TW202207907A - Suppression of cytokine release and cytokine storm - Google Patents

Abstract

The present invention includes methods and compositions for ameliorating symptoms or treating one or more adverse reactions triggered by infectious diseases or disease conditions that trigger a widespread release of cytokines in a subject comprising the steps of: identifying the subject in need of amelioration of symptoms or treatment of the infectious diseases or disease conditions that trigger a widespread release of cytokines; and administering one or more pharmaceutical compositions comprising a therapeutically effective amount of a curcumin extract, curcuminoids or synthetic curcumin (S-curcumin) and derivatives thereof, and lipids dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the level of cytokines in the host.

Description

Cytokine release and inhibition of cytokine storm

The present invention relates generally to the field of infectious diseases and disease conditions that trigger cytokine cascades, and more particularly, to the use of compositions that reduce cytokine cascades.

Without limiting the scope of the present invention, its prior art describes infectious diseases and disease conditions that trigger, for example, the allergic cytokine cascade.

US Patent No. 8,354,276 to Har-Noy, entitled "T-cell compositions that elicit type I cytokine response," describes a method of manipulating allogeneic cells for use in an allogeneic cell therapy regimen. This approach provides a composition of highly activated allogeneic T cells infused into immunocompetent cancer patients to elicit a novel anti-tumor immune mechanism known as the "Mirror Effect". The inventors believe that in contrast to current allogeneic cell therapy regimens in which T cells in the graft mediate beneficial graft-versus-tumor (GVT) and detrimental graft-versus-host (GVH) effects, the allogeneic cells of the present invention stimulate T cells to mediate the "mirror" of these effects. The highly activated allogeneic cells of the present invention are said to stimulate host immunity in the context of complete HLA mismatch in patients who have not had prior bone marrow transplantation or received chemotherapy and/or radiation conditioning regimens.

US Patent No. 8,309,519, issued to Li et al., is entitled "Compositions and methods for inhibiting vascular permeability" and relates to compounds, compositions and methods for inhibiting vascular permeability and pathological angiogenesis. These inventors teach methods for generating and screening compounds and compositions capable of inhibiting vascular permeability and pathological angiogenesis. The described compositions are said to be useful in methods of inhibiting vascular permeability and pathological angiogenesis, including inhibition of vascular permeability and pathology induced by specific angiogenic, permeable, and inflammatory factors such as, for example, VEGF, βFGF, and thrombin Methods of sexual angiogenesis.

US Patent No. 7,479,498 to Keller, entitled "Treatments for viral infections," relates to improved methods and compositions for treating viral infections and other diseases and disorders that induce cytokine storms. Further, the present invention relates to novel compositions comprising quercetin and a combination of anticonvulsants such as phenytoin and multivitamins as antiviral compositions and methods of their use.

U.S. Patent Application No. 20100075329, filed by O'Toole et al., is entitled "Methods For Predicting Production Of Activating Signals By Cross-Linked Binding Proteins" and relates to specific binding to the human interleukin-21 receptor (IL21R) Human binding proteins and antigen-binding fragments thereof, and uses thereof. The present invention is said to include methods for predicting whether the binding proteins of the present invention can exhibit agonistic activity and produce a cytokine storm in vivo. Furthermore, the present invention is said to provide methods for determining whether an anti-IL21R binding protein is a neutralizing anti-IL21R binding protein based on the identification of several IL21 responsive genes. Finally, binding proteins are said to act as antagonists of IL21R activity, thereby modulating immune responses in general, and in particular those mediated by IL21R.

In one embodiment, the present invention includes a method of ameliorating symptoms in a subject or treating one or more adverse reactions in a subject triggered by widespread release of a cytokine, the method comprising the steps of: identifying a condition in need of amelioration of symptoms or treatment of a triggering cytokine Widely released to an individual of one or more infectious diseases or disease conditions; and administration of one or more pharmaceutical compositions comprising a therapeutically effective amount of lipids dissolved or dispersed in a suitable aqueous or non-aqueous amount sufficient to reduce the individual's cytokine concentration in the medium. In one aspect, the broad release of cytokines is by at least one or more selected from viral, bacterial, fungal, helminthic, protozoal, or hemorrhagic infectious agents caused by infectious diseases. In another aspect, the one or more infectious diseases are selected from at least one of the following infections: rhinovirus, coronavirus, Paramyxoviridae, Orthomyxoviridae, adenovirus, Parainfluenza virus, Interstitial pneumonia virus, Respiratory syncytial virus, Influenza virus, Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, Baculoviridae Rhabdoviridae virus, Ebola virus (Ebola), Marburg virus (Marburg), Crimean–Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, Dengue fever, Yellow fever, Rift Valley fever, Omsk hemorrhagic fever virus, Kyasanur Forest, Junin, Machupo, Sabi Subvirus (Sabiá), Guanarito virus (Guanarito), Garissa virus (Garissa), Ilesha virus (Ilesha) or Lassa fever virus. In another aspect, the one or more disease conditions are selected from at least one of the following: cachexia, septic shock syndrome, chronic inflammation, septic shock syndrome, traumatic brain injury, cerebral cytokine storm, transplantation Germ-versus-host disease (GVHD), autoimmune disease, multiple sclerosis, acute pancreatitis or hepatitis. In another aspect, the one or more disease conditions are due to therapy with anti-CD19 chimeric antigen receptor (CAR) T cells or anti-tumor cells, activated dendritic cells, activated macrophages, or activated B cells Adverse reactions caused by treatment. In another aspect, the composition further comprises curcumin extract, curcumin, and curcuminoids configured in lipids, wherein the curcuminoids are selected from at least one of the following: aryl curcumin (Ar- tumerone), methyl curcumin, demethoxycurcumin, bisdemethoxycurcumin, sodium curcumin, dibenzoylmethane, acetyl curcumin, feruloyl methane (feruloyl methane), tetrahydroturmeric 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin 1), 1,7-bis(piperonyl ( piperonyl))-1,6-heptadiene-3,5-dione (piperonyl curcumin), 1,7-bis(2-hydroxynaphthyl)-1,6-heptadiene-2,5- Diketone (2-hydroxynaphthylcurcumin) and 1,1-bis(phenyl)-1,3,8,10-undecatetraene-5,7-dione. In another aspect, the lipid or phospholipid is selected from the group consisting of dimyristyl phospholipid choline (DMPC), dimyrist phospholipid glycerol (DMPG), dipalmitoyl phospholipid choline ( DPPC), Distearyl Phosphatidyl Glycerol (DSPG), Dipalmitoyl Phosphatidyl Glycerol (DMPG), Phosphatidyl Choline, Lysolecithin, Lysophosphatidylethanolamine, lysoDMPC, lysoDMPG, lysoDSPG, lysoDPPC, Phosphatidylserine acid, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, phosphatidylcholine, and dipalmitoyl glycerol, stearylamine, dodecyl Alkylamine, Cetylamine, Acetyl Palmitate, Glycerin Ricinoleate, Cetyl Stearate, Isopropyl Myristate, Amphoteric Acrylic Polymer, Fatty Acid, Fatty Acid Amide, Cholesterol, cholesterol esters, diacylglycerol and diacylglycerol succinate. In another aspect, the therapeutically effective amount includes 50 nM/kg, 10 to 100 nM/kg, 25 to 75 nM/kg, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nM/kg individual body weight. In another aspect, the composition comprises an active agent and has lipid phospholipid to active agent ratios of 3:1, 1:1, 0.3:1, and 0.1:1. In another aspect, the disease is rheumatoid arthritis, psoriasis, multiple sclerosis, relapsing multiple sclerosis, or inflammatory bowel disease.

In another embodiment, the present invention includes a composition for ameliorating symptoms in a subject or treating one or more adverse reactions in a subject triggered by an infectious disease or disease condition of widespread release of cytokines comprising a therapeutically effective The amount of lipid or lysophospholipid is dissolved or dispersed in a suitable aqueous or non-aqueous medium. In one aspect, the one or more infectious diseases are selected from at least one of viral, bacterial, fungal, helminthic, protozoal, or hemorrhagic infectious agents. In another aspect, the one or more infectious diseases are selected from at least one of the following infections: rhinovirus, coronavirus, Paramyxoviridae, Orthomyxoviridae, adenovirus, parainfluenza virus, Interstitial pneumonia virus, respiratory syncytial virus, influenza virus, arenaviridae, filoviridae, Benjaviridae, flaviviridae, baculoviridae, Ebola virus, Marburg virus, Kerry Mia-Congo haemorrhagic fever (CCHF), South American haemorrhagic fever, Dengue fever, Yellow fever, Rift Valley fever, Omsk haemorrhagic fever virus, Kejeldahl disease, Junin virus, Maqiubo virus, Sabia virus, Guana Reto virus, Garissa virus, Ilexia virus or Lyssa virus. In another aspect, the one or more disease conditions are selected from at least one of the following: cachexia, septic shock syndrome, chronic inflammation, septic shock syndrome, traumatic brain injury, cerebral cytokine storm, transplantation Germ-versus-host disease (GVHD), autoimmune disease, multiple sclerosis, acute pancreatitis or hepatitis. In another aspect, the curcumin extract, curcuminoids or synthetic curcuminoids are formulated in lipids. In another aspect, the lipid or lysophospholipid is selected from the group consisting of dimyristophospholipid choline (DMPC), dimyristophospholipid glycerol (DMPG), dipalmitophospholipid choline Alkali (DPPC), Distearyl Phosphatidyl Glycerol (DSPG), Dipalmitoyl Phosphatidyl Glycerol (DMPG), Phosphatidyl Choline, Lysophosphatidylcholine, Lysophosphatidylethanolamine, lysoDMPC, lysoDMPG, lysoDSPG, lysoDPPC, Phosphatidylserine Serine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine, cardiolipin, phosphatidic acid, cerebroside, bicetyl phosphate, phosphatidylcholine, and dipalmitophosphatidylglycerol, stearylamine, Laurylamine, Cetylamine, Acetyl Palmitate, Glycerin Ricinoleate, Cetyl Stearate, Isopropyl Myristate, Amphoteric Acrylic Polymer, Fatty Acid, Fatty Acid Acrylate Amines, cholesterol, cholesterol esters, diacylglycerol and diacylglycerol succinate. In another aspect, the biodegradable polymer is selected from the group consisting of polyester, polylactic acid, polyglycinide, polycaprolactone, polyanhydride, polyamide, polyurethane Ester, polyesteramide, polydioxanone, polyacetal, polyketal, polycarbonate, polyorthocarbonate, polyorthoester, polyphosphate, polyphosphazene, polyhydroxybutyric acid Esters, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acids), poly(amino acids), copolymers, terpolymers, and combinations or mixtures thereof. In another aspect, the composition is suitable for intravenous, subcutaneous, intramuscular or intraperitoneal injection into a subject. In another aspect, the composition further comprises a curcumin or curcuminoid selected from at least one of: aryl curcumin, methyl curcumin, demethoxycurcumin, bisdemethoxycurcumin base curcumin, curcumin sodium, dibenzoylmethane, acetyl curcumin, ferulic acid methane, tetrahydrocurcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-Heptadiene-3,5-dione (Curcumin 1), 1,7-Bis(piperonyl)-1,6-heptadiene-3,5-dione (Piperonyl Curcumin), 1 ,7-bis(2-hydroxynaphthyl)-1,6-heptadiene-2,5-dione (2-hydroxynaphthylcurcumin) and 1,1-bis(phenyl)-1,3, 8,10-undecatetraene-5,7-dione. In another aspect, the composition comprises an active agent and has lipid to active agent ratios of 3:1, 1:1, 0.3:1, and 0.1:1.

In another embodiment, the invention includes a method of determining whether a drug candidate results in amelioration of symptoms in a subject or treats one or more adverse reactions in a subject triggered by an infectious disease or disease condition that triggers widespread release of cytokines, The method comprises: (a) administering an amount of a drug candidate in combination with empty liposomes, and administering a placebo to a second subgroup of patients, wherein the drug candidate is effective to reduce or prevent cytokine levels in the individual (b) measuring individual cytokine concentrations from the first and second cohorts of patients; and (c) determining the combination of the drug candidate and empty liposomes compared to that occurring in patients taking placebo. Whether any reduction in a subgroup of patients statistically significantly improves symptoms or treats one or more adverse reactions triggered by an infectious disease or disease condition that triggers widespread release of cytokines, where a statistically significant reduction indicates that the drug candidate is available In treating a disease state while also reducing or eliminating the overall concentration of cytokines in an individual.

In another embodiment, the invention includes a method of ameliorating symptoms or treating a cytokine storm caused by a therapeutic agent in an individual, the method comprising the steps of: identifying a need to improve symptoms or treat a cytokine storm caused by a therapeutic agent and administration of one or more pharmaceutical compositions comprising a therapeutically effective amount of curcumin extract, curcuminoid or synthetic curcumin and derivatives thereof, or empty liposomes, dissolved or dispersed in sufficient amount to reduce the in aqueous or non-aqueous media suitable for the concentration of cytokines.

Cross-references to related applications

This application is a continuation-in-part of US Patent Application Serial No. 14/983,844 filed on December 30, 2015, which claims US Provisional Application Serial No. 62/098,898, filed on December 31, 2014, and May 22, 2015 Priority to US Provisional Application Serial No. 62/165,567, filed on August 28, 2015, and US Provisional Application Serial No. 62/211,450, filed on August 28, 2015, the entire contents of which are incorporated herein by reference. Federally Funded Research Statement

This invention was made by USAMRIID with US Government support under Project No. 1323839. The government has certain rights in this invention.

Although the making and using of various embodiments of the invention are discussed in detail below, it should be appreciated that the invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention, and do not limit the scope of the invention.

To facilitate understanding of the present invention, a number of terms are defined below. Terms defined herein have the meanings as commonly understood by those of ordinary skill in the art to which the present invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only singular entities, but rather include the general class in which a particular instance may be used for description. The terminology herein is used to describe specific embodiments of the invention, and its use is not intended to limit the invention, except as outlined in the scope of the claims.

As used herein, the term "cytokine storm" refers to the dysregulation of pro-inflammatory cytokines that lead to disease, and has been referred to as "cytokine storm," "cytokine release syndrome," or "inflammatory cascade." Often, a cytokine storm or cascade is referred to as part of a sequence, as one cytokine often leads to the production of multiple other cytokines, which enhance and amplify the immune response. In general, these pro-inflammatory mediators are divided into two subgroups: early mediators and late mediators. Early mediators (such as eg tumor necrosis factor, interleukin-1, interleukin-6) are not sufficient therapeutic targets to restore homeostatic balance because they resolve within the time frame of the patient's visit to the clinic for medical attention. By contrast, so-called "late mediators" have been targeted because it is during this late "inflammatory cascade" that patients become aware of their disease.

Infectious diseases commonly associated with "cytokine storms" include, but are not limited to, malaria, avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS). Certain specific infectious agents include, but are not limited to, infectious diseases selected from at least one of the following: Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, Dengue fever, Yellow fever, Rift Valley fever, Omsk haemorrhagic fever virus, Kejeldahl disease, Junin virus, Maqiubo virus, Sabia virus, Guanarito virus, Garissa virus, Ilexia virus or Raisa fever Virus. Other viruses may include rhinovirus, coronavirus, paramyxoviridae, orthomyxoviridae, adenovirus, parainfluenza virus, interstitial pneumonia virus, respiratory syncytial virus, or influenza virus.

Disease conditions commonly associated with "cytokinin storm" include, but are not limited to: sepsis, systemic inflammatory response syndrome (SIRS), cachexia, septic shock syndrome, traumatic brain injury (eg, brain cytokine storm), graft-versus-host disease (GVHD), or treatment with activated immune cells (eg, IL-2 activated T cells, T cells activated with anti-CD19 chimeric antigen receptor (CAR) T cells).

In general, a cytokine storm is a healthy systemic manifestation of a strong immune system. The present invention can be used to reduce or eliminate some or most of the excessive immune response caused by, for example, rapidly proliferating and highly activated T cells or natural killer (NK) cells leading to the release of a "cytokine storm" that can Includes more than 150 inflammatory mediators (cytokines, oxygen free radicals and coagulation factors). Pro-inflammatory cytokines (such as tumor necrosis factor-alpha, interleukin-1, and interleukin-6) and anti-inflammatory cytokines (such as interleukin-10, and interleukin-1 receptor antagonists (IL- 1RA)) both became substantially elevated eg in serum. It is this excessive release of inflammatory mediators that triggers the "cytokine storm."

In the absence of timely intervention, such as that provided by the present invention, cytokine storms can lead to permanent lung damage and, in many cases, death. End-stage symptoms of cytokine storm include, but are not limited to: hypotension; tachycardia; dyspnea; fever; ischemia or tissue hypoperfusion; uncontrolled bleeding; severe metabolic disorders; and multisystem organ failure. Deaths from infectious diseases such as Ebola virus infection are not caused by the virus itself, but by cytokine storms leading to uncontrolled bleeding; severe metabolic disorders; hypotension; tachycardia; dyspnea; fever; ischemia or tissue hypoperfusion; and multisystem organ failure.

As used herein, the term "curcumin (diferuloid methane; 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)" is a naturally occurring compound whose principal coloration principle is described in the rhizomes of the plant Curcuma longa (US Pat. No. 5,679,864 (Krackov et al.)). In one aspect, the synthetic curcumin is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96% pure diferulomethane. Non-limiting examples of curcuminoids and curcuminoids include, for example, aryl curcumin, methyl curcumin, demethoxy curcumin, bisdemethoxy curcumin, sodium curcumin, dibenzylmethane, acetyl curcumin ferulic acid, ferulic acid methane, tetrahydrocurcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin 1) , 1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin), 1,7-bis(2-hydroxynaphthyl)-1,6-heptane Diene-2,5-dione (2-hydroxynaphthylcurcumin) and 1,1-bis(phenyl)-1,3,8,10-undecatetraene-5,7-dione.

The term "liposome" refers to a capsule in which the walls or membranes are formed of lipids, especially phospholipids, with the addition of sterols, especially cholesterol, if desired. In one specific non-limiting example, these liposome systems are empty liposomes and can be formulated from a single type of phospholipid or a combination of phospholipids. Empty liposomes or lipids may further comprise one or more surface modifications, such as proteins, carbohydrates, glycolipids or glycoproteins, and even nucleic acids, such as aptamers, sulfur-modified nucleic acids, protein nucleic acid mimetics, protein mimetics, stealth agents (stealthing agent), etc. In a specific non-limiting example, the composition also comprises the active agent in or around the liposomes or lipids and the composition has 3:1, 1:1, 0.3:1 and 0.1:1 lipid to active agent Compare.

As used herein, the term "lipid" refers to an amphiphilic biomolecule that is soluble in a non-polar solvent. Lipids are capable of forming liposomes, forming vesicles, forming micelles, forming emulsions, and are substantially nontoxic when administered at the necessary concentrations of liposomes. The lipid composition of the present invention may include, for example, dimyristophospholipid choline (DMPC), dimyristophospholipid choline (DMPG), dipalmitophospholipid choline (DPPC), distearyl phospholipid choline (DPPC), distearyl phospholipid choline (DSPG), Dipalmitophosphatidylglycerol (DMPG), Phosphatidylcholine, Lysolecithin, Lysophosphatidylethanolamine, Phosphatidylserine, Phosphatidylinositol, Sphingomyelin, Phosphatidylethanolamine, Cardiolipin, Phosphatidylserine Acid, Cerebroside, Dicetyl Phosphate, Phosphatidyl Choline, and Dipalmitoyl Phosphatidyl Glycerol, Stearylamine, Laurylamine, Cetylamine, Acetyl Palmitate, Glycerin Castor Sesame oleate, cetyl stearate, isopropyl myristate, amphoteric acrylic polymers, fatty acids, fatty acid amides, cholesterol, cholesterol esters, diacylglycerol and diacylglycerol succinate.

As used herein, the term "in vivo" refers to within the body. The term "in vitro" as used in this application should be understood to refer to operations performed in non-living systems.

As used herein, the term "treatment" refers to the treatment of a disorder referred to herein, particularly in a patient exhibiting symptoms of a disease or disorder. As used herein, the term "treating" refers to any administration of a compound of the invention and includes (i) inhibiting disease in an animal that is experiencing or exhibiting a diseased pathology or symptomatology (ie, preventing the pathology and/or or further development of symptomatology) or (ii) amelioration of disease (ie reversal of pathology and/or symptomology) in animals that are experiencing or exhibiting diseased pathology or symptomology. The term "control" includes prophylactic treatment, eradication, amelioration, or otherwise reducing the severity of a controlled condition.

The term "effective amount" or "therapeutically effective amount" as described herein means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human being sought by a researcher, veterinarian, physician or other clinician. In one example, the therapeutically effective amount includes 50 nM/kg, 10 to 100 nM/kg, 25 to 75 nM/kg, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nM/kg kg body weight.

As used herein, the term "administration of" or "administering a" a compound is to be understood to mean providing a compound of the invention to an individual in need of treatment, introduced in a therapeutically useful form and in a therapeutically useful amount Forms within the body of the individual, including but not limited to: oral dosage forms such as lozenges, capsules, syrups, suspensions and the like; injectable dosage forms such as intravenous (IV), intramuscular (IM) or intraperitoneal (IP) and the like; enteral or parenteral, transdermal dosage forms, including creams, jellies, powders or patches; buccal dosage forms; inhalation powders, sprays, suspensions and the like; and rectal suppositories.

As used herein, the term "intravenous administration" includes injection and other modes of intravenous administration.

The term "pharmaceutically acceptable" as used herein to describe a carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not injurious to its recipient.

The curcumin formulations of the present invention may contain one or more optional pharmaceutical excipients, diluents, prolonged or controlled release agents, lubricants, preservatives, or any combination thereof, and once dissolved, can be added to the injectable antidiabetic drug Or administered on a schedule, depending on the release kinetics of the curcumin formulation. A wide variety of biodegradable polymers can be used in the formulations of the present invention. Non-limiting examples of such polymers include polyester, polylactic acid, polyglycinide, polycaprolactone, polyanhydride, polyamide, polyurethane, polyesteramide, polydioxa Cyclohexanone, polyacetal, polyketal, polycarbonate, polyorthocarbonate, polyorthoester, polyphosphate, polyphosphazene, polyhydroxybutyrate, polyhydroxyvalerate, polyalkylene Oxalates, polyalkylene succinates, poly(malic acids), poly(amino acids), copolymers, terpolymers, and combinations or mixtures thereof. Particular polymers that can be used include acrylic acid, vinylpyrrolidone, N-isopropylacrylamide, or combinations and modifications thereof. The synthetic curcuminoids used include curcumin, curcumin analogs, curcumin derivatives, and any modifications thereof.

Treatment of infectious diseases. The end stage of Ebola and other viral diseases is often the beginning of a cytokine storm, the massive overproduction of cytokines by the body's immune system. The present invention includes curcumin action to inhibit cytokine release and cytokine storm to treat infectious agents that trigger cytokine storm, such as Ebola virus.

Curcumin has been found to block the release of cytokines, most importantly the key pro-inflammatory cytokines, interleukin-1, interleukin-6 and tumor necrosis factor-alpha. Inhibition of cytokine release by curcumin is associated with clinical improvement in experimental models of disease conditions in which cytokine storm plays an important role in mortality. Therefore, curcumin can be used to treat cytokine storm in patients with Ebola virus. In certain instances, intravenous formulations allow therapeutic blood concentrations to be achieved.

The high fatality rate in patients infected with Ebola virus is thought to be due in part to the occurrence of cytokine storms in the late stages of infection ^1-2 . Cytokine storms can occur following a variety of infectious and non-infectious stimuli. In a cytokine storm, large amounts of cytokines (both pro-inflammatory (IL-1, IL-6, TNF-α) and anti-inflammatory (IL-10)) are released, resulting in hypotension, bleeding, and ultimately multiorgan exhaustion. The term "cytokinin storm" is most associated with the 1918 H1N1 influenza pandemic and the recent ^3-5 cases of avian influenza H5N1 infection. In these cases, young people with presumably healthy immune systems die disproportionately from the disease, and the cause is believed to be abnormal activity of their immune systems. This syndrome is also known to occur in SARS ⁶ , Epstein-Barr virus-associated phagocytic lymphohistiocytosis ⁷ , gram negative sepsis ⁸ , malaria ⁹ and many others. In advanced or terminal cases of infectious disease. Cytokine storms can be caused by non-infectious causes such as acute pancreatitis ¹⁰ , severe burns or trauma ¹¹ or acute respiratory distress syndrome ¹² secondary to drug use or inhaled toxins. In a recent phase 1 trial, injection of the monoclonal antibody TGN1412, which binds to the CD28 receptor on T cells, resulted in severe cases of cytokine storm and multiple organ failure in six human volunteers who received the agent. This was despite the fact that a given dose of this agent was 500 times lower than that found in animal ¹³ . Other viruses may include rhinovirus, coronavirus, paramyxoviridae, orthomyxoviridae, adenovirus, parainfluenza virus, interstitial pneumonia virus, respiratory syncytial virus, or influenza virus.

Cytokinin inhibition by curcumin.

Curcumin has been shown to inhibit the release of various cytokines. Abe et al. showed that curcumin inhibited IL-1β, IL-8, TNF-α, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP) in monocytes and macrophages -1α) releases ¹⁴ . Jain et al. showed that curcumin significantly reduced IL-6, IL-8, TNF-α and MCP-1 release from monocytes cultured in a high glucose environment ¹⁵ . These same investigators studied streptozotocin-induced increases in plasma blood glucose concentrations and in rats with markedly elevated concentrations of IL-6, TNF-α, and MCP-1; curcumin significantly decreased these concentrations ¹⁵ . Curcumin has been reported to block IL-6 release from rheumatoid synovial fibroblasts, ¹⁶ IL-8 release from human esophageal epithelial cells, ¹⁷ and alveolar epithelial cells, ¹⁸ and bone marrow stromal cells, ¹⁹ colonic epithelial ^cells20 and human joints. IL-1 release from chondrocytes ²¹ . Curcumin also prevented the release of IL- ^{222, IL-1222-23, interferon-gamma 22-23 , and} many other key cytokines ^24-26 (Tables 1 and 2).

Example 1: Curcumin Cytokinin inhibition is associated with clinical improvement in cytokine storm-related disorders.

Curcumin has positive effects on a variety of disease states in patients and animal systems. Avasarala et al. report the effect of curcumin on cytokine expression and disease progression in a mouse model of virus-induced acute respiratory distress syndrome. Curcumin decreased the expression of key cytokines IL-6, IL-10, interferon gamma and MCP-1 and this was associated with a significant reduction in inflammation and reduced fibrosis ²⁷ . Yu et al. showed that inhibition of TNF-α concentrations by curcumin correlated with a reduction in pancreatic damage in a mouse model of acute pancreatitis ²⁸ . Cheppudira et al. reported that curcumin inhibition of IL-8 and GRO-α and ultimately NF-κB was associated with a reduction in thermal injury in a rat model ²⁹ . Cytokinin inhibition by curcumin was also associated with clinical improvement in a model of severe viral infection. Song et al. show that curcumin administration reduces the expression of IL-1β, IL-6 and TNF-α and ultimately NF-κB, and protects infected mice from coxsackievirus-induced severe myocardial injury ³⁰ . Curcumin has been shown to have activity against a variety of viruses, including coronavirus, HIV-1, HIV-2, HSV, HPV, HTLV-1, HBV, HCV and Japanese encephalitis ^virus31 . The virus may include a rhinovirus, coronavirus, paramyxoviridae, orthomyxoviridae, adenovirus, parainfluenza virus, interstitial pneumonia virus, respiratory syncytial virus, or influenza virus. In addition, curcumin has been shown to have specific activity against H1N1 virus in culture ^32-33 , although cytokine concentrations were not measured in these two studies. Most importantly, curcumin has been shown to stimulate SOCS protein ³⁴ . These proteins have been shown to be critical in protecting influenza virus-infected mice from severe cytokine storms ³⁵ .

The activity of curcumin in inhibiting various cytokines and its activity in experimental models of diseases and disorders associated with cytokine storm suggests that it may be useful in the treatment of patients with Ebola virus and cytokine storm. Curcumin is poorly absorbed from the gut; however, intravenous formulations can achieve therapeutic curcumin blood concentrations in patients diagnosed with cytokine storm. When treating patients with curcumin, the clinical status and concentrations of important cytokines such as IL-1β, IL-6 and TNF-α should be carefully monitored.

Table 1: The effect of curcumin on interleukin Biomolecules key function ↓ IL-1 Major pro-inflammatory cytokines, hematopoesis, CNS development ↓ IL-2 T cell lymphocyte differentiation ↓ ↑ IL-4 B cell proliferation ↓ IL-5 Immunoglobulin secretion, eosinophil function, allergy ↓ IL-6 Major pro-inflammatory cytokines, B cell differentiation, neural cell differentiation ↓ IL-8 Neutrophil chemotaxis, angiogenesis ↑ IL-10 Anti-inflammatory cytokine, also has T cell stimulating effect ↓ IL-11 Induction of acute phase proteins, antigen-antibody response, bone remodeling ↓ IL-12 Defense against intracellular pathogens ↓ IL-13 Induction of matrix metalloproteinases, induction of IgE ↓ IL-17 pro-inflammatory cytokines

Table 2: Inhibition of other key cytokines by curcumin Biomolecules key function ↓ TNF-α Major pro-inflammatory cytokine, insulin resistance, induces secretion of corticotropin-releasing hormone ↓ Interferon gamma Macrophage activation, T and B cell activation and differentiation ↓ MCP-1 (CCL2) Neuroinflammation, monocyte and basophil chemotaxis ↓ MIP-1α (CCL3) Activates granulocytes, induces the synthesis of pro-inflammatory cytokines ↓ GROα (CXCL1) Neutrophil chemokines, angiogenesis, wound healing ↓ GROβ (CXCL2) Neutrophil and Monocyte Chemokines ↓ IP-10 (CXCL10) Monocyte and macrophage chemokines, NK cell chemokines, angiogenesis ↓ SDF-1 (CXCL12) Lymphocyte chemokine, angiogenesis, inhibition of osteoclastogenesis

Test Results. The first study.

Liposomes and liposome-curcumin were prepared as 6 mg/ml solutions. Curcumin (solid) was dissolved in DMSO at 6 mg/ml. All three compounds were tested in the EBOV infection assay with two cell lines, Hela and HFF-1. The study was conducted using two groups with different dilution strategies.

The Hela cell line was used in two independent experiments (replicate 1 and replicate 2) and the HFF-1 line was used in one independent experiment. Liposomes and curcumin-liposomes were diluted in medium 2 hours prior to infection from the highest concentration (final concentration in the assay) of 600 ug/ml in 2-fold step dilutions to generate 10 points for a dose-response curve. 5 ul of each dose was dispensed into assay wells with cells by a PE Janus 384-tip dispenser. Curcumin (solid) was dispensed directly from a 100% DMSO stock solution into assay wells with cells by HP D300. DMSO was normalized to a final 1% in all wells. Each dose was tested 4 times on the plate, n=4.

Test Results. Second study.

Two cell lines were used in one study (replicate 1). Curcumin-liposomes and liposomes were diluted in medium 2 hours before infection from the highest concentration (final concentration in the assay) of 60 ug/ml in 2-fold step dilutions to generate 10 points for the dose-response curve. In this case, the titration is done by manual mixing and changing the tip for each new dose. Curcumin (solid) was manually titrated in DMSO and then equal amounts of each dose were mixed 1/10 diluted in medium. 5 ul of each dose was dispensed into assay wells with cells by a PE Janus 384-tip dispenser. Each dose was tested 4 times on the plate, n=4. For both studies: cells were infected with EBOV (Zaire) at MOI=0.5 for Hela cells and at MOI=3 for HFF-1 and the infection was stopped within 48 hours by fixing cells in formalin solution. To detect infected cells, immunostaining was performed using anti-GP antibody. Images were taken on a PE Opera confocal platform with a 10x objective and analyzed using Acapella software.

GP staining signal was converted to % infection. The % viability of cells was determined using the number of nuclei per well (n=16 compared to infected but untreated control wells). Data were analyzed using GeneData software and % infection was converted to % inhibition (%INH) using plate controls.

Figures 1A and 1B show the percent inhibition and percent survival, respectively, achieved with liposomal curcumin in HeLa cells. Figures 2A and 2B show the percent inhibition and percent survival, respectively, achieved using solid S-curcumin in HeLa cells. Similar results were obtained from the same study using HFF-1 cells.

Figures 3A and 3B show the comparative liposomal curcumin and solid curcumin in the percentage of inhibition and the percentage of survival, respectively, in HeLa cells. Similar results were obtained from the same study using HFF-1 cells.

Anti-EBOV activity was determined in Hela cells and correlated with "cytotoxicity". Briefly, the results are as follows: (1) Curcumin-Liposome EC ₅₀ = 2.5 ± 0.2 ug/ml, Safety Index = 2. Liposome EC ₅₀ = 3.9 ± 0.2 ug/ml, safety index = 4. Curcumin EC ₅₀ = 6.5 ± 0.5 ug/ml, safety index = 1. Thus, liposomes showed _EC50 = 0.6 ± 2 ug/ml, with an excellent safety index of about 50.

Pro-inflammatory cytokines leading to prolongation of the QT interval: role of ceramide and sphingosine-1 phosphate pathways. There is increasing evidence that excessive concentrations of pro-inflammatory cytokines play a major role in the pathogenesis of prolonged QT syndrome. In anticancer trials, QT prolongation was seen as a side effect of cytokines, interferon gamma, and QT prolongation was seen after treatment with interleukin-18. Patients with inflammatory diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease have a high incidence of QT prolongation, and deaths secondary to this complication are more frequent. The degree of QT prolongation has been shown to be directly related to the degree of elevation of the key pro-inflammatory cytokines TNF-α, IL-1β and IL-6. In large studies in normal populations, asymptomatic elevations of these cytokines have been found to be associated with QT prolongation. In a trial of tocilizumab, an IL-6 blocker, in patients with rheumatoid arthritis, a reduction in the duration of the previously prolonged QT interval was noted, and the magnitude of the QT shortening was comparable to serum associated with a reduction in inflammatory markers. Studies in animal models and cultured cardiomyocytes have shown that TNF-α inhibits IKr, IK and Ito. This is thought to be due to the stimulation of reactive oxygen species (ROS). TNF-α and other cytokines have been shown to lead to increased production of ROS. The effects of TNF-alpha can be blocked by administration of anti-TNF-alpha antibodies or by antioxidants. IL-1β and IL-6 have been shown to increase L-type Ca( ²⁺ ) currents (ICaL), and this effect can be blocked by aspirin or indomethacin. The close link between phospholipidosis and prolonged QT provides another hint of the importance of these cytokines. 77% of the agents that can cause phospholipidemia are also hERG channel blockers. Phospholipid cells have been shown to secrete large amounts of TNF-[alpha] and IL-6 following LPS stimulation. It is also speculated that the damage mechanism of drug-induced phospholipidemia is the accumulation of ceramide. Numerous studies have shown that cytokines such as interferon gamma, IL-1 beta and TNF-alpha increase sphingomyelinase activation and increase the production of ceramide, which is known to inhibit hERG currents. Neurolipids are also known to mediate ROS signaling. Cerebrolysine is metabolized to sphingosine and fatty acids, and sphingosine is phosphorylated by sphingosine kinase to form sphingosine-1 phosphate. Cerebrolysine and sphingosine-1 phosphate have opposite effects, cerebrolysine induces apoptosis and sphingosine-1 phosphate promotes cell survival. Fingolimod, a sphingosine analog (which has both agonist and antagonist effects at the sphingosine-1 phosphate-1 receptor) for the treatment of patients with relapsing polyps In patients with sclerosis, inhibition of hERG currents causes QT prolongation and fatal ventricular arrhythmias. Furthermore, studies in a mouse model of influenza-induced cytokine storm have shown that sphingosine-1 phosphate-1 signaling is a major pathway for activation of cytokine storm. Cytokine storm was reversed by feedback inhibition of sphingosine analogs through cytokines, and significant reductions in TNF-α, IL-1α, IL-6, MCP-1, IFN-α, and MIP-1α were seen, and clinical Improve. The sphingosine analogs enabled the mice in this study to survive much longer than with antiviral therapy. It should also be noted that progestogens, statins, liposomal curcumin, resveratrol, and antioxidants, which are clinically found to be inhibitors of QT prolongation, are also strong inhibitors of these inflammatory cytokines. Even beta-blockers, the standard of care for patients with prolonged QT, appear to have inhibitory effects on cytokines as part of their mechanism of action in the treatment of these patients. All of these agents shift the balance from the ceramide pathway towards the sphingosine-1 phosphate pathway. Therefore, excessive concentrations of pro-inflammatory cytokines play a major role in the etiology of prolonged QT syndrome, possibly through stimulation of ROS and the ceramide pathway.

By way of explanation and in no way limiting the invention, a proposed mechanism for the correction of the previously prolonged QT interval by liposomal curcumin and EU8120 is described. Substantial evidence from early phase 2 anticancer trials of pro-inflammatory cytokines and recent studies in patients with rheumatoid arthritis, psoriasis and inflammatory bowel disease suggest that elevated concentrations of pro-inflammatory cytokines lead to prolongation of the QT interval. These patients have a significantly increased incidence of prolonged QT. Furthermore, in a trial of anti-IL-6 antibodies in patients with rheumatoid arthritis, prolonged QT shortening was noted, which was associated with a decrease in cytokines. An association between elevated cytokine concentrations and asymptomatic QT prolongation has also been found in a large-scale study of normal populations. In animal models and ventricular myocytes, TNF-α administration resulted in a decrease in the fast component of delayed rectifier potassium current (IKr), the slow component of delayed rectifier current (IKs), and the transient outward current (Ito). These effects are believed to be due to stimulation of reactive oxygen species (ROS) and can be blocked by administration of anti-TNF-alpha antibodies or by antioxidants. It is known from other studies that TNF-α and other pro-inflammatory cytokines stimulate ROS production. IL-1β and IL-6 also have QT prolonging effects in these models. It is also known from studies of other diseases that ROS increases brain amide production, thus shifting the balance from the sphingosine-1-phosphate (S1P) pathway (protective) to the brain amide pathway (destructive). Several studies in experimental models have shown that ceramide leads to inhibition of hERG currents. Statins that reduce pro-inflammatory cytokine concentrations and lead to prolonged QT shortening have been proposed to stimulate this protective S1P pathway as their underlying mechanism. Other agents that shorten the prolonged QT, including antioxidants such as vitamin E, reduce the concentrations of pro-inflammatory cytokines and ROS, and stimulate S1P. Finally, the inventors have recognized that the list of agents that lead to cytokine inhibition and previously prolonged shortening of the QT interval is remarkably similar to agents that have been shown to reduce cytokine concentrations and secondary brain inflammation in animal models and also reduce the extent of brain damage list. Thus, the present invention can be used to target such diseases that increase ceramide production, thereby shifting the balance from the sphingosine-1-phosphate (S1P) pathway (protective) to the ceramide pathway (destructive).

The inventors have shown that liposomal curcumin and EU8120 reduce IL-1β, IL-6, TNF-α, MCP-1, MIP-1 and Rantes in both in vitro and in vivo models. In other models, liposomes have also been shown to compete for the enzyme sphingomyelinase and reduce ceramide concentrations, thus shifting the ceramide/S1P balance towards S1P as well.

LPS-induced cytokine storm produces QTc prolongation that is prevented by anti-inflammatory lipids. There is increasing evidence that excessive concentrations of pro-inflammatory cytokines play a major role in the pathogenesis of prolonged QT syndrome. Conversely, blocking agents such as tocilizumab (IL-6) or anti-cytokinin antibodies (TNFα) help to shorten the previously prolonged QT interval.

In this study, LPS and Kdo2-lipid-A were used to induce cytokine release in guinea pigs with simultaneous ECG monitoring and blood draw followed by Q-ELISA assay of cytokine production. The guinea pig was chosen for its reliable QTc prolongation due to proarrhythmic excitation, with consistently visible T waves on the ECG. Male adult guinea pigs received 300 µg/kg LPS at time 0, and ECGs were analyzed 1, 2, and 4 hours after LPS, and blood was drawn concurrently. Animals receiving LPS exhibited an 8 msec increase in QTc only 1 hour after LPS, when TNFα concentrations were up to 5.5 times pre-LPS values. A 29-millisecond QTc prolongation at 2 hours after LPS was associated with a 7- and 9-fold increase in IL-1β and IL-6, respectively. When animals were euthanized, QTc prolongation (27 ms) was still present 4 hours after LPS. When 9 mg/kg EU8120 (a lipid blend shown to prevent blockade of IKr channels by various hERG blockers) was administered 1 hr before LPS induction, QTc prolongation was limited to 5 ms after 2 hr, and at Complete arrest was achieved 1 hour and 4 hours after LPS. Plasma concentrations of TNFα, IL1β and IL-6 were significantly reduced in animals administered EU8120. This example demonstrates that EU8120 inhibits QTc prolongation through an anti-inflammatory cytokine effect and not through any interaction with the active agent (LPS).

Synthetic curcumin.

Compositions can be used in the present invention to treat cytokine storm disorders using synthetic curcumin (S-curcumin).

Curcumin, the active ingredient of the turmeric plant, has been synthesized to near-purity (99.2%). It is formulated with liposomes, polymers or PLGM to enable intravenous administration as a bolus injection or as a continuous infusion in combination with other active agents over 1 to 72 hours. Curcumin possesses antioxidant and anti-inflammatory activities, and blocks autonomous intracellular signaling pathways in response to extracellular growth factors, uncontrolled proliferation of cells, and abnormal responses to fibrosis-related and tissue degenerative disorders. Specifically, curcumin negatively responds to components of key signaling pathways that control proliferation, metabolism, survival, and death.

Oral and topical administration of turmeric plant extracts has been used in traditional medicine for over two thousand years. Although oral administration has no systemic toxicity, it also has no systemic therapeutic activity. This is due to blood insolubility, and inactivation of the intestinal wall and liver, ie the oral route has negligible bioavailability for systemic disease. To overcome these limitations, parenteral intravenous curcumin formulations with liposomes, polymers (n-isopropylacrylamide, N-vinylpyrrolidone and acrylic acid) and polylactic acid glycolic acid copolymers Enter preclinical drug development. ⁴

Curcumin is available as a turmeric root extract for use by researchers as a mixture of three curcuminoids and by the public as a food supplement or flavor according to the FDA. The extract was 79.2% curcumin (diferulomethane), 18.27% demethoxycurcumin and 2.53% bisdemethoxycurcumin.

The synthetic curcumin is GMP grade 99.2% pure diferulomethane, which is prepared for non-human experimental research and future Phase I clinical trials. There are significant differences between the C3 three-component extract and the single-component synthetic S-curcumin, and these differences extend to discernible analytical, physicochemical, and biological properties. In certain aspects, the diferulomethane is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96% pure diferulomethane.

The present invention relates to the synthesis of curcumin (S-curcumin) and the comparison of the properties and activities of S-curcumin with liposomal curcumin ^Nanocurc® and PLGA-curcumin (hereinafter referred to as C3-complex).

Liposome Curcumin: A preliminary study of liposomal curcumin was performed using materials purchased as complexes ^6-7 . S-curcumin was studied by Mach CM et al. (2009) ⁸ and Mach CM et al. (2010) ⁹ .

Nanocurc ^® : Preliminary studies of Nanocurc ® were conducted using the product purchased as a compound. Savita Bisht et al. (2007) ¹⁰ used non-sabinsa sources. Since then, the remainder of Nanocurc® has published research using S-curcumin. ^11-13

PLGA-Curcumin: A preliminary study of PLGA-Curcumin was carried out using the product prepared as a C3-complex. ^14-18 Studies include PLGA-curcumin C3 complex and PLGA-S-curcumin pharmacokinetic studies in rat brain.

Comparison of PLGA C3-complex curcumin and PLGA S-curcumin indicates the following differences. The solubility of 99.2% S-curcumin in all four solvents (ethanol, ethyl acetate, acetone and acetonitrile) was significantly different from the C3 complex containing 76% curcumin. Pure materials have greater solubility when normalized to equal concentrations. This confers improved manufacturing capability and is attributable to different pharmacokinetics and pharmacodynamics in the in vivo context (Table 3).

Table 3: Solubility of S-curcumin and C3-complex curcumin in different organic solvents. solvent Weight of Curcumin (mg) Volume of solvent (μL) Concentration (mg/mL) Physical apparent solubility 1 g solvent dissolved in solvent (mL) Lot - C100609, Curcumin = 99.2% Ethyl acetate 20.8 2000 10.4 part 96.2 Ethanol 17.5 4600 3.804 part 263 acetone 46.5 1100 42.3 completely 23.6 Acetonitrile 17.4 3200 5.44 part 184 Batch-C3 Complex, Curcumin = 76% Ethyl acetate 21.5 1800 11.9 completely 83.7 Ethanol 28.4 6500 4.37 completely 229 acetone 39.8 700 56.9 completely 17.6 Acetonitrile 18.2 2900 6.28 completely 159 * Samples were weighed and transferred to scintillation vials. Add a small volume of solvent and shake well, repeating each addition until this solubility.

Table 4: Difference comparison between S-curcumin and C3-complex curcumin. C3-Curcumin S-Curcumin Residue on ignition 0.03% 0.4% loss on drying 0.98% 0.24% melting range 172 to 175C 179.8 to 181.9 C Knocking Packing Density 0.61 g/ml 0.26g/ml loose packing density 0.37g/ml 0.18g/ml Sieve test -40 mesh 100% 64.71% -80 mesh 98.27% 64.0% HPLC curcumin content 79.2% 99.2% total curcuminoids 96.21% 99.2% bisdemethoxycurcumin 2.53% 0 Demethoxycurcumin 18.27 0 heavy metal lead 0.91ppm(ug/g) <0.2ppm arsenic 0.54ppm(ug/g) <0.2ppm cadmium <0.2ppm (ug/g) - HG <0.02ppm (ug/g) - residual solvent meets the meets the Micro – Full Plate Counting <100cfug 10cfu/g Yeast and Mold Count <10cfu/g 15cfu/g Escherichia coli neg/10g neg/10g Salmonella neg/10g neg/10g Staph. aureus neg/10g neg/10g Pseudomonas aeruginosa neg/10g neg/10g

Figure 4A is a graph showing the effect of liposomal curcumin on liver function, including aspartate transaminase (AST) and alanine transaminase (AST) concentrations, during sepsis in a mouse model system. These results demonstrate the effectiveness of liposomal curcumin compared to the standard of care for sepsis in age-matched controls. For AST and ALT, liposomal curcumin showed higher efficacy compared to standard of care.

Figures 4B to 4D are graphs showing the effects of liposomal curcumin on glomerular filtration rate (GFR) on renal function during sepsis, respectively, by measuring creatine, neutrophil gelatinase, a measure of chronic kidney disease progression. Lipocalin (NGAL) and blood urea nitrogen (NGAL) which measures the ability of the kidneys to remove urea from the blood. Specifically and importantly, with respect to NGAL, a measure of chronic kidney disease progression, liposomal curcumin showed higher efficacy in protecting kidney function than standard care.

Figures 4E and 4F are graphs showing the effect of liposomal curcumin on cardiac function, c-troponin, a measure of cardiac tissue damage, and percent ejection fraction, a measure of cardiac tissue damage, and percent ejection fraction, a measure of cardiac function during sepsis. The effectiveness of the heartbeat left ventricle (or right ventricle) pumping blood. Liposome curcumin showed reduced cardiac damage compared to standard of care and was equal to standard of care in percent ejection fraction.

Figure 4G is a graph showing the effect of liposomal curcumin on overall survival during sepsis. Liposome curcumin showed longer survival compared to standard of care treatment.

When compared to the teachings of Yang et al., Protective effect of curcumin against cardiac dysfunction in sepsis rats, Pharmaceutical Biology, 2013; 51(4): 482 to 487, the present invention shows that there is no cardiac toxicity associated with curcumin. The results were significantly improved.

14:0 Lysophospholipid glycerol (Lyso PG)

Myristate Monoglyceride

myristic acid, a free fatty acid

Example 2

Brain injury (TBI) following traumatic brain injury (TBI) is a two-stage process: injury resulting from the initial injury followed by an inflammatory phase in which numerous additional injuries can occur. This inflammation begins within minutes of initial injury and can last for months or years, and is caused by the involvement of cytokines (specifically pro-inflammatory cytokines, interleukin-1β, interleukin-6, and tumor necrosis factor- α) is caused by a series of complex metabolic processes. The concentrations of these cytokines can be increased thousands of times over the corresponding concentrations in serum. Strategies to control the concentration of these pro-inflammatory cytokines and reduce cytokine-induced brain damage are discussed. Substantial evidence from experiments in animal models suggests that inhibition of cytokines is effective in ameliorating nerve damage after TBI. However, the efficacy of this approach remains to be demonstrated in patient trials.

It is increasingly recognized that an abnormal immune system and massive overproduction of pro-inflammatory cytokines ("cytokine storm") are major factors in disease progression and mortality from many diseases. Cytokine storm (also known as "cytokine release syndrome") can be associated with malaria[1], SARS[2], dengue[3], leptospirosis[4], Raisa fever[5], Gram Negative sepsis [6] and after infection with many other infectious diseases (7-10). Cytokine storms are a major cause of death in patients with Ebola virus [11-13]. Patients with cytokine storms can experience Increased vascular permeability, severe bleeding, and multiple organ failure, which can ultimately lead to fatal outcomes [8,13,14]. Significant increases in systemic cytokine concentrations, both pro- and anti-inflammatory cytokines, are seen. It is thought that healthy immune This overproduction of systemic cytokines explains why individuals between the ages of 20 and 40 were more likely to die than older adults during the H1N1 pandemic of 1918 [15,16]. Cytokine storms can occur in severe burns or trauma [ 17], acute pancreatitis [18] or after ARDS [19] secondary to drug use or inhaled toxins. Severe acute graft-versus-host disease can be considered as a cytokine storm [20,21]. Cytokine storm is also It is treated with the commonly used antineoplastic agent rituximab [22] and the monoclonal antibodies tositumomab, alemtuzumab, muromonab and A recognized complication of blinatumomab [23] treatment. Elevated levels of cytokines have been found and are thought to be (Parkinson's disease) [25], autism [26] and multiple sclerosis [27]) and in the acute phase of Guillian-Barre syndrome [28,29] Important cause. Elevated levels of cytokines are associated with worsening of psychiatric disorders [30,31] and lupus encephalopathy [32,33].

TBI represents a major health problem in the United States, with 1.7 million cases, 275 000 hospitalizations, and 52 000 deaths annually [34], and neuropsychiatric sequelae are common, especially after severe injury [35]. It is now known that brain injury after traumatic brain injury occurs in two stages: an initial stage, in which damage occurs due to external mechanical forces, and a secondary inflammatory stage, in which a series of cytokines (such as interleukins ( IL)-1β, IL-6 and tumor necrosis factor (TNF)-α [36]). The increase in the concentration of cytokines in the brain can be enormous, especially after severe TBI. IL-6 is generally not detectable in CSF, or is detected only at very low concentrations (1 to 23 pg/ml) [37,38]. In one study, CSF IL-6 concentrations as high as 35 500 pg/ml were seen following severe TBI [38,39]. These IL-6 concentrations were 40 to 100x higher than the corresponding concentrations in the serum of these patients [40]. Kushi et al. reported substantial increases in IL-6 and IL-8 measured at 24 hours, 72 hours, and 168 hours after severe TBI in 22 patients on admission. In the nine fatal cases, mean IL-6 values in CSF at these times were 15 241, 97 384, 548 225 and 336 500 pg/ml, compared with 102, 176, 873, 3 059 pg/ml in blood Compared with ml, it is a "storm" of cytokines that mainly localize the brain. For the 13 survivors, mean IL-6 CSF values were lower but still much higher than peripheral blood compared with 181, 105, 37 and 26 pg/ml in blood: 5 376, 3 565, 328 and 764 pg /ml [41]. Similar differences were seen for IL-8. Although IL-8 concentrations in CSF are typically extremely low (5 to 72 pg/ml) [37], Kushi et al. reported that CSF IL-8 concentrations were consistently thousands of higher than normal or comparable concentrations in peripheral blood times [41]. The researchers also noted that IL-6 and IL-8 blood levels remained significantly elevated after 72 hours, which was associated with poorer prognosis and higher mortality. Helmy et al found that multiple cytokines, including IL-1α, IL-1β, IL-6, IL-8, IL-10, monocyte chemotaxis, were significantly elevated in brain extracellular fluid after severe TBI in 12 patients. inflammatory protein (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). These concentrations were also significantly elevated compared to corresponding blood concentrations [42]. Similar results have been reported by other investigators and noted that extremely high cytokine concentrations are associated with poor prognosis [43,44]. For example, Arand et al. indicated that IL-6 concentrations were 8-fold higher in patients who died compared to patients who survived. In addition, only patients who died showed elevated concentrations of another pro-inflammatory cytokine, IL-12 [43]. These data further support the hypothesis that cytokine storm causes increased neurological damage after TBI. Numerous studies have shown that some of these same cytokines can have beneficial as well as detrimental effects on the brain [45-47]. However, many studies have shown that blocking these cytokines reduces brain damage after TBI, at least in animal models. A list of key cytokines elevated in brain and CSF after TBI is given in Table 5.

Table 5: Key cytokines showed significant increase in brain and CSF after TBI Cytokines Features TNF-α Mainly pro-inflammatory cytokine, insulin resistance, stimulates apoptosis, also has neuroprotective function IL-1α Pro-inflammatory cytokines that stimulate the release of TNF-α from endothelial cells IL-1β Mainly pro-inflammatory cytokines, regulate the production of IL-2, IL-6, IL-8 and interferon-γ, stimulate phagocytosis and programmed cell death, early CNS development IL-2 Pro-inflammatory cytokines, T cell lymphocyte differentiation IL-4 Anti-inflammatory cytokine, also stimulates B cell proliferation IL-6 Mainly pro-inflammatory cytokines, B cell differentiation, neurogenesis, myokine, also has anti-inflammatory functions (inhibits IL-1 and TNF-α) IL-8 (CXCL8) Major pro-inflammatory cytokines (chemokines), neutrophil chemotaxis IL-10 Mainly anti-inflammatory cytokine, also has T cell stimulating function IL-12 Pro-inflammatory cytokines, T cell differentiation Interferon-gamma Pro-inflammatory cytokines, T and B cell lymphocyte and macrophage activation MCP-1 (CCL2) Pro-inflammatory cytokines, monocyte and basophil chemotaxis MIP-1α (CCL3) Pro-inflammatory cytokines, induces the synthesis of pro-inflammatory cytokines, activates neutrophils TGF-beta Anti-inflammatory cytokines, also have pro-inflammatory properties, may have long-term deleterious effects

Interleukin-1. The IL-1 family is a group of 11 cytokines that are closely involved in the body's response to injury or infection [48,49], and it also plays a key role in tumor angiogenesis [50] and stimulation of cancer stem cells [51] . The most important cytokines in the IL-1 group are IL-1β, IL-1α and the IL-1 receptor antagonist IL-1RA, but the IL-1 group also includes the pro-inflammatory cytokines IL-18, IL-33 and IL -36 and some less studied cytokines. The key cytokine IL-1β is a protein produced by activated macrophages. Among the most important functions are neutrophil activation, regulation of production of other cytokines (IL-2, IL-6, IL-8, interferon-γ), regulation of mitosis, stimulation of phagocytosis, induction of fever, angiogenesis and Induces programmed cell death [48,49]. Elevated IL-1β concentrations were found in the CSF of patients with TBI and were detectable within minutes of acute injury [38,52,53]. Very high concentrations in the CSF of TBI patients are associated with progressively worse prognosis [54,55].

Studies in animal models have also given similar results [56-60]. Kamm et al. showed IL-1β concentrations in rat brain after TBI within the first hour and peaked at 8 hours, with no detectable changes in blood or liver IL-1β concentrations [56]. It has also been shown in animal models that intraventricular administration of IL-1β significantly worsens brain injury [61]. Most importantly, administration of an IL-1β antagonist prevented the damage caused by this cytokine in experimental models. Administration of IL-1RA to rodents has been shown to reduce brain damage following TBI. For example, Yang et al. showed that brain damage caused by middle cerebral artery occlusion was reduced in mice in animals previously transfected with adenoviral vectors to induce IL-1RA overexpression [62]. Jones et al showed that a single intraventricular dose of IL-1RA administered to mice at the time of TBI reduced lesion volume, resulted in functional improvement and resulted in a significant reduction in nitric oxide synthase-2-positive cells in the lesion [63-Jones] ]. Sanderson et al. studied the effect of systemically administered IL-1RA on Sprague Dawley rats after TBI. No effect was seen at low doses. Following high doses, the researchers observed reduced neuronal loss and increased memory and cognitive function in the animals. However, motor function was not improved [64]. Hasturk et al. showed that IL-1RA decreased tissue IL-1β concentrations and increased concentrations of the antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase in rats after TBI [65]. Similar results have been reported by other groups [66,67]. Furthermore, Basu et al. reported that mice lacking the IL-1 receptor experienced less brain damage after traumatic injury [68]. The researchers found decreased basal concentrations of IL-1, IL-6, and COX-2, as well as decreased amoeba-like microglia/macrophages, suggesting that the brain inflammatory cycle is blocked at this critical step. Furthermore, Tehranian et al. have shown that transgenic mice overexpressing human IL-1RA in astrocytes have reduced concentrations of IL-1β, IL-6 and TNF-α compared to wild-type mice, and have more Nerve recovery after Jiazhi head injury [69].

These data suggest that the use of IL-1RA may be an effective strategy for patients with TBI. Human recombinant IL-1RA has been the standard of care for patients with rheumatoid arthritis for several years, and its use has been studied in many diseases in which increased cytokines play a role in the destruction process, including diabetes [70], heart failure [71], multiple myeloma [72] and sepsis [73]. In a randomized phase II trial in patients with acute stroke, IL-1RA-treated patients experienced less loss of cognitive function than controls [74]. Helmy et al. conducted a phase II controlled trial of this agent in 20 patients with severe TBI and were able to conclude that IL-1RA does cross the blood-brain barrier and is safe in this population [75]. It cannot be concluded that IL-1RA administration results in a therapeutic benefit in these patients [75]. While many of these results look promising, IL-1RA may have limited efficacy because it directly blocks only one of the important cytokines involved in inflammation (IL-1RA can indirectly block other cytokines , because IL-1 can lead to increased expression of other cytokines), and this may partly explain the agent's lack of greater success against rheumatoid arthritis. In addition, the use of a combination of IL-1RA and a TNF-α blocker is contraindicated because their concomitant use can lead to severe side effects [76].

Tumor necrosis factor-alpha. The second key pro-inflammatory cytokine is TNF-α. This cytokine plays an important role in the body's response to infection and cancer. Since Helson et al. reported TNF-α in 1975 [77], aberrant TNF-α function has been reported in many diseases, including various conditions such as diabetes [78], cardiovascular disease [79], inflammatory bowel disease [80 ] and Alzheimer's disease [81]. TNF blockers, such as infliximab, etanercept, and adalimumab, are used in patients with rheumatoid arthritis, ankylosing spondylitis, and psoriasis standard therapy. As noted, TNF-α is thought to have both beneficial and detrimental effects in patients with TBI [46]. However, results from experimental models suggest that most of these effects are detrimental, especially when excess concentrations of this cytokine are produced. Knoblach et al. reported the correlation of TNF concentrations with the degree of brain injury and neurological impairment in rats following experimental TBI, with the highest TNF concentrations 1 to 4 hours after injury in rats with the most severe brain injury [ 82]. In addition, studies with the TNF blocker etanercept have consistently shown reduced brain damage in these animals following administration of this agent. Chio et al. reported that etanercept decreased ischemia, increased glutamic acid concentration, decreased neuronal and glial apoptosis and microglial activation, and also decreased TNF-α levels when administered to rats after TBI. high concentrations [83]. In a later report, these researchers concluded that etanercept ameliorated brain injury by reducing the early expression of TNF-α in microglia [84]. Ekici et al. showed that administration of a combination of etanercept and lithium chloride one hour after TBI reduced brain edema, tissue damage, and TNF concentrations [85]. Cheong et al. showed that administration of etanercept to rats immediately after TBI resulted in an increase in 5-bromodeoxyuridine and doublecortin markers in the injured brain, suggesting that increased TNF-α concentrations in the brain may affect the Neural stem cells are toxic and thus interfere with neurogenesis [86]. Wang et al. reported that the use of this agent early after injury promoted the survival of transplanted neural stem cells and facilitated nerve regeneration [87]. Other groups have reported similar results with etanercept or other TNF blockers [88-91].

Although TNF blockers have been extensively studied in animal models, little work has been done to assess the potential efficacy of these agents in patients with TBI [92]. Tobinick et al reviewed the medical records of 617 patients with stroke and 12 patients with TBI who had been treated with etanercept. Significant improvements in neurological function were observed, even in patients treated for more than 10 years after initial injury. The researchers concluded that this supports the idea that chronic inflammation, which may persist for many years, is a major cause of nerve damage in these patients [93]. However, the small number of patients in the TBI group and the lack of a control group make the data in this report difficult to interpret, as it is unclear whether TNF blockade caused the observed improvement. Randomized trials are needed to demonstrate benefit in TBI patients, and TNF blockers can have substantial toxicity. Furthermore, since TNF blockers target only a single cytokine, and since the use of these agents in combination with IL-1 antagonists is contradictory, the use of such blockers may not be the most effective strategy for treating these patients.

Interleukin-6. The third major pro-inflammatory cytokine is IL-6. Like TNF-α, elevated concentrations of IL-6 are thought to play a role in the etiology of many diseases, and, like TNF-α, IL-6 is thought to have beneficial and detrimental effects after TBI [94]. In fact, IL-6 appears to have both beneficial and detrimental effects in a number of neurological disorders [95]. IL-6 plays a key role in the induction of nerve growth factor by astrocytes and thus in the repair of the injured brain [39]. Ley et al. reported that IL-6 knockout mice demonstrated reduced neurological function after TBI compared to normal mice, again indicating that IL-6 is required for neuronal recovery. However, IL-6 knockout mice did show significantly elevated IL-1β concentrations [96]. Studies of IL-6 concentrations in the frontal parenchyma in patients following severe TBI also suggest a neuroprotective effect of IL-6. Significantly higher concentrations of IL-6 were found in survivors compared to those who died, while no differences in IL-1β concentrations were found [97]. However, the numbers in this study were small.

On the other hand, numerous studies have shown that IL-6 has deleterious effects after TBI. Conroy et al. showed that IL-6 was toxic to rodent cerebellar granule neurons in culture [98]. In another study, intranasal administration of IL-6 to rats was found to increase the intensity of seizures, as well as increase mortality [99]. Similar results were seen in transgenic mice with glial fibrillary acidic protein promoter-driven astrocyte IL-6 production [100]. Yang et al. showed that motor coordination deficits in mice following mild TBI were corrected by IL-6 blockade [101]. Similar results were reported in experimental spinal cord injury. Okada et al. showed that anti-IL-6 receptor mouse monoclonal antibody increased functional spinal cord recovery in mice after injury [102]. Nakamura et al. reported that antibodies to IL-6R reduced glial scarring and increased recovery after spinal cord injury [103]. Crack et al. reported that anti-lysophosphatidic acid antibodies significantly reduced brain damage in mice following experimental TBI. The researchers attribute this to a significant reduction in IL-6-induced secondary inflammation. These antibodies had no effect on IL-1β or TNF-α concentrations [104]. Suzuki et al. suggested that the different results seen in these studies could be explained because the inflammatory effects of IL-6 appear to be dominant in the acute phase after TBI, while its neurogenesis effect may be important later [105] ]. Little work has been done on IL-6 blockers in patients with TBI. The anti-IL-6 antibody tocilizumab is available and used to treat patients with rheumatoid arthritis [106], but this agent has not been studied in this population.

Anti-inflammatory cytokines. Anti-inflammatory cytokines, such as IL-4, IL-10, IL-11, IL-13 and transforming growth factor (TGF)-beta, are also significantly elevated in inflammatory conditions. One of the main functions of these cytokines is to inhibit the synthesis of pro-inflammatory cytokines [107]. IL-10 is the most important anti-inflammatory cytokine, and IL-10 concentrations are significantly elevated in brain and CSF after TBI [54,108]. Although IL-10 is also known to have pro-inflammatory functions [107], its main effect after TBI appears to be primarily protection from inflammatory injury. Kumar et al studied cytokine concentrations in 87 patients with severe TBI over a twelve-month period and found that patients with an elevated IL-6/IL-10 ratio at six months had a poor prognosis [109]. Studies in cell cultures and animal models appear to confirm the protective effect of IL-10. Bachis et al. showed that IL-10 blocks caspase-3 and reduces neuronal death in cultured rat cerebellar granulosa cells exposed to toxic doses of glutamic acid [110]. Knoblach et al. showed that intravenous or subcutaneous administration of IL-10 reduced IL-1 synthesis and enhanced neurological recovery in rats following experimental TBI in rats. However, intraventricular administration was ineffective [111]. Chen et al. showed that mice lacking IL-10 did not respond to the beneficial effects of hyperbaric oxygen therapy after TBI (112-X. Chen 2013). Bethea et al. showed that IL-10 reduced TNF-α production and improved motor function after spinal cord injury in rats [113]. Similar neuroprotective effects of IL-10 have been seen in other experimental SCI studies [114,115]. This suggests that another method of treating TBI in a patient may be the administration of anti-inflammatory cytokines such as IL-10. Recombinant human IL-10 (ilodecakin) has been tested in various diseases. However, the results so far have been disappointing [116].

Targets multiple cytokines.

Progesterone. Progestins are well known from studies in animal systems to reduce neuronal damage following TBI [117-121]. A major mechanism of neuroprotection seen with progestogens is the ability of these agents to inhibit pro-inflammatory cytokines. Cutler et al. showed that administration of progesterone in aged male rats after TBI decreased brain IL-6 concentrations at 24, 48 and 72 hours. Decreased concentrations of NF-κB and COX-2 were also seen, and rats demonstrated improved motor skills, reduced cerebral edema, and reduced mortality (122-Cutler). He et al. reported that intraperitoneal administration of progesterone reduced IL-1β and TNF-α 3 hours after injury. Similar results were seen following administration of another progestin, allopregnanolone [123]. Chen et al. reported that administration of progesterone in rats after TBI reduced IL-1β, IL-6, and TNF-α concentrations in the brain, as well as decreased brain apoptosis [124]. Pan et al. showed that intraperitoneal administration of progesterone decreased brain TNF-α and NF-κB concentrations in rats after experimental TBI. Treated rats also had better results on the Neurological Severity Score test [125]. Unfortunately, these results have not been confirmed in patient trials. Xiao et al did report positive results in a randomized trial of progesterone administered within 8 hours of TBI [126]. However, large multicenter trials have not confirmed this. The large phase III trial of progesterone in patients with TBI by The Neurologic Emergencies Treatment Trials Network was prematurely stopped due to lack of efficacy [127]. The second main trial, SYNAPSE, a multinational, placebo-controlled trial of progesterone in 1195 patients with severe TBI, also showed no efficacy. In the progesterone group, only 50.4 percent showed a favorable outcome based on the Glasgow outcome scale, compared with 50.5 percent of patients receiving placebo [128,129].

statin. These are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, which are used to inhibit the production of cholesterol in the liver. These drugs are widely used clinically in patients with hypercholesterolemia. It is also known that statins have significant anti-inflammatory effects. Chen et al. showed that pre-administration of lovastatin to rats with experimental TBI resulted in significant reductions in IL-1β and TNF-α in areas of brain injury at 6 and 96 hours post-injury. Treated rats had significantly reduced FJB-positive degenerating neurons and better functional recovery [130]. Simvastatin was shown to reduce brain IL-1β concentrations and reduce microglia and astrocyte activation in rats after TBI, with functional improvement based on NCS scores. However, no changes in IL-6 or TNF-α concentrations were noted [131]. Atorvastatin was found to reduce IL-6 and TNF-[alpha] in mice after TBI. Hippocampal degeneration and functional neurological deficits were reduced in treated animals compared with controls [132]. There is also evidence that discontinuation of these statins in patients results in increased pro-inflammatory cytokines, including IL-6 [133-135], and discontinuation of these agents after TBI appears to result in worse outcomes [136] . In addition, a retrospective study showed that preinjury statin use was associated with better outcomes [137]. This has led to the recommendation to study these agents in patients with TBI. Only a few small trials are reported. Tapia-Perez et al. studied the effect of rosuvastatin in patients with severe TBI and reported a reduction in forgetfulness in treated patients [138]. However, there was no difference in disability at 3 months. In addition, the trial included only 8 rosuvastatin patients and 13 controls, and 21 of the 43 TBI patients evaluated were deemed ineligible. In another small study, in 19 patients receiving 10 days of rosuvastatin and 17 controls, Sanchez-Aquilar et al reported that rosuvastatin patients had plasma TNF- Significant reduction in alpha concentrations and improvement in disability scores. No effect was seen on IL-1β, IL-6 or IL-10 [139]. Rasras et al studied the effects of a similar agent, simuvastatin, in a randomized trial of 66 patients with severe TBI; however, no differences were found between treatment and control groups [140].

tetracycline. In animal models, tetracycline has been shown to inhibit inflammation and achieve better results in several neurological disorders. Bye et al. showed that minocycline reduced IL-1β and IL-6 expression and microglia and macrophage activation in mice after TBI. Treated mice had better neural function on day 1, however there was no difference between treated and control mice on day 4 [141]. However, a later study in this same group did show relative improvement in the minocycline group by 6 weeks [142]. Shanchez Mejia et al. reported that administration of minocycline in mice after TBI reduced IL-1β by inhibiting caspase-1 activation, resulting in improved neurological function and reduced lesion volume in treated animals [143]. Lee et al. showed that administration of minocycline to rats following spinal cord injury decreased TNF-α, increased IL-10, decreased neuronal cell death, and improved motor function [144]. Yrjanheikki reported that either deoxytetracycline or minocycline reduced IL-1β convertase mRNA induction and protected against neuronal death after ischemic stroke [145]. Other researchers have also reported positive results for tetracycline in animal models of TBI [146-148]. However, Turtzo et al. could demonstrate no benefit in minocycline-treated rats after TBI [149]. Furthermore, in another study, minocycline was found to cause an increase in ischemic brain damage in neonatal mice [150,151].

other anti-inflammatory agents. Many other agents have shown anti-inflammatory activity in animal models of TBI. Melatonin has been reported to decrease TNF-α and IL-1β and increase the number of surviving neurons in mice after TBI. Researchers believe this effect is secondary to dephosphorylation of the m-TOR pathway [152]. Other researchers have also reported positive results for melatonin in animal models [153-156].

Zhu et al. reported that intraperitoneal administration of curcumin administered to mice 15 minutes after TBI significantly reduced IL-1β, IL-6 and MCP-1 concentrations and decreased TLR4-positive microglia/macrophage numbers, resulting in Neuronal apoptosis is reduced [157]. Other researchers have also reported neuroprotective effects of curcumin in animal models of TBI [158-161].

Cyclosporine is an effective immunosuppressant. Due to its broad range of effects on cytokines [162-165] and activity in animal models [166], it has been studied in experiments in patients with TBI. However, randomized, placebo-controlled trials of this agent in patients with TBI showed no activity [167]. Ciclosporine (neurostat) formulations continue to be studied in patients with TBI and other neurological disorders, however recent reports suggest that urelostat has no neuroprotective activity in acute ischemic stroke [168] .

Many other agents that inhibit pro-inflammatory cytokines have also been studied in animal models. Carprofen, a COX-2 inhibitor currently used to treat arthritis in dogs and other animals, was found to significantly reduce IL-1β and IL-6, and improve neuronal function in mice after TBI [169 ]. Triptolide, a diterpene epoxide with anticancer activity in animal models, was found to inhibit IL-1β, IL-6, and TNF-α, increase IL-10 concentrations, and reduce experimental TBI Neuronal apoptosis in Sprague-Dawley rats after [170]. TSG-6 (TNF-α-stimulated gene/protein 6) is an anti-inflammatory agent that inhibits IL-1β, IL-6 and other pro-inflammatory cytokines (MIP-1α, MCP-1) and stimulates anti-inflammatory cells production of hormones such as IL-4 [171]. Watanabe et al showed that administration of the drug to mice after TBI reduced lesion size and improved neurological recovery [172]. Another agent, the CNS penetrating small molecule MW151, which is known to inhibit IL-1β and TNF-α, but not anti-inflammatory cytokines such as IL-10, has been tested in mice after TBI. This agent restores abnormal cytokine concentrations to normal, reduces glial activation and results in improved neural function in treated animals [173]. However, none of these drugs have been tested in patient trials.

Brain damage after TBI may worsen significantly during the subsequent phase of brain inflammation. During this phase, concentrations of key cytokines (specifically IL-1β, IL-6, and TNF-α) increase substantially in a "brain cytokine storm" that can increase relative to their corresponding serum concentrations thousands of times. Although some of these cytokines, such as IL-6 and TNF-alpha, may have beneficial effects, evidence suggests that high concentrations are detrimental, as numerous studies in animal models have shown that blocking these cytokines reduces Brain Injury. Therefore, inhibition of pro-inflammatory cytokines may limit the secondary damage caused by neuroinflammation after TBI.

It is contemplated that any embodiment discussed in this specification may be practiced with respect to any method, kit, reagent or composition of the invention, and vice versa. Furthermore, the compositions of the present invention can be used to achieve the methods of the present invention.

It should be understood that the specific embodiments described herein are presented by way of illustration and not limitation of the invention. The principal features of this invention may be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are deemed to be within the scope of this invention and are covered by the scope of the patent application.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if individual publications or patent applications were specifically and individually indicated to be incorporated by reference.

The use of the word "a" or "an" may mean "an" when used in conjunction with the term "comprising" in the scope of the claims and/or in this specification, but it is also used in conjunction with "one or more", "at least one". ” and “one or more than one” have the same meaning. The use of the term "or" in the scope of the claims is intended to mean "and/or" unless it is expressly stated that only alternatives are meant or that such alternatives are mutually exclusive, although the present invention supports only "alternatives" and " and/or" definition. Throughout this application, the term "about" is used to indicate that a value includes inherent variation in device error, the method used to determine the value, or variation that exists between individuals studied.

As used in this specification and in the scope of the claims, the word "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "includes") or "Containing" (and any form of containing, such as "contains" and "contain") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In any of the composition and method examples provided herein, "comprising" may be replaced by "consisting essentially of" or "consisting of." As used herein, the phrase "consisting essentially of" requires the specified integer or steps and those that do not materially affect the character or function of the claimed invention. As used herein, the term "composition" is used to indicate the presence of only the recited integer (eg, feature, element, characteristic, property, method/process step or limitation) or group of integers (eg, feature, element, characteristic, property, method/process) steps or restrictions).

As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding that term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with the example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Skilled artisans will understand that, unless the context indicates otherwise, there is generally no limit to the number of items or terms in any combination.

As used herein, approximate terms such as, but not limited to, "about," "substantially," or "substantially" mean that conditions when so modified are to be understood as not necessarily absolute or perfect, but are to be considered close enough to Make sure that the general skilled person specifies that the condition exists. The extent to which this description can vary will depend on how much changes can be made and those skilled in the art still recognize that a modified feature still has the desired characteristics and capabilities of an unmodified feature. In general, but limited by the foregoing discussion, numerical values modified by approximations (such as "about") herein may differ from the stated values by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

Additionally, the section headings here are provided to be consistent with recommendations under 37 CFR 1.77 or to otherwise provide organizational cues. These headings shall not limit or characterize the invention set forth in any claims that may be issued by this disclosure. Specifically and by way of example, although these headings refer to the "Field of Invention," these claims should not be limited by the language under which such headings describe the so-called technical field. Furthermore, descriptions of techniques in the "Prior Art" section should not be construed as an admission that the techniques are prior art to any of the inventions in this disclosure. Nor should the "Summary" be taken as a representation of the invention set forth in the issued claims. Furthermore, any reference to "the invention" in this disclosure in the singular should not be used to demonstrate that there is only one point of novelty in this disclosure. The various inventions may be set forth in light of the limitations of the various claims issued by this disclosure, and such claims thus define the inventions protected by them and their equivalents. In all cases, the scope of this claim should be considered on its own merits in light of this disclosure and should not be limited by the headings listed herein.

In accordance with the present invention, all compositions and/or methods disclosed and claimed herein can be made and performed without undue experimentation. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the compositions and/or methods and in steps may be modified without departing from the concept, spirit and scope of the present invention. Variations are applied in or in the order of the steps described herein. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. References – Example 1

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12) Park WY, Goodman RB, Steinberg KP, Ruzinsky JT, Rudella F, Park DR, Pugin J, Skeritt SJ, Hudson LD, Martin TR. Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Amer J Resp Crit Care Med 2001;164:1896-1903.

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40) Kondo A, Mogi M, Koshihara Y, Togani A. Signal transduction system for interleukin-6 and interleukin-11 synthesis stimulated by epinephrine in human osteoblasts and human osteogenic sarcoma cells. Biochemical Pharmacology 2001;61:319-326. Reference Example 2

[1] Clark IA, Alleva LM, Budd AC, Cowden WB. Understanding the role of inflammatory cytokines in malaria and related diseases. Travel Med Infect Dis 2008;6:67-81.

[2] Huang KJ, Siu IJ, Theron M, Wu YC, Liu CC, Lei HY. An interferon-gamma related cytokine storm in SARS patients. J Med Virol 2005;75:185-194.

[3] Espada-Murao LA, Morita K. Dengue and soluble mediators of the innate immune system. Trop Med Health 2011;39:53-62.

[4] Reis EAG, Hagan JE, Ribeiro GS, Teixeira-Carvalho A, Martins-Filho OA, Montgomery RR, et al. Cytokine response signatures in disease progression and development of severe clinical outcomes for leptospirosis. PLoS Negl Trop Dis. 2013;7(9):e2457.

[5] Branco LM, Grove JN, Boisen ML, Shaffer JG, Goba A, Fullah M, Momob M, Grant DS, Garry RF: Emerging trends in Lassa fever. Redefining the role of immunoglobulin M and inflammation in diagnosing acute infection. Virol J 2011;8:478.

[6] Harrison C. Sepsis: calming the cytokine storm. Nat Rev Drug Discov 2010;9:360-361.

[7] Clark IA. The advent of the cytokine storm. Immunol Cell Biol 2007;85:271-273.

[8] D’Elia RV, Harrison K, Oyston PC, Lukaszewski RA, Clark GC. Targeting the 'cytokine storm' for therapeutic benefit. Clin Vaccine Immunol 2013;20:319-327.

[9] Sun Y, Jin C, Zhan F, Wang X, Liang M, Zhang Q, Ding S, Guan X, Huo X, Li C, Qu J, Wang Q, Zhang S, Zhang Y, Wang S, Xu A , BiZ, LiD. Host cytokine storm is associated with disease severity of severe fever with thrombocytopenia syndrome. J Infect Dis 2012;206:1085-1094.

[10] Johnston SC, Johnson JC, Stonier SW, Lin KL, Kisalu NK, Hensley LE, Rimoin AW. Cytokine modulation correlates with severity of monkeypox disease in humans. J Clin Virol 2015;63:42-45.

[11] Wauquier N, Becquart P, Padilla C, Baize S, Leroy EM. Human fatal Zaire Ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis. PLoS Negl Trop Dis 2010;4(10):e837.

[12] Sordillo PP, Helson L. Curcumin suppression of cytokine release and cytokine storm. A potential therapy for patients with Ebola and other severe viral infections. In Vivo. 2015;29:1-4.

[13] Mohamadzadeh M, Chen L, Schmaljohn AL. How Ebola and Marburg viruses battle the immune system. Nat Rev Immunol 2007;7:556-567.

[14] Tisonick JR, Korth MJ, Simmons CP, Farrar J, Martin TR, Katze MG. Into the eye of the cytokine storm. Microbiol Mol Biol Rev 2012;76:16-32.

[15] Teijaro JR, Walsh KB, Rice S, Rosen H, Oldstone MBA. Mapping the innate signaling cascade essential for cytokine storm during influenza virus infection. Proc Natl Acad Sci 2014;111:3799-3804.

[16] Goa R, Bhatnagar J, Blau DM, Greer P, Rollin DC, Denison AM, Deleon-Carnes M, Shieh WJ, Sambhara S, Tumpey TM, Patel M, Liu L, Paddock C, Drew C, Shu Y, Katz JM, Zaki SR. Cytokine and chemokine profiles in lung tissue from fatal cases of 2009 pandemic influenza A (H1N1): Role of the host immune response in pathogenesis. Am J Pathol 2013;183:1258-1268.

[17] Aikawa N. Cytokine storm in the pathogenesis of multiple organ dysfunction syndrome associated with surgical insults. Nihon Geka Gakkei Zasshi 1996;97:771-777.

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For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention taken in conjunction with the accompanying drawings and wherein:

Figures 1A and 1B show the percent inhibition and percent survival, respectively, achieved with liposomal curcumin in HeLa cells.

Figures 2A and 2B show the percent inhibition and percent survival, respectively, achieved using solid S-curcumin in HeLa cells.

Figures 3A and 3B show the comparison of the percentage inhibition and percentage survival of liposomal curcumin and solid curcumin in HeLa cells, respectively.

Figure 4A is a graph showing the effect of liposomal curcumin on liver function including aspartate transaminase (AST) and alanine transaminase (AST) concentrations during sepsis. These results demonstrate the effectiveness of liposomal curcumin compared to the standard of care for sepsis in age-matched controls. For AST and ALT, liposomal curcumin showed higher efficacy compared to standard of care.

Figures 4B-4D are graphs showing the effect of liposomal curcumin on renal function during sepsis as measured by creatine, neutrophil gelatinase-associated lipocalin (NGAL) and blood urea nitrogen (BUN). Specifically and importantly, with respect to NGAL, which measures chronic kidney disease progression, liposomal curcumin was shown to be more effective in maintaining kidney function than standard care.

Figures 4E and 4F are graphs showing the effect of liposomal curcumin on c-troponin and percent ejection fraction on cardiac function during sepsis. Liposome curcumin showed reduced cardiac damage compared to standard of care and was equal to standard of care in percent ejection fraction.

Figure 4G is a graph showing the effect of liposomal curcumin on overall survival during sepsis. Liposome curcumin showed longer survival compared to standard-of-care treatments.

Claims (22)

A method of ameliorating or treating a symptom or one or more adverse reactions triggered by the widespread release of cytokines in an individual, the method comprising the steps of: Identifying individuals in need of amelioration or treatment of symptoms of one or more infectious diseases or disease conditions that trigger widespread release of cytokines; and One or more pharmaceutical compositions are administered comprising a therapeutically effective amount of lipid dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the cytokine concentration in the subject. The method of claim 1, wherein the widespread release of cytokines is by at least one of one or more selected from the group consisting of viral, bacterial, fungal, helminthic, protozoal, or hemorrhagic infectious agents caused by infectious diseases. The method of claim 1, wherein the one or more infectious diseases are selected from at least one of the following infections: rhinoviruses, coronaviruses, Paramyxoviridae, Orthomyxoviridae, adenoviruses, paraendemics Parainfluenza Virus, Metapneumovirus, Respiratory Syncytial Virus, Influenzavirus, Arenaviridae, Filoviridae, the Bunyaviridae, Flaviviridae, Rhabdoviridae virus, Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever Congo hemorrhagic fever; CCHF) virus, South American hemorrhagic fever virus, dengue virus, yellow fever virus, Rift Valley fever virus, Omsk hemorrhagic fever virus ), Kyasanur Forest virus, Junin, Machupo, Sabiá, Guanarito, Garissa, Ilesha or Lassa fever virus. The method of claim 1, wherein the one or more disease conditions are selected from at least one of the following: cachexia, septic shock syndrome, chronic inflammation, septic shock syndrome, traumatic brain injury, cerebral cytokine storm, Graft versus host disease (GVHD), autoimmune disease, multiple sclerosis, acute pancreatitis, or hepatitis. The method of claim 1, wherein the one or more disease conditions are due to therapy with anti-CD19 chimeric antigen receptor (CAR) T cells or anti-tumor cells, activated dendritic cells, activated macrophages, or activated B cells Adverse reactions caused by cell therapy. The method of claim 1, wherein the composition further comprises curcumin extract, curcumin, and curcuminoids configured in lipids, wherein the curcuminoids are selected from at least one of the following: aryl curcumin (Ar -tumerone), methyl curcumin, demethoxycurcumin, bisdemethoxycurcumin, sodium curcumin, dibenzoylmethane, acetyl curcumin, feruloyl methane (feruloyl methane), tetrahydro Curcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin 1), 1,7-bis(piperonyl) (piperonyl))-1,6-heptadiene-3,5-dione (piperonyl curcumin), 1,7-bis(2-hydroxynaphthyl)-1,6-heptadiene-2,5 - Diketone (2-hydroxynaphthylcurcumin) and 1,1-bis(phenyl)-1,3,8,10-undecatetraene-5,7-dione. 9. The method of claim 1, wherein the lipid or phospholipid is selected from the group consisting of: dimyristophospholipid choline (DMPC), dimyristophospholipid glycerol (DMPG), dipalmitophospholipid choline (DPPC), Distearyl Phosphatidyl Glycerol (DSPG), Dipalmito Phosphatidyl Phosphatidyl Glycerol (DMPG), Phosphatidyl Choline, Lysophosphatidylcholine, Lysophosphatidylethanolamine, lysoDMPC, lysoDMPG, lysoDSPG, lysoDPPC, Phosphatidyl Filament Amino acid, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, phosphatidylcholine, and dipalmitophosphatidylglycerol, stearylamine, ten Dialkylamine, Cetylamine, Acetyl Palmitate, Glycerin Ricinoleate, Cetyl Stearate, Isopropyl Myristate, Amphoteric Acrylic Polymer, Fatty Acid, Fatty Acid Amide , cholesterol, cholesterol esters, diacylglycerol, and diacylglycerol succinate. The method of claim 1, wherein the therapeutically effective amount comprises 50 nM/kg, 10 to 100 nM/kg, 25 to 75 nM/kg, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 nM/kg individual body weight. The method of claim 1, wherein the composition comprises an active agent and has a lipid phospholipid to active agent ratio of 3:1, 1:1, 0.3:1 and 0.1:1. The method of claim 1, wherein the disease is rheumatoid arthritis, psoriasis, multiple sclerosis, relapsing multiple sclerosis, or inflammatory bowel disease. A composition for the amelioration or treatment of symptoms or one or more adverse reactions triggered by an infectious disease or disease condition that triggers widespread release of cytokines in an individual, comprising a therapeutically effective amount of a lipid or lysophospholipid dissolved or dispersed in a suitable in aqueous or non-aqueous media. The composition of claim 11, wherein the one or more infectious diseases are selected from at least one of viral, bacterial, fungal, helminthic, protozoal, or hemorrhagic infectious agents. The composition of claim 11, wherein the one or more infectious diseases are selected from at least one of the following infections: rhinovirus, coronavirus, Paramyxoviridae, Orthomyxoviridae, adenovirus, parainfluenza Viruses, Interstitial Pneumonia Virus, Respiratory Fusion Virus, Influenza Virus, Arenaviridae, Filoviridae, Benjaviridae, Yellow Fever Viruses, Baculoviridae, Ebola Virus, Marburg Virus, Crimean-Congo haemorrhagic fever (CCHF) virus, South American haemorrhagic fever virus, Dengue virus, Yellow fever virus, Rift Valley fever virus, Omsk haemorrhagic fever virus, Kejeldahl virus, Junin virus, Maqiubo virus , Sabia virus, Guanarito virus, Garissa virus, Ilexia virus or Lyssa virus. The composition of claim 11, wherein the one or more disease conditions are selected from at least one of the following: cachexia, septic shock syndrome, chronic inflammation, septic shock syndrome, traumatic brain injury, cerebral cytokine storm , Graft Versus Host Disease (GVHD), Autoimmune Disease, Multiple Sclerosis, Acute Pancreatitis, or Hepatitis. The composition of claim 11, wherein the curcumin extract, curcuminoid or synthetic curcumin is formulated in lipids. 11. The composition of claim 11, wherein the lipid or lysophospholipid is selected from the group consisting of: dimyristine phospholipid choline (DMPC), dimyristine phospholipid glycerol (DMPG), dipalmitophospholipid Choline (DPPC), Distearyl Phosphatidyl Glycerol (DSPG), Dipalmito Phosphatidyl Glycerol (DMPG), Phosphatidyl Choline, Lysophosphatidylcholine, Lysophosphatidylethanolamine, lysoDMPC, lysoDMPG, lysoDSPG, lysoDPPC, Phosphatidylserine, Phosphatidylinositol, Sphingomyelin, Phosphatidylethanolamine, Cardiolipin, Phosphatidic Acid, Cerebroside, Dicetyl Phosphate, Phosphatidylcholine, and Dipalmitoyl Phosphatidylglycerol, Stearyl Amine, Laurylamine, Cetylamine, Acetyl Palmitate, Glycerin Ricinoleate, Cetyl Stearate, Isopropyl Myristate, Amphoteric Acrylic Polymers, Fatty Acids, Fatty acid amides, cholesterol, cholesterol esters, diacylglycerol, and diacylglycerol succinate. The composition of claim 11, wherein the biodegradable polymer is selected from the group consisting of polyesters, polylactic acids, polyglycolides, polycaprolactones, polyanhydrides, polyamides Amines, polyurethanes, polyesteramides, poly-dioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyorthoesters, polyphosphoric acids ester, polyphosphazene, polyhydroxybutyrate, polyhydroxyvalerate, polyalkylene oxalate, polyalkylene succinate, poly(malic acid), poly(amino acid), copolymer, Terpolymers, and combinations or mixtures thereof. The composition of claim 11, wherein the composition is suitable for intravenous, subcutaneous, intramuscular or intraperitoneal injection into the individual. The composition of claim 11, wherein the composition further comprises curcumin or curcuminoids selected from at least one of the following: aryl curcumin, methyl curcumin, demethoxy curcumin, bisdemethoxy curcumin base curcumin, curcumin sodium, dibenzoylmethane, acetyl curcumin, ferulic acid methane, tetrahydrocurcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-Heptadiene-3,5-dione (Curcumin 1), 1,7-Bis(piperonyl)-1,6-heptadiene-3,5-dione (Piperonyl Curcumin), 1 ,7-bis(2-hydroxynaphthyl)-1,6-heptadiene-2,5-dione (2-hydroxynaphthylcurcumin) and 1,1-bis(phenyl)-1,3, 8,10-undecatetraene-5,7-dione. The composition of claim 11, wherein the composition comprises an active agent and has a lipid to active agent ratio of 3:1, 1:1, 0.3:1 and 0.1:1. A method of determining whether a drug candidate results in amelioration or treatment of symptoms or one or more adverse effects in an individual triggered by an infectious disease or disease condition that triggers widespread release of cytokines, the method comprising: (a) administering an amount of the drug candidate in combination with empty liposomes, and a placebo to a second subgroup of patients, wherein the drug candidate is provided in an amount effective to reduce or prevent the overall concentration of cytokines in the subject ; (b) measuring individual cytokine concentrations from patients of the first and second groups; and (c) determine whether the combination of the drug candidate and empty liposomes results in a statistically significant improvement or treatment of an infectious disease caused by triggering widespread release of cytokines compared to any reduction occurring in the subgroup of patients taking the placebo A symptom or one or more adverse effects triggered by a disease condition wherein a statistically significant reduction indicates that the drug candidate is useful for treating the disease state while also reducing or eliminating the overall concentration of cytokines in the individual. A method of ameliorating or treating a symptom or cytokine storm caused by a therapeutic agent in an individual, the method comprising the steps of: Identifying individuals in need of amelioration or treatment of symptoms or cytokine storms caused by therapeutic agents; and Administration of one or more pharmaceutical compositions comprising a therapeutically effective amount of curcumin extract, curcuminoid or synthetic curcumin and derivatives thereof, or empty liposomes, dissolved or dispersed in a sufficient amount to reduce cytokines in the individual appropriate concentration in aqueous or non-aqueous media.

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