Necrosis inhibitors

Abstract

The present application provides compositions comprising an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor and a gap junction inhibitor for use in various methods, such as methods for treating or preventing necrosis, cell death, tissue damage, or organ damage. The compositions may also comprise a cell membrane calcium channel inhibitor and / or a calcium chelator. Also provided are compositions comprising a calpain inhibitor and a cathepsin inhibitor for use in various methods. Furthermore, compositions comprising a calcium chelator, preferably at a relatively high dose, for use in various methods are provided.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to a method for treating or preventing necrosis, cell death, tissue damage, or organ damage.

[0002] The present invention relates to the medical use of calcium-active inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors, particularly in the treatment or prevention of necrosis, cell death, tissue damage, or organ damage. The present invention also relates to cosmetic compositions, foods, and food preservatives containing calcium-active inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors, as well as methods of using them to preserve foods, cells, tissues, and organs, and to methods of using them to culture cells, tissues, or organs. For example, the present invention relates to calpain inhibitors in combination with cathepsin inhibitors. For example, the present invention may relate to calcium chelators, especially when the calcium chelators are used at relatively high doses.

[0003] The present invention also relates to advantageous combinations of one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) with one or more gap junction inhibitors. Accordingly, the present invention also relates to the medical combination of one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) with one or more gap junction inhibitors, particularly in the treatment or prevention of necrosis, cell death, tissue damage, or organ damage. The present invention also relates to cosmetic compositions, foods, and food preservatives comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors, as well as methods of using them to preserve foods, cells, tissues, and organs, and to methods of using them to culture cells, tissues, or organs. The combination of one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors can be further combined with (i) one or more plasma membrane calcium channel inhibitors, and / or (ii) one or more calcium chelators.

[0004] [Background technology] Necrosis is one of the two major types of cell death that occur in living organisms, the other being programmed cell death (also known as apoptosis).

[0005] Here, one aspect of the present invention relates to the treatment or prevention of cell death, including the treatment or prevention of necrosis and / or apoptosis.

[0006] Necrosis is characterized as passive, accidental cell death resulting from environmental disturbances (i.e., stress triggers) accompanied by uncontrolled release of cellular contents. It is believed to be mediated by random intracellular events. If necrotic cells within a tissue are left untreated, necrosis spreads to adjacent cells and tissues. This can then lead to irreversible organ damage (e.g., organ failure) and ultimately death. Apoptosis, on the other hand, is described as an active, programmed process of autonomous cellular disassembly. Unlike necrosis, apoptosis does not typically spread to adjacent cells or tissues.

[0007] Current treatments for necrosis target the triggers of necrosis rather than directly halting or preventing the cellular changes associated with necrosis. This is because necrosis is traditionally thought to be mediated by random intracellular events. Because such treatments work solely by attempting to block the trigger, their effectiveness is limited to the extent that the trigger is the sole cause of necrosis, and treatment must be administered early enough to suppress the trigger, which may entail undesirable trade-offs. One such class of drugs is immunosuppressants. Immunosuppressants act by suppressing secondary inflammation that can induce necrosis (e.g., TNF-α inhibition). A trade-off associated with immunosuppressants is an increased risk of infection.

[0008] Therefore, there is a need for improved therapies to directly treat cell death, necrosis, and other pathologies that occur downstream of necrosis.

[0009] Summary of the Invention According to a first aspect of the present invention, there is provided a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor for use in treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject. For example, one or more cathepsin inhibitors may be used in combination with one or more calpain inhibitors. As another example, one or more calcium chelators may be used.

[0010] In accordance with a preferred embodiment of the present invention, there is provided a composition comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject.

[0011] A preferred embodiment of the present invention provides one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) for use in a method for treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, wherein the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor is administered in combination with one or more gap junction inhibitors (e.g., one gap junction inhibitor or two gap junction inhibitors). The endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor(s) and the gap junction inhibitor(s) can be administered simultaneously, separately, or sequentially. For example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor(s) and the gap junction inhibitor(s) can be administered simultaneously.

[0012] A preferred embodiment of the present invention provides one or more gap junction inhibitors for use in a method for treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, wherein the gap junction inhibitors are administered in combination with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists). The endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor(s) and the gap junction inhibitor(s) can be administered simultaneously, separately, or sequentially. For example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor(s) and the gap junction inhibitor(s) can be administered simultaneously.

[0013] The combination of one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors may be further combined with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0014] Another aspect of the present invention provides a method for treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, the method comprising administering to the subject a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to treat or prevent necrosis, tissue damage, organ damage, or cell death. For example, one or more cathepsin inhibitors can be administered in combination with one or more calpain inhibitors. As another example, one or more calcium chelators can be administered.

[0015] A preferred embodiment of the present invention provides a method for treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, the method comprising administering to the subject one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to treat or prevent necrosis, tissue damage, organ damage, or cell death. The endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor(s) and gap junction inhibitor(s) can be administered simultaneously, separately, or sequentially. For example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor(s) and gap junction inhibitor(s) can be administered simultaneously. Additionally, one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators can be administered.

[0016] Another aspect of the present invention provides a method for preventing necrosis or cell death of a cell or tissue, the method comprising contacting the cell or tissue with a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to prevent necrosis or cell death of the cell or tissue. For example, one or more cathepsin inhibitors can be used in combination with one or more calpain inhibitors. As another example, one or more calcium chelators can be used. The method for preventing necrosis or cell death of a cell or tissue can be performed in vivo or in vitro. For in vitro methods, it is preferable to use a relatively high dose of calcium chelator(s).

[0017] According to another preferred embodiment of the present invention, there is provided a method for preventing (i) necrosis or (ii) cell death of a cell or tissue, the method comprising contacting the cell or tissue with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to prevent necrosis or cell death of the cell or tissue. The contacting may further comprise contacting the cell or tissue with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0018] The present invention is based on the discovery that cell and tissue necrosis is mediated by a cascade of events that may occur within and between specific intracellular events. As shown in the Examples (see, in particular, Example 1) and Figure 1, blocking one or more critical steps in the cascade can treat, delay, or prevent necrosis and associated downstream pathologies (e.g., tissue or organ damage) that may occur in a subject. The downstream pathologies depend on the cell or tissue in which necrosis occurs. For example, if necrosis occurs in the kidney, the downstream pathology may be renal failure.

[0019] Preferably, the present invention is used to treat or prevent organ damage in non-nervous organs or organs that are not part of the central nervous system (CNS). Thus, the present invention can be used to treat or prevent damage to one or more or all of the following organs: integumentary organs, skeletal organs, muscular organs, circulatory organs, respiratory organs, digestive organs, urinary organs, immune system organs, endocrine organs, and reproductive organs. Thus, the present invention can be used to treat or prevent damage in organs selected from the group including or consisting of the heart, kidney, liver, skin, spleen, pancreas, intestine, stomach, lung, bladder, eye, capillaries, joints, tendons, arteries, tongue, diaphragm, ovaries, scrotum, thyroid, adrenal glands, ear, larynx, esophagus, trachea, ligaments, penis, thymus, bone, fallopian tubes, lymph nodes, ureters, bronchi, genitals, pharynx, salivary glands, urethra, gallbladder, lymphatic vessels, placenta, skeletal muscle, uterus, bone marrow, oral cavity, prostate, seminal vesicles, vulva, bulbourethral glands, hair follicles, mesentery, pineal gland, subcutaneous tissue, veins, colon, mammary glands, pituitary gland, teeth, vagina, cervix, interstitium, nose, parathyroid glands, tonsils, vas deferens, nails, rectum, testes, and vestigial organs. Additionally, the combinations described herein (e.g., a combination of one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors with one or more gap junction inhibitors) can be used to treat or prevent organ damage in nervous organs or in organs that are part of the central nervous system (CNS).

[0020] The compounds, combinations, and compositions described herein can be used to treat or prevent tissue damage. For example, calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors (e.g., (a) one or more calpain inhibitors combined with one or more cathepsin inhibitors, or (b) one or more calcium chelators) can be used to treat or prevent tissue damage. Preferably, one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors (optionally combined with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators) can be used to treat or prevent tissue damage. The tissue injury can be one or more or all selected from the group consisting of burns, multiple organ dysfunction syndrome, organ failure, surgical trauma, bedsores, chemical burns, radiation burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, physical injury, mechanical trauma, thermal injury, cold injury, chilblains, trench foot, frostbite, avascular necrosis, pressure injury, and skin graft failure. The tissue injury can be injury associated with an infection.

[0021] The compounds, combinations, and compositions described herein can be used to treat or prevent autoimmune diseases, including inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), arthritis, lupus, diabetes, liver disease / injury, kidney disease / injury, and metabolic syndrome.

[0022] The combinations described herein (e.g., a combination of one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors with one or more gap junction inhibitors) may also be used to treat traumatic brain injury (TBI), other forms of head injury (including, for example, concussion, closed head injury, penetrating head injury, diffuse brain injury, including cerebral contusion caused by, for example, depressed or penetrating skull fractures, and cerebral palsy), intracranial hematoma (ICH), hematoma, stroke, cerebral aneurysm, hypoxic and anoxic brain injury, hemorrhage, meningitis, encephalitis, epilepsy, ataxia, motor neuron disease, and polyneuropathy. It can be used to treat or prevent system atrophy, progressive supranuclear palsy, spinal cord injury, neurodegenerative diseases (including, for example, Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases, amyotrophic lateral sclerosis (ALS), Huntington's disease, spinal muscular atrophy, spinocerebellar ataxia), autoimmune diseases (including, for example, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis)), arthritis, lupus, diabetes, multiple sclerosis (MS), liver disease / injury, kidney disease / injury, pancreatic disease / injury, metabolic syndrome, cardiac arrest, and ischemia.

[0023] The compounds, combinations, and compositions described herein can also be used to treat or prevent cancer. For example, the present invention relates to methods of preventing or treating damage (e.g., cellular damage) associated with the development of tumors and / or metastases in a subject.

[0024] Although necrosis is not reversible, the present inventors have found that necrosis can be prevented, attenuated, and / or treated by using the present invention, for example, one or more of the inhibitors, combinations, and compositions described herein. Necrosis can be cell necrosis and / or tissue necrosis. If necrosis persists, organ damage (e.g., organ damage or organ failure) occurs. Therefore, organ damage can be associated with necrosis (e.g., cell or tissue necrosis) or can be caused by necrosis (e.g., cell or tissue necrosis).

[0025] The inventors have identified that a key step in the cascade of intracellular events leading to necrosis is an increase in intracellular calcium, which can be caused by the influx of extracellular calcium through calcium channels (e.g., voltage-gated and / or ligand-gated ion channels) and / or by the efflux of calcium from intracellular stores such as the endoplasmic reticulum (e.g., via ryanodine receptors), and also through gap junctions that connect cells to each other. Such changes in cytosolic calcium levels, as well as changes in calcium levels within internal stores, can further activate receptors / channels (e.g., store-operated calcium channels (SOCCs), also known as calcium release-activated calcium (CRAC) channels), thus amplifying the increase described above.

[0026] As shown in the Examples (see particularly Example 1) and Figures (see particularly Figure 1), the inventors have identified that a key step leading to necrosis is an increase in calcium ions in the cytosol, and that this can be caused by any one of three major routes for calcium ion entry into cells: via cell surface membrane channels, via gap junctions, and via internal intracellular reservoirs (particularly via the endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR)). The inventors have identified that inhibiting ER / SR calcium channels in combination with inhibiting gap junctions is particularly effective in preventing or reducing the increase in calcium ions. Accordingly, the present invention provides a combination therapy of one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors with one or more gap junction inhibitors. The inventors have found that this combination exhibits a synergistic effect beyond the simple additive effect of the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor and gap junction inhibitor used individually. The inventors have found that by simultaneously blocking calcium ion entry through the cell membrane, It has been found that the efficacy of treatment can be further improved by using a calcium chelator and / or preventing or reducing the increase in intracellular calcium ion concentration. Therefore, the present invention further provides a combination therapy of (i) one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors, (ii) one or more gap junction inhibitors, and (iii) one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators. For example, the combination therapy can use one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors, one or more gap junction inhibitors, and one or more plasma membrane calcium channel inhibitors. As another example, the combination therapy can use one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors, one or more gap junction inhibitors, and one or more calcium chelators. As another example, the combination therapy can use one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors, one or more gap junction inhibitors, one or more plasma membrane calcium channel inhibitors, and one or more calcium chelators.As will be apparent, these preferred combinations can be used in each of the medical applications, uses, and methods described herein.

[0027] As will be apparent, the compounds described herein can be used in therapeutically effective amounts. The term "therapeutically effective amount" refers to that amount of compound that produces a desired therapeutic result. That result can be prevention, reduction, amelioration, palliative, relief, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0028] It will also be apparent that references to compounds and agents may also include pharmaceutically acceptable salts and solvates thereof.

[0029] The calcium activity inhibitors referred to herein can be agents that prevent or reduce the increase in the intracellular concentration of free calcium ions or agents that lower the intracellular concentration of free (unbound) calcium ions. That is, calcium activity inhibitors are agents that reduce the availability of free intracellular calcium ions. This can be achieved by chelation (using a chelator), blocking the calcium channel pore (using a calcium channel blocker), blocking the ligand binding site of the calcium channel (using a calcium channel antagonist), blocking the ryanodine receptor (using an antagonist), or blocking the inositol triphosphate receptor (InsP3R) (using an antagonist). Thus, calcium activity inhibitors can be calcium chelators, calcium channel blockers or calcium channel antagonists, ryanodine receptor antagonists, and / or InsP3R antagonists.

[0030] The calcium activity inhibitor can be a calcium chelator (an agent that sequesters free calcium ions).Calcium chelating agents include EDTA (ethylenedioxy-diethylene-dinitrilo-tetraacetic acid), derivatives of EDTA (e.g., tetrasodium EDTA tetrahydrate, EDTA-d12, DMNP-EDTA), EGTA (ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid), derivatives of EGTA (e.g., tetrasodium EGTA and DMNP-EGTA), EGTA-AM, NP-EGTA (nitrophenyl-EGTA), NP-EGTA-AM, DTPA (diethylenetriaminepentaacetic acid), HEDTA (N-(2-hydroxy- (diethyl)ethylenediamine-N,N',N'-triacetic acid trisodium salt), NTA (nitrilotriacetic acid), BAPTA ((1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), BAPTA derivatives (BAPTA tetrasodium, BAPTA tetrapotassium, BAPTA tetracesium salt, 5,5'-dimethylBAPTA tetrapotassium, 5-nitroBAPTA tetramethyl ester, 5-nitroBAPTA, 5,5'-dibromoBAPTA tetrapotassium, 5,5'-dimethylBAPTA, and 5,5'-difluoroBAPTA), BAPTA AM (2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetic acid acetyloxymethyl ester), BAPTA AM derivatives (e.g., 5,5'-dimethyl BAPTA AM, 5,5'-difluoro BAPTA AM, and 5,5'-dinitro BAPTA AM), citric acid, TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), DMSA (dimercaptosuccinic acid), Fura-5F AM, Fura-4F AM, Fluo-3AM, Mag-Fluo-4AM, diethylenetriaminepentaacetic acid trisodium calcium hydrate, DP-b99, Quin-2AM, ethylenediaminetetraacetic acid-d16, sodium citrate, calcimycin, 4-bromo A-23187 (4-bromo-calcimycin), diazo-2, DTPA ITC (1-(p-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid), and ionomycin.For example, calcium chelating agents include EDTA (ethylenedioxy-diethylene-dinitrilo-tetraacetic acid), EGTA (ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), HEDTA (N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid trisodium salt), NTA (nitrilotriacetic acid), BAPTA ((1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), BAPTA It may be one or more selected from the group consisting of AM (2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetic acid acetyloxymethyl ester), citric acid, TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), and DMSA (dimercaptosuccinic acid).

[0031] Preferably, the calcium chelator is specific for calcium (e.g., BAPTA or BAPTA AM). Preferably, the calcium chelator is one or more selected from the group including BAPTA, BAPTA AM, EGTA, EDTA, citric acid, and DMSA. Preferably, the calcium chelator is selected from the group including EGTA, EDTA, and BAPTA AM. Most preferably, the calcium chelator is selected from DTPA, BAPTA AM, sodium citrate, and citric acid.

[0032] A calcium activity inhibitor can be an agent that regulates the intracellular concentration of free calcium ions. Thus, a calcium activity inhibitor can be a calcium modulator, such as calreticulin (also known as calregulin) or a stromal interaction molecule (STIM).

[0033] The calcium channel blockers referred to herein are voltage-dependent Ca 2+The increase in the intracellular concentration of free calcium ions can be prevented by blocking the voltage-gated Ca2+ channel, Orai channel, and / or store-operated calcium channel. Calcium channel blockers include one or more selected from the group consisting of L-type, N-type, T-type, P / Q-type, and R-type. + The calcium channel blocker may block L-type, N-type, T-type, P-type, or Q-type voltage-gated calcium channels. Most preferably, the calcium channel blocker blocks L-type voltage-gated calcium channels. The calcium channel blocker may be a dihydropyridine, a non-dihydropyridine, or a gabapentinoid. The dihydropyridine blocks L-type voltage-gated calcium channels. The non-dihydropyridine may be a phenylalkylamine or a benzothiazepine. The non-dihydropyridine may be one or more selected from the group including or consisting of diltiazem, mibefradil, bepridil, fendiline, flunarizine, fluspirilene, gabapentin, and pregabalin.

[0034] The endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors referred to herein are preferably one or more ryanodine receptor antagonists. As will be apparent, endoplasmic reticulum / sarcoplasmic reticulum inhibitors are also referred to herein as endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors.

[0035] The ryanodine receptor antagonist referred to herein may be one or more selected from the group consisting of dantrolene, dibromide DHBP, cis-Ned19, trans-Ned19, ryanodine, SKF86365 hydrochloride, ruthenium red, procaine, and tetracaine. Preferably, the ryanodine receptor antagonist is dantrolene, ryanodine, trans-Ned19, cis-Ned19, procaine, or ruthenium red. Preferably, the ryanodine receptor antagonist is dantrolene and / or ryanodine. Most preferably, the ryanodine receptor antagonist is dantrolene. The ryanodine receptor antagonist may be a xestospondin-based antagonist, including hydroxylated xestospondin.

[0036] The InsP3R antagonists referred to herein may be one or more selected from the group consisting of macrocyclic 1-oxaquinolizidines, 2-aminoethoxydiphenyl borate, aromatic polyphosphates, and tetrakisphosphates (e.g., myo-inositol 1,3,4,5-tetrakisphosphate). The aromatic polyphosphate or its derivative may be benzene 1,2,4-triphosphate [Bz(1,2,4)P3], the biphenyl derivative BiPh(2,3',4,5',6)P5, or dimeric benzene phosphate. The macrocyclic 1-oxaquinolizidine may be a xestospondin (e.g., xestospondin A, B, C, or D), alaguspondin B, alaguspondin C, 7S-hydroxyxestospondin A (7-OHXeA), and demethylxestospondin B (DMXeB). The InsP3R antagonist referred to herein may be heparin, caffeine, IP3R binding protein released with inositol 1,4,5-triphosphate (IRBIT), or ci-IP3 / PM. Preferably, the InsP3R antagonist is heparin, caffeine, 2-APB (2-aminoethoxydiphenyl borate), xestospondin A, xestospondin B, xestospondin C, or xestospondin D.

[0037] Calpains are members of the calcium-activated cysteine ​​protease protease family. These enzymes contain an 80 kD catalytic subunit and a 20 kD regulatory subunit that stabilizes the catalytic subunit. The family members are listed in the table below.

[0038] [Table 1]

[0039] Calpains tend to be located in the cytoplasm and nucleus. As a result, elevated intracellular calcium activates calpains, leading to autolysis. Therefore, a calpain inhibitor can be any agent that inhibits the proteolytic activity of calpain enzymes. Calpain inhibitors can block the active site of the enzyme. Calpain inhibitors can allosterically inhibit the proteolytic activity of the enzyme. Preferably, calpain inhibitors block calpain 1 (mu-type) and / or calpain 2 (m-type). The calpain inhibitor referred to herein can be one or more selected from the group including or consisting of calpastatin, MG101, MG132, bidupiprant, α-mercaptoacrylate, 5-azolone, carboxamide, and α-helical cysteine ​​protease inhibitor. Calpain inhibitors can be agents that specifically bind to the calpain hexapeptide CYGRKK or the calpain 5-amino acid peptide CYGAK. The calpain inhibitor can be an agent that also inhibits cathepsins, such as E64 ([L-trans-3-carboxyoxirane-2-carbonyl]-L-Leu-agmatine), E64d, or calpeptin. Obviously, agents that inhibit calpains and cathepsins can also be referred to as "non-specific calpain / cathepsin inhibitors." The calpain inhibitor can be an agent specific for calpain, such as MG101, MG132, or PD150606. The calpain inhibitor can be MDL28170. For example, the calpain inhibitor can be MG101, bidupiprant, calpeptin, E64, PD150606, or MDL28170. Calpain inhibition can be measured by cleavage of the substrate Ac-LLY-AFC, which emits blue light (λmax=400 nm) when cleaved by calpain. Free AFC emits yellow-green fluorescence (λmax = 505 nm), which can be quantified using a fluorometer or a fluorescence plate reader. Comparing the fluorescence intensity from treated samples with that of normal controls allows for the measurement of changes in calpain activity.

[0040] A cathepsin inhibitor can be any drug that inhibits cathepsin enzyme activity. It can block the active site of an enzyme. It can allosterically inhibit the activity of an enzyme. Preferably, the cathepsin inhibitor inhibits cathepsin L, B, and / or D. The cathepsin inhibitor referred to herein can be one or more selected from the group including cystatins, triterpenes, and thiosemicarbazones. The cathepsin inhibitor can be one or more of dexamethasone, rifampicin, E64, E64d, calpeptin, CA074, SID26681509, L006235, bafilomycin, alloxistatin, astaxanthin, chloroquine, clofazimine, dec-RVKR, or dexamethasone. The cathepsin inhibitor can also be a drug that inhibits calpain enzymes, such as E64, E64d, or calpeptin. Cathepsin inhibitors can be cathepsin-specific agents such as CA074 ([L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester), SID26681509, L006235, or bafilomycin. Cathepsin inhibition can be measured by performing a fluorometric assay using a suitable substrate. For example, the substrate sequence for cathepsin B is RR labeled with AFC (amino-4-trifluoromethylcoumarin). Cell lysates or other samples containing cathepsin B cleave the synthetic substrate RR-AFC, releasing free AFC. The released AFC can be easily quantified using a fluorometer or fluorescence plate reader at Ex / Em 400 / 505 nm. The substrate sequence for cathepsin D is GKPILFFRLK(Dnp)-DR-NH2 labeled with MCA. Cell lysates or other samples containing cathepsin D cleave the synthetic substrate, releasing fluorescence, which can then be easily quantified using a fluorometer or fluorescence plate reader at Ex / Em = 328 / 460 nm.

[0041] For example, a combination of one or more calpain inhibitors and one or more cathepsin inhibitors (eg, PD150606 in combination with dexamethasone, or PD150606 in combination with rifampicin) can be used.

[0042] In embodiments of the invention that include a combination of one or more ER / SR endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors with one or more gap junction inhibitors, the composition or combination may not further include a calpain inhibitor and / or may not further include a cathepsin inhibitor.

[0043] Gap junctions are the membrane junctions that allow ions (e.g., Ca) to pass through. 2+ ) and small molecules. Therefore, an increase in intracellular calcium in one cell can spread to adjacent cells through corresponding gap junctions, potentially causing necrosis in the adjacent cells. Therefore, gap junction inhibitors can be used to treat or prevent necrosis. In a particularly preferred embodiment of the present invention, one or more gap junction inhibitors are used in combination with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors to treat or prevent necrosis.

[0044] The gap junction inhibitors referred to herein may be vitamin A products (e.g., retinoic acid, glycyrrhetinic acid, and its derivative carbenoxolone), long-chain alcohols (e.g., heptanol and octanol), common halogenated volatile anesthetics (e.g., halothane), fatty acids (e.g., linoleic acid, arachidonic acid, and oleic acid), fatty acid amides (e.g., oleamide), fenamic acids (arylaminobenzoates, e.g., flufenamic acid, niflumic acid, and meclofenamic acid), quinine, quinidine, and quinine derivatives (e.g., mefloquine). The gap junction inhibitors referred to herein may be fatty acids (preferably fatty acids containing 13 to 21 carbon atoms), polyamines, or cyclodextrins. The gap junction inhibitors referred to herein may be pannexin inhibitors and / or connexin inhibitors, preferably connexin inhibitors. The gap junction inhibitors referred to herein may be retinoids, which are derivatives of vitamin A.

[0045] Preferably, the gap junction inhibitor referred to herein can be selected from one or more (e.g., one or two) of 18α-glycyrrhetinic acid, 18β-glycyrrhetinic acid, carbenoxolone disodium, heptanol, octanol, halothane, oleic acid, oleamide, linoleic acid, arachidonic acid, palmitoleic acid, flufenamic acid, myristic acid, lauric acid, quinine, quinidine, dihydroquinidine, mefloquine, meclofenamic acid, probenecid, niflumic acid, flufenamic acid, astaxanthin, and retinoic acid. Preferably, the gap junction inhibitor is one or more (e.g., one or two) of retinoic acid, oleic acid, linoleic acid, octanol, heptanol, arachidonic acid, palmitoleic acid, flufenamic acid, myristic acid, halothane, astaxanthin, and quinine. Preferably, the gap junction inhibitor is one or more of retinoic acid, oleic acid, linoleic acid, octanol, and heptanol. Most preferably, the gap junction inhibitor is one or more of retinoic acid, oleic acid, and linoleic acid.

[0046] When two or more gap junction inhibitors are present, the gap junction inhibitors can be two or more (e.g., two) of retinoic acid, oleic acid, linoleic acid, octanol, heptanol, arachidonic acid, palmitoleic acid, flufenamic acid, myristic acid, halothane, astaxanthin, and quinine. Preferably, the gap junction inhibitors are two of heptanol, octanol, oleic acid, retinoic acid, linoleic acid, palmitoleic acid, astaxanthin, and quinine. For example, it can be retinoic acid combined with one or more of oleic acid, linoleic acid, octanol, and heptanol, or it can be octanol combined with one or more of retinoic acid, oleic acid, linoleic acid, and heptanol. In another preferred example, the gap junction inhibitor can be two or more of retinoic acid, oleic acid, and linoleic acid.

[0047] Measurement of fluorescent dye tracer transfer between adjacent cells by techniques such as fluorescence recovery after photobleaching (FRAP) or flow cytometry can be used as an assay to measure gap junction inhibition.

[0048] The cell membrane calcium channel inhibitor referred to herein may be one or more of dihydropyridines, non-dihydropyridines (e.g., phenylalkylamines and benzothiazepines), non-selective calcium channel inhibitors, and gabapentinoids. The cell membrane calcium channel inhibitor may be selected from one or more of amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, efonidipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, fendiline, gallopamil, verapamil, diltiazem, mibefradil, bepridil, flunarizine, fluspirilene, fendiline, gabapentin, and pregabalin. Preferably, the cell membrane calcium channel inhibitor can be selected from one or more of alandipine, clevidipine, efonidipine, felodipine, isradipine, lercanidipine, manidipine, nifedipine, nimodipine, nitrendipine, gallopamil, verapamil, diltiazem, mibefradil, fluspirilene, gabapentin, and pregabalin.

[0049] Thus, calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors can be used to treat or prevent necrosis, tissue damage, or organ damage in a subject.

[0050] The present invention may include one or more selections of calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors. The present invention may include two or more selections of calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors. Thus, the present invention may include a combination of a calcium activity inhibitor and a cathepsin inhibitor. The present invention may include a combination of a calcium activity inhibitor and a calpain inhibitor. In a preferred embodiment, the present invention may include a combination of a cathepsin inhibitor and a calpain inhibitor. In another preferred embodiment, the present invention includes a combination of one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors (optionally further combined with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators).

[0051] The present invention may include one endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor and one or more gap junction inhibitors. In a preferred embodiment, the present invention may include one endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor and two or more gap junction inhibitors. For example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor can be dantrolene, and the two or more gap junction inhibitors can be selected from heptanol, octanol, oleic acid, retinoic acid, linoleic acid, palmitoleic acid, astaxanthin, and quinine. As another example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor can be dantrolene, and the two or more gap junction inhibitors can be selected from oleic acid, retinoic acid, and linoleic acid (i.e., the two or more gap junction inhibitors can be selected from (i) oleic acid and retinoic acid, (ii) linoleic acid and retinoic acid, and (iii) oleic acid and linoleic acid). As another example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor can be dantrolene, and the two or more gap junction inhibitors can be selected from retinoic acid, heptanol, and octanol. As another example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor can be dantrolene, and the two or more gap junction inhibitors can be selected from (i) retinoic acid and heptanol, and (ii) heptanol and octanol. In another preferred embodiment, the present invention can include two or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and two or more gap junction inhibitors. For example, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor can be dantrolene and ryanodine, and the two or more gap junction inhibitors can be selected from any of the combinations listed above (e.g., two or more of oleic acid, retinoic acid, and linoleic acid).

[0052] The present invention may include a selection of three or more of calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors. For example, the present invention may include one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., dantrolene), one or more gap junction inhibitors (e.g., retinoic acid, oleic acid, and / or linoleic acid), and one or more calcium chelators (e.g., DTPA, BAPTA-AM, sodium citrate, and / or citric acid). Alternatively, the present invention may include one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., dantrolene), one or more gap junction inhibitors (e.g., retinoic acid, oleic acid, and / or linoleic acid), and one or more plasma membrane calcium channel inhibitors (e.g., aranidipine, clevidipine, nimodipine, pranidipine, verapamil, nisoldipine, efonidipine, gabapentin, gallopamil, and / or felodipine). Alternatively, the present invention may include one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (e.g., dantrolene), one or more gap junction inhibitors (e.g., retinoic acid, oleic acid, and / or linoleic acid, etc.), one or more calcium chelators (e.g., DTPA, BAPTA-AM, sodium citrate, and / or citric acid), and one or more plasma membrane calcium channel inhibitors (e.g., aranidipine, clevidipine, nimodipine, pranidipine, verapamil, nisoldipine, efonidipine, gabapentin, gallopamil, and / or felodipine).

[0053] The present invention may further include an SOCC inhibitor (e.g., 2-aminoethoxydiphenyl borate, GNF362, leflunomide, roflumilast, teriflunomide, tolvaptan, xestospondin C, and / or zinc chloride). In some embodiments of the present invention, the composition or combination does not include an SOCC inhibitor.

[0054] In embodiments in which the invention includes calcium-active inhibitors, the invention referred to herein may include one or more of the following classes of agents: calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The invention may include two or more of the following classes of agents: calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Most preferably, the invention includes a ryanodine receptor antagonist and a gap junction inhibitor. The invention may include three or more of the following classes of agents: calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Preferably, the invention includes two or more different classes of agents selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Preferably, the present invention includes a calcium chelator, a ryanodine receptor antagonist, and a gap junction inhibitor.

[0055] In one embodiment, calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors can be used to treat or prevent necrosis in a subject. Accordingly, calcium activity inhibitors (e.g., calcium chelators, calcium channel blockers or antagonists, ryanodine receptor antagonists, and / or InsP3R antagonists) can be used to treat or prevent necrosis. Accordingly, calcium chelators can be used to treat or prevent necrosis. Calcium channel blockers or calcium channel antagonists can be used to treat or prevent necrosis. Ryanodine receptor antagonists can be used to treat or prevent necrosis. InsP3R antagonists can be used to treat or prevent necrosis. Calpain inhibitors can be used to treat or prevent necrosis in a subject. Cathepsin inhibitors can be used to treat or prevent necrosis in a subject. Calpain inhibitors can be used in combination with cathepsin inhibitors to treat or prevent necrosis in a subject. Gap junction inhibitors can be used to treat or prevent necrosis. Preferably, one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (preferably one or more ryanodine receptor antagonists) and one or more gap junction inhibitors (optionally further in combination with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators) can be used to treat or prevent necrosis.

[0056] In one embodiment, calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors can be used to treat or prevent tissue damage in a subject. Accordingly, calcium activity inhibitors (e.g., calcium chelators, calcium channel blockers or antagonists, ryanodine receptor antagonists, and / or InsP3R antagonists) can be used to treat or prevent tissue damage. Accordingly, calcium chelators can be used to treat or prevent tissue damage. Calcium channel blockers or calcium channel antagonists can be used to treat or prevent tissue damage. Ryanodine receptor antagonists can be used to treat or prevent tissue damage. InsP3R antagonists can be used to treat or prevent tissue damage. Calpain inhibitors can be used to treat or prevent tissue damage. Cathepsin inhibitors can be used to treat or prevent tissue damage. Calpain inhibitors can be used in combination with cathepsin inhibitors to treat or prevent tissue damage. Gap junction inhibitors can be used to treat or prevent tissue damage. Preferably, one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (preferably one or more ryanodine receptor antagonists) and one or more gap junction inhibitors (optionally further combined with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators) can be used to treat or prevent tissue damage. Tissue damage can occur in one or more organs mentioned herein.Tissue damage can occur in one or more tissues selected from the group including or consisting of: (i) epithelial tissues (e.g., squamous epithelial tissue, cuboidal epithelial tissue, columnar epithelial tissue, simple epithelial tissue, stratified epithelial tissue, pseudostratified epithelial tissue, and tissue proper); (ii) connective tissues (e.g., collagen, reticular tissue, and elastic tissue, as well as tissues classified as proper (dense, loose), embryonic (mesenchymal, mucosal), and specialized (cartilage, fat, bone, blood)); and (iii) muscle tissues (e.g., skeletal muscle, cardiac muscle (gap junctions, intercalated discs), smooth muscle, striatum, and nonmuscle). Therefore, calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors can be used to treat or prevent tissue damage in epithelial tissues, connective tissues, and / or muscle tissues. Therefore, one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors can be used to treat or prevent tissue damage in epithelial tissues, connective tissues, and / or muscle tissues. This combination therapy can also be used to treat or prevent tissue damage in nervous tissue (e.g., neurons (soma, dendrites, axons, ganglia (PNS), nuclei (CNS)) and / or glia (astrocytes, oligodendrocytes, Schwann cells, and microglia)).

[0057] In one embodiment, calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors can be used to treat or prevent organ damage in a subject. Accordingly, calcium activity inhibitors (calcium chelators, calcium channel blockers or antagonists, ryanodine receptor antagonists, and / or InsP3R antagonists) can be used to treat or prevent organ damage. Accordingly, calcium chelators can be used to treat or prevent organ damage. Calcium channel blockers or calcium channel antagonists can be used to treat or prevent organ damage. Ryanodine receptor antagonists can be used to treat or prevent organ damage. InsP3R antagonists can be used to treat or prevent organ damage. Calpain inhibitors can be used to treat or prevent organ damage. Cathepsin inhibitors can be used to treat or prevent organ damage. Calpain inhibitors can be used in combination with cathepsin inhibitors to treat or prevent organ damage. Gap junction inhibitors can be used to treat or prevent organ damage. One or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (preferably one or more ryanodine receptor antagonists) and one or more gap junction inhibitors (optionally further combined with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators) can be used to treat or prevent organ damage. The organ damage can be multiple organ failure.Organ damage may occur in one or more organs selected from the group consisting of heart, kidney, liver, skin, spleen, pancreas, intestine, stomach, lung, bladder, eye, capillary, joint, tendon, artery, tongue, diaphragm, ovary, scrotum, thyroid, adrenal gland, ear, larynx, esophagus, trachea, ligament, penis, thymus, bone, fallopian tube, lymph node, ureter, bronchi, genitalia, pharynx, salivary gland, urethra, gallbladder, lymphatic vessel, placenta, skeletal muscle, uterus, bone marrow, oral cavity, prostate, seminal vesicle, vulva, bulbourethral gland, hair follicle, mesentery, pineal gland, subcutaneous tissue, vein, colon, mammary gland, pituitary gland, tooth, vagina, cervix, interstitium, nose, parathyroid gland, tonsil, vas deferens, nail, rectum, testis, and vestigial organs. Preferably, the present invention is used to treat organ damage in non-nervous or non-central nervous system (CNS) organs. Thus, the present invention can be used in integumentary, skeletal, muscular, respiratory, digestive, urinary, immune, endocrine, and reproductive organs. Additionally, the combinations described herein can be used to treat or prevent organ damage in nervous organs or in organs that are part of the central nervous system (CNS). Additionally, the combinations described herein can be used to treat or prevent organ damage in the brain, spinal cord, and / or nerves connected to both that make up the central nervous system.

[0058] In one embodiment, calcium activity inhibitors (e.g., calcium chelators) can be used to treat or prevent one or more or all selected from the group consisting of burns, multiple organ dysfunction syndrome, surgical trauma, bedsores, chemical burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, physical injuries, mechanical trauma, thermal injuries, cold injuries, chilblains, trench foot, frostbite, avascular necrosis, pressure injuries, and skin graft failure.

[0059] In another embodiment, a calpain inhibitor (e.g., a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an InsP3R antagonist) can be used to treat or prevent one or more or all of the following conditions: burns, multiple organ dysfunction syndrome, surgical trauma, bedsores, chemical burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, physical injuries, mechanical trauma, thermal burns, cold injuries, chilblains, trench foot inflammation, frostbite, avascular necrosis, pressure injuries, and skin graft failure. Preferably, the calpain inhibitor is used in combination with a cathepsin inhibitor.

[0060] In another embodiment, a cathepsin inhibitor can be used to treat or prevent one or more or all of the following: burns, multiple organ dysfunction syndrome, organ failure, surgical trauma, bedsores, chemical burns, radiation burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, physical injuries, mechanical trauma, thermal burns, cold injuries, chilblains, trench foot, frostbite, avascular necrosis, pressure injuries, and skin graft failure. Preferably, a cathepsin inhibitor is used in combination with a calpain inhibitor.

[0061] In another embodiment, gap junction inhibitors can be used to treat or prevent one or more or all selected from the group consisting of burns, multiple organ dysfunction syndrome, organ failure, surgical trauma, bedsores, chemical burns, radiation burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, physical injuries, mechanical trauma, thermal injuries, cold injuries, chilblains, trench foot, frostbite, avascular necrosis, pressure injuries, and skin graft failure.

[0062] In another embodiment, one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors (optionally in combination with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators) can be used to treat or prevent one or more or all selected from the group consisting of burns, multiple organ dysfunction syndrome, organ failure, surgical trauma, bedsores, chemical burns, radiation burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, physical injuries, mechanical trauma, thermal injuries, cold injuries, chilblains, trench foot, frostbite, avascular necrosis, pressure injuries, and skin graft failure.

[0063] According to another aspect, there is provided a composition comprising one or more selected from a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor.

[0064] In one embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more selected from a calcium activity inhibitor (e.g., a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an InsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. In one embodiment, the composition comprises a calcium chelator. In one embodiment, the composition comprises a calpain inhibitor and a cathepsin inhibitor.

[0065] In another embodiment, a composition is provided comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. For example, the composition may comprise one endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor and one gap junction inhibitor, or one endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor and two or more gap junction inhibitors, or two or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and two or more gap junction inhibitors. Such a composition may further comprise one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators. For example, the composition may comprise an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor. As another example, the composition may comprise an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor, a gap junction inhibitor, and a calcium chelator. As another example, the composition may comprise an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor, a gap junction inhibitor, a plasma membrane calcium channel inhibitor, and a calcium chelator. Each of these compositions may further comprise a pharmaceutically acceptable carrier.

[0066] In another embodiment, the composition comprises two or more of a calcium activity inhibitor (e.g., a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an InsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. In one embodiment, the composition comprises three or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. The present invention may comprise two or more different classes of agents selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists.

[0067] The present invention may include one or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include two or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include three or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Preferably, the present invention includes two or more or three or more different subclasses of drugs selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists.

[0068] The compositions can be formulated as a powder, liquid, capsule, or non-capsule. The compositions can be incorporated into food products or provided as food additives or supplements.

[0069] When skin tissue is exposed to a trigger, necrosis can occur. The compositions or inhibitors described herein can be used to prevent skin (tissue) necrosis.

[0070] The compounds, combinations and compositions described herein can be administered by any suitable route known in the art.Therefore, the compounds, combinations and compositions of the present invention can be administered by injection, infusion, continuous infusion, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, sublingually, intravenously, mucosally, intrapericardially, intraumbilically, intraocularly, orally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, locally, topically, by inhalation (for example, aerosol inhalation), via catheter, via irrigation, orally, rectally, vaginally, via ophthalmic route, via otologic route, via nasal route, by nebulization, via skin, systemically, transdermally, or by any other method or any combination of the above, as known to those skilled in the art.

[0071] Each of the compounds, combinations, and compositions described herein may be provided as a pharmaceutical composition, which may further comprise a pharmaceutically acceptable carrier. As known to those skilled in the art, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof.

[0072] Each of the compounds described herein can be provided as a pharmaceutical composition, and the pH of the pharmaceutical composition is adjusted. For example, any of the acidic compounds described herein (e.g., citric acid) may require pH adjustment of the composition.

[0073] Cosmetic composition Each of the compounds, combinations, and compositions described herein may be provided as a cosmetic composition.The cosmetic composition can be used for the cosmetic treatment of skin aging, for example, to treat or prevent one or more signs of skin aging, such as the presence of fine lines and wrinkles, loss of elasticity, uneven skin, and blemishes.

[0074] Thus, according to another embodiment, there is provided a cosmetic composition comprising a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor, and a cosmetically acceptable carrier. For example, the cosmetic composition may include one or more calcium chelators, optionally in relatively high doses (e.g., the doses set forth below in embodiment A).

[0075] In another preferred embodiment, a cosmetic composition is provided comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, and optionally further comprising one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0076] In another embodiment, the composition comprises two or more of a calcium activity inhibitor (e.g., a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an InsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. For example, the composition may comprise a calpain inhibitor and a cathepsin inhibitor. In one embodiment, the composition comprises three or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. The present invention may comprise agents from two or more different classes selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists.

[0077] The present invention may include one or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include two or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include three or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Preferably, the present invention includes two or more or three or more different subclasses of drugs selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists.

[0078] The cosmetic composition can be a liquid, cream, or powder. The cosmetic composition can be for topical use (e.g., on the skin).

[0079] According to another embodiment, there is provided a method for preparing a cosmetic composition, the method comprising contacting one or more selected from calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors with a cosmetically acceptable carrier to prepare the cosmetic composition. In another preferred embodiment, there is provided a method for preparing a cosmetic composition, the method comprising contacting one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors with a cosmetically acceptable carrier to prepare the cosmetic composition. The method may further comprise contacting one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0080] In another embodiment, the composition comprises two or more of a calcium activity inhibitor (e.g., a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an InsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. For example, the composition may comprise a calpain inhibitor and a cathepsin inhibitor. In one embodiment, the composition comprises three or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. The present invention may comprise agents from two or more different classes selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists.

[0081] The present invention may include one or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include two or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include three or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Preferably, the present invention includes two or more or three or more different subclasses of drugs selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Most preferably, the present invention comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally further comprises one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators.

[0082] According to another aspect, there is provided a method of using a cosmetic composition, the method comprising applying a cosmetic composition according to the present invention to the skin of a subject.

[0083] Food preservatives and foods Each of the compounds, combinations, and compositions described herein can be provided as a food preservative.

[0084] As food ages, the cells contained therein eventually begin to die due to necrosis, and the compositions disclosed herein can therefore be used to prevent necrosis in food products and thus extend their shelf life.

[0085] Another embodiment provides a food preservative comprising one or more selected from calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and gap junction inhibitors. The food preservative can include one or more calcium chelators, and preferably, the calcium chelator(s) are used at relatively high doses (e.g., the doses set forth below in embodiment A).

[0086] Another preferred embodiment provides a food preservative comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, and optionally further comprising one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0087] Another preferred embodiment provides a food preservative comprising a combination of over-the-counter compounds, such as any combination of the over-the-counter compounds described herein.For example, the food preservative may comprise two or more of citric acid, arachidonic acid, palmitoleic acid, quinine, oleic acid, linoleic acid, retinoic acid, and astaxanthin.For example, the food preservative may comprise two or more of oleic acid, linoleic acid, retinoic acid, and astaxanthin.For example, the food preservative may comprise three or more of citric acid, arachidonic acid, palmitoleic acid, quinine, oleic acid, linoleic acid, retinoic acid, and astaxanthin.For example, the food preservative may comprise three of oleic acid, linoleic acid, retinoic acid, and astaxanthin, such as linoleic acid, retinoic acid, and astaxanthin.

[0088] According to another aspect, there is provided a food product comprising a food preservative according to the present invention. The food product may, for example, be for use as a sports supplement.

[0089] The food preservative may comprise two or more of a calcium activity inhibitor (e.g., a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an InsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. The food preservative may preferably comprise a calpain inhibitor and a cathepsin inhibitor. In one embodiment, the food preservative comprises three or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor. The present invention may comprise two or more different classes of agents selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists.

[0090] The present invention may include one or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include two or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. The present invention may include three or more of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists. Preferably, the present invention includes two or more or three or more different subclasses of drugs selected from the group consisting of calcium chelators, calcium channel blockers, calcium channel antagonists, ryanodine receptor antagonists, and InsP3R antagonists.

[0091] The food product can be any food product that can be eaten or drunk.

[0092] According to another aspect, there is provided a method for preserving food, the method comprising contacting the food with a food preservative according to the present invention to preserve the food. Preferably, the method comprises contacting the food with a food preservative comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors (optionally further comprising one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators) to preserve the food.

[0093] Method for preserving living tissue or organs Each of the compounds, combinations, and compositions described herein can be used in methods for preserving living tissues or organs.

[0094] According to another aspect, there is provided a method of preserving a living tissue or organ, the method comprising maintaining or culturing the tissue or organ in a medium comprising a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to preserve the living tissue or organ.

[0095] In accordance with a preferred embodiment, there is provided a method for preserving a living tissue or organ, the method comprising maintaining or culturing the tissue or organ in a medium comprising one or more calcium chelators (e.g., DTPA, sodium citrate, citric acid, and / or BAPTA-AM) to preserve the living tissue or organ.

[0096] The calcium chelator(s) can be used at relatively high doses to effectively prevent necrosis and thus preserve viable tissues or organs. The inventors have found that the use of calcium chelators as single-agent treatments is particularly effective for ex vivo and in vitro use, especially at relatively high doses, while such relatively high doses may be less suitable for in vivo use due to potentially associated toxicity issues in vivo.

[0097] The term "relatively high dose" as used herein with respect to a compound or agent may refer to a dose that is about 4 times, or about 5 times, or about 6 times, or about 7 times, or preferably about 8 times the dose typically used in the art for the compound or agent.

[0098] For example, the term "relatively high dose" as used herein may refer to one of the following doses (and such doses are referred to herein as "Aspect A"):

[0099] When the calcium chelator is BAPTA or a derivative of BAPTA (e.g., tetrasodium BAPTA, tetrapotassium BAPTA, tetracesium BAPTA, tetrapotassium 5,5'-dimethylBAPTA, tetrapotassium 5-nitroBAPTA tetramethyl ester, 5-nitroBAPTA, tetrapotassium 5,5'-dibromoBAPTA, tetrapotassium 5,5'-dimethylBAPTA, 5,5'-difluoroBAPTA), the chelator may be present (e.g., in the medium) in an amount of at least about 50 μM, or at least about 60 μM, or at least about 70 μM, or at least about 80 μM, or at least about 90 μM, or at least about 100 μM.

[0100] When the calcium chelator is BAPTA-AM or a derivative of BAPTA-AM (e.g., 5,5'-dimethylBAPTA AM, 5,5'-difluoroBAPTA AM, 5,5'-dinitroBAPTA AM), the chelator may be present (e.g., in the medium) in an amount of at least about 10 μM, or at least about 15 μM, or at least about 20 μM, or at least about 25 μM, e.g., at least about 50 μM, or at least about 100 μM, or at least about 150 μM.

[0101] When the calcium chelator is EGTA or a derivative of EGTA (e.g., tetrasodium EGTA or DMNP-EGTA), the chelator may be present (e.g., in the medium) in an amount of at least about 500 μM, or at least about 1000 μM, or at least about 1500 μM, or at least about 2000 μM, or at least about 2250 μM, or at least about 2500 μM.

[0102] When the calcium chelator is EGTA-AM, NP-EGTA, or NP-EGTA-AM, the chelator may be present (e.g., in the medium) in an amount of at least about 10 μM, or at least about 20 μM, or at least about 50 μM.

[0103] When the calcium chelator is EDTA or a derivative of EDTA (e.g., tetrasodium EDTA tetrahydrate or EDTA-d12), the chelator may be present (e.g., in the medium) in an amount of at least about 500 μM, or at least about 750 μM, or at least about 850 μM.

[0104] When the calcium chelator is Fura-5F AM or Fura-4F AM, the chelator may be present (eg, in the medium) in an amount of at least about 20 μM, or at least about 30 μM, or at least about 50 μM.

[0105] When the calcium chelator is Fluo-3AM or Mag-Fluo-4AM, the chelator may be present (eg, in the medium) in an amount of at least about 20 μM, or at least about 30 μM, or at least about 50 μM.

[0106] When the calcium chelator is diethylenetriaminepentaacetic acid trisodium calcium hydrate, the chelator may be present (e.g., in the medium) in an amount of at least about 500 μM, or at least about 750 μM, or at least about 850 μM.

[0107] When the calcium chelator is DP-b99, the chelator may be present (eg, in the medium) in an amount of at least about 20 μM, or at least about 30 μM, or at least about 50 μM.

[0108] When the calcium chelator is Quin-2AM, the chelator may be present (eg, in the medium) in an amount of at least about 20 μM, or at least about 30 μM, or at least about 50 μM.

[0109] When the calcium chelator is ethylenediaminetetraacetic acid-d16, the chelator may be present (eg, in the medium) in an amount of at least about 100 μM, or at least about 125 μM, or at least about 150 μM.

[0110] When the calcium chelator is DTPA, the chelator may be present (e.g., in the medium) in an amount of at least about 200 μM, or at least about 500 μM, or at least about 1000 μM, or at least about 1500 μM, or at least about 2000 μM, or at least about 2500 μM, or at least about 3000 μM, or at least about 3500 μM, or at least about 4000 μM, e.g., at least about 8000 μM, or at least about 10,000 μM.

[0111] When the calcium chelator is HEDTA, the chelator may be present (eg, in the medium) in an amount of at least about 200 μM, or at least about 500 μM, or at least about 1000 μM.

[0112] When the calcium chelator is NTA, the chelator may be present (eg, in the medium) in an amount of at least about 200 μM, or at least about 500 μM, or at least about 1000 μM.

[0113] When the calcium chelator is citrate, the chelator may be present (eg, in the medium) in an amount of at least about 200 μM, or at least about 500 μM, or at least about 1000 μM.

[0114] When the calcium chelator is sodium citrate, the chelator may be present (eg, in the medium) in an amount of at least about 200 μM, or at least about 500 μM, or at least about 1000 μM.

[0115] When the calcium chelator is TPEN, the chelator may be present (eg, in the medium) in an amount of at least about 10 μM, or at least about 20 μM, or at least about 50 μM.

[0116] When the calcium chelator is DMSA, the chelator may be present (e.g., in the medium) in an amount of at least about 50 μM, or at least about 60 μM, or at least about 70 μM, or at least about 80 μM, or at least about 90 μM, or at least about 100 μM.

[0117] When the calcium chelator is calcimycin, the chelator may be present (eg, in the medium) in an amount of at least about 0.02 μM, or at least about 0.03 μM, or at least about 0.05 μM.

[0118] When the calcium chelator is 4-bromo A-23187, the chelator may be present (eg, in the medium) in an amount of at least about 20 μM, or at least about 30 μM, or at least about 50 μM.

[0119] When the calcium chelator is diazo-2, the chelator can be present (eg, in the medium) in an amount of at least about 200 μM, or at least about 500 μM, or at least about 1000 μM.

[0120] When the calcium chelator is DTPA ITC, the chelator can be present (eg, in the medium) in an amount of at least about 10 μM, or at least about 20 μM, or at least about 50 μM.

[0121] When the calcium chelator is ionomycin, the chelator may be present (eg, in the medium) in an amount of at least about 10 μM, or at least about 20 μM, or at least about 50 μM.

[0122] According to another preferred embodiment, there is provided a method for preserving a living tissue or organ, the method comprising maintaining or culturing the tissue or organ in a medium comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, the medium optionally further comprising one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0123] According to another preferred embodiment, there is provided a method for preserving a living tissue or organ, the method comprising maintaining or culturing the tissue or organ in a medium comprising one or more calpain inhibitors and one or more cathepsin inhibitors.

[0124] Methods of preserving living tissues or organs described herein include methods that involve maintaining or culturing the tissue or organ at room temperature (e.g., about 20°C). Additionally or alternatively, the methods involve maintaining or culturing the tissue or organ under cryopreservation conditions (e.g., a temperature of about 4°C). Additionally or alternatively, the methods involve maintaining or culturing the tissue or organ by cryopreservation (e.g., at -80°C, -196°C, or temperatures intermediate therebetween).

[0125] Methods of preserving living tissue or organs described herein can include (i) simple cold storage (SCS), and / or (ii) machine perfusion (MP) storage (sometimes referred to as dynamic storage). Machine perfusion storage can include hypothermic machine perfusion (HMP), normothermic machine perfusion (NMP), and / or oxygen perfusion (OP).

[0126] The medium used in the method for preserving living tissues or organs (preferably organs) can be any medium known in the art, and in particular can be the University of Wisconsin (UW) solution, which is commonly used for organ preservation. This solution can contain potassium lactobionate (about 100 mM), KH2PO4 (about 25 mM), MgSO4 (about 5 mM), raffinose (about 30 mM), adenosine (about 5 mM), glutathione (about 3 mM), allopurinol (about 1 mM), and hydroxyethyl starch (about 50 g / L).

[0127] Thus, the invention may involve maintaining the organ in a medium (e.g., the UW solution described above) comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally further comprising one or more cell membrane calcium channel inhibitors and / or optionally one or more calcium chelators.

[0128] Also provided herein is a medium suitable for organ or tissue preservation, the medium comprising University of Wisconsin (UW) solution, one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists), and one or more gap junction inhibitors, and optionally further comprising one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0129] Also provided herein is a medium suitable for organ or tissue preservation, the medium comprising University of Wisconsin (UW) solution, one or more calpain inhibitors, and one or more cathepsin inhibitors.

[0130] Also provided herein is a medium suitable for organ or tissue preservation, the medium comprising University of Wisconsin (UW) solution and one or more calcium chelating agents, preferably wherein the calcium chelating agent(s) are present in the amount described in embodiment A above.

[0131] Methods for Manipulating Tissues or Organs One of the biggest challenges in engineering tissues in vitro is the low surface area to volume ratio. As a result, when engineering / growing large tissues, the tissues begin to develop necrotic cores. The inhibitors, combinations, or compositions described herein can be used to overcome this problem.

[0132] According to another aspect, there is provided a method of manipulating a tissue or organ, the method comprising contacting the organ or tissue with a medium comprising a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor.

[0133] In accordance with a preferred embodiment, there is provided a method of manipulating a tissue or organ, the method comprising contacting the organ or tissue with a medium comprising one or more calcium chelators (e.g., citric acid, sodium citrate, DTPA, and / or BAPTA-AM).

[0134] To effectively prevent necrosis and thus maximize tissue or organ growth and operation, the calcium chelator(s) can be used in relatively high doses, e.g., the calcium chelator(s) can be used in the doses described in aspect A above.

[0135] According to another preferred embodiment, there is provided a method of engineering a tissue or organ, the method comprising contacting the tissue or organ with a medium comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors. The medium may further comprise one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0136] According to another preferred embodiment, there is provided a method of manipulating a tissue or organ, the method comprising contacting the tissue or organ with a medium comprising one or more cathepsin inhibitors and one or more calpain inhibitors.

[0137] Contacting may refer to synthesizing an organ or tissue in a medium. Thus, the method may include synthesizing the tissue or organ in a medium containing a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor. For example, the method may include synthesizing the tissue or organ in a medium containing one or more calcium chelators (e.g., citric acid, sodium citrate, DTPA, and / or BAPTA-AM). Additionally or alternatively, the method may include synthesizing the tissue or organ in a medium containing one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. The medium may further contain one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators. Additionally or alternatively, the method may include synthesizing the tissue or organ in a medium containing one or more calpain inhibitors and one or more cathepsin inhibitors.

[0138] In one embodiment, the method comprises contacting the tissue or organ with a medium comprising two or more of a calcium activity inhibitor (e.g., a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an InsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor. For example, a combination of a calpain inhibitor and a cathepsin inhibitor can be used. In one embodiment, the method comprises contacting the tissue or organ with a medium comprising three or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor. The present invention may comprise contacting the tissue or organ with a medium comprising two or more different classes of agents selected from the group consisting of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an InsP3R antagonist.

[0139] The present invention may include contacting a tissue or organ with a medium containing one or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an InsP3R antagonist. The present invention may include contacting a tissue or organ with a medium containing one or more calcium chelators. Additionally or alternatively, the present invention may include contacting a tissue or organ with a medium containing one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. The medium may further contain one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators. The present invention may include contacting a tissue or organ with a medium containing two or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an InsP3R antagonist. The present invention may comprise contacting a tissue or organ with a medium comprising three or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an InsP3R antagonist. Preferably, the present invention comprises contacting a tissue or organ with a medium comprising two or more or three or more different subclasses of agents selected from the group consisting of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an InsP3R antagonist.

[0140] Preferably, the method comprises contacting the tissue or organ with a medium comprising a calcium-active inhibitor and a calpain inhibitor. Preferably, the method comprises contacting the tissue or organ with a medium comprising a calcium-active inhibitor and a cathepsin inhibitor. Preferably, the method comprises contacting the tissue or organ with a medium comprising a cathepsin inhibitor and a calpain inhibitor. Most preferably, the method comprises contacting the tissue or organ with a medium comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally further comprising one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0141] The medium described herein can be any medium known in the art, and in particular, can be the University of Wisconsin (UW) solution, which is commonly used for organ preservation and can contain potassium lactobionate (about 100 mM), KH2PO4 (about 25 mM), MgSO4 (about 5 mM), raffinose (about 30 mM), adenosine (about 5 mM), glutathione (about 3 mM), allopurinol (about 1 mM), and hydroxyethyl starch (about 50 g / L).

[0142] Thus, the present invention can involve contacting a tissue or organ with a medium (e.g., the UW solution described above) comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, and optionally further comprising one or more cell membrane calcium channel inhibitors and / or optionally one or more calcium chelators.

[0143] Alternatively, the invention may involve contacting a tissue or organ with a medium comprising one or more calcium chelators, preferably in which the one or more calcium chelators are present in a relatively high dose (e.g., the amount described in Embodiment A above).

[0144] Alternatively, the invention may involve contacting a tissue or organ with a medium (eg, the UW solution described herein above) containing one or more calpain inhibitors and one or more cathepsin inhibitors.

[0145] Methods for storing or culturing blood cells According to another aspect of the present invention, there is provided a method for preserving or culturing blood cells, the method comprising contacting a medium containing blood cells with a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor, and preserving or culturing the blood cells.

[0146] The method can include contacting a medium containing the blood cells with a calpain inhibitor and a cathepsin inhibitor to preserve or culture the blood cells.The method can include contacting a medium containing the blood cells with one or more calcium chelators (preferably at a relatively high dose) to preserve or culture the blood cells.

[0147] Additionally or alternatively, there is provided a method for preserving or culturing blood cells, the method comprising contacting a medium containing the blood cells with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to preserve or culture the blood cells. The medium may further comprise one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0148] The medium may be blood, plasma, or serum. Preferably, the medium is blood or plasma. The blood cells may be white blood cells (or leukocytes), red blood cells (erythrocytes), and / or platelets. The white blood cells may be one or more selected from the group consisting of neutrophils, monocytes, lymphocytes, granulocytes, and natural killer cells. The lymphocytes may be B lymphocytes or T lymphocytes.

[0149] The calcium chelator can be used in a relatively high dose to effectively prevent necrosis and thus effectively preserve or culture blood cells. For example, in the method for preserving or culturing blood cells, the calcium chelator(s) can be used in the doses described in embodiment A above.

[0150] Methods for storing blood, DNA, or reproductive samples According to another aspect of the present invention, there is provided a method for preserving a blood sample, DNA sample, or reproductive sample, the method comprising contacting the sample with a calcium-active inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to preserve the sample. The method may include contacting the sample with a calpain inhibitor and a cathepsin inhibitor. The method may include contacting the sample with one or more calcium chelators (preferably at a relatively high dose).

[0151] Additionally or alternatively, a method for preserving a blood sample, DNA sample, or reproductive sample is provided, comprising contacting the sample with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to preserve the sample. Optionally, the contacting further comprises contacting the sample with one or more cell membrane calcium channel inhibitors. Optionally, the contacting further comprises contacting the sample with one or more calcium chelators.

[0152] Calcium chelators can be used in relatively high doses to effectively prevent necrosis and thus effectively preserve blood, DNA, or reproductive samples. For example, in methods for preserving blood, DNA, or reproductive samples, calcium chelators(s) can be used in the doses described in embodiment A above.

[0153] Methods for preserving blood, DNA, or reproductive samples can be particularly useful to allow for preservation of biological reagent(s) and / or biological kit(s).

[0154] How to preserve the graft According to another aspect of the present invention, there is provided a method for preserving a graft, the method comprising contacting the graft with a calcium-active inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to preserve the graft. The method may comprise contacting the sample with one or more calpain inhibitors and one or more cathepsin inhibitors. Additionally or alternatively, the method may comprise contacting the sample with one or more calcium chelators.

[0155] Additionally or alternatively, a method for preserving a graft is provided, the method comprising contacting the graft with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to preserve the graft. Optionally, the contacting further comprises contacting the graft with one or more cell membrane calcium channel inhibitors. Additionally or alternatively, the contacting optionally further comprises contacting the graft with one or more calcium chelators.

[0156] As can be seen, the compounds, combinations, and compositions described herein can protect the graft from necrosis and / or cell death, thus preserving the graft before it is implanted into the patient.

[0157] Methods of preserving grafts described herein include cryopreservation, cryopreservation, or vitrification followed by thawing and / or washing the graft. For example, the method can include cryopreservation under low temperature conditions (e.g., at a temperature of about 4°C). Alternatively, the method can include cryopreservation (e.g., at -80°C, -196°C, or temperatures intermediate therebetween).

[0158] The calcium chelator can be used in a relatively high dose to effectively prevent necrosis and thus effectively preserve the graft. For example, in the method of preserving the graft, the calcium chelator(s) can be used in the doses described in embodiment A above.

[0159] bioreactor Clearly, cell death is a significant problem in the bioindustry, as it is common in bioreactors due to a variety of stress factors. Such stresses therefore have a negative impact on culture longevity and overall product yield. Therefore, methods for improving product yield in bioreactors are desirable.

[0160] Another aspect of the present invention provides a method for preserving or culturing cells during cell line cultivation in a bioreactor. The method may include contacting the cells with a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor, and preserving or culturing the cells in a production bioreactor. For example, the method may include contacting the cells with one or more calpain inhibitors and one or more cathepsin inhibitors. For example, the method may include contacting the cells with one or more calcium chelators (preferably at a relatively high dose, e.g., the dose described in Embodiment A above). Preferably, the method includes contacting the cells with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, and preserving or culturing the cells. Optionally, the contacting further includes contacting the cells with one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators.

[0161] Therefore, also provided herein are methods for increasing bioreactor efficiency by enhancing cell survival and / or function. The methods may include introducing a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor into the bioreactor to increase bioreactor efficiency. For example, the methods may include introducing one or more calcium chelators. For example, the methods may include introducing one or more calpain inhibitors and one or more cathepsin inhibitors. Preferably, the methods may include introducing one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors into the bioreactor to increase bioreactor efficiency. Optionally, one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators may be introduced.

[0162] The inhibitors referred to herein may be specific for calcium channel pores, calcium channel receptors, calcium ions, calpains, cathepsins, and / or gap junctions. The inhibitors referred to herein may be selective for calcium ions. Preferred inhibitors referred to herein are endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and gap junction inhibitors (particularly in combination).

[0163] Calcium chelators can be used in relatively high doses to effectively prevent necrosis and thus effectively preserve or culture cells or increase the efficiency of a bioreactor. For example, in the methods described above, calcium chelators(s) can be used in the doses described in embodiment A above.

[0164] Cell culture preservation or propagation According to another aspect of the present invention, there is provided a method for preserving or expanding a cell culture, the method comprising contacting the culture medium with a calcium-active inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to preserve or expand the cells.

[0165] The method can include contacting the medium with a calpain inhibitor and a cathepsin inhibitor. The method can include contacting the medium with one or more calcium chelators (preferably at relatively high doses).

[0166] Preferably, a method for preserving or expanding a cell culture is provided, the method comprising contacting a medium with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors (e.g., one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to preserve or expand the cell culture. The contacting may further comprise contacting the medium with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelators.

[0167] The calcium chelating agent can be used in a relatively high dose to effectively prevent necrosis and thus maximize the preservation or growth of the cell culture. For example, in the method for preserving or culturing a cell culture, the calcium chelating agent(s) can be used in the doses described in embodiment A above.

[0168] definition The term "tissue damage" may refer to dysfunction of (biological) tissue and / or failure of (biological) tissue. Preferably, the tissue damage is within an organ as referred to herein. Similarly, the term "organ damage" may refer to organ dysfunction and / or failure of an organ.

[0169] The organs referred to herein may be one or more or all selected from the group consisting of kidney, liver, skin, spleen, pancreas, intestine, stomach, lung, bladder, eye, capillary, joint, tendon, artery, tongue, diaphragm, ovary, scrotum, thyroid, adrenal gland, ear, larynx, esophagus, trachea, ligament, penis, thymus, bone, fallopian tube, lymph node, ureter, bronchi, genitalia, pharynx, salivary gland, urethra, gallbladder, lymphatic vessel, placenta, skeletal muscle, uterus, bone marrow, oral cavity, prostate, seminal vesicle, vulva, bulbourethral gland, hair follicle, mesentery, pineal gland, subcutaneous tissue, vein, colon, mammary gland, pituitary gland, tooth, vagina, cervix, interstitium, nose, parathyroid gland, tonsil, vas deferens, nail, rectum, testis, and vestigial organs.

[0170] The tissue referred to herein may be one or more tissues selected from the group including or consisting of epithelial tissue (e.g., squamous epithelial tissue, cuboidal epithelial tissue, columnar epithelial tissue, simple epithelial tissue, stratified epithelial tissue, pseudostratified epithelial tissue, and tissue proper); (ii) connective tissue (e.g., collagen, reticular tissue, and elastic tissue, and tissues classified as proper (dense, loose), embryonic (mesenchymal, mucosal), and specialized (cartilage, fat, bone, blood)); (iii) muscle tissue (e.g., skeletal muscle, cardiac muscle (gap junctions, intercalated discs), smooth muscle, striatum, and muscleless).

[0171] Preferably, the organs referred to herein are one or more or all selected from the group consisting of kidney, liver, pancreas, intestine, stomach, and lung.

[0172] The tissue referred to herein may be derived from an organ selected from the group consisting of or including: (i) epithelial tissue, (ii) connective tissue, and (iii) muscle tissue.

[0173] The method according to the invention can be carried out in vitro or in vivo.

[0174] The term "burn" can be defined as injury caused by exposure to heat or flame. Cell necrosis is the primary cause of cell and tissue damage due to heat or flame. The term "multiple organ dysfunction syndrome (MODS)" (formerly known as multiple organ failure) can be defined as sequential, progressive organ failure accompanied by a persistent, widespread inflammatory response, tissue damage, and inflammatory conditions. Examples of such inflammatory conditions include pancreatitis, any form of shock (i.e., a medical condition in which an individual experiences cellular and tissue hypoxia due to reduced oxygen delivery, increased oxygen consumption, inadequate oxygen utilization, or a combination of these three processes; such shock includes different types of shock, such as distributive shock, hypovolemic shock, cardiogenic shock, and vaso-occlusive shock), and sepsis. Cell necrosis is the primary cause of MODS. The term "organ failure" can be defined as organ dysfunction to the extent that normal homeostasis cannot be maintained without external clinical intervention. It can be classified by cause, or, if the cause is unknown, by whether the onset is chronic or acute. The primary cause of organ failure (e.g., kidney failure, liver failure) is cell necrosis. The term "surgical trauma" can be defined as any injury resulting from or associated with surgery. Necrosis contributes significantly to surgical trauma, for example, due to hypoxia or mechanical forces. The term "bedsore" can be defined as damage to an area of ​​skin caused by constant pressure on that area for a long period of time. This pressure can reduce blood flow to the affected area, causing necrosis and subsequent soreness. The term "chemical burn" can be defined as tissue damage caused by strong acids, drain cleaners, paint thinners, gasoline, and other chemicals. Such chemicals cause cell necrosis, which then leads to chemical burns. The term "radiation burn" can be defined as tissue damage caused by exposure to radiation, including ultraviolet light, X-rays, or radiation therapy. Cell necrosis is the primary cause of tissue damage after radiation exposure.The term "gangrene" can be defined as a severe condition in which body tissue dies due to loss of blood supply. Necrosis contributes to the death of tissue in gangrene. The term "alopecia (hair loss)" can be defined as the absence or loss of hair from areas of the body where hair is normally present. Necrosis contributes to hair loss. The term "solar erythema (sunburn)" can be defined as a short-term skin reaction to excessive amounts of ultraviolet (UV) radiation, primarily from the sun or tanning lamps. Necrosis contributes to solar erythema. The term "acute tubular necrosis" can be defined as kidney damage involving damage to kidney cells that can lead to acute renal failure. Necrosis contributes to acute tubular necrosis. The term "ulcer" can be defined as a break in the epithelium, skin, organ epithelium, or on the surface of tissue. Necrosis contributes to ulcers. The term "physical injury" can be defined as damage / injury to the body due to mechanical trauma, heat and cold, electrical discharge, pressure changes, and radiation. Necrosis contributes to or complicates physical injury. The term "mechanical trauma" can be defined as injury / damage to the body due to a bruise, contusion, cut, or penetrating wound. Necrosis contributes to or complicates mechanical trauma. The term "burn" can be defined as injury / injury to the body due to a heat source that causes an increase in local tissue temperature. Necrosis contributes to or complicates a burn. The term "cold injury" can be defined as injury / injury to the body due to cold exposure, and includes peripheral cold injuries primarily localized to the extremities and exposed skin, including periphery cold injury, trench foot, and frostbite. Necrosis due to a drop in temperature contributes to or complicates cold injury. The term "chilosing rash" can be defined as small swellings on the skin that occur in response to low temperatures. Necrosis due to a drop in temperature contributes to chilblains. The term "trench foot" can be defined as a painful condition of the foot caused by prolonged immersion in cold water or mud and characterized by the darkening and death of surface tissue. Necrosis due to the drop in temperature and / or stress from prolonged immersion in water contributes to trench foot. The term "frostbite" can be defined as damage / injury caused by freezing of the skin and underlying tissue.Necrosis due to temperature drop contributes to frostbite. The term "avascular necrosis" can be defined as a condition in which blood flow to bone tissue is lost, causing bone death. Necrosis contributes to avascular necrosis. The term "pressure injury" can be defined as an area or areas of necrosis and often ulceration (also called pressure sores) caused by mechanical pressure in which soft tissue is compressed and not relieved with friction, shear, and moisture. Necrosis contributes to pressure injuries. The term "skin graft failure" can be defined as the failure of a patch of skin and its derivatives (hair, nails, sweat glands, and sebaceous glands) to be surgically removed from one area of ​​the body and applied to another area. Necrosis contributes to skin graft failure. The "subject" referred to herein can be a vertebrate, mammal, or domestic animal. The subject can be any animal of veterinary importance, for example, a cat, dog, horse, sheep, or cow. However, it is preferred that the subject is a mammal, such as a human. The term "epilepsy" can be defined as a condition in which sudden bursts of electrical activity in the brain cause seizures or convulsions. The term "neurodegenerative disease" can be defined as a disease characterized by neuronal loss. The term "Alzheimer's disease (AD)" can be defined as a progressive degenerative disease of the brain characterized, among other things, by confusion, disorientation, memory loss, speech disorders, and eventual dementia. The term "Parkinson's disease (PD)" can be defined as a chronic, progressive neurological disease characterized, among other things, by resting muscle tremors, rigidity, bradykinesia, balance disorders, and a shuffling gait. The term "prion disease" can be defined as a transmissible brain disease in mammals. The term "amyotrophic lateral sclerosis (ALS)" can be defined as a neurological disorder affecting motor neurons and nerve cells in the brain and spinal cord that control voluntary muscle movement and breathing. The term "Huntington's disease" can be defined as a genetic brain disorder that is a progressive neurodegenerative disorder associated with nerve cell loss or atrophy, particularly characterized by impaired thinking and reasoning, emotional and behavioral disturbances, and the involuntary jerky movements of chorea.The term "spinal muscular atrophy" can be defined as a group of genetic disorders that can damage and kill specialized nerve cells (motor neurons) in the brain and spinal cord. The term "spinocerebellar ataxia" can be defined as a group of dominantly inherited, primarily late-onset cerebellar ataxias. The term "ataxia" can be defined as a group of genetic disorders characterized by slowly progressive loss of coordination in walking, often accompanied by poor coordination of hand, speech, and eye movements. The term "motor neuron disease" can be defined as a group of progressive neurological disorders that destroy motor neurons. The term "multiple system atrophy" can be defined as a rare disease of the nervous system that gradually damages nerve cells in the brain. The term "progressive supranuclear palsy" can be defined as a rare neurological disease that can cause problems with balance, movement, vision, speech, and swallowing. The term "spinal cord injury" can be defined as damage to the nerve bundles and fibers that send and receive signals from the brain. The term "acute liver disease / injury" can be defined as rapidly progressive loss of liver function over days to months. The term "acute kidney disease / injury" can be defined as a rapidly progressive loss of kidney function over days to months. The term "chronic liver disease / injury" can be defined as a disease in which kidney function deteriorates over time, including related diseases such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), and cirrhosis. The term "chronic kidney disease / injury" can be defined as a disease in which kidney function deteriorates over time. The term "pancreatic disease" can be defined as a disease that results in the loss of pancreatic function (e.g., due to alcohol or inflammation). The term "metabolic syndrome" can be defined as a disease that includes a set of risk factors specific to cardiovascular disease. These metabolic factors include abdominal obesity, hypertension, impaired fasting glucose, high triglyceride levels, and low HDL cholesterol levels. The term "autoimmune disease" can be defined as a disease associated with an overactive immune system. The term "inflammatory bowel disease" can be defined as an autoimmune disease that affects part or all of the gastrointestinal system, including diseases such as Crohn's disease, inflammatory bowel syndrome, and ulcerative colitis.The term "arthritis" can be defined as a disease of joint inflammation. The term "lupus" can be defined as a long-term autoimmune disease that causes joint pain, skin rashes, and fatigue. The term "diabetes" can be defined as a chronic metabolic disease characterized by high blood glucose (or blood sugar). The term "multiple sclerosis (MS)" can be defined as a disorder in which the body's immune system attacks the protective covering of nerve cells. The term "traumatic brain injury (TBI)" can be defined as a form of acquired brain injury that occurs when sudden trauma causes damage to the brain. The term "head injury" can be any injury that occurs to the scalp, skull, brain, and underlying tissues and blood vessels of the head. The term "concussion" can be defined as a sudden but short-term loss of mental function that occurs after a blow or other injury to the head. The term "close head injury" can be defined as injury that occurs when there is impact without fracture of the skull. The term "penetrating head injury" can be defined as injury that occurs when an object fractures the skull and enters brain tissue. The term "diffuse brain injury" can be defined as damage that occurs when the brain moves within the skull when the head is shaken, and can cause damage in several areas where the brain hits the skull. The term "cerebral contusion" can be defined as bruising or swelling of brain tissue that occurs when the skull cracks or splits. The term "depressed skull fracture" can be defined as skull fragments pressing against the brain. The term "penetrating skull fracture" can be defined as bone fragments entering brain tissue. The term "cerebral palsy" can be defined as a group of lifelong disorders that affect movement and coordination. The term "intracranial hematoma (ICH)" can be defined as a collection of blood within the skull. The term "hematoma" can be defined as a solid swelling of clotted blood within tissue. The term "stroke" can be defined as a disorder in which blood supply to a portion of the brain is interrupted or reduced. The term "cerebral aneurysm" can be defined as a bulge in a blood vessel caused by a weakness in the vessel wall.The term "hypoxic brain injury" can be defined as a disease when a part of the brain receives reduced oxygen. The term "anoxic brain injury" can be defined as a disease when a part of the brain receives no oxygen. The term "hemorrhage" can be defined as the outflow of blood from a ruptured blood vessel. The term "meningitis" can be defined as an infection of the protective membranes (meninges) that surround the brain and spinal cord. The term "encephalitis" can be defined as inflammation of the brain caused by an infection or an allergic reaction. The term "cancer" is caused by the uncontrolled division of abnormal cells in a part of the body. The term "tumor" may be defined as a group of abnormal cells that form a lump or growth. The term "infection" may be defined as an illness caused by a pathogen or its toxic products. The term "cardiac arrest" may be defined as the sometimes temporary cessation of heart function. The term "ischemia" may be defined as an inadequate blood supply to an organ or other part of the body.

[0175] In certain embodiments, the term "comprising" can refer to "consisting of" or "consisting essentially of."

[0176] All of the embodiments and features described in this specification (including any accompanying sections, claims, abstract, and drawings), and / or all of the steps of any method or process disclosed, can be combined with any of the above aspects or embodiments in any combination, unless a specific combination is specifically mentioned, e.g., a combination in which at least some of such features and / or steps are mutually exclusive.

[0177] For a better understanding of the present invention, and to show how embodiments of the same may be carried out, reference will now be made, by way of example, to the accompanying drawings in which:

[0178] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates predicted vulnerability points in cell physiology that predispose cells to necrosis. These vulnerability points include (i) an increase in cytosolic calcium due to calcium ion movement into the cytosol through plasma membrane calcium ion channels; (ii) an increase in cytosolic calcium due to calcium ion movement from organelles, particularly the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR), through ER / SR calcium ion channels; and (iii) an increase in cytosolic calcium due to the movement of calcium ions and other damaging factors from one cell to another through gap junctions. Other vulnerability points include excessive activation of calpains, excessive activation of cathepsins, and the opening of store-operated calcium channels (also called calcium release-activated calcium channels) after ER calcium depletion. The most important of these steps is calcium ion entry through ER / SR calcium ion channels and gap junctions, followed by entry through plasma membrane calcium ion channels, as described in more detail in Example 1 below. Figures 2-28 show the formation of a necrotic core in HepG2s spheroids under different stressors and treatment conditions, as described below. Spheroids are spherical cell aggregates that maintain cell-cell and cell-matrix interactions in an environment that mimics real-world conditions. Spheroids cannot be maintained for long periods of time or grown large due to the formation of a necrotic core (cells in the center of the spheroid lack oxygen and adequate nutrients, and hypoxia primarily causes cell death via necrosis). Figure 2 shows that the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in Hepg2 spheroids was reduced 7 days after the addition of a relatively high dose of a calcium chelator. Treatments were various doses of different chelators. The red dotted line represents the mean value of the control. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to the control. Figure 3 shows that the addition of various plasma membrane calcium channel inhibitors does not reduce the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Treatments were different plasma membrane calcium channel inhibitors. The inhibitors were added at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control by two-way ANOVA (Dunnett's correction). Figure 4 shows that the addition of various endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors did not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments were endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three replicates (n = 2 per replicate). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control by two-way ANOVA (Dunnett's correction). Figure 5 shows that the addition of various gap junction inhibitors does not reduce the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Treatments were gap junction inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three replicates (n = 2 per replicate). The violin plot shows the distribution of data with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 6 shows that the addition of various store-operated calcium channel (SOCC) inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments were SOCC inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three replicates (n = 2 per replicate). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control by two-way ANOVA (Dunnett's correction). Figure 7 shows that the addition of various calpain inhibitors does not reduce the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Treatments were calpain inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 8 shows that the addition of a nonspecific calpain / cathepsin inhibitor does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. The treatment was a nonspecific calpain / cathepsin inhibitor at a dose predicted to be the maximum dose at which toxicity was minimal. The red dotted line represents the mean control value. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 9 shows that the addition of various cathepsin inhibitors does not reduce the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Treatments were cathepsin inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 10 shows that the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included different doses of dantrolene alone, different doses of retinoic acid alone, and different doses of dantrolene and retinoic acid in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control by two-way ANOVA (Dunnett's correction). Figure 11 shows that the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor oleic acid significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included dantrolene and oleic acid, both alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment) were performed. The violin plot shows the distribution of data, with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 12 shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included dantrolene and various gap junction inhibitors, both alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control by two-way ANOVA (Dunnett's correction). Figure 13 shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene in combination with two or more gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. The treatment consisted of a combination of dantrolene and two or more gap junction inhibitors, and the individual components of the combination were also tested separately. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 14 shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included ryanodine and various gap junction inhibitors, both alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control by two-way ANOVA (Dunnett's correction). Figure 15 shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor trans-Ned19 in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included trans-Ned19 and various gap junction inhibitors, alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 16 shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor cis-Ned19 in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included cis-Ned19 and various gap junction inhibitors, both alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. Figure 17 shows that the addition of either the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor procaine or the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ruthenium red in combination with the gap junction inhibitor retinoic acid significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were procaine and the gap junction inhibitor retinoic acid, alone and in combination. (b) Treatments were ruthenium red and the gap junction inhibitor retinoic acid, alone and in combination. While the individual component treatments for the combination (both (a) and (b)) were not significantly different from the control, the combination (both (a) and (b)) exerted a greater-than-additive effect. The red dotted line represents the mean value for the control. Three experiments (n = 2 per experiment) were performed. The violin plot shows the distribution of data with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control. Figure 18 shows that the addition of a combination of the gap junction inhibitor retinoic acid and various plasma membrane calcium channel inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. The treatments were a combination of the gap junction inhibitor retinoic acid and various plasma membrane calcium channel inhibitors. The combinations showed no significant differences from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control. Figure 19 shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene in combination with various plasma membrane calcium channel inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. The treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene in combination with various plasma membrane calcium channel inhibitors. The combinations showed no significant differences from the control. The red dotted line represents the control mean. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control. Figure 20a shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Figure 20b shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Figure 20c shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Figure 20d shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) The treatment agents were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid, used alone, and their combination with the cell membrane calcium channel inhibitor alandipine.The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) is more effective than the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exert a greater-than-additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three studies (n = 2 per study) were performed. The violin plot shows the distribution of the data, with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). Figure 21 shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a calcium chelator, significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combinations with a series of different calcium chelators. The triple-compound combination was more effective than the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor alone. As can be seen, both the two-compound combination and the triple-compound combination exerted a greater than additive effect compared to the individual components of the combination compared to the control (see Figures 2, 4, and 5 for individual components showing no significant difference from the control. Figures 2, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean control value. The gray dotted line represents the mean for the combination of two compounds, one of which was a gap junction inhibitor and the other an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control and to the combination of two compounds (i.e., an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). Figure 22a shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin non-specific protease inhibitors, and SOCC inhibitors) does not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in Hepg2s spheroids. Figure 22b shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin non-specific protease inhibitors, and SOCC inhibitors) does not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in Hepg2s spheroids. Figure 22c shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin non-specific protease inhibitors, and SOCC inhibitors) does not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in Hepg2s spheroids. Figure 22d shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin non-specific protease inhibitors, and SOCC inhibitors) does not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in Hepg2s spheroids. (a) The treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid, alone and in combination with a series of different calpain inhibitors.(b) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different cathepsin inhibitors. (c) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different calpain / cathepsin nonspecific protease inhibitors. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different SOCC inhibitors. All three combinations were as effective, if not worse, than the combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker and the gap junction blocker alone. The red dotted line represents the mean control value. The gray dotted line represents the mean for the combination of two compounds, one of which is a gap junction inhibitor and the other an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three trials were performed (n = 2 per trial). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared with the combination of two compounds (i.e., an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). Figure 23 shows that the addition of a combination of a calpain inhibitor and a cathepsin inhibitor reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids, but not other combinations (e.g., a combination of two or more calpain inhibitors, a combination of two or more calpain inhibitors, or a combination with a nonspecific calpain / cathepsin inhibitor). (a) Treatments are (i) a combination of different calpain inhibitors, or (ii) a combination of a calpain inhibitor and a nonspecific calpain / cathepsin inhibitor. (b) Treatments are combinations of different cathepsin inhibitors. (a, b) The combination has no significant effect compared to the control. (c) Treatments are combinations of different cathepsin inhibitors and calpain inhibitors. The combination exerts a significant beneficial effect over the control, and the effect exceeds the effects of the individual components, which do not show a significant difference from the control. The combination of the two exerts a supra-phasic effect compared to the individual components for the combination versus the control (see Figures 7 and 9 for the individual components showing no significant difference from the control. Figures 7 and 9 and this figure can be directly compared to each other in parallel studies). The red dotted line is the mean value for the control. Three studies (n=2 per study). The violin plot shows the distribution of the data with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to the control. Figure 24 shows that the use of a combination of over-the-counter (OTC) compounds that inhibit targets associated with predicted weak points in cell physiology that predispose cells to necrosis reduces the formation of a necrotic core induced by hypoxia and nutrient deprivation stress in HepG2 spheroids. Treatment with OTC compounds inhibiting targets associated with predicted weak points in cell physiology that predispose cells to necrosis was performed (see Figure 1). Notably, such compounds have low toxicity, allowing them to be combined without causing a significant increase in cell death due to chemical toxicity. Spheroids are spherical cell aggregates that maintain cell-cell and cell-matrix interactions in an environment that mimics real-world conditions. Spheroids cannot be maintained for long periods or grown large due to the formation of a necrotic core (cells in the center of the spheroid lack oxygen and adequate nutrients, and hypoxia primarily triggers cell death via necrosis). While the individual components of the combination did not show significant differences from the control, the combination exerted a more-than-additive effect. The red dotted line represents the mean control value. Three trials (n=2 per trial). Violin plot shows distribution of data with dotted quartiles. P=0.07 indicates minimal reduction in core formation compared to control by two-way ANOVA (Dunnett's correction). Figure 25 shows a time series of HepG2s spheroids treated with a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid, demonstrating a significant reduction in necrotic core formation induced by hypoxia and nutrient deprivation stress over time. The treatments (added on day 2) were dantrolene and retinoic acid. This combination was followed over a time course for up to 7 days, revealing a strong suppression of necrotic core formation in all three experiments. Top: Brightfield images of spheroids in a multiwell plate well, over fluorescence (DRAQ7™, a viability dye (red) that stains necrotic cells). Center: Bar graphs with standard deviations (std) showing the intensity of cell death stain DRAQ7™ over time in treated and control groups. The left bar represents the treated group, and the right bar represents the control group. Bottom: Example of a fluorescence (DRAQ7™, a viability dye (red) that stains necrotic cells) image of a spheroid in a well of a multiwell plate. Three replicates (n=2 per replicate). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA (Bonferroni correction). Figure 26 shows a time series of HepG2s spheroids treated with a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid, demonstrating no significant toxic effects (spheroid formation, growth, size, and appearance appear normal). The treatments (added on day 2) were dantrolene and retinoic acid. This combination was followed over time for up to 7 days, and no significant effects on spheroid formation, growth, size, or appearance were observed. Top: Brightfield images of spheroids in a multiwell plate well. Center: Violin plot of spheroid size over time, a marker of formation and proliferation. Data distribution is indicated by dotted quartiles. The plot on the left represents the treatment ("drug") group, and the plot on the right represents the control group. Bottom: An example of a brightfield image of a spheroid in a multiwell plate well, showing normal morphology and appearance, indicating that the treatment did not compromise spheroid integrity. Three trials (n=2 per trial). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA (Bonferroni correction). Figure 27 shows a time series of HEK293 spheroids treated with a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid, demonstrating a significant reduction in necrotic core formation induced by hypoxia and nutrient deprivation stress over time. The treatments (added on day 2) were dantrolene and retinoic acid. This combination was followed over a time course for up to 5 days, revealing a strong suppression of necrotic core formation in all three experiments. Top: Brightfield images of spheroids in a well of a multiwell plate, over fluorescence (DRAQ7™, a viability dye (red) that stains necrotic cells). Center: Bar graph with standard deviation (std) showing cell death stain DRAQ7™ intensity over time in treated and control groups. The left bar represents the treated group, and the right bar represents the control group. Bottom: Example of a fluorescence (DRAQ7™, a viability dye (red) that stains necrotic cells) image of a spheroid in a well of a multiwell plate. Three replicates (n=2 per replicate). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA (Bonferroni correction). Figure 28 shows a time series of HEK293 spheroids treated with a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid, demonstrating no significant toxic effects (spheroid formation, growth, size, and appearance appear normal). The treatments (added on day 2) were dantrolene and retinoic acid. This combination was followed over time for up to 5 days, and no significant effects on spheroid formation, growth, size, or appearance were observed. Top: Brightfield images of spheroids in a multiwell plate well. Center: Violin plot of spheroid size over time, a marker of formation and proliferation. Data distribution is indicated by dotted quartiles. The plot on the left represents the treatment ("drug") group, and the plot on the right represents the control group. Bottom: An example of a brightfield image of a spheroid in a multiwell plate well, showing normal morphology and appearance, indicating that the treatment did not compromise spheroid integrity. Three trials (n=2 per trial). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA (Bonferroni correction). Figure 29 shows that the necrosis induced by the necrotic core due to hypoxia and nutrient deprivation stress in Hepg2s spheroids, as measured by the lactate dehydrogenase LDH-Glo™ assay, was significantly reduced by the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid. Treatment was a combination of dantrolene and retinoic acid. Three experiments (n=2 per experiment). Bar graphs with standard error (sem) showing cell death. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by unpaired two-tailed t-test. Figure 30 shows that treatment (as a preventative measure) of primary human intestinal fibroblast spheroids with either (i) a combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, or (ii) a combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor and a plasma membrane calcium channel inhibitor significantly (beyond additive effects) reduced oxidative stress-induced necrosis, as measured by the standard viability dye calcein AM / ethidium homodimer-1, whereas other combinations did not. Red: combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor. Specifically, the combinations shown in red are labeled "D100;RA10," "D100;RA20," and "D100;RA40." D refers to dantrolene, and RA refers to retinoic acid. Numbers indicate dose; for example, 100 refers to treatment with 100 μM of compound. Blue: Combination of endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors with gap junction inhibitors and plasma membrane calcium channel inhibitors. Specifically, the combinations shown in blue are labeled "D100;R20;N10." N stands for nifedipine. Stress was a 6-hour exposure to hydrogen peroxide (800 μM). Top: Data before stress; bottom: Data after stress. Left: Raw data of the ratio of ethidium homodimer-1 intensity to calcein AM intensity. Right: Data normalized to Triton X-100, which kills all cells, allowing the rate of necrosis to be assessed. (i) Combination of endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors with gap junction inhibitors, and (ii) combination of endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors with gap junction inhibitors and plasma membrane calcium channel inhibitors exerted significant, supra-additive effects on necrosis compared to single compounds (in the combination) that showed no significant difference or worse effects compared to the control.The combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker with a gap junction blocker and a plasma membrane calcium channel blocker is more effective than the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker with a gap junction blocker. Other combinations, including (i) an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker with a plasma membrane calcium channel blocker, and (ii) a gap junction blocker with a plasma membrane calcium channel blocker, are not significantly different from stressed controls. Boxplots (10th–90th percentiles). Two replicates (n=31 per replicate). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Kruskal-Wallis test (Dunn's multiple comparisons correction). Figure 31 shows that treatment of primary human intestinal fibroblast spheroids with a combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor (as both a preventative and therapeutic measure) significantly reduced necrosis due to prolonged mild oxidative stress, as measured by the standard viability dye calcein AM / ethidium homodimer-1. The ER / SR calcium channel inhibitor used was dantrolene, and the gap junction inhibitor used was retinoic acid. The stress was a 6-hour exposure to hydrogen peroxide (500 μM). Line graphs show the mean and standard error (sem). Two experiments (n = 31 per experiment) were performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Dunn's multiple comparison correction. For each data point (i.e., day 1, day 2, and day 3), asterisks are as follows: Top: stress control versus combined stress treatment (i.e., day 1****, day 2***, and day 3****); middle: stress control versus untreated control (i.e., day 1*, day 2**, day 3****); bottom: stress treatment versus untreated control (i.e., day 1 ns, day 2 ns, and day 3****). Figure 32 shows that treatment of primary human intestinal fibroblast spheroids with a combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor (as both preventative and therapeutic measures) significantly reduced necrosis induced by long-term moderate oxidative stress, as measured by the standard viability dye calcein AM / ethidium homodimer-1. The ER / SR calcium channel inhibitor used was dantrolene, and the gap junction inhibitor used was retinoic acid. The stress was a 6-hour exposure to hydrogen peroxide (800 μM). Line graphs show the mean and standard error (sem). Two experiments (n = 31 per experiment) were performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Dunn's multiple comparison correction. For each data point (i.e., day 1, day 2, and day 3), asterisks are as follows: Top: stress control versus combined stress treatment (i.e., day 1****, day 2****, and day 3****); middle: stress control versus untreated control (i.e., day 1****, day 2****, and day 3****); bottom: stress treatment versus untreated control (i.e., day 1****, day 2****, and day 3****). Figure 33 shows that treatment of primary human intestinal fibroblast spheroids with a combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor (as both preventative and therapeutic measures) significantly reduced necrosis induced by a short, 3-hour period of intense oxidative stress, as measured by the standard viability dye calcein AM / ethidium homodimer-1. The ER / SR calcium channel inhibitor used was dantrolene, and the gap junction inhibitor used was retinoic acid. The stress was a 3-hour exposure to hydrogen peroxide (1 mM). Line graphs show the mean and standard error (sem). Two experiments (n = 31 per experiment) were performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Dunn's multiple comparison correction. For each data point (i.e., day 1, day 2, and day 3), asterisks are as follows: Top: stress control versus combined stress treatment (i.e., day 1 ns, day 2 ****, and day 3 ****); middle: stress control versus untreated control (i.e., day 1 ****, day 2 ****, day 3 ****); bottom: stress treatment versus untreated control (i.e., day 1 ****, day 2 ***, and day 3 ***). Figure 34 shows examples of images demonstrating that treatment of primary human intestinal fibroblast spheroids with a combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor (as both a preventative and therapeutic measure) significantly reduced necrosis induced by long-term moderate oxidative stress, as measured by the standard viability dye calcein AM / ethidium homodimer-1. The ER / SR calcium channel inhibitor used was dantrolene, and the gap junction inhibitor used was retinoic acid. The stress was a 6-hour exposure to hydrogen peroxide (800 μM). Green: live cells stained with calcein AM; red: dead / necrotic cells stained with ethidium homodimer-1. Approximately 16 spheroids are observed per image. Images of Triton X-100, which kills all cells, are displayed, allowing the percentage of necrosis to be assessed. All images were taken on day 3 of the experiment (see Figure 32 for related images). Figure 35 shows primary human intestinal fibroblast spheroids treated with the combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid, demonstrating that this combination does not produce significant toxic effects (spheroid formation, growth, and size, as well as ATP levels, are maintained). (a) Images of cells aggregate to form spheroids by day 1. Spheroids are spherical cell aggregates that maintain cell-cell and cell-matrix interactions in an environment that mimics real-world conditions. (b) Control spheroid size shows no significant change from day 1 to day 3. (c) The treatments added on day 0 or day 1 were dantrolene and retinoic acid. This combination was followed over time for up to 3 days, and no significant effect on spheroid size or growth (i.e., change in size over time) was observed. Three experiments (n=30 per experiment) (d) ATP abundance was measured on day 3 using the CellTiter-Glo® 3D Cell Viability Assay, and treatments added on day 1 did not decrease ATP levels. Bar graphs of raw data points are shown. Duplicate experiments (n=31 per replicate). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by one-way ANOVA with Sidak's multiple comparisons correction. Figure 36 shows images of primary human intestinal fibroblast spheroids treated with a combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress at day 6, as measured by the standard viability dye calcein AM / ethidium homodimer-1. The ER / SR calcium channel inhibitor used was dantrolene (100 μM), and the gap junction inhibitor used was retinoic acid (10 μM). Top: Boxplots (10th–90th percentiles). Data normalized to Triton X-100, which kills all cells. Duplicate experiments (n=31 per experiment). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Kruskal-Wallis test with Dunn's multiple comparisons correction). Bottom: Example images. Green: Viable cells stained with calcein AM, Red: Dead / necrotic cells stained with ethidium homodimer-1. Approximately 16 spheroids are visible per image. Figure 37 shows that E. coli and S. pombe bacteria are prevented from dying by agents that inhibit necrosis after oxidative stress (stress of 1 mM H2O2 for 50 minutes). (a) E. coli bacteria. The treatment agent was 2500 μM EGTA during stress. After stress, the bacteria were washed with buffer, diluted 1:500 with MilliQ water, and plated on agar plates containing bacto-peptone, then incubated overnight at 37°C. Colony-forming units (CFUs) were then counted. N = 3 experiments. (b) S. pombe yeast. The treatment agent was 2500 μM EGTA during stress. After stress, the yeast were washed and stained with propidium iodide (PI (red)), which enters necrotic cells. Boxplot (Tukey's) of % necrotic cells (N = 6-7 images, data from two experiments combined). *P < 0.05 by unpaired two-tailed t-test. Figure 38 shows that treatment with drugs that inhibit necrosis prevents death of C. elegans subjected to various stressors, including heat, cold / freeze, oxidative, and toxic stress. Left to right: cold stress (-80°C for 1.5 minutes), heat stress (37°C for 4.5 hours), oxidative stress (2 M hydrogen peroxide for 5 minutes), and toxic stress (1.3% (v / v) tert-butyl hydroperoxide (tBOOH) for 5 minutes). Treatments were 2500 μM liposomal EGTA given as a preventative measure 3 hours before stress or 2500 μM liposomal EGTA given as a treatment 3 hours after stress. Box plot (Tukey). Three experiments (n=10 per experiment). *P<0.05, ***P<0.001, ****P<0.0001 by one-way ANOVA with Dunnett's correction. Figure 39 shows that treatment with drugs that inhibit necrosis reduces motor dysfunction in mutants for necrosis after various stressors, including heat, cold / freeze, oxidative, and toxic stress. Left to right: cold stress (-80°C for 1.5 min), heat stress (37°C for 4.5 h), oxidative stress (2 M hydrogen peroxide for 5 min), and toxic stress (1.3% (v / v) tert-butyl hydroperoxide (tBOOH) for 5 min). Treatment was 2500 μM EGTA in liposomes given 3 h before stress. Box plot (Tukey). Three experiments (n=10 per experiment). *P<0.05, ***P<0.001, ****P<0.0001 by one-way ANOVA with Dunnett's correction. Worms that exhibit slowed movement (usually stationary and only slightly moving or not moving at all when prodded with a nematode pick) are scored and the percentage of hypokinetic worms is displayed (y-axis). Figure 40 shows that treatment with drugs that inhibit necrosis prevents death of D. melanogaster (Drosophila melanogaster) after cold / freeze stress. The stress consisted of holding flies at 0°C for 10 minutes. Seven-day-old flies (D. melanogaster W1118) were starved for 8 hours and then fed a diet containing 0 or 2500 μM EGTA for 12 hours. This was followed by exposure to cold / freeze stress, and survival was measured every 12 hours thereafter. Treatments showed significant differences from the control. *P<0.05 by log-rank analysis. Figure 41 shows that necrosis and cell death in zebrafish (Danio reio) livers after oxidative stress (7 minutes in 1 mM H2O2) are prevented by drugs that inhibit necrosis. The treatment is EGTA2 500 μM during stress. After stress, livers are washed and stained with propidium iodide (PI, red), which enters necrotic cells. Tissues are also stained with DAPI (4'6-diamidino-2-phenylindole, blue), which enters all cells and allows analysis of the total number of cells present in the image. Percentage of necrotic cells (PI stained) relative to total cells present (DAPI stained). Box plot (Tukey). Triplicate tests. P=0.07 by unpaired two-tailed t-test. Figure 42 shows that necrosis and cell death in mouse organs are prevented after hypoxic stress (2.5 hours in a buffer solution bubbled with N2 gas) by drugs that inhibit necrosis. Organotypic sections of tissues were prepared and placed in a buffer mimicking cerebrospinal fluid (CSF) for brain tissue or Ringer's buffer for other tissues. The section thicknesses were as follows: heart: 0.210 mm, brain: 0.300 mm, and spleen: 300 mm. The treatment agent was EGTA 2500 μM during stress. After stress, sections were washed and stained with propidium iodide (PI, red), which enters necrotic cells. Tissues were also stained with DAPI (blue), which enters all cells, allowing analysis of the total number of cells present in the image. Percentage of necrotic cells (PI stained) relative to total cells observed (DAPI stained) is shown as a boxplot (Tukey's). Data are combined from two experiments. ***P<0.001, ****P<0.0001 by unpaired two-tailed t-test. Figure 43 shows that necrosis and cell death are prevented by anti-necrotic drugs regardless of cell type, cell culture, or stress type. (a) HEK293 suspension culture after oxidative stress (1 M H2O2 for 20 seconds). Treatment was EGTA 2500 μM 2 hours before stress and EGTA 2500 μM during stress. After stress, cells were washed and stained with propidium iodide (PI, red), which enters necrotic cells. Cells were also stained with DAPI (blue), which enters all cells, allowing analysis of the total number of cells present in the image. Percentage of necrotic cells (PI stained) relative to total cells (DAPI stained) observed. Box and whisker plot (Tukey's). Data from two experiments combined. ****P<0.0001 by unpaired two-tailed t-test. (b) HepG2s spheroids grown by the hanging drop method and placed on agarose pads did not exhibit a necrotic core even 5 days after the addition of anti-necrotic drugs. The treatment was EGTA 2500 μM or EGTA 5000 μM in spheroid cultures. Spheroids (spherical cell aggregates that maintain cell-cell and cell-matrix interactions in an environment that mimics real-world conditions) used in tissue engineering cannot be maintained long-term or allowed to grow large due to the formation of a necrotic core (cells in the center of the spheroid are starved of oxygen and nutrients, which leads to cell death via necrosis). Boxplot (Tukey). Data from three experiments combined. *P<0.05, ***P<0.001, ****P<0.0001 by unpaired two-tailed t-test. Figure 44 shows that the addition of a necrosis-suppressing drug (a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid) to University of Wisconsin (UW) medium, a representative organ / graft preservation medium, can increase cell viability, reduce cell necrosis during warm and cold storage, and improve organ / graft quality. After acclimation, mice were anesthetized with isoflurane and perfused at 5 mL / min with 50 mL of UW buffer containing either no treatment (i.e., control group) or treatment (100 μM dantrolene and 10 μM retinoid acid). Kidneys were incubated in 5 mL of UW buffer at 4°C for 24 hours, followed by 2 hours at 37°C, after which single-cell isolation was performed from the whole kidney. After washing, cells were stained with Annexin V and propidium iodide (PI) to determine the ratio of necrotic (PI + Annexin V-) to viable (PI-) cells and subjected to flow cytometry. Left (Figure 44a): Example of a flow cytometry image. Center and right (Figures 44b and 44c): Non-viable dead and necrotic cells in the control and treatment groups. Four replicates (n=1 kidney per replicate). *P=0.057 by unpaired two-tailed t-test.

[0179] [Mode for Carrying Out the Invention] Materials and Methods Spheroid formation and drug screening of HEK293 and HepG2 cells in U-bottom plates: Hypoxic stress Static cell culture of HEK293T and HepG2 cells was performed in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were then transferred to either 96-well Nunc Sphera or 384-well PrimeSurface® 3D culture spheroid plates (ultra-low attachment (ULA) plates) at an initial cell density of 500 cells. After spheroid formation in the plates, treatment compound(s) were dissolved in DMSO (dimethyl sulfoxide) to a final assay DMSO concentration of 1% v / v and then added to the ULA plate wells. Medium changes were performed every other day. Brightfield, phase-contrast, and fluorescence images were acquired periodically up to 6 days after treatment using an Incucyte® S3 Live Cell Analysis System. The standard viability dye DRAQ7™ (which stains only the nuclei of dead and permeabilized cells) was used as a marker of cell death. All conditions were tested in duplicate on three separate occasions (i.e., three independent experiments were performed). A DMSO control was added to each plate. The size of the necrotic core was quantified as a percentage change from the average of the DMSO control wells based on the fluorescent area normalized to spheroid size. As a secondary indicator of cell death, a lactate dehydrogenase (LDH-Glo™) assay was performed on the supernatants from treated and control wells.

[0180] Spheroid formation and drug screening of primary human fibroblasts in U-bottom plates: Oxidative and hypoxic stresses Static cell culture of primary human intestinal fibroblasts was performed. Cells were then transferred to 384-multiwell plates (Gri3D Product No. Gri3D-96P-S-96-800). The seeding density per well was 125,000 cells, resulting in the formation of 4,000 cells per spheroid in the well. After spheroid formation in the plate, treatment compounds were dissolved in DMSO to a final assay DMSO concentration of 0.5% v / v and then added to the plate wells. Medium changes were performed every other day. Triton X-100 was used as a positive control marker for cell death. Drug treatments were performed overnight before stress. Brightfield, phase contrast, and fluorescence images were acquired periodically immediately after stress and on subsequent days up to day 3 after stress. Brightfield, phase contrast, and fluorescence images were acquired using a Tecan Spark instrument (model number Spark Cyto 600) for brightfield and a GE Healthcare IN Cell instrument (model number IN Cell Analyzer 2200) for fluorescence. Images were processed using a pipeline by Doppl. Stress was exposure to various levels of hydrogen peroxide for different durations, as indicated in the figure legends. A standard viability calcein AM / ethidium homodimer-1 staining kit (Thermo Molecular Probes) was used as a marker of cell death (incubated at 37°C for 3 hours before stress). To confirm the presence of metabolically active live cells in the 3D cell cultures, quantification of the ATP levels present was performed using the CellTiter-Glo® 3D Cell Viability Assay. For this, 150 μl of supernatant was collected per well, followed by the addition of 50 μl of CTG and mixing for 5 minutes. Luminescence was then measured after 30 minutes of incubation at room temperature on days 1, 3, and 6. All conditions were performed in duplicate. Each replicate used 31 organoids per condition. The information recorded was resistance to hydrogen peroxide stress and necrotic core formation (i.e., hypoxic stress) over time, and cell viability as measured by metabolic activity (i.e., ATP levels). A DMSO control was evaluated in parallel on each plate.The level of dead organoid tissue was quantified as a percentage change from DMSO control wells based on fluorescent area normalized to spheroid size.

[0181] Viability analysis of perfused mouse kidneys by flow cytometry Eight mice were acclimated to the vivarium 7 days before the start of the study. After acclimation, mice were randomized by weight, sacrificed, and cardiac perfused with UW (University of Wisconsin) preservation medium with or without treatment compound. Intact right kidneys were then removed from the mice and maintained at low temperature (4°C) for 24 hours, followed by 2 hours at 37°C, before being processed for analysis. Kidneys were processed by manual dissociation and digestion into a single-cell suspension using Liberase™, collagenase, dispase, and DNase I. Cells were then passed through a 70 μM filter to remove debris and obtain a single-cell suspension suitable for flow cytometry. For flow cytometry analysis, kidney cells were stained with FITC-annexin V (AnnV) and propidium iodide (PI) and analyzed for: (i) AnnV-PI for necrotic cells, and (ii) PI for nonviable dead cells. Cells were then detected on a BD Accuri C6 flow cytometer with FL-1 for annexin V-FITC and FL-3 for PI.

[0182] Oxidative stress in HEK293 suspension cultures HEK293 cells were detached using trypsin, washed, and placed in cell culture medium. Cells were pretreated with or without EGTA (500 μM, control) for 2 hours before and during stress. Cells were stressed by placing them in a 1 M hydrogen peroxide solution (in cell culture medium) for 20 seconds. Cells were then washed in cell culture medium, stained with PI and DAPI to visualize all cell nuclei (including intact cell nuclei), placed on a microscope slide, covered with a coverslip, and imaged.

[0183] Preparation of 3D HepG2 cell culture spheroids Multicellular spheroids were prepared using the hanging drop method. A methylcellulose (MC) stock solution was prepared by dissolving 1.2 g of MC powder (4000 cP, Sigma-Aldrich) in 100 mL of DMEM. The solution was magnetically stirred overnight at 4°C and then centrifuged at 4,000 g for 3 hours. HepG2s cells were resuspended in cell culture medium containing 20% ​​MC stock solution at a concentration of 20,000 cells per 50 μL spheroid. 50 μL drops were then pipetted into non-adherent Petri dishes (Greiner), the lids were replaced, the dishes were inverted, and incubated overnight at 37°C. Multicellular spheroids were either harvested individually with a wide-bore pipette or collectively by adding 5 mL of cell culture medium to the dish, collecting the suspension, centrifuging at 300 g for 5 minutes, and resuspending the spheroids in cell culture medium. Spheroids were then cultured in 48-well plates containing 2.5% agarose gel and 400 μL cell culture medium for 5 days. EGTA was added to both the spheroid precursor solution and the cell culture medium at 0 μM and 2500 μM. Spheroids were imaged and evaluated on days 3 and 5.

[0184] Preparation and culture of mouse organotypic slices Sagittal hippocampal brain slices for organotypic culture were harvested from wild-type mice. After decapitation, all organs were rapidly removed and placed in ice-cold buffer (artificial cerebrospinal fluid (ACSF) (mmol / L): NaCl 125, KCl 2.4, NaHCO3 26, NaH2PO4 1.25, glucose 25, CaCl22 (0.5 mmol / L for dissection), MgCl21 (5 mmol / L for dissection), bubbled with carbogen) for the brain, and Ringer's buffer for all other organs. Most dissections were performed using high-magnesium (5 mmol / L) / low-calcium (0.5 mmol / L) ACSF to reduce the rate of cell death. The brain was hemisected and manually cut into approximately 100° sections from the midline. The hemisphere was then fixed to a slice stage with superglue, and 300 μm hippocampal slices were cut using a vibrating slicer and transferred to an incubation chamber containing circulating ACSF (bubbled with carbogen). All other organs were treated similarly, except that Ringer's buffer was used instead of ACSF. For the heart and spleen, cross-sections were obtained across the entire organ. The slices were incubated at 35°C for approximately 1 hour, after which the incubator was turned off and the bath was allowed to cool to room temperature.

[0185] To apply stress, the sections were transferred to beakers (containing ACSF or Ringer's buffer for the brain and other organs, respectively) that had been bubbled with N2 instead of carbogen for 3 hours prior to treatment. They were left in the beakers without gas bubbling to mimic hypoxia. In the treatment beakers, drugs were dissolved at a given concentration in buffer solution bubbled with N2. Following this, the sections were stained with PI and DAPI to visualize all cell nuclei (including intact cell nuclei). The sections were then placed on microscope slides, covered with coverslips, and imaged.

[0186] Preparation of zebrafish liver Freshly culled zebrafish (Danio reio) were dissected and the livers were rapidly removed. The livers were then placed in PBS buffer and immediately stressed with 1 mM hydrogen peroxide for 7 minutes in the presence or absence of 2500 μM EGTA. The livers were then washed in PBS to remove stress and stained with PI and DAPI to visualize all cell nuclei (including intact cell nuclei). The livers were then placed on microscope slides, covered with a coverslip, and imaged.

[0187] C. elegans (nematode) growth method C. elegans were maintained on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50 according to standard growth methods (S. Brenner, 1974). A C. elegans (N2H) hermaphrodite strain was used as the wild type. Hermaphrodite strains were used for experiments on day 1, and all maintenance and testing was performed at 20°C unless otherwise specified.

[0188] C. elegans stress assay Cold / freeze stress For each test, 100 worms were placed in a 1.5 ml Eppendorf tube (30 worms per tube) containing 100 μl of M9 buffer and then transferred to a -80°C incubator for 1.5 minutes. After stress, the worms were transferred to a 20°C incubator and maintained as usual on OP50NGM plates.

[0189] heat stress For each test, 100 worms were placed on NGM plates in an incubator at 37°C for 4.5 hours. After stress, worms were transferred to an incubator at 20°C and maintained as usual on OP50 NGM plates.

[0190] oxidative stress For each test, 100 worms were transferred to a 1.5 ml Eppendorf tube (20 worms per tube) containing 300 μL of 2 M hydrogen peroxide for 5 minutes. After stress, worms were transferred to a 20°C incubator and maintained as usual on OP50NGM plates.

[0191] Toxic stress For each test, 100 nematodes were transferred to 1.5 ml Eppendorf tubes (20 nematodes per tube) containing 300 μl of 1.3% (v / v) tert-butyl hydroperoxide (tBOOH) for 5 min. After stress, nematodes were transferred to a 20°C incubator and maintained as usual on OP50NGM plates.

[0192] Post-stress movement analysis in C. elegans Movement was scored according to an adapted method (from Herndon et al., 2002). Briefly, worms were recorded as either moving normally or as having poor movement. Poor movement was defined as the worm remaining motionless unless touched with a pick, in which case it would move forward only a few centimeters and then stop again, or would not move at all. 100 worms were used per test, and the percentage of worms with poor movement was scored.

[0193] Liposome-mediated drug delivery in C. elegans Drugs were dissolved in MilliQ water and encapsulated into liposomes according to the protocol of Shibamura et al., 2009. The drug used was EGTA at a final concentration of 2500 μM. Briefly, liposomes were prepared using L-α-phosphatidylcholine (LαPC) by first dissolving LαPC in distilled water at a concentration of 96 mg / ml. A 2x concentration of drug solution was mixed with LαPC in a 1:1 ratio and heated to 65°C. The drug-LαPC solution was then extruded into liposomes using a mini-Avanti extruder and a 100 nm pore size polycarbonate membrane. Liposome-encapsulated MilliQ water was used as an empty vector control.

[0194] After preparation, 25 μl of the liposome drug solution was spread onto a 25 μl spotted OP50 layer on an NGM plate, and then nematodes were placed on the plate.

[0195] Drosophila growth and stress Drosophila were grown under standard conditions. On day 1 of adulthood, they were starved for 8 hours and then fed + / - 2500 μM EGTA for 12 hours. Following this, the flies were transferred to 0°C for 10 minutes, then removed and viability was measured.

[0196] Oxidative stress in E. coli Two loopfuls of stationary-phase E. coli cells grown for 24 hours were collected in buffer and plated onto agar plates containing bactopeptone (80 μL from a 12-hour culture). The cells were then stressed for 50 minutes using 1 mM hydrogen peroxide. The treatment was EGTA 2500 μM during stress. The cells were then washed in buffer. After stress, the bacteria were washed in buffer and then diluted 1:500 with MilliQ water and plated onto agar plates containing bactopeptone and left overnight at 37°C. Following this, colony-forming units (CFUs) were counted. Three replicates were performed.

[0197] Oxidative stress in S. pombi (yeast) Cultures of S. pombe were exposed to 1 mM hydrogen peroxide for 50 minutes. Following this, the yeast cells were washed in buffer, stained with the dye propidium iodide (PI), placed on microscope slides, covered with a coverslip, and imaged using Nomarski epifluorescence microscopy to determine the percentage of necrotic yeast cells. The treatment was 2500 μM EGTA during stress. Two replicates were performed.

[0198] [Example] Data collection was carried out by Doppl SA (Swiss Federal Institute of Technology, Lausanne, affiliated with EPFL), Domainex (Cambridge, UK), and ICHOR Life Sciences (NY, US).

[0199] Example 1. Necrosis can be prevented and / or treated by blocking "predicted weak points" in cell physiology. One such measure is the use of relatively high doses of calcium chelators.

[0200] Traditionally, necrosis has been viewed as a chaotic process that cannot be intervened: it has been speculated that necrosis is the result of a number of random events that cause a loss of cellular homeostasis.

[0201] As mentioned above, we believe that any stress is followed by many random events that can lead to the loss of cellular homeostasis and necrosis. However, there are certain initiating events that make cells more vulnerable to stress (i.e., cellular weak points that cause greater damage than other processes). Inhibiting such predicted weak points should make cells more resistant to stress (i.e., necrosis can be prevented and / or treated). As a result, the amount of stress required for other random events to induce cell death should be much greater. Therefore, downstream conditions, such as tissue and organ damage, can also be prevented and / or treated. The predicted weak points shown in Figure 1 are: (1a) an increase in the intracellular cytoplasmic concentration of free calcium ions due to calcium movement across the cell plasma membrane, and (1b) an increase in the intracellular cytoplasmic concentration of free calcium ions due to movement from internal compartments where calcium is stored (primarily the endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR)). Following calcium entry, many proteins are activated in undesirable ways. As part of this futile protein activation, (2) calpain proteins are activated. Calpains then disrupt internal compartments, including lysosomes, resulting in (3) the activation and release of harmful components of internal compartments such as lysosomes, including the release of cathepsin proteins. Finally, (4) the movement of calcium ions across gap junctions to neighboring cells leads to the disruption of calcium homeostasis and the induction of necrosis in those neighboring cells. Store-operated calcium channels (SOCCs), also known as calcium release-activated calcium (CRAC) channels, are another source of calcium entry into cells. However, SOCC channels only become relevant after calcium depletion of the ER / SR; therefore, they are less important because they are expected to be less vulnerable than steps (1) through (4) described herein.

[0202] The inventors have discovered that a key step in the development of necrosis is an increase in cytosolic calcium ions, which can result from any one of three major sites of calcium ion entry into cells: via cell surface membrane channels, via gap junctions, and via the endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR). Because the cell membrane is in contact with the environment surrounding the cell, it is believed to exhibit a higher level of strengthening than ER / SR calcium channels and gap junctions. Furthermore, for cells in certain tissues with very tightly packed cells, calcium movement via ER / SR calcium channels and gap junctions becomes more threatening during stress.

[0203] Thus, the present inventors have surprisingly discovered that using a combination of compounds that block ER / SR calcium channels and gap junctions is particularly advantageous for preventing and / or treating necrosis. This combination can be further enhanced by additionally blocking calcium ion entry through the cell membrane. Thus, the present invention provides a combination therapy of an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor (e.g., a ryanodine receptor antagonist), a gap junction inhibitor, and a cell membrane calcium channel inhibitor. Additionally or alternatively, this combination can be further enhanced by other means that exhibit an effect similar to that of reducing cytoplasmic calcium ion concentration, for example, using a calcium chelator that sequesters calcium (or using a competitive inhibitor of calcium ions (e.g., other cations of similar charge and size, such as magnesium ions)). That is, the present inventors have made the surprising discovery that the most effective combination for preventing and / or treating necrosis is the simultaneous inhibition of ER / SR calcium channels and gap junctions, and that this combination can be enhanced with the addition of a plasma membrane calcium channel inhibitor and / or a calcium chelator (or a competitive inhibitor of calcium ions). Thus, the present invention provides a method for preventing and / or treating necrosis by inhibiting ER / SR calcium channels and gap junctions. The present invention provides a combination therapy using an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor (e.g., a ryanodine receptor antagonist) and a gap junction inhibitor. The present invention also provides a combination therapy using an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor (e.g., a ryanodine receptor inhibitor or antagonist), a gap junction inhibitor, and a plasma membrane calcium channel inhibitor. The present invention also provides a combination therapy using an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor (e.g., a ryanodine receptor inhibitor or antagonist), a gap junction inhibitor, and a calcium chelator. The present invention also provides a combination therapy using an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor (e.g., a ryanodine receptor inhibitor or antagonist), a gap junction inhibitor, a plasma membrane calcium channel inhibitor, and a calcium chelator.

[0204] The rationale for the combination therapy of the present invention can be further explained by considering what may occur when other combinations of calcium ion channel blockers are used: The inventors believe that if only calcium membrane channels are blocked, necrosis may still occur significantly, since calcium ions may still enter via the ER / SR and gap junctions; if only ER / SR channels are blocked, necrosis may still occur significantly, since calcium ions may still enter primarily via gap junctions; if only gap junctions are blocked, necrosis may still occur significantly, since calcium ions may still enter primarily via the ER / SR channels; if the combination of ER / SR channels and plasma membrane calcium ion channels is blocked, necrosis may still occur significantly, since calcium ions may still enter primarily via gap junctions; if the combination of gap junctions and plasma membrane calcium ion channels is blocked, necrosis may still occur significantly, since calcium ions may still enter primarily via the ER / SR channels.

[0205] Blockade of calpains and cathepsins (Figure 1) represents another therapeutic target for treating and / or preventing necrosis. Therefore, the present invention provides a combination of calpain and cathepsin inhibitors. However, these are only two of many downstream mediators of the destruction that occurs after cytoplasmic calcium ion elevation during cell necrosis. Therefore, inhibition of calpains and cathepsins may not be the most effective form of intervention.

[0206] In some of the following examples, human 3D cell cultures were employed using liver Hepg2 cells. 3D cell cultures are known to accurately recapitulate the cells' native environment. During embryonic development, cells aggregate and self-organize into complex multicellular structures. The same aggregation process can occur in in vitro culture systems as well, when cell-cell adhesion is favored over cell-to-cell adhesion to the substrate. The resulting 3D cell culture aggregates are known as spheroids. Spheroids exhibit extensive cell-to-cell adhesion, typically retain their endogenous extracellular matrix, and possess properties that closely resemble the corresponding in vivo tissues in both structure and function. A standard method for spheroid formation in U-bottom low-attachment plates was used in the examples presented herein.

[0207] In particular, spheroids typically cannot grow to large sizes. This is because, as spheroids grow, only peripheral cells obtain oxygen and nutrients from the cell culture medium. Cells within the spheroid die through apoptosis and necrosis, forming a large necrotic core (observable using standard viability stains). Thus, the formation of the necrotic core represents naturally occurring hypoxic and nutrient stress. This is similar to various forms of shock in humans, in which blood flow carrying nutrients and oxygen is interrupted to specific cells, tissues, and / or organs. To demonstrate that necrosis can be prevented and / or treated by the compositions and compounds of the present invention, some of the examples presented herein utilize such naturally formed necrotic cores and demonstrate that treatment results in pharmacological inhibition of necrotic core formation.

[0208] For example, Hepg2 cell spheroids generated at an initial seeding density of 500 cells in U-bottom low-attachment plates proliferate, form spheroids, and exhibit a prominent necrotic core by day 7, as observed using the standard viability dye DRAQ7™ (see Figure 25, control), which stains only the nuclei of dead and permeabilized cells and is therefore a marker of cell death.

[0209] Increased cytosolic calcium ion levels are a key vulnerability point for inducing necrosis. The present inventors have found that relatively high doses of chelators (i.e., agents that sequester cytosolic calcium) are effective agents for preventing cell death, such as necrosis, during stress. The chelators EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), and BAPTA AM (2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetic acid acetyloxymethyl ester) were tested in this example. As known in the art, cell culture doses of approximately 50 μM, 50 μM, and 1-3 μM, respectively, are considered to be the maximum doses at which toxicity is minimized, and therefore these doses are appropriate. When an eightfold higher dose of BAPTA-AM (i.e., 25 μM) was used, significant inhibition of necrosis was observed at day 7 (Fig. 2a). However, this dose was somewhat less toxic in some studies at earlier time points (Fig. 2a, middle row). A 50-fold higher dose of EGTA (i.e., 2500 μM) and an 80-fold higher dose of DTPA (i.e., 4000 μM) were required to inhibit necrosis (Fig. 2c). These latter two chelators also showed somewhat milder, but not statistically significant, toxicity at earlier time points.

[0210] Thus, it has been shown that the use of relatively high doses of chelating agents can inhibit necrosis. However, given potential toxic side effects, such relatively high doses of chelating agents may not be desirable for in vivo treatment. However, this discovery of the effectiveness of using high doses of chelating agents to treat or prevent necrosis indicates benefits for ex vivo use. Such uses include in vitro cell culture, the expansion of such cell cultures in the laboratory and in tissue engineering, and the preservation of transplants (including cells, tissues, and / or organs).

[0211] As will be apparent, the discussion in Example 1 and in the following examples generally applies to the present invention as a whole.

[0212] Example 2. Blocking individual steps associated with vulnerable points in cellular physiology does not significantly prevent and / or treat necrosis.

[0213] We blocked individual steps (Figure 1) related to predicted weak points in cell physiology that predispose cells to necrosis. In line with the above discussion of Hepg2 cell spheroids, we analyzed the formation of the necrotic core.

[0214] Inhibition of individual steps was found not to significantly reduce the level of cell necrosis compared to controls. The doses used in Example 2 (as shown in the associated figures) were based on literature estimates of the maximum dose that would produce minimal toxicity in cell culture. No significant preventive and / or therapeutic effect on necrosis was observed compared to controls in the following cases:

[0215] 1.) We tested a full range of individual plasma membrane calcium channel inhibitors, including dihydropyridines, non-dihydropyridines (phenylalkylamines and benzothiazepines), nonselective calcium channel inhibitors, and gabapentinoids, covering inhibition of L-, N-, P-, Q-, and / or T-type voltage-gated ion channels (Figure 3).

[0216] 2.) Various ER / SR channel inhibitors were tested (Figure 4).

[0217] 3.) A variety of gap junction inhibitors were tested (Figure 5), including vitamin A products such as retinoic acid, glycyrrhetinic acid, and its derivative carbenoxolone, long-chain alcohols such as heptanol and octanol, common halogenated volatile anesthetics such as halothane, fatty acids such as linoleic acid, arachidonic acid, and oleic acid, fatty acid amides such as oleamide, fenamic acids (arylaminobenzoates) such as flufenamic acid, niflumic acid, and meclofenamic acid, quinine, quinidine, and quinine derivatives such as mefloquine, and other inhibitors.

[0218] 4.) Various SOCC inhibitors were tested (Figure 6).

[0219] 5.) Various calpain inhibitors were tested (Figure 7).

[0220] 6.) Calpain / cathepsin non-selective protease inhibitors were tested (Figure 8).

[0221] 7.) Various cathepsin inhibitors were tested (Figure 9).

[0222] Example 3. With regard to potent efficacy and minimal toxic side effects, the best method for preventing and / or treating necrosis is to use a combination of a gap junction inhibitor and an endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR) calcium channel inhibitor.

[0223] Next, we tested compound combinations that simultaneously inhibited multiple targets in (Figure 1). Surprisingly, gap junction inhibition combined with ER / SR calcium channel inhibition resulted in significant inhibition of necrosis.

[0224] In particular, the gap junction inhibitor retinoic acid was tested in combination with the ER / SE calcium channel inhibitor dantrolene. Various doses of these two compounds were used individually as well as in combination. The individual compounds did not show a significant effect on necrosis inhibition (see Figure 10). In fact, the mean DRAQ7™ intensity values ​​exhibited by some of the individual compound doses were higher than those of the control, suggesting potential toxicity (e.g., 20 μM retinoic acid on day 7). However, in combination, a surprisingly strong inhibition of cell necrosis was observed for each combined dose (Figure 10). The observed effect was synergistic and exceeded the simple additive effect of a single compound that had no significant effect (Figure 10). Furthermore, no increased toxicity was observed with the combination prior to day 7 (Figure 10).

[0225] Surprisingly, a similar synergistic effect was observed when combining dantrolene and oleic acid, as with dantrolene and retinoic acid. In this example, oleic acid was used as a gap junction inhibitor instead of retinoic acid. The combination of dantrolene and oleic acid resulted in a significant reduction in cell necrosis (Figure 11). This effect exceeded additive effects. In fact, the individual dantrolene compounds had no significant effect on cell necrosis compared to the control, and the individual oleic acid compounds had no significant effect on cell necrosis compared to the control (Figure 11).

[0226] To further demonstrate this advantageous combination of ER / SR inhibitors and gap junction inhibitors, we used a variety of gap junction inhibitors in combination with dantrolene (Figure 12). The gap junction inhibitors included glycyrrhetinic acid, long-chain alcohols such as heptanol and octanol, common halogenated volatile anesthetics such as halothane, fatty acids, fatty acid amides, fenamates (arylaminobenzoates), and other inhibitors. As shown in Figure 12, individual gap junction inhibitors and the individual compound dantrolene had no significant effect. In many cases, administration of individual gap junction inhibitors resulted in higher levels of cell death than control. However, a surprisingly potent, more-than-additive, inhibition of necrosis was achieved by administering a gap junction inhibitor in combination with the ER / SR calcium channel inhibitor dantrolene. This effect was significant in all cases (see Figure 12).

[0227] Notably, this beneficial effect on necrosis suppression (i.e., this synergistic effect) was also observed when multiple gap junction inhibitors were used in combination with the ER / SR inhibitor dantrolene. Indeed, when multiple gap junction inhibitors were used in combination with dantrolene, almost no cell death or necrosis was observed, even at day 7 (Figure 13). Therefore, the combination of two or more gap junction inhibitors with an ER / SR calcium channel blocker is a particularly effective therapeutic strategy.

[0228] Next, we replaced the ER / SR calcium channel inhibitor dantrolene with the ER / SR calcium channel inhibitor ryanodine. Ryanodine was tested with and in combination with different gap junction inhibitors. Similar to dantrolene, ryanodine alone showed no significant difference from the control; in fact, most ryanodine tests showed higher levels of cell death than the control (Figure 14). Similarly, gap junction inhibitors alone showed no significant difference from the control. However, the combination of ryanodine with any gap junction inhibitor showed surprisingly potent, greater-than-additive, inhibition of cell death and necrosis (Figure 14). Notably, even with compounds such as ryanodine and octanol or linoleic acid, which showed higher toxicity compared to the control when used alone in most tests, the combination of one ER / SR calcium channel inhibitor and the other gap junction inhibitor showed significant inhibition of cell death and necrosis compared to the control (Figure 14).

[0229] Next, we used various other ER / SR calcium channel inhibitors, including trans-Ned19 (Figure 15), cis-Ned19 (Figure 16), procaine (Figure 17a), and ruthenium red (Figure 17b), in combination with a series of different gap junction inhibitors. Again, despite being potent gap junction inhibitors, and even more potent ER / SR calcium channel inhibitors, these compounds had no significant effect on the levels of cell death or necrosis when used individually. However, when the compounds were combined, i.e., an ER / SR channel inhibitor and a gap junction inhibitor, a surprisingly strong, supra-additive effect on necrosis suppression was observed (Figures 15, 16, and 17).

[0230] Example 4. Other combinations of two compounds, including a plasma membrane calcium channel inhibitor, a gap junction inhibitor, and an (ER) / sarcoplasmic reticulum (SR) inhibitor, do not exert significant effects on the prevention and / or treatment of cell necrosis.

[0231] Other combinations of cell membrane inhibitors, gap junction inhibitors, and (ER) / SR inhibitors were investigated. Compounds with high levels of efficacy and favorable safety profiles were used. The best compounds were dantrolene as an ER / SR calcium channel inhibitor and retinoic acid as a gap junction inhibitor. As shown herein, the specific combination of a gap junction inhibitor with an ER / SR inhibitor had a strong effect on necrosis inhibition. However, other combinations were not found to have a beneficial effect. (i.e., simultaneous blockade of ER / SR calcium ion channels and gap junctions was found to be advantageous for achieving significant effects on necrosis inhibition, and treatments that do not simultaneously block these two targets may result in cell necrosis when cells are stressed.) This is illustrated in the examples where an ER / SR calcium ion channel inhibitor was used in combination with a gap junction inhibitor (Figures 10-17).

[0232] When the gap junction inhibitor retinoic acid was used in combination with a series of different cell membrane inhibitors (e.g., dihydropyridines, non-dihydropyridines (phenylalkylamines and benzothiazepines), nonselective calcium channel inhibitors, and gabapentinoids, as well as inhibition of L-, N-, P-, Q-, and / or T-type voltage-gated ion channels), no significant effect on necrosis suppression was observed compared to controls (Figure 18).

[0233] When the ER / SR calcium channel binding inhibitor dantrolene was used in combination with a series of different cell membrane inhibitors (e.g., dihydropyridines, non-dihydropyridines (phenylalkylamines and benzothiazepines), nonselective calcium channel inhibitors, and gabapentinoids, as well as inhibition of L-, N-, P-, Q-, and / or T-type voltage-gated ion channels), no significant effect on necrosis inhibition was observed compared to controls (Figure 19).

[0234] Example 5. The combination of a gap junction inhibitor and an (ER) / sarcoplasmic reticulum (SR) inhibitor can suppress necrosis and is potentiated by a cell membrane calcium channel inhibitor.

[0235] The present inventors have found that simultaneous blockade of ER / SR calcium ion channels and gap junctions is advantageous for achieving significant effects on necrosis inhibition. This is because they discovered that an important step in the development of necrosis is an increase in cytosolic calcium ions, which can be attributed to one of three major sites of calcium ion entry into cells: cell surface membrane channels, gap junctions, and ER / SR calcium channels. The most susceptible sites of entry are, in particular, calcium ion entry via gap junctions and endoplasmic ER / SR. Because the cell membrane is in contact with the environment surrounding the cell, it is believed that cell membrane calcium ion channels exhibit a higher level of potentiation than ER / SR calcium channels and gap junctions.

[0236] Based on this, the most important therapeutic combination for necrosis inhibition is the combination of one or more ER / SR calcium channel inhibitors with one or more gap junction inhibitors. We have found that this combination can be further enhanced by the addition of one or more cell membrane inhibitors. This is supported herein. We further combined a gap junction inhibitor and an ER / SR calcium channel inhibitor, namely, dantrolene in combination with retinoic acid, with various cell membrane calcium channel inhibitors. Surprisingly, this combination resulted in potent inhibition of necrosis. This effect was statistically significant when compared with the control and with the combination of dantrolene and retinoic acid alone (Figure 20a). Notably, the three classes of combinations, namely, the combination of a gap junction inhibitor with an ER / SR calcium channel inhibitor and a cell membrane calcium channel inhibitor, were more effective than the additive effect of any of the three compounds administered alone (see Figure 20a for the combinations, and Figures 3, 4, and 5, where the individual compounds have no significant effect on the control; all figures are comparable to three independent tests of each compound performed as part of the same experiment).

[0237] This benefit was further demonstrated by using other gap junction inhibitors and different ER / SR calcium channel inhibitors. The same effect was observed (Figure 20b, c, d). This strongly supports the idea that, regardless of the compound, the combination of a gap junction inhibitor with an ER / SR calcium channel inhibitor and a plasma membrane calcium channel inhibitor results in a potent, statistically significant, additive inhibitory effect on necrosis. In fact, the effect was so strong that even by day 7, when significant necrosis and necrotic cores were observed in the control, the triple combination of a gap junction inhibitor with an ER / SR calcium channel inhibitor and a plasma membrane calcium channel inhibitor produced little increase in necrosis (Figure 20).

[0238] Example 6. The combination of a gap junction inhibitor and an (ER) / sarcoplasmic reticulum (SR) inhibitor can suppress necrosis and is potentiated by a calcium chelator.

[0239] Another means of reducing cytoplasmic calcium ion concentration is by using drugs that capture calcium ions, i.e., calcium chelators. The addition of a calcium chelator to the combination of a gap junction inhibitor and an ER / SR calcium channel blocker has been shown to have an additional beneficial effect. The inventors believe that this is because, by acting on a different target, efficacy is increased while toxicity is less likely to increase.

[0240] When this was tested with different chelators, it was found that the addition of a calcium chelator to the combination of a gap junction inhibitor and an ER / SR calcium channel blocker had an additional beneficial effect in inhibiting necrosis without any additional toxic effects (Figure 21). Indeed, the toxic effects previously shown to cause variability between studies with chelators (Figure 2) were not observed with this combination of a gap junction inhibitor with an ER / SR calcium channel blocker and a calcium chelator.

[0241] Example 7. Other compounds, including calpain inhibitors, cathepsin inhibitors, and SOCC inhibitors, do not potentiate the combination of gap junction inhibitors and (ER) / sarcoplasmic reticulum (SR) inhibitors.

[0242] To further test our hypothesis, we investigated the combination of a gap junction inhibitor with an ER / SR calcium channel inhibitor (i.e., dantrolene combined with retinoic acid) along with other combinations of three compounds, based on the key targets of vulnerability in cellular physiology that predispose cells to cell death and necrosis under stress (see Figure 1). Other compounds tested in combination here are a calpain inhibitor, a cathepsin inhibitor, and a SOCC inhibitor.

[0243] The data was as follows:

[0244] 1.) When various calpain inhibitors were added to dantrolene in combination with retinoic acid, no further reduction in cell death or necrosis was observed compared to the combination of dantrolene alone with retinoic acid. In fact, the three combinations here were more variable, with many tests producing levels of cell death greater than the maximal level observed with the combination of dantrolene and retinoic acid alone (Figure 22a). This is in contrast to the addition of cell membrane inhibitors and chelators, which potentiated the effect of the combination of dantrolene and retinoic acid alone (Figures 20 and 21).

[0245] 2.) The same trend was observed when various cathepsin inhibitors were added to the dantrolene and retinoic acid combination (Figure 22b), and these three combinations did not significantly enhance the effects of dantrolene and retinoic acid alone. Again, this is in contrast to the addition of cell membrane inhibitors and chelators, which enhanced the effects of the dantrolene and retinoic acid combination alone (Figures 20 and 21).

[0246] 3.) The same trend was observed when a calpain / cathepsin nonspecific protease inhibitor was added to the dantrolene and retinoic acid combination (Figure 22c), and this triple combination did not significantly enhance the effects of the dantrolene and retinoic acid combination alone. Again, this is in contrast to the addition of cell membrane inhibitors and chelators, which enhanced the effects of the dantrolene and retinoic acid combination alone (Figures 20 and 21).

[0247] 4.) The same trend was observed when various SOCC inhibitors were added to the dantrolene and retinoic acid combination (Figure 22d), and these three combinations did not significantly enhance the effects of dantrolene and retinoic acid alone. Again, this is in contrast to the addition of cell membrane inhibitors and chelators, which enhanced the effects of dantrolene and retinoic acid alone (Figures 20 and 21).

[0248] Example 8. Combinations of cathepsin inhibitors with calpain inhibitors (but not combinations of two or more calpain-only inhibitors or two or more cathepsin-only inhibitors) exert beneficial effects that are greater than additive.

[0249] Calpains and cathepsins are only two of many downstream mediators of the destruction that follows cytosolic calcium ion elevation during cell necrosis. Therefore, inhibiting either calpains alone or cathepsins alone may have limited efficacy, as shown in this example, with no significant effect on cell death / necrosis compared to controls (Figures 7 and 9). Furthermore, nonspecific calpain / cathepsin inhibitors run the risk of causing off-target toxic effects. Indeed, when tested, nonspecific calpain / cathepsin inhibitors did not demonstrate significantly effective inhibition of cell death / necrosis compared to controls (Figure 8).

[0250] The data was as follows:

[0251] 1.) When calpain-only inhibitors were combined, no beneficial additive effect was observed, and the combination showed no significant difference from the control (Figure 23a).

[0252] 2.) When cathepsin-only inhibitors were combined, no beneficial additive effect was observed and the combination was not significantly different from the control (Figure 23b).

[0253] 3.) Combining a calpain inhibitor with a cathepsin inhibitor showed a surprising beneficial and supra-additive effect, with the combination showing significant differences from the control (Figure 23c, data can be compared with Figures 7 and 9 for compounds tested alone which showed no significant differences from the control).

[0254] Example 9. Utilizing the safety profile of over-the-counter (OTC) compound combinations.

[0255] It may be advantageous to use combinations of compounds known to minimize toxicity when used alone. This can be particularly important when the compounds are available without a prescription. Examples of such compounds include linoleic acid and retinoic acid, as well as over-the-counter (OTC) compounds such as astaxanthin (which exhibits dual inhibitory effects as a gap junction inhibitor and a cathepsin inhibitor) and other similar carotenoids. By combining such OTC compounds associated with different targets in Figure 1, it is possible to achieve necrosis inhibition with minimal toxicity. This example demonstrates that the combination of astaxanthin with retinoic acid and linoleic acid resulted in a modest, yet surprisingly beneficial, inhibition of cell death and necrosis (Figure 24). This is particularly useful in vivo, where toxicity is a major concern, and when considering supplements to improve overall health and performance and reduce damage in individuals at risk for cell death and necrosis (e.g., individuals engaged in strenuous exercise or individuals with other underlying conditions associated with cell death and necrosis).

[0256] Example 10. The combination of gap junction inhibitors and endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR) calcium channel inhibitors used to prevent cell death and necrosis demonstrates robust safety and efficacy across multiple cell types.

[0257] Further analysis was performed in this example to demonstrate that the combination described herein is effective across multiple cell types and robustly blocks cell death and necrosis without significant toxic side effects.

[0258] First, we performed an analysis on HepG2 spheroids. After the addition of a combination of a gap junction inhibitor and an ER / SR calcium channel blocker, we examined the level of cell death over time, as well as the variation in spheroid size and the overall morphology and appearance of the spheroids. The combination used here was dantrolene in combination with retinoic acid. As mentioned above, to identify the necrotic core, we used the standard viability dye DRAQ7™ (a cell death marker), which stains only the nuclei of dead and permeabilized cells.

[0259] In contrast, when the combination was added to spheroids on day 2 of spheroid formation, no necrotic core was observed, even up to 7 days after spheroid formation, whereas large necrotic core areas were observed in the control spheroids (Figure 25).

[0260] Notably, when spheroid size was analyzed over time (where spheroid size is an indicator of spheroid growth), no significant difference was observed with the added combination compared to the control (Figure 26). Furthermore, compared to the control, the added combination did not significantly affect spheroid morphology; that is, there was no impairment in spheroid integrity, formation, or growth (Figure 26). Therefore, the combination of a gap junction inhibitor and an ER / SR calcium channel inhibitor did not cause significant toxicity.

[0261] Next, we repeated the same experiment using a different cell line, HEK293 cells, which grow faster in culture. In HEK293 cells, necrotic cores were clearly visible in the control by day 5 of culture (Figure 27). Even in this different cell type, the combination of a gap junction inhibitor and an ER / SR calcium channel inhibitor significantly suppressed necrosis compared to the control (Figure 27).

[0262] Taken together, these findings demonstrate that the combined addition of gap junction inhibitors and ER / SR calcium channel blockers significantly suppresses cell death and necrosis in multiple cell types.

[0263] Spheroid size was again analyzed over time, and no significant differences were observed between the added combinations across the different cell types compared to the control (Figure 28). Furthermore, the added combinations did not significantly affect spheroid morphology compared to the control; that is, there was no impairment in spheroid integrity, formation, or growth (Figure 28). Taken together, this demonstrates that the combination of gap junction inhibitors and ER / SR calcium channel inhibitors did not result in significant toxicity across multiple cell types.

[0264] Finally, in additional experiments, a lactate dehydrogenase (LDH-Glo™) assay was performed on HepG2 spheroids using supernatants from control-treated wells and combination-treated wells on day 7 as a secondary indicator of cell death. Spheroids treated with a combination of a gap junction inhibitor and an ER / SR calcium channel inhibitor exhibited significantly lower levels of cell death than controls (Figure 29). This demonstrates that the same effect is observed regardless of the marker of cell death. Therefore, compositions comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors are effective for use in methods of treating or preventing necrosis and cell death, as well as downstream pathologies such as tissue or organ damage, in subjects.

[0265] Example 11. The combination of a gap junction inhibitor and an endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR) calcium channel inhibitor + / - a cell membrane inhibitor exerts potent preventive and therapeutic effects against cell death and necrosis across a variety of stressors.

[0266] Thus far, the examples have focused on the stressors of hypoxia and nutrient deprivation. To test other stressors, this example used an oxidative / toxic stress model in which primary human intestinal fibroblast (HIF) spheroids were exposed to hydrogen peroxide. Standard viability calcein AM / ethidium homodimer-1 staining was used as a marker of cell death. After 6 hours of exposure to 800 μM hydrogen peroxide, just over 25% of the cells in the control group died (i.e., compared to Triton X-100, which killed all cells). The addition of a gap junction inhibitor in combination with an ER / SR calcium channel inhibitor resulted in a surprisingly large and statistically significant decrease in the level of cell death and necrosis compared to the control group (Figure 30). Notably, this effect exceeded the additive effects of the individual components of the combination alone (Figure 30).

[0267] An even better effect was observed when the combination of gap junction inhibitors and ER / SR calcium channel blockers was further enhanced by adding a plasma membrane calcium channel blocker to the combination. After stress, this combination of the three compounds showed little increase in necrosis compared to unstressed controls. A surprisingly large and statistically significant reduction in cell death and necrosis was obtained compared to stressed controls (Figure 30). This effect clearly exceeded additive effects (Figure 30).

[0268] In contrast, the other combinations tested did not show significant preventative effects on necrosis inhibition. This included the combination of an ER / SR calcium channel inhibitor with a plasma membrane calcium channel inhibitor, which did not show significant improvement over the control (Figure 30), and the combination of a gap junction inhibitor with a plasma membrane calcium channel inhibitor, which did not show significant improvement over the control (Figure 30). Thus, ER / SR calcium channel inhibitors must be used in combination with gap junction inhibitors to achieve the most effective treatment.

[0269] To further characterize the effect of combining an ER / SR calcium channel inhibitor with a gap junction inhibitor as a therapeutic (and preventative) strategy following stress, we tracked stress cell death in spheroids over a time course of up to 3 days. Various stress conditions were used, including a 6-hour exposure to 500 μM hydrogen peroxide (Figure 31), a 6-hour exposure to 800 μM hydrogen peroxide (Figure 32), and a 3-hour exposure to 1 mM hydrogen peroxide (Figure 33). When the 6-hour exposure on day 1 exceeded 800 μM hydrogen peroxide, the majority of cells in the control group died by day 3 (Figure 34). In all cases, adding a combination of a gap junction inhibitor and an ER / SR calcium channel inhibitor during and after stress significantly reduced cell death and necrosis over time (Figures 31, 32, 33, and 34).

[0270] Taken together, these results demonstrate that the combination of gap junction inhibitors and endoplasmic reticulum ER / SR calcium channel inhibitors (+ / - plasma membrane inhibitors) exerts potent preventive and therapeutic effects against cell death and necrosis across a variety of stressors.

[0271] Example 12. The combination of gap junction inhibitors and endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR) calcium channel inhibitors used to prevent cell death and necrosis exhibits a robust safety profile in primary human cells.

[0272] The potential toxic effects of combining gap junction inhibitors with ER / SR inhibitors were assessed in primary HIF cell spheroids by (i) assessing spheroid growth over time when the combination was added either on day 0 before spheroid formation or on day 1 after spheroid formation, and (ii) quantifying ATP levels using a standard CellTiter-Glo® 3D cell viability assay.

[0273] After cell seeding on day 0, spheroids formed at 4,000 cells per spheroid by day 1. Except for some spheroid compaction, which was largely offset by low levels of cell proliferation, there was little change in spheroid size from day 1 to day 3 (Figures 35a and 35b). Notably, the addition of a gap junction inhibitor in combination with an ER / SR inhibitor (administered either on day 0 before spheroid formation or on day 1 after spheroid formation) did not significantly decrease spheroid size, indicating that the combination did not have any severe toxic effects (Figure 35c).

[0274] ATP levels are a marker of cell viability, with higher levels indicating higher cell viability. When ATP levels were assessed on day 3 in cells treated with the combination on day 1 of spheroid formation, no significant decrease in ATP levels was observed (in fact, the average ATP levels were higher in treated cells than in controls) (Figure 35d).

[0275] In summary, these results suggest that the combination of gap junction inhibitors and ER / SR inhibitors has a robust safety profile in primary human cells.

[0276] Example 13. A combination of a gap junction inhibitor and an endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR) calcium channel inhibitor can inhibit cell death, necrosis, and necrotic core following nutrient deprivation and hypoxic stress in primary human cells.

[0277] Primary HIF cells showed spontaneous necrotic core formation 6 days after spheroid formation. These cores formed due to oxygen and nutrient deficiency inside the spheroids (Figure 36). Notably, the combination of a gap junction inhibitor with an endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR) calcium channel inhibitor inhibited the formation of this necrotic core (Figure 36). This example demonstrates the robust beneficial effects of this combination against multiple stressors.

[0278] Example 14. The benefits of blocking "predicted weak points" in cell physiology to prevent cell necrosis, increase survival, and reduce activity impairment are observed across multiple species.

[0279] We tested whether the anti-necrosis effect of inhibiting predicted weak points was maintained across species ranging from simple unicellular bacteria and yeast to roundworms, fruit flies, fish, and other mammals.

[0280] The bacterium E. coli and yeast S. pombe were subjected to a stress of the toxin hydrogen peroxide (1 mM H2O2 for 50 min) in the presence or absence of a high dose (2500 μM, see Figure 2) of the chelating agent EGTA (an inhibitor of a predicted vulnerable point for the loss of calcium ion homeostasis in the cytosol).

[0281] After stress, bacterial cells were placed on agar plates and grown overnight at 37°C, and then colony forming units (CFU) were counted.

[0282] For yeast, cells were stained with the standard viability dye propidium iodide (PI), which enters only necrotic cells, placed on microscope slides, covered with a coverslip, and imaged using Nomarski epifluorescence microscopy to determine the percentage of cells that were necrotic (total cells present can be counted by Nomarski microscopy).

[0283] Notably, the use of high levels of calcium chelators resulted in reduced levels of necrosis in both bacteria and yeast. In bacteria, this was observed as an increase in CFUs, which represent a higher proportion of viable cells (Figure 37a). In yeast, this effect was observed as a decrease in the proportion of cells stained with PI (Figure 37b).

[0284] Overall, these results indicate that the use of high doses of chelating agents in unicellular organisms such as yeast and bacteria, as well as mammalian cells, can prevent cell necrosis during stress by targeting and inhibiting key predicted vulnerable points in cell physiology.

[0285] Next, we used Caenorhabditis elegans (nematode) as a model to score survival after stress. Because it is difficult to directly administer free drugs to this model (C. elegans is a filter feeder that preferentially ingests particulate matter), we used liposomes as vectors. We exposed the nematodes to various stresses. These included heat stress (placed at 37°C for 4.5 hours; the optimal temperature for this species is approximately 18–20°C; temperatures above this are more stressful, and even 25°C can adversely affect reproduction); cold / freeze stress (placed at -80°C for 1.5 minutes); oxidative stress (placed in 2 M hydrogen peroxide for 5 minutes); and toxic stress (placed in 1.3% (v / v) tert-butyl hydroperoxide (tBOOH) for 5 minutes).

[0286] High doses of chelating agents in liposomes were administered to nematodes 3 hours before stress (i.e., as a preventative measure) or 3 hours after stress (i.e., as a post-stress treatment). In all cases, across all stresses, a significant increase in nematode survival was observed with the high dose of chelating agent compared to controls, regardless of the timing of treatment administration (Figure 38). These results demonstrate a robust reduction in overall necrosis during stress, regardless of the type of stress, and no significant toxic effects were observed from the treatment. Thus, the inhibitors described herein are effective in preventing necrosis and improving survival after stress. These results also demonstrate a robust reduction in overall necrosis when treatment was administered after stress, regardless of the stressor, and no significant toxic effects were observed from the treatment. Thus, the inhibitors described herein are effective in treating necrosis and improving survival after stress. These results also demonstrate that the inhibitors described herein can be administered via a variety of routes, including the use of vectors such as liposomes.

[0287] Specifically, after stress, C. elegans exhibited motor impairment, with the worms exhibiting slowed and uncoordinated movements. The percentage of worms with this motor impairment was recorded after stress. Worms administered high levels of the chelator before stress showed a marked and statistically significant improvement in behavior, regardless of the stressor (FIG. 39). Thus, these results demonstrate that administration of the inhibitors described herein reliably reduces overall motor impairment after stress.

[0288] Drosophila melanogaster fruit flies were similarly subjected to heat and cold stress. Heat stress consisted of exposure to the toxin hydrogen peroxide. The flies were starved for a short period of 8 hours and then fed a high dose of a calcium chelator, yeast, for 12 hours to ensure sufficient drug delivery. The doses used were 0 or 2500 μM EGTA. Then, cold / freeze stress was applied, in which the flies were placed at 0°C for 10 minutes, after which fly survival was recorded every 12 hours. Significant protection was observed with 2500 μM of the calcium chelator EGTA (Figure 40). Thus, as with unicellular yeast and bacteria, and nematodes, whole-organism protection by blocking predicted vulnerable points for cell death and necrosis was observed in Drosophila.

[0289] Moving to more complex animals, intact livers from freshly culled and dissected zebrafish (Danio reio) were placed in PBS buffer and immediately exposed to stress with the toxin hydrogen peroxide in the presence or absence of EGTA. The livers were then washed in PBS and stained with PI and DAPI to visualize all cell nuclei (including intact cell nuclei). The livers were then placed on microscope slides, covered with coverslips, and imaged, and the percentage of PI-stained cells was recorded (relative to all cells visible in the image, i.e., DAPI-stained cells). EGTA-treated livers showed a reduced proportion of necrotic (PI-stained) cells (Figure 41).

[0290] Moving to mammals, organotypic slices were prepared from various organs, including mouse brain (hippocampal slices), spleen, and heart, and placed in the appropriate oxygenated buffer. Following this, the oxygen supply was changed to nitrogen gas to induce hypoxic stress in the presence or absence of EGTA in the buffer. Cells were then stained with PI and DAPI, and the percentage of necrotic (PI-stained) cells was recorded. The presence of high concentrations of the calcium chelator EGTA resulted in significant improvements in all three tissues (Figure 42), demonstrating protective effects at the cellular, tissue, and organ levels.

[0291] Example 15. Further evidence in human cells and tissue engineering applications of blocking "predicted weak points" in cell physiology to inhibit cell necrosis.

[0292] Furthermore, the protective effect of inhibiting fragile points predicted to predispose cells to necrosis in cell culture was examined in other forms of cell culture: (i) HEK293 suspension culture and (ii) different techniques used to generate Hepg2 spheroids, namely, hanging drop culture and culture on agarose.

[0293] HEK293 suspension cultures were treated with high doses of EGTA for 2 hours before and during stress. The stress consisted of exposure to 1 M hydrogen peroxide, along with an untreated control. Cells were then stained with DAPI and PI and Nomarski epifluorescence imaging was performed. The percentage of necrotic cells (PI staining relative to total DAPI stained cells) was significantly reduced in EGTA-treated cells (Figure 43a), indicating that the protective effect was maintained regardless of cell culture morphology.

[0294] Next, we performed 3D cell culture using Hepg2 cells and standard hanging drop protocols for spheroid formation. A particular challenge in tissue engineering is the formation of necrotic cores in 3D cell cultures, which means that organoids, organs, and the like cannot currently be grown to relatively large sizes in the laboratory. Because necrotic cores disrupt physiological function, the formation of vasculature, which occurs more quickly than necrotic core formation, presents a challenge. To investigate the usefulness of our compounds in tissue engineering, we plated 20,000 cells in the presence or absence of EGTA (0, 250, and 500 mM) and then analyzed necrotic core formation by imaging the spheroids after 5 days. Note that 20,000-cell spheroids are at risk of developing cores as early as 1 day. The presence of 250 mM and 500 mM EGTA significantly improved the necrotic core in treated spheroids, with no visible necrotic cores even at day 5 (Figure 43b). Therefore, in conjunction with the data in (Figures 2-36), the usefulness of necrosis inhibition in tissue engineering can be evaluated.

[0295] Example 16. Preservation of cells, tissues, and organs in University of Wisconsin (UW) organ preservation medium supplemented with a combination of gap junction inhibitors and endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR) calcium channel blockers results in reduced cell death and necrosis.

[0296] UW solution is a typical organ preservation medium. Kidneys were removed from culled mice and perfused with UW supplemented with or without a combination of gap junction inhibitors and endoplasmic reticulum (ER / SR) calcium channel inhibitors (i.e., dantrolene and retinoic acid). The kidneys were then kept at 4°C for 36 hours to mimic cold storage, and then rewarmed to 37°C to mimic warm storage / reperfusion. Following this, the levels of viable cells (i.e., neither apoptotic nor necrotic) and necrotic cells were measured using flow cytometry. Using a sample size of only four kidneys per condition, a marginally significant difference was again observed (p = 0.057), with an average 35% reduction in necrotic death and a 33% reduction in nonviable dead cells (Figure 44).

[0297] Example clauses The invention is further described by the following numbered clauses:

[0298] 1. A calcium activity inhibitor, calpain inhibitor, cathepsin inhibitor, and / or gap junction inhibitor for use in the treatment or prevention of (i) necrosis, (ii) tissue damage, or (iii) organ damage in a subject.

[0299] 2. A method of treating or preventing (i) necrosis, (ii) tissue damage, or (iii) organ damage in a subject, comprising: The method comprises administering to the subject a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to treat or prevent necrosis, tissue damage, or organ damage.

[0300] 3. A method for preventing cell or tissue necrosis, comprising: The method comprises contacting the cells or tissue with a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to prevent necrosis of the cells or tissue.

[0301] 4. The calcium activity inhibitor, calpain inhibitor, cathepsin inhibitor, and / or gap junction inhibitor for use according to clause 1 or the method according to clause 2, wherein the tissue damage is one or more or all selected from the group consisting of burns, multiple organ dysfunction syndrome, organ failure, surgical trauma, bedsores, chemical burns, radiation burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, physical injury, mechanical trauma, thermal injury, cold injury, chilblains, trench foot, frostbite, avascular necrosis, pressure injuries, and skin graft failure.

[0302] 5. The calcium activity inhibitor, calpain inhibitor, cathepsin inhibitor, and / or gap junction inhibitor for use according to clause 1 or 4, or the method according to any one of clauses 2 to 4, wherein the tissue is one or more tissues selected from the group comprising or consisting of epithelial tissue, connective tissue, and muscle tissue.

[0303] 6. A composition comprising one or more selected from calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and gap junction inhibitors.

[0304] 7. The composition of clause 6, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more selected from a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor.

[0305] 8. A cosmetic composition comprising a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor and a cosmetically acceptable carrier.

[0306] 9. The composition of clause 8, wherein the composition comprises two or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and a gap junction inhibitor.

[0307] 10. A method for producing a cosmetic composition, comprising: The method comprises contacting one or more selected from calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and / or gap junction inhibitors with a cosmetically acceptable carrier to produce a cosmetic composition.

[0308] 11. A method of using a cosmetic composition, comprising: The method comprises applying the cosmetic composition of clause 8 or clause 9 to the skin of a subject.

[0309] 12. A food preservative comprising one or more selected from calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors, and gap junction inhibitors.

[0310] 13. Foods containing food preservatives as defined in clause 12.

[0311] 14. A method of preserving food, comprising contacting the food with a food preservative according to clause 12 to preserve the food.

[0312] 15. A method for preserving a living tissue or organ, comprising culturing or maintaining said tissue or organ in a medium comprising a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor to preserve said tissue or organ.

[0313] 16. A method of manipulating a tissue or organ, comprising contacting said organ or tissue with a medium comprising a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor.

[0314] 17. A method for storing or culturing blood cells, comprising contacting a medium containing the blood cells with a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor, and / or a gap junction inhibitor.

[0315] 18. The method of clause 17, wherein the medium is blood, plasma, or serum.

[0316] 19. A calcium activity inhibitor, calpain inhibitor, cathepsin inhibitor, and / or gap junction inhibitor for use according to any one of clauses 1, 4, and 5, wherein the calcium activity inhibitor is a calcium chelator, calcium channel blocker or antagonist, ryanodine receptor antagonist, and / or InsP3R antagonist; a method according to any one of clauses 2 to 5, 10, 11, 14 to 18, a composition according to any one of clauses 6 to 9, a food preservative according to clause 12, and a food according to clause 13.

[0317] 20. The calcium channel blocker is a voltage-dependent Ca 2+ 19. A calcium activity inhibitor, calpain inhibitor, cathepsin inhibitor, and / or gap junction inhibitor for use according to clause 19, which blocks the increase in intracellular concentration of free calcium ions by blocking calcium channels, Orai channels, and / or store-operated calcium channels; the method according to clause 19, the composition according to clause 19, the food preservative according to clause 19, and the food according to clause 19.

[0318] 21. A calcium activity inhibitor, calpain inhibitor, cathepsin inhibitor, and / or gap junction inhibitor for use according to clause 19, the method according to clause 19, the composition according to clause 19, the food preservative according to clause 19, and the food according to clause 19, wherein the calcium channel blocker is a dihydropyridine, a non-dihydropyridine, or a gabapentinoid.

[0319] 22. A calcium activity inhibitor, calpain inhibitor, cathepsin inhibitor, and / or gap junction inhibitor for use according to clause 21, the method according to clause 21, the composition according to clause 21, the food preservative according to clause 21, and the food according to clause 21, wherein the non-dihydropyridine is a phenylalkylamine or a benzothiazepine. [Brief explanation of the drawings]

[0320] [Figure 1]Figure 2 illustrates predicted vulnerability points in cell physiology that predispose cells to necrosis. These vulnerability points include (i) an increase in cytosolic calcium due to calcium ion movement into the cytosol through plasma membrane calcium ion channels; (ii) an increase in cytosolic calcium due to calcium ion movement from organelles, particularly the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR), through ER / SR calcium ion channels; and (iii) an increase in cytosolic calcium due to the movement of calcium ions and other damaging factors from one cell to another through gap junctions. Other vulnerability points include excessive activation of calpains, excessive activation of cathepsins, and the opening of store-operated calcium channels (also called calcium release-activated calcium channels) after ER calcium depletion. The most important of these steps is calcium ion entry through ER / SR calcium ion channels and gap junctions, followed by entry through plasma membrane calcium ion channels, as described in more detail in Example 1 below. Figures 2-28 show the formation of a necrotic core in HepG2s spheroids under different stressors and treatment conditions, as described below. Spheroids are spherical cell aggregates that maintain cell-cell and cell-matrix interactions in an environment that mimics real-world conditions. Spheroids cannot be maintained for long periods of time or grown large due to the formation of a necrotic core (cells in the center of the spheroid lack oxygen and adequate nutrients, and hypoxia primarily causes cell death via necrosis). [Figure 2] This figure shows that the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in Hepg2 spheroids was reduced after 7 days by the addition of a relatively high dose of a calcium chelator. Treatments were various doses of different chelators. The red dotted line represents the mean value of the control. Three experiments (n=2 per experiment). The violin plot shows the distribution of data with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 3]This figure shows that the addition of various plasma membrane calcium channel inhibitors does not reduce the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Treatments were different plasma membrane calcium channel inhibitors. The inhibitors were added at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 4] This figure shows that the addition of various endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments were endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three replicates (n = 2 per replicate). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control by two-way ANOVA (Dunnett's correction). [Figure 5] This figure shows that the addition of various gap junction inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments were gap junction inhibitors at doses predicted to be the maximal dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three replicates (n = 2 per replicate). The violin plot shows the distribution of data with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 6]This figure shows that the addition of various store-operated calcium channel (SOCC) inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatment was with SOCC inhibitors at a dose predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three replicates (n = 2 per replicate). The violin plot shows the distribution of data with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 7] This figure shows that the addition of various calpain inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments were calpain inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three replicates (n = 2 per replicate). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 8] This figure shows that the addition of a nonspecific calpain / cathepsin inhibitor does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. The treatment was a nonspecific calpain / cathepsin inhibitor at a dose predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 9]This figure shows that the addition of various cathepsin inhibitors does not reduce the formation of necrotic cores in HepG2s spheroids induced by hypoxia and nutrient deprivation stress. Treatments were cathepsin inhibitors at doses predicted to be the maximum dose at which minimal toxicity was observed. The red dotted line represents the mean control value. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 10] This figure shows that the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included different doses of dantrolene alone, different doses of retinoic acid alone, and different doses of dantrolene and retinoic acid in combination. The combination exerts a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 11]This figure shows that the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor oleic acid significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments were dantrolene and oleic acid, alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 12] This figure shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included dantrolene and various gap junction inhibitors, alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The dotted red line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 13]This figure shows that the addition of dantrolene, an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor, in combination with two or more gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. The treatment consisted of a combination of dantrolene and two or more gap junction inhibitors; the individual components of the combination were also tested separately. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment) were performed. The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 14] This figure shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included ryanodine and various gap junction inhibitors, alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The dotted red line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 15]This figure shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor trans-Ned19 in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included trans-Ned19 and various gap junction inhibitors, alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The dotted red line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 16] This figure shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor cis-Ned19 in combination with various gap junction inhibitors significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included cis-Ned19 and various gap junction inhibitors, alone and in combination. The combination exerted a supra-additive effect, whereas the individual component treatments showed no significant difference from the control. The dotted red line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to control. [Figure 17]This figure shows that the addition of either the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor procaine or the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ruthenium red, in combination with the gap junction inhibitor retinoic acid, significantly reduced the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were procaine and the gap junction inhibitor retinoic acid, alone and in combination. (b) Treatments were ruthenium red and the gap junction inhibitor retinoic acid, alone and in combination. While the individual component treatments for the combination (both (a) and (b)) were not significantly different from the control, the combination (both (a) and (b)) exerted a greater-than-additive effect. The red dotted line represents the mean value for the control. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control. [Figure 18] This figure shows that the addition of a combination of the gap junction inhibitor retinoic acid and various plasma membrane calcium channel inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments were a combination of the gap junction inhibitor retinoic acid and various plasma membrane calcium channel inhibitors. The combinations showed no significant differences from the control. The red dotted line represents the mean value of the control. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted lines representing quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control. [Figure 19]This figure shows that the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene in combination with various plasma membrane calcium channel inhibitors does not reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. The treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene in combination with various plasma membrane calcium channel inhibitors. The combinations showed no significant differences from the control. The red dotted line represents the control mean. Three experiments (n = 2 per experiment). The violin plot shows the distribution of data with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control. [Figure 20a-1]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20a-2]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20a-3]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20b-1]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20b-2]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20b-3]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20c-1]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20c-2]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20c-3]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20d-1]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20d-2]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 20d-3]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a plasma membrane calcium channel inhibitor, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different plasma membrane calcium channel inhibitors. (b) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor palmitoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (c) Treatments: the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor linoleic acid alone, and their combination with the plasma membrane calcium channel inhibitor verapamil. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid alone, and their combination with the plasma membrane calcium channel inhibitor alandipine. The combination of three compounds (i.e., an ER / SR calcium channel inhibitor, a gap junction inhibitor, and a plasma membrane calcium channel inhibitor) was more effective than the combination of an ER / SR calcium channel inhibitor and a gap junction inhibitor alone. However, it is clear that both the two-compound combination and the three-compound combination exerted a greater than additive effect compared to the individual components of the combination versus the control (see Figures 3, 4, and 5 for individual components showing no significant difference from the control. Figures 3, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the two-compound combination, where one compound is a gap junction inhibitor and the other is an ER / SR calcium channel inhibitor. Three trials (n=2 per trial). Violin plot shows the distribution of data with dotted lines representing quartiles.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with control and compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 21-1] This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a calcium chelator, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different calcium chelators. The triple-compound combination was more effective than the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor alone. As can be seen, both the two-compound combination and the triple-compound combination exerted a greater than additive effect compared to the individual components of the combination compared to the control (see Figures 2, 4, and 5 for individual components showing no significant difference from the control. Figures 2, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean control value. The gray dotted line represents the mean for the combination of two compounds, one of which was a gap junction inhibitor and the other an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control and to the combination of two compounds (i.e., an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 21-2]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a calcium chelator, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different calcium chelators. The triple-compound combination was more effective than the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor alone. As can be seen, both the two-compound combination and the triple-compound combination exerted a greater than additive effect compared to the individual components of the combination compared to the control (see Figures 2, 4, and 5 for individual components showing no significant difference from the control. Figures 2, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean control value. The gray dotted line represents the mean for the combination of two compounds, one of which was a gap junction inhibitor and the other an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control and to the combination of two compounds (i.e., an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 21-3]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor, plus a calcium chelator, significantly reduces the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. Treatments included the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and their combination with a series of different calcium chelators. The triple-compound combination was more effective than the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor alone. As can be seen, both the two-compound combination and the triple-compound combination exerted a greater than additive effect compared to the individual components of the combination compared to the control (see Figures 2, 4, and 5 for individual components showing no significant difference from the control. Figures 2, 4, and 5 and this figure can be directly compared with parallel studies). The red dotted line represents the mean control value. The gray dotted line represents the mean for the combination of two compounds, one of which was a gap junction inhibitor and the other an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three experiments were performed (n = 2 per experiment). The violin plot shows the distribution of data, with dotted quartiles. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA (Dunnett's correction) compared to the control and to the combination of two compounds (i.e., an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor). [Figure 22a-1]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin nonspecific protease inhibitors, and SOCC inhibitors) did not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different calpain inhibitors. (b) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different cathepsin inhibitors. (c) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different calpain / cathepsin nonspecific protease inhibitors. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different SOCC inhibitors. All three combinations were as effective, if not worse, than the combination of an ER / SR calcium channel blocker and a gap junction blocker alone. The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the combination of two compounds, one of which is a gap junction blocker and the other is an ER / SR calcium channel blocker. Three trials were performed (n = 2 per trial). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor). [Figure 22a-2]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin nonspecific protease inhibitors, and SOCC inhibitors) did not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different calpain inhibitors. (b) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different cathepsin inhibitors. (c) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different calpain / cathepsin nonspecific protease inhibitors. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different SOCC inhibitors. All three combinations were as effective, if not worse, than the combination of an ER / SR calcium channel blocker and a gap junction blocker alone. The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the combination of two compounds, one of which is a gap junction blocker and the other is an ER / SR calcium channel blocker. Three trials were performed (n = 2 per trial). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor). [Figure 22a-3]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin nonspecific protease inhibitors, and SOCC inhibitors) did not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different calpain inhibitors. (b) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different cathepsin inhibitors. (c) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different calpain / cathepsin nonspecific protease inhibitors. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different SOCC inhibitors. All three combinations were as effective, if not worse, than the combination of an ER / SR calcium channel blocker and a gap junction blocker alone. The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the combination of two compounds, one of which is a gap junction blocker and the other is an ER / SR calcium channel blocker. Three trials were performed (n = 2 per trial). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor). [Figure 22b-1]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin nonspecific protease inhibitors, and SOCC inhibitors) did not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different calpain inhibitors. (b) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different cathepsin inhibitors. (c) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different calpain / cathepsin nonspecific protease inhibitors. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different SOCC inhibitors. All three combinations were as effective, if not worse, than the combination of an ER / SR calcium channel blocker and a gap junction blocker alone. The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the combination of two compounds, one of which is a gap junction blocker and the other is an ER / SR calcium channel blocker. Three trials were performed (n = 2 per trial). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor). [Figure 22b-2]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin nonspecific protease inhibitors, and SOCC inhibitors) did not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different calpain inhibitors. (b) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different cathepsin inhibitors. (c) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different calpain / cathepsin nonspecific protease inhibitors. (d) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel blocker dantrolene and the gap junction blocker retinoic acid alone, and in combination with a series of different SOCC inhibitors. All three combinations were as effective, if not worse, than the combination of an ER / SR calcium channel blocker and a gap junction blocker alone. The red dotted line represents the mean value for the control. The gray dotted line represents the mean value for the combination of two compounds, one of which is a gap junction blocker and the other is an ER / SR calcium channel blocker. Three trials were performed (n = 2 per trial). The violin plot shows the distribution of data, with dotted lines representing quartiles. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two-way ANOVA (Dunnett's correction) compared with the combination of two compounds (i.e., the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor). [Figure 22b-3]This figure shows that the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor with drugs targeting other vulnerable points in cell physiology that predispose cells to necrosis (e.g., calpain inhibitors, cathepsin inhibitors, calpain / cathepsin nonspecific protease inhibitors, and SOCC inhibitors) did not further reduce the formation of necrotic cores induced by hypoxia and nutrient deprivation stress in HepG2s spheroids. (a) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different calpain inhibitors. (b) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor dantrolene and the gap junction inhibitor retinoic acid alone, and in combination with a series of different cathepsin inhibitors. (c) Treatments were the endoplasmic reticulum / sarcoplasmic reticulum (ER / S...

Claims

1. A composition comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject.

2. An endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, wherein the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor is to be administered in combination with one or more gap junction inhibitors.

3. A gap junction inhibitor for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, wherein the gap junction inhibitor is to be administered in combination with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors.

4. A method for treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, To treat or prevent necrosis, tissue damage, organ damage, or cell death, administer one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject, The method comprising administering one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors simultaneously, separately, or sequentially.

5. A method for preventing (i) necrosis or (ii) cell death of cells or tissues, The method comprising contacting the cells or tissue with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors in order to prevent necrosis or cell death of the cells or tissue.

6. A composition for use according to claim 1, comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and two or more gap junction inhibitors; an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to claim 2; a gap junction inhibitor for use according to claim 3; or the method according to any one of claims 4 to 5.

7. A composition for use according to claim 1 or 6, comprising two or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and two or more gap junction inhibitors; an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to claim 2 or 6; a gap junction inhibitor for use according to claim 3 or 6; or the method according to any one of claims 4 to 6.

8. The aforementioned endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor is a ryanodine receptor antagonist, Depending on the circumstances, the ryanodine receptor antagonist may be selected from one or more of dantrolene, DHBP dibromide, cis-Ned19, trans-Ned19, ryanodine, SKF86365 hydrochloride, ruthenium red, procaine, and tetracaine. In some cases, the ryanodine receptor antagonist is selected from dantrolene and / or ryanodine, comprising the composition for use according to any one of claims 1 or 6-7, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 2 or 6-7, the gap junction inhibitor for use according to any one of claims 3 or 6-7, or the method according to any one of claims 4-7.

9. The composition for use according to any one of claims 1 or 6 to 8, wherein the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor is dantrolene, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 2 or 6 to 8, the gap junction inhibitor for use according to any one of claims 3 or 6 to 8, or the method according to any one of claims 4 to 8.

10. The gap junction inhibitor is selected from (i) one or more of fatty acids, polyamines, and cyclodextrins, wherein the fatty acid optionally comprises 13 to 21 carbon atoms, or the gap junction inhibitor is selected from (ii) a connexin inhibitor and / or a panexin inhibitor, preferably a connexin inhibitor, the composition for use according to any one of claims 1 or 6 to 9, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 2 or 6 to 9, the gap junction inhibitor for use according to any one of claims 3 or 6 to 9, or the method according to any one of claims 4 to 9.

11. The gap junction inhibitor is selected from one or more of 18α-glycyrrhetinic acid, 18β-glycyrrhetinic acid, carbenoxolone disodium, heptanol, octanol, halothane, oleic acid, oleamide, linoleic acid, arachidonic acid, palmitoleic acid, flufenamic acid, myristic acid, lauric acid, quinine, quinidine, dihydroquinidine, mefloquine, meclofenamic acid, probenecid, nifluminic acid, flufenamic acid, astaxanthin, and retinoic acid, and is a composition for use according to any one of claims 1 or 6 to 10, an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 2 or 6 to 10, a gap junction inhibitor for use according to any one of claims 3 or 6 to 10, or the method according to any one of claims 4 to 10.

12. The composition for use according to claim 11, wherein the gap junction inhibitor is one or more of retinoic acid, oleic acid, linoleic acid, octanol, heptanol, arachidonic acid, palmitoleic acid, flufenamic acid, myristic acid, halothane, astaxanthin, and kinin, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to claim 11, the gap junction inhibitor for use according to claim 11, or the method according to claim 11.

13. The composition for use according to claim 12, wherein the gap junction inhibitor is one or more of retinoic acid, oleic acid, and linoleic acid, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to claim 12, the gap junction inhibitor for use according to claim 12, or the method according to claim 12.

14. The gap junction inhibitor is selected from two or more of oleic acid, linoleic acid, palmitoleic acid, kinin, astaxanthin, and retinoic acid, and is a composition for use according to any one of claims 1 or 6 to 13, an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 2 or 6 to 13, a gap junction inhibitor for use according to any one of claims 3 or 6 to 13, or the method according to any one of claims 4 to 13.

15. The gap junction inhibitor is selected from (i) retinoic acid and linoleic acid, (ii) retinoic acid and oleic acid, and (iii) linoleic acid and oleic acid, the composition for use according to claim 14, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to claim 14, the gap junction inhibitor for use according to claim 14, or the method according to claim 14.

16. A composition for use according to any one of claims 1 or 6 to 15, further comprising one or more cell membrane calcium channel inhibitors; an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 2 or 6 to 15; a gap junction inhibitor for use according to any one of claims 3 or 6 to 15; or the method according to any one of claims 4 to 15.

17. A composition for use according to any one of claims 1 or 6 to 16, further comprising one or more calcium chelating agents; an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 2 or 6 to 16; a gap junction inhibitor for use according to any one of claims 3 or 6 to 16; or the method according to any one of claims 4 to 16.

18. The cell membrane calcium channel inhibitor is selected from one or more of the following: amlodipine, alanidipine, azelnidipine, barnidipine, benidipine, cilnidipine, crebidipine, efonidipine, felodipine, isradipine, lasidipine, relcanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, fendiline, garopamil, verapamil, diltiazem, mibefuradil, bepridil, flunarizine, fluspirylene, fendiline, gabapentin, and pregabalin. Preferably, the cell membrane calcium channel inhibitor is selected from one or more of alandipine, clebidipine, efonidipine, felodipine, isradipine, relcanidipine, manidipine, nifedipine, nimodipine, nitrendipine, garopamil, verapamil, diltiazem, mibefradil, fluspirylene, gabapentin, and pregabalin, to form a composition for use according to claim 16 or 17, an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to claim 16 or 17, a gap junction inhibitor for use according to claim 16 or 17, or the method according to claim 16 or 17.

19. The calcium chelating agents mentioned above are EDTA (ethylenedioxydiethylene-dinitrilotetraacetic acid), EGTA (ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), HEDTA (N-(2-hydroxyethyl)ethylenediamine-N,N',N'-trisodium acetate), NTA (nitrilotriacetic acid), BAPTA ((1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), BAPTA One or more of the following are selected: AM (2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetyloxymethyl acetate), citric acid, TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), sodium citrate, and DMSA (dimercaptosuccinic acid). Preferably, the calcium chelating agent is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid, the composition for use according to any one of claims 17 to 18, the endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 17 to 18, the gap junction inhibitor for use according to any one of claims 17 to 18, or the method according to any one of claims 17 to 18.

20. The tissue injury is one or more selected from the group consisting of burns, multiple organ failure syndrome, organ failure, surgical trauma, bedsores, chemical burns, radiation burns, gangrene, alopecia, solar erythema, acute tubular necrosis, ulcers, bodily injury, mechanical trauma, burns, cold injuries, chilblains, trench foot inflammation, frostbite, avascular necrosis, pressure wounds, and skin graft failure, a composition for use according to any one of claims 1 or 6 to 19, an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 1 or 6 to 19, a gap junction inhibitor for use according to any one of claims 1 or 6 to 19, or the method according to any one of claims 4 to 19.

21. The aforementioned tissue includes or is one or more tissues selected from the group consisting of epithelial tissue, connective tissue, muscle tissue, and nerve tissue. Preferably, the tissue comprises or is selected from epithelial tissue, connective tissue, and muscle tissue, a composition for use according to any one of claims 1 or 6 to 20, an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 1 or 6 to 20, a gap junction inhibitor for use according to any one of claims 1 or 6 to 20, or the method according to any one of claims 4 to 20.

22. The organ damage is located in a nervous organ or in an organ that is part of the central nervous system (CNS). The composition for use according to any one of claims 1 or 6 to 20, an endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor for use according to any one of claims 1 or 6 to 20, a gap junction inhibitor for use according to any one of claims 1 or 6 to 20, and the method according to any one of claims 4 to 20.

23. A food preservative comprising one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, The food preservative further comprises, in some cases, one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

24. A food containing the food preservative described in claim 23.

25. A method for preserving food, comprising preserving the food by bringing it into contact with a food preservative described in claim 23.

26. A method for preserving living tissue or organ, comprising culturing or holding the tissue or organ in a culture medium containing one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors for the purpose of preserving the tissue or organ, The method, wherein the culture medium further comprises one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

27. The method according to claim 26, wherein the culture medium is the University of Wisconsin (UW) solution.

28. A method for manipulating tissue or organ, comprising contacting the tissue or organ with a culture medium containing one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, The method, wherein the culture medium further comprises one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

29. A method for preserving or culturing blood cells, comprising contacting a culture medium containing blood cells with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, The method, in some cases, further comprises contacting the culture medium with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

30. The method according to claim 29, wherein the culture medium is blood, plasma, or serum.

31. A method for preserving blood samples, DNA samples, or reproductive samples, comprising contacting the samples with one or more endoplasmic reticulum / muscular endoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to preserve the samples. The method, in some cases, further comprises contacting the sample with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

32. A method for preserving a graft, comprising preserving the graft by contacting it with one or more endoplasmic reticulum / muscular endoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, The method, in some cases, further comprises contacting the graft with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

33. A method for preserving or culturing cells while culturing a cell line in a bioreactor, comprising contacting the cells with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to preserve or cultivate the cells, The method, in some cases, further comprises bringing the cells into contact with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

34. A method for preserving or growing a cell culture, comprising contacting the culture medium with one or more endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to preserve or grow the cell culture, The method, in some cases, further comprises contacting the culture medium with one or more cell membrane calcium channel inhibitors and / or one or more calcium chelating agents.

35. The endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor and / or the gap junction inhibitor and / or the cell membrane calcium channel inhibitor and / or the calcium chelating agent is as described in any one of claims 6 to 19, the food preservative according to claim 23, the food according to claim 24, or the method according to any one of claims 25 to 34.

36. A method for preserving living tissue or organ, comprising culturing or holding the tissue or organ in a culture medium containing one or more calcium chelating agents for the purpose of preserving the tissue or organ.

37. The method according to claim 36, wherein the culture medium is the University of Wisconsin (UW) solution.

38. A method for manipulating tissue or organs, comprising contacting the tissue or organ with a culture medium containing one or more calcium chelating agents.

39. The method according to any one of claims 36 to 38, wherein the culture medium contains one or more calcium chelating agents in a relatively high dose, optionally the culture medium contains the calcium chelating agent in an amount about four times the dose normally used, and optionally the culture medium contains one or more calcium chelating agents in the amount described in embodiment A.

40. A composition comprising one or more calpain inhibitors and one or more cathepsin inhibitors for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject.

41. The composition for use according to claim 40, wherein the one or more calpain inhibitors include or are selected from one or more selected from the group consisting of calpastatin, MG101, MG132, vidupiplant, α-mercaptoacrylate, 5-azolone, carboxamide, and α-helix cysteine ​​protease inhibitors.

42. The composition for use according to claim 40 or 41, wherein the one or more cathepsin inhibitors are selected from one or more of dexamethasone, rifampicin, E64, E64d, carpeptin, CA074, SID26681509, L006235, bafilomycin, alloxistatin, astaxanthin, chloroquine, clofazimine, dec-RVKR, and dexamethasone.

43. A combination comprising dantrolene, or a pharmaceutically acceptable salt or solvate thereof, and quinine, or a pharmaceutically acceptable salt or solvate thereof.

44. The combination according to claim 43, further comprising one or more additional endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitors.

45. The additional endoplasmic reticulum / sarcoplasmic reticulum calcium channel inhibitor is a ryanodine receptor antagonist; Optionally, additional ryanodine receptor antagonists are selected from one or more of the following: DHBP dibromide, cis-Ned19, trans-Ned19, ryanodine, SKF86365 hydrochloride, ruthenium red, procaine, and tetracaine; The combination according to claim 44, optionally the additional ryanodine receptor antagonist being ryanodine.

46. The combination according to any one of claims 43 to 45, further comprising one or more additional gap junction inhibitors.

47. The combination according to claim 46, wherein the additional gap junction inhibitor is selected from (i) one or more of fatty acids, polyamines, and cyclodextrins, optionally wherein the fatty acid contains 13 to 21 carbon atoms, or (ii) a connexin inhibitor and / or a panexin inhibitor, preferably a connexin inhibitor, or (iii) one or more of 18α-glycyrrhetinic acid, 18β-glycyrrhetinic acid, carbenoxolone disodium, heptanol, octanol, halothane, oleic acid, oleamide, linoleic acid, arachidonic acid, palmitoleic acid, flufenamic acid, myristic acid, lauric acid, quinidine, dihydroquinidine, mefloquine, meclofenamic acid, probenecid, nifluminic acid, flufenamic acid, astaxanthin, and retinoic acid.

48. The combination according to any one of claims 43 to 47, further comprising one or more cell membrane calcium channel inhibitors.

49. The cell membrane calcium channel inhibitor is selected from one or more of the following: amlodipine, alanidipine, azelnidipine, barnidipine, benidipine, cilnidipine, crebidipine, efonidipine, felodipine, isradipine, lasidipine, relcanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, pranidipine, fendiline, garopamil, verapamil, diltiazem, mibefradil, bepridil, flunarizine, fluspirylene, fendiline, gabapentin, and pregabalin; Preferably, the combination according to claim 48, wherein the cell membrane calcium channel inhibitor is selected from alandipine, clebidipine, efonidipine, felodipine, isradipine, relcanidipine, manidipine, nifedipine, nimodipine, nitrendipine, garopamil, verapamil, diltiazem, mibefradil, fluspirylene, gabapentin, or pregabalin.

50. The combination according to any one of claims 43 to 49, further comprising one or more calcium chelating agents.

51. The calcium chelating agent is EDTA (ethylenedioxydiethylene-dinitrilotetraacetic acid); EGTA (ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid); DTPA (diethylenetriaminepentaacetic acid); HEDTA (N-(2-hydroxyethyl)ethylenediamine-N,N',N'-trisodium acetate); NTA (nitrilotriacetic acid); BAPTA ((1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)); BAPTA AM (2-[N-[2-(acetyloxymethoxy)-2-oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetyloxymethyl acetate); citric acid; TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine); sodium citrate; and one or more selected from DMSA (dimercaptosuccinic acid); Preferably, the calcium chelating agent is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid, as per claim 50.

52. A pharmaceutical composition comprising dantrolene, or a pharmaceutically acceptable salt or solvate thereof, and kinin, or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject.

53. A pharmaceutical composition comprising dantrolene or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, wherein the pharmaceutical composition is administered in combination with kinin or a pharmaceutically acceptable salt or solvate thereof.

54. A pharmaceutical composition comprising kinin, or a pharmaceutically acceptable salt or solvate thereof, for use in a method of treating or preventing (i) necrosis, (ii) tissue damage, (iii) organ damage, or (iv) cell death in a subject, wherein the pharmaceutical composition is administered in combination with dantrolene, or a pharmaceutically acceptable salt or solvate thereof.

55. The pharmaceutical composition according to any one of claims 53 to 54, wherein the combination is as defined in any one of claims 44 to 51.

56. The pharmaceutical composition according to any one of claims 52 to 55, comprising administering dantrolene, or a pharmaceutically acceptable salt or solvate thereof, and kinin, or a pharmaceutically acceptable salt or solvate thereof, simultaneously, separately, or in succession.

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