Necrosis inhibitors and their uses

Abstract

Provided herein are methods comprising administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. For example, the methods include methods of preventing or treating organ ageing. The compositions may also comprise a plasma membrane calcium channel inhibitor and / or a calcium chelator.

Description

[0001] NECROSIS INHIBITORS AND THEIR USES

[0002] Field of the Invention

[0003] The present invention relates to compositions comprising one or more necrosis inhibitors, and the uses of such compositions. For instance, the compositions may be used to prevent or treat organ ageing, reduce toxicity, reduce the side-effects of space radiation and / or gravitational environments associated with space, and / or reduce downstream effects of necrosis. The compositions may be used to prevent or treat medical conditions associated with necrosis.

[0004] The compositions may comprise one or more calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors and / or gap junction inhibitors. The compositions described herein may be used in any of the methods, uses and medical uses described herein. For example, the composition may comprise one or more calpain inhibitors in combination with one or more cathepsin inhibitors. As another example, the composition may comprise one or more calcium chelators, particularly where the calcium chelators are used at relatively high doses.

[0005] Preferably, the compositions comprise (i) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and / or (ii) one or more gap junction inhibitors. Most preferably, the compositions comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors. The composition may further comprise (iii) one or more plasma membrane calcium channel inhibitors, and / or (iv) one or more calcium chelators.

[0006] Necrosis is one of two main types of cell death that occur in living organisms, the other being programmed cell death (also known as apoptosis). Methods of treating or preventing cell death, such as treating or preventing necrosis and / or apoptosis, are taught in PCT / IB2024 / 050203, the entire disclosure of which is incorporated herein by reference.

[0007] Necrosis has been characterized as passive, accidental cell death resulting from environmental perturbations (i.e., a stressful trigger) with uncontrolled release of cellular contents. It is believed to be mediated by random intracellular events. If necrotic cells in a tissue are left untreated, the necrosis will spread to neighbouring cells and tissues. This, in turn, can lead to irreversible organ damage (e.g., organ failure), and even death. Apoptosis, on the other hand, is described as an active, programmed process of autonomous cellular dismantling. Unlike necrosis, apoptosis does not typically spread to neighbouring cells or tissues.

[0008] Current treatments for necrosis target the triggers of necrosis, rather than trying to directly stop / prevent the cellular changes associated with necrosis; this is because necrosis is traditionally believed to be mediated by random intracellular events. As these work by only attempting to block triggers, their effectiveness is limited to the extent that the trigger is the sole cause of necrosis, treatment must be given early enough to supress the trigger, and the treatment may be associated with an unwanted trade off. One such class of drugs is immunosuppressants. They work by supressing secondary inflammation that can trigger necrosis, e.g., TNF- a inhibition. A trade-off associated with immunosuppressants is the increased risk of infection.

[0009] Medical conditions may occur downstream of necrosis. There is therefore a need for an improved therapy for treating medical conditions that occur downstream of necrosis. Necrosis may also occur as a side-effect to other medical conditions, as a result of medical treatments, and / or as a result of administration of therapeutic agents or therapy. There is therefore a need for an improved adjunctive therapy for treating necrosis which may occur as a result of other medical treatments and / or administration of other therapeutic agents or therapy.

[0010] Statements of the invention

[0011] The present inventors have found that the compositions of the present invention may treat or prevent necrosis. Thus, the skilled person will appreciate that the compositions may be used prophylactically and / or curatively (i.e. during and after the onset of necrosis).

[0012] The compositions are further defined herein. The compositions may comprise one or more calcium activity inhibitors, calpain inhibitors, cathepsin inhibitors and / or gap junction inhibitors. For example, the composition may comprise one or more calpain inhibitors in combination with one or more cathepsin inhibitors. As another example, the composition may comprise one or more calcium chelators, particularly where the calcium chelators are used at relatively high doses.

[0013] As a preferred example, the compositions comprise (i) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and / or (ii) one or more gap junction inhibitors. Most preferably, the compositions comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors. Most preferably, the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor is dantrolene. Most preferably, the gap junction inhibitors are one or more of retinoic acid, oleic acid, linoleic acid, and quinine (such as one or more of retinoic acid, oleic acid and linoleic acid). Most preferably, the gap junction inhibitor is quinine. Thus, most preferably, the compositions comprise dantrolene and quinine. The compositions may further comprise (iii) one or more plasma membrane calcium channel inhibitors, and / or (iv) one or more calcium chelators. Most preferably, the plasma membrane calcium channel inhibitor is amlodipine. Most preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0014] The compositions of the present invention can surprisingly be used as a therapy for treating medical conditions that occur downstream of necrosis, and / or as an adjunctive therapy for treating necrosis whilst also treating other primary medical conditions. The compositions of the present invention can surprisingly be used in any of the methods, medical uses and uses described herein.

[0015] Alcohol toxicity

[0016] The consumption of alcohol can cause necrosis via multiple mechanisms including, in particular, oxidative stress and hypoxia. Specifically, alcohol metabolism increases oxygen consumption by the liver, leading to hypoxia. Hypoxia can cause ATP depletion and subsequent necrosis due to insufficient oxygen for cellular metabolism. Additionally, alcohol metabolism generates oxygen radicals, and chronic alcohol consumption increases the levels of free iron in organs such as the liver, both of which can contribute to oxidative stress. Oxidative stress can directly cause cell damage, and can modify DNA, lipids, and / or proteins, leading to necrosis. It can also reduce the levels of antioxidants like glutathione and vitamin E, which are crucial for neutralizing oxygen radicals. It would be desirable to treat or prevent the side-effects (such as treating or preventing necrosis) associated with alcohol consumption in a subject.

[0017] The present invention provides a method of treating or preventing the side-effects (such as treating or preventing necrosis) associated with alcohol consumption in a subject, wherein the method comprises administering a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject.

[0018] Excess alcohol consumption has been shown to be associated with development of necrosis, such as osteonecrosis (ON). Osteonecrosis relates to necrotic changes in the bone related to either the sudden, permanent, or temporary depletion of the blood supply.

[0019] Other alcohol associated diseases are associated with the development of necrosis, such as:

[0020] • Liver diseases: including steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. Necrosis plays a critical role in the progression of steatosis to steatohepatitis and ultimately to cirrhosis and cancer, where damaged liver cells are killed and are replaced by scar tissue or cancerous cells;

[0021] • Pancreatitis: acute and chronic inflammation of the pancreas, often leading to fibrosis. Alcohol- induced necrosis of pancreatic acinar cells disrupts normal pancreatic function, leading to inflammation and long-term damage;

[0022] • Cardiovascular diseases: including hypertension, ischemic heart disease, stroke, cardiomyopathy, myocarditis, and various arrhythmias. Alcohol-induced necrosis of cardiac muscle cells can impair heart function and lead to one or more of these conditions;

[0023] • Neuropsychiatric disorders: including major depression and other mental health issues which may be linked to alcohol abuse. Excessive alcohol consumption can lead to brain damage and cognitive impairment via necrosis, which may contribute to these conditions;

[0024] • Tumors and cancer: alcohol-induced cellular necrosis can lead to mutations and uncontrolled cell proliferation resulting in these types of cancers;

[0025] • Glomerulonephritis and kidney damage: including inflammation and damage to the kidneys, potentially leading to kidney graft failure. Alcohol-induced necrosis of renal cells can impair kidney function and lead to these conditions;

[0026] • Reproductive and developmental issues: including impaired fertility, premature birth, low-weight births, and fetal alcohol syndrome spectrum disorders. Alcohol-induced cellular necrosis during pregnancy can lead to developmental abnormalities in the fetus. It would be desirable to provide a method for treating or preventing one or more alcohol associated diseases, such as one or more of the alcohol associated diseases set out above.

[0027] The present invention provides a method of treating or preventing an alcohol associated disease (such as any of the alcohol associated diseases mentioned herein) in a subject, wherein the method comprises administering a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject.

[0028] Preventing cell death associated with cancer, cancer therapies and / or chemotherapeutic agents Tumours may exert various stressors upon surrounding healthy tissue. For example, tumours can lead to the redirection of blood supply, which results in hypoxic damage to healthy cells and induces oxidative stress. These stressors may kill healthy tissue via necrosis.

[0029] Necrosis is a feature of many tumours, such as aggressive fast-growing tumours. These tumours may include breast, renal, prostate, and endometrial carcinomas. In tumours, necrosis plays a key role in poor prognosis and treatment resistance, and can lead to a markedly increased risk of metastasis. In more detail, in cancers, necrosis typically arises within the central regions of dense-growing tumours due to, for example, poor blood supply, nutrient deprivation, and metabolic stress. The hypoxic peri-necrotic region (rich in cytokines such as TNF-alpha, interleukin- (IL-) 1a, IL-16, IL-33, oxidative stress, and DAMPs) may promote angiogenesis, genetic instability, and immune dysfunction. Additionally, as seen in most tumour cancers, tumour dissemination and metastasis strongly correlates with necrosis, which is typically localized to dilated peri-necrotic vessels within the interior of the tumour. Aside from increased vascular density and proinflammatory infiltrate (e.g., focal macrophage infiltration), this may be explained by necrotic regions having loose ready-to-detach cells due to reduced cell-cell and cell-matrix adhesion, which promotes tumour dissemination.

[0030] The inventors have found that compositions of the present invention protect against necrosis, and thus can be used to prevent the death of healthy tissue associated with cancer and / or tumours.

[0031] Additionally, compositions of the present invention can enhance the effectiveness of cancer therapies. Tumor cells exhibit poorer DNA repair capabilities compared to normal cells, which contributes significantly to their ability to accumulate DNA lesions and evolve into cancerous states. In contrast, normal cells possess robust DNA repair machinery that allows them to recognize and repair DNA lesions effectively. This poorer DNA repair capability of tumour cells is a double-edged sword; although it increases the risk of tumours and hence can lead to cancer, the inability of tumour cells to efficiently repair DNA lesions makes them more susceptible to the cytotoxic effects of cancer therapies, such as chemotherapy and radiation, which target DNA repair mechanisms. Notably, cancer therapies induce stress and cell death through a variety of different mechanisms, not just by causing DNA lesions. These alternative mechanisms include inducing damage through oxidative stress, hypoxia, and deleterious changes in physiology (loss of fluid, and change in blood supply etc). The compositions of the present invention will protect all cells against these alternative mechanisms, and increase the ability of normal cells to survive DNA lesions.

[0032] The present invention provides a method of treating or preventing necrosis associated with cancer and / or tumours in a subject, wherein the method comprises administering a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject.

[0033] The present invention also provides a method of treating or preventing the cell death (such as necrosis) associated with therapy for one or more cancers and / or for one or more precancerous lesions of a subject, wherein the method comprises administering a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject. The therapy for one or more cancers and / or for one or more precancerous lesions may be chemotherapy or radiation therapy, such as radiation therapy.

[0034] The present invention also provides a method of treating or preventing the cell death (such as necrosis) associated with the administration of one or more chemotherapeutic agents to a subject, wherein the method comprises co-administering to a subject the chemotherapeutic agent in combination with a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject.

[0035] Thus, the present invention provides a method of treating or preventing cancer, wherein the method comprises administering a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject.

[0036] The cancer may be breast carcinoma, endometrial carcinoma, prostate carcinoma and / or renal carcinoma.

[0037] Reducing toxicity associated with administration of a toxic agent

[0038] Toxic agents may kill cells via cellular necrosis directly or indirectly. Indirect death caused by a toxic agent may result from a lack of oxygen and nutrient / blood supply to healthy tissue and induced oxidative stress. The inventors have found that compositions of the present invention protect against necrosis, and thus can be used to protect against both direct cell death and indirect cell death via hypoxia, nutrient deprivation stress and oxidative stress.

[0039] The present invention provides a method of treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more toxic agents, wherein the method comprises co-administering to a subject the toxic agent in combination with a composition of the present invention. For example, the method may comprise co-administering the toxic agent in combination with a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject.

[0040] Reducing toxicity associated with administration of a pharmaceutical agent

[0041] Existing and newly synthesised pharmaceutical agents may lead to toxic effects, or toxic side-effects. Pharmaceutical agents may therefore be a type of “toxic agent” as discussed herein. The inventors have found that such toxic effects and / or toxic side-effects can be reduced by at least the following two ways.

[0042] Firstly, combining necrosis inhibitors with existing and / or new pharmaceutical agents can reduce the toxicity of the pharmaceutical agent. In more detail, pharmaceutical agents typically come with a trade-off to cell vitality, where a trade-off is necrosis that may give rise to general toxicity or local (e.g. liver and / or kidney) toxicity. Specifically, pharmaceutical agents may cause necrosis induced toxicity by increasing oxidative stress on cells, as well as increasing hypoxic stress. The inventors have found that compositions of the present invention block this necrosis, and thus can be used to protect or reduce the toxicity associated with administration of pharmaceutical agents.

[0043] The present invention provides a method of treating or preventing a toxic effect (such as necrosis or necrosis induced toxicity) associated with the administering of one or more pharmaceutical agents, wherein the method comprises co-administering to a subject the pharmaceutical agent in combination with a composition of the present invention. For example, the method may comprise co-administering the pharmaceutical agent in combination with a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject.

[0044] Secondly, a first-in-class means to block cellular necrosis via the combined inhibition of specific molecular targets is outlined in PCT / IB2024 / 050203, the entire disclosure of which is incorporated herein by reference. In particular, as discussed in Example 1 and Figure 1 , the inventors discovered that a critical step in the occurrence of necrosis is the calcium ion increase in the cytosol, which may be due to any one of the three main points of calcium ion entry of into a cell: i.e., via cell surface membrane channels, gap junctions, and the endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR). It is believed that, because plasma membranes are in contact with the environment surrounding the cell, they will show greater levels of re-enforcement compared to ER / SR calcium channels and gap junctions. Moreover, for cells in certain tissue types that are very tightly packed, calcium movement via ER / SR calcium channels and gap junctions will pose a greater threat during a stress. Therefore, the inventors have made the surprising discovery that using a combination of compounds to block the ER / SR calcium channels and gap junctions is particularly advantageous for preventing and / or treating necrosis. This combination can be further improved by also blocking calcium ion entry via plasma membranes, e.g., by additionally using a plasma membrane calcium channel inhibitor in the combination. Additionally, or alternatively, the combination can be further improved by the use of other means to mimic reduction of cytosolic calcium ion concentration, such as through use of calcium chelating agents that sequester calcium (or use competitive inhibitors of calcium ions, such as other cations of a similar charge and size such as magnesium ions). The inventors have discovered that, if administration of a pharmaceutical agent already inhibits one or more of these molecular targets (e.g., directly or indirectly), compositions of the present invention can be co-administered with the pharmaceutical agent in order to reduce toxicity because the combination of the pharmaceutical agent and the compositions of the present invention treat or prevent necrosis.

[0045] The present invention also provides a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject one or more pharmaceutical agents in combination with a composition of the present invention. Preferably, the composition of the present invention comprises one or more necrosis inhibitors which block different molecular targets involved in the necrosis pathway than the pharmaceutical agent. For example, where the pharmaceutical agent inhibits endoplasmic / sarcoplasmic reticulum calcium channels, the composition comprises one or more gap junction inhibitors (e.g., quinine). As another example, where the pharmaceutical agent inhibits the gap junction, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (e.g., dantrolene).

[0046] Reducing toxicity associated with administration of a biologically active agent

[0047] As discussed above, means to block cellular necrosis via the combined inhibition of specific molecular targets is outlined in PCT / IB2024 / 050203, the entire disclosure of which is incorporated herein by reference. The inventors have discovered that any means to inhibit the targets of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions will provide the beneficial effect of preventing necrosis. This inhibition of necrosis may be further improved by any means to inhibit plasma membrane calcium channels and / or inhibit cytosolic calcium levels.

[0048] Thus, the inventors have discovered that, if administration of a biologically active agent already blocks one of these targets (e.g., directly or indirectly), compositions of the present invention can be co-administered with the biologically active agent in order to prevent or treat necrosis because the combination of the biologically active agent and the compositions of the present invention prevent or treat necrosis.

[0049] The biologically active agent may be any substance which can affect any physical or biochemical properties of a subject. For example, biologically active agents may include any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active agents include peptides, proteins, nucleic acids, antibodies, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, nanoparticles and micelles, such as peptides, proteins, nucleic acids, antibodies, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of biologically active agents that are suitable for use include drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbially derived toxins, and the like. For example, imaging agents may include contrast dyes, including, for example, iodinated contrast agents (which may be used in CT scans and angiography), high- osmolar contrast media (such as diatrizoate (Hypaque, Renografin)), low-osmolar contrast media (such as lohexol (Omnipaque), lopamidol (Isovue), and loversol (Optiray)), iso-osmolar contrast media (such as lodixanol (Visipaque)), gadolinium-based contrast agents (which may be used in MRI), linear agents (such as gadodiamide (Omniscan) and gadopentetate dimeglumine (Magnevist)), and macrocyclic agents (such as gadoteridol (ProHance), gadobutrol (Gadavist), and gadoterate meglumine (Dotarem)).

[0050] The biologically active agent may be the pharmaceutical agent discussed herein. Alternatively, the biologically active agent may be a biologic agent such as an antibody, protein, peptide, or nucleic acid.

[0051] The biologically active agent is preferably suitable for inhibiting the targets of endoplasmic / sarcoplasmic reticulum calcium channels or gap junctions.

[0052] The present invention provides a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject one or more biologically active agents in combination with a composition of the present invention. Preferably, the composition of the present invention comprises one or more necrosis inhibitors which block different molecular targets involved in the necrosis pathway than the biologically active agent. For example, where the biologically active agent inhibits endoplasmic / sarcoplasmic reticulum calcium channels, the composition comprises one or more gap junction inhibitors (e.g., quinine). As another example, where the biologically active agent inhibits the gap junction, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (e.g., dantrolene).

[0053] Administration of contrast dyes can, in some cases, lead to acute kidney injury, also known as contrast-induced nephropathy. Thus, according to a preferred aspect of the invention, there is provided a method of treating of preventing necrosis contrast-induced nephropathy, wherein the method comprises administering a composition of the present invention to the subject.

[0054] Reducing damage associated with medical diagnostics and interventions

[0055] Medically invasive interventions and / or medical diagnostic procedures may cause mechanical damage where tissue is compressed, such that blood supply is reduced and hypoxia ensues and / or such that oxidative stress is caused via inflammatory response pathways. Hypoxia and oxidative stress can, in turn, induce irreversible organ and tissue damage via cellular necrosis.

[0056] Medically invasive interventions and / or medical diagnostic procedures include surgery, dialysis, and invasive diagnostics such as catheterization, balloon angioplasty and / or atherectomy, stenting, vascular angioplasty, atherectomy, stents, IVC filter placement and / or removal, thrombectomy, ablation (such as vein ablation, radiofrequency ablation and / or cryothermal ablation), phlebectomy procedures, pacemakers, echocardiography, defibrillator insertion, subcutaneous ICD and / or appendage closure.

[0057] The present invention provides a method of treating or preventing the cell death (such as necrosis) associated with one or more medically invasive interventions and / or medical diagnostic procedures, wherein the method comprises administering a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject who has been subjected to, or will be subjected to, one or more medically invasive interventions and / or medical diagnostic procedures. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject who has been subjected to, or will be subjected to, one or more medically invasive interventions and / or medical diagnostic procedures.

[0058] Use in space

[0059] On space missions (such as on missions to Earth’s moon, to Mars, or elsewhere), the Earth’s atmosphere and magnetosphere are not available to protect the crew of the mission from sources of ionizing radiation. Thus, the crew of a space mission is at risk of the side-effects of exposure to ionizing radiation.

[0060] Exposure of living cells and tissues to ionizing radiation can cause necrosis via direct and indirect mechanisms. Examples of such mechanisms include: (i) vascular injury where ionizing radiation primarily damages small arteries, leading to necrosis via hypoxia as well as coagulative necrosis, (ii) direct damage to cells as radiation can directly injure cells, including oligodendroglia in the brain, contributing to tissue necrosis (it will be appreciated that the white matter is more vulnerable because oligodendrocytes, which myelinate nerve fibers, are sensitive to radiation), (iii) alterations in blood-brain barrier and fluid dynamics as radiation may impact the blood-brain barrier, leading to increased permeability and altered fluid dynamics, and (iv) immune responses, causing oxidative damage. These mechanisms may collectively lead to the development of radiation necrosis, a severe complication that can occur days to years after radiation exposure. Thus, it would be desirable to provide a method for protecting a subject from the side-effects of space radiation, such as ionizing radiation.

[0061] The present invention provides a method of protecting a subject from one or more side-effects of space radiation, the method comprising administering a composition of the invention to the subject. Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists, such as dantrolene) in combination with one or more gap junction inhibitors (such as quinine). Preferably, the space radiation is ionizing radiation. Preferably, the side-effect is radiation necrosis.

[0062] Furthermore, during space missions and / or during preparation for a space mission (e.g. in a simulation), the crew of the mission may be exposed to different gravitational environments. For example, the crew may be exposed to an environment wherein the gravity is much lower than Earth’s gravitational environment. As another example, the crew may be exposed to an environment wherein the gravity is much higher than Earth’s gravitational environment. Different gravitational environments have been shown to lead to cell death, via necrosis. For example, cells under microgravity conditions might become more susceptible to various stressors, including oxidative stress, and hypoxia, leading to cell injury and necrosis. Thus, it would be desirable to provide a method for protecting a subject from the side-effects of exposure to different gravitational environments.

[0063] The present invention also provides a method of protecting a subject from one or more side-effects (such as necrosis) associated with a gravitational environment which is different to Earth’s gravitational environment, the method comprising administering a composition of the invention to the subject. Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors in combination with one or more gap junction inhibitors. Preferably, the composition comprises dantrolene and quinine.

[0064] The complex set of environmental stressors associated with space missions, particularly altered gravity (such as microgravity and hypergravity) and higher doses of cosmic radiation, may converge to heighten cellular susceptibility to necrosis. These factors are linked to over thirty specific health risks ranging from acute inmission effects to those emerging later in life, and including accelerated changes mimicking those seen in aging, such as kidney damage, muscle atrophy, bone loss, and immune cell loss. Specific underlying mechanisms include the combined effects of hemodynamic stress (such as posterior cerebral hyperperfusion, promoting minor blood-brain barrier disruption), consequential ischemic stress, and systemic oxidative stress that contribute to and are further exasperated by chronic inflammatory pathways. Many of these changes culminate in disrupted redox homeostasis, triggering necrosis via disrupted cell membrane integrity, with especially sensitive cells to necrosis including kidney tubular epithelial cells, neuronal white matter cells, innate immune cells, and retinal cells.

[0065] The present invention therefore provides a method of treating or preventing kidney damage, muscle atrophy, bone loss, and / or immune cell loss, wherein the method comprises administering a composition of the present invention. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. For example, the method may comprise administering a composition comprising dantrolene and quinine to the subject. The kidney damage, muscle atrophy, bone loss, and / or immune cell loss may be associated or induced by the environmental stressors associated with space missions.

[0066] Radiation damage Ionizing radiation can be produced by weapons such as surface-burst nuclear weapons, air burst nuclear weapons, and radiological dispersion devices. Ionizing radiation can also be produced during medical therapies such as cancer / tumour radiation therapy. For example, ionizing radiation may include x-rays, gamma rays, particle bombardment (such as neutron beams, electron beams, protons, mesons, and others), and particle rays (such as alpha particles, beta particles, and neutrons). Exposure of living cells and tissues to ionizing radiation may cause necrosis via direct and indirect mechanisms, as discussed herein. These mechanisms collectively lead to the development of radiation necrosis, a severe complication that can occur days to years after radiation exposure. Thus, it would be desirable to provide a method for protecting a subject from the side-effects of ionizing radiation.

[0067] The present invention provides a method of protecting a subject from one or more side-effects associated with ionizing radiation, the method comprising administering a composition of the invention to the subject. Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors in combination with one or more gap junction inhibitors. For example, the composition comprises dantrolene and quinine. Preferably, the side-effect is radiation necrosis.

[0068] Defence against weapons

[0069] In addition to the ionizing radiation discussed hereinabove, injuries caused by weapons may also induce necrosis via physical trauma, kinetic energy, chemical trauma and / or thermal trauma. These stressors may directly or indirectly (via hypoxia by affecting blood supply) induce cell necrosis. Thus, it would be desirable to provide a method for protecting a subject from the side-effects of an injury caused by a weapon.

[0070] The present invention provides a method of protecting a subject from one or more side-effects of an injury caused by a weapon, the method comprising administering a composition of the invention to the subject. Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors in combination with one or more gap junction inhibitors. For example, the composition comprises dantrolene and quinine. Preferably, the side-effect is necrosis.

[0071] Necrosis causes local and general accelerated ageing via cellular senescence, vascular rarefaction, presence of a chronic inflammatory infiltrate, and / or fibrosis, which in turn causes a loss of tissue and organ structure, a loss of tissue and organ function, and accelerates ageing. In particular, ageing caused by necrosis may be seen in the kidney. In addition to the above, tubular loss and glomerulosclerosis can also be seen in the kidney. Thus, it would be desirable to provide a method for preventing or treating ageing, particularly for preventing or treating organ ageing (such as kidney ageing, such as accelerated kidney ageing).

[0072] The present invention provides a method of preventing or treating ageing, wherein the method comprises administering a composition of the invention to the subject. Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors in combination with one or more gap junction inhibitors. For example, the composition comprises dantrolene and quinine. Preferably, the method prevents or treats organ ageing, preferably kidney ageing, preferably accelerated kidney ageing. Preferably, the method prevents or treats one or more of cellular senescence, vascular rarefaction, presence of a chronic inflammatory infiltrate, glomerulosclerosis and fibrosis.

[0073] The method may be a therapeutic method. Alternatively, the method may be a cosmetic method.

[0074] Artificially produced meat

[0075] It will be appreciated that cell death is a major problem in bio-industry, as cell death often occurs within bioreactors due to various stressors. These stressors therefore negatively affect the culture longevity and the overall product yield.

[0076] Bioreactors are used to produce artificial meat and cultivated meat. For example, cultivated meat is typically made by a process wherein stem cells are acquired from an animal. These cells are then grown in bioreactors at high densities and volumes, and are differentiated into the skeletal muscle, fat, and connective tissues that make up meat. The differentiated cells are then harvested, prepared, and packaged into final cultivated meat products. However, one of the challenges with growing cells in this way is that, as the larger cells are engineered / grown, they begin to develop necrotic cores. In order to improve the cell longevity, overall product yield, and / or to enable scaling, it would be desirable to reduce the cell death and to reduce the formation of necrotic cores which may occur during production of artificial meat and cultivated meat.

[0077] The present invention provides a method for producing artificial meat or cultivated meat, the method comprising contacting the cells with a composition of the present invention to preserve or culture the cells. The composition of the present invention preferably comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

[0078] Genome-editing technologies

[0079] Gene editing can be defined as creating a genomic modification in a targeted manner. Any gene editing strategy known to one of ordinary skill in the art may be used in the present invention. For example, some genome editing strategies involve creating a targeted DNA double-strand break (DSB) in genomic DNA near the site of desired change. This can be achieved using several different nuclease platforms including meganucleases, zinc finger nucleases (ZFNs), transcription activator- 1 ike effector nucleases (TALENs), and CRISPR-Cas9. Other transcription and translation affecting techniques may also be used, including technologies involving RNA Interference (RNAi), siRNAs (Small Interfering RNAs), shRNAs (Short Hairpin RNAs) and miRNAs (MicroRNAs).

[0080] As discussed above, means to block cellular necrosis via the combined inhibition of specific molecular targets is outlined in PCT / IB2024 / 050203, the entire disclosure of which is incorporated herein by reference. The inventors have discovered that any means to inhibit the targets of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions will provide the beneficial effect of preventing necrosis. Thus, the present inventors have discovered that gene editing to provide dual inhibition of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions advantageously treats or prevents necrosis.

[0081] Any means that simultaneously inhibit the targets of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions will provide the beneficial effect of preventing necrosis, and thus may be used in the present invention. For example, the inventors have discovered that any one of the following techniques may be utilized in any one of the methods described herein: gene editing using CRISPR-Cas9, base editing, prime editing, zinc finger nucleases (ZFNs), TALENs, gene therapy through gene replacement, gene silencing, antisense oligonucleotides (ASOs), epigenetic modifiers (like DNA methylation modifiers and histone deacetylase inhibitors (HDAC inhibitors)), RNA interference via siRNA, shRNA, miRNA mimics and inhibitors, antisense oligonucleotides (ASOs) which may block translation or alter splicing, mRNA therapy, small molecule drugs (which may activate or inhibit enzymes, stabilize or degrade proteins, or block protein-protein interactions), PROTACs (Proteolysis Targeting Chimeras), splice modifiers for exon skipping or inclusion, RNA editing using ADAR enzymes, engineered cells (like CAR-T cells and iPSCs), peptide or protein therapy (e.g., insulin, enzyme replacement), RNA aptamers, decoy oligonucleotides which may trap transcription factors, ribozymes which may cleave RNA, trans-splicing to repair RNA, DNAzymes with catalytic activity against RNA, synthetic mRNA switches, nanobody therapeutics, intron retention modulators, exosome-based delivery systems, gene knockdown with morpholinos, tethered protein degraders such as molecular glues, lipid nanoparticles (LNPs) for RNA delivery, and / or monoclonal antibodies which may neutralize, block, or mark proteins for immune destruction. Any one of these techniques may provide inhibition of endoplasmic / sarcoplasmic reticulum calcium channels and / or gap junctions which advantageously treats or prevents necrosis. It will be readily apparent to a person of ordinary skill in the art that these compounds or techniques may be used in any one or more of the methods described separately herein.

[0082] The present invention provides a therapeutic agent that has been modified or produced by gene editing so as to provide dual inhibition of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions. The therapeutic agent can be used in a method for treating or preventing necrosis. The therapeutic agent can be used in any of the methods, uses and medical uses described herein. The therapeutic agent can also be used in any of the methods, uses and medical uses described in PCT / IB2024 / 050203, the entire disclosure of which is incorporated herein by reference.

[0083] Cell therapies

[0084] Cell therapy is a therapy in which living cells are administered to a subject in order to treat or prevent a disease. Necrosis is a significant challenge in the development of primary or stem cell therapies (therapies in which donor cell populations are injected into diseased or damaged tissue) because these therapies need for densely packed cells that may induce hypoxic conditions and metabolic stress and hence may lead to necrosis.

[0085] For example, cell therapy includes autologous cell therapy using a patient’s own cells, allogeneic cell therapy using donor cells, hematopoietic stem cell transplantation (HSCT), mesenchymal stem cell (MSC) therapy, induced pluripotent stem cell (iPSC) therapy, embryonic stem cell (ESC) therapy, CAR-T cell therapy (chimeric antigen receptor T cells), CAR-NK cell therapy (engineered natural killer cells), TCR-T cell therapy (T-cell receptor engineered T cells), tumor-infiltrating lymphocyte (TIL) therapy, dendritic cell therapy for cancer immunotherapy, regulatory T cell (Treg) therapy for immune modulation, macrophage-based cell therapy, myoblast or satellite cell therapy for muscular disorders, neural stem cell therapy, retinal pigment epithelial (RPE) cell therapy, pancreatic islet cell transplantation for diabetes, fibroblast therapy for wound healing or skin regeneration, endothelial progenitor cell therapy for vascular repair, astrocyte or glial progenitor therapy for neurodegenerative diseases, iPSC-derived cardiomyocyte therapy for heart failure, genetically engineered stem cells for gene-corrected transplantation, universal donor and / or hypoimmunogenic cell therapy using gene-edited cells to avoid immune rejection.

[0086] The present invention provides a method of improving cell therapy, wherein the method comprises administering a composition of the invention. Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors in combination with one or more gap junction inhibitors. For example, the composition comprises dantrolene and quinine. Preferably, the method increases cell survival, and / or the method increases the cell yield (e.g. the cell yield %), and / or the method increases the cell quality (e.g. the viability and / or potency of the cells) in cell therapy. Preferably, the method reduces the necrosis associated with cell therapy.

[0087] The inventors have discovered that the compositions prevent cellular damage associated with necrosis in cell therapy, such that improved cell therapies are achieved when the composition is administered.

[0088] Thus, the present invention provides a method of treating or preventing necrosis associated with cell therapy. The method may comprise administering a composition of the invention, in combination with a cell therapy. For example, the method may comprise administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject, in combination with a cell therapy. For example, the method may comprise administering a composition comprising dantrolene and quinine, in combination with a cell therapy.

[0089] The method of improving cell therapy and the method of treating or preventing necrosis associated with cell therapy may comprise administering the composition of the invention to the subject. Alternatively, the methods may comprise administering the composition of the invention ex vivo, e.g. the composition may be introduced to cells ex vivo or may be introduced to the cell therapy agent.

[0090] Detailed description

[0091] The compositions of the present invention may comprise a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor. Where multiple inhibitors are present, the inhibitors may be administered simultaneously, separately or sequentially. The compositions described herein may be used in any of the medical uses, uses, and methods described herein. For example, the composition may comprise one or more cathepsin inhibitors in combination with one or more calpain inhibitors. As another example, the composition may comprise one or more calcium chelators. For in vitro methods (such as methods for producing artificial or cultivated meat), the calcium chelator(s) are preferably used in relatively high doses.

[0092] The invention is based on the discovery that cellular and tissue necrosis are mediated by a cascade of specific intracellular and potentially intercellular events. As shown in the Examples (in particular, see Example 1) and in Figure 1 , blocking one or more key steps in the cascade enables one to treat, delay or prevent necrosis and associated downstream conditions which may occur in a subject. Necrosis is not reversible, but the inventors have discovered that it can be prevented, attenuated and / or treated by using the invention, e.g., by using one or more of the inhibitors, combinations, and compositions recited herein.

[0093] The inventors have identified that a key step in a cascade of intracellular events that lead to necrosis is a rise in intracellular calcium, which may be caused by an influx of extracellular calcium through calcium channels (e.g., voltage- and / or ligand-gated ion channels) and / or an efflux of calcium from intracellular stores, such as the endoplasmic reticulum (e.g., via ryanodine receptors) as well as through the gap junctions that connect cells to one another. These changes in the cytosolic calcium levels, as well as changes to levels of calcium within internal stores, may further activate receptors / channels (e.g. store-operated calcium channels (SOCC), also called calcium release-activated calcium (CRAC) channels) and thus amplify the increase.

[0094] As shown in the Examples (in particular, see Example 1) and the Figures (in particular, see Figure 1), the inventors have identified that a critical step that leads to necrosis is an increase of calcium ions in the cytosol, which may be caused by any one of the three main pathways of calcium ion entry into a cell: via cell surface membrane channels, via gap junctions, and via internal stores in the cell, in particular the endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR). The inventors have identified that inhibiting the ER / SR calcium channels in combination with inhibiting the gap junctions is particularly effective for preventing or reducing an increase in calcium ions.

[0095] Preferably, therefore, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists, such as dantrolene) and one or more gap junction inhibitors (such as quinine). The inventors have found that this combination is synergistic beyond simple additive effects of the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor and the gap junction inhibitor used individually.

[0096] For example, the composition may comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and (i) one gap junction inhibitor or (ii) two gap junction inhibitors. The endoplasmic / sarcoplasmic reticulum calcium channel inhibitor(s) and the gap junction inhibitor(s) may be administered simultaneously, separately or sequentially. For example, the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor(s) and the gap junction inhibitor(s) may be administered simultaneously.

[0097] The inventors have found that also simultaneously blocking calcium ion entry via plasma membranes, and / or by preventing or reducing a rise in the intercellular concentration of calcium ions by using a calcium chelator, further improves the efficacy of the treatment and uses described herein. The combination of the one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and the one or more gap junction inhibitors may therefore be further combined with one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0098] Thus, the composition may comprise (i) one or more endoplasmic / 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 composition may comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors, one or more gap junction inhibitors, and one or more plasma membrane calcium channel inhibitors. As another example, the composition may comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors, one or more gap junction inhibitors, and one or more calcium chelators. As another example, the composition may comprise one or more endoplasmic / 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. It will be appreciated that these preferred compositions can be used for each of the medical uses, uses, and methods described herein.

[0099] It will be appreciated that the compounds described herein may be used in a therapeutically effective amount. The term “therapeutically effective amount” refers to an amount of a compound that provides the desired therapeutic result. That result can be the prevention, reduction, amelioration, palliation, lessening, delaying, 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.

[0100] It will also be appreciated that references to compounds and agents may also include pharmaceutically acceptable salts and solvates thereof.

[0101] The calcium activity inhibitor referred to herein may be an agent that prevents or reduces a rise in the intracellular concentration of free calcium ions or lowers the intracellular concentration of free (unbound) calcium ions. In other words, the calcium activity inhibitor is an agent that reduces the availability of free intracellular calcium ions. This may be achieved by chelation (using a chelator), blocking the pore of a calcium channel (using a calcium channel blocker), blocking the ligand binding site of a calcium channel (using a calcium channel antagonist), blocking a ryanodine receptor (using an antagonist), or blocking an inositol triphosphate receptor (lnsP3R) (using an antagonist). Thus, the calcium activity inhibitor may be a calcium chelator, a calcium channel blocker or antagonist, a ryanodine receptor antagonist, and / or an lnsP3R antagonist. The calcium activity inhibitor may be a calcium chelator (an agent that sequesters free calcium ions). The calcium chelator may be one or more members selected from the group consisting of: EDTA (ethylenedioxy- diethylene-dinitrilo-tetraacetic acid); derivatives of EDTA (such as EDTA tetrasodium tetrahydrate; EDTA-d12; DMNP-EDTA); EGTA (ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid); derivatives of EGTA (such as EGTA tetrasodium and DMNP-EGTA); EGTA-AM; NP-EGTA (nitrophenyl-EGTA); NP-EGTA-AM; 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)); a derivative of BAPTA (such as BAPTA tetrasodium; BAPTA tetrapotassium; BAPTA tetracesium salt; 5,5’-dimethyl BAPTA tetrapotassium; 5-nitro BAPTA tetramethyl ester; 5-nitro BAPTA, 5,5’-dibromo BAPTA tetrapotassium; 5,5’-dimethyl BAPTA; and 5,5’-difluoro BAPTA); BAPTA AM (2-[N-[2-(acetyloxymethoxy)-2- oxoethyl]-2-[2-[2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]anilino]acetic acid acetyloxymethyl ester); a derivative of BAPTA AM (such as 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 (dimercapto succinic acid); Fura-5F AM; Fura-4F AM; Fluo-3AM; Mag-Fluo-4AM; calcium trinatrium diethylenetriaminepentaacetic acid 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, the calcium chelator may be one or more members selected from the group consisting of: 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 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 (dimercapto succinic acid).

[0102] Preferably the calcium chelator is specific for calcium (e.g., BAPTA or BAPTA AM). Preferably the calcium chelator is one or more members selected from the group comprising BAPTA, BAPTA AM, EGTA, EDTA, citric acid and DMSA. Preferably, the calcium chelator is selected from the group comprising EGTA, EDTA and BAPTA AM. Most preferably, the calcium chelator is selected from DTPA, BAPTA AM, sodium citrate, and citric acid.

[0103] The calcium chelator(s) may be used at relatively high doses to effectively prevent necrosis. The inventors have found that using calcium chelators as a monotherapy is particularly effective for ex-v / vo and in vitro uses, particularly in relatively high doses, but that these relatively high doses may be less suitable for in vivo uses due to potential associated toxicity issues in vivo. In particular, for the methods for producing artificial meat or cultivated meat described herein, the composition of the present invention preferably comprises one or more calcium chelators, particularly in relatively high doses. The term “relatively high dose” used herein for a compound or agent may refer to a dose which is about four times, or about five times, or about six times, or about seven times, or preferably about eight times, the dose typically used in the art for said compound or agent for said use.

[0104] For example, the term “relatively high dose” used herein in the context of calcium chelators may refer to one of the following doses (and these doses are referred to herein as “aspect A”):

[0105] • When the calcium chelator is BAPTA or a derivative of BAPTA (such as BAPTA tetrasodium; BAPTA tetrapotassium; BAPTA tetracesium salt; 5,5’-Dimethyl BAPTA tetrapotassium; 5-Nitro BAPTA tetramethyl ester; 5-Nitro BAPTA, 5,5’-Dibromo BAPTA tetrapotassium; 5,5’-dimethyl BAPTA; 5,5’- difluoro BAPTA), the chelator may be present in an amount of at least about 50 pM, or at least about 60 pM, or at least about 70 pM, or at least about 80 pM, or at least about 90 pM, or at least about 100 pM.

[0106] • When the calcium chelator is BAPTA-AM or a derivative of BAPTA-AM (such as 5,5’-dimethyl BAPTA AM; 5,5’-difluoro BAPTA AM; 5,5’-Dinitro BAPTA AM), the chelator may be present in an amount of at least about 10 pM, or at least about 15 pM, or at least about 20 pM, or at least about 25 pM, for example at least about 50 pM or at least about 100 pM or at least about 150 pM.

[0107] • When the calcium chelator is EGTA or a derivative of EGTA (such as EGTA tetrasodium or DMNP- EGTA), the chelator may be present in an amount of at least about 500 pM, or at least about 1000 pM, or at least about 1500 pM, or at least about 2000 pM, or at least about 2250 pM, or at least about 2500 pM.

[0108] • When the calcium chelator is EGTA-AM, NP-EGTA, or NP-EGTA-AM, the chelator may be present in an amount of at least about 10 pM, or at least about 20 pM, or at least about 50 pM.

[0109] • When the calcium chelator is EDTA or a derivative of EDTA (such as EDTA tetrasodium tetrahydrate or EDTA-d12), the chelator may be present in an amount of at least about 500 pM, or at least about 750 pM, or at least about 850 pM.

[0110] • When the calcium chelator is Fura-5F AM or Fura-4F AM, the chelator may be present in an amount of at least about 20 pM, or at least about 30 pM, or at least about 50 pM.

[0111] • When the calcium chelator is Fluo-3AM or Mag-Fluo-4AM, the chelator may be present in an amount of at least about 20 pM, or at least about 30 pM, or at least about 50 pM.

[0112] • When the calcium chelator is Calcium trinatrium diethylenetriaminepentaacetic acid hydrate, the chelator may be present in an amount of at least about 500 pM, or at least about 750 pM, or at least about 850 pM.

[0113] • When the calcium chelator is DP-b99, the chelator may be present in an amount of at least about 20 pM, or at least about 30 pM, or at least about 50 pM.

[0114] • When the calcium chelator is Quin-2AM, the chelator may be present in an amount of at least about 20 pM, or at least about 30 pM, or at least about 50 pM.

[0115] • When the calcium chelator is Ethylenediaminetetraacetic acid-d16, the chelator may be present in an amount of at least about 100 pM, or at least about 125 pM, or at least about 150 pM.

[0116] • When the calcium chelator is DTPA, the chelator may be present in an amount of at least about 200 pM, or at least about 500 pM, or at least about 1000 pM, or at least about 1500 pM, or at least about 2000 pM, or at least about 2500 pM, or at least about 3000 pM, or at least about 3500 pM, or at least about 4000 pM, for example at least about 8000 pM or at least about 10,000 pM.

[0117] • When the calcium chelator is HEDTA, the chelator may be present in an amount of at least about 200 pM, or at least about 500 pM, or at least about 1000 pM.

[0118] • When the calcium chelator is NTA, the chelator may be present in an amount of at least about 200 pM, or at least about 500 pM, or at least about 1000 pM.

[0119] • When the calcium chelator is citric acid, the chelator may be present in an amount of at least about 200 pM, or at least about 500 pM, or at least about 1000 pM.

[0120] • When the calcium chelator is sodium citrate, the chelator may be present in an amount of at least about 200 pM, or at least about 500 pM, or at least about 1000 pM.

[0121] • When the calcium chelator is TPEN, the chelator may be present in an amount of at least about 10 pM, or at least about 20 pM, or at least about 50 pM.

[0122] • When the calcium chelator is DMSA, the chelator may be present in an amount of at least about 50 pM, or at least about 60 pM, or at least about 70 pM, or at least about 80 pM, or at least about 90 pM, or at least about 100 pM.

[0123] • When the calcium chelator is calcimycin, the chelator may be present in an amount of at least about 0.02 pM, or at least about 0.03 pM, or at least about 0.05 pM.

[0124] • When the calcium chelator is 4-bromo A-23187, the chelator may be present in an amount of at least about 20 pM, or at least about 30 pM, or at least about 50 pM.

[0125] • When the calcium chelator is diazo-2, the chelator may be present at a concentration of at least about 200 pM, or at least about 500 pM, or at least about 1000 pM.

[0126] • When the calcium chelator is DTPA ITC, the chelator may be present in an amount of at least about 10 pM, or at least about 20 pM, or at least about 50 pM.

[0127] • When the calcium chelator is ionomycin, the chelator may be present in an amount of at least about 10 pM, or at least about 20 pM, or at least about 50 pM.

[0128] The calcium activity inhibitor may be an agent that modulates intracellular concentration of free calcium ions. Thus, the calcium activity inhibitor may be a calcium modulator, such as calreticulin (also known as calregulin) or a stromal interaction molecule (STIM).

[0129] The calcium channel blocker referred to herein may prevent a rise in the intracellular concentration of free calcium ions by blocking voltage-dependent Ca2+channels, orai channels and / or store-operated calcium channels. The calcium channel blocker may block voltage-dependent Ca2+channels including one or more selected from the group consisting of the L-, N-, T-, P / Q-, and R-type. Preferably, the calcium channel blocker blocks 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. Dihydropyridines block L-type voltage gated calcium channels. The non-dihydropyridine may be a phenylalkylamine or a benzothiazepine. The non- dihydropyridine may be one or more members selected from the group comprising or consisting of: diltiazem, mibefradil, bepridil, fendiline, flunarizine, fluspirilene, gabapentin, and pregabalin. The endoplasmic / sarcoplasmic reticulum calcium channel inhibitors referred to herein are preferably one or more ryanodine receptor antagonists. It will be appreciated that endoplasmic / sarcoplasmic reticulum inhibitors are also referred to herein as endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors.

[0130] The ryanodine receptor antagonist referred to herein may be one or more members selected from the group comprising or consisting of: dantrolene, DHBP dibromide, cis-Ned 19, trans-Ned 19, ryanodine, SKF 86365 hydrochloride, ruthenium red, procaine, and tetracaine. Preferably the ryanodine receptor antagonist is dantrolene, ryanodine, trans-Ned 19, cis-Ned 19, procaine or ruthenium red. Preferably the ryanodine receptor antagonist is dantrolene, ryanodine, trans-Ned 19, cis-Ned 19, or ruthenium red. Preferably the ryanodine receptor antagonist is dantrolene and / or ryanodine. Most preferably the ryanodine receptor antagonist is dantrolene. Thus, most preferably, the compositions of the present invention comprise dantrolene, and these compositions are most preferred for each of the uses, medical uses and methods of the invention. It will be appreciated that any derivative, analog or variant of the ryanodine receptor antagonist (e.g. of dantrolene) may be used instead of the ryanodine receptor antagonist in the compositions of the present invention.

[0131] The ryanodine receptor antagonist may be a xestospongin, including a hydroxylated xestospongin.

[0132] The lnsP3R antagonist referred to herein may be one or more members selected from the group comprising or consisting of: a macrocyclic 1-oxaquinolizidine, 2-aminoethoxydiphenyl borate, an aromatic polyphosphate, and a tetrakisphosphate (e.g., a myo-lnositol 1 ,3,4,5-tetrakisphosphate). The aromatic polyphosphate or derivatives thereof may be benzene 1 ,2,4-trisphosphate [Bz(1 ,2,4)P3]; biphenyl derivative BiPh(2,3',4,5',6)P5; or a dimeric benzene phosphate. The macrocyclic 1-oxaquinolizidine may be a xestospongin (such as xestospongin A, B, C or D), araguspongine B, araguspongine C, 7S-Hydroxyxestospongin A (7-OHXeA); and demethylxestospongin B (DMXeB). The lnsP3R antagonist referred to herein may be heparin, caffeine, IP3R- Binding protein released with Inositol 1 ,4,5-Trisphosphate (IRBIT), or ci-IP3 / PM. Preferably the lnsP3R antagonist is heparin, caffeine, 2-APB (2-aminoethoxydiphenyl borate), xestospongin A, xestospongin B, xestospongin C or xestospongin D. The skilled person would appreciate that heparin may also be used as one of the one or more gap junction inhibitors in each of the compositions (e.g. in combination with one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors, such as dantrolene) of the present invention.

[0133] A calpain is a member of a family of calcium-activated cysteine protease proteolytic enzymes. They comprise an 80kD catalytic subunit and a 20kD regulatory subunit that stabilises the catalytic subunit. Members of the family are listed in the table below.

[0134] Table 1 - Calpain family members

[0135] Calpains tend to be located in the cytoplasm and the nucleus. Consequently, a rise in intracellular calcium causes activation of calpains and thus leads to autolysis. Thus, a calpain inhibitor may be any agent that inhibits calpain enzyme proteolytic activity. The calpain inhibitor may block the active site of the enzyme. The calpain inhibitor may allosterically inhibit the proteolytic activity of the enzyme. Preferably the calpain inhibitor blocks calpain 1 (mu-type) and / or calpain 2 (m-type). The calpain inhibitor referred to herein may be one or more members selected from the group comprising or consisting of: a calpastatin, MG101 , MG132, vidupiprant, an a-mercaptoacrylate, a 5-azolone, a carboxamide and a a-helical cysteine protease inhibitor. The calpain inhibitor may be an agent that binds (specifically) to the calpain hexapeptide, CYGRKK, or the calpain 5-mer peptide CYGAK. The calpain inhibitor may be an agent that also inhibits cathepsins, such as E64 ([L-trans-3-Carboxyoxirane-2-carbonyl]-L-Leu-agmatine), E64d, or calpeptin. It will be understood that agents that inhibit calpains and cathepsins may also be referred to as “non-specific calpain / cathepsin inhibitors”. The calpain inhibitor may be an agent that is specific for calpains, such as MG101 , MG132, or PD150606. The calpain inhibitor may be MDL 28170. For example, the calpain inhibitor may be MG101 , vidupiprant, calpeptin, E64, PD150606 or MDL 28170. Calpain inhibition may be measured by cleavage of the substrate, Ac-LLY-AFC, which emits blue light (Amax = 400 nm) upon cleavage of the substrate by calpain. Free AFC emits a yellow-green fluorescence (Amax = 505 nm), which can be quantified using a fluorometer or a fluorescence plate reader. Comparison of the fluorescence intensity from a treated sample with a normal control allows determination of the changes in calpain activity.

[0136] A cathepsin inhibitor may be any agent that inhibits cathepsin enzyme activity. The cathepsin inhibitor may block the active site of the enzyme. The cathepsin inhibitor may allosterically inhibit the activity of the enzyme. Preferably the cathepsin inhibitor inhibits cathepsins L, B, and / or D. The cathepsin inhibitor referred to herein may be one or more members selected from the group comprising: a cystatin, a triterpene, and a thiosemicarbazone. The cathepsin inhibitor may be one or more of dexamethasone, rifampicin, E64, E64d, calpeptin, CA 074, SID26681509, L006235, bafilomycin, aloxistatin, astaxanthin, chloroquine, clofazimine, dec-RVKR, or dexamethasone. The cathepsin inhibitor may be an agent that also inhibits calpain enzymes, such as E64, E64d, or calpeptin. The cathepsin inhibitor may be an agent that is specific for cathepsins, such as CA 074 ([L-3-trans-(Propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester), SID26681509, L006235, or bafilomycin. Cathepsin inhibition may be measured by performing a fluorometric assay with a preferred substrate. For example, the sequence of a cathepsin-B substrate is RR labelled with AFC (amino-4-trifluoromethyl coumarin). Cell lysates or other samples that contain cathepsin-B will cleave the synthetic substrate RR-AFC to release free AFC. The released AFC can easily be quantified using a fluorometer or fluorescence plate reader at Ex / Em 400 / 505 nm. The sequence of a cathepsin-D substrate is GKPILFFRLK(Dnp)-D-R-NH2) labelled with MCA. Cell lysates or other samples that contain cathepsin-D will cleave the synthetic substrate to release fluorescence, which can then easily be quantified using a fluorometer or fluorescence plate reader at Ex / Em = 328 / 460 nm.

[0137] For example, a combination of one or more calpain inhibitors and one or more cathepsin inhibitors can be used, such as PD150606 in combination with dexamethasone, or PD150606 in combination with rifampicin.

[0138] In compositions of the present invention which comprise the combination of one or more ER / SR endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, the composition or combination may not further comprise a calpain inhibitor and / or may not further comprise a cathepsin inhibitor.

[0139] Gap junctions are intercellular channels that permit cell-cell transfer of ions (such as Ca2+) and small molecules. Thus, a rise in intracellular calcium in one cell may spread to neighbouring cells through corresponding gap junctions and thus cause necrosis in neighbouring cells. Thus, a gap junction inhibitor may be used to treat or prevent necrosis, and thus may be useful for treating other medical conditions that occur downstream of necrosis, and / or may be useful for adjunctive therapies for treating necrosis whilst also treating other primary medical conditions. In particularly preferred embodiments of the invention, one or more gap junction inhibitors are used in combination with one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors.

[0140] The gap junction inhibitor referred to herein may be a product of vitamin A (such as retinoic acid, glycyrrhetinic acid and its derivative carbenoxolone), long-chain alcohols (such as heptanol and octanol), halogenated general volatile anaesthetics (such as halothane), fatty acids (such as linoleic acid, arachidonic acid and oleic acid), fatty acid amides (such as oleamide), fenamates (arylaminobenzoates, such as flufenamic acid, niflumic acid and meclofenamic acid), quinine, quinidine and quinine-derivatives (such as mefloquine). The gap junction inhibition may be a fatty acid (preferably a fatty acid containing 13-21 carbons), a polyamine or a cyclodextrin. The gap junction inhibitor referred to herein may be a pannexin inhibitor and / or a connexin inhibitor, preferably a connexin inhibitor. The gap junction inhibitor referred to herein may be a retinoid which is a derivative of vitamin A. Preferably, the gap junction inhibitor referred to herein may be selected from one or more (e.g., one or two) of 18a-glycyrrhetinic acid, 18p-glycyrrhetinic acid, carbenoxolone (such as carbenoxolone disodium), heptanol (such as 1-heptanol), heptanoic (oenanthic) acid, octanol (such as 1-octanol), halothane, oleic acid (such as cis-oleic acid), oleamide, linoleic acid, arachidonic acid, palmitic acid, palmitoleic acid, flufenamic acid, myristic acid, lauric acid, quinine, quinidine, quinidine gluconate, dihydroquinidine, mefloquine, mefloquine hydrochloride, meclofenamic acid, 2-aminoethoxydiphenyl borate, probenecid, beta-cyclodextrin, acridone 3, spermine, spermidine, putrescine, boldine, 5-hydroxytryptophan (5-HTP), niflumic acid, flufenamic acid, astaxanthin, and retinoic acid (such as tretinoin (all-trans-retinoic acid), isotretinoin (13-cis-retinoic acid), and / or 9-cis-retinoic acid). Preferably, the gap junction inhibitor referred to herein may be selected from one or more (e.g., one or two) of 18a-glycyrrhetinic acid, 18p-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. For example, the gap junction inhibitor may be retinoic acid or oleic acid. Most preferably, the gap junction inhibitor is quinine.

[0141] Another gap junction inhibitor is tonabersat. As such, the disclosure herein of aspects, embodiments, claims and statements of invention that are presented in relation to the gap junction inhibitors listed above (e.g. quinine) give rise to corresponding aspects, embodiments, claims and statements of invention in which tonabersat is used as the gap junction inhibitor. Tonabersat is particularly preferred as a gap junction inhibitor when the composition of the invention is used to treat or prevent a central nervous system and / or neuronal disease or disorder. For example, tonabersat may be used in any one of the methods and compositions disclosed herein or disclosed in PCT / IB2024 / 050203 (which is incorporated herein by reference) including, for example, in methods or compositions used to treat or prevent traumatic brain injury (TBI); other forms of head injury including concussion, closed head injury, penetrating head injury, diffuse brain injury, brain contusion caused for example by a depressed skull fracture ora penetrating skull fracture, and cerebral palsy; intracranial hematoma (ICH); hematoma; stroke; brain aneurysm; hypoxic and anoxic brain injury; haemorrhage; meningitis; encephalitis; epilepsy; ataxia; motor neuron disease; multiple system atrophy; progressive supranuclear palsy; spinal cord injury; neurodegenerative disease including Alzheimer’s disease (AD), Parkinson’s disease (PD), prion diseases, amyotrophic lateral sclerosis (ALS), Huntington’s disease, spinal muscular atrophy, spinocerebellar ataxia; and multiple sclerosis (MS).

[0142] Where there are two or more gap junction inhibitors, the gap junction inhibitor may 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 inhibitor is two of heptanol, octanol, oleic acid, retinoic acid, linoleic acid, palmitoleic acid, astaxanthin and quinine. For example, it may be retinoic acid in combination with one or more of oleic acid, linoleic acid, octanol and heptanol, or it may be octanol in combination with one or more of retinoic acid, oleic acid, linoleic acid, and heptanol. As another preferred example, the gap junction inhibitor may be two or more of retinoic acid, oleic acid and linoleic acid.

[0143] Determination of the transfer of fluorescent dye tracers between adjacent cells via techniques like fluorescence recovery after photobleaching (FRAP) or flow cytometry can be used to as an assay to measure gap junction inhibition.

[0144] The plasma membrane calcium channel inhibitors referred to herein may be one or more of dihydropyridines, nondihydropyridines (such as phenylalkylamines and benzothiazepines), non-selective calcium channel inhibitors and gabapentinoids. The plasma membrane calcium channel inhibitors 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 plasma membrane calcium channel inhibitors may be selected from one or more of amlodipine, arandipine, clevidipine, efonidipine, felodipine, isradipine, lercanidipine, manidipine, nifedipine, nimodipine, nitrendipine, gallopamil, verapamil, diltiazem, mibefradil, fluspirilene, gabapentin and pregabalin. Preferably, the plasma membrane calcium channel inhibitor is amlodipine.

[0145] Thus, a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor can be used to treat or prevent necrosis, and thus may be useful for treating other medical conditions that occur downstream of necrosis, and / or may be useful for adjunctive therapies for treating necrosis whilst also treating other primary medical conditions.

[0146] The invention may comprise a selection of one or more of a calcium-activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor. The invention may comprise a selection of two or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor. Thus, the invention may comprise a combination of a calcium activity inhibitor and a cathepsin inhibitor. The invention may comprise a combination of a calcium activity inhibitor and a calpain inhibitor. The invention may comprise a combination of a cathepsin inhibitor and a calpain inhibitor. In a preferred embodiment, the invention comprises a combination of one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors (optionally also in combination with one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators). For example, the invention comprises a combination of dantrolene and quinine (optionally also in combination with one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0147] The invention may comprise one endoplasmic / sarcoplasmic reticulum calcium channel inhibitor and one or more gap junction inhibitors. In one preferred embodiment, the invention may comprise one endoplasmic / sarcoplasmic reticulum calcium channel inhibitor and two or more gap junction inhibitors. For example, the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor may be dantrolene, and the two or more gap junction inhibitors may be selected from heptanol, octanol, oleic acid, retinoic acid, linoleic acid, palmitoleic acid, astaxanthin and quinine. As another example, the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor may be dantrolene, and the two or more gap junction inhibitors may be selected from oleic acid, retinoic acid and linoleic acid (i.e., the two or more gap junction inhibitors may 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 / sarcoplasmic reticulum calcium channel inhibitor may be dantrolene, and the two or more gap junction inhibitors may be selected from retinoic acid, heptanol and octanol. As another example, the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor may be dantrolene, and the two or more gap junction inhibitors may be selected from (i) retinoic acid and heptanol, and (ii) heptanol and octanol.

[0148] As another preferred embodiment, the invention may comprise two or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and two or more gap junction inhibitors. For example, the endoplasmic / sarcoplasmic reticulum calcium channel inhibitors may be dantrolene and ryanodine, and the two or more gap junction inhibitors may be selected from any of the combinations recited herein (such as two or more of oleic acid, retinoic acid and linoleic acid).

[0149] The invention may comprise a selection of three or more of a calcium activity-inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor. For example, the invention may comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as dantrolene), one or more gap junction inhibitors (such as retinoic acid, oleic acid, and / or linoleic acid), and one or more calcium chelators (such as DTPA, BAPTA-AM, sodium citrate, and / or citric acid). Alternatively, the invention may comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as dantrolene), one or more gap junction inhibitors (such as retinoic acid, oleic acid, and / or linoleic acid), and one or more plasma membrane calcium channel inhibitors (such as amlodipine, aranidipine, clevidipine, nimodipine, pranidipine, verapamil, nisoldipine, efonidipine, gabapentin, gallopamil and / or felodipine, preferably amlodipine). Alternatively, the invention may comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as dantrolene), one or more gap junction inhibitors (such as retinoic acid, oleic acid, and / or linoleic acid), one or more calcium chelators (such as DTPA, BAPTA-AM, sodium citrate, and / or citric acid), and one or more plasma membrane calcium channel inhibitors (such as amlodipine, aranidipine, clevidipine, nimodipine, pranidipine, verapamil, nisoldipine, efonidipine, gabapentin, gallopamil and / or felodipine, preferably amlodipine).

[0150] The invention may further comprise an SOCC inhibitor, such as 2-aminoethoxydiphenyl borate, GNF362, Leflunomide, Roflumilast, Teriflunomide, Tolvaptan, Xestospongin C and / or zinc chloride. In some embodiments of the present invention, the composition or combination does not comprise an SOCC inhibitor.

[0151] In embodiments in which the invention comprises a calcium activity inhibitor, the invention referred to herein may comprise one or more of the following classes of agent: a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. The invention may comprise two or more of the following classes of agent: calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. Most preferably, the invention comprises a ryanodine receptor antagonist and a gap junction inhibitor. The invention may comprise three or more of the following classes of agent: a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. Preferably the invention comprises two or more different classes agents selected from the group consisting of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. Preferably, the invention comprises a calcium chelator, a ryanodine receptor antagonist, and a gap junction inhibitor.

[0152] The compounds, combinations and compositions described herein can be administered via any suitable route known in the art. Thus, the compounds, combinations and compositions of the present invention can be administered by injection, infusion, continuous infusion, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intravitreally, intravag inally, intra rectally, topically, intratumourally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivaly, sublingually, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, topically, locally, via inhalation (e.g. aerosol inhalation), via a catheter, via a lavage, buccally, rectally, vaginally, via the ocular route, via the otic route, nasally, by nebulization, cutaneously, systemically, transdermally, or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art.

[0153] Each of the compounds, combinations and compositions described herein may be provided as a pharmaceutical composition, which may further comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, and antifungal agents), isotonic agents, absorption delaying agents, salts, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.

[0154] Each of the compounds described herein may be provided as a pharmaceutical composition, wherein the pH of the pharmaceutical composition has been adjusted. For example, any acidic compounds described herein (such as citric acid) may require the pH of the composition to be adjusted.

[0155] Each of the compounds, combinations and compositions described herein may be provided in a drug delivery device. Thus, the invention provides a drug delivery device comprising any of the compounds, combinations and compositions described herein (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators). The drug delivery device may be selected from an inhalation device (including nebulizers and metered-dose inhalers), injection device (including autoinjectors, injector pens, jet injectors, safety syringes, syringes, pre-filled syringes, nanoneedles, and microneedle drug delivery devices), implant and stent (including drug-eluting stents), intrauterine device, anaesthetic machine, anesthetic vaporizer, analytical nebulizer, aptargroup, aquasome, cervical drug delivery device, coiled-coil drug delivery device, convection enhanced delivery device, douche, infusion pump, stimuli- responsive drug delivery system, stretch-triggered drug delivery device, and / or a mechanical sensation delivery device.

[0156] Each of the compounds, combinations and compositions described herein may be provided in safety equipment. Thus, the invention provides safety equipment comprising any of the compounds, combinations and compositions described herein (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators). The safety equipment may be selected from car airbags, protective vests, airbag vests, ankle braces, protective eyewear, body armor, protective clothing, gloves, masks, personal protective equipment, goggles, face shields, and gowns.

[0157] Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing the side-effects associated with alcohol consumption in a subject, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject. It will be appreciated that the subject will be a person or persons who consume or have consumed alcohol.

[0158] The side-effect may be necrosis. Thus, each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing necrosis associated with alcohol consumption in a subject, wherein the method comprises administering the compound(s), combination^) or composition^) to the subject.

[0159] According to a preferred aspect of the invention, there is provided a method of treating or preventing the sideeffects (such as necrosis) associated with alcohol consumption in a subject, wherein the method comprises administering one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, the method comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0160] The compounds, combinations or compositions may be administered to a subject priorto alcohol consumption, to a subject who is concurrently consuming alcohol, and / or to a subject who has previously consumed alcohol. In an aspect, the compounds, combinations or compositions are administered immediately before a subject consumes alcohol, or before an undesired amount of alcohol is reached in the body. In an aspect, the compounds, combinations or compositions are administered concurrently with a subject consuming alcohol. In an aspect, the compounds, combinations or compositions are administered after a subject has consumed alcohol, or after an undesired amount of alcohol has been reached in the body. Preferably, the compounds, combinations or compositions are administered to the body whilst alcohol is in the subject’s blood.

[0161] Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing an alcohol associated disease in a subject, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject. The alcohol associated disease may be selected from one or more of: osteonecrosis, liver diseases (such as steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma), pancreatitis, cardiovascular diseases (such as hypertension, ischemic heart disease, stroke, cardiomyopathy, myocarditis, and various arrhythmias), neuropsychiatric disorders (such as major depression), tumours, cancers, glomerulonephritis, kidney damage, and reproductive and developmental issues (such as impaired fertility, premature birth, low-weight births, and fetal alcohol syndrome spectrum disorders). Preferably, the alcohol associated disease may be liver disease, pancreatitis, cardiovascular disease, and / or kidney damage.

[0162] According to a preferred aspect of the invention, there is provided a method of treating or preventing an alcohol associated disease in a subject, wherein the method comprises administering one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, the method comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0163] Cell death associated with therapy

[0164] Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing necrosis associated with cancer and / or tumours in a subject, wherein the method comprises administering the compound(s), combination(s) or composition(s).

[0165] Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing the cell death associated with therapy of a subject, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject who is undergoing, or who has undergone, therapy.

[0166] The method may comprise simultaneously, separately, or sequentially administering (i) one or more doses of therapy, and (ii) one or more doses of the compound(s), combination(s) or composition(s).

[0167] According to a preferred aspect of the invention, there is provided a method of treating or preventing the cell death (such as necrosis) associated with therapy of a subject, wherein the method comprises administering one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject who is undergoing, or who has undergone, therapy. Preferably, the method comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0168] The method may comprise simultaneously, separately, or sequentially administering (i) one or more doses of therapy, and (ii) one or more doses of a composition comprising endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors, and / or optionally one or more calcium chelators.

[0169] The cell death is preferably necrosis. The therapy is preferably therapy for one or more cancers and / or for one or more precancerous lesions. The cell death (such as necrosis) may be associated with one or more of the following therapies:

[0170] (i) External beam radiation therapy (EBRT), such as 3D conformal radiation therapy;

[0171] (ii) Intensity-modulated radiation therapy (IMRT);

[0172] (iii) Arc-based radiotherapy;

[0173] (iv) Image-guided radiotherapy (IGRT);

[0174] (v) Particle therapy;

[0175] (vi) Stereotactic radiosurgery, such as Gamma Knife surgery, Stereotactic body radiation therapy (SBRT), and / or Intraoperative radiation (IORT); and / or

[0176] (vii) internal radiation therapy, such as brachytherapy and / or systemic therapy.

[0177] Reducing toxicity associated with administration of a toxic agent

[0178] Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing toxicity (such as necrosis) in a subject, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject.

[0179] Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing a toxic effect (such as necrosis) in a subject, wherein the method comprises administering the compounds, combinations or compositions described herein.

[0180] There is also provided a method of treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more toxic agents, wherein the method comprises co-administering to a subject the toxic agent in combination with the compounds, combinations or compositions described herein. According to a preferred aspect, there is provided a method of treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more toxic agents, wherein the method comprises co-administering to the subject (i) the toxic agent, and (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. Preferably, (ii) comprises one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0181] Suitable toxic agents are described herein. Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more of the toxic agents described herein, wherein the method comprises coadministering the toxic agents with the compound(s), combination(s) or composition(s).

[0182] The toxic agent may be one or more poisons. For example, the toxic agent may be one or more of carbon monoxide, a heavy metal (such as lead, mercury, and arsenic), tobacco smoke, asbestos, benzene, cadmium, formaldehyde, radon, vinyl chloride, cocaine, heroin, methamphetamine, a blood flow altering narcotic, strychnine, cyanide, ricin, botulinum toxin, tetrodotoxin, snake venom, scorpion venom, bee venom, insect venom, and / or mushroom toxins (such as amatoxins).

[0183] Thus, a preferred method comprises treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more poisons, wherein the method comprises co-administering to the subject (i) the poisons, and (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, (ii) comprises one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0184] The toxic agent may be a chemotherapeutic agent. For example, the chemotherapeutic agent may be one or more of an alkylating agent, antimetabolite, antimicrotubule agents, anthracyclines, platinum compounds, topoisomerase inhibitors, antibiotics, enzymes, and antineoplastic drugs.

[0185] Suitable alkylating agents include Bendamustine, Busulfan, Carmustine, Chlorambucil, Cyclophosphamide, Dacarbazine, Estramustine, Hydroxyurea, Ifosfamide, Lomustine, Mechlorethamine, Melphalan, Oxaliplatin, Procarbazine, Streptozocin, Temozolomide, Thiotepa and Treosulfan.

[0186] Suitable antimetabolites include Azacitidine, Capecitabine, Cladribine, Cytarabine, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, Pralatrexate, Pemetrexed, Pentostatin, Raltitrexed, Thioguanine, and Trifluridine-Tipiracil. Suitable antimicrotubule agents include Cabazitaxel, Docetaxel, Eribulin, Ixabepilone, Paclitaxel, Vinblastine, Vincristine, and Vinorelbine. For example, taxanes may be used, such as Paclitaxel. For example, vinca alkaloids may be used, such as Vinblastine and Vincristine.

[0187] Suitable a nth racy clines include Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, and Mitoxantrone.

[0188] Suitable platinum compounds include Cisplatin, Carboplatin, and Oxaliplatin.

[0189] Suitable topoisomerase inhibitors include Etoposide, Irinotecan, Teniposide, and Topotecan.

[0190] Suitable antibiotics include Actinomycin D, Bleomycin, and Mitomycin.

[0191] Suitable enzymes include Asparaginase.

[0192] Suitable antineoplastic drugs include All-trans-retinoic acid, Arsenic trioxide, Mitotane, Omacetaxine, Pegaspargase, Romidepsin, and Vorinostat.

[0193] Thus, a preferred method comprises treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more chemotherapeutic agents, wherein the method comprises co-administering to the subject (i) the chemotherapeutic agents, and (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, (ii) comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0194] The toxic agent may be a corticosteroid, such as Prednisolone and Dexamethasone.

[0195] Thus, a preferred method comprises treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more corticosteroids, wherein the method comprises co-administering to the subject (i) the corticosteroid, and (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, (ii) comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. The toxic agent may be an agent which decreases calcium ion levels across the plasma membrane and / or which decreases calcium ion cytosol levels.

[0196] For example, the toxic agent may be a carbonic anhydrase inhibitor, such as Acetazolamide, Dorzolamide, and / or Rinzolamide. Carbonic anhydrase inhibitors are compounds which inhibit carbonic anhydrase. This may lead to bicarbonate reabsorption in the kidney, which can indirectly affect the acid-base balance and calcium handling. It may therefore be desirable to alter the calcium ion levels in a subject who has been, or who will be, administered a carbonic anhydrase inhibitor, in order to treat or prevent the undesirable calcium imbalances induced by the carbonic anhydrase inhibitors.

[0197] Thus, the invention provides a method of treating or preventing a toxic effect (such as a calcium imbalance) associated with administering of one or more carbonic anhydrase inhibitors (such as Acetazolamide, Dorzolamide, and / or Rinzolamide), wherein the method comprises co-administering to a subject the carbonic anhydrase inhibitor in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0198] For example, the toxic agent may be a histamine H2 receptor antagonist, such as Famotidine, Ranitidine, and / or Cimetidine. Histamine H2 receptor antagonists are compounds which block histamine H2 receptors, which can reduce gastric acid secretion, which can in turn affect the calcium absorption in the gut. It may therefore be desirable to improve or modify calcium absorption in the gut of a subject who has been administered a histamine H2 receptor antagonist, in order to treat or prevent the undesirable changes to the calcium absorption induced by the histamine H2 receptor antagonist.

[0199] Thus, the invention provides a method of treating or preventing a toxic effect (such as modified calcium absorption) associated with administering of one or more histamine H2 receptor antagonists (such as Famotidine, Ranitidine, and / or Cimetidine), wherein the method comprises co-administering to a subject the histamine H2 receptor antagonist in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0200] For example, the toxic agent may be a proton pump inhibitor (PPI), such as Omeprazole, Pantoprazole, and / or Esomeprazole. PPIs are compounds which inhibit the gastric proton pump. This can reduce gastric acid secretion, which can in turn affect the calcium absorption in the gut. It may therefore be desirable to improve or modify calcium absorption in the gut of a subject who has been administered a PPI, in order to treat or prevent the undesirable changes to the calcium absorption induced by the PPI. Thus, the invention provides a method of treating or preventing a toxic effect (such as modified calcium absorption) associated with administering of one or more PPIs (such as Omeprazole, Pantoprazole, and / or Esomeprazole), wherein the method comprises co-administering to a subject the PPI in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0201] For example, the toxic agent may be a diuretic, such as Furosemide, Spironolactone and / or Thiazides. Diuretics are known to increase urine output, which can affect electrolyte balance and indirectly influence cytosolic calcium levels. It may therefore be desirable to improve or modify the cytosolic calcium levels in a subject who has been administered a diuretic, in order to treat or prevent the undesirable changes to the cytosolic calcium levels resulting from the diuretic.

[0202] Thus, the invention provides a method of treating or preventing a toxic effect (such as modified cytosolic calcium levels) associated with administering of one or more diuretics (such as Furosemide, Spironolactone and / or Thiazides), wherein the method comprises co-administering to a subject the diuretic in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0203] For example, the toxic agent may be a bicarbonate, such as sodium bicarbonate. Bicarbonates are known to buffer hydrogen ions, which may affect the acid-base balance and calcium handling in cells. It may therefore be desirable to alter the calcium ion levels in a subject who has been, or who will be, administered a bicarbonate, in order to treat or prevent the undesirable calcium imbalances induced by the bicarbonate.

[0204] Thus, the invention provides a method of treating or preventing a toxic effect (such as a calcium imbalance) associated with administering of one or more bicarbonates (such as sodium bicarbonate), wherein the method comprises co-administering to a subject the bicarbonate in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0205] For example, the toxic agent may be a phosphodiesterase inhibitor, such as Sildenafil, Tadalafil and / or Vardenafil. Phosphodiesterase inhibitors are known to increase cyclic AMP levels, which can modulate calcium release from intracellular stores. It may therefore be desirable to modify the calcium release from intracellular stores in a subject who has been, or who will be, administered a phosphodiesterase inhibitor, in order to treat or prevent the undesirable calcium release modulation caused by the phosphodiesterase inhibitor.

[0206] Thus, the invention provides a method of treating or preventing a toxic effect (such as a modified calcium release from interacellular stores) associated with administering of one or more phosphodiesterase inhibitors (such as Sildenafil, Tadalafil and / or Vardenafil), wherein the method comprises co-administering to a subject the phosphodiesterase inhibitor in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0207] For example, the toxic agent may be a sarco / endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, such as Thapsigargin. SERCA inhibitors block the sarco / endoplasmic reticular Ca2+-ATPase, thereby preventing reuptake of calcium into the ER and thus increasing cytosolic calcium levels. It may therefore be desirable to modulate the uptake of calcium into the ER in a subject who has been, or who will be, administered a SERCA inhibitor, in order to counteract the increased cystosolic calcium levels induced by the SERCA inhibitor.

[0208] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the increased cystosolic calcium levels) associated with administering of one or more SERCA inhibitors (such as Thapsigargin), wherein the method comprises co-administering to a subject the SERCA inhibitor in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0209] For example, the toxic agent may be a PMCA inhibitor, such as Calmidazolium. PMCA inhibitors are known to block the plasma membrane calcium ATPase, which may prevent calcium efflux from the cytosol and hence increase cytosolic calcium levels. It may therefore be desirable to modulate the calcium efflux from the cytosol and / or decrease cytosolic calcium levels in a subject who has been, or who will be, administered a PMCA inhibitor, in order to counteract the increased cystosolic calcium levels induced by the PMCA inhibitor.

[0210] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the increased cystosolic calcium levels) associated with administering of one or more PMCA inhibitors (such as Calmidazolium), wherein the method comprises co-administering to a subject the PMCA inhibitor in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0211] For example, the toxic agent may be a TRPV channels modulator, such as Capsaicin. TRPV channels modulators may affects transient receptor potential vanilloid channels, which can influence calcium influx into cells. It may therefore be desirable to improve or modify the calcium influx into cells in a subject who has been administered a TRPV channels modulator, in order to treat or prevent the undesirable changes to the calcium influx into cells resulting from the TRPV channels modulator.

[0212] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing changes to the calcium influx or cells) associated with administering of one or more TRPV channels modulators (such as Capsaicin), wherein the method comprises co-administering to a subject the TRPV channels modulator in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0213] For example, the toxic agent may be a calcium-sensing receptor agonist, such as Cinacalcet. Calcium-sensing receptor agonist may mimic the effect of high calcium levels on the calcium-sensing receptor, reducing PTH secretion and hence lowering calcium levels in the body. It may therefore be desirable to improve or increase the calcium levels in the body of a subject who has been administered a calcium-sensing receptor agonist, in order to treat or prevent the undesirable lowering of the calcium levels in the body resulting from administration of the calcium-sensing receptor agonist.

[0214] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing a lowering in the calcium levels in the body) associated with administering of one or more calcium-sensing receptor agonists (such as Cinacalcet), wherein the method comprises co-administering to a subject the calcium-sensing receptor agonist in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0215] For example, the toxic agent may be a vitamin D analogue, such as Calcitriol and / or Paricalcitol. Vitamin D anaologues may act as agonists for vitamin D receptors, which can reduce intestinal calcium absorption and hence promote the renal excretion of calcium. It may therefore be desirable to improve or increase the intestinal calcium absorption and / or reduce renal excretion of calcium in a subject who has been administered a vitamin D analogue, in order to treat or prevent the reduced intestinal calcium absorption and / or promoted renal excretion of calcium resulting from administration of the vitamin D analogue.

[0216] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing reduced intestinal calcium absorption and / or treating or preventing the promoted renal excretion of calcium) associated with administering of one or more vitamin D analogues (such as Calcitriol and / or Paricalcitol), wherein the method comprises co-administering to a subject the vitamin D analogue in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0217] For example, the toxic agent may be a bisphosphonate, such as Alendronate, Risedronate and / or Zoledronic Acid. Bisphosphonates may inhibit bone resorption, and may reduce the release of calcium into the bloodstream. It may therefore be desirable to improve or increase the bone resorption and / or increase the release of calcium into the bloodstream in a subject who has been administered a bisphosphonate, in order to treat or prevent the inhibited bone resorption and / or reduced calcium release resulting from administration of the bisphosphonate.

[0218] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the inhibited bone resorption and / or treating or preventing the reduced calcium release into the bloodstream) associated with administering of one or more bisphosphonates (such as Alendronate, Risedronate and / or Zoledronic Acid), wherein the method comprises co-administering to a subject the bisphosphonate in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0219] For example, the toxic agent may be a Calcitonin Gene-Related Peptide (CGRP) Antagonist, such as Telcagepant. CGRP antagonists are known to block CGRP receptors, thereby reducing vasodilation and potentially affecting calcium handling in vascular smooth muscle. It may therefore be desirable to improve or increase vasodilation and / or modulate calcium handling in vascular smooth muscle in a subject who has been administered a CGRP antagonist, in order to treat or prevent the reduced vasodilation and impaired calcium handling in vascular smooth muscle resulting from administration of the CGRP antagonist.

[0220] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the reduced vasodilation and / or impaired calcium handling in vascular smooth muscle) associated with administering of one or more CGRP antagonists (such as Telcagepant), wherein the method comprises coadministering to a subject the CGRP antagonist in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0221] For example, the toxic agent may be a natriuretic peptide, such as an atrial natriuretic peptide (ANP) or a brain natriuretic peptide (BNP). Such natriuretic peptides are known to increase the urinary excretion of sodium and water, which may affect electrolyte balance and indirectly influence cytosolic calcium levels. It may therefore be desirable to modulate the cytosolic calcium levels in a subject who has been, or who will be, administered a natriuretic peptide, in order to counteract the undesirable changes to the cystosolic calcium levels induced by the natriuretic peptide.

[0222] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the impaired or modulated cystosolic calcium levels) associated with administering of one or more natriuretic peptides, wherein the method comprises co-administering to a subject the natriuretic peptide in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators). For example, the toxic agent may be a chloride channel blocker, such as dihydroindenyl oxyacetic acid (DIOA). Chloride channel blockers are known to block chloride channels, which may affect cell volume and potentially influence calcium handling. It may therefore be desirable to alter the calcium ion levels in a subject who has been, or who will be, administered a chloride channel blocker, in order to treat or prevent the undesirable changes to calcium handling induced by the chloride channel blocker.

[0223] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing a calcium imbalance or impaired calcium handling) associated with administering of one or more chloride channel blockers (such as DIOA), wherein the method comprises co-ad ministering to a subject the chloride channel blocker in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0224] For example, the toxic agent may be a potassium channel activator, such as Minoxidil. Potassium channel activators open potassium channels, thereby leading to hyperpolarization and inhibition of voltage-gated calcium channels. This can reduce cytosolic calcium levels. It may therefore be desirable to increase the cytosolic calcium levels in a subject who has been, or who will be, administered a potassium channel activator, in order to counteract the reduced cytosolic calcium levels induced by the potassium channel activator.

[0225] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the reduced cytosolic calcium levels) associated with administering of one or more potassium channel activators (such as Minoxidil), wherein the method comprises co-ad ministering to a subject the potassium channel activator in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0226] For example, the toxic agent may be a sodium-potassium ATPase inhibitor, such as Ouabain. Sodiumpotassium ATPase inhibitors inhibit the sodium-potassium pump, thereby disrupting the electrochemical gradient and affecting calcium handling. It may therefore be desirable to modulate calcium handling in a subject who has been administered a sodium-potassium ATPase inhibitor, in order to treat or prevent any impaired calcium handling resulting from administration of the sodium-potassium ATPase inhibitor.

[0227] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing impaired calcium) associated with administering of one or more sodium-potassium ATPase inhibitors (such as Ouabain), wherein the method comprises co-administering to a subject the sodium-potassium ATPase inhibitor in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators). For example, the toxic agent may be a sodium channel blocker, such as Lidocaine and / or Mexiletine. Sodium channel blockers prevent sodium influx, which can affect membrane potential and indirectly influence calcium channel activity. It may therefore be desirable to modulate calcium channel activity in a subject who has been administered a sodium channel blocker, in order to treat or prevent any impaired or undesirable calcium channel activity resulting from administration of the sodium channel blocker.

[0228] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing impaired or undesirably affected calcium channel activity) associated with administering of one or more sodium channel blockers (such as Lidocaine and / or Mexiletine), wherein the method comprises co-administering to a subject the sodium channel blocker in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0229] For example, the toxic agent may be a potassium channel opener, such as Nicorandil and / or Pinacidil. Potassium channel openers increase potassium efflux from cells, leading to hyperpolarization of the membrane and inhibition of voltage-gated calcium channels, thereby reducing cytosolic calcium levels. It may therefore be desirable to increase the cytosolic calcium levels in a subject who has been, or who will be, administered a potassium channel opener, in order to counteract the reduced cytosolic calcium levels induced by the potassium channel opener.

[0230] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the reduced cytosolic calcium levels) associated with administering of one or more potassium channel openers (such as Nicorandil and / or Pinacidil), wherein the method comprises co-administering to a subject the potassium channel opener in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0231] For example, the toxic agent may be an adrenergic receptor agonist / antagonist, such as Isoproterenol (agonist) and / or Propranolol (antagonist). In one embodiment, the toxic agent is an adrenergic receptor agonist, such as Isoproterenol. In another embodiment, the toxic agent is an adrenergic receptor antagonist, such as Propranolol. The adrenergic receptor agonist / antagonist may activate or block adrenergic receptors, thereby influencing intracellular signaling pathways that regulate calcium release from internal stores or calcium influx through channels. It may therefore be desirable to modulate the calcium release from internal stores and / or modulate the calcium influx through channels in a subject who has been, or who will be, administered an adrenergic receptor agonist / antagonist, in order to counteract any undesirable changes to the calcium release from internal stores or calcium influx through channels induced by the adrenergic receptor agonist / antagonist. Thus, the invention provides a method of treating or preventing a toxic effect (such as modulating the calcium release from internal stores and / or the calcium influx through channels) associated with administering of one or more adrenergic receptor agonist / antagonists, wherein the method comprises co-administering to a subject the adrenergic receptor agonist / antagonist in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0232] For example, the toxic agent may be a magnesium supplement, such as magnesium sulfate (also commonly known as epsom salt). Magnesium supplements may act as a natural calcium channel blocker, reducing calcium influx into cells and this can lower cytosolic calcium levels. It may therefore be desirable to increase the cytosolic calcium levels in a subject who has been, or who will be, administered a magnesium supplement, in order to counteract the reduced cytosolic calcium levels induced by the magnesium supplement.

[0233] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing the reduced cytosolic calcium levels) associated with administering of one or more magnesium supplements (such as magnesium sulfate), wherein the method comprises co-administering to a subject the magnesium supplement in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0234] For example, the toxic agent may be a sodium supplement, such as sodium chloride (also commonly known as table salt). Sodium supplements may increase sodium levels, which can affect membrane potential and indirectly influence calcium channel activity. It may therefore be desirable to modulate calcium channel activity in a subject who has been administered a sodium supplement, in order to treat or prevent any impaired or undesirable calcium channel activity resulting from administration of the sodium supplement.

[0235] Thus, the invention provides a method of treating or preventing a toxic effect (such as treating or preventing impaired or undesirably affected calcium channel activity) associated with administering of one or more sodium supplements (such as sodium chloride), wherein the method comprises co-administering to a subject the sodium supplement in combination with a composition of the invention (such as a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators).

[0236] The toxic agent(s) and the composition(s) of the invention described herein may be administered simultaneously, separately or sequentially. In one embodiment, equipment suitable for delivering the toxic agent(s) is provided, wherein the equipment comprises the composition(s) of the present invention. For example, the equipment may include inhalation devices (including nebulizers and metered-dose inhalers), injection devices (including autoinjectors, injector pens, jet injectors, safety syringes, syringes, pre-filled syringes, nanoneedles, microneedle drug delivery devices), implants and stents (including drug-eluting stents), intrauterine devices, anaesthetic machines, anesthetic vaporizers, analytical nebulizers, aptargroups, aquasomes, cervical drug delivery devices, coiled- coil drug delivery devices, convection enhanced delivery devices, douches, infusion pumps, stimuli-responsive drug delivery systems, stretch-triggered drug delivery devices, and / or mechanical sensation delivery devices. Thus, any one of these equipment may be provided, wherein the equipment comprises the composition(s) of the present invention. The equipment comprising the composition(s) of the present invention may then be used to co-administer the toxic agent(s) with the composition(s) of the present invention.

[0237] Reducing toxicity associated with administration of a pharmaceutical agent

[0238] Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing a toxic effect (such as necrosis) associated with administering of one or more pharmaceutical agents, wherein the method comprises co-administering to a subject the pharmaceutical agent(s) with the compound(s), combination(s) or composition(s).

[0239] According to a preferred aspect of the invention, there is provided a method of treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more pharmaceutical agents, wherein the method comprises co-administering to a subject (i) the pharmaceutical agent, and (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors. Preferably, (ii) comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0240] Suitable pharmaceutical agents are described herein. Each ofthe compounds, combinations and compositions described herein may be used in a method of treating or preventing a toxic effect (such as necrosis) associated with the administering of one or more of the pharmaceutical agents described herein, wherein the method comprises co-administering the pharmaceutical agents with the compound(s), combination^) or composition(s).

[0241] For example, the pharmaceutical agent may be a Tumor Necrosis Factor (TNF) inhibitor. Such inhibitors are typically prescribed for conditions like rheumatoid arthritis, and they riskthe side effect of necrosis. Specifically, TNF inhibitors can suppress the immune system, leading to an increased risk of infections, including opportunistic infections that can cause necrotizing lesions or tissues. Additionally, the suppression of the immune system can exacerbate existing infections, potentially leading to necrosis if not properly managed. TNF inhibitor drugs include Humira, Enbrel, Remicade, Simponi, Cimzia, Inflectra, Avsola, Renflexis, Amjevita, Hadlima, Erelzi, Hyrimoz, Hulio, Zymfentra, Yusimry, Yuflyma, Simlandi, Ixifi, Idacio, Eticovo, Cyltezo, Abrilada. Thus, the invention provides a method of treating or preventing necrosis associated with the administering of one or more of the TNF inhibitors described herein, wherein the method comprises coadministering the TNF inhibitor with the compound(s), combination(s) or composition^) of the invention. Preferably the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and may optionally further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0242] For example, the pharmaceutical agent may be a Target of Rapamycin (mTOR) inhibitor, such as Sirolimus, Everolimus, and Temsirolimus. Thus, the invention provides a method of treating or preventing necrosis associated with the administering of one or more of the mTOR inhibitors described herein, wherein the method comprises co-administering the mTOR inhibitor with the compound(s), combination(s) or composition(s) of the invention. Preferably the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and may optionally further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0243] Some pharmaceutical agents may induce liver injury through (at least in part) necrosis. This includes pharmaceutical agents such as Acetaminophen, Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) (such as ibuprofen, diclofenac, and naproxen), antiarrhythmic medication (such as Amiodarone), Anabolic Steroids, Birth Control Pills, antipsychotic medication (such as Chlorpromazine), antibiotics (such as Erythromycin, Sulfa Drugs, Isoniazid, Tetracyclines, Amoxicillin-clavulanate), anaesthetics (such as Halothane), Blood pressure medication (such as Methyldopa), rheumatoid arthritis drugs (such as Methotrexate), Statins, Anti-Seizure Medicines, Disulfiram used to treat alcoholism, supplements (such as Niacin, also known as Vitamin B3), immunosuppressive drugs (such as Azathioprine), and antifungals (such as Ketoconazole). Thus, the invention provides a method of treating or preventing necrosis associated with the administering of one or more of these pharmaceutical agents, wherein the method comprises co-administering the pharmaceutical agent with the compound(s), combination(s) or composition(s) of the invention. Preferably the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and may optionally further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0244] Some pharmaceutical agents may induce kidney injury through (at least in part) necrosis. This includes pharmaceutical agents such as Angiotensin-Converting Enzyme (ACE) Inhibitors, Aminoglycoside Antibiotics (such as gentamicin), other antibiotics (such as Vancomycin), HIV Medications (such as Tenofovir disoproxil fumarate (Viread), Truvada, and Atazanavir (Reyataz)), Metformin and Beta blockers. Thus, the invention provides a method of treating or preventing necrosis associated with the administering of one or more of these pharmaceutical agents, wherein the method comprises co-administering the pharmaceutical agent with the compound(s), combination(s) or composition(s) of the invention. Preferably the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and may optionally further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0245] As discussed hereinabove, the inventors have discovered that, if administration of a pharmaceutical agent already inhibits one or more of the molecular targets associated with cellular necrosis (e.g., directly or indirectly via off target side effects), compositions of the present invention can be co-administered with the pharmaceutical agent in order to reduce toxicity because the combination of the pharmaceutical agent and the compositions of the present invention treat or prevent necrosis. Thus, depending on the molecular target inhibited by the pharmaceutical agent, the necrosis inhibitors present in the compositions of the present invention can be tailored accordingly.

[0246] For example, the pharmaceutical agent may be a pharmaceutical agent which inhibits gap junction activity and / or which inhibits gap junctions. This includes pharmaceutical agents that act as calcium inhibitors, such as pharmaceutical agents that act via competitive inhibition (e.g., lanthanides, such as gadolinium ions (Gd3+) and lanthanum ions (La3+)), and / or pharmaceutical agents that act as inhibitors to other similar cations to calcium (e.g. magnesium ions (Mg2+)) that have comparable ionic radii and metal coordination chemistry to calcium, and / or anesthetics (e.g., heptanol, halothane and isoflurane, preferably halothane). This also includes gap junction inhibitors as described herein. In this instance, necrosis may be prevented by also administering to the subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists). The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0247] Thus, according to this aspect of the invention, there is provided a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject (i) a pharmaceutical agent which inhibits gap junction activity in combination with (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors, and optionally (iii) one or more plasma membrane calcium channel inhibitors, and / or optionally (iv) one or more calcium chelators.

[0248] As another example, the pharmaceutical agent may be a pharmaceutical agent which inhibits calcium ion movement out of the endoplasmic reticulum / sarcoplasmic reticulum and / or which inhibits endoplasmic / sarcoplasmic reticulum calcium channels (such as one or more ryanodine receptor antagonists). This includes pharmaceutical agents that act as calcium inhibitors, such as pharmaceutical agents that act via competitive inhibition (e.g. lanthanides, such as gadolinium ions (Gd3+) and lanthanum ions (La3+)) and / or pharmaceutical agents that act as inhibitors to other similar cations to calcium (e.g. magnesium ions (Mg2+)) that have comparable ionic radii and metal coordination chemistry to calcium. This also includes the endoplasmic / sarcoplasmic reticulum calcium channel inhibitors as described herein. In this instance, necrosis may be prevented by also administering to the subject a composition comprising one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Thus, according to this aspect of the invention, there is provided a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject (i) a pharmaceutical agent which inhibits endoplasmic / sarcoplasmic reticulum calcium channels in combination with (ii) one or more gap junction inhibitors, and optionally (iii) one or more plasma membrane calcium channel inhibitors, and / or optionally (iv) one or more calcium chelators.

[0249] As another example, the pharmaceutical agent may be a pharmaceutical agent which inhibits calcium ion movement via plasma membrane calcium ion channels and / or which reduces calcium ion influx / movement into cells via the plasma membrane. This includes pharmaceutical agents that reduce calcium ion influx / movement into cells via the plasma membrane by affecting other cellular ion gradients. This is due to the intimate relationships between the ionic and electrical properties of the membrane (e.g. potassium ions (K+), sodium ions (Na+), hydrogen ions (H+), chloride ions (Cl ), magnesium ions (Mg2+), or phosphate ions (PO43). One example of this is via hyperpolarisation or depolarisation of the membrane, which can then reduce the calcium ion influx / movement into cells. This includes pharmaceutical agents that act as calcium inhibitors, such as pharmaceutical agents that act via competitive inhibition (e.g. lanthanides, such as gadolinium ions (Gd3+) and lanthanum ions (La3+)) and / or pharmaceutical agents that act as inhibitors to other similar cations to calcium (e.g. magnesium ions (Mg2+)) that have comparable ionic radii and metal coordination chemistry to calcium. This also includes pharmaceutical agents such as: dihydropyridines (such as Amlodipine (Norvasc)), benzothiazepines (such as Diltiazem (Cardizem, Tiazac, Tiazac XC), Felodipine (Plendil), Nifedipine XL (Adalat XL) and Verapamil (Isoptin, Isoptin SR, Verelan). This also includes plasma membrane calcium channel inhibitors as described herein. In this instance, necrosis may be prevented by also administering to the subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists), and / or one or more gap junction inhibitors, and / or one or more calcium chelators.

[0250] Thus, according to this aspect of the invention, there is provided a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject (i) a pharmaceutical agent which inhibits calcium ion movement via plasma membrane calcium ion channels in combination with (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally (iii) one or more calcium chelators.

[0251] As another example, the pharmaceutical agent may be a pharmaceutical agent which is a chelator that sequesters calcium ions in the cytoplasm, within compartments like the endoplasmic or extracellular space. This includes pharmaceutical agents such as DTPA, BAPTA AM, BAPTA, sodium citrate, and citric acid. In this instance, necrosis may be prevented by also administering to the subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and / or one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors. Thus, according to this aspect of the invention, there is provided a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject (i) a pharmaceutical agent which is a chelator that sequesters calcium ions in the cytoplasm in combination with (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, and optionally (iii) one or more plasma membrane calcium channel inhibitors.

[0252] As another example, the pharmaceutical agent may be a pharmaceutical agent which is a protease inhibitor. In this instance, necrosis may be prevented by also administering to the subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0253] Thus, according to this aspect of the invention, there is provided a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject (i) a protease inhibitor in combination with (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, and optionally (iii) one or more plasma membrane calcium channel inhibitors, and / or optionally (iv) one or more calcium chelators. The method may further comprise administration of more than one protease inhibitor in order to achieve inhibition of broad classes of protease inhibitors.

[0254] As another example, the dosage of a pharmaceutical agent may be increased, e.g., relative to a previously known dosage and / or relative to a dosage which has previously been approved by a regulatory body. The dosage of the pharmaceutical agent may be increased because the co-administration of the compositions of the present invention ensure that any toxic effect (such as necrosis) induced by the increased dosage is treated and / or prevented by the compositions of the present invention. Thus, the invention provides a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject (i) an increased dose of a pharmaceutical agent (such as an increased dose of a calcium inhibitor) in combination with (ii) one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors, and / or one or more gap junction inhibitors, and / or one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably (ii) is one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, optionally further comprising one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0255] In one embodiment, equipment suitable for delivering the pharmaceutical agent(s) is provided, wherein the composition(s) of the present invention is present within the equipment. For example, the equipment may include inhalation devices (including nebulizers and metered-dose inhalers), injection devices (including autoinjectors, injector pens, jet injectors, safety syringes, syringes, pre-filled syringes, nanoneedles, microneedle drug delivery devices), implants and stents (including drug-eluting stents), intrauterine devices, anaesthetic machines, anesthetic vaporizers, analytical nebulizers, aptargroups, aquasomes, cervical drug delivery devices, coiled-coil drug delivery devices, convection enhanced delivery devices, douches, infusion pumps, stimuli-responsive drug delivery systems, stretch-triggered drug delivery devices, and / or mechanical sensation delivery devices. Thus, any one of these devices may be provided, wherein the device comprises the composition(s) of the present invention. The device comprising the composition (s) of the present invention may then be used to co-administer the pharmaceutical agent(s) with the composition (s) of the present invention.

[0256] Reducing toxicity associated with administration of a biologically active agent

[0257] Biologically active agents may block one or more of the molecular targets associated with necrosis, as discussed herein. For example, biologically active agents may block one or more of the predicted weak spots in cell physiology that make cells susceptible to cellular necrosis. The weak spots include: increase in cytosolic calcium via (i) movement of calcium ions into the cytosol via plasma membrane calcium ion channels; (ii) movement of calcium ions from within organelles with the cell, especially the endoplasmic / sarcoplasmic (ER / SR) reticulum via ER / SR calcium ion channels; and (iii) movement of calcium ions and other damaging agents from cell to cell via gap junctions. Other weak spots include calpain hyper-activation, cathepsin hyperactivation and the opening of store-operated calcium channels (also called calcium release-activated calcium channels) post ER calcium depletion. The most pertinent of these steps are the calcium ion entry via ER / SR calcium ion channels and gap junctions, and followed by entry via the plasma membrane calcium ion channels, as discussed in further detail in Example 1 below.

[0258] In these circumstances, i.e., wherein administration of a biologically active agent already blocks one or more of these targets, compositions of the present invention can be co-administered with the biologically active agent in order to prevent or treat necrosis because the combination of the biologically active agent and the compositions of the present invention prevent or treat necrosis.

[0259] The present invention provides a method of treating or preventing necrosis, wherein the method comprises co-administering to a subject a biologically active agent in combination with a composition of the present invention. Preferably, the composition of the present invention comprises one or more necrosis inhibitors which block different molecular targets involved in the necrosis pathway than the biologically active agent.

[0260] For example, where the biologically active agent inhibits endoplasmic / sarcoplasmic reticulum calcium channels, the composition comprises one or more gap junction inhibitors. For example, the biologically active agent may be ryanodine, a plant alkaloid and / or anti-RyR antibodies. For example, the gap junction inhibitors may be one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0261] For example, where the biologically active agent inhibits the gap junction, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors. For example, the biologically active agent may be anti-pannexin antibodies and / or anti-connexin antibodies (such as AT-2, CT-360, MHC, A-CAM, Cx32 164-189, EL-46, EL-186). For example, the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor may be dantrolene. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0262] Suitable biologically active agents are described herein, and are known in the art. Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing a toxic effect (such as necrosis), wherein the method comprises co-administering the biologically active agent(s) with the compound(s), combination^) or composition(s).

[0263] For example, the biologically active agent may be one or more of: Monoclonal Antibodies (MABs) (including Whole Antibody, Fab' Fragment, Single-Chain Variable Fragments (scFv), Dimeric Single-Chain Variable Fragments (di-scFv), Single-Domain Antibodies (sdAb)), Bispecific Monoclonal Antibodies (BsAb), Trifunctional Antibodies, Bi-Specific T-Cell Engagers (BiTE)), Viral Antibodies (e.g. Ansuvimab, Atoltivimab (+maftivimab / odesivimab), Avdoralimab, Bamlanivimab (+etesevimab), Bebtelovimab, Casirivimab (+imdevimab), Cilgavimab (+tixagevimab), Diridavumab, Etesevimab, Exbivirumab, Foravirumab, Imdevimab (+casirivimab), Libivirumab, Maftivimab, Nirsevimab, Odesivimab, Pemivibart, Rafivirumab, Regavirumab, Regdanvimab, Sevirumab, Sotrovimab, Suptavumab, Tixagevimab (+cilgavimab),Tuvirumab), Humanized Antibodies (e.g. Felvizumab, Lenvervimab, Motavizumab, Palivizumab, Suvizumab), Chimeric Antibodies(e.g. Cosfroviximab, Larcaviximab, Porgaviximab, Vilobelimab) and Fusion proteins (e.g. Etanercept, Bevacizumab, Rituximab).

[0264] The biologically active agent(s) and the composition(s) of the invention described herein may be administered simultaneously, separately or sequentially.

[0265] In one embodiment, equipment suitable for delivering the biologically active agent(s) is provided, wherein the equipment comprises the composition(s) of the present invention. For example, the equipment may include inhalation devices (including nebulizers and metered-dose inhalers), injection devices (including autoinjectors, injector pens, jet injectors, safety syringes, syringes, pre-filled syringes, nanoneedles, microneedle drug delivery devices), implants and stents (including drug-eluting stents), intrauterine devices, anaesthetic machines, anesthetic vaporizers, analytical nebulizers, aptargroups, aquasomes, cervical drug delivery devices, coiled-coil drug delivery devices, convection enhanced delivery devices, douches, infusion pumps, stimuli-responsive drug delivery systems, stretch-triggered drug delivery devices, and / or mechanical sensation delivery devices. Thus, any one of these equipment may be provided, wherein the equipment comprises the composition(s) of the present invention. The equipment comprising the composition^) of the present invention may then be used to co-administer the biologically active agent(s) with the composition (s) of the present invention.

[0266] Reducing damage associated with medical diagnostics and interventions Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing the side-effects associated with medical diagnostics and / or medical interventions in a subject. The subject is a subject who has been subjected to, or will be subjected to, one or more medically invasive interventions and / or medical diagnostic procedures.

[0267] The side-effect may be cell death, such as necrosis. Thus, each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing necrosis associated with medical diagnostics and interventions in a subject, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject.

[0268] According to a preferred aspect of the invention, there is provided a method of treating or preventing the sideeffects (such as necrosis) associated with medical diagnostics and interventions in a subject, wherein the method comprises administering one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, the method comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0269] The compounds, combinations or compositions may be administered to a subject prior to the medically invasive intervention and / or medical diagnostic procedure, to a subject during the medically invasive intervention and / or medical diagnostic procedure, and / or to a subject who has previously received medically invasive intervention and / or medical diagnostic procedure.

[0270] In an aspect, the compounds, combinations or compositions are administered immediately or shortly before a subject undergoes a medically invasive intervention and / or medical diagnostic procedure (e.g., no more than 24 hours before, no more than 12 hours before, no more than 6 hours before, or no more than 4 hours before). The compounds, combinations or compositions may additionally, or alternatively, be administered during and / or post-surgery. For example, the compounds, combinations or compositions may be administered for up to 6 months after, up to 4 months after, up to 2 months after, up to 1 month after, up to 2 weeks after, up to 1 week after, or up to 24 hours after the surgery.

[0271] The medically invasive interventions and / or medical diagnostic procedures include surgery, dialysis, and invasive diagnostics such as catheterization, balloon angioplasty and / or atherectomy, stenting, vascular angioplasty, atherectomy, stents, IVC filter placement and / or removal, thrombectomy, ablation (such as vein ablation, radiofrequency ablation and / or cryothermal ablation), phlebectomy procedures, pacemakers, echocardiography, defibrillator insertion, subcutaneous ICD and / or appendage closure. Preferably, the medically invasive intervention is surgery, such as major surgery. Thus, according to a preferred aspect of the invention, there is provided a method of treating or preventing the side-effects (such as necrosis) associated with surgery in a subject, wherein the method comprises administering one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, the method comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators.

[0272] The major surgery may be one or more of a cesarean section, organ replacement, joint replacement, full hysterectomy, heart surgeries, bariatric surgeries, including gastric bypass, gallbladder surgery, appendectomy, breast surgery, colon & rectal surgery, endocrine surgery, general surgery, gynecological surgery, hand surgery, hernia surgery, minimally invasive surgery, neurosurgery, orthopedic surgery, ophthalmological surgery, outpatient surgery, pediatric surgery, plastic, reconstructive, hand and micro- vascular surgery, robotic surgery, thoracic surgery, trauma surgery, urologic surgery, and vascular surgery.

[0273] The surgery may be one of more of bariatric surgery, breast surgery, colon surgery, cardiac surgery, cardiothoracic surgery, rectal surgery, endocrine surgery, general surgery, gynecological surgery, hand surgery, head surgery, neck surgery, hernia surgery, minimally invasive surgery, neurosurgery, orthopedic surgery, ophthalmological surgery, outpatient surgery, oral maxillofacial surgery, paediatric surgery, plastic surgery, reconstructive surgery, hand surgery, micro-vascular surgery, otorhinolaryngology (ENT) surgery, robotic surgery, thoracic surgery, trauma surgery, urologic surgery, orthopaedic surgery, and vascular surgery. Preferably, the surgery is cardiac surgery. Preferably the subject may have cardiac surgery associated acute kidney injury.

[0274] The surgery may be minor surgery. Minor surgery includes Wound Treatment and Debridement (e.g. removing foreign objects from the skin, treating skin growths or lesions, preventing wounds from enlarging or scarring substantially); Sutures / Stitches; diagnostic and identification procedures (e.g. biopsies, abdominal issues assessment). Specific minor surgeries (e.g. cataract surgery, dental restorations, circumcision, breast biopsy (excision or open biopsy), arthroscopy, laparoscopy, burn excision and debridement procedures, removal of foreign objects, and incision & drainage.

[0275] For example, when treating or preventing necrosis associated with a major surgery (such as cardiac surgery, or cardiac surgery associated acute kidney injury), the method may comprise administering the compound(s), combination^) or composition(s) to the subject prior to surgery, during the surgery, and after the surgery. For example, an oral form of the compound(s), combination(s) or composition (s) may be administered prior to the surgery, and then an IV form of the compound(s), combination(s) or composition (s) may be administered during and / or after the surgery. For example, when treating or preventing necrosis associated with a minor surgery, the method may comprise administering the compound(s), combination(s) or composition(s) to the subject prior to surgery, during the surgery, and afterthe surgery. For example, an oral form of the compound(s), combination(s) or composition(s) may be administered prior to the surgery, and then an IV form of the compound(s), combination^) or composition(s) may be administered during and / or after the surgery to the local area that has undergone surgery. Alternatively, an oral form of the compound(s), combination(s) or composition(s) may be administered prior to the surgery, during and after the surgery.

[0276] Necrosis associated with ischemia-reperfusion injury (IRI) is a principal driver of acute kidney injury (AKI). Necrosis is also central to cardiovascular events such as myocardial infarctions (heart attacks) and strokes. As with the kidney, these conditions may be characterized by cellular membrane destabilization and damage. More generally, during IRI, cellular membrane destabilization and necrosis arise from disrupted blood flow followed by abrupt restoration. This process exerts stress in the form of hypoxia, nutrient deprivation, and oxidative stress due to reactive oxygen and nitrogen species (ROS / RNS), including hydroxyl radicals (’OH), superoxide anions (O2“), and hydrogen peroxide (H2O2), leading to macromolecule oxidation. This results in the loss of Ca2+gradients and overload, associated protease activation, ATP depletion, and mitochondrial permeability transition pore (mPTP) opening. Also, as in the kidney, the resulting necrotic damage fuels inflammation and secondary cell death, exacerbating disease outcomes. Delaying or preventing necrosis, therefore, has potential therapeutic applications in broader IRI events, where it could extend the therapeutic window for resuscitation, mitigate tissue degeneration, and disrupt damage cascades, thereby allowing tissue regeneration to predominate.

[0277] Thus, according to a preferred aspect of the invention, there is provided a method of treating or preventing necrosis associated with ischemia-reperfusion injury in a subject, wherein the method comprises administering one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, the method comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0278] According to a preferred aspect of the invention, there is provided a method of treating or preventing acute kidney injury in a subject (for example by treating or preventing necrosis associated with acute kidney injury), wherein the method comprises administering one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject. Preferably, the method comprises administering one or more ryanodine receptor antagonists (preferably dantrolene) and one or more gap junction inhibitors (preferably one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine). The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0279] Combination therapy

[0280] As discussed herein, the inventors have made the surprising discovery that using a combination of compounds to block the ER / SR calcium channels and gap junctions is particularly advantageous for preventing and / or treating necrosis. Thus, the invention provides the combination therapy of an endoplasmic / sarcoplasmic reticulum calcium channel inhibitor (such as a ryanodine receptor inhibitor or antagonist) and a gap junction inhibitor for use in treating or preventing necrosis. The monotherapy (i.e., the use of endoplasmic / sarcoplasmic reticulum calcium channel inhibitor alone, or the use of a gap junction inhibitor alone) is not efficient.

[0281] In some embodiments, monotherapy using an endoplasmic / sarcoplasmic reticulum calcium channel inhibitor may exert stress on a cell. Thus, according to an aspect of the invention, there is provided a method of treating or preventing the undesirable side-effects associated with administering of one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors, wherein the method comprises administering to a subject the one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors in combination with one or more gap junction inhibitors. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine. The method may further comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0282] In some embodiments, monotherapy using a gap junction inhibitor may exert stress on a cell. Thus, according to an aspect of the invention, there is provided a method of treating or preventing the undesirable side-effects associated with administering of one or more gap junction inhibitors, wherein the method comprises administering to a subject one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with the one or more gap junction inhibitors. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine. The method may further comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0283] Use in space

[0284] Each of the compounds, combinations and compositions described herein may be used in a method of protecting a subject from one or more side-effects of space radiation, wherein the method comprises administering the compound(s), combination(s) or composition (s) to the subject. The side-effect may be radiation necrosis. Each of the compounds, combinations and compositions described herein may be used in a method of protecting a subject from radiation necrosis, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject. For example, each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing radiation necrosis, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject.

[0285] The space radiation may be ionizing radiation. For example, the ionizing radiation may be one or more of galactic cosmic radiation, solar energetic particles (SEPs), trapped radiation, solar flares and cosmic rays.

[0286] Galactic cosmic radiation is a form of radiation which comes from supernovae and / or exploding stars. The radiation may be formed of high energy particles that cannot be effectively shielded by current spacecraft design technologies.

[0287] Solar Energetic Particles (SEPs) are released by the sun during solar particle events. Although SEPs tend to be lower in energy than galactic cosmic radiation, they still pose a significant risk and can induce radiation necrosis.

[0288] Trapped radiation may occur when radiation becomes trapped in Earth's magnetic field.

[0289] Solar flares are intense bursts of radiation from the Sun. Solar flares can also release SEPs.

[0290] Cosmic rays are high-energy particles originating from outer space, and they may include both ionizing and non-ionizing components.

[0291] According to an aspect of the invention, there is provided a method of protecting a subject from one or more side-effects (such as radiation necrosis) of space radiation (such as ionizing radiation), the method comprising administering to a subject one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0292] The one or more side-effects may include cell death, such as necrosis, such as radiation necrosis.

[0293] The compounds, combinations or compositions may be administered to a subject prior to exposure to space radiation, to a subject during exposure to space radiation, or to a subject who has previously been exposed to space radiation. Preferably, the compounds, combinations or compositions are administered after a subject has been exposed to space radiation.

[0294] Each of the compounds, combinations and compositions described herein may be used in a method of protecting a subject from one or more side-effects associated with a gravitational environment which is different to Earth’s gravitational environment, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject.

[0295] Thus, according to an aspect of the invention, there is provided a method of protecting a subject from one or more side-effects associated with a gravitational environment which is different to Earth’s gravitational environment, the method comprising administering to a subject one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0296] The side-effect may be necrosis. Each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing necrosis in a subject, wherein the necrosis has been caused by a gravitational environment which is different to Earth’s gravitational environment, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject. For example, each of the compounds, combinations and compositions described herein may be used in a method of treating or preventing necrosis, wherein the method comprises administering the compound(s), combination^) or composition (s) to the subject.

[0297] The gravitational environment may be a reduced gravitational environment compared to Earth’s gravitational environment, such as microgravity, partial gravity, and / or martian gravity. Alternatively, the gravitational environment may be an increased gravitational environment compared to Earth’s gravitational environment, such as hypergravity or artificial gravity simulations.

[0298] Microgravity may be defined as a gravity of between about Og and about 1 x10'6g, such as about Og, or about 1 xi o-6g. Microgravity may be experienced during spaceflights, especially in the International Space Station (ISS) and / or during the journey to Mars.

[0299] Partial gravity may be defined as a gravity of about 0.16g to about 0.38g. Partial gravity may be simulated during parabolic flights and may be used to study the effects of Martian gravity. These simulations may involve brief periods of reduced gravity, providing insights into how humans would adapt to and perform in Martian gravity conditions. Martian gravity may be defined as a gravity of about 0.38g. This gravity would be expected on the surface of Mars, where the gravity is approximately 38% of Earth's gravity. Studies and simulations aim to understand the physiological adaptations and potential health risks associated with living and working in this gravity environment.

[0300] Hypergravity may be defined as greater than about 1g.

[0301] Artificial gravity (increased) simulations are theoretical spacecraft designs exploring artificial gravity. The simulations may involve rotating the spacecraft to create a centrifugal force that simulates gravity.

[0302] These gravitational environments may occur during space flight and / or simulations. Thus, the subject is a subject who has gone, or will go, on at least one space flight and / or who has experienced, or will experience, at least one flight simulation.

[0303] According to an aspect of the invention, there is provided a method of treating or preventing necrosis in a subject, wherein the necrosis has been caused by a gravitational environment which is different to Earth’s gravitational environment, the method comprising administering to a subject one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The method may also comprise administering one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0304] In-space tissue / orpan engineering

[0305] One of the biggest challenges with engineering tissue in vitro is the small surface area to volume ratio. Consequently, as large tissues are engineered / grown they begin to develop necrotic cores. Moreover, current tissue engineering techniques may suffer from low yield, low purity and / or low survival rate of the tissue.

[0306] Thus, in-space tissue engineering is being explored. This is because numerous advantages are associated with tissue engineering under microgravitational environments. Such advantages may include:

[0307] • Reduced mechanical stress: under microgravitational conditions, organs and tissues would experience less mechanical stress (compared to earth's gravity), which contributes to better preservation of organ function over time;

[0308] • Improved cell culture conditions: microgravitational environments can enhance the quality of cultured cells. For example, studies have shown that simulated microgravity increases the yield, purity, and survival rate of cardiomyocytes; • Facilitation of fluid dynamics: the absence of gravity alters fluid dynamics within biological systems, affecting nutrient distribution and waste removal; and / or

[0309] • Fabrication of soft tissues, which usually collapse under their own weight, can be accelerated under microgravity conditions because external forces are reduced or eliminated.

[0310] According to an aspect of the invention, there is provided a method of engineering a tissue or an organ in space, the method comprising contacting the organ or tissue with a culture media comprising a composition of the present invention.

[0311] According to another aspect, there is provided a method of preserving a live tissue or an organ in space, the method comprising storing or culturing the tissue or organ in a culture media comprising a composition of the present invention.

[0312] According to an aspect of the invention, there is provided a method of engineering a tissue or an organ, wherein at least some of the method steps occur under a microgravitational environment, the method comprising contacting the organ or tissue with a culture media comprising a composition of the present invention.

[0313] According to an aspect of the invention, there is provided a method of preserving a live tissue or an organ, wherein at least some of the method steps occur under a microgravitational environment, the method comprising contacting the organ or tissue with a culture media comprising a composition of the present invention.

[0314] The microgravitational environment may occur during spaceflight, or during an appropriate simulation. For example, the microgravitational environment may be imparted by a rotating bioreactor. A rotating bioreactor may simulate microgravity by gently moving the growth medium while growing cells are kept in suspension by a constant ‘free fall’ effect. Suitable rotating bioreactors are known in the art.

[0315] Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0316] Thus, the culture media may comprise one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors. The culture media optionally further comprises one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators. The methods of engineering a tissue or an organ, and the methods of preserving a live tissue or an organ, discussed herein include methods comprising storing or culturing the tissue or organ at room temperature (such as at around 20 °C). Additionally, or alternatively, the methods comprise storing or culturing the tissue or organ under hypothermic conditions (e.g., a temperature of between about 0 °C and about 4 °C, such as about 0 °C, about 1 °C, about 2 °C, about 3 °C, or about 4 °C). Additionally, or alternatively, the methods comprise storing or culturing the tissue or organ via cryopreservation (e.g., at a temperature of between about -200 °C to about - 15 °C, such as at about - 20 °C, about -80 °C, about -196 °C or any intermediate temperature(s)).

[0317] The methods of engineering a tissue or an organ, and the methods of preserving a live tissue or an organ, discussed herein may comprise (i) simple cold storage preservation (SCS), and / or (ii) machine perfusion (MP) preservation (which may also be called dynamic preservation method). Machine perfusion preservation may comprise hypothermic machine perfusion (HMP), normothermic machine perfusion (NMP) and / or oxygen persufflation (OP).

[0318] The culture media used in the methods of engineering a tissue or an organ or in the methods of preserving a live tissue or an organ may be any culture media known in the art, in particular the University of Wisconsin (UW) solution which is generally used for organ preservation. This solution may comprise 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).

[0319] Thus, the invention may comprise storing the organ in a culture media (such as the UW solution described hereinabove) with one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, and optionally one or more plasma membrane calcium channel inhibitors and / or optionally one or more calcium chelators.

[0320] Also provided herein is a culture media suitable for engineering a tissue or an organ, or suitable for preserving an organ or tissue, wherein the culture media comprises the University of Wisconsin (UW) solution, one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors. The culture media optionally further comprises one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators.

[0321] Also provided herein is a culture media suitable for engineering a tissue or an organ, or suitable for preserving an organ or tissue, wherein the culture media comprises the University of Wisconsin (UW) solution, one or more calpain inhibitors, and one or more cathepsin inhibitors.

[0322] Also provided herein is a culture media suitable for engineering a tissue or an organ, or suitable for preserving an organ or tissue, wherein the culture media comprises the University of Wisconsin (UW) solution and one or more calcium chelators, preferably wherein the calcium chelator(s) are present in the amounts recited hereinabove in aspect A.

[0323] Radiation damage

[0324] Each of the compounds, combinations and compositions described herein may be used in a method of protecting a subject from one or more side-effects associated with radiation, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject.

[0325] Thus, according to an aspect of the invention, there is provided a method of protecting a subject from one or more side-effects associated with radiation, the method comprising administering to a subject a composition of the invention.

[0326] The radiation may be ionizing radiation. Ionizing radiation can be produced by weapons such as surface-burst nuclear weapons, air burst nuclear weapons, and radiological dispersion devices. Ionizing radiation can also be produced during medical therapies such as cancer / tumour radiation therapy. For example, ionizing radiation may include x-rays, gamma rays, particle bombardment (such as neutron beams, electron beams, protons, mesons, and others), and particle rays (such as alpha particles, beta particles, and neutrons). Ionizing radiation can also be produced by nuclear events and / or nuclear weapons.

[0327] The one or more side-effects of the radiation damage may be radiation necrosis.

[0328] Thus, according to an aspect of the invention, there is provided a method of protecting a subject from the radiation necrosis associated with ionizing radiation, the method comprising administering to a subject a composition of the invention. For example, the method may prevent or treat radiation necrosis associated with ionizing radiation.

[0329] Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0330] Defence against weapons

[0331] Each of the compounds, combinations and compositions described herein may be used in a method of protecting a subject from one or more side-effects of an injury, such as an injury caused by a weapon, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject. Thus, according to an aspect of the invention, there is provided a method of protecting a subject from one or more side-effects of an injury, such as an injury caused by a weapon, the method comprising administering to a subject a composition of the invention.

[0332] The injury may be a brain injury, for example a brain injury caused by a low-level blast from a heavy weapon. The inventors have realised that low-level blasts from heavy weapons may cause the body to be subjected to shock waves and / or high-pressure air molecules / pulse of high pressure that cause brain injury. The injury may be a gun shot. The brain injury and / or gunshot injury may lead to cell death, such as necrosis. Thus, cell death, such as necrosis, may be a side-effect of these injuries.

[0333] The one or more side-effects of the injury, such as an injury caused by a weapon, may be necrosis. Thus, according to an aspect of the invention, there is provided a method of protecting a subject from the necrosis caused by an injury, the method comprising administering a composition of the invention to the subject. For example, the method treats or prevents the necrosis caused by an injury.

[0334] Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0335] Ageing

[0336] Each of the compounds, combinations and compositions described herein may be used in a method of preventing or treating ageing, wherein the method comprises administering the compound(s), combination(s) or composition(s) to the subject.

[0337] Thus, according to an aspect of the invention, there is provided a method of preventing or treating ageing, wherein the method comprises administering a composition of the present invention to the subject.

[0338] These methods may comprise preventing or treating one or more signs of skin ageing, such as the presence of fine lines and wrinkles, loss of elasticity, uneven skin and blotchiness. Such methods may be cosmetic methods.

[0339] Alternatively, the method comprises preventing or treating accelerated ageing phenotypes. Additionally, or alternatively, the method comprises preventing or treating organ ageing (such as kidney ageing, such as accelerated kidney ageing). Additionally, or alternatively, the method comprises preventing or treating cellular senescence, vascular rarefaction, presence of a chronic inflammatory infiltrate, and / or fibrosis. Such methods may be therapeutic methods. These methods may comprise preventing or treating ageing caused by direct damage associated with the necrosis pathway. Alternatively, the methods may comprise preventing or treating ageing caused by indirect damage associated with the necrosis pathway, such as ageing caused by maladaptive repair including cellular senescence, inflammation and / or fibrosis. The method may prevent or treat one or more of cellular senescence, vascular rarefaction, presence of a chronic inflammatory infiltrate, glomerulosclerosis and fibrosis.

[0340] Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine, such as quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0341] Increased cellular susceptibility to necrosis, leading to frailty (e.g. leading to a decline in physical function and strength) and diminished resilience to stress (e.g. leading to a reduced ability to withstand, adapt to, or recover from stress arising from physiological deterioration, including structural and functional impairments), is a significant characteristic of the aging process. Necrosis and necrosis-associated cellular damage influence aging at both local and systemic levels, contributing to frailty, diminished regenerative capacity, loss of resilience, and disrupted tissue architecture. These effects are both direct and compounding. Low-grade necrosis-related cellular damage drives key aging processes within cells, including genomic instability, loss of proteostasis, telomere shortening, and increased oxidative-inflammatory-nitrosative stress, alongside epigenetic changes. In turn, these factors heighten susceptibility to tissue-level loss of resilience, compounding damage. Targeting necrosis therefore leads to therapies effective against multiple diseases and, crucially, aging itself.

[0342] As the skilled person would appreciate, each of the compounds, combinations and compositions described herein for use in a method of preventing or treating aging may be provided as a cosmetic composition.

[0343] The cosmetic composition may comprise 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 comprise one or more calcium chelators, optionally in a relatively high dose (such as the doses recited hereinbelow in aspect A).

[0344] In another preferred aspect, the cosmetic composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors, optionally further comprising one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators. In another embodiment, the cosmetic 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 lnsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor and a gap junction inhibitor. For example, the cosmetic composition may comprise a calpain inhibitor and a cathepsin inhibitor. In one embodiment, the cosmetic composition comprises three or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and a gap junction inhibitor. The cosmetic composition may comprise two or more different classes of agent selected from the group consisting of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist.

[0345] The cosmetic composition may comprise one or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. The cosmetic composition may comprise two or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. The cosmetic composition may comprise three or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. Preferably the cosmetic composition comprises two or more or three or more different subclasses of agent selected from the group consisting of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist.

[0346] The cosmetic composition may be a liquid, a cream, or a powder. The cosmetic composition may be for topical use (e.g., use on skin).

[0347] According to another aspect, there is provided a method of making a cosmetic composition, the method comprising: contacting a selection of one or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor with a cosmetically acceptable carrier to create a cosmetic composition. In another preferred aspect, there is provided a method of making a cosmetic composition, the method comprising: contacting one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors with a cosmetically acceptable carrier to create a cosmetic composition. The method may further comprise contacting one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators.

[0348] In another embodiment, the cosmetic 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 lnsP3R antagonist), a calpain inhibitor, a cathepsin inhibitor and a gap junction inhibitor. For example, the cosmetic composition may comprise a calpain inhibitor and a cathepsin inhibitor. In one embodiment, the cosmetic composition comprises three or more of a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and a gap junction inhibitor. The cosmetic composition may comprise two or more different classes of agent selected from the group consisting of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. The cosmetic composition may comprise one or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. The cosmetic composition may comprise two or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. The cosmetic composition may comprise three or more of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. Preferably the cosmetic composition comprises two or more or three or more different subclasses of agent selected from the group consisting of a calcium chelator, a calcium channel blocker, a calcium channel antagonist, a ryanodine receptor antagonist, and an lnsP3R antagonist. Most preferably, the cosmetic composition 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.

[0349] According to another aspect, there is provided a method of using a cosmetic composition, the method comprising: applying a cosmetic composition according to the invention to the skin of a subject.

[0350] Producing artificial meat or cultivated meat

[0351] Each of the compounds, combinations and compositions described herein may be used in a method for producing artificial meat or cultivated meat, wherein the method comprises the cultivation of cell lines in a bioreactor, and contacting the cells with any of the compound(s), combination(s) or composition (s). The step of contacting the cells with the compound(s), combination(s) or composition(s) would preserve or culture the cells.

[0352] Thus, according to an aspect of the invention, there is provided a method for producing artificial meat or cultivated meat, the method comprising the cultivation of cell lines in a bioreactor, and contacting the cells with a composition of the present invention to preserve or culture the cells.

[0353] Preferably, the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) in combination with one or more gap junction inhibitors. The composition may further comprise one or more plasma membrane calcium channel inhibitors, and / or one or more calcium chelators. Preferably, the one or more ryanodine receptor antagonists is dantrolene. Preferably the one or more gap junction inhibitors is one or more of retinoic acid, oleic acid, linoleic acid, and quinine. Preferably, the plasma membrane calcium channel inhibitor is amlodipine. Preferably, the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

[0354] Methods for producing artificial meat or cultivated meat involve the cultivation of cell lines in a bioreactor. It will be appreciated that cell death is a major problem in bio-industry, as cell death often occurs within bioreactors due to various stressors. These stressors therefore negatively affect the culture longevity and the overall product yield. Thus, methods of improving the product yield in bioreactors are desired. There is therefore provided a method of preserving or culturing cells during the production of artificial meat or cultivated meat. The method may comprise contacting the cells with a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor to preserve or culture the cells in the production bioreactor. For example, the method may comprise contacting the cells with one or more calpain inhibitors and one or more cathepsin inhibitors. For example, the method may comprise contacting the cells with one or more calcium chelators (preferably at relatively high doses, such as the doses recited hereinabove in aspect A). Preferably, the method comprises contacting the cells with one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to preserve or culture the cells. Optionally, the contacting step further comprises contacting the cells with one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators.

[0355] Thus, also provided herein is a method of increasing bioreactor efficiency by enhancing cell survival and / or function during the production of artificial meat or cultivated meat. The method may comprise introducing a calcium activity inhibitor, a calpain inhibitor, a cathepsin inhibitor and / or a gap junction inhibitor to the bioreactor to increase bioreactor efficiency. For example, the method may comprise introducing one or more calcium chelators. For example, the method may comprise introducing one or more calpain inhibitors and one or more cathepsin inhibitors. Preferably, the method may comprise introducing one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors (such as one or more ryanodine receptor antagonists) and one or more gap junction inhibitors to the bioreactor to increase bioreactor efficiency. Optionally, one or more plasma membrane calcium channel inhibitors and / or one or more calcium chelators may also be introduced.

[0356] 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. The preferred inhibitors referred to herein are endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and gap junction inhibitors, particularly in combination.

[0357] The calcium chelators may be used at relatively high doses to effectively prevent necrosis and hence effectively preserve or culture cells, or increase bioreactor efficiency. For example, in these methods, the calcium chelator(s) may be used at the doses recited hereinabove in aspect A.

[0358] As the skilled person would appreciate, the methods may further comprise a step of removing the composition of the present invention prior to production of the final artificial meat or cultivated meat product.

[0359] Genome-editing technologies

[0360] The present invention provides a therapeutic agent that has been modified or produced by gene editing so as to provide dual inhibition of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions.

[0361] Any gene editing strategy known to one of ordinary skill in the art may be used to provide a therapeutic agent that exhibits dual inhibition of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions. For example, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and / or CRISPR-Cas9 techniques may be used. Other transcription and translation affecting techniques may also be used, including technologies involving RNA Interference (RNAi), siRNAs (Small Interfering RNAs), shRNAs (Short Hairpin RNAs) and miRNAs (MicroRNAs).

[0362] For example, in order to provide inhibition of gap junctions, connexin 43 (GJAI) knockout therapeutic agents or connexin 43 (GJAI) knockdown therapeutic agents may be prepared. Indeed, connexin 43 (GJA1) is known to play a critical role in various tissues, including heart, brain, and skin. Knocking out GJA1 has been shown to be successful in differentiating human induced pluripotent stem cells (iPSCs) into cells of all three germ layers without significant compensatory upregulation of other connexins. This suggests that connexin 43 is dispensable during directed iPSC lineage specification, indicating that targeting GJA1 could effectively block gap junctions in a broad range of contexts. Additionally, connexin 43 is known to play an integral role in allowing calcium ions to move between cells.

[0363] As another example, in order to provide inhibition of endoplasmic / sarcoplasmic reticulum calcium channels, ryanodine receptor RyR1 knockout therapeutic agents or ryanodine receptor RyR1 knockdown therapeutic agents may be prepared. RyR1 is believed to be directly responsible for mediating calcium release from the ER stores.

[0364] Thus, the present invention provides a therapeutic agent wherein both GJAI and RyR1 are simultaneously knocked down and / or knocked out. The therapeutic agent would thus provide dual inhibition of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions.

[0365] The therapeutic agent can be used in any of the methods, uses and medical uses described herein. The therapeutic agent can be used in a method of treating or preventing necrosis, cell death, tissue damage, or organ damage. Preferably, the therapeutic agent is used in a method for treating or preventing necrosis. The therapeutic agent can be used in a method of treating or preventing medical conditions that occur downstream of necrosis.

[0366] For example, the therapeutic agent can be used to treat or prevent organ damage.

[0367] The organ damage may be in a non-neuronal organ or an organ that is not part of the central nervous system (CNS). Thus, the therapeutic agent may be used to treat or prevent organ damage in a selection of one, more, or all of the group consisting of an integumentary organ, a skeletal organ, a muscular organ, a circulatory organ, a respiratory organ, a digestive organ, a urinary organ, an immune system organ, an endocrine organ, and a reproductive organ. Thus, the therapeutic agent may be used to treat or prevent damage in an organ selected from the group comprising or consisting of the heart, the kidneys, the liver, the skin, the spleen, the pancreas, the intestines, the stomach, lungs, bladder, eye, capillaries, joints, tendons, arteries, tongue, diaphragm, ovaries, scrotum, thyroid, adrenal glands, ears, larynx, oesophagus, trachea, ligaments, penis, thymus gland, bones, fallopian tubes, lymph nodes, ureters, bronchi, genitals, pharynx, salivary glands, urethra, gallbladder, lymphatic vessel, placenta, skeletal muscles, uterus, bone marrow, mouth, prostate, seminal vesicles, vulva, bulbourethral glands, hair follicle, 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 organ. Furthermore, the therapeutic agent may be used to treat or prevent organ damage in a neuronal organ or an organ that is part of the central nervous system (CNS).

[0368] The therapeutic agent described herein may be used to treat or prevent tissue damage.

[0369] The tissue damage may be a selection of one, more or all of 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, heat injury, cold injury, chilblain, trench foot, frostbite, avascular necrosis, pressure injury, and skin graft failure.

[0370] The tissue damage may be damage associated with infectious disease.

[0371] The tissue damage may occur in one or more organs referred to herein. For example, the tissue damage may occur in one or more tissues selected from the group comprising or consisting of: (i) epithelial tissue, (e.g., squamous, cuboidal, columnar, simple, stratified, pseudostratified, and specialized tissue); (ii) connective tissue (e.g., collagen, reticular and elastic, and tissue classified as proper (dense, loose), embryonic (mesenchyme, mucous), and specialized (cartilage, adipose, bone, blood)); and (iii) muscle tissue (e.g., skeletal, cardiac (gap junctions, intercalated discs), smooth, striated, and nonstriated). Therefore, the therapeutic agent may be used to treat or prevent tissue damage in epithelial tissue, connective tissue, and / or muscle tissue.

[0372] The therapeutic agent may 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).

[0373] Exposure of skin tissue to a trigger may cause it experience necrosis. The therapeutic agent may be used to prevent necrosis in skin (tissue).

[0374] The therapeutic agent described herein may be used to treat or prevent autoimmune disease including inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); arthritis; lupus; diabetes; liver disease / injury; kidney disease / injury; and metabolic syndrome.

[0375] The therapeutic agent described herein may also be used to treat or prevent traumatic brain injury (TBI); other forms of head injury including concussion, closed head injury, penetrating head injury, diffuse brain injury, brain contusion caused for example by a depressed skull fracture or a penetrating skull fracture, and cerebral palsy; intracranial hematoma (ICH); hematoma; stroke; brain aneurysm; hypoxic and anoxic brain injury; haemorrhage; meningitis; encephalitis; epilepsy; ataxia; motor neuron disease; multiple system atrophy; progressive supranuclear palsy; spinal cord injury; neurodegenerative disease including Alzheimer’s disease (AD), Parkinson’s disease (PD), prion diseases, amyotrophic lateral sclerosis (ALS), Huntington’s disease, spinal muscular atrophy, spinocerebellar ataxia; autoimmune disease including inflammatory bowel disease (such as 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.

[0376] As seen in breast carcinomas in particular, tumor dissemination and metastasis strongly correlate temporally with necrosis in animal models and in human cancer patients. Aside from increased vascular density and proinflammatory infiltrate (e.g., focal macrophage infiltration), this may be explained by necrotic regions having loose ready-to-detach cells due to reduced cell-cell and cell-matrix adhesion, which promotes tumor dissemination. Endometrial and renal carcinomas show a similar trend.

[0377] The therapeutic agent described herein may also be used to treat or prevent cancer, such as breast carcinomas, endometrial carcinomas and / or renal carcinomas. For example, the present invention relates to methods of preventing or treating the damage (such as the cellular damage) associated with the development of tumours and / or metastases in a subject.

[0378] Also provided is a method for creating tissue that is resistant to necrosis, wherein the method comprises creating or producing the tissue by gene editing. The tissue may be replacement tissue. The method may comprise tissue engineering and / or 3D cell culture, as discussed herein.

[0379] Definitions

[0380] The term “tissue damage” can refer to (biological) tissue dysfunction and / or (biological) tissue failure. Preferably the tissue damage is within an organ referred to herein. Similarly, the term “organ damage” can refer to organ dysfunction and / or organ failure.

[0381] The organ referred to herein may be a selection of one more or all of the following group consisting of: the kidneys, the liver, the skin, the spleen, the pancreas, the intestines, the stomach, lungs, bladder, eye, capillaries, joints, tendons, arteries, tongue, diaphragm, ovaries, scrotum, thyroid, adrenal glands, ears, larynx, oesophagus, trachea, ligaments, penis, thymus gland, bones, fallopian tubes, lymph nodes, ureters, bronchi, genitals, pharynx, salivary glands, urethra, gallbladder, lymphatic vessel, placenta, skeletal muscles, uterus, bone marrow, heart, mouth, prostate, seminal vesicles, vulva, bulbourethral glands, hair follicle, 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 organ.

[0382] The tissue referred to herein may be one or more tissues selected from the group comprising or consisting of: epithelial tissue (e.g., squamous, cuboidal, columnar, simple, stratified, pseudostratified, and specialized tissue); (ii) connective tissue (e.g., collagen, reticularand elastic, and tissue classified as proper (dense, loose), embryonic (mesenchyme, mucous), and specialized (cartilage, adipose, bone, blood)); and (iii) muscle tissue (e.g., skeletal, cardiac (gap junctions, intercalated discs), smooth, striated, and nonstriated).

[0383] Preferably, the organ referred to herein is a selection of one more or all of the following group consisting of: the kidneys, the liver, the pancreas, the intestines, the stomach, and lungs.

[0384] The tissue referred to herein may be from an organ selected from the group consisting of or comprising (i) epithelial tissue, (ii) connective tissue, and (iii) muscle tissue.

[0385] The methods according to the invention may be performed in vitro, or in vivo.

[0386] The term “burn” can be defined as an injury caused by exposure to heat or flame. A major contributor to cell and tissue damage following heat or flame is cellular necrosis. The term “multiple organ dysfunction syndrome (MODS)”, previously known as multiple organ failure, can be defined as sequential and progressive organ dysfunction, associated with a sustained, massive inflammatory response, and tissue injury, inflammatory states such as pancreatitis, any form of shock (i.e. a medical condition in which an individual is in a state of cellular and tissue hypoxia due to reduced oxygen delivery, increased oxygen consumption, inadequate oxygen utilization, or a combination of these three processes; this includes different types of shock such as distributive shock, hypovolemic shock, cardiogenic shock, and obstructive shock) and insults such as sepsis. A major contributor to MODS is cellular necrosis. The term “organ failure” can be defined as organ dysfunction to such a degree that normal homeostasis cannot be maintained without external clinical intervention. It can be classified by the cause, but when the cause is not known, it can also be classified by whether the onset is chronic or acute. A major contributor to organ failure is cellular necrosis, e.g., kidney failure, liver failure. The term “surgical trauma” can be defined as any injury produced by or related to surgery. Necrosis greatly contributes to surgical trauma due to for example hypoxia or mechanical force. The term “bedsores” can be defined as damage to an area of the skin caused by constant pressure on the area for a long time. This pressure can lessen blood flow to the affected area, which causes necrosis that then produces the sore. The term “chemical bum” can be defined as tissue damage caused by strong acids, drain cleaners, paint thinner, gasoline and other chemical substances. Such chemicals cause cell necrosis that then cause the chemical burn. The term “radiation burn” can be defined as tissue damage caused by exposure to radiation, including UV-rays, X-rays or radiation therapy. A major contributor to tissue damage following exposure to radiation is cellular necrosis. The term “gangrene” can be defined as a serious condition where a loss of blood supply causes body tissue to die. Necrosis contributes to the death of the tissue in gangrene. The term “alopecia (hair loss)” can be defined as the lack or loss of hair from areas of the body where hair is usually found. Necrosis contributes to hair loss. The term “solar erythema (sun burn)” can be defined as a short-term skin response to excessive amounts of ultraviolet (UV) radiation, mostly from the sun or from tanning lamps. Necrosis contributes to solar erythema. The term “acute tubular necrosis” can be defined as a kidney disorder involving damage to the cells of the kidneys, which can lead to acute kidney failure. Necrosis contributes to acute tubular necrosis. The term “ulcer” can be defined as a break in the epithelium, skin, in the lining of an organ, or on the surface of a tissue. Necrosis contributes to an ulcer. The term “physical injury” can be defined as damage / injury to the body from mechanical trauma, heat and cold, electrical discharges, changes in pressure, and radiation. Necrosis contributes to or complicates physical injury. The term “mechanical trauma” can be defined as damage / injury to the body from a blow, crush, cut, or penetrating wound. Necrosis contributes to or complicates mechanical trauma. The term “heat injury” can be defined as damage / injury to the body from a heat source, causing an increase in the temperature of local tissue. Necrosis contributes to or complicates thermal injury. The term “cold injury” can be defined as damage / injury to the body from cold exposure, including primarily peripheral cold injuries that are localized to the extremities and exposed skin and includes chilblain, trench foot and frostbite. Necrosis due to the decrease in temperature contributes to or complicates cold injury. The term “chilblain” can be defined as small swellings on the skin that occur as a reaction to cold temperatures. Necrosis due to the decrease in temperature contributes to chilblain. The term “trench foot” can be defined as a painful condition of the feet caused by long immersion in cold water or mud and marked by blackening and death of surface tissue. Necrosis due to the decrease in temperature and / or stress from prolonged water immersion, contributes to trench foot. The term “frostbite” can be defined as damage / injury caused by freezing of the skin and underlying tissues. Necrosis due to a decrease in temperature contributes to frostbite. The term “avascular necrosis” can be defined as a condition in which there is a loss of blood flow to bone tissue, which causes the bone to die. Necrosis contributes to avascular necrosis. The term “pressure injury” can be defined as an area(s) of necrosis and often ulceration (also called pressure ulcers) where soft tissues are compressed, and are caused by unrelieved mechanical pressure in combination with friction, shearing forces, and moisture. Necrosis contributes to pressure injury. The term “skin graft failure" can be defined as the failure of transplantation of one patch of skin and its derivatives (hair, nails, sweat and oil glands) that is surgically removed from one area of the body and attached to another area. Necrosis contributes to skin graft failure. The “subject” referred to herein may be a vertebrate, a mammal, or a domestic animal. The subject may be any animal of veterinary interest, for instance, a cat, a dog, a horse, a sheep or a cow. However, it is preferred that the subject is a mammal, such as a human. The term “epilepsy” can be defined as a condition where sudden bursts of electrical activity in the brain cause seizures or fits. The term “neurodegenerative disease" can be defined as disease characterized by neuron loss. The term “Alzheimer’s disease (AD)" can be defined as a progressive, degenerative disease of the brain marked especially by confusion, disorientation, memory failure, speech disturbances, and eventual dementia. The term “Parkinson’s disease (PD)" can be defined as a chronic progressive neurological disease marked especially by tremor of resting muscles, rigidity, slowness of movement, impaired balance, and a shuffling gait. The term “prion diseases" can be defined as transmissible brain diseases of mammals. The term “amyotrophic lateral sclerosis (ALS)" can be defined as a neurological disorder that affects motor neurons, the 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 hereditary brain disorder that is a progressive, neurodegenerative condition marked especially by impairments in thinking and reasoning, disturbances of emotion and behaviour, and the involuntary spasmodic movements of chorea and that is associated with the loss or atrophy of nerve cells. The term “spinal muscular atrophy” can be defined as a group of hereditary diseases that can damage and kill specialized nerve cells in the brain and spinal cord (motor neurons). The term “spinocerebellar ataxia" can be defined as a group of dominantly inherited, predominately late-onset, cerebellar ataxias. The term “ataxia" can be defined as one of a group of genetic disorders characterized by slowly progressive incoordination of gait and is often associated with poor coordination of hands, 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 condition of the nervous system that causes gradual damage to nerve cells in the brain. The term “progressive supranuclear palsy” can be defined as a rare neurological condition that can cause problems with balance, movement, vision, speech and swallowing. The term “spinal cord injury” can be defined as damage to the bundle of nerves and nerve fibres that sends and receives signals from the brain. The term “acute liver disease / injury” can be defined as a loss of liver function that progresses quickly in days to a few months. The term “acute kidney disease / injury” can be defined as a loss of kidney function that progresses quickly in days to a few months. The term “chronic liver disease / injury” can be defined as a condition where kidney function gets worse over time and includes associated conditions like non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and cirrhosis. The term “chronic kidney disease / injury” can be defined as a condition where kidney function gets worse overtime. The term “pancreatic disease" can be defined as a condition that results in loss of pancreatic function (e.g. due to alcohol or inflammation). The term “metabolic syndrome" can be defined as a condition that includes a cluster of risk factors specific for cardiovascular disease. The cluster of metabolic factors include abdominal obesity, high blood pressure, impaired fasting glucose, high triglyceride levels, and low HDL cholesterol levels. The term “autoimmune disease" can be defined as a condition associated with a hyperactive immune system. The term “inflammatory bowel disease" can be defined as an autoimmune condition affecting part or all of the gastrointestinal system, and includes conditions like Crohn's disease, inflammatory bowel syndrome and ulcerative colitis. The term “arthritis" can be defined as a condition of joint inflammation. The term “lupus" can be defined as a long-term autoimmune condition that causes joint pain, skin rashes and tiredness. The term “diabetes" can be defined as a chronic, metabolic disease characterized by elevated levels of 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 the nerve cells. The term “traumatic brain injury (TBI)" can be defined as a form of acquired brain injury which occurs when a sudden trauma causes damage to the brain. The term “head injury” can be any injury that occurs to the scalp, skull, brain, and underlying tissue and blood vessels in the head. The term “concussion" can be defined as the sudden but short-lived loss of mental function that occurs after a blow or other injury to the head. The term “closed head injury” can be defined as an injury that occurs where there is impact without breaking the skull. The term “penetrating head injury” can be defined as an injury that occurs when an object fractures the skull and enters brain tissue. The term “diffuse brain injury” can be defined as an injury that occurs when the brain moves within the skull when the head is shaken, and damage might be caused in several areas where the brain hits the skull. The term “brain contusion" can be defined as bruised or swollen brain tissue that occurs when the skull cracks or breaks. The term “depressed skull fracture" can be defined as fragments of a broken skull pressing against the brain. The term “penetrating skull fracture" can be defined as bone fragments entering the brain tissue. The term “cerebral palsy” can be defined as a group of lifelong conditions that affect movement and co-ordination. 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 the tissues. The term “stroke" can be defined as a condition when the blood supply to part of the brain is interrupted or reduced. The term “brain aneurysm" can be defined as a bulge in a blood vessel caused by a weakness in the blood vessel wall. The term “hypoxic brain injury” can be defined as a condition when part of the brain receives reduced oxygen. The term “anoxic brain injury" can be defined as a condition when part of the brain receives no oxygen. The term “haemorrhage" can be defined as an escape of blood from a ruptured blood vessel. The term “meningitis" can be defined as an infection of the protective membranes that surround the brain and spinal cord (meninges). The term “encephalitis" can be defined as inflammation of the brain, caused by infection or an allergic reaction. The term “cancer3’ can be defined as disease caused by an uncontrolled division of abnormal cells in a part of the body. The term “tumours" can be defined as groups of abnormal cells that form lumps or growths. The term “infectious disease" can be defined as an illness due to a pathogen or its toxic product. The term “cardiac arrest” can be defined as a, sometimes temporary, cessation of the heart’s functioning. The term “ischemia” can be defined as an inadequate blood supply to an organ or other part of the body.

[0387] In certain embodiments, the term “comprising” can refer to “consisting of’ or “consisting essentially of’.

[0388] In certain embodiments, the term “co-administering” refers to administration of multiple components. The components may be administered simultaneously, separately or sequentially. For example, one or more of the components may be administered prior to, simultaneously with, or following administration of other component(s). The components may be administered at the same site or at a different site. The components may be administered via the same or via different administration routes. The components may be administered in any order.

[0389] The term “co-administering” is not intended to imply that the components must be physically mixed or administered at the same time and / or formulated for delivery together, although these methods of delivery and corresponding formulations are within the scope described herein. It will further be appreciated that the components may be administered together in a single composition (e.g., if the components are co-formulated in any suitable form, such as a solid or liquid form) or administered separately in different compositions.

[0390] All of the embodiments and features described herein (including any accompanying clauses, claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects or embodiments in any combination, unless stated otherwise with reference to a specific combination, for example, combinations where at least some of such features and / or steps are mutually exclusive.

[0391] For a better understanding of the invention, and to show embodiments of the invention may be put into effect, reference will now be made, by way of example, to the accompanying drawings, in which:-

[0392] Figure 1 is an illustration of the predicted weak spots in cell physiology that make cells susceptible to cellular necrosis. The weak spots include: increase in cytosolic calcium via (i) movement of calcium ions into the cytosol via plasma membrane calcium ion channels; (ii) movement of calcium ions from within organelles with the cell, especially the endoplasmic / sarcoplasmic (ER / SR) reticulum via ER / SR calcium ion channels; and (iii) movement of calcium ions and other damaging agents from cell to cell via gap junctions. Other weak spots include calpain hyper-activation, cathepsin hyper-activation and the opening of store-operated calcium channels (also called calcium release-activated calcium channels) post ER calcium depletion. The most pertinent of these steps are the calcium ion entry via ER / SR calcium ion channels and gap junctions, and followed by entry via the plasma membrane calcium ion channels, as discussed in further detail in Example 1 below.

[0393] Figures 2-28 show necrotic core formation in Hepg2s spheroids under different stressors and treatment conditions, as discussed below. Spheroids are spherical cellular aggregates supporting cell-cell and cell-matrix interactions in an environment that mimics the real-world situation. Spheroids cannot be supported for long periods of time or grown large due to necrotic core formation (cells in the middle of the spheroid are starved of oxygen and adequate nutrients, with primarily hypoxia causing cell death via necrosis).

[0394] Figure 2 shows that Hepg2s spheroids show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, after 7 days with the addition of relatively high doses of calcium chelators. Treatment is different chelating agents at varying dosages. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0395] Figure 3 shows that Hepg2s spheroids do not show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a range of plasma membrane calcium channel inhibitors. Treatment is different plasma membrane calcium channel inhibitors. The inhibitors were added at doses which were expected to be the maximum doses showing minimal toxicity. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines.*P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0396] Figure 4 shows that Hepg2s spheroids do not show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a range of endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors. Treatment is endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitors at doses which were expected to be the maximum doses showing minimal toxicity. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two- way ANOVA (Dunnett’s correction) relative to control.

[0397] Figure 5 shows that Hepg2s spheroids do not show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a range of gap junction inhibitors. Treatment is gap junction inhibitors at doses which were expected to be the maximum doses showing minimal toxicity. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0398] Figure 6 shows that Hepg2s spheroids do not show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a range of store-operate calcium channels (SOCC) inhibitors. Treatment is SOCC inhibitors at doses which were expected to be the maximum doses showing minimal toxicity. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0399] Figure 7 shows that Hepg2s spheroids do not show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a range of calpain inhibitors. Treatment is calpain inhibitors at doses which were expected to be the maximum doses showing minimal toxicity. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0400] Figure 8 shows that Hepg2s spheroids do not show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of calpain / cathepsin non-specific inhibitors. Treatment is calpain / cathepsin non-specific inhibitors at doses which were expected to be the maximum doses showing minimal toxicity. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines.*P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0401] Figure 9 shows that Hepg2s spheroids do not show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a range of cathepsin inhibitors. Treatment is cathepsin inhibitors at doses which were expected to be the maximum doses showing minimal toxicity. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines.*P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0402] Figure 10 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with 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 is Dantrolene in different dosages individually, retinoic acid in different dosages individually, and the combination of Dantrolene and retinoic acid in different dosages. The combination has a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control. Figure 11 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor oleic acid. Treatment is Dantrolene and oleic acid used individually and in combination. The combination has a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. ,*P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0403] Figure 12 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and a range of gap junction inhibitors. Treatment is Dantrolene and a range of gap junction inhibitors used individually and in combination. The combination has a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. ,*P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0404] Figure 13 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene combined with more than one gap junction inhibitor. Treatment is Dantrolene and more than one gap junction inhibitor combined, as well as the individual components of the combination tested separately. The combination has a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines.*P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0405] Figure 14 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and a range of gap junction inhibitors. Treatment is ryanodine and a range of gap junction inhibitors used individually and in combination. The combination has a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control. Figure 15 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor trans-Ned 19 and a range of gap junction inhibitors. Treatment is trans-Ned 19 and a range of gap junction inhibitors used individually and in combination. The combination has a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0406] Figure 16 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor cis-Ned 19 and a range of gap junction inhibitors. Treatment is cis-Ned 19 and a range of gap junction inhibitors used individually and in combination. The combination has a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0407] Figure 17 shows that Hepg2s spheroids show greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination 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, with the gap junction inhibitor retinoic acid, (a) Treatment is procaine and the gap junction inhibitor retinoic acid used individually and in combination, (b) Treatment is ruthenium red and the gap junction inhibitor retinoic acid used individually and in combination. The combinations (of both (a) and (b)) have a more than additive effect compared to treatment with the individual components of the combination that show no significant difference from the controls. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0408] Figure 18 shows that Hepg2s spheroids show no reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of the gap junction inhibitor retinoic acid and a range of plasma membrane calcium channel inhibitors. Treatment is a combination of the gap junction inhibitor retinoic acid and a range of plasma membrane calcium channel inhibitors. The combinations show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****p<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0409] Figure 19 shows that Hepg2s spheroids show no reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and a range of plasma membrane calcium channel inhibitors. Treatment is a combination the Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and a range of plasma membrane calcium channel inhibitors. The combinations show no significant difference from the control. Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Two-way ANOVA (Dunnett’s correction) relative to control.

[0410] Figure 20 shows that reduction in Hepg2s spheroid necrotic core formation, induced by hypoxic and nutrient deprivation stress, is even greater when the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor is further combined with a plasma membrane calcium channel inhibitor, (a) Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, used alone and in combination with a range of different plasma membrane calcium channel inhibitors, (b) Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor palmitoleic acid, used alone and in combination with the plasma membrane calcium channel inhibitor Verapamil, (c) Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor linoleic acid, used alone and in combination with the plasma membrane calcium channel inhibitor Verapamil, (d) Treatment is the Endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor ryanodine and the gap junction inhibitor retinoic acid, used alone and in combination with the plasma membrane calcium channel inhibitor Arandipine. The combination of the 3 compounds (i.e., the ER / SR calcium channel inhibitor, the gap junction inhibitor and the plasma membrane calcium channel inhibitor) has a greater effect than the combination of only the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with the gap junction inhibitor. However, it will be appreciated that both the combination of 2 compounds, and the combination of 3 compounds, have a more than additive effect compared to the individual components of the combination relative to the control (c.f. Figures 3,4 and 5 of the individual components that show no significant difference to the control; Figures 3,4 and 5 and this figure are directly comparable with trials done alongside each other). The dotted lines show the mean of the control orthe mean of the combination of 2 compounds, where one is the gap junction inhibitor and the other the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control and relative to the combination of 2 compounds, i.e., to the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with the gap junction inhibitor.

[0411] Figure 21 shows that reduction in Hepg2s spheroid necrotic core formation, induced by hypoxic and nutrient deprivation stress, is even greater when the combination of an endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor is further combined with a calcium chelator. Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, used alone and in combination with a range of different calcium chelators. The combination of the 3 compounds has a greater effect than the combination of only the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with the gap junction inhibitor. It will be appreciated that both the combination of 2 compounds, and the combination of 3 compounds, have a more than additive effect compared to the individual components of the combination relative to the control (c.f. Figures 2,4 and 5 of the individual components that show no significant difference to control; Figures 2,4 and 5 and this figure are directly comparable with trials done alongside each other). The dotted lines show the mean of control or the mean of the combination of 2 compounds, where one is the gap junction inhibitor and the other the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control and relative to the combination of 2 compounds, i.e., to the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with the gap junction inhibitor.

[0412] Figure 22 shows that there is no further reduction in Hepg2s spheroid necrotic core formation, induced by hypoxic and nutrient deprivation stress, when the combination of an endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor and a gap junction inhibitor is further combined with drugs associated with other targets of weak spots in cell physiology that make cells susceptible to necrosis (including calpain inhibitors, cathepsin inhibitors, calpain / cathepsin nonspecific protease inhibitors and SOCC inhibitors), (a) Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, used alone and in combination with a range of different calpain inhibitors, (b) Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, used alone and in combination with a range of different cathepsin inhibitors, (c) Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, used alone and in combination with a range of different calpain / cathepsin non-specific protease inhibitors, (d) Treatment is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, used alone and in combination with a range of different SOCC inhibitors. The combination of 3 has the same, if not a worse, effect than the combination of only the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with the gap junction inhibitor. The dotted lines show the mean of control or the mean of the combination of 2 compounds, where one is the gap junction inhibitor and the other is the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Two-way ANOVA (Dunnett’s correction) relative to the combination of the 2 compounds, i.e., to the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with the gap junction inhibitor.

[0413] Figure 23 shows that Hepg2s spheroids show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, with the addition of a combination of a calpain inhibitor with a cathepsin inhibitor, but not other combinations such as a combination of 2 or more calpain inhibitors, a combination of 2 or more calpain inhibitors or combinations with non-specific calpain / cathepsin inhibitors, (a) Treatment is a combination of different (i) calpain inhibitors, and (ii) calpain inhibitors combined with non-specific calpain / cathepsin inhibitors, (b) Treatment is a combination of different cathepsin inhibitors. (a,b) The combination has no significant effect relative to the control, (c) Treatment is a combination of different calpain inhibitors combined with cathepsin inhibitors; the combination has a significant beneficial effect relative to the control, and the effect is more than compared of the individual components of the combination that show no significant difference from control. The combination of 2 has a more than additive effect compared to the individual components of the combination relative to the control, (c.f. Figures 7 and 9 of the individual components that show no significant difference to control; Figures 7 and 9 and this figure are directly comparable with trials done alongside each other). Dotted line: mean of control. Three trials (n=2 per trial). Violin plot showing the distribution of the data with quartiles as dotted lines. *P<0.05, **P<0.01 , ***P<0.001 , ****p<0.0001 by two-way ANOVA (Dunnett’s correction) relative to control.

[0414] Figure 24 shows that Hepg2s spheroids show reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, when combinations of over-the-counter compounds are used that inhibit targets associated with the predicted weak spots in cell physiology that make cells susceptible to cellular necrosis. Treatment is over the counter compounds that inhibit targets associated with the predicted weak spots in cell physiology (c.f. Figure 1) that make cells susceptible to cellular necrosis. Notably, the low toxicity of these compounds allows them to be mixed in combinations without a severe increase in cell death from chemical toxicity being observed. Spheroids are spherical cellular aggregates supporting cell-cell and cell-matrix interactions in an environment that mimics the real-world situation. Spheroids cannot be supported for long periods of time or grown large due to necrotic core formation (cells in the middle of the spheroid are starved of oxygen and adequate nutrients, with primarily hypoxia causing cell death via necrosis). The combination has a more than additive effect compared of the individual components of the combination that show no significant difference from control. Dotted line: mean of control. Three trials (n=2 per trials). Violin plot showing the distribution of the data with quartiles as dotted lines. P=0.07 showing marginal reduced core formation by two-way ANOVA (Dunnett’s correction) relative to control.

[0415] Figure 25 is a time series of Hepg2s spheroids with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, and shows greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, through time. Treatment (added on day 2) is Dantrolene and retinoic acid. The combination is followed through time up to 7 days and shows robust necrotic core suppression in all 3 trials. Top: Bright field images above fluorescence (DRAQ7™; viability dye that stains necrotic cells; red) images of spheroids in the well of multi-welled plates. Middle: Bar graphs with s.t.d showing cell death stain DRAQ7™ intensity through time in the treatment and control group. The left-hand bar shows the treatment group, and the right-hand bar shows the control group. Bottom: example fluorescence (DRAQ7™; viability dye that stains necrotic cells; red) images of spheroids in the well of multi-welled plates. 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 26 is a time series of Hepg2s spheroids with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, and shows no significant toxic effect of the combination; spheroid formation, growth, size and appearance appears normal. Treatment (added on day 2) is Dantrolene and retinoic acid. The combination is followed through time up to 7 days and shows no significant effect on spheroid formation, growth, size or appearance. Top: Bright field images of spheroids in the well of multi-welled plates. Middle: Violin plot of spheroid size through time, a marker of formation and growth, showing the distribution of the data with quartiles as dotted lines. The left-hand plot shows the treatment (“drug”) group, and the right-hand plot shows the control group. Bottom: example bright filed images of spheroids in the well of multi-welled plates, showing a normal morphology and appearance and no loss of integrity of the spheroid with the treatment. Three trials (n=2 per trial). *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Bonferroni correction).

[0416] Figure 27 is a time series of HEK293 spheroids with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, and shows greatly reduced necrotic core formation, induced by hypoxic and nutrient deprivation stress, through time. Treatment (added on day 2) is Dantrolene and retinoic acid. The combination is followed through time up to 5 days and shows robust necrotic core suppression in all 3 trials. Top: Bright field images above fluorescence (DRAQ7™; viability dye that stains necrotic cells; red) images of spheroids in the well of multi-welled plates. Middle: Bar graphs with s.t.d showing cell death stain DRAQ7™ intensity through time in the treatment and control group. The left-hand bar shows the treatment group, and the right-hand bar shows the control group. Bottom: example fluorescence (DRAQ7™; viability dye that stains necrotic cells; red) images of spheroids in the well of multi-welled plates. Three trials (n=2 per trial). *p<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Bonferroni correction).

[0417] Figure 28 is a time series of HEK293 spheroids with the addition of a combination of the endoplasmic reticulum / sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, and shows no significant toxic effect of the combination; spheroid formation, growth, size and appearance appears normal. Treatment (added on day 2) is Dantrolene and retinoic acid. The combination is followed through time up to 5 days and shows no significant effect on spheroid formation, growth, size or appearance. Top: Bright field images of spheroids in the well of multi-welled plates. Middle: Violin plot of spheroid size through time, a marker of formation and growth, showing the distribution of the data with quartiles as dotted lines. The left-hand plot shows the treatment (“drug”) group, and the right-hand plot shows the control group. Bottom: example bright filed images of spheroids in the well of multi-welled plates, showing a normal morphology and appearance and no loss of integrity of the spheroid with the treatment. Three trials (n=2 per trial). *p<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by two-way ANOVA (Bonferroni correction).

[0418] Figure 29 shows that Hepg2s spheroids show greatly reduced necrosis as measured by an lactate dehydrogenase LDH-Glo™ assay, induced by necrotic cores from hypoxic and nutrient deprivation stress, with 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 is Dantrolene and retinoic acid used in combination. Three trials (n=2 per trial). Bar graphs with s.e.m. showing cell death. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by unpaired two-tailed t-test.

[0419] Figure 30 shows human intestinal fibroblast primary cell spheroids show a (more than additive) large reduction in necrosis, as measured by the standard viability stain Calcein AM / Ethidium homodimer-1 , following oxidative stress when treated with either (i) a combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with 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 as a preventative measure, but not other combinations. Fig 30 shows a combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor. Specifically, these combinations are those labelled “D 100; RA 10”, “D 100; RA 20” and “D 100; RA 40”. D refers to Dantrolene, and RA refers to retinoic acid. The numerical value refers to the dosage, e.g., 100 refers to treatment with 100pM of the compound. Fig 30 also shows 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. Specifically, this combination is labelled “D 100; RA 20; N 10”. N refers to Nifedipine. Stress is exposure to hydrogen peroxide (800pM) for 6 hrs. Top: pre stress data and Bottom: post-stress data. Left: raw data of the ratio of Ethidium homodimer-1 over Calcein AM intensity. Right: data normalised to Triton X- 100 that kills all cells, so the percentage of necrosis can be gauged. A combination of (i) an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor, and (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 have a significant and more than additive effect on necrosis, compared to the single compounds in the combination that show either no significant difference or a worse effect to control. The combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor and a plasma membrane calcium channel inhibitor is better than the combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor. Other combinations, including (i) the combination an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a plasma membrane calcium channel inhibitor, and (ii) the combination a gap junction inhibitor with a plasma membrane calcium channel inhibitor, show no significant difference to stressed control. Box and whiskers plots (10-90 percentile). Two trials (n=31 per trial). *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Kruskal-Wallis test (Dunn’s multiple comparisons correction).

[0420] Figure 31 shows human intestinal fibroblast primary cell spheroids show a large reduction in necrosis, as measured by the standard viability stain Calcein AM / Ethidium homodimer-1 , following mild oxidative stress over a long duration when treated with a combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor as a preventative and treatment measure. The ER / SR calcium channel inhibitor used was Dantrolene, and the gap junction inhibitor used was retinoic acid. Stress is exposure to hydrogen peroxide (500pM) for 6 hrs. Line graphs showing mean and s.e.m. Two trials (n=31 per trial). *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Two-way ANOVA (Dunn’s multiple comparisons correction). At each data point (i.e., at day 1 , day 2 and day 3), the stars are as follows: top, stressed control vs stressed treatment with combo (i.e., **** at day 1 , *** at day 2, and **** at day 3); middle, stressed control vs untreated control (i.e., * at day 1 , ** at day 2, and **** at day 3); bottom, stressed treatment vs untreated control (i.e., ns at day 1 , ns at day 2 and **** at day 3).

[0421] Figure 32 shows human intestinal fibroblast primary cell spheroids show a large reduction in necrosis, as measured by the standard viability stain Calcein AM / Ethidium homodimer-1 , following moderate oxidative stress over a long duration when treated with a combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor as a preventative and treatment measure. The ER / SR calcium channel inhibitor used was Dantrolene, and the gap junction inhibitor used was retinoic acid. Stress is exposure to hydrogen peroxide (800pM) for 6 hrs. Line graphs showing mean and s.e.m. Two trials (n=31 per trial). *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Two-way ANOVA (Dunn’s multiple comparisons correction). At each data point (i.e., at day 1 , day 2 and day 3), the stars are as follows: top, stressed control vs stressed treatment with combo (i.e., **** at day 1 , **** at day 2, and **** at day 3); middle, stressed control vs untreated control (i.e., **** at day 1 , **** at day 2, and **** at day 3); bottom, stressed treatment vs untreated control (i.e., **** at day 1 , **** at day 2, and **** at day 3).

[0422] Figure 33 shows human intestinal fibroblast primary cell spheroids show a large reduction in necrosis, as measured by the standard viability stain Calcein AM / Ethidium homodimer-1 , following severe oxidative stress over a moderate 3 hr duration when treated with a combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor as a preventative and treatment measure. The ER / SR calcium channel inhibitor used was Dantrolene, and the gap junction inhibitor used was retinoic acid. Stress is exposure to hydrogen peroxide (1 mM) for 3 hrs. Line graphs showing mean and s.e.m. Two trials (n=31 per trial). *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001 by Two-way ANOVA (Dunn’s multiple comparisons correction). At each data point (i.e., at day 1 , day 2 and day 3) the stars are as follows: top, stressed control vs stressed treatment with combo (i.e., ns at day 1 , **** at day 2, and **** at day 3); middle, stressed control vs untreated control (i.e., **** at day 1 , **** at day 2, and **** at day 3); bottom, stressed treatment vs untreated control (i.e., **** at day 1 , *** at day 2, and *** at day 3).

[0423] Figure 34 shows exemplary images of human intestinal fibroblast primary cell spheroids showing a large reduction in necrosis, as measured by the standard viability stain Calcein AM / Ethidium homodimer-1 , following moderate oxidative stress over a long duration when treated with a combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor as a preventative and treatment measure. The ER / SR calcium channel inhibitor used was Dantrolene, and the gap junction inhibitor used was retinoic acid. Stress is exposure to hydrogen peroxide (800pM) for 6 hrs. Green: viable cells stained with Calcein AM; Red: dead necrotic cells stained with Ethidium homodimer-1. Around 16 spheroids can be seen per image. Triton X-100 that kills all cells images displayed, so the percentage of necrosis can be gauged. All images taken on day 3 of the experiment (c.f. Figure 32 that these images relate to).

[0424] Figure 35 showing human intestinal fibroblast primary cell spheroids with the addition of a combination of the Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid, and shows no significant toxic effect of the combination; spheroid formation, growth and size, as well as ATP levels are maintained, (a) Image of cells aggregating to form spheroids by day 1 . Spheroids are spherical cellular aggregates supporting cell-cell and cell-matrix interactions in an environment that mimics the real-world situation, (b) Spheroid size in the control shows no major change from day 1 to day 3. (c) T reatment added on day 0 or day 1 is Dantrolene and retinoic acid. The combination is followed through time up to 3 days and shows no significant effect on spheroid size or growth, i.e. change in size with time. Three trials (n=30 per trial), (d) ATP presence measured on day 3 using a CellTiter-Glo® 3D Cell Viability Assay shows no reduction in ATP levels with treatment added on day 1. Bar graph with raw data points displayed. Two trials (n=31 per trial). *p<0.05, **P<0.01 , ***P<0.001 , ****p<0.0001 by One-way ANOVA (Sidak’s multiple comparisons correction).

[0425] Figure 36 shows images of human intestinal fibroblast primary cell spheroids showing a large reduction in necrotic core formation, induced by hypoxic and nutrient deprivation stress, on day 6, as measured by the standard viability stain Calcein AM / Ethidium homodimer-1 , when treated with a combination of an Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor with a gap junction inhibitor. The ER / SR calcium channel inhibitor used was Dantrolene (100 pm), and the gap junction inhibitor used was retinoic acid (10 pm). Top: Box and whiskers plots (10-90 percentile); data normalised to Triton X-100 that kills all cells. Two trials (n=31 per trial). *P<0.05, **P<0.01 , ***P<0.001 , ****p<0.0001 by Kruskal-Wallis test (Dunn’s multiple comparisons correction). Bottom: Example images. Green: viable cells stained with Calcein AM; Red: dead necrotic cells stained with Ethidium homodimer-1. Around 16 spheroids can be seen per image.

[0426] Figure 37 shows that E. coli and S. pombi death is prevented with drug that supresses necrosis following oxidative stress (stress is for 50 min 1 mM H2O2). (a) E. coli bacteria. Treatment is EGTA 2500 pM during stress. Following stress, the bacteria is washed in buffer, diluted 1 :500 in MilliQ water, seeded onto agar plates containing bactopeptone and then left at 37 °C overnight, followed by the counting of colony forming units (CFU). N=3 trials, (b) S. pombi yeast. Treatment is EGTA 2500 pM during stress. Following stress, the yeast is washed and stained with Propidium iodide (PI; red) which enters necrotic cells. Box and whiskers (Tukey) of % of necrotic cells. (N=6-7 images, combined data of 2 trials). *P<0.05 by unpaired two- tailed t-test.

[0427] Figure 38 shows that C. elegans death is prevented following a variety of stressors including heat, cold / freeze, oxidative and toxin stress, when treated with drug that supresses necrosis. From left to right: Cold stress (-80°C for 1 .5 min); Heat stress (37°C for 4.5 hrs); Oxidative stress (2M hydrogen peroxide for 5 min); and toxin stress (1 ,3%(v / v) tert-butylhydroperoxide (tBOOH) for 5 min). Treatment is EGTA 2500 pM in liposomes given as a preventative measure 3 hrs prior to stress or as a treatment 3 hrs post stress. Box and whiskers (Tukey) plots. 3 trails (n=10 per trial). *P<0.05, ***P<0.001 , ****P<0.0001 , by One-way ANOVA (Dunnett correction).

[0428] Figure 39 shows that necrosis mutants show reduced movement defects post a variety of stressors including heat, cold / freeze, oxidative and toxin stress, when treated with drug that supresses necrosis. From left to right: Cold stress (-80°C for 1 .5 min); Heat stress (37°C for 4.5 hrs); Oxidative stress (2M hydrogen peroxide for 5 min); and toxin stress (1 ,3%(v / v) tert-butylhydroperoxide (tBOOH) for 5 min). Treatment is EGTA 2500 pM in liposomes given 3 hrs prior stress. Box and whiskers (Tukey) plots. 3 trails (n=10 pertrial). *P<0.05, ***P<0.001 , ****P<0.0001 by One-way ANOVA (Dunnett correction). Worms that show slowed movement (are typically still and only move when poked with a worm pick or may not move at all), are scored and the percentage of animals with a movement defect displayed (y-axis).

[0429] Figure 40 shows that D. melanogaster death is prevented following cold / freeze stress, when treated with drug that supresses necrosis. Stress is keeping animals at 0 degrees for 10 min. D. melanogaster W1 118 animals are aged to day 7, starved for 8 hrs and then fed for 12 hrs with food containing 0 or 2500pM EGTA. This is followed by the cold / freeze stress and then survival measured every 12 hrs following. Treatment showed a significant difference to control. *P<0.05 by log-rank analysis

[0430] Figure 41 shows that Zebra fish (Danio rerio) liver shows necrosis cell death is prevented by drug that supress necrosis following oxidative stress (7 min in 1 mM H2O2). Treatment is 2500pM EGTA during stress. Following stress, liver is washed and stained with Propidium iodide (PI; red)which enters necrotic cells. Tissue is also stained with DAPI (4’6-diamidino-2-phenylindole) that enters all cells (blue) so total number of cells present in the image can be analysed. % of necrotic cells (PI stain), relative to all cells seen (DAPI stain); Box and whiskers plot (Tukey). 3 trials. P = 0.07 by unpaired two-tailed t-test.

[0431] Figure 42 shows that mouse organs show necrosis cell death is prevented by drug that supress necrosis following hypoxia stress (2.5 hrs in N2gas bubbled buffer). Organotypic slices of tissue are prepared and placed in buffer mimicking Cerebrospinal fluid (CSF) for brain tissue or Ringer’s buffer for other tissue. Thickness of slices is as follows: heart: 0.210 mm; brain: 0.300 mm; Spleen: 300 mm. Treatment is 2500pM EGTA during stress. Following stress, slices are washed and stained with Propidium iodide (PI; red) which enters necrotic cells. Tissue is also stained with DAPI that enters all cells (blue) so total number of cells present in the image can be analysed. % of necrotic cells (PI stain), relative to all cells seen (DAPI stain). Box and whiskers plot (Tukey). Combined data of 2 trials. ***P<0.001 , **** P<0.0001 by unpaired two-tailed t-test.

[0432] Figure 43 shows that regardless of the type of cell, type of cell culture or stress, necrosis cell death is prevented by drugs that supress necrosis, (a) HEK293 suspension culture following oxidative stress (20 sec 1 M H2O2). Treatment is 2500pM EGTA 2 hrs prior as well as during stress. Following stress, cells are washed and stained with Propidium iodide (PI; red) which enters necrotic cells. Cells are also stained with DAPI that enters all cells (blue) so total number of cells present in the image can be analysed. % of necrotic cells (PI stain), relative to all cells seen (DAPI stain). Box and whiskers plot (Tukey). Combined data of 2 trials. **** P<0.0001 by unpaired two-tailed t-test. (b) Hepg2s spheroids made by hanging drop technique and placed on agarose pads show no necrotic core even after 5 days with the addition of drug that supress necrosis. Treatment is 2500pM EGTA or 5000pM EGTA in spheroid culture. Spheroids, used in tissue engineering (spherical cellular aggregate supporting cell-cell and cell-matrix interactions in an environment that mimics the real-world situation), cannot be supported for long periods of time or grown large due to necrotic core formation (cells in the middle of the spheroid are starved of oxygen and nutrients and this causes cell death via necrosis). Box and whiskers plot (Tukey). Combined data of 3 trials. *P<0.05, ***P<0.001 , **** P<0.0001 by unpaired two-tailed t-test.

[0433] Figure 44. Drugs to supress necrosis (a combination of the Endoplasmic reticulum / Sarcoplasmic reticulum (ER / SR) calcium channel inhibitor Dantrolene and the gap junction inhibitor retinoic acid) can be added to the gold standard organ / graft preservation media, University of Wisconsin (UW) media to increase cell viability and reduce cell necrosis during warm and cold storage and so increase organ / graft quality. Following acclimation, mice were anesthetized with isoflurane and cardiac perfusion was performed with 50mL UW buffer at 5mL / min containing either no treatment (i.e. control group) or treatment: 100pM Dantrolene and 10 pM Retinoid Acid. Kidneys were incubated in 5mL 4°C for 24h followed by 2h at 37°C prior to single cell isolation from whole kidney. Following washing, cells were stained with Annexin V and Propidium Iodide (PI) to determine the ratio of necrotic (PI+ Annexin V-) and viable (PI-) cells and flow cytometry performed. Left (Fig 44a): example images of flow cytometry. Middle and right (Figs 44b and 44c): Non-viable dead cells and necrotic cells in control and treatment groups. 4 trials (n=1 kidney per trial). *P=0.057 by unpaired two-tailed t-test.

[0434] Examples

[0435] Materials and methods

[0436] HEK293 and HepG2 cell spheroid production and drug screen in U-bottom plates: hypoxic stress.

[0437] Stand cell culture of HEK293T and HepG2 cells was performed (10% Fetal bovine serum in Dulbecco's Modified Eagle Medium). Cells were then transferred to either 96-well Nunc Sphera or 384-well PrimeSurface® 3D Culture Spheroid plates (ultra-low attachment (ULA) plates) using an initial cell density of 500 cells. Following spheroid formation in the plates, treatment compound(s) were solubilised in DMSO (dimethylsulfoxide) to get a final assay DMSO concentration of 1 % v / v and then added to the ULA plate wells. Media changes were on alternative days. Brightfield phase contrast and fluorescent image acquisition was performed at regular intervals up until 6 days post-treatment using an Incucyte® S3 live-cell analysis system. The standard viability dye DRAQ7™ (that only stains the nuclei in dead and permeabilised cells) was used as a marker of cell death. All conditions were tested in duplicate on three separate occasions (i.e. three independent trials were performed). DMSO controls were added on each plate. Necrotic core size was quantified as the percentage change from the average of DMSO control wells based on fluorescence area normalised to spheroid size. As a secondary indicator of cell death, a lactate dehydrogenase LDH-Glo™ assay was performed on supernatants from the treated well and control wells. Human fibroblast primary cells cell spheroid production and drug screen in U-bottom plates: oxidative stress and hypoxic stress.

[0438] Stand cell culture of Human intestinal fibroblast primary cells was performed. Cells were then transferred to 384-multi well plates (Gri3D product number Gri3D-96P-S-96-800). Seeding density per well was 125,000 cells, with 4000 cells per spheroid within the wells forming. Following spheroid formation in the plates, treatment compounds were solubilised in DMSO to get a final assay DMSO concentration of 0.5% v / v and then added to the plate wells. Media changes were on alternative days. Triton X-100 was used as a positive control marker of cell death. Drug treatment was done overnight prior stress. Brightfield phase contrast and fluorescent image acquisition was performed at regular intervals immediately post stress, and on subsequent days post stress up to day 3 using a Tecan Spark instrument for brightfield (model number Spark Cyto 600) and a GE Healthcare IN Cell instrument for fluorescence (model number IN Cell Analyzer 2200); images were processed using Doppl's proprietary pipelines. Stress was exposure to varying levels of hydrogen peroxide for different time durations as stated in the figure legends. The standard viability Calcein AM / Ethidium homodimer-

[0439] 1 staining kit (Thermo Molecular Probes) was used as a marker of cell death (3 hrs incubation at 37°C prior stress). To determine the presence of metabolically active viable cells in 3D cell culture, a quantitation of the level of ATP present was performed using the CellTiter-Glo® 3D Cell Viability Assay. For this, 150pl of supernatant is collected per well, followed by the addition of 50pl CTG and mixing for 5 minutes. Luminescence was then measured after 30 minutes of incubation at r.t.p. on day 1 and day 3 or 6. Two repeats of all conditions were performed. Each repeat used 31 organoids per condition. Readouts were resistance against hydrogen peroxide stress and necrotic core formation (i.e. hypoxic stress) through time; and cell viability gauged from metabolic activity, i.e. ATP levels. DMSO controls were assessed in parallel on each plate. Level of dead organoid tissue was quantified as percentage change from DMSO control wells based on fluorescence area normalised to spheroid size.

[0440] Viability Analysis of Perfused Mouse Kidneys by Flow Cytometry.

[0441] 8 animals were acclimated to a vivarium 7 days prior to the study initiation. After acclimation, animals were randomized by body weight, and culled and cardiac perfused with UW (University of Wisconsin) preservation media with and without treatment compounds added. Intact right kidneys were then extracted from the animal and stored in UWwith or without treatment compounds in hypothermic (4°C) conditions for 24 hrs, followed by

[0442] 2 hrs at 37°C before processing for analysis. Kidney Processing was done by dissociation manually by mincing and digestion into a single cell suspension using Liberase TM, collagenase, dispase, and Dnase I. Cells were then passed through a 70 pM filter to remove debris and generate 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 the following: (i) AnnV-PI+ for necrotic cells; and (ii) PI+ for non-viable dead cells. Cells were then acquired on a BD Accuri C6 Flow Cytometer, on FL-1 for Annexin V-FITC and FL-3 for PI.

[0443] HEK293 suspension culture oxidative stress

[0444] HEK293 cells were detached using trypsin, washed and placed in cell culture medium. Cells were pre-treated for 2 hrs before and during stress with or without (control) EGTA 2500 pM. Cells were stressed by placing them in a 1 M hydrogen peroxide solution (in cell culture medium) for 20 sec. Cells were then washed in cell culture medium and stained with PI as well as DAPI to visualise all cell nuclei (including intact cell nuclei), and placed on a microscope slide under a coverslip and imaged.

[0445] Preparation of 3D HepG2 cell culture spheroids

[0446] Multi-cellular spheroids were prepared using a hanging drop method. A stock solution of methylcellulose (MC) 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 subsequently centrifuged at 4,000 g for 3 hrs. HepG2s cells were re-suspended in cell medium containing 20 % MC stock solution at a concentration of 20,000 cells per 50 pL spheroid. 50 pL drops were subsequently pipetted onto a non-adherent petri dish (Greiner) and, replacing the lid, the dish was turned upside-down and incubated overnight at 37°C. Multi-cellular spheroids were harvested individually with a wide bore pipette or collectively by adding 5 mL cell medium to the dish, collecting and centrifuging the suspension at 300 g for 5 min and resuspending the spheroids in cell medium. Spheroids were then cultured for 5 days in 48 well plates containing 2.5 % agarose gel and 400 uL cell medium. EGTA was added at 0 and 2500 pM to both the spheroid pre-cursor solution and the cell culture medium. At day 3 and day 5 the spheroids were imaged and assessed.

[0447] Mouse organotypic slice preparation and culture

[0448] For organotypic cultures, sagittal hippocampal brainslices were taken from wild type mice. Following decapitation, all organs were quickly removed, placed in ice-cold buffer (artificial cerebrospinal fluid (ACSF) [in mmol / L]:NaC1 125, KCI 2.4, NaHCO326, NaH2PO4 1.25, glucose 25, CaCI22 [0.5 mmol / L for dissection], MgCI21 [5 mmol / L for dissection]; bubbled with carbogen) for brain, and for all other organs Ringers buffer. For brain, to reduce the rate of cell death most dissections were carried out with high-magnesium (5 mmol / L) / low-calcium (0.5 mmol / L) ACSF. The brain was hemisected and a section was cut away by hand that was approximately 100° from the midline surface. The hemispheres were then mounted with superglue on to a slicing stage and 300 pm hippocampal slices were cut with a vibroslicer and transferred to an incubating chamber with circulating ACSF (bubbled with carbogen). All other organs were treated similarly but Ringer’s buffer was used instead of ACSF. For the heart and spleen, cross sectional slices going fully across the organ were taken. The slices were incubated at 35°C for approximately 1 hrs before the incubator was switched off and the bath was allowed to cool to room temperature.

[0449] For stress, slices were transferred to beakers (containing ACSF or Ringers buffer, for brain and other organs, respectively) that had N2bubbled through for 3 hrs prior rather than carbogen. They were left in the beakers with no gas bubbling to mimic hypoxia. Treated beakers had drug solubilised at the given concentration in the N2bubbled buffer. Following this, the slices were stained with PI as well as DAPI to visualise all cell nuclei (including intact cell nuclei). Slices were then placed on a microscope slide under a coverslip and imaged.

[0450] Preparation of Zebra fish liver

[0451] Freshly culled zebrafish (Danio rerid) was dissected to quickly remove the liver which was then placed in PBS buffer and immediately stressed with 1 mM hydrogen peroxide for 7 min in with or without 2500 pM EGTA. The liver was then washed in PBS to stop the stress and stained with PI as well as DAPI to visualise all cell nuclei (including intact cell nuclei). The liver was then placed on a microscope slide under a coverslip and imaged.

[0452] C. elegans culture methods

[0453] C. elegans was maintained on nematode growth medium (NGM) agar plates seeded with Escherichia coll OP50, using standard culture methodologies (S. Brenner, 1974). A C. elegans (N2H) hermaphrodite stock was used as wild-type. Day 1 hermaphrodites were used in experiments and all maintenance and trials were conducted at 20°C unless otherwise stated.

[0454] C. elegans stress assays

[0455] • Cold / freeze stress

[0456] Per trial, 100 animals were placed in 1 .5 ml Eppendorf tubes (30 animals per tube) containing 100 pl M9 buffer, and then transferred to a -80°C incubator for 1 .5 min. Following the stress, animals were transferred to a 20°C incubator and maintained as usual on OP50 NGM plates.

[0457] • Heat stress

[0458] Per trial, 100 animals were placed in a 37°C incubator for 4.5 hrs on NGM plates. Following the stress, animals were transferred to a 20°C incubator and maintained as usual on OP50 NGM plates.

[0459] • Oxidative stress

[0460] Per trial, 100 animals were transferred into 1.5 ml Eppendorf tubes (20 animals per tube) containing 300 pL of 2M hydrogen peroxide for 5 min. Following the stress, animals were transferred to a 20°C incubator and maintained as usual on OP50 NGM plates.

[0461] • Toxin stress

[0462] Per trial, 100 animals were transferred into 1.5 ml Eppendorf tubes (20 animals per tube) containing 300 pL of 1 ,3%(v / v) tert-butylhydroperoxide (tBOOH) for 5 min. Following the stress, animals were transferred to a 20°C incubator and maintained as usual on OP50 NGM plates.

[0463] Movement analysis post stress in C. elegans

[0464] Movement was scored according to an adapted methodology (from Herndon et al., 2002). Briefly, animals were marked as either: normal movement; or defective movement. Defective movement was defined as worms that are stationary unless touched with a pick, in which case they may only move a few centimetres forward before stopping again or they may not move at all. 100 animals were used per trial and the percentage of worms with defective movement was scored.

[0465] Liposome mediated drug delivery in C. elegans

[0466] According to the protocol in Shibamura et al., 2009, drug was dissolved in MilliQ water and encapsulated in liposomes. Drug used was EGTA, at a final concentration of 2500 pM. Briefly, L-a-phosphatidylcholine (LaPC) was used to prepare liposomes by first dissolving LaPC in distilled water at the concentration of 96 mg / ml. A x2 concentration of drug solution was mixed with LaPC to a 1 :1 ratio and heated to 65°C. Drug-LaPC solution was then converted into liposomes using a mini-Avanti extruder a 100 nm pore size polycarbonate membrane. MilliQ water encapsulated in liposomes was used a as the empty vector control.

[0467] Following preparation, 25 pl of the liposome drug solution was spread onto a 25 pl spot OP50 lawn on NGM plates and worms were then picked onto the plates.

[0468] Drosophila culture and stress.

[0469] Animals were under standard culture conditions. On day 1 of adulthood, they were starved for 8 hrs, then fed 12 hrs + / - EGTA 2500 pM. Following this, the animals were moved to 0°C for 10 min and then recovered and survival measured.

[0470] E. coll oxidative stress.

[0471] 2 loops of E. coll stationary-phase bacterial cells collected post seeding (80 pL from a culture grown for 12 hrs) on agar plates containing bactopeptone and allowing to grow for 24 hrs was collected in buffer. Bacterial cells were then stressed for 50 min using 1 mM hydrogen peroxide. Treatment is EGTA 2500 pM during stress. Cells were then washed in buffer. Following stress, bacteria was washed in buffer and then diluted 1 :500 in MilliQ water and seeded onto agar plates containing bactopeptone and left at 37 °C overnight. This was followed by counting colony forming units (CFU) counted. Three repeats were performed.

[0472] S. pombi oxidative stress.

[0473] A culture of S. pombi was exposed to 1 mM hydrogen peroxide for 50 min. Following this the yeast cells were washed in buffer and stained with the dye Propidium iodide (PI) and placed on a microscope slide under a coverslip and imaged using Nomarski and epifluorescence microscopy to identify the percentage of yeast cells that underwent necrosis. Treatment is EGTA 2500 pM during stress. Two repeats were performed.

[0474] Doppl SA, (affiliated with EPFL, the Swiss Federal Institute of Technology in Lausanne); Domainex, Cambridge UK; and ICHOR life sciences NY, US performed data collection.

[0475] Traditionally necrosis is viewed as a chaotic process that cannot be intervened in. It has been assumed that necrosis is the consequence of numerous random events that cause loss of cell homeostasis.

[0476] The inventors believe that: following any stress, many random events follow that may culminate in loss of cell homeostasis and necrosis. However, there are certain initiated events that make a cell more vulnerable to stress (i.e., cellular weak spots that cause more damage than other processes). Suppression of these predicted weak spots should make a cell more resistant to stress, i.e., necrosis can be prevented, and / or treated; a much greater amount of stress would then be required for any other random events to induce cell death. Downstream conditions (such as tissue damage, organ damage, toxic effects, alcohol associated diseases, necrosis associated with cancer, tumours or therapy, radiation necrosis etc.), can therefore also be prevented and / or treated. The predicted weak spots, illustrated in Figure 1, are: an increase in intracellular cytoplasmic concentration of free calcium ions due to movement of calcium (1 a) across the cell plasma membrane, (1 b) and from internal compartments where it is stored, primarily the endoplasmic reticulum (ER)Zsarcoplasmic reticulum (SR). Following calcium entry, many proteins are activated in an unwanted manner. As part of this futile protein activation, (2) calpain proteins are activated. Calpains then destroy internal compartments including lysosomes, resulting in the activation and release of hazardous components of internal compartments like the lysosomes, including release of (3) cathepsin proteins. Finally, (4) movement of calcium ions across to neighbouring cells via gap junctions, results in the disruption of calcium homeostasis and triggering of necrosis in those neighbouring cells. Store-Operated Calcium Channels (SOCC), also called Calcium release-activated calcium (CRAC) channels, are another source of calcium entry into the cells. SOCC channels, however, only become relevant post ER / SR calcium depletion, and hence SOCC is less of a focus because SOCC is expected to be less of a weak spot than steps (1) - (4) discussed herein.

[0477] The inventors have discovered that a critical step in the occurrence of necrosis is the calcium ion increase in the cytosol, which may be due to any one of the 3 main points of calcium ion entry of into a cell: i.e., via cell surface membrane channels, gap junctions, and the endoplasmic reticulum (ER) / sarcoplasmic reticulum (SR). It is believed that, because plasma membranes are in contact with the environment surrounding the cell, they will show greater levels of re-enforcement compared to ER / SR calcium channels and gap junctions. Moreover, for cells in certain tissue types that are very tightly packed, calcium movement via ER / SR calcium channels and gap junctions will pose a greater threat during a stress.

[0478] Therefore, the inventors have made the surprising discovery that using a combination of compounds to block the ER / SR calcium channels and gap junctions is particularly advantageous for preventing and / or treating necrosis. Thus, this combination can be used to treat or prevent necrosis associated with any one of: alcohol consumption, cancer, tumours, therapy, medically invasive interventions, medical diagnostic procedures, administration of one or more toxic agents, administration of one or more pharmaceutical agents, space radiation and ageing. This combination can also be used to treat or prevent an alcohol associated disease.

[0479] This combination can be further improved by also blocking calcium ion entry via plasma membranes. Thus, the invention provides the combination therapy of an endoplasmic / sarcoplasmic reticulum calcium channel inhibitor (such as a ryanodine receptor antagonist), a gap junction inhibitor, and a plasma membrane calcium channel inhibitor. Additionally, or alternatively, the combination can be further improved by the use of other means to mimic reduction of cytosolic calcium ion concentration, such as through use of calcium chelating agents that sequester calcium (or use competitive inhibitors of calcium ions, such as other cations of a similar charge and size such as magnesium ions). In other words, the inventors made the surprising discovery that the most efficacious combination for the methods, uses and medical uses described herein is the simultaneous inhibition of ER / SR calcium channels and gap junctions, and this combination can be enhanced with the addition of plasma membrane calcium channel inhibitors and / or calcium chelating agents (or competitive inhibitors of calcium ions). Thus, the invention provides the combination therapy of an endoplasmic / sarcoplasmic reticulum calcium channel inhibitor (such as a ryanodine receptor inhibitor or antagonist) and a gap junction inhibitor. The invention also provides the combination therapy of an endoplasmic / sarcoplasmic reticulum calcium channel inhibitor (such as a ryanodine receptor inhibitor or antagonist), a gap junction inhibitor, and a plasma membrane calcium channel inhibitor. The invention also provides the combination therapy of an endoplasmic / sarcoplasmic reticulum calcium channel inhibitor (such as a ryanodine receptor inhibitor or antagonist), a gap junction inhibitor, and a calcium chelator. The invention also provides the combination therapy of an endoplasmic / sarcoplasmic reticulum calcium channel inhibitor (such as a ryanodine receptor inhibitor or antagonist), a gap junction inhibitor, a plasma membrane calcium channel inhibitor and a calcium chelator. Each of these combination therapies can be used in each of the methods, uses, and medical uses described herein.

[0480] The rationale of the combination therapy of the present invention can be further illustrated by considering what may happen when other combinations of calcium ion channel blockers are used, as follows. The inventors believe that, if only calcium plasma membrane channels are blocked, necrosis may still occurto a large degree because calcium ions may still enter via the ER / SR and via gap junctions. If only ER / SR channels are blocked, necrosis may still occur to a large degree because calcium ions may still enter primarily via gap junctions. If only gap junctions are blocked, necrosis may still occur to a large degree because calcium ions may still enter primarily via ER / SR channels. If a combination of ER / SR channels and plasma membrane calcium ion channels are blocked, necrosis may still occur to a large degree because calcium ions still enter primarily via gap junctions. If a combination of gap junctions and plasma membrane calcium ion channels are blocked, necrosis may still occur to a large degree as calcium ions still enter primarily via ER / SR channels.

[0481] It follows that, if a pharmaceutical agent and / or biologically active agent is used which blocks one of these pathways, then co-administering said pharmaceutical agent and / or biologically active agent with an inhibitor which blocks one of the other pathways, then necrosis can be prevented or treated. For example, if a pharmaceutical agent which blocks ER / SR channels is used as a monotherapy, necrosis may still occur to a large degree because calcium ions may still enter primarily via gap junctions. Thus, co-administering the pharmaceutical agent with one or more gap junction inhibitors would be advantageous to prevent or treat necrosis.

[0482] Blocking calpains and cathepsins (Figure 1) represents another therapeutic target for treating and / or preventing necrosis. Thus, a combination of a calpain inhibitor and a cathepsin inhibition is provided by the present invention. However, these are only two of the many downstream mediators of destruction following a rise in cytosolic calcium ion during cell necrosis. Inhibition of calpains and cathepsins may therefore not be the most efficacious form of intervention.

[0483] In some of the following Examples, human 3D cell culture was employed using liver Hepg2 cells. 3D cell culture is known to accurately replicate a cell’s native environment. During embryonic development, cells aggregate and self-assemble into complex multi-cellular structures. The same aggregation process can likewise occur in in vitro culture systems if cell-cell cohesion is favoured over cell attachment to a substrate. The resulting 3D cell culture aggregates are known as spheroids. Spheroids exhibit extensive cell-cell adhesion, typically retain their endogenous extracellular matrix, and have properties that closely mimic their in vivo tissue counterparts in both structure and function. A standard method of spheroid formation in U-bottom low adhesion plates was used in the Examples herein.

[0484] Notably, spheroids typically cannot be grown to large sizes. This is because, as the spheroids grow, only the peripheral cells obtain oxygen and nutrients from the cell culture medium. The inner cells die from cell death necrosis, with the formation of large necrotic cores (observable though use of standard live dead stains). Thus, necrotic core formation represents a naturally occurring hypoxic and nutrient stress. This resembles multiple forms of shock in humans where blood flow carrying nutrients and oxygen is disrupted to certain cells, tissues and / or organs. To show that necrosis can be prevented and / or treated by the compositions and compounds of the present invention, some of the Examples herein make use of these naturally forming necrotic cores, in order to show that treatment results in pharmacological inhibition of necrotic core formation.

[0485] For instance, Hepg2 cell spheroids made in U-bottom low adhesion plates with an initial seeding cell density of 500 cells proliferate and form spheroids and by the 7th day show prominent necrotic cores, as seen with the standard viability dye DRAQ7™ that is a marker of cell death as it only stains the nuclei of dead and permeabilised cells (Figure 25, see control).

[0486] An increase in the level of cytosolic calcium ions is a key weak spot that induces necrosis. The inventors have found that relatively high doses of chelating agents (i.e. agents that sequester cytosolic calcium) are effective agents at blocking cell death, such as necrosis, during stress. The chelating agents EGTA (ethylene glycol- bis(p-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. It is known in the art that cell culture dosages of around 50 pM, 50 pM and 1-3 pM, respectively are appropriate because they are considered maximum dosages which result in minimum toxicity. When a x8 dosage of BAPTA-AM (i.e., 25 pM) was used, significant necrosis suppression was seen on day 7 (Figure 2a). However, this dosa...

Claims

CLAIMS1 . A method of preventing or treating organ ageing, wherein the method comprises administering to a subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

2. The method of claim 1 , wherein the organ ageing is kidney ageing.

3. The method of claim 1 or claim 2, wherein the organ ageing is accelerated kidney ageing.

4. A method of preventing of treating acute kidney injury in a subject, wherein the method comprises administering to a subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

5. A method of protecting a subject from one or more side-effects of space radiation, the method comprising administering to a subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

6. The method of claim 5 wherein the space radiation is ionizing radiation.

7. The method of claim 5 or claim 6 wherein the side-effect of space radiation is radiation necrosis.

8. A method of protecting a subject from one or more side-effects associated with a gravitational environment which is different to Earth’s gravitational environment, the method comprising administering to a subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

9. A method of engineering a tissue or organ, or preserving a live tissue or organ, wherein at least one of the method steps occurs under a microgravitational environment, the method comprising contacting the organ or tissue with a culture media comprising one or more calcium activity inhibitors, one or more calpain inhibitors, one or more cathepsin inhibitors and / or one or more gap junction inhibitors; optionally wherein the culture media comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

10. A method of treating or preventing necrosis associated with therapy of a subject, wherein the method comprises administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject who is undergoing, or who has undergone, therapy.11 . The method of claim 10 wherein the therapy is a medically invasive intervention and / or medical diagnostic procedure, such as surgery, dialysis, invasive diagnostics, balloon angioplasty, atherectomy, stenting, vascular angioplasty, atherectomy, stents, IVC filter placement and / or removal, thrombectomy, ablation, phlebectomy procedures, pacemakers, echocardiography, defibrillator insertion, subcutaneous ICD and / or appendage closure, optionally surgery, optionally cardiac surgery.

12. The method of claim 10 or claim 11 , wherein the therapy is a therapy for one or more cancers and / or for one or more precancerous lesions.

13. The method of any one of claims 10-12, wherein the therapy is chosen from one of more of the following:(i) External beam radiation therapy (EBRT);(ii) Intensity-modulated radiation therapy (IMRT);(iii) Arc-based radiotherapy;(iv) Image-guided radiotherapy (IGRT);(v) Particle therapy;(vi) Stereotactic radiosurgery; and / or(vii) Internal radiation therapy.

14. A method of treating or preventing necrosis associated with administering of one or more toxic agents, wherein the method comprises co-administering to a subject the toxic agent in combination with a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

15. The method of claim 14 wherein the toxic agent is one or more chemotherapeutic agents.

16. The method of claim 15 wherein the chemotherapeutic agent is one or more of an alkylating agent, antimetabolite, antimicrotubule agent, anthracycline, platinum compound, topoisomerase inhibitor, antibiotic, enzyme, and antineoplastic drug.

17. The method of claim 14 wherein the toxic agent is one or more pharmaceutical agents.

18. The method of claim 14 wherein the toxic agent is one or more biologically active agents.

19. A method of treating or preventing necrosis, wherein the method comprises co-administering to a subject one or more pharmaceutical agents in combination with a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

20. The method of claim 17 or claim 19 wherein the pharmaceutical agent is selected from one or more of a chemotherapeutic agent, a TNF inhibitor, a mTOR inhibitor, acetaminophen, NSAIDs, antiarrhythmic medication, Anabolic Steroids, Birth Control Pills, antipsychotic medication, antibiotics, anaesthetics, Blood pressure medication, rheumatoid arthritis drugs, Statins, Anti-Seizure Medicines, Disulfiram, supplements, immunosuppressive drugs, antifungals, ACE Inhibitors, Aminoglycoside Antibiotics, HIV Medications, Metformin and Beta blockers.21 . A method of treating or preventing necrosis, wherein the method comprises co-administering to a subject one or more biologically active agents in combination with a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

22. The method of claim 18 or claim 21 wherein the biologically active agent is selected from one or more of Monoclonal Antibodies, Bispecific Monoclonal Antibodies, Trifunctional Antibodies, BiSpecific T-Cell Engagers, Viral Antibodies, Humanized Antibodies, Chimeric Antibodies, Fusion proteins, anti-RyR antibodies, anti-pannexin antibodies and anti-connexin antibodies.

23. The method of claim 14 wherein the toxic agent is one or more of a carbonic anhydrase inhibitor, a histamine H2 receptor antagonist, a proton pump inhibitor, a diuretic, a bicarbonate, a phosphodiesterase inhibitor, a SERCA inhibitor, a PMCA inhibitor, a TRPV channels modulator, a calcium-sensing receptor agonist, a vitamin D analogue, a bisphosphonate, a calcitonin gene-related peptide (CGRP) antagonist, a natriuretic peptide, a chloride channel blocker, a potassium channel activator, a sodium-potassium ATPase inhibitor, a sodium channel blocker, a potassium channel opener, an adrenergic receptor agonist / antagonist, a magnesium supplement and / or a sodium supplement.

24. A method of treating or preventing necrosis associated with cancer and / or tumours in a subject, wherein the method comprises administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors, optionally wherein the cancer is breast carcinoma, endometrial carcinoma and / or renal carcinoma.

25. A method of protecting a subject from one or more side-effects associated with ionizing radiation, the method comprising administering to a subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

26. The method of claim 25 wherein the side-effect is radiation necrosis.

27. A method of protecting a subject from one or more side-effects of an injury, such as an injury caused by a weapon, the method comprising administering to a subject a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

28. A method of treating or preventing necrosis associated with alcohol consumption in a subject, wherein the method comprises administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors to the subject.

29. A method of treating or preventing an alcohol associated disease in a subject, wherein the method comprises administering a composition comprising one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

30. The method of claim 29, wherein the alcohol associated disease is selected from osteonecrosis, liver disease, pancreatitis, fibrosis, cardiovascular disease, neuropsychiatric disorder, tumors, cancer, glomerulonephritis, kidney damage, and / or reproductive and developmental issues; optionally wherein the alcohol associated disease is selected from liver disease, pancreatitis, cardiovascular disease, and / or kidney damage.31 . The method of claim 14 wherein the toxic agent is one or more poisons.

32. The method of claim 31 wherein the poison is one or more of carbon monoxide, a heavy metal, tobacco smoke, asbestos, benzene, cadmium, formaldehyde, radon, vinyl chloride, cocaine, heroin, methamphetamine, a blood flow altering narcotic, strychnine, cyanide, ricin, botulin toxin, tetrodotoxin, snake venom, scorpion venom, bee venom, insect venom, and mushroom toxin.

33. The method of claim 14 wherein the toxic agent is one or more cell membrane channel inhibitors.

34. A method of producing artificial meat or cultivated meat, the method comprising contacting cells with a composition comprising one or more calcium activity inhibitors, one or more calpain inhibitors, one or more cathepsin inhibitors and / or one or more gap junction inhibitors to preserve or culture the cells, optionally wherein the composition comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and one or more gap junction inhibitors.

35. The method of any preceding claim, wherein the composition or culture media further comprises one or more plasma membrane calcium channel inhibitors.

36. The method of claim 35, wherein the plasma membrane calcium channel inhibitor is 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; optionally wherein the plasma membrane calcium channel inhibitor is selected from one or more of amlodipine, arandipine, clevidipine, efonidipine, felodipine, isradipine, lercanidipine, manidipine, nifedipine, nimodipine, nitrendipine, gallopamil, verapamil, diltiazem, mibefradil, fluspirilene, gabapentin or pregabalin.

37. The method of claim 35 or claim 36, wherein the plasma membrane calcium channel inhibitor is amlodipine.

38. The method of any preceding claim, wherein the composition or culture media further comprises one or more calcium chelators.

39. The method of claim 38, wherein the calcium chelator is selected from one or more of 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 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); sodium citrate; and DMSA (dimercapto succinic acid); optionally wherein the calcium chelator is one or more of DTPA, BAPTA AM, sodium citrate, and citric acid.

40. The method of any preceding claim, wherein the composition or culture media comprises one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and two or more gap junction inhibitors.

41. The method of any preceding claim, wherein the composition or culture media comprises two or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and two or more gap junction inhibitors.

42. The method of any preceding claim, wherein the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor is a ryanodine receptor antagonist; optionally wherein the ryanodine receptor antagonist is selected from one or more of dantrolene, DHBP dibromide, cis-Ned 19, trans-Ned 19, ryanodine, SKF 86365 hydrochloride, ruthenium red, procaine, and tetracaine;optionally wherein the ryanodine receptor antagonist is selected from one or more of dantrolene, DHBP dibromide, cis-Ned 19, trans-Ned 19, ryanodine, SKF 86365 hydrochloride, ruthenium red, and tetracaine; optionally wherein the ryanodine receptor antagonist is selected from dantrolene and / or ryanodine.

43. The method of any preceding claim wherein the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor is dantrolene.

44. The method of any preceding claim wherein the gap junction inhibitor is selected from (i) one or more of a fatty acid, a polyamine and a cyclodextrin, optionally wherein the fatty acid contains 13-21 carbons, or (ii) a connexin inhibitor and / or a pannexin inhibitor, preferably a connexin inhibitor.

45. The method of any preceding claim wherein the gap junction inhibitor is selected from one or more of 18a-glycyrrhetinic acid, 18p-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; optionally 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 quinine; optionally wherein the gap junction inhibitor is one or more of retinoic acid, oleic acid, and linoleic acid.

46. The method of any preceding claim, wherein the gap junction inhibitor is retinoic acid.

47. The method of any one of claims 1 to 45, wherein the gap junction inhibitor is oleic acid.

48. The method of any one of claims 1 to 45, wherein the gap junction inhibitor is linoelic acid.

49. The method of any one of claims 1 to 45, wherein the gap junction inhibitor is quinine.

50. The method of any one of claims 1 to 45 and 49, wherein the endoplasmic / sarcoplasmic reticulum calcium channel inhibitor is dantrolene and the gap junction inhibitor is quinine.51 . The method of any one of claims 1 to 45 wherein the gap junction inhibitor is selected from two or more of oleic acid, linoleic acid, palmitoleic acid, quinine, astaxanthin, and retinoic acid; optionally wherein the gap junction inhibitor is selected from (i) retinoic acid and linolic acid, (ii) retinoic acid and oleic acid, and (iii) linoleic acid and oleic acid.

52. The method of any preceding claim, wherein the one or more endoplasmic / sarcoplasmic reticulum calcium channel inhibitors and the one or more gap junction inhibitors are administered simultaneously, separately or sequentially.

53. A therapeutic agent that has been modified or produced by gene editing so as to provide dual inhibition of endoplasmic / sarcoplasmic reticulum calcium channels and gap junctions.

54. The therapeutic agent of claim 53, wherein the therapeutic agent has both GJAI and RyR1 simultaneously knocked down and / or knocked out.

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