyde is excreted in urine as cinnamic acid, an oxidized form of cinnamaldehyde.

Additional names for cinnamaldehyde include (2E)-3-Phenyl-2-propenal, (2E)-3- Phenylacrylaldehyde, (e)-3-Phenyl-2-propenal, (e)-3-Phenylpropenal, (e)-3-Phenyl-propenal, (e)- Cinnamaldehyde, (e)-Cinnamic aldehyde, (e)-Phenylvinyl aldehyde, 3-Fenylpropenal, 3-Phenyl- 2-propen-l-al, 3-Phenyl-2-propenaldehyde, 3-Phenylacrolein, 3-Phenylacrylaldehyde, 3- Phenylprop-2-enal, 3-Phenylprop-2-enaldehyde, 3-Phenylpropenal, Benzylideneacetaldehyde, beta-Phenylacrolein, beta-Phenylcrolein, Cinnamal, Cinnamic aldehyde, Cinnamic aldehyde, (e)- isomer, Cinnamyl aldehyde, Cinnamylaldehyde, fv-Cinnemaldehyde, Supercinnamaldehyde, trans-3-Phenyl-2-propenal, trans-3-Phenylprop-2-enaldehyde, trans-Cinnamaldehyde, trans- Cinnamic aldehyde, and trans-Cinnamylaldehyde.

Combinations with a Metal

In some cases, a method for increasing lifespan, for preventing or treating an aging-related disorder, for reducing a symptom of aging, and/or boosting an immune system in a mammal comprises administering the mammal a first composition and a second composition. The first composition comprises a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g., disulfiram) and the second composition comprises a therapeutically effective amount of a metal (e.g, copper and zinc).

In embodiments, the first composition and the second composition are administered contemporaneously. By contemporaneously is meant administering the two compositions together or by administering the two compositions at nearly the same time. In embodiments, the first composition is administered before the second composition is administered. In embodiments, the second composition is administered before the first composition is administered.

The first composition and the second composition may be combined into a single unit does, e.g. , a single pill, capsule, and tablet.

In embodiments, the metal is selected from copper, copper (II), zinc, arsenic, titanium, bismuth, vanadium, chromium, molybdenum, manganese, iron, ruthenium, osmium, cobalt, nickel, platinum, silver, gold, gold (II), cadmium, and mercury. Disulfiram has been shown to chelate certain metals and/or to be useful in the context of cancer treatments. See, e.g, Viola-Rhenals et al. “Recent Advances in Antabuse (Disulfiram): The Importance of its Metal -binding Ability to its Anticancer Activity”, Curr Med Chem. 2018 Feb 12; 25(4): 506-524; W02018081309A1; and W02019094053A1. The contents of each of which is incorporated herein by reference in its entirety.

The metal may be in the form of any metal salt or metal ester described in the present FDA’s list of food additives.

In embodiments, copper is in the form of copper or copper (II) gluconate, copper chloride, copper glycinate, copper carbonate, copper hydroxide, copper orthophosphate, copper oxide, copper pyrophosphate, copper sulfate, or (cuprous) iodide.

In embodiments, a zinc is in the form of zinc or zinc (II) acetate, zinc carbonate, zinc chloride, zinc oxide, zinc sulfate, zinc methionine sulfate, or zinc stearate.

In embodiments, an iron is in the form of iron ascorbate, iron carbonate, iron chloride, iron citrate, iron fumarate, iron gluconate, iron lactate, iron oxide, iron phosphate, iron pyrophosphate, iron sodium pyrophosphate, iron sulfate, reduced iron, iron ammonium citrate, iron-choline citrate complex.

In embodiments, a cadmium is in the form of cadmium sulfide.

In embodiments, a cobalt is in the form of cobalt acetate, cobalt carbonate, cobalt chloride, cobalt oxide, or cobalt sulfate.

In embodiments, a manganese is in the form of manganese acetate, manganese carbonate, manganese chloride, manganese citrate, manganese gluconate, manganese glycerophosphate, manganese ortho-phosphate, manganese dibasic phosphate, manganese hypophosphite, manganese sulfate, or manganese oxide.

The dosage of the first composition may comprise 0.1 - 60 units of the active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram)and the second composition may comprise about 1 unit of a metal (e.g, copper or zinc), where active ingredient ranges from 50 - 500 mg. The effective amount of a metal (is an above-described form) may between 0.1 mg to 30 mg. In an embodiment, the amount of the metal is between 1.5 mg and 3 mg. In embodiments, the metal is copper or zinc and approximately 1.5 mg of the metal is administered.

The first composition and/or the second composition may be administered to a subject in need thereof once per day, twice per day, or thrice per day.

The administration route of the first composition and the second composition may be the same or may be different. In one example, the first composition and the second composition are administrated orally. In one example, the first composition and the second composition are administrated by inhalation. In another example, the first composition is administrated orally and the second composition is by injection, inhalation, or topically. In yet another example, the first composition is administrated by inhalation and the second composition is by injection, orally, or topically.

Boosting the immune system

Another aspect of the present disclosure is a method for boosting the immune system in a mammal.

The method comprising administering to a mammal in need a composition comprising a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor ( e.g ., disulfiram). The therapeutically effective amount the active ingredient is sufficient to boost the immune system in the mammal.

The method for boosting an immune system in a mammal may comprise administering to the mammal a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g., disulfiram) and as potentiating ingredient (e.g, TBHQ).

The method for boosting an immune system may comprise additionally administering to a mammal a second composition comprising a therapeutically effective amount of a metal (e.g, copper and zinc).

In embodiments, boosting the immune system increases an effective immune response against an infectious agent. In embodiments, the infectious agent is a virus, a bacterium, a fungus, a protozoan, a helminth, a prion, or a parasite. In embodiments, the bacterium is Bordatella pertussis or Streptococcus pneumoniae. In embodiments, the virus is selected from Alphavirus, BK virus, Bunyaviridae, Chickenpox virus, Colorado tick fever virus (CTFV), Coronaviruses, Crimean- Congo hemorrhagic fever virus, Cytomegalovirus, Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) , Ebolavirus (EBOV), Enteroviruses, mainly Coxsackie A virus and enterovirus 71 (EV71), Epstein-Barr virus (EBV), Flaviviruses, Guanarito virus, Heartland virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D Virus, Hepatitis E virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7), Human immunodeficiency virus (HIV), Human metapneumovirus (hMPV), Human papillomaviruses (HPV), Human parainfluenza viruses (HPIV), Influenza, Japanese encephalitis virus, JC virus, Junin virus, Lassa virus, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Marburg virus, Measles virus, Middle East respiratory syndrome coronavirus, Molluscum contagiosum virus (MCV), Monkeypox virus, Mumps virus, Nipah virus, Norovirus, Orthomyxoviridae species, Parvovirus B19, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, SARS coronavirus, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Shingles virus, Sin Nombre virus, Smallpox , Varicella zoster virus (VZV), Variola major or Variola minor, Venezuelan equine encephalitis virus, West Nile virus, Yellow fever virus, and Zika virus. In embodiments, the Coronavirus is Sars-CoV-2.

In embodiments, when the infectious agent affects the mammal’s respiratory system or is transmitted via the mammal’s respiratory system and wherein the administering is by inhalation.

In embodiments, the administering is oral or by inhalation.

In some cases, the mammal has a healthy immune system. In other cases, the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

In embodiments, the term dysfunctional immune system may be an overactive immune system, e.g., resulting in a cytokine storm; such overactive immune systems are observed in certain viral infections, e.g, in some severe coronavirus patients. In these cases, “boosting the immune system”, relates to “boosting” a proper immune response. That is, minimizing an overactive immune response.

In embodiments, the mammal is nearing or has reached halfway to its expected lifespan for the mammal’s species, size, sex, age, and/or health status. In embodiments, the aged mammal has reached an age that is at least 60%, 70%, 80%, 90%, or 100% of its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

Of course, any mammal may benefit from a boost in the immune system. Thus, the active ingredients, compositions, and methods for boosting the immune system can be used with aged and with non-aged mammals.

In embodiments, the administering is oral, by injection, inhalation, or topical. The administering may comprise intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

In embodiments, the ALDH modulator for boosting an immune system is an ALDH modulator or an ALDH agonist. In embodiments, the ALDH modulator inhibits ALDHl, ALDHIAI, ALDHl A2, ALDHl A3, ALDH2, and/or ALDH3 A1. In embodiments, the ALDH agonist activates ALDH2. In embodiments, the ALDH modulator for boosting an immune system is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3- Hydroxybenzaldehyde, or ANS-66372. In embodiments, the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, or Daidzin. In embodiments, the ALDH modulator is disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

In embodiments, the ALDH agonist for boosting an immune system is Alda-1.

In embodiments, the gasdermin D inhibitor for boosting an immune system is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082.

In embodiments, the gasdermin D inhibitor for boosting an immune system is disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

In certain embodiments, the active ingredient is disulfiram. The disulfiram is administered at a daily dosage of about 5 mg to about 500 mg per day. For example, disulfiram is administered at a total daily dosage of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg per day, and any total dosage therebetween. As examples, the daily dosage may be 5-10 mg, 10- 15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50-55 mg, 55- 60 mg, 60-65 mg, 65-70 mg, 70-75 mg, 75-80 mg, 80-85 mg, 85-90 mg, 90-95 mg, 95-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275- 300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 400-425 mg, 425-450 mg, 450-475 mg, or 475-500 mg. The daily dosage may be greater than 500 mg per day, e.g., 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,

640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg,

750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg,

860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,

970 mg, 980 mg, 990 mg, or 1000 mg. The daily dosage may be 0.5-1.0 g, 1.0-1.5 g, 1.5-2.0 g, 2.0-2.5 g, 2.5-3.0 g, 3.0-3.5 g, 3.5-4.0 g, 4.0-4.5 g, or 4.5-5.0 g. The disulfiram may be administered lx, 2x, or 3x per day to achieve the daily dosage. Thus, for a daily dose of 5 mg with a once per day administration, only a single administration of 5 mg will be given; for a daily dose of 5 mg with a twice per day administration, two administrations of about 2.5 mg will be given; and for a daily dose of 5 mg with a thrice per day administration, three administrations of about 1.7 mg will be given. Similarly, for a daily dose of 500 mg with a once per day administration, only a single administration of 500 mg will be given; for a daily dose of 500 mg with a twice per day administration, two administrations of about 250 mg will be given; and for a daily dose of 500 mg with a thrice per day administration, three administrations of about 170 mg will be given.

Improving a vaccine response

Another aspect of the present disclosure is a method for improving effectiveness of a vaccine in a mammal in need thereof. The method comprises administering a composition comprising a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram). The mammal may contemporaneously and/or subsequently be administered a vaccine.

In embodiments, the active ingredient and the vaccine are administered contemporaneously. In embodiments, the vaccine is administered subsequent to the active ingredient’s administration.

The method for improving effectiveness of a vaccine in a mammal may comprise administering to the mammal a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) and as potentiating ingredient (e.g, TBHQ).

The method for improving a vaccine response may further comprise administering to a mammal a composition comprising a therapeutically effective amount of a metal (e.g, copper and zinc).

In embodiments, the therapeutically effective amount the active ingredient boosts the immune system in the mammal. In embodiments, boosting the immune system increases an immune response against a component contained in the vaccine. In embodiments, the increased immune response promotes future immunity against the component contained in the vaccine.

It has been reported in the art that aged mammals respond less strongly to vaccines than mammal who are less aged. Accordingly, methods for improving effectiveness of a vaccine in an aged mammal are needed.

In embodiments, an aged mammal is nearing or has reached halfway to its expected lifespan for the mammal’s species, size, sex, age, and/or health status. In embodiments, the aged mammal has reached an age that is at least 60%, 70%, 80%, 90%, or 100% of its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

Influenza is problematic in older adults with increased risk for serious complications and hospitalization. In addition, approximately 90% of flu-related deaths occur in this population, with influenza and pneumonia being the eighth leading cause of death among persons over 65 years of age in the United States. Even when death is avoided, older adults have an increased risk for secondary complications and morbidities from flu infection. Depending on how successful the WHO predicts the influenza strains causing seasonal epidemics, the produced vaccines show efficacy rates between 60% and 90%. However, vaccine effectiveness in adults aged 65 and older is usually significantly lower, ranging from an average of 28% protection against fatal and nonfatal complications (with large dispersion), 39% protection against typical influenza-like illness, and 49% protection against disease with confirmed virus infection. Influenza vaccine effectiveness is a significant problem in elderly as compared to young individuals and is associated with high rates of complicated illness including pneumonia, heart attacks, and strokes in the >65 -year-old population.

Furthermore, the outbreak of the novel coronavirus (SARS-CoV-2) has had devastating effects on the aged and those with pre-existing health conditions. A mammal would particularly benefit from a composition of the present disclosure and methods of administering the same to improve effectiveness of a SARS-CoV-2 vaccine.

Of course, mammals who are not aged may benefit from improved effectiveness of a vaccine. Thus, the active ingredients, compositions, and methods for improving effectiveness of a vaccine can be used with non-aged mammals.

In some cases, the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system. In other cases, the mammal has a healthy immune system.

In embodiments, the term dysfunctional immune system may be an overactive immune system, e.g., resulting in a cytokine storm; such overactive immune systems are observed in certain viral infections, e.g, in some severe coronavirus patients. In these cases, “improving a vaccine response”, relates to “improving” a proper immune response to a vaccine and, later, when a subject is contacted with an infectious agent. That is, minimizing an overactive immune response and promoting a proper immune response.

In embodiments, the increased immune response promotes future immunity against the component contained in the vaccine. In embodiments, the component contained in the vaccine is an antigen obtained from, related to, homologous to, or expressed by an infectious agent.

In embodiments, the vaccine is a Chickenpox vaccine, Coronavirus vaccine, Diphtheria vaccine, Hepatitis A vaccine, Hepatitis B vaccine, Haemophilus influenzae type b vaccine, Human Immunovirus (HIV) vaccine, Human papillomavirus vaccine, influenza vaccine, Japanese encephalitis vaccine, Measles, mumps, or rubella (including MMR combined vaccine) vaccine, Meningococcal disease vaccine, Pneumococcal disease vaccine, Polio vaccine, Rabies vaccine, Respiratory syncytial virus (RSV) vaccine, Rotavirus vaccine, Shingles vaccine, Smallpox vaccine, Tetanus vaccine, Varicella virus vaccine, Whooping cough (part of the DTaP combined vaccine) vaccine, or Yellow fever vaccine. In embodiments, the vaccine is a coronavirus vaccine, e.g., directed against Sars-CoV-2.

In embodiments, the ALDH modulator for improving effectiveness of a vaccine is an ALDH modulator or an ALDH agonist.

In embodiments, the ALDH modulator for improving effectiveness of a vaccine inhibits ALDH1, ALDHIAI, ALDH1A2, ALDH 1 A3, ALDH2, and/or ALDH3A1. In embodiments, the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4- Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, or ANS-66372. In embodiments, the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, or Daidzin. In embodiments, the ALDH modulator is disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

In embodiments, the ALDH agonist for improving effectiveness of a vaccine activates ALDH2. In embodiments, the ALDH agonist is Alda-1.

In embodiments, the gasdermin D inhibitor is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082. In embodiments, the gasdermin D inhibitor is disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

In embodiments, administering of the active ingredient is oral, by injection, inhalation, or topical. In embodiments, administering of the vaccine is oral, by injection, inhalation, or topical. In embodiments, the injection is intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

In certain embodiments, the active ingredient is disulfiram. The disulfiram is administered at a daily dosage of about 5 mg to about 500 mg per day. For example, disulfiram is administered at a total daily dosage of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg per day, and any total dosage therebetween. As examples, the daily dosage may be 5-10 mg, 10- 15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50-55 mg, 55- 60 mg, 60-65 mg, 65-70 mg, 70-75 mg, 75-80 mg, 80-85 mg, 85-90 mg, 90-95 mg, 95-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275- 300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 400-425 mg, 425-450 mg, 450-475 mg, or 475-500 mg. The daily dosage may be greater than 500 mg per day, e.g., 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,

640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg,

750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg,

860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,

970 mg, 980 mg, 990 mg, or 1000 mg. The daily dosage may be 0.5-1.0 g, 1.0-1.5 g, 1.5-2.0 g, 2.0-2.5 g, 2.5-3.0 g, 3.0-3.5 g, 3.5-4.0 g, 4.0-4.5 g, or 4.5-5.0 g. The disulfiram may be administered lx, 2x, or 3x per day to achieve the daily dosage. Thus, for a daily dose of 5 mg with a once per day administration, only a single administration of 5 mg will be given; for a daily dose of 5 mg with a twice per day administration, two administrations of about 2.5 mg will be given; and for a daily dose of 5 mg with a thrice per day administration, three administrations of about 1.7 mg will be given. Similarly, for a daily dose of 500 mg with a once per day administration, only a single administration of 500 mg will be given; for a daily dose of 500 mg with a twice per day administration, two administrations of about 250 mg will be given; and for a daily dose of 500 mg with a thrice per day administration, three administrations of about 170 mg will be given.

Reducing the predicted age of cell

Yet another aspect of the present disclosure is a method for reducing a predicted biological age of a cell. The method comprising contacting the cell with a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram).

The method for reducing a predicted biological age of a cell may comprises administering to the mammal a therapeutically effective amount of an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) and as potentiating ingredient (e.g, TBHQ).

The method for reducing a predicted biological age of a cell may further comprise contacting the cell with a metal (e.g, copper and zinc).

In embodiments, the ALDH modulator for reducing a predicted biological age of a cell is an ALDH modulator selected from Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, and ANS-66372.

In embodiments, the ALDH modulator for reducing a predicted biological age of a cell is an ALDH agonist that is Alda-1. In embodiments, the ALDH modulator is disulfiram itself and any of its metabolites and/or their derivatives and variants thereof. In embodiments, the gasdermin D inhibitor for reducing a predicted biological age of a cell is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082. In embodiments, the gasdermin D inhibitor is disulfiram itself and any of its metabolites and/or their derivatives and variants thereof.

In embodiments, the cell is in vitro, ex vivo, or in vivo.

Assays and formulations used in methods for reducing a predicted biological age of a cell may be related to those described in US20190228840, the entire contents of which is incorporated by reference its entirety.

In certain embodiments, the active ingredient is disulfiram. The disulfiram is administered at a daily dosage of about 5 mg to about 500 mg per day. For example, disulfiram is administered at a total daily dosage of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg per day, and any total dosage therebetween. As examples, the daily dosage may be 5-10 mg, 10- 15 mg, 15-20 mg, 20-25 mg, 25-30 mg, 30-35 mg, 35-40 mg, 40-45 mg, 45-50 mg, 50-55 mg, 55- 60 mg, 60-65 mg, 65-70 mg, 70-75 mg, 75-80 mg, 80-85 mg, 85-90 mg, 90-95 mg, 95-100 mg, 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, 200-225 mg, 225-250 mg, 250-275 mg, 275- 300 mg, 300-325 mg, 325-350 mg, 350-375 mg, 375-400 mg, 400-425 mg, 425-450 mg, 450-475 mg, or 475-500 mg. The daily dosage may be greater than 500 mg per day, e.g., 510 mg, 520 mg, 530 mg, 540 mg, 550 mg, 560 mg, 570 mg, 580 mg, 590 mg, 600 mg, 610 mg, 620 mg, 630 mg,

640 mg, 650 mg, 660 mg, 670 mg, 680 mg, 690 mg, 700 mg, 710 mg, 720 mg, 730 mg, 740 mg,

750 mg, 760 mg, 770 mg, 780 mg, 790 mg, 800 mg, 810 mg, 820 mg, 830 mg, 840 mg, 850 mg,

860 mg, 870 mg, 880 mg, 890 mg, 900 mg, 910 mg, 920 mg, 930 mg, 940 mg, 950 mg, 960 mg,

970 mg, 980 mg, 990 mg, or 1000 mg. The daily dosage may be 0.5-1.0 g, 1.0-1.5 g, 1.5-2.0 g, 2.0-2.5 g, 2.5-3.0 g, 3.0-3.5 g, 3.5-4.0 g, 4.0-4.5 g, or 4.5-5.0 g. The disulfiram may be administered lx, 2x, or 3x per day to achieve the daily dosage. Thus, for a daily dose of 5 mg with a once per day administration, only a single administration of 5 mg will be given; for a daily dose of 5 mg with a twice per day administration, two administrations of about 2.5 mg will be given; and for a daily dose of 5 mg with a thrice per day administration, three administrations of about 1.7 mg will be given. Similarly, for a daily dose of 500 mg with a once per day administration, only a single administration of 500 mg will be given; for a daily dose of 500 mg with a twice per day administration, two administrations of about 250 mg will be given; and for a daily dose of 500 mg with a thrice per day administration, three administrations of about 170 mg will be given. Aging-related disorders

The herein-disclosed active ingredients, compositions, and methods treat, prevent, reduce the severity of, and/or delay the onset of various aging-related disorders, e.g., chronic diseases and disabilities/conditions of aging. Illustrative aging-related disorders include actinic keratosis, age- related macular degeneration (AMD), Alzheimer’s disease, arthritis, atherosclerosis and cardiovascular disease, benign prostatic hyperplasia (BPH), bone atrophy, cachexia, cancer, cardiomyopathy, cataracts, chronic obstructive pulmonary disease (COPD), constipation, decrease in overall energy, decrease in visual acuity, delirium, dementia, depression, dermal atrophy (thinning of the skin), diminished peripheral vision, greater risk of heat stroke or hypothermia, hearing loss, hypertension, increased susceptibility to infection (including influenza and pneumonia), lentigines (aging spots), liver conditions (e.g, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and cirrhosis), memory loss, metabolic syndrome, muscle atrophy (e.g, Sarcopenia and myopenia), frailty, muscle repair or rejuvenation deficiency, muscular dystrophy, osteoarthritis, osteoporosis, periodontitis, photoaging, reduced metabolism (including increased risk for obesity), reduced reflexes and coordination including difficulty with balance, respiratory disease (including acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI) and pulmonary fibrosis (e.g, idiopathic pulmonary fibrosis)), rheumatoid arthritis, sarcopenic obesity, sexual dysfunction, shingles, type 2 diabetes, urologic changes (including incontinence), vaginal atrophy, whitening or graying of hair, wrinkling/sagging skin (including loss of skin elasticity), and xerosis cutis (skin dryness). Aged non-human subjects experience similar, homologous, and/or equivalent aging-related disorders.

Without wishing to be bound by theory, an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) mitigates dysfunction of or rejuvenates a signaling pathway disrupted by aging where the dysfunction can ultimately lead to aging-related disorders. Moreover, the combination of the active ingredient of the present disclosure (e.g, disulfiram) and a potentiating ingredient (e.g, TBHQ) mitigates dysfunction of or rejuvenates a signaling pathway disrupted by aging where the dysfunction can ultimately lead to aging-related disorders.

Subjects

In embodiments, the subject is a mammal, e.g, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the mammal is a non-rodent. In embodiments, the mammal is a dog. In embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet, e.g., a dog. In another specific embodiment, the non-human animal is a livestock animal. In embodiments, the mammal is a human.

In embodiments, the mammal has reached maturity. As used herein, the term mature or maturity, and the like, refers to a mammal that is capable of sexual reproduction and/or a mammal that has achieved its adult height and/or length.

In embodiments, the mammal is nearing or has reached halfway to its expected lifespan for the mammal’s species, size, sex, age, and/or health status. The mammal may have reached an age that is at least 60%, 70%, 80%, 90%, or 100% of its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

In embodiments, the human is an adult human. In embodiments, the human has an age in a range of from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old, or older.

In some cases, the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system. In other cases, the mammal has a healthy immune system.

DEFINITIONS

The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting.

As used herein, unless otherwise indicated, the terms “a”, “an” and “the” are intended to include the plural forms as well as the single forms, unless the context clearly indicates otherwise.

The terms “comprise”, “comprising”, “contain,” “containing,” “including”, “includes”, “having”, “has”, “with”, or variants thereof as used in either the present disclosure and/or in the claims, are intended to be inclusive in a manner similar to the term “comprising.”

By preventing is meant, at least, avoiding the occurrence of a disease and/or reducing the likelihood of acquiring the disease; the term “preventing” and the like includes limiting the progression of the disease. By treating is meant, at least, ameliorating or avoiding the effects of a disease, including reducing a sign or symptom of the disease; the term “treating” and the like includes limiting the progression of the disease.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean 10% greater than or less than the stated value. In another example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.

By “ALDH modulator” is meant an agent, drug, chemical, or compound, capable of increasing ALDH activity or by reducing ALDH activity. ALDH modulators that reduce ALDH activity may be identified as ALDH modulators and ALDH modulators that increase ALDH activity may be identified as ALDH agonists.

By “gasdermin D inhibitor” is meant an agent, drug, chemical, or compound, capable of reducing gasdermin D activity.

Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

EMBODIMENTS

Embodiment Al. A method for increasing lifespan in a mammal, for preventing or treating an aging-related disorder in a mammal, for reducing a symptom of aging in a mammal, and/or boosting an immune system in a mammal comprising: administering to the mammal a composition comprising a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator.

Embodiment A2. The method of Embodiment Al, wherein the ALDH modulator is an ALDH modulator or an ALDH agonist.

Embodiment A3. The method of Embodiment A2, wherein the ALDH modulator inhibits ALDHl, ALDHIAI, ALDH1A2, ALDHl A3, ALDH2, and/or ALDH3A1.

Embodiment A4. The method of Embodiment A2 or Embodiment A3, wherein the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4- Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, or ANS-66372.

Embodiment A5. The method of Embodiment A4, wherein the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, or Daidzin.

Embodiment A6. The method of Embodiment A2, wherein the ALDH agonist activates ALDH2.

Embodiment A7. The method of Embodiment A2 or Embodiment A6, wherein the ALDH agonist is Alda-1.

Embodiment A8. The method of any one of Embodiments A1 to A7, wherein the administering is oral, by injection, inhalation, or topical.

Embodiment A9. The method of Embodiment A8, wherein the injection is intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

Embodiment A10. The method of any one of Embodiments A1 to A9, wherein the mammal is near or has reached maturity.

Embodiment Al l. The method of any one of Embodiments A1 to A10, wherein the mammal is nearing or has reached halfway to its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

Embodiment A12. The method of Embodiment Al l, wherein the mammal has reached an age that is at least 60%, 70%, 80%, 90%, or 100% of its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

Embodiment A13. The method of any one of Embodiments A1 to A12, wherein the mammal is a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.

Embodiment A14. The method of Embodiment A13, wherein the mammal is a human.

Embodiment A15. The method of any one of Embodiments A1 to A14, wherein increasing lifespan comprises an at least 5% increase in lifespan relative to the expected or median lifespan of a mammal of similar species, sex, age, and/or health status.

Embodiment A16. The method of Embodiment A15, wherein increasing lifespan comprises an at least 10%, at least 15%, at least 20%, or at least 25% increase in lifespan.

Embodiment A17. The method of any one of Embodiments A1 to A16, wherein the aging-related disorder or symptom of aging selected from one or more of actinic keratosis, age-related macular degeneration (AMD), Alzheimer’s disease, arthritis, atherosclerosis and cardiovascular disease, benign prostatic hyperplasia (BPH), bone atrophy, cachexia, cancer, cardiomyopathy, cataracts, chronic obstructive pulmonary disease (COPD), constipation, decrease in overall energy, decrease in visual acuity, delirium, dementia, depression, dermal atrophy (thinning of the skin), diminished peripheral vision, greater risk of heat stroke or hypothermia, hearing loss, hypertension, increased susceptibility to infection (including influenza and pneumonia), lentigines (aging spots), liver conditions ( e.g ., nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and cirrhosis), memory loss, metabolic syndrome, muscle atrophy (e.g., Sarcopenia and myopenia), frailty, muscle repair or rejuvenation deficiency, muscular dystrophy, osteoarthritis, osteoporosis, periodontitis, photoaging, reduced metabolism (including increased risk for obesity), reduced reflexes and coordination including difficulty with balance, respiratory disease (including acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI) and pulmonary fibrosis (e.g, idiopathic pulmonary fibrosis)), rheumatoid arthritis, sarcopenic obesity, sexual dysfunction, shingles, type 2 diabetes, urologic changes (including incontinence), vaginal atrophy, whitening or graying of hair, prolonged/inefficient wound healing, wrinkling/sagging skin (including loss of skin elasticity), and xerosis cutis (skin dryness).

Embodiment A18. The method of Embodiment A17, wherein the aging-related disorder or symptom of aging is actinic keratosis, dermal atrophy (thinning of the skin), lentigines (aging spots), photoaging, vaginal atrophy, prolonged/inefficient wound healing, wrinkles, and/or xerosis cutis (skin dryness) and wherein the administering is oral or topical.

Embodiment A19. The method of Embodiment A17 or Embodiment A18, wherein the mammal has at least one aging-related disorder or symptom of aging.

Embodiment A20. The method of any one of Embodiments A1 to A19, wherein the therapeutically effective amount of the ALDH modulator boosts the immune system in the mammal.

Embodiment A2T The method of Embodiment A20, wherein boosting the immune system increases an effective immune response against an infectious agent.

Embodiment A22. The method of Embodiment A21, wherein the infectious agent is a virus, a bacterium, a fungus, a protozoan, a helminth, a prion, or a parasite.

Embodiment A23. The method of Embodiment A22, wherein the bacterium is Bordatella pertussis or Streptococcus pneumoniae or the virus is a Chickenpox virus, Coronavirus, Hepatitis A virus, Hepatitis B virus, Human papillomavirus, Human immunodeficiency virus (HIV), influenza, Japanese encephalitis virus, Measles, mumps, or rubella virus, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rotavirus, Shingles virus, Smallpox, Varicella virus, or Yellow fever virus.

Embodiment A24. The method of Embodiment A23, wherein the Coronavirus is Sars-CoV-2. Embodiment A25. The method of any one of Embodiments A20 to A24, wherein the administering is oral or by inhalation.

Embodiment A26. The method of any one of Embodiments A21 to A25, wherein the infectious agent affects the mammal’s respiratory system or is transmitted via the mammal’s respiratory system and wherein the administering is by inhalation.

Embodiment A27. The method of any one of Embodiments A1 to A26, wherein the mammal has a healthy immune system.

Embodiment A28. The method of any one of Embodiments A1 to A27, wherein the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

Embodiment A29. A method for improving effectiveness of a vaccine in a mammal in need thereof comprising administering a composition comprising a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator, wherein the mammal contemporaneously and/or subsequently will be administered a vaccine.

Embodiment A30. The method of Embodiment A29, wherein the ALDH modulator and the vaccine are administered contemporaneously.

Embodiment A31. The method of Embodiment A29 or Embodiment A30, wherein the vaccine is administered subsequent to the ALDH modulator’s administration.

Embodiment A32. The method of any one of Embodiments A29 to A31, wherein the therapeutically effective amount the ALDH modulator boosts the immune system in the mammal.

Embodiment A33. The method of Embodiment A32, wherein boosting the immune system increases an immune response against a component contained in the vaccine.

Embodiment A34. The method of Embodiment A33, wherein the increased immune response promotes future immunity against the component contained in the vaccine.

Embodiment A35. The method of Embodiment A34, wherein the component contained in the vaccine is an antigen obtained from, related to, homologous to, or expressed by an infectious agent.

Embodiment A36. The method of any one of Embodiments A29 to A35, wherein the vaccine is a Chickenpox vaccine, Coronavirus vaccine, Diphtheria vaccine, Hepatitis A vaccine, Hepatitis B vaccine, Haemophilus influenzae type b vaccine, Human Immunovirus (HIV) vaccine, Human papillomavirus vaccine, influenza vaccine, Japanese encephalitis vaccine, Measles, mumps, or rubella (including MMR combined vaccine) vaccine, Meningococcal disease vaccine, Pneumococcal disease vaccine, Polio vaccine, Rabies vaccine, Respiratory syncytial virus (RSV) vaccine, Rotavirus vaccine, Shingles vaccine, Smallpox vaccine, Tetanus vaccine, Varicella virus vaccine, Whooping cough (part of the DTaP combined vaccine) vaccine, or Yellow fever vaccine.

Embodiment A37. The method of Embodiment A36, wherein the vaccine is a coronavirus vaccine.

Embodiment A38. The method of Embodiment A37, wherein the coronavirus vaccine is directed against Sars-CoV-2.

Embodiment A39. The method of any one of Embodiments A29 to A38, wherein the mammal has a healthy immune system.

Embodiment A40. The method of any one of Embodiments A29 to A38, wherein the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

Embodiment A41. The method of any one of Embodiments A29 to A40, wherein the ALDH modulator is an ALDH modulator or an ALDH agonist.

Embodiment A42. The method of Embodiment A41, wherein the ALDH modulator inhibits ALDHl, ALDHIAI, ALDH1A2, ALDH 1 A3, ALDH2, and/or ALDH3A1.

Embodiment A43. The method of Embodiment A41 or Embodiment A42, wherein the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4- Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, or ANS-66372.

Embodiment A44. The method of Embodiment A43, wherein the ALDH modulator is Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, or Daidzin.

Embodiment A45. The method of Embodiment A41, wherein the ALDH agonist activates ALDH2.

Embodiment A46. The method of Embodiment A41 or Embodiment A45, wherein the ALDH agonist is Alda- 1.

Embodiment A47. The method of any one of Embodiments A29 to A46, wherein the administering of the ALDH modulator is oral, by injection, inhalation, or topical.

Embodiment A48. The method of any one of Embodiments A29 to A47, wherein the administering of the vaccine is oral, by injection, inhalation, or topical.

Embodiment A49. The method of Embodiment A47 or Embodiment A48, wherein the injection is intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

Embodiment A50. The method of any one of Embodiments A1 to A49 further comprising administering to the mammal a composition comprising a therapeutically effective amount of a metal.

Embodiment A51. The method of Embodiment A50, wherein the composition comprising a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator and the composition comprising a therapeutically effective amount of a metal are administered contemporaneously .

Embodiment A52. The method of Embodiment A50, wherein the composition comprising a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator is administered before the composition comprising a therapeutically effective amount of a metal is administered.

Embodiment A53. The method of Embodiment A50, wherein the composition comprising a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator is administered after the composition comprising a therapeutically effective amount of a metal is administered.

Embodiment A54. The method of any one of Embodiments A50 to A53, wherein the metal is copper, copper (II), zinc, arsenic, titanium, bismuth, vanadium, chromium, molybdenum, manganese, iron, ruthenium, osmium, cobalt, nickel, platinum, silver, gold, gold (II), cadmium, or mercury.

Embodiment A55. The method of Embodiment A54, wherein the metal is copper which is in the form of copper or copper (II) gluconate, copper chloride, copper glycinate, copper carbonate, copper hydroxide, copper orthophosphate, copper oxide, copper pyrophosphate, copper sulfate, or (cuprous) iodide.

Embodiment A56. The method of Embodiment A54, wherein the metal is zinc which is in the form of zinc or zinc (II) acetate, zinc carbonate, zinc chloride, zinc oxide, zinc sulfate, zinc methionine sulfate, or zinc stearate.

Embodiment A57. The method of any one of Embodiments A50 to A56, wherein the effective amount of the metal is between about 0.1 mg to about 30 mg.

Embodiment A58. The method of Embodiment A57, wherein the effective amount of the metal is between about 1.5 mg and 3 mg.

Embodiment A59. The method of Embodiment A58, wherein the metal is copper or zinc and approximately 1.5 mg of the metal is administered per dose.

Embodiment A60. A method for reducing a predicted biological age of a cell comprising contacting the cell with a therapeutically effective amount of an aldehyde dehydrogenase (ALDH) modulator.

Embodiment A61. The method of Embodiment A60, wherein the ALDH modulator is an ALDH modulator selected from Disulfiram, NCT-501, 4-Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3-Hydroxybenzaldehyde, and ANS-66372.

Embodiment A62. The method of Embodiment A60, wherein the ALDH modulator is an ALDH agonist that is Alda-1. Embodiment A63. The method of any one of Embodiments A60 to A62, wherein the cell is in vitro, ex vivo, or in vivo.

Embodiment A64. A composition comprising an ALDH modulator for use in the method of any one of Embodiments A1 to A63.

Embodiment B 1. A method for increasing lifespan in a mammal, for preventing or treating disease including an aging-related disorder in a mammal, for reducing a symptom of aging in a mammal, and/or boosting an immune system in a mammal comprising: administering to the mammal a composition comprising a therapeutically effective amount of a gasdermin D inhibitor.

Embodiment B2. An in vivo, in vitro, or ex vivo method for increasing lifespan of a cell and/or boosting activity of an immune cell comprising contacting the cell or the immune cell with a gasdermin D inhibitor.

Embodiment B3. The method of Embodiment B1 or Embodiment B2, wherein the gasdermin D inhibitor is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082.

Embodiment B4. The method of Embodiment B3, wherein the gasdermin D inhibitor is disulfiram. Embodiment B5. The method of any one of Embodiments B1 to B4, wherein the administering is oral, by injection, inhalation, or topical.

Embodiment B6. The method of Embodiment B5, wherein the injection is intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

Embodiment B7. The method of any one of Embodiments B1 to B6, wherein the mammal is near or has reached maturity.

Embodiment B8. The method of any one of Embodiments B1 to B7, wherein the mammal is nearing or has reached halfway to its expected lifespan for the mammal’s species, size, sex, age, and/or health status. Embodiment B9. The method of Embodiment B8, wherein the mammal has reached an age that is at least 60%, 70%, 80%, 90%, or 100% of its expected lifespan for the mammal’s species, size, sex, age, and/or health status.

Embodiment B10. The method of any one of Embodiments B1 to B9, wherein the mammal is a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.

Embodiment B11. The method of Embodiment B10, wherein the mammal is a human.

Embodiment B 12. The method of any one of Embodiments B1 toBl 1, wherein increasing lifespan comprises an at least 5% increase in lifespan relative to the expected or median lifespan of a mammal of similar species, sex, age, and/or health status.

Embodiment B 13. The method of Embodiment B12, wherein increasing lifespan comprises an at least 10%, at least 15%, at least 20%, or at least 25% increase in lifespan.

Embodiment B 14. The method of any one of Embodiments B1 to B13, wherein the aging-related disorder or symptom of aging selected from one or more of actinic keratosis, age-related macular degeneration (AMD), Alzheimer’s disease, arthritis, atherosclerosis and cardiovascular disease, benign prostatic hyperplasia (BPH), bone atrophy, cachexia, cancer, cardiomyopathy, cataracts, chronic obstructive pulmonary disease (COPD), constipation, decrease in overall energy, decrease in visual acuity, delirium, dementia, depression, dermal atrophy (thinning of the skin), diminished peripheral vision, greater risk of heat stroke or hypothermia, hearing loss, hypertension, increased susceptibility to infection (including influenza and pneumonia), lentigines (aging spots), liver conditions ( e.g ., nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and cirrhosis), memory loss, metabolic syndrome, muscle atrophy (e.g., Sarcopenia and myopenia), frailty, muscle repair or rejuvenation deficiency, muscular dystrophy, osteoarthritis, osteoporosis, periodontitis, photoaging, reduced metabolism (including increased risk for obesity), reduced reflexes and coordination including difficulty with balance, respiratory disease (including acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI) and pulmonary fibrosis (e.g, idiopathic pulmonary fibrosis)), rheumatoid arthritis, sarcopenic obesity, sexual dysfunction, shingles, type 2 diabetes, urologic changes (including incontinence), vaginal atrophy, whitening or graying of hair, prolonged/inefficient wound healing, wrinkling/sagging skin (including loss of skin elasticity), and xerosis cutis (skin dryness); or the disease includes asthma, deafness, or a viral infections and/or a symptom of the disease comprises sepsis. Embodiment B15. The method of Embodiment B14, wherein the aging-related disorder or symptom of aging is actinic keratosis, dermal atrophy (thinning of the skin), lentigines (aging spots), photoaging, vaginal atrophy, prolonged/inefficient wound healing, wrinkles, and/or xerosis cutis (skin dryness) and wherein the administering is oral or topical.

Embodiment B16. The method of Embodiment B14 or Embodiment B15, wherein the mammal has at least one aging-related disorder or symptom of aging.

Embodiment B 17. The method of any one of Embodiments B1 to B16, wherein the therapeutically effective amount of the gasdermin D inhibitor boosts the immune system in the mammal.

Embodiment B18. The method of Embodiment B17, wherein boosting the immune system increases an effective immune response against an infectious agent.

Embodiment B19. The method of Embodiment B18, wherein the infectious agent is a virus, a bacterium, a fungus, a protozoan, a helminth, a prion, or a parasite.

Embodiment B20. The method of Embodiment B 19, wherein the bacterium is Bordatella pertussis or Streptococcus pneumoniae or the virus is a Chickenpox virus, Coronavirus, Hepatitis A virus, Hepatitis B virus, Human papillomavirus, Human immunodeficiency virus (HIV), influenza, Japanese encephalitis virus, Measles, mumps, or rubella virus, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rotavirus, Shingles virus, Smallpox, Varicella virus, or Yellow fever virus.

Embodiment B21. The method of Embodiment B20, wherein the Coronavirus is Sars-CoV-2.

Embodiment B22. The method of any one of Embodiments B5 to B21, wherein the administering is oral or by inhalation.

Embodiment B23. The method of any one of Embodiments B18 to B22, wherein the infectious agent affects the mammal’s respiratory system or is transmitted via the mammal’s respiratory system and wherein the administering is by inhalation.

Embodiment B24. The method of any one of Embodiments B1 to B23, wherein the mammal has a healthy immune system.

Embodiment B25. The method of any one of Embodiments B1 to B24, wherein the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

Embodiment B26. A method for improving effectiveness of a vaccine in a mammal in need thereof comprising administering a composition comprising a therapeutically effective amount of a gasdermin D inhibitor, wherein the mammal contemporaneously and/or subsequently will be administered a vaccine.

Embodiment B27. The method of Embodiment B26, wherein the gasdermin D inhibitor and the vaccine are administered contemporaneously.

Embodiment B28. The method of Embodiment B26 or Embodiment B27, wherein the vaccine is administered subsequent to the gasdermin D inhibitor’s administration.

Embodiment B29. The method of any one of Embodiments B26 to B28, wherein the therapeutically effective amount the gasdermin D inhibitor boosts the immune system in the mammal.

Embodiment B30. The method of Embodiment B29, wherein boosting the immune system increases an immune response against a component contained in the vaccine.

Embodiment B31. The method of Embodiment B30, wherein the increased immune response promotes future immunity against the component contained in the vaccine.

Embodiment B32. The method of Embodiment B31, wherein the component contained in the vaccine is an antigen obtained from, related to, homologous to, or expressed by an infectious agent.

Embodiment B33. The method of any one of Embodiments B26 to B32, wherein the vaccine is a Chickenpox vaccine, Coronavirus vaccine, Diphtheria vaccine, Hepatitis A vaccine, Hepatitis B vaccine, Haemophilus influenzae type b vaccine, Human Immunovirus (HIV) vaccine, Human papillomavirus vaccine, influenza vaccine, Japanese encephalitis vaccine, Measles, mumps, or rubella (including MMR combined vaccine) vaccine, Meningococcal disease vaccine, Pneumococcal disease vaccine, Polio vaccine, Rabies vaccine, Respiratory syncytial virus (RSV) vaccine, Rotavirus vaccine, Shingles vaccine, Smallpox vaccine, Tetanus vaccine, Varicella virus vaccine, Whooping cough (part of the DTaP combined vaccine) vaccine, or Yellow fever vaccine.

Embodiment B34. The method of Embodiment B33, wherein the vaccine is a coronavirus vaccine.

Embodiment B35. The method of Embodiment B34, wherein the coronavirus vaccine is directed against Sars-CoV-2.

Embodiment B36. The method of any one of Embodiments B26 to B35, wherein the mammal has a healthy immune system.

Embodiment B37. The method of any one of Embodiments B26 to B35, wherein the mammal has an unhealthy immune system, dysfunctional immune system, and/or weakened immune system. Embodiment B38. The method of Embodiment B37, wherein the gasdermin D inhibitor is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082.

Embodiment B39. The method of Embodiment B38, wherein the gasdermin D inhibitor is disulfiram.

Embodiment B40. The method of any one of Embodiments B26 to B39, wherein the administering of the gasdermin D inhibitor is oral, by injection, inhalation, or topical.

Embodiment B41. The method of any one of Embodiments B26 to B40, wherein the administering of the vaccine is oral, by injection, inhalation, or topical.

Embodiment B42. The method of Embodiment B40 or Embodiment B41, wherein the injection is intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection.

Embodiment B43. The method of any one of Embodiments B1 to B42 further comprising administering to the mammal a composition comprising a therapeutically effective amount of a metal.

Embodiment B44. The method of Embodiment B43, wherein the composition comprising a therapeutically effective amount of a gasdermin D inhibitor and the composition comprising a therapeutically effective amount of a metal are administered contemporaneously.

Embodiment B45. The method of Embodiment B43, wherein the composition comprising a therapeutically effective amount of a gasdermin D inhibitor is administered before the composition comprising a therapeutically effective amount of a metal is administered.

Embodiment B46. The method of Embodiment B43, wherein the composition comprising a therapeutically effective amount of a gasdermin D inhibitor is administered after the composition comprising a therapeutically effective amount of a metal is administered.

Embodiment B47. The method of any one of Embodiments B43 to B46, wherein the metal is copper, copper (II), zinc, arsenic, titanium, bismuth, vanadium, chromium, molybdenum, manganese, iron, ruthenium, osmium, cobalt, nickel, platinum, silver, gold, gold (II), cadmium, or mercury.

Embodiment B48. The method of Embodiment B47, wherein the metal is copper which is in the form of copper or copper (II) gluconate, copper chloride, copper glycinate, copper carbonate, copper hydroxide, copper orthophosphate, copper oxide, copper pyrophosphate, copper sulfate, or (cuprous) iodide.

Embodiment B49. The method of Embodiment B47, wherein the metal is zinc which is in the form of zinc or zinc (II) acetate, zinc carbonate, zinc chloride, zinc oxide, zinc sulfate, zinc methionine sulfate, or zinc stearate.

Embodiment B50. The method of any one of Embodiments B43 to B49, wherein the effective amount of the metal is between about 0.1 mg to about 30 mg.

Embodiment B51. The method of Embodiment B50, wherein the effective amount of the metal is between about 1.5 mg and 3 mg.

Embodiment B52. The method of Embodiment B51, wherein the metal is copper or zinc and approximately 1.5 mg of the metal is administered per dose.

Embodiment B53. A method for reducing a predicted biological age of a cell comprising contacting the cell with a therapeutically effective amount of a gasdermin D inhibitor.

Embodiment B54. The method of Embodiment B53, wherein the gasdermin D inhibitor is disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082.

Embodiment B55. The method of Embodiment B53 or Embodiment B54, wherein the cell is in vitro, ex vivo, or in vivo.

Embodiment B56. A composition comprising a gasdermin D inhibitor for use in the method of any one of Embodiments B1 to B55.

Embodiment C 1. A method for inhibiting and/or reducing pyroptotic cell death in a cell, the method comprising contacting the cell with an active agent that is disulfiram and a potentiating ingredient that is tert-Butylhydroquinone (TBHQ), thereby inhibiting and/or reducing pyroptotic cell death.

Embodiment C2. The method of Embodiment Cl, wherein disulfiram and TBHQ are included in one composition and the composition consists essentially of disulfiram and TBHQ.

Embodiment C3. The method of Embodiment Cl, wherein the method comprises contacting the cell with a first composition consisting essentially of disulfiram and contacting the cell with a second composition consisting essentially TBHQ.

Embodiment C4. The method of any one of Embodiments Cl to C3, wherein inhibiting and/or reducing pyroptotic cell death in the cell increases the lifespan of the cell. Embodiment C5. The method of any one of Embodiments Cl to C4, wherein the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is from about 0.02% to about 56% by weight of disulfiram.

Embodiment C6. The method of any one of Embodiments Cl to C5, wherein the TBHQ further potentiates disulfiram’ s ability to treat acute lung injury (ALI), acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI), pulmonary fibrosis ( e.g idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, dry eye, actinic keratosis, alopecia, and/or skin cancer.

Embodiment C7. The method of any one of Embodiments Cl to C6, wherein the TBHQ further potentiates disulfiram’ s ability to inhibit and/or reduce a pathological inflammatory response, alter a T-cell’s age, and/or alter mitochondrial function in the cell.

Embodiment C8. A method for boosting activity of an immune cell, the method comprising contacting the immune cell with an active agent that is disulfiram and a potentiating ingredient that is tert-Butylhydroquinone (TBHQ), thereby boosting activity of an immune cell.

Embodiment C9. The method of Embodiment C8, wherein disulfiram and TBHQ are included in one composition and the composition consists essentially of disulfiram and TBHQ.

Embodiment CIO. The method of Embodiment C8, wherein the method comprises contacting the cell with a first composition consisting essentially of disulfiram and contacting the cell with an at least second composition consisting essentially TBHQ.

Embodiment Cl 1. The method of any one of Embodiments C8 to CIO, wherein boosting activity of an immune cell increases an effective immune response against an infectious agent and/or an atypical cell.

Embodiment Cl 2. The method of any one of Embodiments C8 to Cl l, wherein boosting activity of an immune cell improves the immune cell’s response against a component contained in a vaccine, wherein the component contained in the vaccine is an antigen obtained from, related to, homologous to, or expressed by an infectious agent.

Embodiment C13. The method of any one of Embodiments C8 to C12, wherein boosting activity of an immune cell comprises inhibiting a pathological immune response.

Embodiment Cl 4. The method of any one of Embodiments C8 to C13, wherein boosting activity of an immune cell minimizes overactive immune cell activity. Embodiment C15. The method of any one of Embodiments C8 to C14, wherein the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is from about 0.02% to about 56% of the weight of the disulfiram.

Embodiment Cl 6. A composition consisting essentially of an active agent that is disulfiram and a potentiating ingredient that is tert-Butylhydroquinone (TBHQ), wherein the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is from about 0.02% to about 56% of the of the disulfiram.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Identification of ALDH modulators useful in methods of the present disclosure In this example, aldehyde dehydrogenase (ALDH) modulators capable of increasing lifespan in a mammal, of preventing or treating an aging-related disorder in a mammal, of reducing a symptom of aging in a mammal, and/or of boosting an immune system were identified.

Sets of cultured cells — fibroblasts, peripheral blood mononuclear cells (PBMCs including lymphocytes and monocytes, and/or myoblasts — having either characteristics of young cells or characteristics of old cells are contacted with known ALDH modulators, either ALDH modulators or ALDH agonists. The ability of the ALDH modulator to reverse aging in the cells, e.g., reducing the characteristics of old cells and promoting characteristics of a young cells was assayed (also known as reducing the predicted age of the cells). ALDH modulators were used at various concentrations ranging from 0.000005 to 20 mM. ALDH modulator showing the ability to reverse aging were further validated.

The following ALDH modulators were tested: Disulfiram, NCT-501, 4- Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, and 3-Hydroxybenzaldehyde. Among which, at least, Disulfiram, NCT-501, 4- Diethylaminobenzaldehyde, and Daidzin were shown to have the ability to reverse aging. Disulfiram provided the greatest ability in these assays. Alda-1, an ALDH agonist, was tested and was shown to have the ability to reverse aging.

Assays and formulations used in this example are related to those described in US20190228840, the entire contents of which is incorporated by reference its entirety.

Example 2: Methods comprising administering a composition comprising an ALDH modulator In this example, compositions comprising an ALDH modulator is administered to a mammal for increasing lifespan, for preventing or treating an aging-related disorder, for reducing a symptom of aging, and/or boosting an immune system ( e.g ., for treating an infection).

The compositions comprise one of the following ALDH modulators: Disulfiram, NCT-501, 4- Diethylaminobenzaldehyde, Daidzin, CM10, EN40, 4-Hydroxynonenal, RV01, CVT-10216, 3- Hydroxybenzaldehyde, ANS-66372, or Alda-1.

Administration of the composition is by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the composition is administered orally, by inhalation, or topically.

The mammal’s lifespan is measured and the presence, absence, and/or severity of various aging- associated conditions are determined; these are compared to control mammals and/or to historical controls to determine the effectiveness of the composition administered.

The mammal may be aged or not aged.

The mammal may have a healthy immune system or the mammal may have an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

Assays and formulations used in this example are related to those described in US20190228840, the entire contents of which is incorporated by reference its entirety.

Example 3: Identification of gasdermin D inhibitor useful in methods of the present disclosure In this example, gasdermin D inhibitors capable of increasing lifespan in a mammal, of preventing or treating a disease including an aging-related disorder in a mammal, of reducing a symptom of aging in a mammal, and/or of boosting an immune system were identified.

Sets of cultured cells — fibroblasts, peripheral blood mononuclear cells (PBMCs including lymphocytes and monocytes, and/or myoblasts — having either characteristics of young cells or characteristics of old cells are contacted with known gasdermin D inhibitor. The ability of the gasdermin D inhibitor to reverse aging in the cells, e.g., reducing the characteristics of old cells and promoting characteristics of a young cells was assayed (also known as reducing the predicted age of the cells). Gasdermin D inhibitors were used at various concentrations ranging from 0.000005 to 20 mM. Gasdermin D inhibitors showing the ability to reverse aging were further validated.

The following gasdermin D inhibitors were tested: disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, and BAY 11-7082. Among which, at least, disulfiram, aloe emodin, 2-iodomelatonin, and LDC7559 were shown to have the ability to reverse aging. Disulfiram provided the greatest ability in these assays.

Assays and formulations used in this example are related to those described in US20190228840, the entire contents of which is incorporated by reference its entirety.

Example 4: Methods comprising administering a composition comprising a gasdermin D inhibitor

In this example, compositions comprising a gasdermin D inhibitor is administered to a mammal for increasing lifespan, for preventing or treating a disease including an aging-related disorder, for reducing a symptom of aging, and/or boosting an immune system ( e.g for treating an infection).

The compositions comprise one of the following gasdermin D inhibitors: disulfiram, aloe emodin, 2-iodomelatonin, LDC7559, necrosulfonamide, tasimelteon, ramelteon, melatonin, emodin, or BAY 11-7082.

Administration of the composition is by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the composition is administered orally, by inhalation, or topically.

The mammal’s lifespan is measured and the presence, absence, and/or severity of various aging- related disorders are determined; these are compared to control mammals and/or to historical controls to determine the effectiveness of the composition administered.

The mammal may be aged or not aged.

The mammal may have a healthy immune system or the mammal may have an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

Illustrative diseases treated in this example may be asthma, deafness, or a viral infections and an illustrative symptom thereof may be sepsis. Assays and formulations used in this example are related to those described in US20190228840, the entire contents of which is incorporated by reference its entirety.

Example 5: Methods for improving a vaccine response

In this example, a composition comprising an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) is administered to a mammal for improving effectiveness of a vaccine that is administered to the mammal.

The composition comprising the active ingredient may be administered by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the composition may be administered orally, by inhalation, or topically.

Administration of the vaccine may be by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the vaccine may be administered orally, by inhalation, or topically.

In some cases, the composition comprising the active ingredient and the vaccine are administered contemporaneously. In other cases, the vaccine is administered subsequent to the administration of the composition. In some cases, the vaccine is administered before the administration of the composition comprising the active ingredient. A subject may be administered vaccines and/or compositions comprising the active ingredient multiple times and in any order.

The vaccine may be a Chickenpox vaccine, Coronavirus vaccine, Diphtheria vaccine, Hepatitis A vaccine, Hepatitis B vaccine, Haemophilus influenzae type b vaccine, Human immunodeficiency virus (HIV) vaccine, Human papillomavirus vaccine, influenza vaccine, Japanese encephalitis vaccine, Measles, mumps, or rubella (including MMR combined vaccine) vaccine, Meningococcal disease vaccine, Pneumococcal disease vaccine, Polio vaccine, Rabies vaccine, Respiratory syncytial virus (RSV) vaccine, Rotavirus vaccine, Shingles vaccine, Smallpox vaccine, Tetanus vaccine, Varicella virus vaccine, Whooping cough (part of the DTaP combined vaccine) vaccine, or Yellow fever vaccine. In embodiments, the vaccine is a coronavirus vaccine. In embodiments, the coronavirus vaccine is directed against Sars-CoV-2.

The mammal may be aged or not aged.

The mammal may have a healthy immune system or the mammal may have an unhealthy immune system, dysfunctional immune system, and/or weakened immune system. The mammal’s ability to fend off a subsequent infection is determined and compared to mammals and/or historical controls who were only administered the vaccine.

The mammal’s ability to later produce antibodies directed to an infectious agent (related to the vaccine) is determined and compared to mammals and/or historical controls who were only administered the vaccine.

Example 6: Methods for treating skin disorders

In this example, a composition comprising an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) is administered to a mammal for improving treating a skin disorder.

The composition comprising the active ingredient may be administered orally or topically, with a dose depending on the quantity of composition needing to be administered. Alternately, the composition may be administered by injection or by inhalation.

The mammal has a skin disorder, e.g, wrinkles, which may be a result of photoaging or related to actinic keratosis. Other skin disorders the mammal may have includes dermal atrophy (thinning of the skin), lentigines (aging spots), vaginal atrophy, prolonged/inefficient wound healing, and/or xerosis cutis (skin dryness). In examples, the mammal has moderate skin aging (i.e., Glogau Classification III).

The composition’s or compositions’ ability to treat a skin disorder, e.g, wrinkles, is determined and compared to mammals and/or historical controls who were not administered the composition or compositions. For example, the determination relates to a change in the Glogau Classification.

Example 7: Illustrative assays

In this example, an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) was shown to modify quantifiable parameters in virally- infected cells from older donors that reflects those observed in cells from younger donor.

Whole blood from younger and older donors was collected into EDTA tubes and then diluted with an equal volume of phosphate-buffered saline (PBS) + 2% fetal bovine serum (FBS) and layered over Ficoll using SepMate™-50 tubes (STEMCELL Technologies Inc., Vancouver, Canada). Cells were centrifuged at 1200g for 10 min at room temperature, and the top plasma layer was removed peripheral blood mononuclear cells (PBMCs) were collected, washed with PBS + 2% FBS, and counted using acridine orange/propidium iodide using a Cellometer® Vision CBA (Nexcelom Bioscience, Lawrence, MA, USA). PBMCs were cryopreserved in CryoStor® CS10 (BioLife Solutions, Bothell, WA, USA), frozen using CoolCell® FTS30 freezing containers (BioCision, San Rafael, CA, USA), and stored in the liquid nitrogen vapor phase until use.

Specific cell types were isolated from the PBMC fraction using the following kits (STEMCELL Technologies Inc.) per manufacturer’s recommendations: EasySep™ Human T Cell Enrichment Kit (T cells); EasySep™ Human B Cell Enrichment Kit (B cells); EasySep™ Human NK Cell Enrichment Kit (NK cells); and EasySep™ Human Monocyte Enrichment Kit (monocytes). T cells (CD3+), B cells (CD19+), natural killer (NK) cells (CD59+), and monocytes (CD14+) were isolated based on their customarily defined gene expression markers. Isolated cells were counted using acridine orange/propidium iodide on a Cellometer Vision CBA and then cryopreserved as described above until use.

After cells undertook a 30-min adhesion onto a 384-well assay plate, 10 pL of 5 trigger medium (including vesicular stomatitis virus encoding a red fluorescent protein (rVSV-AG-mCherry), DMSO, test compound, and FBS) was added to the assay plate using a 384-well pipetting head to achieve a final concentration of rVSV-AG-mCherry at 10x MOI, 0.1% DMSO, 10% FBS, and 0.33 mM or 5.3 pM compound concentration. The assay plate was centrifuged for 1 min at 138xg and incubated for 24 h at 37 °C with 5% humidity. rVSV-AG-mCherry infected monocytes and macrophages, which subsequently created a highly inflamed environment for the lymphocytes and other cells. rVSV-AG-mCherry was used because of its ability to model the innate immune activation pathways of prevalent respiratory RNA viruses and because it was safe to use in a high-throughput laboratory with biosafety level one.

Virally-infected cells were contacted different concentrations of the active ingredient disulfiram (e.g., 0.00 pM, 0.02 pM, 0.10 pM, 0.33 pM, 1.65 pM, 6.25 pM, and 25.02 pM).

Cytokine levels in the cellular supernatant were evaluated using the FirePlex-HT assay system with the Human Cytokines FirePlex-HT Panel 1 (ab234897; Abeam). Morphological/cellular phenotypes were assayed.

The active ingredient disulfiram demonstrated notable effects on cellular phenotypes in older immune cells that were suggestive of a rejuvenation of the response to infection.

Disulfiram significantly reduced several proinflammatory cytokines when compared with old untreated controls. MCP1, IL-Ib, IL-6, and TNFa all significantly decreased in at least 2 doses (FIG. 1A). Again, the reduction in MCP1 followed a downward dose response (0.02 pM [P=.35]; 0.1 pM [P=.04]; 0.33 pM [P< 001]; 1.65 pM [P< 001]; 6.25 pM [P< 001]; 25.02 pM [P< 001]; FIG. 1A). IL-Ib was significantly reduced after the second lowest dose, then stayed significantly low for all higher doses (0.02 mM [P= 28]; 0.1 mM [P= 005]; 0.33 mM [R= 001]; 1.65 mM [P= 004]; 6.25 mM [P=.004]; 25.02 mM [P<.001]; FIG. 1A). These anti-inflammatory effects caused disulfiram showing its potential to improve older viral immune responses to infection.

Disulfiram improved the appearance of virally-infected cells. Untreated and treated PBMCs were exposed to 10x MOI rVSV, yet, at several doses, disulfiram made the cells appear like they were responding to a lower viral load. Compared with old controls, which appeared to be responding to 10x MOI rVSV, the higher doses of disulfiram made the cells appear to be responding to a lower viral load (0.02 mM [P= 63]; 0.1 mM [P=.01]; 0.33 mM [P= 43]; 1.65 mM [P=.05]; 6.25 mM [P< 001]; 25.02 mM [P< 001]). The higher doses of disulfiram also significantly reduced the percentage of the mitochondria with a reticular shape (0.02 mM [P=.04]; 0.1 mM [P=.42]; 0.33 mM [P< 001]; 1.65 mM [P< 001]; 6.25 mM [P<.001]; 25.02 mM [P<.001]; FIG. IB). Both of these effects restore important phenotypes of the older viral immune response.

T cells treated with disulfiram also exhibited a much younger phenotype compared with old untreated controls. The first two doses of disulfiram saw a significant rejuvenating effect on the T- cell age scores in the multi -phenotype aging profiles (0.02 mM [ < 001]; 0.1 mM [.P=.005]; FIG. 1C). The higher doses of disulfiram also saw significant, beneficial reductions in the T-cell age score (0.33 mM [fU.OOl]; 1.65 mM [fU.OOl]; 6.25 mM [fU.OOl]; 25.02 mM [ < 001]; FIG. 1C).

Similar trends were seen with the “on-age” and “off-age” scores. Compared with controls, the “on- age” T-cell score significantly shifted in the young direction after treatment with disulfiram (0.02 mM [ =.003]; 0.1 mM [ =.l l]; 0.33 mM [ .001]; 1.65 mM [ .001]; 6.25 mM [ .001]; 25.02 mM [/ ^J <.001 ]; FIG. ID). The “off-age” score also had a significant effect but only in the higher doses (0.02 mM [ =.26]; 0.1 mM [ =.44]; 0.33 mM [ <.001]; 1.65 mM [ =.008]; 6.25 mM [ =.003]; 25.02 mM [ =.01]; FIG. ID). All these beneficial shifts in age-related phenotypes show the strong beneficial potential of disulfiram for treating dysfunction in the older viral immune response.

Machine learning model predictions of the immune response resulting from different viral loads (at O.lx, l x, and 10x MOI) were notably different with disulfiram treatment compared with untreated control cells. At higher disulfiram concentrations, the model indicated that monocytes responded at a lower MOI than did untreated control cells.

Together, disulfiram restored multiple aspects of the viral immune response of older adults to a younger state demonstrating its usefulness in therapeutic methods of the present disclosure.

Disulfiram treatment induced apoptosis. On average, the three highest concentrations of disulfiram increased dead cells and infected monocytes by 12% and decreased T cells by 13%. This trend is in the opposite direction of a young phenotype.

Disulfiram also appeared to operate via several anti-inflammatory mechanisms, including an ability to inhibit NLRP3 inflammasome-mediated pyroptotic cell death. The inflammasome- blocking mechanisms of disulfiram ultimately inhibits pyroptosis by stopping formation of pores in the cell membrane that lead to cell lysis and release of proinflammatory molecules such as IL- 1b and IL-18. The data presented herein show that disulfiram restores aspects of the old viral immune response.

These data demonstrated significant anti-inflammatory and rejuvenating effects by disulfiram. Disulfiram reduced the proinflammatory cytokines MCP1, IL-Ib, IL-6, and TNFa while also rejuvenating several other features in aging profile such as T-cell age score, viral load response, and mitochondrial function. These immunomodulatory mechanisms could be related to the ability of disulfiram to block the final step in inflammasome-mediated pyroptosis and cytokine release. Disulfiram may also operate by ultimately reducing pore formation on the cell membrane of neutrophils, which would allow for the release of neutrophil extracellular traps (NETs), a process known as NETosis. Both of these mechanisms make disulfiram an attractive treatment for hyperinflammatory infections such as COVID-19 and sepsis. Disulfiram has already been shown to protect mice from lethal lipopolysaccharide-induced septic shock. By targeting any of these mechanisms, disulfiram may be a useful agent for treating infection, e.g., caused by a virus such as SARS-CoV2.

Sera from patients with severe COVID-i9 demonstrated increased NETs and an autopsy of a lung specimen from a patient with COVID-i9 showed extensive neutrophil infiltration. Proinflammatory cytokines in patients with severe COVID-i9 were significantly higher than in moderate cases. This includes elevated levels of IL-Ib that result from inflammasome activation. Given the relationship between NETosis, the inflammasome, and COVID-19 pathology, the ability of disulfiram to target these pathways, treatments with disulfiram could provide substantial clinical benefit.

In addition to improving host response, disulfiram may have antiviral effects on SARS-CoV2. Disulfiram has been shown to inhibit papain-like proteases of deadly coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoVl, which may disrupt the replication and IFN suppression mechanisms of these viruses. Example 8: Identification of combinations of disulfiram and potentiating ingredients useful in methods of the present disclosure

In this example, disulfiram and potentiating ingredients capable of increasing lifespan in a mammal, of preventing or treating a disease including an aging-related disorder in a mammal, of reducing a symptom of aging in a mammal, and/or of boosting an immune system were identified.

Sets of cultured cells — fibroblasts, peripheral blood mononuclear cells (PBMCs including lymphocytes and monocytes, and/or myoblasts — having either characteristics of young cells or characteristics of old cells were contacted with a combination of disulfiram and one or more potentiating ingredients. The ability of disulfiram and one or more potentiating ingredients to reverse aging in the cells, e.g., reducing the characteristics of old cells and promoting characteristics of a young cells was assayed (also known as reducing the predicted age of the cells). Disulfiram and one or more potentiating ingredients were used at various concentrations ranging from 0.000005 to 80 mM. In some cases, the concentration of disulfiram was as high as 80 pM and the concentration of the potentiating ingredient was as high as 25 pM. Combinations of disulfiram and one or more potentiating ingredients showing the ability to reverse aging were further validated.

Assays and formulations used in this example are related to those described in US20190228840, the entire contents of which is incorporated by reference its entirety.

In this experiment, TBHQ was identified as a potentiating ingredient.

Example 9: Synergistic effect of disulfiram and TBHQ

A luciferase assay was performed as described briefly: 125k THP1 pyroptosis reporter cells/ml final density were plated on 384 well plates and incubated with disulfiram, TBHQ, or disulfiram and TBHQ for 20 minutes. 1 pg/ml LPS was added to the wells, and the plate was incubated for 3 hours, followed by addition of 10 pM nigericin. After three hours, supernatants were harvested and the presence of HMGB1 luciferase reporter protein was quantified using QUANTI-Luc.

FIG. 2 shows the effect on pyroptosis of the combination of disulfiram and TBHQ as compared to each individual compound. FIG. 3 shows the potentiating effects of varying constant concentrations of TBHQ added to disulfiram titration curves.

Inhibition of pyroptosis was assessed by inducing inflammasome activation in THPl HMGB1 Lucia cells (Invivogen). These cells code for a luciferase reporter protein (HMGB 1) that is released from cells during pyroptosis. HMGB1 levels in the supernatant can therefore be used to quantify pyroptosis. Concentration matrices of disulfiram titration curves mixed with titration curves of TBHQ were tested. Disulfiram curves at each concentration of TBHQ were plotted and fit using a log(agonist) vs. response variable slope (four parameters) least squares fit model. The diagonal of each matrix was representative of a dose response curve of disulfiram plus TBHQ combinations titrated at a constant ratio.

Synergy Assessment of Disulfiram Combinations

Synergy fold ratio was measured by calculating the ratio of actual effect over expected effect: [(TBHQ+disulfiram) / no treatment] / [(disulfiram alone / no treatment) x (TBHQ alone / no treatment)] at each concentration combination. Values greater than 1 were considered synergistic.

Synergy using the Loewe additivity model was calculated using synergyfmder package on R (software environment for statistical computing and graphics; see the World Wide Web (at) /bioconductor.org/packages/release/bioc/vignettes/synergyfin der/inst/doc/synergyfinder.pdf, the contents of which is incorporated by reference in its entirety). The Loewe additivity model assumes the null hypothesis that a combination of drugs is the same as increasing the concentration of either drug alone {i.e. the effect is simply additive rather than the drugs interacting to produce a greater effect). y _e is the effect as if a drug is combined with itself, i.e., y ^e = yi(xi + X2) = y2(xi + X2).

Using R, the synergy score is calculated using ye, and determined to be the difference between the observed effect and the expected effect. Score negativity or positivity determines whether the combination is synergistic or antagonist, respectively. Loewe S. (1953), The problem of synergism and antagonism of combined drugs. Arzneimittelforschung 3, 285-290; Loewe, S. (1928). Die quantitativen probleme der pharmakologie. Ergebnisse Physiol. 27, 47-187, the contents of which is incorporated by referenced in its entirety.

FIG. 4A to FIG. 4C show the synergistic effect of the combination of disulfiram and TBHQ mediated inhibition of pyroptosis.

IC50 Calculations

IC50s were calculated using the log(agonist) vs. response variable slope (four parameters) least squares fit model from the GraphPad Prism software package.

The percent difference between disulfiram alone and each disulfiram +TBHQ curve was calculated, and two replicates for each condition were averaged. Statistical significance was determined using a standard Student’s t-test (unpaired, 2-tailed). Individual curves for disulfiram alone, TBHQ alone, and the combination of the two were plotted as log of the relative concentrations of each. In these experiments, disulfiram and TBHQ were titrated equally with different starting concentrations of disulfiram and TBHQ and so the relative ratio of disulfiram: TBHQ was maintained across the entire curve. The concentrations were normalized so that 1 is set as the median for each curve, and the curves are plotted as 1.5x dilution series.

Curves of disulfiram + constant concentrations of TBHQ were plotted using the log of disulfiram concentration.

FIG. 5 is a table showing the percent leftward shift in IC50 mediated by TBHQ relative to disulfiram alone in a dose-dependent matter, while TBHQ alone has no effect on pyroptosis inhibition or IC50. Accordingly, these data show synergistic effects on pyroptotic inhibition when cells are subjected to a combination of disulfiram and TBHQ.

Example 10: Methods that comprise administering disulfiram and TBHQ In this example, a composition comprising disulfiram and TBHQ, or distinct compositions of a first composition comprising disulfiram and a second composition comprising TBHQ are administered to a mammal, e.g., a human, for increasing lifespan, for preventing or treating a disease including an aging-related disorder, for reducing a symptom of aging, and/or boosting an immune system (e.g, for treating an infection).

The dose of disulfiram is from about 5 mg to about 500 mg. The dose of the TBHQ is from about 0.001 mg to about 280 mg. The amount of disulfiram may be from about 5 mg to about 500 mg and the amount of tert-Butylhydroquinone (TBHQ) may be from about 0.02% to about 56% by weight of disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is up to about 37 mg. In various cases, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg. In a specific case, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg.

Administration of the compositions is by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the compositions are administered orally, by inhalation, or topically. Combinations of administration routes may be used.

The mammal’s lifespan is measured and the presence, absence, and/or severity of various aging- related disorders are determined; these are compared to control mammals and/or to historical controls to determine the effectiveness of the composition administered. The mammal may be aged or not aged.

The mammal may have a healthy immune system or the mammal may have an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

Illustrative diseases treated in this example may be asthma, deafness, or a viral infections and an illustrative symptom thereof may be sepsis.

Assays and formulations used in this example are related to those described in US20190228840, the entire contents of which is incorporated by reference its entirety.

Example 11: Methods for preventing and/or treating a respiratory disease or disorder In this example, a composition comprising disulfiram and TBHQ, or distinct compositions of a first composition comprising disulfiram and a second composition comprising TBHQ are administered to a mammal, e.g., a human, for preventing and/or treating a respiratory disease or disorder, e.g, acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) with or without acute lung injury (ALI).

The dose of disulfiram is from about 5 mg to about 500 mg. The dose of the TBHQ is from about 0.001 mg to about 280 mg. The amount of disulfiram may be from about 5 mg to about 500 mg and the amount of tert-Butylhydroquinone (TBHQ) may be from about 0.02% to about 56% by weight of disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is up to about 37 mg. In various cases, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg. In a specific case, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg.

The amounts of disulfiram and TBHQ limit the progression of acute respiratory distress syndrome (ARDS) in patients with or without acute lung injury (ALI), e.g, limit the progression of ARDS in patients with ALI.

Administration of the compositions is by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the compositions are administered orally, by inhalation, or topically. Combinations of administration routes may be used.

Treatment is identified as an improvement in the administered mammal in one or more of the following symptoms severe shortness of breath, labored and unusually rapid breathing, low blood pressure, and confusion and extreme tiredness. The improvement may be relative to the pre administration state for the mammal. The mammal may be aged or not aged.

The underlying cause for the ALI and/or ARDS may be sepsis ( e.g ., a serious and widespread infection of the bloodstream); inhalation of a harmful substance (e.g., smoke, chemical fumes, asbestos, dust, particulates, vomit, and water); viral or bacterial pneumonia (which may affect up to all five lobes of the lungs) and other respiratory disorders including those caused by a coronavirus (e.g, SARS, MERS, and COVID-19), influenzas (influenza A, influenza B, or parainfluenza), pneumococcal infection, adenovirus, respiratory syncytial virus (RSV), enterovirus and/or other respiratory viral infections; and a head, chest or other major injury; or another cause (e.g, pancreatitis which is inflammation of the pancreas, a massive blood transfusion, and severe burns).

In some embodiments, ALI differs from ARDS in that ALI exists during early stage of a respiratory disease and ARDS exists during a later state of the respiratory disease.

In some embodiments, the composition or compositions prevent or treat pulmonary fibrosis (e.g, idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, dry eye, actinic keratosis, alopecia, and/or skin cancer.

Example 12: Methods for improving a vaccine response

In this example, a composition comprising disulfiram and TBHQ, or distinct compositions of a first composition comprising disulfiram and a second composition comprising TBHQ are administered to a mammal, e.g, a human, for improving effectiveness of a vaccine that is administered to the mammal.

The dose of disulfiram is from about 5 mg to about 500 mg. The dose of the TBHQ is from about 0.001 mg to about 280 mg. The amount of disulfiram may be from about 5 mg to about 500 mg and the amount of tert-Butylhydroquinone (TBHQ) may be from about 0.02% to about 56% by weight of disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is up to about 37 mg. In various cases, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg. In a specific case, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg.

The compositions may be administered by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the composition may be administered orally, by inhalation, or topically. Combinations of administration routes may be used. Administration of the vaccine may be by intravenous injection or infusion, intraperitoneal injection, intramuscular injection, or subcutaneous injection, with a dose depending on the quantity of composition needing to be administered. Alternately, the vaccine may be administered orally, by inhalation, or topically.

In some cases, the composition(s) comprising the combination of disulfiram and/or TBHQ and the vaccine are administered contemporaneously. In other cases, the vaccine is administered subsequent to the administration of the combination(s) of disulfiram and/or one or more additional ingredients. In some cases, the vaccine is administered before the administration of the combination(s) of disulfiram and/or TBHQ. A subject may be administered vaccines and/or combination(s) of disulfiram and/or one TBHQ multiple times and in any order.

The vaccine may be a Chickenpox vaccine, Coronavirus vaccine, Diphtheria vaccine, Hepatitis A vaccine, Hepatitis B vaccine, Haemophilus influenzae type b vaccine, Human immunodeficiency virus (HIV) vaccine, Human papillomavirus vaccine, influenza vaccine, Japanese encephalitis vaccine, Measles, mumps, or rubella (including MMR combined vaccine) vaccine, Meningococcal disease vaccine, Pneumococcal disease vaccine, Polio vaccine, Rabies vaccine, Respiratory syncytial virus (RSV) vaccine, Rotavirus vaccine, Shingles vaccine, Smallpox vaccine, Tetanus vaccine, Varicella virus vaccine, Whooping cough (part of the DTaP combined vaccine) vaccine, or Yellow fever vaccine. In embodiments, the vaccine is a coronavirus vaccine. In embodiments, the coronavirus vaccine is directed against Sars-CoV-2.

The mammal may be aged or not aged.

The mammal may have a healthy immune system or the mammal may have an unhealthy immune system, dysfunctional immune system, and/or weakened immune system.

The mammal’s ability to fend off a subsequent infection is determined and compared to mammals and/or historical controls who were only administered the vaccine.

The mammal’s ability to later produce antibodies directed to an infectious agent (related to the vaccine) is determined and compared to mammals and/or historical controls who were only administered the vaccine.

Example 13: Methods for treating skin disorders

In this example, a composition comprising disulfiram and TBHQ, or distinct compositions of a first composition comprising disulfiram and a second composition comprising TBHQ are administered to a mammal, e.g., a human, for treating a skin disorder. The dose of disulfiram is from about 5 mg to about 500 mg. The dose of the TBHQ is from about 0.001 mg to about 280 mg. The amount of disulfiram may be from about 5 mg to about 500 mg and the amount of tert-Butylhydroquinone (TBHQ) may be from about 0.02% to about 56% by weight of disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is up to about 37 mg. In various cases, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg. In a specific case, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg.

The compositions may be administered orally or topically, with a dose depending on the quantity of composition needing to be administered. The compositions may be formulated as a gel, lotion, ointment, cream, suspension, paste, liniment, powder, tincture, or aerosol or administered via an impregnated solid support ( e.g ., a patch)). Alternately, the composition may be administered by injection or by inhalation. The composition(s) may be administered orally. Combinations of administration routes may be used.

The mammal has a skin disorder, e.g., wrinkles, which may be a result of photoaging or related to actinic keratosis. Other skin disorders the mammal may have includes dermal atrophy (thinning of the skin), lentigines (aging spots), vaginal atrophy, prolonged/inefficient wound healing, and/or xerosis cutis (skin dryness). In examples, the mammal has moderate skin aging ( i.e ., Glogau Classification III).

The composition’s or compositions’ ability to treat a skin disorder, e.g, wrinkles, is determined and compared to the mammal before administration and/or to historical controls who were not administered the composition or compositions. For example, the determination relates to a change in the Glogau Classification.

Example 14: Methods for treating dry eye

In this example, a composition comprising disulfiram and TBHQ, or distinct compositions of a first composition comprising disulfiram and a second composition comprising TBHQ are administered to a mammal, e.g, a human, for treating dry eye.

The dose of disulfiram is from about 5 mg to about 500 mg. The dose of the TBHQ is from about 0.001 mg to about 280 mg. The amount of disulfiram may be from about 5 mg to about 500 mg and the amount of tert-Butylhydroquinone (TBHQ) may be from about 0.02% to about 56% by weight of disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is up to about 37 mg. In various cases, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg. In a specific case, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg.

The compositions may be administered topically, with a dose depending on the quantity of composition needing to be administered. The compositions may be formulated as eye drops or as eye ointments.

The composition’s or compositions’ ability to treat dry eyes, is determined and compared to the mammal before administration and/or to historical controls who were not administered the composition or compositions.

Example 15: Methods for treating alopecia

In this example, a composition comprising disulfiram and TBHQ, or distinct compositions of a first composition comprising disulfiram and a second composition comprising TBHQ are administered to a mammal, e.g., a human, for treating alopecia.

The dose of disulfiram is from about 5 mg to about 500 mg. The dose of the TBHQ is from about 0.001 mg to about 280 mg. The amount of disulfiram may be from about 5 mg to about 500 mg and the amount of tert-Butylhydroquinone (TBHQ) may be from about 0.02% to about 56% by weight of disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is up to about 37 mg. In various cases, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg. In a specific case, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg.

The compositions may be administered topically, with a dose depending on the quantity of composition needing to be administered. The compositions may be formulated as a gel, lotion, ointment, cream, suspension, paste, liniment, powder, tincture, or aerosol or administered via an impregnated solid support (e.g, a patch)). Alternately, the composition may be administered by injection or by inhalation. The composition(s) may be administered orally. Combinations of administration routes may be used.

The composition’s or compositions’ ability to treat alopecia, is determined and compared to the mammal before administration and/or to historical controls who were not administered the composition or compositions.

Example 16: Methods for treating a skin cancer

In this example, a composition comprising disulfiram and TBHQ, or distinct compositions of a first composition comprising disulfiram and a second composition comprising TBHQ are administered to a mammal, e.g., a human, for treating a skin cancer, e.g, (e.g, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)).

The dose of disulfiram is from about 5 mg to about 500 mg. The dose of the TBHQ is from about 0.001 mg to about 280 mg. The amount of disulfiram may be from about 5 mg to about 500 mg and the amount of tert-Butylhydroquinone (TBHQ) may be from about 0.02% to about 56% by weight of disulfiram. In some cases, the amount of disulfiram is from about 5 mg to about 500 mg and the amount of TBHQ is up to about 37 mg. In various cases, the amount of disulfiram is about 500 mg and the amount of TBHQ is up to about 37 mg. In a specific case, the amount of disulfiram is 500 mg and the amount of TBHQ is 37 mg.

The compositions may be administered topically, with a dose depending on the quantity of composition needing to be administered. The compositions may be formulated as a gel, lotion, ointment, cream, suspension, paste, liniment, powder, tincture, or aerosol or administered via an impregnated solid support (e.g, a patch)). Alternately, the composition may be administered by injection or by inhalation. The composition(s) may be administered orally. Combinations of administration routes may be used.

The composition’s or compositions’ ability to treat the skin cancer, e.g, BCC and SCC, is determined and compared to the mammal before administration and/or to historical controls who were not administered the composition or compositions.

Example 17: Methods comprising administering a first composition and a second composition In this example, a first composition comprises a therapeutically effective amount of a first agent that is an active ingredient of the present disclosure, i.e., an ALDH modulator or Gasdermin D inhibitor (e.g, disulfiram) and a second composition comprises a therapeutically effective amount of a metal (e.g, copper and zinc in a salt or ester form) are administered to a mammal for increasing lifespan, for preventing or treating an aging-related disorder, for reducing a symptom of aging, and/or boosting an immune system.

The first composition is administered orally, by inhalation, injection, or topically. The second composition is administered orally, by inhalation, injection, or topically. The administration route of the first composition and the second composition may be the same or may be different.

The first composition may be administered before the second composition is administered.

The first composition may be administered after the second composition is administered. The first composition and the second composition may be administered contemporaneously (either by combining the two compositions or by administering the two compositions at nearly the same time).

The mammal’s lifespan is measured and the presence, absence, and/or severity of various aging- associated conditions are determined; these are compared to control mammals and/or to historical controls to determine the effectiveness of the first composition administered.