(en)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, or empty liposomes, dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the level of cytokines in the host.

1.ApplicationNumber: US-201514983844-A
1.PublishNumber: US-2016184330-A1
2.Date Publish: 20160630
3.Inventor: SORDILLO PETER P.
HELSON LAWRENCE
BURGESS STEPHEN W.
SHAW WALTER A.

4.Inventor Harmonized: SORDILLO PETER P(US)
HELSON LAWRENCE(US)
BURGESS STEPHEN W(US)
SHAW WALTER A(US)

5.Country: US
6.Claims:
(en)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, or empty liposomes, dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the level of cytokines in the host.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Ser. No. 62/098,898 filed Dec. 31, 2014, U.S. Provisional Application Ser. No. 62/165,567 filed May 22, 2015, and U.S. Provisional Application Ser. No. 62/211,450 filed Aug. 28, 2015, the entire contents of which are incorporated herein by reference.


STATEMENT OF FEDERALLY FUNDED RESEARCH
This invention was made with U.S. Government support by the USAMRIID under Project No. 1323839. The government has certain rights in this invention.


TECHNICAL FIELD OF THE INVENTION
The present invention relates in general to the field of infectious diseases and disease conditions that trigger a cytokine cascade, and more particularly, to the use of compositions that reduce the cytokine cascade.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in connection with infectious diseases and disease conditions that trigger, e.g., an anaphylactic cytokine cascade.
U.S. Pat. No. 8,354,276, issued to Har-Noy, entitled, “T-cell compositions that elicit type I cytokine response”, relates to a method of manipulating allogeneic cells for use in allogeneic cell therapy protocols is described. The method provides a composition of highly activated allogeneic T-cells, which are infused into immunocompetent cancer patients to elicit a novel anti-tumor immune mechanism called the “Mirror Effect”. The inventors argue that, in contrast to current allogeneic cell therapy protocols where T-cells in the graft mediate the beneficial graft vs. tumor (GVT) and detrimental graft vs. host (GVH) effects, the allogeneic cells of the invention stimulate host T-cells to mediate the “mirror” of these effects. The highly activated allogeneic cells of the invention are said to stimulate host immunity in a complete HLA mis-matched setting in patients that have not had a prior bone marrow transplant or received chemotherapy and/or radiation conditioning regimens.
U.S. Pat. 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 pathologic angiogenesis. These inventors teach methods for producing and screening compounds and compositions capable of inhibiting vascular permeability and pathologic angiogenesis. It is said that the compositions described are useful in, methods of inhibiting vascular permeability and pathologic angiogenesis, including methods of inhibiting vascular permeability and pathologic angiogenesis induced by specific angiogenic, permeability and inflammatory factors, such as, for example VEGF, βFGF and thrombin.
U.S. Pat. No. 7,479,498, issued to Keller, is entitled “Treatments for viral infections” and relates to improved methods and compositions for treating viral infections and other diseases and conditions that induce a cytokine storm. It is further said that the invention relates to novel compositions comprising quercetin, and an anti-convulsant, such as phenytoin, in combination with multivitamins as an anti-viral composition and methods of use thereof.
United States 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 human binding proteins and antigen-binding fragments thereof that specifically bind to the human interleukin-21 receptor (IL21R), and uses therefore. The invention is said to include methods to predict whether the binding proteins of the invention may take on agonistic activities in vivo and produce a cytokine storm. In addition, the 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, it is said that the binding proteins can act as antagonists of IL21R activity, thereby modulating immune responses in general, and those mediated by IL21R in particular.
SUMMARY OF THE INVENTION
In one embodiment, the present invention includes a method of 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 liposome or a lysophosphatidyl-monoglyceride-fatty acid eutectic, dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the level of cytokines in the subject. In one aspect, the one or more infectious diseases are selected from at least one of viral, bacterial, fungal, helminthic, protozoan, or hemorrhagic infectious agents. In another aspect, the one or more infectious diseases is selected from at least one of infection with an Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae virus. In another aspect, the one or more infectious diseases is selected from at least one of Ebola, Marburg, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Rift Valley fever, Omsk hemorrhagic fever virus, Kyasanur Forest, Junin, Machupo, Sabia, Guanarito, Garissa, Ilesha, or Lassa fever viruses. In another aspect, the one or more disease conditions is selected from at least one of cachexia, septic shock syndrome, a chronic inflammatory response, septic shock syndrome, graft versus host disease (GVHD), traumatic brain injury (e.g., cerebral cytokine storm), autoimmune diseases, multiple sclerosis, acute pancreatitis, or hepatitis. In another aspect, the one or more disease conditions is an adverse reaction caused by the treatment with anti-CD19 Chimeric Antigen Receptor (CAR) T cells or antitumor cell therapy, activated dendritic cells, activated macrophages, or activated B cells. In another aspect, the composition further comprises curcumin extract, curcuminoids or synthetic curcumin are disposed in the liposome and comprise a lipid or a phospholipid wall. In another aspect, the lipid or the phospholipid is selected from the group consisting of phosphatidylcholine (lecithin), lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine, and dipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine, hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, fatty acid, fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, and diacylglycerolsuccinate. In another aspect, 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 of body weight of the subject. In another aspect, the synthetic curcumin is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96% pure diferuloylmethane. In another aspect, the composition further comprises a curcumin or curcuminoids are selected from at least one of Ar-tumerone, methylcurcumin, demethoxy curcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane, tetrahydrocurcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1), 1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin) 1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxyl naphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dione. In another aspect, the composition comprises a liposome or liposome precursors comprising a lysophosphatidyl compound, a monoglyceride, and free fatty acid, and in certain aspects the ratios of the composition are 1:4:2, 1:3:3, 2:4:2, or 1:2:4 mole percent lysophosphatidyl compound:monoglyceride:free fatty acid. In another aspect, the composition comprises a eutectic mixture comprising a LysoPG, a myristoyl monoglyceride, and a myristic acid. In another aspect, the composition comprises an active agent and the composition has a ratio of phospholipids to active agent of 3:1, 1:1, 0.3:1, and 0.1:1. In another aspect, the eutectic does not interact with the active agent in vivo. In another aspect, the eutectic is provided separately from the active agent to the subject. In another aspect, the eutectic is provided in an amount that reduces QT prolongation in the subject. In another aspect, the eutectic is provided in an amount that reduces an anti-inflammatory response. In another aspect, the eutectic is provided in an amount that treats rheumatoid arthritis, psoriasis, multiple sclerosis, relapsing multiple sclerosis, or inflammatory bowel disease. In another aspect, the eutectic is provided in an amount that reduces the expression or activity of at least one of IL-1β, IL-6, TNF-α, MCP-1, MIP-1, or Rantes. In another aspect, the subject is suspected of having a traumatic brain injury and the eutectic is provided in an amount that reduces the expression or activity of at least one of TNF-α, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12, Interferon-γ, MCP-1 (CCL2), MIP-1α (CCL3), or TGF-β in the brain.
In another embodiment, the present invention includes a composition 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 a therapeutically effective amount of empty liposomes or a lysophosphatidyl-monoglyceride-fatty acid eutectic, dissolved or dispersed in a suitable aqueous or non-aqueous medium. In another aspect, the one or more infectious diseases are selected from at least one of viral, bacterial, fungal, helminthic, protozoan, or hemorrhagic infectious agents. In another aspect, the one or more infectious diseases is selected from at least one of infection with an Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae virus. In another aspect, the one or more infectious diseases is selected from at least one of Ebola, Marburg, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Rift Valley fever, Omsk hemorrhagic fever virus, Kyasanur Forest, Junin, Machupo, Sabia, Guanarito, Garissa, Ilesha, or Lassa fever viruses. In another aspect, the one or more disease conditions is selected from at least one of cachexia, septic shock syndrome, a chronic inflammatory response, septic shock syndrome, traumatic brain injury (e.g., cerebral cytokine storm), graft versus host disease (GVHD), autoimmune diseases, multiple sclerosis, acute pancreatitis, or hepatitis. In another aspect, the one or more disease conditions is an adverse reaction caused by the treatment with anti-CD19 Chimeric Antigen Receptor (CAR) T cells or antitumor cell therapy, activated dendritic cells, activated macrophages, or activated B cells. In another aspect, the composition further comprises a curcumin extract, curcuminoids or synthetic curcumin are disposed in a liposome and comprise a lipid or a phospholipid wall. In another aspect, the liposome comprises a lipid or phospholipid, wherein lipid or the phospholipid is selected from the group consisting of phosphatidylcholine (lecithin), lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, phosphatidylcholine, and dipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine, hexadecyl-amine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, fatty acid, fatty acid amides, cholesterol, cholesterol ester, diacylglycerol, and diacylglycerolsuccinate. In another aspect, the composition further comprises a biodegradable polymer selected from the group consisting of polyesters, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyorthoesters, polyphosphoesters, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), copolymers, terpolymers, and combinations or mixtures thereof. In another aspect, the composition adapted for intravenous, subcutaneous, intramuscular, or intraperitoneal injection in the subject. In another aspect, the composition further comprises a synthetic curcumin is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96% pure diferuloylmethane. In another aspect, the curcumin or curcuminoids are selected from at least one of Ar-tumerone, methylcurcumin, demethoxy curcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane, tetrahydrocurcumin, 1, 7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione (curcumin1), 1,7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin) 1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxyl naphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dione. In another aspect, the composition comprises a liposome or liposome precursors comprising a lysophosphatidyl compound, a monoglyceride, and free fatty acid, and in certain aspects the ratios of the composition are 1:4:2, 1:3:3, 2:4:2, or 1:2:4 mole percent lysophosphatidyl compound:monoglyceride:free fatty acid. In another aspect, the composition comprises a eutectic mixture comprising a LysoPG, a myristoyl monoglyceride, and a myristic acid. In another aspect, the composition comprises an active agent and the composition has a ratio of phospholipids to active agent of 3:1, 1:1, 0.3:1, and 0.1:1. In another aspect, the eutectic does not interact with the active agent in vivo. In another aspect, the eutectic is provided separately from the active agent to the subject. In another aspect, the eutectic is provided in an amount that reduces QT prolongation in the subject. In another aspect, the eutectic is provided in an amount that reduces an anti-inflammatory response. In another aspect, the eutectic is provided in an amount that treats rheumatoid arthritis, psoriasis, multiple sclerosis, relapsing multiple sclerosis, or inflammatory bowel disease. In another aspect, the eutectic is provided in an amount that reduces the expression or activity of at least one of IL-1β, IL-6, TNF-α, MCP-1, MIP-1, or Rantes. In another aspect, the subject is suspected of having a traumatic brain injury and the eutectic is provided in an amount that reduces the expression or activity of at least one of TNF-α, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12, Interferon-γ, MCP-1 (CCL2), MIP-1α (CCL3), or TGF-β in the brain.
In another aspect, the present invention includes a method of ameliorating symptoms or treating one or more diseases or conditions that comprise a cytokine storm in a subject comprising the steps of: identifying the subject in need of amelioration of symptoms or treatment of the diseases or conditions triggered by a cytokine storm; and administering one or more pharmaceutical compositions comprising a therapeutically effective amount of an empty liposome or a lysophosphatidyl-monoglyceride-fatty acid eutectic, dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the level of cytokines in the subject, wherein the liposome comprises a eutectic mixture comprising a LysoPG, a myristoyl monoglyceride, and a myristic acid. In another aspect, the composition comprises a liposome or liposome precursors comprising a lysophosphatidyl compound, a monoglyceride, and free fatty acid, and in certain aspects the ratios of the composition are 1:4:2, 1:3:3, 2:4:2, or 1:2:4 mole percent lysophosphatidyl compound:monoglyceride:free fatty acid. In another aspect, the composition comprises a eutectic mixture comprising a LysoPG, a myristoyl monoglyceride, and a myristic acid. In another aspect, the composition comprises an active agent and the composition has a ratio of phospholipids to active agent of 3:1, 1:1, 0.3:1, and 0.1:1. In another aspect, the eutectic does not interact with the active agent in vivo. In another aspect, the eutectic is provided separately from the active agent to the subject. In another aspect, the eutectic is provided in an amount that reduces QT prolongation in the subject. In another aspect, the eutectic is provided in an amount that reduces an anti-inflammatory response. In another aspect, the eutectic is provided in an amount that treats rheumatoid arthritis, psoriasis, multiple sclerosis, relapsing multiple sclerosis, or inflammatory bowel disease. In another aspect, the eutectic is provided in an amount that reduces the expression or activity of at least one of IL-1β, IL-6, TNF-α, MCP-1, MIP-1, or Rantes. In another aspect, the subject is suspected of having a traumatic brain injury and the eutectic is provided in an amount that reduces the expression or activity of at least one of TNF-α, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12, Interferon-γ, MCP-1 (CCL2), MIP-1α (CCL3), or TGF-β in the brain.
In another embodiment, the present invention includes a method of determining if a candidate drug causes an amelioration symptoms or treats one or more adverse reactions triggered by an infectious disease or a disease condition that trigger a widespread release of cytokines in a subject, the method comprising: (a) administering an amount of the candidate drug in combination with empty liposomes or a lysophosphatidyl-monoglyceride-fatty acid eutectic, and a placebo to a second subset of the patients, wherein the candidate drug is provided in an amount effective to reduce or prevent the overall level of cytokines in the subject; (b) measuring the level of cytokines in the subject from the first and second set of patients; and (c) determining if the candidate drug in combination with empty liposomes ameliorates symptoms or treats one or more adverse reactions triggered by infectious diseases or disease conditions that trigger a widespread release of cytokines is statistically significant as compared to any reduction occurring in the subset of patients that took the placebo, wherein a statistically significant reduction indicates that the candidate drug is useful in treating a disease state while also reducing or eliminating the overall level of cytokines in the subject. In another aspect, the composition comprises a liposome or liposome precursors comprising a lysophosphatidyl compound, a monoglyceride, and free fatty acid, and in certain aspects the ratios of the composition are 1:4:2, 1:3:3, 2:4:2, or 1:2:4 mole percent lysophosphatidyl compound:monoglyceride:free fatty acid. In another aspect, the composition comprises a eutectic mixture comprising a LysoPG, a myristoyl monoglyceride, and a myristic acid. In another aspect, the composition comprises an active agent and the composition has a ratio of phospholipids to active agent of 3:1, 1:1, 0.3:1, and 0.1:1. In another aspect, the eutectic does not interact with the active agent in vivo. In another aspect, the eutectic is provided separately from the active agent to the subject. In another aspect, the eutectic is provided in an amount that reduces QT prolongation in the subject. In another aspect, the eutectic is provided in an amount that reduces an anti-inflammatory response. In another aspect, the eutectic is provided in an amount that treats rheumatoid arthritis, psoriasis, multiple sclerosis, relapsing multiple sclerosis, or inflammatory bowel disease. In another aspect, the eutectic is provided in an amount that reduces the expression or activity of at least one of IL-1β, IL-6, TNF-α, MCP-1, MIP-1, or Rantes. In another aspect, the subject is suspected of having a traumatic brain injury and the eutectic is provided in an amount that reduces the expression or activity of at least one of TNF-α, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12, Interferon-γ, MCP-1 (CCL2), MIP-1α (CCL3), or TGF-β in the brain.
In another embodiment, the present invention includes a method of ameliorating symptoms or treating a cytokine storm caused by a therapeutic agent in a subject comprising the steps of: identifying the subject in need of amelioration of symptoms or treatment of the cytokine storm caused by a therapeutic agent; 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, empty liposomes or a lysophosphatidyl-monoglyceride-fatty acid eutectic, dissolved or dispersed in a suitable aqueous or non-aqueous medium sufficient to reduce the level of cytokines in the subject. In one aspect, the therapeutic agent is selected from at least one of anti-CD19 Chimeric Antigen Receptor (CAR) T cells, an antitumor cell therapy, activated dendritic cells, activated macrophages, a tissue graft, or activated B cells. In another aspect, the composition comprises a liposome or liposome precursors comprising a lysophosphatidyl compound, a monoglyceride, and free fatty acid, and in certain aspects the ratios of the composition are 1:4:2, 1:3:3, 2:4:2, or 1:2:4 mole percent lysophosphatidyl compound:monoglyceride:free fatty acid. In another aspect, the composition comprises a eutectic mixture comprising a LysoPG, a myristoyl monoglyceride, and a myristic acid. In another aspect, the composition comprises an active agent and the composition has a ratio of phospholipids to active agent of 3:1, 1:1, 0.3:1, and 0.1:1. In another aspect, the eutectic does not interact with the active agent in vivo. In another aspect, the eutectic is provided separately from the active agent to the subject. In another aspect, the eutectic is provided in an amount that reduces QT prolongation in the subject. In another aspect, the eutectic is provided in an amount that reduces an anti-inflammatory response. In another aspect, the eutectic is provided in an amount that treats rheumatoid arthritis, psoriasis, multiple sclerosis, relapsing multiple sclerosis, or inflammatory bowel disease. In another aspect, the eutectic is provided in an amount that reduces the expression or activity of at least one of IL-1β, IL-6, TNF-α, MCP-1, MIP-1, or Rantes. In another aspect, the subject is suspected of having a traumatic brain injury and the eutectic is provided in an amount that reduces the expression or activity of at least one of TNF-α, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12, Interferon-γ, MCP-1 (CCL2), MIP-1α (CCL3), or TGF-β in the brain.



BRIEF DESCRIPTION OF THE DRAWINGS
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 along with the accompanying figures and in which:
FIGS. 1A and 1B show the percent inhibition and percent viability, respectively, achieved with liposomal curcumin in HeLa cells.
FIGS. 2A and 2B show the percent inhibition and percent viability, respectively, using solid S-curcumin curcumin in HeLa cells.
FIGS. 3A and 3B show the percent inhibition and percent viability, respectively, comparing liposomal curcumin and solid curcumin in HeLa cells.
FIG. 4 shows the cytokine data (IL-6 and TNF-α) with empty liposomes compared with EU8120.



DETAILED DESCRIPTION OF THE INVENTION
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present 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 delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
As used herein, the term “cytokine storm” refers to the dysregulated of pro-inflammatory cytokines leading to disease has been referred to as a “cytokine storm,” “cytokine release syndrome” or “inflammatory cascade”. Often, a cytokine storm or cascade is referred to as being part of a sequence because one cytokine typically leads to the production of multiple other cytokines that can reinforce and amplify the immune response. Generally, these pro-inflammatory mediators have been divided into two subgroups: early mediators and late mediators. Early mediators, such as e.g., tumor-necrosis factor, interleukin-1, interleukin-6, are not sufficient therapeutic targets for re-establishing homeostatic balance because they are resolved within the time frame of a patient's travel to a clinic to receive medical attention. In contrast, the so-called “late mediators” have been targeted because it is during this later “inflammatory cascade” that the patient realizes that he or she has fallen ill.
Infectious diseases commonly associated with a “cytokine storm” include but at 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 is selected from at least one of Ebola, Marburg, Crimean-Congo hemorrhagic fever (CCHF), South American hemorrhagic fever, dengue, yellow fever, Rift Valley fever, Omsk hemorrhagic fever virus, Kyasanur Forest, Junin, Machupo, Sabiá, Guanarito, Garissa, Ilesha, or Lassa fever viruses.
Disease conditions commonly associated with a “cytokine storm” include but at not limited to: sepsis, systemic inflammatory response syndrome (SIRS), cachexia, septic shock syndrome, traumatic brain injury (e.g., cerebral cytokine storm), graft versus host disease (GVHD), or the result of treatment with activated immune cells, e.g., IL-2 activated T cells, T cells activated with anti-CD19 Chimeric Antigen Receptor (CAR) T cells.
Generally, a cytokine storm is a healthy systemic expression of a vigorous immune system. The present invention can be used to reduce or eliminate some or most of an exaggerated immune response caused by, e.g., rapidly proliferating and highly activated T-cells or natural killer (NK) cells that results in the release of the “cytokine storm” that can include more than 150 inflammatory mediators (cytokines, oxygen free radicals, and coagulation factors). Both pro-inflammatory cytokines (such as Tumor Necrosis Factor-α, Interleukin-1, and Interkeukin-6) and anti-inflammatory cytokines (such as Interleukin-10, and Interleukin-1 receptor antagonist (IL-1RA)) become greatly elevated in, e.g., serum. It is this excessive release of inflammatory mediators that triggers the “cytokine storm.”
In the absence of prompt intervention, such as that provided by the present invention, a cytokine storm can result in permanent lung damage and, in many cases, death. The end stage symptoms of the cytokine storm include but are not limited to: hypotension; tachycardia; dyspnea; fever; ischemia or insufficient tissue perfusion; uncontrollable hemorrhage; severe metabolism dysregulation; and multisystem organ failure. Deaths from infectious diseases such as Ebola virus infection are not caused by the virus itself, but rather, the cytokine storm that causes uncontrollable hemorrhaging; severe metabolism dysregulation; hypotension; tachycardia; dyspnea; fever; ischemia or insufficient tissue perfusion; and multisystem organ failure.
As used herein the term “Curcumin (diferuloyl methane; 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)” is a naturally occurring compound which is the main coloring principle found in the rhizomes of the plant Curcuma longa (U.S. 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 diferuloylmethane. Non-limiting examples of curcumin and curcuminoids include, e.g., Ar-tumerone, methylcurcumin, demethoxy curcumin, bisdemethoxycurcumin, sodium curcuminate, dibenzoylmethane, acetylcurcumin, feruloyl methane, tetrahydrocurcumin, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (curcumin1), 1, 7-bis(piperonyl)-1,6-heptadiene-3,5-dione (piperonyl curcumin) 1,7-bis(2-hydroxy naphthyl)-1,6-heptadiene-2,5-dione (2-hydroxyl naphthyl curcumin) and 1,1-bis(phenyl)-1,3,8,10 undecatetraene-5,7-dione.
The term “liposome” refers to a capsule wherein the wall or membrane thereof is formed of lipids, especially phospholipid, with the optional addition therewith of a sterol, especially cholesterol. In one specific non-limiting example the liposomes are empty liposomes and can be formulated from a single type of phospholipid or combinations of phospholipids. The empty liposomes can further includes one or more surface modifications, such as proteins, carbohydrates, glycolipids or glycoproteins, and even nucleic acids such as aptamers, thio-modified nucleic acids, protein nucleic acid mimics, protein mimics, stealthing agents, etc. In one embodiment, the liposome is a liposome or a liposome precursor comprising a lysophosphatidyl-monoglyceride-fatty acid eutectic, such as a eutectic that includes: lysophosphatidyl compound, a monoglyceride, and free fatty acid, and in certain aspects the ratios of the composition are 1:4:2, 1:3:3, 2:4:2, or 1:2:4 mole percent lysophosphatidyl compound:monoglyceride:free fatty acid. The composition may comprise a eutectic mixture comprising a LysoPG, a myristoyl monoglyceride, and a myristic acid. In one specific, non-limiting example the composition also comprises an active agent in or about the liposome and the composition has a ratio of phospholipids to active agent of 3:1, 1:1, 0.3:1, and 0.1:1.
As used herein, the term “in vivo” refers to being inside the body. The term “in vitro” as used in the present application is to be understood as indicating an operation carried out in a non-living system.
As used herein, the term “treatment” refers to the treatment of the conditions mentioned herein, particularly in a patient who demonstrates symptoms of the disease or disorder. As used herein, the term “treating” refers to any administration of a compound of the present invention and includes (i) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology) or (ii) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology). The term “controlling” includes preventing treating, eradicating, ameliorating or otherwise reducing the severity of the condition being controlled.
The terms “effective amount” or “therapeutically effective amount” described herein means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. In one example, 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 of body weight of the subject.
The terms “administration of” or “administering a” compound as used herein should be understood to mean providing a compound of the invention to the individual in need of treatment in a form that can be introduced into that individual's body in a therapeutically useful form and therapeutically useful amount, including, but not limited to: oral dosage forms, such as tablets, 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 deleterious to the recipient thereof.
The curcumin formulation of the present invention may comprise one or more optional pharmaceutical excipients, diluents, extended or controlled release agents, lubricants, preservatives or any combination thereof, and once solubilized may be added to injectable anti-diabetic medications or administered in a schedule depending upon the release kinetics of the curcumin formulation. A large number of biodegradable polymers may be used in the formulation of the present invention. Non-limiting examples of these polymers include polysesters, polylactides, polyglycolides, polycaprolactones polyanhydrides, polyamides, polyurethanes, polyesteramides, polydiaxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyorthoesters, polyphosphoesters, polyphosphazenes, polyhydroxybuterates, polyhydroxyvalerates, polyalkelene oxalates, polyalkylene succinates, poly(malic)acid, poly(amino)acids, copolymers, terpolymers, and combinations or mixtures thereof. Specific polymers that may be used include an acrylic acid, a vinylpyrolidinome, a N-isopropylacrylamide or combinations and modifications thereof. The synthesized curcumin that is used includes curcumin, curcumin analogues, curcumin derivatives and any modifications thereof.
Treatment of Infectious Diseases. The terminal stage of Ebola and other viral diseases is often the onset of cytokine storm, the massive overproduction of cytokines by the body's immune system. The present invention includes the treatment of infectious agents that trigger a cytokine storm, such as Ebola virus, with curcumin actions to suppress cytokine release and cytokine storm.
It was found that curcumin blocks cytokine release, most importantly the key pro-inflammatory cytokines, interleukin-1, interleukin-6 and tumor necrosis factor-α. Curcumin's suppression of cytokine release correlates with clinical improvement in experimental models of disease conditions where cytokine storm plays a significant role in mortality. Thus, curcumin can be used to treat the cytokine storm of patients with Ebola. In certain examples, intravenous formulations allow achievement of therapeutic blood levels.
The high fatality rate in patients infected with the Ebola virus is thought to be due partly to the onset of cytokine storm in the advanced stages of the infection 1-2 . Cytokine storm can occur after a wide variety of infectious and non-infectious stimuli. In cytokine storm, numerous cytokines, both pro-inflammatory (IL-1, IL-6, TNF-α) and anti-inflammatory (IL-10), are released, resulting in hypotension, hemorrhage, and, ultimately, multi-organ failure. The term “cytokine storm” is most associated with the 1918 H1N1 influenza pandemic and the more recent cases of bird flu H5N1 infection 3-5 . In these cases, young people, with presumably healthy immune systems, died disproportionally from the disease, and aberrant activity of their immune systems is thought to be the cause. This syndrome has also been known to occur in advanced or terminal cases of SARS 6 , Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis 7 , gram negative sepsis 8 , malaria 9 and numerous other infectious diseases. Cytokine storm can occur from non-infectious causes, such as acute pancreatitis 10 , severe burns or trauma 11 or acute respiratory distress syndrome secondary to drug use or inhalation of toxins 12 . 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 multi-organ failure in the 6 human volunteers who received this agent. This was despite the fact that the dose of this agent given was 500 times lower than had been found to be safe in animals 13 .
Curcumin suppression of Cytokines.
Curcumin has been shown to inhibit the release of numerous cytokines. Abe et al showed that curcumin suppresses IL-1β, IL-8, TNF-α, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) release from monocytes and macrophages 14 . Jain et al., showed that curcumin markedly reduced the release of IL-6, IL-8, TNF-α and MCP-1 from monocytes that had been cultured in a high glucose environment 15 . These same investigators studied rats with streptozotocin-induced elevated plasma blood sugar levels and significantly elevated levels of IL-6, TNF-α and MCP-1; these levels were markedly reduced by curcumin 15 . Curcumin has been reported to block the release of IL-6 in rheumatoid synovial fibroblasts 16 , of IL-8 in human esophageal epithelial cells 17 and alveolar epithelial cells 18 , and of IL-1 in bone marrow stromal cells 19 , colonic epithelial cells 20 and human articular chondrocytes 21 . Curcumin also prevents release of IL-2 22 , IL-12 22-23 , Interferon-γ 22-23 and many other key cytokines 24-26 (Tables 1 and 2).
Example 1
Curcumin Cytokine Suppression Correlates with Clinical Improvement in Conditions Associated with Cytokine Storm
Curcumin has positive effects on numerous disease conditions in patients and in animal systems. Avasarala et al reported on curcumin's effects on cytokine expression and disease progression in a mouse model of viral-induced acute respiratory distress syndrome. Curcumin reduced the expression of key cytokines IL-6, IL-10, interferon γ and MCP-1, and this correlated with a marked decrease in inflammation and reduction in fibrosis 27 . Yu et al showed curcumin's suppression of TNF-α levels was associated with decreased pancreatic injury in an acute pancreatitis mouse model 28 . Cheppudira et al reported that curcumin's suppression of IL-8 and GRO-α, and ultimately with NF-κB, correlated with reduction in thermal injury in a rat model 29 . Curcumin suppression of cytokines also correlates with clinical improvement in models of severe viral infection. Song et al showed that curcumin administration reduced expression of IL-1β, IL-6 and TNF-α and ultimately NF-κB, and protected against coxsackie virus-induced severe myocardial damage in infected mice 30 . Curcumin has been shown to have activity against numerous viruses, including HIV-1, HIV-2, HSV, HPV, HTLV-1, HBV, HCV, and Japanese encephalitis virus 31 . In addition, curcumin has been shown to have specific activity against the H1N1 virus in culture 32-33 , although cytokine levels were not measured in these two studies. Most importantly, curcumin has been shown to stimulate the SOCS proteins 34 . These proteins have been shown to be crucial in protecting against severe cytokine storm in mice infected with influenza virus 35 .
Curcumin's activity in suppressing multiple cytokines, and its activity in experimental models of diseases and conditions associated with cytokine storm, suggest it may be useful in the treatment of patients with Ebola and cytokine storm. Curcumin is poorly absorbed from the intestinal tract; however intravenous formulations may allow therapeutic curcumin blood levels to be achieved in patients diagnosed with cytokine storm. Clinical status and levels of important cytokines, such as IL-1β, IL-6 and TNF-α, should be monitored carefully when patients are treated with curcumin.





TABLE 1



Curcumin Effect on Interleukins




Biomolecule
Key Functions



↓ IL-1
Major pro-inflammatory cytokine, 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 cytokine, B-cell differentiation,


nerve cell differentiation

↓ IL-8
Neutrophil chemotaxis, angiogenesis

↑ IL-10
Anti-inflammatory cytokine, also has T-cell stimulatory


effects

↓ IL-11
Induces acute phase proteins, antigen-antibody reactions,


bone remodeling

↓ IL-12
Defense against intracellular pathogens

↓ IL-13
Induces matrix metalloproteinases, induces IgE

↓ IL-17
Pro-inflammatory cytokine








































TABLE 2



Curcumin Suppression of Other Key Cytokines




Biomolecules
Key Functions



↓ TNF-α
Major pro-inflammatory cytokine, insulin resistance,


induces secretion of corticotropin releasing hormone

↓ Interferon γ
Macrophage activation, T and B cell activation


and differentiation

↓ MCP-1
Neuroinflammation, monocyte and basophil chemotaxis

(CCL2)

↓ MIP-1α
Activates granulocytes, induces synthesis of

(CCL3)
pro-inflammatory cytokines

↓ GROα
Neutrophil chemoattractant, angiogenesis, wound healing

(CXCL1)

↓ GROβ
Neutrophil and monocyte chemoattractant

(CXCL2)

↓ IP-10
Monocyte and macrophage chemoattractant, NK cell

(CXCL10)
chemoattractant, angiogenesis

↓ SDF-1
Lymphocyte chemoattractant, angiogenesis, suppresses

(CXCL12)
osteoclastogenesis




































Test results. First study.
Liposomes and Liposomal-Curcumin were prepared as a 6 mg/ml solutions. Curcumin (solid) was solubilized in DMSO at 6 mg/ml. All three compounds were tested in EBOV infection assay with two cell lines Hela and HFF-1. There were two sets for studies done with different dilution strategy.
Hela cell lines were used in two independent experiments (replica 1 and replica 2) and HFF-1 in one. 2 hours prior infection Liposome And Curcumin-Liposomal were diluted in media from highest concentration of 600 ug/ml (final in assay) to generate 10 points for dose response curve with 2 fold step dilution. 5 ul of each dose was dispensed by PE Janus 384-tip dispenser into assay wells with cells. The Curcumin (solid) was dispensed by HP D300 directly from the 100% DMSO stock into assay wells with cells. DMSO was normalized in all wells to final 1%. Each dose were tested 4 times on the plate n=4.
Test results. Second study.
Both cell line were used in one study (replicate 1). 2 hours prior infection Curcumin-Lip and Lip. were diluted in Media from highest concentration of 60 ug/ml (final in assay) to generate 10 points for dose response curve with 2 fold step dilution. In this case titration was done with manual mixing and changing tips for each new dose. Curcumin (solid) was tittered manually in DMSO and then equal amount for each dose was diluted 1/10 in media with mixing. 5 ul of each dose was dispensed by PE Janus 384-tip dispenser into assay wells with cells. Each dose were 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 MOI=3 for HFF-1 and Infection was stopped in 48 h by fixing cells in formalin solution. To detect infected cells the immunostaining was done using anti-GP antibody. Images were taken by PE Opera confocal platform with 10× objective, analyzed using Acapella software.
Signal for GP-staining was converted into % infection. The number of nuclei per well was used to determine % viability of cells (in comparison to infected but untreated controls well n=16). Data was analyzed using GeneData software and % Infection converted into % Inhibition (% INH) using plate controls.
FIGS. 1A and 1B show the percent inhibition and percent viability, respectively, achieved with liposomal curcumin in HeLa cells. FIGS. 2A and 2B shows the percent inhibition and percent viability, respectively, using solid S-curcumin curcumin in HeLa cells. Similar results were obtained with the same study using HFF-1 cells.
FIGS. 3A and 3B show the percent inhibition and percent viability, respectively, comparing liposomal curcumin and solid curcumin in HeLa cells. Similar results were obtained with the same study using HFF-1 cells.
The anti-EBOV activity was determined in Hela cells and it was correlating with “cytotoxicity”. Briefly, the results are as follows: (1) Curcumin-liposomal EC 50 =2.5±0.2 ug/ml, safety index=2. Liposomes EC 50 =3.9±0.2 ug/ml, safety index=4. Curcumin EC 50 =6.5±0.5 ug/ml, safety index=1. As such, liposomes show EC 50 =0.6±2 ug/ml with very good safety index ˜50.
Pro-Inflammatory Cytokines in the Causation of The Prolonged QT Interval: Role of the Ceramide and Sphingosine-1 Phosphate Pathways. There is increasing evidence that excess levels of pro-inflammatory cytokines play a major role in the pathogenesis of the prolonged QT syndrome. In anti-cancer trials, QT prolongation was noted as a side effect of the cytokine, interferon γ, and QT prolongation has been 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 die more frequently secondary to this complication. It has been shown that the degree of QT prolongation correlates directly with the extent of elevation of the key pro-inflammatory cytokines, TNF-α, IL-1β and IL-6. In large-scale studies of normal populations, it was found that asymptomatic elevations of these cytokines correlate with QT prolongation. In trials of tocilizumab, an IL-6 blocker, in patients with rheumatoid arthritis, a shortening of the duration of the previously prolonged QT interval was noted, and the degree of QT shortening correlated with the decrease in serum inflammatory markers. Studies in animal models and in cultured cardiomyocytes have shown that TNF-α suppressed IKr, IKs and Ito. This was thought to be due to the stimulation of reactive oxygen species (ROS). TNF-α and other cytokines have been shown to cause increased production of ROS. The effects of TNF-α could be blocked by administration of an anti-TNF-α antibody or by an anti-oxidant. IL-1β and IL-6 have been shown to increase the L-type Ca(2 + ) current (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 phospholipidosis also are hERG channel blockers. Phospholipidotic cells have been shown to secrete large amounts of TNF-α and IL-6 after LPS stimulation. It has also been speculated that the mechanism of damage from drug-induced phospholipidosis is accumulation of ceramides. Numerous studies have shown that cytokines such as interferon γ, IL-1β and TNF-α increase sphingomyelinase activation, and increase production of ceramides, which are known to suppress hERG current. It is also known that sphingolipids mediate ROS signaling. Ceramides are metabolized to sphingosine and fatty acids, and sphingosine is phosphorylated by sphingosine kinases to form sphingosine-1 phosphate. Ceramides and sphingosine-1 phosphate have opposite effects, ceramides causing apoptosis and sphingosine-1 phosphate promoting cell survival. Fingolimod, a sphingosine analogue (which has both agonist and antagonist effects on the sphingosine-1 phosphate-1 receptor), is used to treat patients with relapsing multiple sclerosis, causes QT prolongation through inhibition of the hERG current, as well as fatal ventricular arrhythmias. Further, studies in the mouse model of influenza-induced cytokine storm have shown sphingosine-1 phosphate-1 signaling to be the primary pathway for activation of the cytokine storm. The cytokine storm was reversed by a sphingosine analogue through feedback inhibition of cytokines, with marked reductions in TNF-α, IL-1α, IL-6, MCP-1, interferon α and MIP-1α, and clinical improvement seen. The survival of the mice in the study was much higher with the sphingosine analogue than with anti-viral therapy. It is also noted that agents found clinically to be suppressors of QT prolongation, progestins, statins, liposomal curcumin, resveratrol, anti-oxidants, are also strong suppressors of these inflammatory cytokines. Even β-blockers, the standard treatment for patients with prolonged QT, seem to have suppression of cytokines as part of their mechanism of action in treating these patients. All these agents shift the balance away from the ceramide pathway toward the sphingosine-1 phosphate pathway. Thus, excess levels of pro-inflammatory cytokines play a major role in the causation of the prolonged QT syndrome, probably through stimulation of ROS and the ceramide pathway.
By way of explanation, and in no way a limitation of the present invention, a proposed mechanism for correction of the previously prolonged QT interval by liposomal curcumin and EU8120 is described. There is a great deal of evidence from early phase 2 anti-cancer trials of pro-inflammatory cytokines, and from recent studies on patients with rheumatoid arthritis, psoriasis and inflammatory bowel disease, suggesting increased levels of pro-inflammatory cytokines cause prolongation of the QT interval. These patients have a markedly increased incidence of prolonged QT. In addition, in trials of an anti-IL-6-antibody in patients with rheumatoid arthritis, shortening of the prolonged QT was noted, which correlated with the decrease in cytokines. The correlation of increased cytokine levels and asymptomatic QT prolongation is also found in large-scale studies of normal populations. In animal models and in ventricular myocytes, TNF-α administration causes a decrease in the rapid component of the delayed rectifier potassium current (IKr), in the slow component of the delayed rectifier current (IKs) and in the transient outward current (Ito). These effects are thought to be due to stimulation of reactive oxygen species (ROS) and can be blocked by administration of an anti-TNF-α antibody 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 in other diseases that ROS increases ceramide production, thus shifting the balance from the sphingosine-1-phosphate (S1P) pathway (protective) to the ceramide pathway (destructive). Multiple studies in experimental models have shown that ceramides cause suppression of the hERG current. It has been proposed that statins, which reduce levels of pro-inflammatory cytokines and cause shortening of the prolonged QT, may have as their underlying mechanism the stimulation of this protective S1P pathway. Other agents that shorten the prolonged QT, including anti-oxidants, such as vitamin E, reduce levels of pro-inflammatory cytokines and of ROS, and stimulate S1P. Finally, the present inventors have recognized that the list of agents which cause both cytokine suppression and shortening of the previously prolonged QT interval is strikingly similar to the list of agents that have been shown in animal models to reduce cytokine levels and secondary brain inflammation and also reduce the degree of brain damage. Thus, the present invention can be used to target those diseases that increase ceramide production, thus shifting the balance from the sphingosine-1-phosphate (S1P) pathway (protective), to the ceramide pathway (destructive).
The present inventors have shown, in both in vitro and in vivo models, that Liposomal Curcumin and EU8120 reduce IL-1β, IL-6, TNF-α, MCP-1, MIP-1 and Rantes. Liposomes have also been shown, in other models, to compete for the enzyme sphingomyelinase and to reduce levels of ceramides, thus also shifting the ceramide/S1P balance toward S1P.
LPS-induced cytokine storm produces QTc prolongation, which is prevented by an anti-inflammatory eutectic blend. There is increasing evidence that excess levels of pro-inflammatory cytokines play a major role in the pathogenesis of the prolonged QT syndrome. Inversely, blockers such as tocilizumab (IL-6), or anti-cytokine antibodies (TNFα) contribute to a shortening of the previously-prolonged QT interval.
In this study, LPS and Kdo2-Lipid-A were used to induce cytokine release in guinea-pigs with concomitant ECG monitoring and blood draws, followed by Q-ELISA measurement of cytokine production. The guinea pig was selected because it yields reliable QTc prolongation as a result of pro-arrhythmic challenge, with consistently visible T-waves on the ECG. Male adult guinea pigs received 300 μg/kg LPS at time 0, and had ECGs analyzed at 1 h, 2 h, and 4 hours post-LPS, with simultaneous blood draw. Animals receiving LPS only exhibited a 8-msec increase in QTc after 1 h post-LPS, when TNFα levels were maximal at 5.5-fold the pre-LPS values. A 29-msec QTc prolongation 2 h post-LPS correlated with 7- and 9-fold increases in IL-1β and IL-6, respectively. The QTc prolongation remained (27 msec) after 4 hours post-LPS, when the animals were euthanized. When 9 mg/kg EU8120 (a lipid blend shown to prevent IKr-channel block by a variety of hERG blockers) was given 1 hour prior to LPS-induction, QTc prolongation was limited to 5 ms after 2 hours, and completely prevented at 1 and 4 hours post-LPS. Plasma levels of TNFα, IL1β, and IL-6 were significantly lower in EU8120-administered animals. This example demonstrates that EU8120 suppresses QTc prolongation via an anti-inflammatory cytokine-effect and not by any interaction with the active agent (LPS).
Synthetic Curcumin.
The present invention can use the compositions to treat the cytokine storm disorders using synthetic curcumin (S-curcumin).
Curcumin is the active principle of the turmeric plant, which has been synthesized to near purity (99.2%). It is formulated with liposomes, polymers, or PLGM to render it capable of being administered intravenously as a bolus or as a continuous infusion over 1-72 hours in combination with other active agents. Curcumin has antioxidant and anti-inflammatory activity, and can block autonomous intracellular signaling pathways abnormally responsive to extracellular growth factors, uncontrolled proliferation of cells and fibrosis-associated and tissue degenerative conditions. Specifically, Curcumin reacts negatively with components of key signaling pathways commanding proliferation, metabolism, survival and death.
Oral and topical administration of the extract of the turmeric plant has been used in traditional medicine for over two thousand years. While oral administration is devoid of systemic toxicity it is also devoid of systemic therapeutic activity. This is due to blood insolubility, and intestinal wall and hepatic inactivation, i.e. it has negligible bioavailability for systemic diseases by the oral route. To overcome these limitations, parenteral intravenous curcumin formulations with liposomes, polymers (n-isopropylacrylamide, N-vinylpyrrolidione and acrylic acid) and polylactic glycolic acid copolymer were entered into in pre-clinical drug development. 4
Curcumin as an extract of turmeric root is available to researchers as a mixture of three curcuminoids and to the public as a food supplement or spice according to the FDA. The extract is 79.2% curcumin (diferuloylmethane), 18.27% demethoxycurcumin, and 2.53% bisdemethoxycurcumin.
Synthesized curcumin is GMP grade 99.2% pure diferuloylmethane produced for non-human experimental study and future Phase I clinical trials. There are obvious differences between the C3 three component extract and the single component synthesized S-curcumin that extend to discernable analytic, physicochemical, and biological characteristics. In certain aspects, the diferuloylmethane is 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96% pure diferuloylmethane.
The present invention relates to synthetic curcumin (S-curcumin) and compares the properties and the activity of S-curcumin with liposomal curcumin, NANOCURC®, and PLGA-curcumin (hereinafter C3-complex).
Liposomal curcumin: The initial studies of liposomal curcumin were done using material bought as the complex. 6-7 Studies with S-curcumin are Mach C M, et al (2009) 8 and Mach C M et al (2010) 9 .
NANOCURC®: The initial study of Nanocurc® was done using product bought as the complex Savita Bisht et al (2007) 10 used a non-sabinsa source. Since then studies with S-curcumin are used in the remainder of Nanocurc® publications. 11-13
PLGA-curcumin: The initial studies of PLGA-curcumin were done using product manufactured as the C3-complex. 14-18 Studies included PLGA-curcumin C3 complex and PLGA-S-curcumin pharmacokinetic studies in rat brains.
Comparison of PLGA C3-complex-curcumin vs PLGA S-curcumin indicated the following differences. The solubilities of 99.2% S-Curcumin in all four solvents. Ethanol, Ethyl acetate, Acetone, and Acetonitrile, differed significantly from the C3 complex containing 76% curcumin. When normalized to equal concentrations, the pure material has greater solubility. This confers improved manufacturing capability, and attributes to different pharmacokinetics and pharmacodynamics in in vivo settings (Table 3).





TABLE 3



Solubilities of S-curcumin and C3-complex curcumin

in different organic solvents.












Physical
1 g


Wt. of
Vol. of
Conc.
appearance
of solvent


Curcumin
solvent
(mg/
of
solubilize in

Solvent
(mg)
(μL)
mL)
solubility
solvent (mL)





Batch-C100609, Curcumin = 99.2%








Ethyl
20.8
2000
10.4
Partial
96.2

acetate

Ethanol
17.5
4600
3.804
Partial
263

Acetone
46.5
1100
42.3
Fully
23.6

Acetonitrile
17.4
3200
5.44
Partial
184



Batch-C3 complex, Curcumin = 76%








Ethyl
21.5
1800
11.9
Fully
83.7

acetate

Ethanol
28.4
6500
4.37
Fully
229

Acetone
39.8
700
56.9
Fully
17.6

Acetonitrile
18.2
2900
6.28
Fully
159



*Weight the sample and transfer into a scintillation vial. Add small volume of solvent and shake well repeat for each addition till the solubility.




















































TABLE 4



A comparison of differences 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
172-175 C.
179.8-181.9 C.


Tapped bulk density
0.61 g/ml
0.26 g/ml


Loose bulk density
0.37 g/ml
0.18 g/ml


Sieve test


−40 mesh
100%
64.71%


−80 mesh
98.27%
64.0%


HPLC content curcumin
79.2%
99.2%


Total curcuminoids
96.21%
99.2%


Bisdemethoxycurcumin
2.53%
0


Demethoxycurcumin
18.27
0


Heavy Metals


Lead
0.91 ppm (ug/g)
<0.2 ppm


Arsenic
0.54 ppm (ug/g)
<0.2 ppm


Cadmium
<0.2 ppm (ug/g)
—


Mercury
<0.02 ppm (ug/g)
—


Residual solvents
complies
complies


Micro-total Plate count
<100 cfug
10 cfu/g


Yeast and mold count
<10 cfu/g
15 cfu/g



Esherichia coli

neg/10 g
neg/10 g



Salmonella

neg/10 g
neg/10 g



Staph aureus

neg/10 g
neg/10 g



Pseudomonas aeruginosa

neg/10 g
neg/10 g

















































FIG. 4 shows the cytokine data (IL-6 and TNF-α) with empty liposomes compared with the eutectic EU8120. The EU8120 used was constituted of a 1:4:2 ratio of 14:0 LysoPG/myristoyl monoglyceride/myristic fatty acid chain. EU was dissolved in water (stock 4 mM) and extensively vortexted before being added to the cells. RAW264 macrophages were pre-incubated for 24 h with empty liposomes or EU8120 before being stimulated for 24 h with KDO2 (10 ng/ml) or LPS (100 ng/ml). These data show that EU8120 like empty liposomes inhibited IL-6 and TNF-α production in KDO2 and LPS-stimulated macrophage. Empty liposomes and EU8120 were used at concentrations from 1-20 microM.
As used herein, the term “eutectic blend” refers to a mixture of chemical compounds or elements that have a chemical composition that solidifies at a lower temperature than other composition made up of the same ingredients. For example, one blended eutectic (referred to herein as EU8120) is composed of three components (14:0 Lysophosphatidylglycerol (Lyso PG), Myristoyl monoglyceride, and Myristic acid, a free fatty acid). In one non-limiting example, the blended eutectic can be made to enhance the oral bioavailability of LysoPG.
14:0 Lysophosphatidylglycerol (Lyso PG)









Myristoyl Monoglyceride









Myristic Acid, a Free Fatty Acid









In one non-limiting example, EU8120 was constituted of a 1:4:2 ratio of 14:0 LysoPG/myristoyl monoglyceride/myristic acid chain (also referred to as myristic fatty acid). The ration can be changed to have a constituent ratio of, e.g., 2:4:2, 3:4:2, 4:4:2 (i.e., increasing the LysoPG content of EU8120).
Example 2
Additional Studies
Brain damage after traumatic brain injury (TBI) is a two-stage process: the injury caused by the initial insult is followed by a stage of inflammation where a great deal of additional damage may occur. This inflammation begins within minutes of the initial insult and can continue for months or years, and results from a complex series of metabolic processes involving marked increases in cytokines, particularly the pro-inflammatory cytokines, interleukin-1β, interleukin-6 and tumor necrosis factor-α. Levels of these cytokines may increase thousands of times more than the corresponding levels in serum. Strategies to control the levels of these pro-inflammatory cytokines and to reduce the cytokine-induced brain damage are discussed. There is extensive evidence from experiments in animal models that suppression of cytokines is effective in ameliorating neurologic damage after TBI. However, the efficacy of this approach remains to be proven in patient trials.
It is increasingly recognized that an aberrant immune system and a massive overproduction of pro-inflammatory cytokines, a ‘cytokine storm’, is a major factor in the disease progression and the mortality from numerous diseases. Cytokine storm, also known as ‘cytokine release syndrome,’ can occur after infection with malaria [1], SARS [2], dengue [3], leptospirosis [4], Lassa fever [5], gram-negative sepsis [6] as well as with numerous other infectious diseases (7-10]. Cytokine storm is a major cause of death in patients with Ebola [11-13]. Patients with cytokine storm may experience increased vascular permeability, severe hemorrhage and multi-organ failure, which may ultimately be the cause of a fatal outcome [8, 13, 14]. Marked increases in systemic cytokine levels, of both pro-inflammatory and anti-inflammatory cytokines, are seen. It is thought that this overproduction of cytokines by healthy immune systems is the explanation for why individuals from 20 to 40 were more likely to die than the elderly during the 1918 H1N1 pandemic [15, 16]. Cytokine storm can occur after severe burns or trauma [17], with acute pancreatitis [18], or with ARDS secondary to drug use or inhalation of toxins [19]. Severe acute graft vs. host disease can be considered a cytokine storm [20, 21]. Cytokine storm is also a recognized complication of treatment with the commonly-used antineoplastic agent rituximab [22], as well as of treatment with the monoclonal antibodies, tositumomab, alemtuzumab, muromonab and blinatumomab [23]. Elevated levels of cytokines are found and are thought to be an important cause of the pathology in many neurological conditions, including Alzheimer's disease [24], Parkinson's disease [25], autism [26], and multiple sclerosis [27], as well as in the acute phase of Guillian-Barre syndrome [28, 29]. Increased cytokine levels have been linked to exacerbations of psychiatric illnesses [30, 31], and of 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 each year [34], and neuropsychiatric sequalae are common, especially after severe injury [35]. It is now understood that cerebral damage after traumatic brain injury occurs in two stages: an initial stage where damage occurs from the external mechanical force, and a secondary inflammatory stage where damage can occur due to a cascade of processes involving cytokines such as interleukin (IL)-1β, IL-6, and tumour necrosis factor (TNF)-α [36]. The increases in cytokine levels in the brain can be massive, especially after severe TBI. IL-6 is not usually detectable in CSF, or is detectable in only very low concentrations (1-23 pg/ml) [37, 38]. In one study, CSF levels of IL-6 as high as 35 500 pg/ml were seen after severe TBI [38, 39]. These IL-6 levels were 40-100× greater than the corresponding levels in the serum of these patients [40]. Kushi et al reported very large increases in both IL-6 and IL-8, measured on admission, at 24 hours, at 72 hours and at 168 hours after severe TBI in 22 patients. In the nine fatalities, average IL-6 values at these times in the CSF were 15 241, 97 384, 548 225 and 336 500 pg/ml compared to 102, 176, 873, 3 059 pg/ml in the blood, a ‘storm’ of cytokines mostly localized to the brain. For the 13 survivors, average IL-6 CSF values were lower, but still much greater than in the peripheral blood: 5 376, 3 565, 328 and 764 pg/ml compared to 181, 105, 37 and 26 pg/ml in the blood [41]. Similar differences were seen for IL-8. Whereas IL-8 levels in the CSF are normally very low (5-72 pg/ml) [37], Kushi et al reported CSF IL-8 levels that were consistently elevated thousands of times more than normal levels or comparable levels in the peripheral blood [41]. These investigators also noted that IL-6 and IL-8 blood levels that remained markedly elevated after 72 hours correlated with a worse prognosis and high fatality rate. Helmy et al found marked elevations of multiple cytokines, including IL-1α, IL-1β, IL-6, IL-8, IL-10, monocyte chemotactic protein (MCP-1) and macrophage inflammatory protein-la (MIP-1α), in brain extracellular fluid after severe TBI in 12 patients. These levels were also significantly elevated compared to the corresponding blood levels [42]. Other investigators have reported similar results, and have noted that very high cytokine levels correlate with a poor prognosis [43, 44]. For example, Arand et al noted that IL-6 levels were eight-fold higher in patients who died compared to those who survived. In addition, only patients who died showed increased levels of another pro-inflammatory cytokine, IL-12 [43]. These data further support, the hypothesis that a cytokine storm is responsible for increased neurological damage after TBI. A number of studies suggest that some of these same cytokines can have beneficial as well as harmful effects on the brain [45-47]. However, it has been shown in numerous studies that blockage of these cytokines, at least in animal models, can reduce the cerebral damage after TBI. A list of key cytokines that are elevated in the brain and CSF after TBI is given in Table 5.





TABLE 5



Key cytokines show marked increases in brain and CSF after TBI




Cytokine
Function



TNF-α
Major pro-inflammatory cytokine, insulin resistance, stimulates apoptosis,


may also have neuroprotective functions

IL-1α
Pro-inflammatory cytokine, stimulates TNF-α release from endothelial


cells

IL-1β
Major pro-inflammatory cytokine, regulates production of IL-2, IL-6, IL-8


and interferon-γ, stimulation of phagocytosis and programmed cell death,


early CNS development

IL-2
Pro-inflammatory cytokine, T-cell lymphocyte differentiation

IL-4
Anti-inflammatory cytokine, also stimulates B-cell proliferation

IL-6
Major pro-inflammatory cytokine, B-cell differentiation, neurogenesis,


myokine, also has anti-inflammatory functions (suppression of IL-1 and


TNF-α)

IL-8
Major pro-inflammatory cytokine (chemokine), neutrophil chemotaxis

(CXCL8)

IL-10
Major anti-inflammatory cytokine, also has T-cell stimulatory functions

IL-12
Pro-inflammatory cytokine, T-cell differentiation

Interferon-γ
Pro-inflammatory cytokine, T and B cell lymphocyte and macrophage


activation

MCP-1
Pro-inflammatory cytokine, monocyte and basophil chemotaxis

(CCL2)

MIP-1α
Pro-inflammatory cytokine, induces synthesis of pro-inflammatory

(CCL3)
cytokines, activates neutrophils

TGF-β
Anti-inflammatory cytokine, also has pro-inflammatory functions, may


have long-term harmful effects












































Interleukin-1. The IL-1 family is a group of 11 cytokines which are intimately involved in the body's response to injury or infection [48, 49], and which also play a key role in tumour angiogenesis [50] and stimulation of cancer stem cells [51]. The most important cytokines of 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, as well as several less well-studied cytokines. The key cytokine IL-1β is a protein produced by activated macrophages. Among its 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 induction of programmed cell death [48, 49]. Increased levels of IL-1β have been found in the CSF of patients with TBI, and may be detected within minutes of acute injury [38, 52, 53]. Very high levels in the CSF of TBI patients have been associated with a worsening prognosis [54, 55].
Studies in animal models have given similar results [56-60]. Kamm et al showed that IL-1β levels appeared in the rat brain after TBI within the first hour and peaked at 8 hours, with no detectable change in IL-1β levels in the blood or liver [56]. It has also been shown, in animal models, that intraventricular administration of IL-1β significantly worsens cerebral damage [61]. Most importantly, administration of an IL-1β antagonist can prevent the damage caused by this cytokine in experimental models. Administration of IL-1RA to rodents has been shown to reduce brain damage after TBI. For example, Yang et al showed that the cerebral damage caused by middle cerebral artery occlusion in mice was reduced in those animals that were previously transfected with an adenoviral vector to induce IL-1RA overexpression [62]. Jones et al showed that a single intracerebroventricular dose of IL-1RA administered to mice at the time of TBI reduced lesion volume, resulted in functional improvement and caused a major decrease in nitric oxide synthase-2-positive cells in the lesion [63-Jones]. Sanderson et al studied the effect of systemically-administered IL-1RA to Sprague Dawley rats after TBI. No effect was seen at low doses. After high-dose administration, the investigators observed decreased neuronal loss and an increase in memory and cognitive function in the animals. No improvement was seen in motor function, however [64]. Hasturk et al showed IL-1RA reduced tissue IL-1β levels and increased levels of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase in rats after TBI [65]. Other groups have reported similar results [66, 67]. In addition, Basu et al reported that mice lacking the IL-1 receptor experience less brain injury after a traumatic insult [68]. The investigators found decreased basal levels of IL-1, IL-6 and COX-2, as well as fewer amoeboid microglia/macrophages, suggesting the cycle of brain inflammation was prevented at this crucial step. Further, Tehranian et al have shown that transgenic mice who overexpress human IL-IRA in astrocytes have decreased levels of IL-1β, IL-6 and TNF-α compared to wild type mice, and have better neurological recovery after head injury [69].
These data suggest that the use of IL-1RA might be an effective strategy in patients with TBI. Human recombinant IL-1RA has been a standard medication for patients with rheumatoid arthritis for several years, and its use has been investigated in a number of diseases where increased cytokines play a role in the destructive process, including diabetes [70], heart failure [71], multiple myeloma [72] and sepsis [73]. In a randomized phase II trial of patients with acute stroke, there was less loss of cognitive function in patients treated with IL-1RA compared to the control group [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]. They were unable to conclude that IL-1RA administration resulted in therapeutic benefit in these patients [75]. While many of these results seem promising, however, the efficacy of IL-1RA may be limited, as it directly blocks only one of the important cytokines involved in the inflammation (IL-1RA may block other cytokines indirectly since IL-1 can cause increased expression of other cytokines), and this may be part of the explanation for the failure of this agent to have a greater than limited success against rheumatoid arthritis. Further, the use of IL-1RA in combination with TNF-α blockers is contraindicated, as severe side effects may result from their concomitant use [76].
Tumor necrosis factor-α. A second key pro-inflammatory cytokine is TNF-α. This cytokine plays an important role in the body's response to infections and to cancer. Since the report on TNF-α by Helson et al in 1975 [77], aberrant TNF-α function has been reported in numerous diseases, including conditions as diverse as diabetes [78], cardiovascular disease [79], inflammatory bowel disease [80] and Alzheimer's disease [81]. TNF blockers, such as infliximab, etanercept, and adalimumab, are standard therapies for patients with rheumatoid arthritis, ankylosing spondylitis and psoriasis. As noted, TNF-α is thought to have both beneficial and detrimental effects in patients with TBI [46]. However, results in experimental models suggest that these effects are mostly detrimental, especially when excessive levels of this cytokine are produced. Knoblach et al reported the correlation of TNF levels and the degree of brain injury and neurological impairment in rats after experimental TBI, with the highest levels of TNF at 1-4 hours after injury in rats with the most severe brain injury [82]. In addition, studies with the TNF-blocker, etanercept, have consistently shown reduction of brain damage in these animals after administration of this agent. Chio et al reported that etanercept, when given to rats after TBI reduced ischemia, increased glutamate levels, reduced neuronal and glial apoptosis and microglial activation, while also reducing the increased levels of TNF-α [83]. In a later report, these investigators concluded etanercept ameliorates brain injury by decreasing the early expression of TNF-α by microglia [84]. Ekici et al showed that the combination of etanercept and lithium chloride administered one hour after TBI reduced cerebral edema, tissue damage and TNF levels [85]. Cheong et al showed that etanercept administered to rats immediately after TBI resulted in increased 5-bromodeoxyuridine and doublecortin markers in the injured brain, suggesting that the increased TNF-α levels in the brain may be toxic to neural stem cells, thus interfering with neurogenesis [86]. Wang et al reported that the early use of this agent after injury promoted the survival of transplanted neural stem cells and facilitated neural regeneration [87]. Other groups have reported similar results using etanercept or other TNF blockers [88-91].
Although TNF blockers have been studied extensively 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 with TBI who had been treated with etanercept. Marked improvement in neurological function was observed, even for patients treated more than 10 years after the initial insult. The investigators concluded that this supported the view that long-term inflammation, perhaps lasting many years, was a major cause of neurological impairment 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 not clear that TNF blockade was responsible for the observed improvement. Randomized trials are needed to prove benefit in TBI patients, and TNF blockers may have substantial toxicity. In addition, since TNF blockers target only a single cytokine, and since the use of these agents is contradicted in combination with IL-1 antagonists, the use of these blockers may not be the most effective strategy in treatment of these patients.
Interleukin-6. A third major pro-inflammatory cytokine is IL-6. As with TNF-α, elevated levels of IL-6 have been thought to have a role in the causation of numerous diseases, and like TNF-α, IL-6 is thought to have beneficial as well as harmful effects after TBI [94]. Indeed, IL-6 appears to have both a beneficial and a deleterious role in a number of neurological conditions [95]. IL-6 plays a key role in 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 suggesting IL-6 is necessary for neuronal recovery. The IL-6 knockout mice did, however, show significantly elevated levels of IL-1β [96]. The neuroprotective role of IL-6 was also suggested in a study of frontal lobe parenchymal IL-6 levels in patients after severe TBI. Markedly elevated IL-6 levels were found in survivors compared to those who died, while levels of IL-1β were not different [97]. However, the numbers in this study were small.
On the other hand, numerous studies have suggested that IL-6 has harmful 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 to 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 after mild TBI could be corrected by IL-6 blockade [101]. Similar results were reported in experimental spinal cord injury. Okada et al showed that an anti-IL-6 receptor mouse monoclonal antibody could increase functional spinal cord recovery in mice after injury [102]. Nakamura et al reported that an antibody to IL-6R decreased glial scar formation and increased recovery after spinal injury [103]. Crack et al reported that anti-lysophosphatidic acid antibodies markedly reduced brain damage in mice after experimental TBI. The investigators attributed this to a dramatic reduction in IL-6 induced secondary inflammation. The antibodies had no effect on levels of IL-1β or TNF-α [104]. Suzuki et al have suggested that the divergent results seen in these studies might be explained because IL-6's inflammatory effect seems to dominate in the acute phase after TBI, while its effect on neurogenesis may be important later on [105]. Little work has been done to investigate IL-6 blockers in patients with TBI. An anti-IL-6 antibody, tocilizumab, is available, and is used for treatment of 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)-β, can also be markedly elevated in inflammatory conditions. One of the major functions of these cytokines is to inhibit synthesis of pro-inflammatory cytokines [107]. IL-10 is the most important anti-inflammatory cytokine, and IL-10 levels are markedly elevated in the brain and CSF after TBI [54, 108]. Although IL-10 is known to also have pro-inflammatory functions [107], its main effect after TBI appears to be primarily protective against inflammatory damage. Kumar et al studied cytokine levels 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 culture and in animal models seem to confirm the protective effect of IL-10. Bachis et al showed IL-10 blocks caspase-3 and reduces neuronal death after exposure of rat cerebellar granule cells in culture to toxic doses of glutamate [110]. Knoblach et al showed that either intravenous or subcutaneous administration of IL-10 after experimental TBI in rats could reduce synthesis of IL-1 and enhance neurological recovery in the animals. Intracerebroventricular administration was not effective, however [111]. Chen et al showed that mice deficient in IL-10 failed to respond to the beneficial effects of hyperbaric oxygen treatment 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 were also seen in other studies of experimental spinal cord injury [114, 115]. This suggests another approach to the treatment of TBI in patients might be administration of an anti-inflammatory cytokine like IL-10. Trials of recombinant human IL-10 (ilodecakin) have been done in a number of diseases. However, results have so far been disappointing [116].
Targeting Multiple Cytokines.
Progestins. It is well known, from studies in animal systems, that progestins can reduce neuronal damage after TBI [117-121]. A major mechanism for the neuroprotection seen with progestins is the ability of these agents to suppress pro-inflammatory cytokines. Cutler et al showed that progesterone given to aged male rats after TBI reduced brain levels of IL-6 at 24, 48 and 72 hours. Decreased levels of NF-κB and COX-2 were also seen, and the rats demonstrated improved motor skills, decreased cerebral edema and decreased mortality (122-Cutler). He et al reported that intraperitoneal administration of progesterone could reduce IL-1β and TNF-α at 3 hours after injury. Similar results were seen after administration of another progestin, allopregnanolone [123]. Chen et al reported that progesterone given to rats following TBI decreased levels of IL-1β, IL-6 and TNF-α in the brain, as well as reducing apoptosis of brain tissue [124]. Pan et al showed intraperitoneal administration of progesterone reduced brain levels of TNF-α and NF-κB 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 given within 8 hours of TBI [126]. However, large multicentre trials have not confirmed this. A large phase III trial of progesterone in patients with TBI conducted by The Neurologic Emergencies Treatment Trials Network was stopped early because of lack of efficacy [127]. A second major trial, SYNAPSE, a multinational, placebo-controlled trial of progesterone in 1195 patients with severe TBI, also showed no efficacy. Among the progesterone group, only 50.4% showed a favorable outcome on the Glasgow outcome scale, compared to 50.5% of patients who received placebo [128, 129].
Statins. These are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, which are used to inhibit cholesterol production in the liver. These drugs are widely utilized clinically in patients with hypercholesterolemia. Statins are also known to have marked anti-inflammatory effects. Chen et al showed that lovastatin pre-administered to rats with experimental TBI caused marked decreases in IL-1β and TNF-α in the areas of brain injury at 6 hours and at 96 hours post-injury. Treated rats had significantly reduced FJB-positive degenerating neurons, and better functional recovery [130]. Simvastatin was shown to decrease brain levels of IL-1β and to reduce microglial and astrocyte activation in rats after TBI, with functional improvement on the NCS score. No change in IL-6 or TNF-α levels was noted, however [131]. Atorvastatin was found to lower both IL-6 and TNF-α in mice after TBI. Hippocampal degeneration and functional neurological deficits were reduced in the treated animals compared to controls [132]. There is also evidence that discontinuation of these statins in patients may lead to an increase in pro-inflammatory cytokines, including IL-6 [133-135], and that stopping these medications after TBI seems to lead to a worse prognosis [136]. Further, a retrospective study suggests pre-injury statin use is associated with better outcomes [137]. This has led to the suggestion that these agents be studied in patients with TBI. Only a few small trials have been reported. Tapia-Perez et al investigated the effect of rosuvastatin in patients with severe TBI and reported there was a reduction in amnesia time in the treated patients [138]. However, there was no difference in disability at 3 months. Further, this trial included only 8 rosuvastatin patients and 13 controls, while 21 of the 43 assessed TBI patients were deemed ineligible. In another small study, of 19 patients receiving 10 days of rosuvastatin and 17 controls, Sanchez-Aquilar et al reported that the rosuvastatin patients had a dramatic decrease in plasma levels of TNF-α compared to placebo and an improvement in disability scores. No effect was seen on IL-1β, IL-6 or IL-10 [139]. Rasras et al investigated the effects of a similar agent, simuvastatin, in a randomized trial of 66 patients with severe TBI; however, no difference was found between the treated and the control groups [140].
Tetracyclines. Tetracyclines have been shown, in animal models, to suppress inflammation and better outcomes in several neurological conditions. Bye et al showed that minocycline could reduce IL-1β and IL-6 expression and microglial and macrophage activation in mice after TBI. Neurological functioning was better at day 1 in treated mice, although there was no difference between treated mice and controls at day 4 [141]. Later studies by this same group did show, however, comparative improvement in the minocycline group by 6 weeks [142]. Shanchez Mejia et al reported that minocycline given to mice after TBI reduced IL-1β by inhibiting caspase-1 activation, resulting in improved neurological function and decreased lesion volume in the treated animals [143]. Lee et al showed that minocycline given to rats after spinal cord injury reduced TNF-α, increased IL-10, reduced neuronal cell death and improved motor function [144]. Yrjanheikki reported that either doxycycline or minocycline could reduce mRNA induction of IL-1β converting enzyme and protect against neuronal death after ischemic stroke [145]. Other investigators have also reported positive results with tetracyclines in animal models of TBI [146-148]. However, Turtzo et al could demonstrate no benefit in rats treated with minocycline after TBI [149]. Further, in another study, minocycline was found to cause increased ischemic brain injury in the neonatal mouse [150, 151].
Other anti-inflammatory agents. A number of other agents have shown anti-inflammatory activity in animal models of TBI. Melatonin was reported to decrease TNF-α and IL-1β and increase the number of surviving neurons in mice after TBI. The investigators felt this effect was secondary to dephosphorylation of the m-TOR pathway [152]. Other investigators have also reported positive results with melatonin in animal models [153-156].
Zhu et al reported that intraperitoneal administration of curcumin given to mice 15 minutes after TBI markedly decreased levels of IL-1β, IL-6 and MCP-1 and reduced the number of TLR4-positive microglia/macrophages, resulting in decreased neuronal apoptosis [157]. Other investigators have also reported neuroprotective effects of curcumin in animal models of TBI [158-161].
Cyclosporine is a potent, immunosuppressant drug. Because of its wide-ranging effects on cytokines [162-165], and activity in animal models [166], it has been studied in trials of patients with TBI. However, a randomized, placebo-controlled, trial of this agent in patients with TBI showed no activity [167]. A formulation of cyclosporine (neurostat) continues to be investigated in patients with TBI and other neurological conditions, although a recent report showed neurostat had no neuroprotective activity in acute ischemic stroke [168].
Many other agents which suppress pro-inflammatory cytokines have also been studied in animal models. Carprofen, a COX-2 inhibitor, which is currently used to treat arthritis in dogs and other animals, was found to markedly reduce IL-1β and IL-6, and to improve neurological functioning in mice after TBI [169]. Triptolide, a diterpenoid epoxide, which has anti-cancer activity in animal models, was found to suppress IL-1β, IL-6 and TNF-α, to increase IL-10 levels and to reduce neuronal apoptosis in Sprague-Dawley rats after experimental TBI [170]. TSG-6 (TNF-α stimulated gene/protein 6) is an anti-inflammatory agent which can suppress IL-1β, IL-6 and other pro-inflammatory cytokines (MIP-1α, MCP-1), and stimulate production of anti-inflammatory cytokines like IL-4 [171]. Watanabe et al showed that administration of this agent to mice after TBI decreased lesion size and improved neurological recovery [172]. Another agent, the CNS-penetrating, small molecule, MW151, which is known to suppress IL-1β and TNF-α, but not to affect anti-inflammatory cytokines like IL-10, has been tested in mice after TBI. This agent restored abnormal cytokine levels to normal, reduced glial activation and caused improvement in neurologic functioning in the treated animals [173]. None of these agents have been tested in patient trials, however.
The brain damage after TBI may be markedly worsened during a succeeding phase of brain inflammation. During this phase, massive increases occur in the levels of key cytokines, particularly IL-1β, IL-6 and TNF-α, a ‘cerebral cytokine storm’ where levels may increase thousands of times compared to their corresponding levels in serum. Although some of these cytokines, such as IL-6 and TNF-α, may have beneficial actions, evidence suggests excessive levels are harmful, since numerous studies in animal models have shown blockade of these cytokines can reduce brain injury. Thus, suppression of pro-inflammatory cytokines can limit the secondary damage caused by neuro-inflammation after TBI.
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can 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, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the 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 herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, 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 “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. 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.
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