RU2843587C1 - Preventing and treating inflammatory conditions - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to the prevention and treatment of inflammatory conditions. Disclosed is the use of miltefosine in the manufacture of a medicament for alleviating or preventing at least one inflammatory condition in a subject, where the amount of miltefosine in the medicament is sufficient to activate type II NKT cells in the subject and where the inflammatory condition is selected from the group consisting of fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis A, B and C, inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis and amyotrophic lateral sclerosis. Also disclosed is use of isolated CD8αα⁺, TCRαβ⁺ T-cells and miltefosine in the manufacture of a medicament for relieving or preventing at least one inflammatory condition in a subject.

EFFECT: group of inventions widens the range of tools for a certain purpose.

24 cl, 18 dwg, 4 tbl, 9 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/089,690, entitled “PREVENTION AND TREATMENT OF INFLAMMATORY CONDITIONS,” filed December 9, 2014, the contents of which are incorporated herein by reference in their entirety.

LEVEL OF TECHNOLOGY

Region

[0002] Some embodiments of the present invention relate to compositions and methods for modulating type II and/or type I NKT cells to prevent and treat inflammatory conditions of the liver.

Description of related technical field

[0003] Excessive alcohol consumption is the leading cause of liver disease in Western countries. Evidence of liver injury has been observed in individuals who consume four or more standard alcoholic drinks per day (four 12-ounce (~355 ml) drinks of beer, four glasses of wine, or four ounces (120 ml) of spirits for men and half that amount for women). Although the mechanism of alcohol-induced liver injury is not fully understood, chronic alcohol consumption results in the secretion of proinflammatory cytokines (TNF-alpha, IL6, and IL8), oxidative stress, lipid peroxidation, and acetaldehyde toxicity, which induce inflammation, apoptosis, and ultimately fibrosis of liver cells.

[0004] Alcoholic liver disease (ALD) has three main phases: alcoholic fatty liver disease, alcoholic hepatitis, and cirrhosis. Alcoholic fatty liver disease, which is characterized by a buildup of fatty acids in the liver, is usually asymptomatic and reverses if the individual abstains from alcohol for several weeks. In severe cases, weakness, nausea, abdominal pain, loss of appetite, and general malaise may be experienced. Although fatty liver disease is present to some degree in most heavy drinkers, with some cases of daily drinking only requiring a strong urge to drink for periods of less than a week, only one in five heavy drinkers develops alcoholic hepatitis and one in four develops cirrhosis. Alcoholic hepatitis is characterized by inflammation of the liver cells and is generally reversible with alcohol abstinence. Cirrhosis, which is characterized by inflammation, fibrosis (hardening of cells), and membrane damage that prevents the body from detoxifying various chemicals and ultimately scarring and necrosis, is generally irreversible.

[0005] The cellular and molecular mechanisms underlying the various phases of liver tissue injury in alcoholic liver disease are poorly understood. Although there have been advances in some areas, there is no effective therapeutic approach to treat the disease at the time of filing. This is partly due to the fact that the liver is a unique organ in terms of immunology as well as anatomy. For example, while parenchymal liver cells have metabolic functions, non-parenchymal cells perform immunological functions. In addition to parenchymal hepatocytes, the liver contains several types of non-parenchymal cells, such as LSECs, Kupffer cells, dendritic cells, NK cells, and NKT cells, all of which may participate in immunity. How the regulation of the immune response determines tolerance or immunity to alcoholic injury is unknown.

BRIEF DESCRIPTION

[0006] According to some embodiments of the present invention, a method of alleviating or preventing at least one inflammatory condition in a subject is provided. The method may include administering an amount of an NKT-2 activator that is sufficient to activate type II NKT cells in the subject.

[0007] According to some embodiments of the present invention, a method of alleviating or preventing at least one inflammatory condition in a subject is provided. The method may comprise administering an amount of an NKT-2 activator and an amount of sulfatide to the subject, wherein, taken together, the amount of NKT-2 activator and the amount of sulfatide are sufficient to activate type II NKT cells in the subject.

[0008] According to some embodiments of the present invention, a method of suppressing tissue damage mediated by type I NKT cells in a subject is provided. The method may include administering an effective amount of an NKT-2 activator to the subject, resulting in a decrease in the activation of type I NKT cells.

[0009] According to some embodiments of the present invention, a composition is provided for use in alleviating or preventing at least one inflammatory condition. The composition may comprise an amount of an NKT-2 activator that is sufficient to activate type II NKT cells in a subject.

[0010] According to some embodiments of the present invention, a composition is provided for use in alleviating or preventing at least one inflammatory condition. The composition may comprise an amount of an NKT-2 activator and an amount of sulfatide, wherein, taken together, the amount of NKT-2 activator and the amount of sulfatide are sufficient to activate type II NKT cells in a subject.

[0011] The present invention also provides several alternative embodiments:

[0012] Alternative embodiment 1 includes a method of alleviating or preventing at least one inflammatory condition in a subject. The method may include administering an amount of an NKT-2 activator that is sufficient to activate type II NKT cells in the subject.

[0013] Alternative embodiment 2 includes the method of alternative embodiment 1, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0014] Alternative embodiment 3 includes the method of any one of alternative embodiments 1-2, wherein the method further comprises administering to the subject an amount of an RAR agonist that is sufficient to suppress activation of type I NKT cells in the subject.

[0015] Alternative embodiment 4 includes the method of alternative embodiment 3, wherein the NKT-2 activator and the RAR agonist are administered simultaneously.

[0016] Alternative embodiment 5 includes the method of alternative embodiment 3, wherein the NKT-2 activator and the RAR agonist are administered separately.

[0017] Alternative embodiment 6 includes the method of alternative embodiment 3, wherein the NKT-2 activator and the RAR agonist are administered sequentially.

[0018] Alternative embodiment 7 includes the method according to any one of alternative embodiments 1-6, wherein the NKT-2 activator is administered orally.

[0019] Alternative embodiment 8 includes the method according to any one of alternative embodiments 1-7, wherein the NKT-2 activator comprises at least one of miltefosine, a miltefosine analog, or a phospholipid.

[0020] Alternative embodiment 9 includes the method of alternative embodiment 8, wherein the phospholipid comprises lysophosphatidylcholine (LPC), an LPC analog, lyso-platelet activating factor (LPAF), lysosphingomyelin (LSM), or an LSM analog.

[0021] Alternative embodiment 10 includes the method of alternative embodiment 9, wherein the LPC comprises one of LPC(C18:0), LPC(C16:0), LPC(C18:1(9Z)), LPC(C18:1(1-oleyl)) or LignA(LPC)(C24:0).

[0022] Alternative embodiment 11 includes the method of any one of alternative embodiments 1-7, wherein the NKT-2 activator comprises at least one of miltefosine or a miltefosine analog.

[0023] Alternative Embodiment 12 includes the method of any one of Alternative Embodiments 8-11, wherein the miltefosine analogue comprises a compound listed in Table 2.1 or Table 2.2.

[0024] Alternative Embodiment 13 includes the method of any one of Alternative Embodiments 8-11, wherein the miltefosine analogue comprises a compound listed in Table 2.1.

[0025] Alternative embodiment 14 includes the method according to any one of alternative embodiments 1-7, wherein the NKT-2 activator comprises miltefosine.

[0026] Alternative embodiment 15 includes the method of any one of alternative embodiments 3-14, wherein the RAR agonist comprises ATRA or isotretinoin.

[0027] Alternative embodiment 16 includes the method of any one of alternative embodiments 3-14, wherein the RAR agonist comprises tazarotene.

[0028] Alternative embodiment 17 includes the method of any one of alternative embodiments 3-14, wherein the RAR agonist comprises retinol or a retinol ester.

[0029] Alternative embodiment 18 includes the method according to any one of alternative embodiments 1-17, wherein the activation of type II NKT cells includes at least one process selected from: production of IL-2 by type II NKT cells, proliferation of type II NKT cells, or expression of CD69 on the surface of type II NKT cells.

[0030] Alternative embodiment 19 includes the method according to any one of alternative embodiments 3-18, wherein suppressing activation of type I NKT cells comprises suppressing accumulation of alpha-GalCer/CD1d-tetramer ⁺ TCR-beta ⁺ cells.

[0031] Alternative embodiment 20 includes a method of alleviating or preventing at least one inflammatory condition in a subject, comprising administering an amount of an NKT-2 activator and an amount of sulfatide to the subject, wherein, taken together, the amount of NKT-2 activator and the amount of sulfatide are sufficient to activate type II NKT cells in the subject.

[0032] Alternative embodiment 21 includes the method of alternative embodiment 20, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0033] Alternative embodiment 22 includes the method of alternative embodiment 20 or alternative embodiment 21, wherein the sulfatide and the NKT-2 activator are administered simultaneously.

[0034] Alternative embodiment 23 includes the method of alternative embodiment 20 or alternative embodiment 21, wherein the sulfatide and the NKT-2 activator are administered separately.

[0035] Alternative embodiment 24 includes the method according to any one of alternative embodiments 20-23, wherein the NKT-2 activator is administered orally.

[0036] Alternative embodiment 25 includes the method of any one of alternative embodiments 20-24, wherein the amount of NKT-2 activator is sufficient to activate type II NKT cells in the subject, and the amount of sulfatide is sufficient to activate type II NKT cells in the subject.

[0037] Alternative embodiment 26 includes the method of any one of alternative embodiments 20-25, wherein the method further comprises administering to the subject an amount of an RAR agonist that is sufficient to suppress activation of type I NKT cells in the subject.

[0038] Alternative embodiment 27 includes the method of alternative embodiment 26, wherein the NKT-2 activator and the RAR agonist are administered simultaneously.

[0039] Alternative embodiment 28 includes the method of alternative embodiment 26, wherein the sulfatide and the NKT-2 activator are administered separately.

[0040] Alternative embodiment 29 includes the method of alternative embodiment 26, wherein the NKT-2 activator and the RAR agonist are administered sequentially.

[0041] Alternative embodiment 30 includes the method of any one of alternative embodiments 20-29, wherein the NKT-2 activator comprises at least one of miltefosine, a miltefosine analog, or a phospholipid.

[0042] Alternative embodiment 31 includes the method of alternative embodiment 30, wherein the phospholipid comprises lysophosphatidylcholine (LPC), an LPC analog, lyso-platelet activating factor (LPAF), lysosphingomyelin (LSM), or an LSM analog.

[0043] Alternative embodiment 32 includes the method of alternative embodiment 31, wherein the LPC comprises one of LPC(C 18:0), LPC(C 16:0), LPC(C18:1(9Z)), LPC(C18:1(1-oleyl)) or LignA(LPC)(C24:0).

[0044] Alternative embodiment 33 includes the method of any one of alternative embodiments 20-29, wherein the NKT-2 activator comprises at least one of miltefosine or a miltefosine analog.

[0045] Alternative embodiment 34 includes the method of any one of alternative embodiments 30-32, wherein the miltefosine analogue comprises a compound listed in Table 2.1 or Table 2.2.

[0046] Alternative embodiment 35 includes the method of any one of alternative embodiments 30-32, wherein the miltefosine analogue comprises a compound listed in Table 2.1.

[0047] Alternative embodiment 36 includes the method of any one of alternative embodiments 20-29, wherein the NKT-2 activator comprises miltefosine.

[0048] Alternative embodiment 37 includes the method according to any one of alternative embodiments 20-36, wherein the sulfatide has the following chemical structure:

wherein R1 is selected from the group consisting of a bond, hydrogen, _C1 - _C30 alkyl, _C1 - _C30 substituted alkyl, _C1 - _C30 alkenyl, _C1 - _C30 substituted alkenyl, and _C5 - _C12 sugar; _R2 is selected from the group consisting of hydrogen, hydroxy, methoxy, and alkoxy; _R3 is selected from the group consisting of hydrogen, hydroxy, methoxy, ethoxy, and alkoxy; _R4 is selected from the group consisting of hydrogen, hydroxy, and alkoxy; _R5 is selected from the group consisting of hydrogen, hydroxy, carbonyl, alkoxy, and a bond; R ₆ is selected from the group consisting of C ₁ -C ₄₀ alkyl, C ₁ -C ₄₀ substituted alkyl, C ₁ -C ₄₀ alkenyl, C ₁ -C ₄₀ substituted alkenyl and C ₁ -C ₄₀ alkynyl; R ₇ is selected from the group consisting of C ₁ -C ₄₀ alkyl, C ₁ -C ₄₀ substituted alkyl, C ₁ -C ₄₀ alkenyl, C ₁ -C ₄₀ substituted alkenyl and C ₁ -C ₄₀ alkynyl; and R ₈ is selected from the group consisting of hydrogen, hydroxyl group, carbonyl, alkoxy group and a bond.

[0049] Alternative embodiment 38 includes the method of any one of alternative embodiments 20-36, wherein the sulfatide has the following chemical structure:

[0050] Alternative embodiment 39 includes the method of any one of alternative embodiments 20-35, wherein the RAR agonist comprises ATRA or isotretinoin.

[0051] Alternative embodiment 40 includes the method according to any one of alternative embodiments 20-38, wherein the RAR agonist comprises tazarotene.

[0052] Alternative embodiment 41 includes the method of any one of alternative embodiments 20-38, wherein the RAR agonist comprises retinol or a retinol ester.

[0053] Alternative embodiment 42 includes a method of suppressing tissue damage mediated by type I NKT cells in a subject, comprising administering an effective amount of a phospholipid to the subject, resulting in a decrease in type I NKT cell activation.

[0054] Alternative embodiment 43 includes the method of alternative embodiment 42, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0055] Alternative embodiment 44 includes the method of any one of alternative embodiments 42-43, wherein the phospholipid comprises at least one of miltefosine, a miltefosine analog, or a phospholipid.

[0056] Alternative embodiment 45 includes the method of alternative embodiment 44, wherein the phospholipid comprises lysophosphatidylcholine (LPC), an LPC analog, lyso-platelet activating factor (LPAF), lysosphingomyelin (LSM), or an LSM analog.

[0057] Alternative embodiment 46 includes the method of alternative embodiment 45, wherein the LPC comprises one of LPC(C18:0), LPC(C16:0), LPC(C18:1(9Z)), LPC(C18:1(1-oleyl)) or LignA(LPC)(C24:0).

[0058] Alternative embodiment 47 includes the method of any one of alternative embodiments 42-43, wherein the phospholipid comprises at least one of miltefosine or a miltefosine analog.

[0059] Alternative embodiment 48 includes the method of any one of alternative embodiments 44-47, wherein the miltefosine analogue comprises a compound listed in Table 2.1 or Table 2.2.

[0060] Alternative embodiment 49 includes the method of any one of alternative embodiments 44-47, wherein the miltefosine analogue comprises a compound listed in Table 2.1.

[0061] Alternative embodiment 50 includes the method of any one of alternative embodiments 42-43, wherein the NKT-2 activator comprises miltefosine.

[0062] Alternative embodiment 51 includes a composition for use in alleviating or preventing at least one inflammatory condition in a subject, comprising an amount of an NKT-2 activator that is sufficient to activate type II NKT cells in the subject.

[0063] Alternative embodiment 52 comprises the composition of alternative embodiment 51 further comprising an amount of an RAR agonist that is sufficient to suppress activation of type I NKT cells in the subject.

[0064] Alternative embodiment 53 includes a composition according to any one of alternative embodiments 48-49, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C and others), inflammatory hepatitis associated with hepatobiliary carcinoma, autoimmune hepatitis, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0065] Alternative embodiment 54 includes a composition according to any one of alternative embodiments 51-53, wherein the NKT-2 activator comprises at least one of miltefosine, a miltefosine analog, or a phospholipid.

[0066] Alternative embodiment 55 includes the composition of alternative embodiment 54, wherein the phospholipid comprises lysophosphatidylcholine (LPC), an LPC analog, lyso-platelet activating factor (LPAF), lysosphingomyelin (LSM), or an LSM analog.

[0067] Alternative embodiment 56 comprises the composition of alternative embodiment 55, wherein the LPC comprises one of LPC(C18:0), LPC(C16:0), LPC(C18:1(9Z)), LPC(C18:1(1-oleyl)) or LignA(LPC)(C24:0).

[0068] Alternative embodiment 57 includes a composition according to any one of alternative embodiments 51-53, wherein the phospholipid comprises at least one of miltefosine or a miltefosine analog.

[0069] Alternative embodiment 58 includes a composition according to any one of alternative embodiments 54-57, wherein the miltefosine analogue includes a compound listed in Table 2.1 or Table 2.2.

[0070] Alternative embodiment 59 includes a composition according to any one of alternative embodiments 51-57, wherein the miltefosine analogue includes a compound listed in Table 2.1.

[0071] Alternative embodiment 60 comprises a composition according to any one of alternative embodiments 51-53, wherein the NKT-2 activator comprises miltefosine.

[0072] Alternative embodiment 61 includes a composition according to any one of alternative embodiments 52-60, wherein the RAR agonist comprises ATRA or isotretinoin.

[0073] Alternative embodiment 62 includes a composition according to any one of alternative embodiments 52-60, wherein the RAR agonist comprises tazarotene.

[0074] Alternative embodiment 63 includes a composition according to any one of alternative embodiments 52-60, wherein the RAR agonist comprises retinol or a retinol ester.

[0075] Alternative embodiment 64 includes a composition according to any one of alternative embodiments 51-63, wherein the composition is for oral administration.

[0076] Alternative embodiment 65 includes a composition for use in alleviating or preventing at least one inflammatory condition in a subject, comprising an amount of an NKT-2 activator and an amount of sulfatide, wherein, taken together, the amount of NKT-2 activator and the amount of sulfatide are sufficient to activate type II NKT cells in the subject.

[0077] Alternative embodiment 66 includes the composition of alternative embodiment 61, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, autoimmune hepatitis, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0078] Alternative embodiment 67 includes the composition of any one of alternative embodiments 65-66, wherein the amount of NKT-2 activator is sufficient to activate type II NKT cells in the subject, and the amount of sulfatide is sufficient to activate type II NKT cells in the subject.

[0079] Alternative embodiment 68 includes a composition according to any one of alternative embodiments 65-67, wherein the NKT-2 activator comprises at least one of miltefosine, a miltefosine analog, or a phospholipid.

[0080] Alternative embodiment 69 includes the composition of alternative embodiment 68, wherein the phospholipid comprises lysophosphatidylcholine (LPC), an LPC analog, lyso-platelet activating factor (LPAF), lysosphingomyelin (LSM), or an LSM analog.

[0081] Alternative embodiment 70 comprises the composition of alternative embodiment 69, wherein the LPC comprises one of LPC(C 18:0), LPC(C 16:0), LPC(C18:1(9Z)), LPC(C18:1(1-oleyl)) or LignA(LPC)(C24:0).

[0082] Alternative embodiment 71 includes a composition according to any one of alternative embodiments 65-67, wherein the NKT-2 activator comprises at least one of miltefosine or a miltefosine analog.

[0083] Alternative Embodiment 72 comprises a composition according to any one of Alternative Embodiments 68-71, wherein the miltefosine analogue comprises a compound listed in Table 2.1 or Table 2.2.

[0084] Alternative Embodiment 73 comprises a composition according to any one of Alternative Embodiments 68-71, wherein the miltefosine analogue comprises a compound listed in Table 2.1.

[0085] Alternative embodiment 74 comprises a composition according to any one of alternative embodiments 65-67, wherein the NKT-2 activator comprises miltefosine.

[0086] Alternative embodiment 75 includes a composition according to any one of alternative embodiments 65-74, wherein the sulfatide has the following chemical structure:

wherein R1 is selected from the group consisting of a bond, hydrogen, _C1 - _C30 alkyl, _C1 - _C30 substituted alkyl, _C1 - _C30 alkenyl, _C1 - _C30 substituted alkenyl, and _C5 - _C12 sugar; _R2 is selected from the group consisting of hydrogen, hydroxy, methoxy, and alkoxy; _R3 is selected from the group consisting of hydrogen, hydroxy, methoxy, ethoxy, and alkoxy; _R4 is selected from the group consisting of hydrogen, hydroxy, and alkoxy; _R5 is selected from the group consisting of hydrogen, hydroxy, carbonyl, alkoxy, and a bond; R ₆ is selected from the group consisting of C ₁ -C ₄₀ alkyl, C ₁ -C ₄₀ substituted alkyl, C ₁ -C ₄₀ alkenyl, C ₁ -C ₄₀ substituted alkenyl and C ₁ -C ₄₀ alkynyl; R ₇ is selected from the group consisting of C ₁ -C ₄₀ alkyl, C ₁ -C ₄₀ substituted alkyl, C ₁ -C ₄₀ alkenyl, C ₁ -C ₄₀ substituted alkenyl and C ₁ -C ₄₀ alkynyl; and R ₈ is selected from the group consisting of hydrogen, hydroxyl group, carbonyl, alkoxy group and a bond.

[0087] Alternative embodiment 76 includes a composition according to any one of alternative embodiments 65-74, wherein the sulfatide has the following chemical structure:

[0088] Alternative embodiment 77 includes a composition according to any one of alternative embodiments 65-76, wherein the composition further comprises an amount of an RAR agonist that is sufficient to suppress activation of type I NKT cells in the subject.

[0089] Alternative embodiment 78 includes the composition of alternative embodiment 77, wherein the RAR agonist comprises ATRA or isotretinoin.

[0090] Alternative embodiment 79 includes the composition of alternative embodiment 77, wherein the RAR agonist comprises tazarotene.

[0091] Alternative embodiment 80 includes the composition of alternative embodiment 77, wherein the RAR agonist comprises retinol or a retinol ester.

[0092] Alternative embodiment 81 includes the method of any one of alternative embodiments 1-50, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0093] Alternative embodiment 82 includes the method of any one of alternative embodiments 1-50 or 81, wherein the amount of NKT-2 activator is sufficient to activate CD8αα ⁺ T cells in the liver of the subject.

[0094] Alternative embodiment 83 includes the method of any one of alternative embodiments 1-50 or 81-82, further comprising administering the isolated CD8αα ⁺ , TCRαβ ⁺ T cells to the subject.

Alternative embodiment 84 includes the method according to any one of alternative embodiments 82-83, wherein the isolated CD8αα ⁺ , TCRαβ ⁺ T cells are administered to the subject prior to administration of the NKT-2 activator.

Alternative embodiment 85 includes the method according to any one of alternative embodiments 82-83, wherein the isolated CD8αα ⁺ , TCRαβ ⁺ T cells are administered to the subject simultaneously with an NKT-2 activator.

Alternative embodiment 86 includes the method according to any one of alternative embodiments 82-83, wherein the isolated CD8αα ⁺ , TCRαβ ⁺ T cells are administered to the subject following an NKT-2 activator.

[0095] Alternative embodiment 87 includes the method of any one of alternative embodiments 82-87, further comprising obtaining a cell sample from a mammal, isolating CD8αα ⁺ , TCRαβ ⁺ T cells from the cell sample, and growing the isolated T cells.

[0096] Alternative embodiment 88 includes the method of alternative embodiment 87, wherein the mammal is the subject.

[0097] Alternative embodiment 89 includes the method of alternative embodiment 87, wherein the mammal is an organism other than the subject.

[0098] Alternative embodiment 90 includes the method of any one of alternative embodiments 82-89, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ .

[0099] Alternative embodiment 91 includes the method according to any one of alternative embodiments 82-89, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD122 ⁺ .

[0100] Alternative embodiment 92 includes the method according to any one of alternative embodiments 82-89, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ , CD122 ⁺ .

[0101] Alternative embodiment 93 includes the method of any one of alternative embodiments 82-92, wherein the isolated T cells are a mixture of two or more cell types selected from CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ CD122 ⁺ T cells.

[0102] Alternative embodiment 94 includes a method of alleviating or preventing at least one inflammatory condition in a subject, comprising administering isolated CD8αα ⁺ , TCRαβ ⁺ T cells to the subject and administering an amount of an NKT-2 activator that is sufficient to activate type II NKT cells in the subject.

[0103] Alternative embodiment 95 includes the method of alternative embodiment 94, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0104] Alternative embodiment 96 includes the method of any one of alternative embodiments 94-95, wherein the NKT-2 activator comprises at least one of miltefosine or a miltefosine analog.

[0105] Alternative embodiment 97 includes the method of any one of alternative embodiments 94-95, wherein the NKT-2 activator comprises miltefosine.

[0106] Alternative embodiment 98 includes the method of any one of alternative embodiments 94-97, wherein the amount of NKT-2 activator is sufficient to activate CD8αα ⁺ T cells in the liver of the subject.

[0107] Alternative embodiment 99 includes the method of any one of alternative embodiments 94-98, wherein the isolated CD8αα ⁺ , TCRαβ ⁺ T cells are administered to the subject prior to administration of the NKT-2 activator.

[0108] Alternative embodiment 100 includes the method of any one of alternative embodiments 94-98, wherein the isolated CD8αα ⁺ , TCRαβ ⁺ T cells are administered to the subject simultaneously with an NKT-2 activator.

[0109] Alternative embodiment 101 includes the method of any one of alternative embodiments 94-98, wherein the isolated CD8αα ⁺ , TCRαβ ⁺ T cells are administered to the subject following an NKT-2 activator.

[0110] Alternative embodiment 102 includes the method of any one of alternative embodiments 94-101, further comprising obtaining a cell sample from a mammal, isolating CD8αα ⁺ , TCRαβ ⁺ T cells from the cell sample, and growing the isolated T cells.

[0111] Alternative embodiment 103 includes the method of alternative embodiment 102, wherein the mammal is the subject.

[0112] Alternative embodiment 104 includes the method of alternative embodiment 102, wherein the mammal is an organism other than the subject.

[0113] Alternative embodiment 105 includes the method of any one of alternative embodiments 94-104, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ .

[0114] Alternative embodiment 106 includes the method of any one of alternative embodiments 94-104, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD122 ⁺ .

[0115] Alternative embodiment 107 includes the method of any one of alternative embodiments 94-104, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ , CD122 ⁺ .

[0116] Alternative embodiment 108 includes the method of any one of alternative embodiments 94-104, wherein the isolated T cells comprise a mixture of two or more cell types selected from CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ CD122 ⁺ T cells.

[0117] Alternative embodiment 109 includes a composition according to any one of alternative embodiments 51-80, wherein the inflammatory condition is selected from the group consisting of: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0118] Alternative embodiment 110 includes a composition according to any one of alternative embodiments 51-80 or 109, wherein the amount of NKT-2 activator is sufficient to activate CD8αα ⁺ T cells in the liver of the subject.

[0119] Alternative embodiment 111 comprises a composition according to any one of alternative embodiments 51-80 or 109 or 110 further comprising isolated CD8αα ⁺ , TCRαβ ⁺ T cells, for use in administration to a subject.

[0120] Alternative embodiment 112 comprises the composition of alternative embodiment 111, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ .

[0121] Alternative embodiment 113 comprises the composition of alternative embodiment 111, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD122 ⁺ . Alternative embodiment 114 comprises the composition of alternative embodiment 111, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ , CD122 ⁺ .

[0122] Alternative embodiment 115 comprises the composition of alternative embodiment 111, wherein the isolated T cells comprise a mixture of two or more cell types selected from CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ , TCRαβ ⁺ CD200 ⁺ CD122 ⁺ T cells.

BRIEF DESCRIPTION OF DRAWINGS

[0123] Figure 1 is a graph depicting the stimulation of type II NKT cells by miltefosine according to some embodiments of the present invention.

[0124] Figure 2A is a graph depicting the suppression of type I NKT cell growth following injection of miltefosine according to some embodiments of the present invention.

[0125] Figure 2B is a series of graphs depicting flow cytometry data showing suppression of type I NKT cell growth following injection of miltefosine according to some embodiments of the present invention.

[0126] Figure 3A is a graph depicting suppression of ConA-induced hepatitis following treatment with miltefosine according to some embodiments of the present invention.

[0127] Figure 3B is a series of microscopic images showing suppression of ConA-induced hepatitis following treatment with miltefosine according to some embodiments of the present invention. Figure 3C is a black and white reproduction of Figure 3B.

[0128] Figure 4A is a graph depicting the suppression of type I NKT cell accumulation in alcoholic liver disease (ALD) in mice treated with miltefosine according to some embodiments of the present invention.

[0129] Figure 4B is a series of graphs depicting flow cytometry data showing suppression of type I NKT cell accumulation in alcoholic liver disease (ALD) in mice treated with miltefosine according to some embodiments of the present invention.

[0130] Figure 4C is a graph depicting the suppression of activated (CD69 ⁺ ) type I NKT cell accumulation in alcoholic liver disease (ALD) in mice treated with miltefosine according to some embodiments of the present invention.

[0131] Figure 4D is a graph depicting the suppression of neutrophil accumulation in alcoholic liver disease (ALD) in mice treated with miltefosine according to some embodiments of the present invention.

[0132] Figure 5 is a graph depicting the results of treatment of chronic and relapsing experimental autoimmune encephalomyelitis (EAE), a prototype of multiple sclerosis, using miltefosine according to some embodiments of the present invention.

[0133] Figure 6 is a graph showing the results of treatment of chronic experimental autoimmune encephalomyelitis (EAE), a prototype of multiple sclerosis, with sulfatide.

[0134] Figure 7 is a graph showing that in vitro growth of type I NKT cells in human PBMCs is suppressed following treatment with miltefosine according to some embodiments of the present invention.

[0135] Figures 8A and 8B are a pair of graphs depicting the mean disease indices in mice injected with varying amounts of expanded T _reg cell populations as a function of the number of days after inducing EAE by injection of MBPAc1-9 (myelin basic protein) according to some embodiments of the present invention. Figure 8A depicts the results in mice injected with 2D11 cells. Figure 8B depicts the results in mice injected with a p42-50 reactive T cell line.

[0136] Figures 9A-9C are a series of graphs showing that treatment of mice with an agonist mAb to Vβ6 in vivo results in activation of CD8αα ⁺ Vβ6 ⁺ T cells and prevention of EAE according to some embodiments of the present invention. Figure 9A depicts cell characteristics determined by flow cytometry. Figure 9B depicts the relative amounts of Vβ6 and Vβ8.2 determined by real-time PCR data. Figure 9C depicts the relative percentage of TL tetramers/CD8+ T cells.

DETAILED DESCRIPTION

[0137] According to some embodiments of the present invention, compositions comprising an amount of an NKT-2 activator that is sufficient to activate type II NKT cells can be used to alleviate or prevent any inflammatory conditions. Without being bound by theory, it is believed that when activated, type II NKT cells can suppress the activation of type I NKT cells and, thus, can alleviate or prevent type I NKT cell-mediated injury associated with inflammation. Accordingly, in some embodiments, methods for alleviating or preventing inflammatory conditions include administering a composition comprising an amount of an NKT-2 activator, such as miltefosine or a miltefosine analog, that is sufficient to activate type II NKT cells. The composition can optionally comprise an amount of sulfatide that is sufficient to activate type II NKT cells. The composition can optionally comprise an amount of a RAR agonist that is sufficient to suppress the activation of type I NKT cells.

NKT cells

[0138] The liver contains a number of specialized cells of the innate immune system, including Kupffer cells, natural killer (NK) cells, natural killer T (NKT) cells, and dendritic cells. NKT cells are unique in that they share the same cell surface receptors as NK cells (e.g., NK1.1) and express T cell receptors (TCRs), which enable them to recognize lipid antigens in the context of CD1d molecules and mediate the transition of innate immune responses to adaptive immunity. NKT cells have the ability to regulate the activity of other cells that are responsible for tissue inflammation and associated cellular damage. When activated, NKT cells rapidly secrete large amounts of interferon-gamma, interleukin 4 (IL-4), granulocyte-macrophage column stimulating factor, and a variety of other cytokines and chemokines. Because NKT cells can secrete Th1 and Th2 cytokines, the consequences of NKT cell activation in vivo can be difficult to predict. Depending on the conditions, NKT cell activation triggers cascades of events that stimulate or suppress various immune responses. Under some conditions, NKT cell activation leads to activation of NK cells, dendritic cells (DCs), and B cells. Aspects of NKT cells are discussed in Kumar, "NKT-cell subsets: Promoters and protectors in inflammatory liver disease" Journal of Hepatology, 2013, 59: 618-620, the contents of which are incorporated herein by reference in their entirety.

[0139] NKT cells recognize lipid antigens presented in the context of a monomorphic MHC class I–like molecule, CD1d. CD1d-restricted NKT cells are divided into type I cells (also referred to as "type I NKT cells" or "NKT-1 cells") and type II cells (also referred to as "type II NKT cells" or "NKT-2 cells"), which recognize different lipid antigens presented by CD1d molecules. Although both NKT cell subsets are primarily NK1.1+ (in mice) or CD161+/CD56+ (in humans), their relative abundance differs between mice and humans: thus, while mice contain primarily type I NKT cells, humans contain a predominantly type II NKT cell subset.

[0140] Type I cells, also known as invariant NKT cells, express a semi-invariant T cell receptor (TCR) characterized by Vα14-Jα18 and Vβ8.2, Vβ7, or Vβ2 in mice or Vα24-JαQ and Vβ11 in humans, are highly reactive with the glycolipid alpha-galactosylceramide isolated from marine sponges ("alpha-GalCer" or "αGalCer"), and are detected by flow cytometry as alpha-GalCer/CD1d tetramers. Type I NKT cells also recognize lipid-based antigens such as bacterial lipids and the host glycolipid isoglobotrigexosyl-ceramide (iGb3). Type I NKT cells have memory markers and are uniquely capable of storing previously produced cytokine mRNA. Mice lacking the Jα18 gene (Jα18 mice) are deficient in type I NKT cells only.

[0141] Type II NKT cells, which are distinct from type I NKT cells, are regulatory cells that can modulate the activity of several other cell subsets, including type I NKT cells. Type II NKT cells recognize the host glycolipid sulfatide (3'-sulfogalactosyl-ceramide) in mice and humans. The major subset of type II NKT cells, which can be identified by sulfatide/CD1d tetramers by flow cytometry, primarily utilize the Vβ8.1/Vβ3.1-Jβ2.7 and Vα1/Vα3-Jα7 gene segments and are reactive for sulfatide. Activation of type II NKT cells can be assessed by determining the proliferative response of type II NKT cells in vitro to a candidate agent, as well as by determining CD69 expression and a cytokine secretion profile by intracellular cytokine staining or real-time PCR for IFN-gamma, IL-4, or IL-13. Additionally, the ability of activated type II NKT cells to suppress type I NKT cells can be assessed by determining the proliferative response of type I NKT cells to alpha-GalCer (a potent activator of type I NKT cells) using a CF8E dilution assay and intracellular cytokine staining in alpha-GalCer/CO1d tetrameric cells. Activation of NKT cells (type I or type II) according to some embodiments of the present invention can be quantified using a stimulation ratio determined by thymidine incorporation (see, for example, Figure 2A).

[0142] As used herein, "NKT-2 activators," including variations of this general term, refer to compounds that, upon contact with type II NKT cells, induce at least one process selected from the secretion of IL-2 by type II NKT cells, proliferation of type II NKT cells, or increased expression of CD69 on the surface of type II NKT cells. Exemplary NKT-2 activators suitable for use in the methods, kits, and compositions provided herein include, but are not limited to, miltefosine, miltefosine analogs (e.g., the compounds listed in Table 2.1 and Table 2.2), phospholipids such as lysophosphatidylcholine (LPC) (e.g., LPC (C18:0), LPC (C16:0), LPC (C18:1(9Z)), LPC (C18:1(1-oleyl)) and/or LignA (LPC) (C24:0)), an LPC analog, lyso-platelet activating factor (LPAF), lysosphingomyelin (LSM), and/or an LSM analog. Without being limited by theory, it is believed that NKT-2 activators according to some embodiments of the present invention may thus activate type II NKT cells, which in turn may suppress the activation of type I NKT cells.

[0143] In some embodiments, activation of type II NKT cells comprises secretion of IL-2 by type II NKT cells, proliferation of type II NKT cells, increased expression of CD69 on the surface of type II NKT cells, any two of these processes, any three of these processes, or all four of these processes.

[0144] In some embodiments, suppressing type I NKT cell activation comprises suppressing type I NKT cell proliferation, reducing type I NKT cell accumulation, reducing expression of CD69 on the surface of type I NKT cells, any two of these processes, or all three of these processes.

NKT-2 activators and NKT cells type I and type II after ischemia and reperfusion injury

[0145] Reperfusion injury occurs when blood flow to an organ or tissue is restored following ischemia. Liver reperfusion injury typically occurs following surgery or trauma and plays an important role in the quality and function of graft tissue following liver transplantation. The development of ischemia-reperfusion injury (IRI) involves at least two phases: an initial period (approximately 1 to approximately 6 hours after reperfusion), which is determined primarily by Kupffer cell activation, release of reactive oxygen species, recruitment of CD4 ⁺ cells, and secretion of proinflammatory cytokines, and a late period (after the initial phase, approximately 6 to approximately 48 hours after reperfusion), which is characterized by neutrophil accumulation and induction of necrosis. Typically, type I NKT cell activation and IFN-gamma secretion also occur following ischemia-reperfusion. It has been shown that administration of NKT-2 activators such as miltefosine can stimulate type II NKT cells (see Figure 1) and suppress the growth of type I NKT cells (see Figure 2A and Figure 2B), thus it is suggested that NKT-2 activators such as miltefosine and miltefosine analogs and phospholipids, according to some embodiments of the present invention, can be used to suppress the activation of type I NKT cells associated with ischemic reperfusion.

[0146] Accordingly, in some embodiments, a composition is provided comprising an amount of an NKT-2 activator, such as miltefosine or a miltefosine analog or a phospholipid, that is sufficient to activate type II NKT cells, for use in treating, ameliorating, preventing, or reducing the risk of developing ischemic and reperfusion injury. The composition optionally further comprises an amount of a RAR agonist, such as tazarotene, that is sufficient to suppress type I NKT cells. The composition optionally further comprises sulfatide in an amount that is sufficient to activate type II NKT cells. In some embodiments, the composition is administered to a subject suffering from or at risk of developing ischemic and reperfusion injury.

[0147] In some embodiments, a composition is provided comprising an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) and an amount of sulfatide that, taken together, are sufficient to activate type II NKT cells, for use in treating, ameliorating, preventing, or reducing the risk of developing ischemic and reperfusion injury. The composition optionally further comprises an amount of a RAR agonist (e.g., tazarotene) that is sufficient to suppress type I NKT cells. In some embodiments, the composition is administered to a subject suffering from or at risk of developing ischemic and reperfusion injury. The composition is optionally intended for oral administration.

[0148] In some embodiments, an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) that is sufficient to activate type II NKT cells is administered to a subject suffering from or at risk for an IRI. Optionally, the subject is administered an amount of an RAR agonist (e.g., tazarotene) that is sufficient to suppress the activation of type I NKT cells. The administration is optionally oral. Optionally, the subject is further administered an amount of sulfatide that is sufficient to activate type II NKT cells. In some embodiments, the NKT-2 activator and the RAR agonist are administered simultaneously. In some embodiments, the NKT-2 activator and the RAR agonist are administered separately. In some embodiments, the NKT-2 activator is administered first, followed by the RAR agonist. In some embodiments, the RAR agonist is administered first, followed by the NKT-2 activator. In some embodiments, alternating administration of a RAR agonist and an NKT-2 activator is performed.

NKT-2 activators and activated type I and type II NKT cells in liver disease

[0149] According to the disclosure of U.S. Patent No. 8,044,029 and U.S. Patent Application Publication No. 2011/0118197 and U.S. Patent Application Publication No. 2014/0187504, the contents of each of which are incorporated herein by reference in their entireties, administration of a host glycolipid ligand, sulfatide, as well as synthetic analogs of sulfatide, can modulate the activity of type I and type II NKT cells. Without being bound by theory, it is believed that CD1d-dependent recognition of sulfatide activates type II NKT cells and, primarily, plasmacytoid dendritic cells (pDCs), but not typical dendritic cell (cDC) populations (which are normally activated by type I NKT cells), resulting in rapid IL-12- and MIP2-mediated recruitment of type I NKT cells to the liver. However, recruited type I NKT cells are not activated, do not secrete cytokines, and are dormant. In vivo administration of sulfatide has been shown to a) suppress type I NKT cell proliferation in vivo in response to alpha-GalCer stimulation, and b) increase the proportion of sulfatide/CD1d tetramer+ cells with intracytoplasmic IFN-gamma staining.

[0150] IFN-gamma secretion by type I NKT cells in the liver during the early phase of ischemic and reperfusion injury was found to be significantly reduced in sulfatide-treated mice compared to untreated animals. Furthermore, similar to Jα18 ^-/- mice that lack type I NKT cells, liver injury following ischemia/reperfusion was significantly reduced in sulfatide-treated mice. This observation suggests a pathogenic role for type I NKT cells in mediating liver ischemia and reperfusion injury. Without being bound by theory, it is believed that the role of IFN-gamma secretion by type I NKT cells may be a general hallmark of ischemic organ injury, as renal ischemia and reperfusion injury is also attenuated in Jα18 ^-/- mice that lack type I NKT cells.

[0151] Similar to IRP, administration of sulfatide or an NKT-2 activator (e.g., miltefosine or a miltefosine analog or phospholipid) can provide protection against concanavalin A (ConA)-induced hepatitis, as indicated by reduced liver cell necrosis and reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, which are markers of liver cell injury. Furthermore, administration of miltefosine 12 hours before ConA lowered serum ALT levels at 24 hours (Figure 3A) and reduced cell necrosis as assessed by hemotoxin-eosin staining of liver sections (Figure 3B). Without being bound by any theory, these observations are believed to indicate a pathogenic role for type I NKT cells in ConA-induced hepatitis and a protective role for sulfatide and/or phospholipid (eg, miltefosine or a miltefosine analog).

[0152] Without being bound by theory, the data obtained in two models of liver injury, IRI and ConA-induced hepatitis, are believed to be consistent with an immunoregulatory pathway in which type I NKT cells play a negative role and type II NKT cells capable of interacting with sulfatide/NKT activator-2 play a protective role in liver inflammation arising from various causes. This protective effect is associated with the inhibition of cytokine secretion by type I NKT cells (suppressed phenotype) and a significant reduction in the recruitment of immature myeloid cells (CD11b+Gr-1+) CD11b+Gr-1- and NK cells. Suppression of type I NKT cells is also associated with tolerance or modification of conventional dendritic cells (cDCs), and they, together with suppressed type I NKT cells, suppress the activation/growth of adaptive Th1/Th17 CD4+/CD8+ T cells. This leads to a model in which administration of sulfatide and/or an NKT-2 activator (e.g., miltefosine or a miltefosine analogue or a phospholipid) stimulates type II NKT cell activity, which in turn suppresses type I NKT cell activity and liver injury induced by type I NKT cell-mediated inflammation.

[0153] Accordingly, in some embodiments, a composition is provided comprising an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) that is sufficient to activate type I NKT cells, for use in the treatment of liver injury or liver disease. The composition optionally further comprises an amount of a RAR agonist (e.g., tazarotene) that is sufficient to suppress the activation of type I NKT cells. The composition optionally further comprises an amount of sulfatide that is sufficient to activate type II NKT cells. In some embodiments, the composition is administered to a subject suffering from liver injury or at risk of developing liver injury or liver disease, such as fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C and others), inflammatory hepatitis associated with hepatobiliary carcinoma.

[0154] In some embodiments, a composition is provided comprising an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) and an amount of sulfatide that, taken together, are sufficient to activate type II NKT cells, for use in treating, ameliorating, preventing, or reducing the risk of developing liver injury or liver disease. The composition optionally further comprises an amount of a RAR agonist (e.g., tazarotene) that is sufficient to suppress type I NKT cell activation. In some embodiments, the composition is administered to a subject suffering from or at risk of developing liver injury or liver disease, such as fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C and others), inflammatory hepatitis associated with hepatobiliary carcinoma.

[0155] In some embodiments, an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) that is sufficient to activate type II NKT cells is administered to a subject suffering from or at risk of liver injury or liver disease. Optionally, the subject is administered an amount of an RAR agonist (e.g., tazarotene) that is sufficient to suppress the activation of type I NKT cells. The administration is optionally oral. Optionally, the subject is further administered an amount of sulfatide that is sufficient to activate type II NKT cells. In some embodiments, the NKT-2 activator and the RAR agonist are administered simultaneously. In some embodiments, the NKT-2 activator and the RAR agonist are administered separately. In some embodiments, the NKT-2 activator is administered first, followed by the RAR agonist. In some embodiments, the RAR agonist is administered first, followed by the NKT-2 activator. In some embodiments, alternating administration of a RAR agonist and an NKT-2 activator is performed. In some embodiments, the subject suffers from or is at risk of developing liver injury or liver disease, such as fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C and others), inflammatory hepatitis associated with hepatobiliary carcinoma.

NKT cells in alcoholic liver disease

[0156] Alcoholic liver disease (ALD) results from liver cell injury caused by excessive alcohol consumption. Several episodes of injury may be required for ALD to become clinically evident. However, chronic alcohol consumption has been shown to be associated with significantly increased numbers of type I NKT cells (alpha-GalCer/CD1d-tetramer+ cells), CD4+ cells, and CD11b+Gr-1+ myeloid cells, but not type II NKT cells or CD11b+Gr-1- cells. Increased expression of the CD69 marker provides further evidence that at least some of the type I NKT cells are activated. Thus, even in the absence of any clinical signs of disease (pre-disease stage), cells that mediate the inflammatory response accumulate in the liver with excessive alcohol consumption. In the absence of type I NKT cells (in Jα18-/- mice), the accumulation of CD11b+Gr-1+ myeloid cells in the liver is significantly reduced by chronic alcohol consumption. Without being bound by any theory, these data suggest the involvement of type I NKT cells in mediating the pre-disease phase of ALD.

[0157] Consistent with data indicating a role for type I NKT cells in the pre-disease phase of ALD, clinical manifestations of liver injury following excessive alcohol consumption in type I NKT cell-deficient (Jα18-/-) mice are significantly reduced as measured by, for example, histological methods and liver enzyme analysis. Without wishing to be bound by any theory, these data are believed to suggest an important role for type I NKT cells in alcohol-induced liver injury.

[0158] NKT cells express T cell receptors that allow them to recognize lipid antigens in the context of CD1d molecules. Without being bound by theory, it is believed that type I NKT cells are activated in the liver following alcohol consumption by local accumulation of oxidized host lipids resulting from the activity of CD1d-expressing antigen-presenting cells (APCs). Activated type I NKT cells then initiate a multistep process that leads to Kupffer cell activation, granulocyte recruitment/activation, and subsequent inflammatory injury to hepatocytes.

[0159] As shown in Figure 4A and Figure 4B, co-administration of miltefosine with ethanol in a mouse model of alcoholic liver disease suppressed the accumulation of type I NKT cells compared to ethanol alone. In addition, as shown in Figure 4C, co-administration of miltefosine with ethanol also resulted in a decrease in the level of activated (CD69 ⁺ ) NKT cells compared to ethanol alone. In addition, as shown in Figure 4D, co-administration of miltefosine with ethanol also decreased the level of neutrophils compared to ethanol alone.

[0160] The interaction of NKT cell subtypes following ethanol consumption sets the stage for liver injury and, in severe cases, alcoholic liver disease (ALD). Several embodiments described herein relate to methods and compositions for regulating the opposing actions of type I NKT cells and type II NKT cells and their interactions with other liver cells to treat, alleviate, or prevent liver injury following alcohol consumption.

[0161] Treatment with sulfatide provides virtually complete protection against liver injury resulting from excess alcohol consumption. Without being bound by theory, it is believed that the protective effect of sulfatide may be mediated by direct activation of type II NKT cells, which mediate inhibitory effects on type I NKT cells and cDCs.

[0162] Accordingly, some embodiments of the present invention relate to modulating the activity of NKT cell subtypes to treat, reduce, and/or prevent alcohol-induced liver injury. As described herein, administration of NKT-2 activators (e.g., miltefosine), sulfatides, or retinoic acid receptor (RAR) agonists suppresses alcohol-induced liver injury. For example, oral administration of miltefosine reduces the accumulation and activation of type I NKT cells and the accumulation of neutrophils induced by ethanol (see Figures 4A, 4B, C, and 4D).

[0163] Several embodiments relate to the protective effect of a major subset of type II NKT cells in alcohol-induced liver injury that are reactive with sulfatide. Activation of this subset of NKT cells by sulfatide suppresses type I NKT cell-mediated injury following excessive alcohol consumption. Sulfatide-mediated protection is associated with activation of type II NKT cells and suppression of IFN-gamma secretion by type I NKT cells in the liver and suppression of type I NKT cell-mediated myeloid cell recruitment, such as the CD11b ⁺ Gr-1 ^int and Gr-1 ^- subsets, and NK cells in the liver.

[0164] Accordingly, in some embodiments, a composition is provided comprising an amount of NKT-2 activators (e.g., miltefosine or a miltefosine analog) that is sufficient to activate type II NKT cells, for use in treating, ameliorating, or preventing alcoholic liver disease. The composition optionally further comprises an amount of a RAR agonist (e.g., tazarotene) that is sufficient to suppress type I NKT cell activation. The composition is optionally for oral administration.

[0165] In some embodiments, a composition is provided comprising an amount of NKT-2 activators (e.g., miltefosine or a miltefosine analog) and an amount of sulfatide that, taken together, are sufficient to activate type II NKT cells, for use in treating, ameliorating, preventing, or reducing the risk of alcoholic liver disease. The composition optionally further comprises an amount of a RAR agonist (e.g., tazarotene) that is sufficient to suppress the activation of type I NKT cells. The composition is optionally intended for oral administration.

[0166] In some embodiments, an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog) that is sufficient to activate type II NKT cells is administered to a subject suffering from or at risk of developing alcoholic liver disease. Optionally, the subject is administered an amount of an RAR agonist (e.g., tazarotene) that is sufficient to suppress the activation of type I NKT cells. The administration is optionally oral. Optionally, the subject is further administered an amount of sulfatide that is sufficient to activate type II NKT cells. In some embodiments, the NKT-2 activator and the RAR agonist are administered simultaneously. In some embodiments, the NKT-2 activator and the RAR agonist are administered separately. In some embodiments, the NKT-2 activator is administered first, followed by the RAR agonist. In some embodiments, the RAR agonist is administered first, followed by the NKT-2 activator. In some embodiments, alternating administration of a RAR agonist and an NKT-2 activator is performed.

Sulfatides

[0167] In some embodiments, the sulfatide comprises, for example, bovine brain sulfatide, which is a mixture of about 20 different compounds, obtained from Sigma Inc. (Chicago, IL, USA). In other embodiments, the sulfatide is semi-synthetic and comprises individual sulfatide compounds, for example, cis-tetracosenoyl sulfatide or lysosulfatide, obtained from Maitreya Inc. (Pleasant Gap, PA, USA). In other embodiments, the sulfatide may be a fully synthetic sulfatide.

[0168] Sulfatide isolated from bovine brain myelin consists of a 2:1 mixture of saturated/unsaturated basic acyl chains ( _C24 ), where the unsaturated site is at _C19 . Several embodiments relate to the use of synthetic analogs of sulfatide, such as analogs with a saturated acyl chain ( _C24 ) and unsaturated chains ( _C24 :1), as well as analogs consisting of fatty acid acyl chains of various lengths or sphingosine moieties (shorter as well as longer, e.g., _C18 , _C32 ), and positional isomers containing a 3' and 4'-sulfated group on a galactose moiety (3'-803 and 4'-803).

[0169] In some embodiments, the sulfatide has the following chemical formula I:

[0170] wherein R ₁ may be a bond, hydrogen, C ₁ -C ₃₀ alkyl, C ₁ -C ₃₀ substituted alkyl, C ₁ -C ₃₀ alkenyl, C ₁ -C ₃₀ substituted alkenyl, or C ₅ -C ₁₂ sugar; R ₂ may be hydrogen, hydroxy, methoxy, or alkoxy; R ₃ may be hydrogen, hydroxy, methoxy, ethoxy, or alkoxy; R ₄ may be hydrogen, hydroxy, or alkoxy; R ₅ may be hydrogen, hydroxy, carbonyl, alkoxy, or a bond; _R6 may be _C1 - _C40 alkyl, _C1 - _C40 substituted alkyl, _C1 - _C40 alkenyl, _C1 - _C40 substituted alkenyl or _C1 - _C40 alkynyl; R7 may be _C1 - _C40 alkyl, _C1 - _C40 substituted alkyl, _C1 - _C40 alkenyl, _C1 - _C40 substituted alkenyl or _C1 - _C40 alkynyl; and _R8 may be hydrogen, hydroxy, carbonyl, alkoxy or a bond.

[0171] In other embodiments, the sulfatide has the following chemical formula II:

[0172] where R ₁ is selected from the group consisting of C ₁ -C ₄₀ alkyl, C ₁ -C ₄₀ substituted alkyl, C ₁ -C ₄₀ alkenyl, C ₁ -C ₄₀ substituted alkenyl, and C ₁ -C ₄₀ alkynyl; and R ₂ is selected from the group consisting of hydrogen, hydroxyl, carbonyl, alkoxy, and a bond.

[0173] In other embodiments, the sulfatide has the following chemical structure:

[0174] As used herein, the term "alkyl" means any straight or branched saturated hydrocarbon. The term "substituted alkyl" means any straight or branched substituted saturated hydrocarbon. Cyclic compounds, including cyclic hydrocarbons and cyclic compounds containing heteroatoms, are included within the term "alkyl".

[0175] As used herein, the term “substituted” refers to any substitution of a hydrogen atom by a functional group.

[0176] As used herein, the term “functional group” has the generally accepted definition and refers to chemical moieties preferably selected from the group consisting of a halogen atom, _C1 - _C20 alkyl, substituted _C1 - _C20 alkyl, perhalogenated alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, benzyl, heteroaryl, substituted heteroaryl, cyano and nitro.

[0177] As used herein, the terms “halogen” and “halogen atom” refer to any of the stable atoms of Group 17 of the periodic table of elements, preferably fluorine, chlorine, bromine or iodine, with fluorine and chlorine atoms being particularly preferred.

[0178] As used herein, the term "alkenyl" means any straight or branched substituted or unsubstituted unsaturated hydrocarbon. The term "substituted alkenyl" means any straight or branched substituted unsaturated hydrocarbon that is substituted by one or more functional groups, with the definition of "substituted alkyl" including straight _C2 - _C6 alkenyl secondary amines, substituted _C2 - _C6 secondary alkenyl amines, and straight _C2 - _C6 alkenyl tertiary amines. Cyclic compounds, including unsaturated cyclic hydrocarbons and cyclic compounds containing heteroatoms, are included within the term "alkenyl".

[0179] As used herein, the term "alkoxy" refers to any straight or branched substituted or unsubstituted saturated or unsaturated ether.

[0180] As used herein, the term "sulfatide" has its generally accepted meaning and refers to a sulfuric acid ester of cerebroside containing one or more sulfate groups on the sugar moiety of the molecule.

[0181] As used herein, the term "cerebroside" refers to any lipid compound containing a sugar and generally of the type normally found in brain and nervous tissue.

[0182] The compounds of formula (I), (II) and (III) may be in the form of pharmaceutically acceptable non-toxic salts. Salts of formula (I), (II) and (III) include acid addition salts such as salts with inorganic acids (e.g. hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid) or organic acids (e.g. acetic acid, propanoic acid, maleic acid, oleic acid, palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, lauryl sulfonic acid, methanesulfonic acid and phthalic acid).

[0183] Compounds of formula (I), (II) and (III) may be in the form of solvates (eg hydrates).

[0184] Compounds of formula (I), (II) and (III) can be prepared by any method for the targeted synthesis of sulfatides.

[0185] Compounds of formula (I), (II) and (III) can also be isolated from natural products (e.g. from biological organisms) and purified by column chromatography, etc.

[0186] In one embodiment, sulfatide has a chemical formula: (2S,3R,4E)-N-nervonat-1-[-D-(3-sulfate)galactopyranosyl]-2-aminooctadecen-3-ol. This chemical formula also applies to cysteine tracosenoyl sulfatide.

[0187] In some embodiments, the specific amount of sulfatide administered to a patient may vary depending on the disease or condition being treated, as well as the age, body weight, and gender of the patient being treated. Generally, to provide the specified final concentration, for example, in the intestine or blood, the amount of phosphatide molecule in a dosage composition according to embodiments of the present invention generally ranges from about 0.1 milligrams to about 100 milligrams, preferably from about 2.0 milligrams to about 60 milligrams, more preferably from about 20 milligrams to about 50 milligrams. Similarly, the amount of a secondary therapeutic compound in an oral dosage composition according to embodiments of the present invention generally ranges from about 0.01 milligrams to about 1000 milligrams, more preferably from about 0.1 milligrams to about 100 milligrams. Obviously, the exact dosage may vary depending on the disease or disorder being treated, and preferred ranges can be readily determined. In some embodiments, the patient is a human.

[0188] In some embodiments, the subject is administered 0.1-10 mg sulfatide/kg body weight, such as about 0.1 mg/kg body weight, 0.2 mg/kg body weight, 0.3 mg/kg body weight, 0.5 mg/kg body weight, 1 mg/kg body weight, 2 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 9 mg/kg body weight, or an amount in a range such as

[0189] 0.2-0.5 mg/kg body weight, 0.2-1 mg/kg body weight, 0.2-5 mg/kg body weight, 0.2-10 mg/kg body weight, 0.5-1 mg/kg body weight, 0.5-5 mg/kg body weight, 0.5-10 mg/kg body weight, 1-5 mg/kg body weight, 1-10 mg/kg body weight, 2-5 mg/kg body weight, 2-10 mg/kg body weight, or 5-10 mg/kg body weight. Preferably, the said dosage is repeated as needed. Optionally, the dosage may be repeated five times a day, four times a day, three times a day, twice a day, once a day, or less frequently, such as once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, including ranges between any two of the listed values, such as every 1-2 days, every 1-3 days, every 1-5 days, every 1-10 days, every 1-20 days, every 2-3 days, every 3-5 days, every 3-10 days, every 3-20 days, every 5-10 days, or every 5-20 days.

Retinoic acid receptor (RAR) agonists

[0190] According to the present disclosure, administration of the retinoic acid receptor agonist, all-trans-retinoic acid (ATRA), significantly suppresses liver injury induced by alcohol consumption. Ethanol consumption depletes liver retinyl esters and alters physiological levels of all-trans-retinoic acid (ATRA), a biologically active form of vitamin A that supports many biological functions and can increase the production, stability, and differentiation function of naive CD4+ T cells into FoxP3+ Tregs even in the presence of IL-6. Several embodiments described herein relate to the fact that ATRA suppresses type I NKT cell effector function, including suppression of cytokine secretion in these cells. In addition, ATRA has a direct inhibitory effect on type I NKT cell activity that occurs in the absence of antigen-presenting cells. In addition, ATRA does not directly inhibit the activity of typical MHC-restricted CD4+ T cells that recognize protein antigens such as myelin basic protein or MBPAc1-9. Without wishing to be bound by a particular theory, these data suggest that ATRA exerts a protective effect against alcohol-induced liver injury by suppressing type I NKT cells.

[0191] According to the present disclosure, RAR agonists induce suppression of type I NKT cells. In addition, liver injury caused by type I NKT cell-mediated inflammation in excess alcohol consumption can be prevented, reduced, or attenuated by administering an RAR agonist. Liver inflammation can be exacerbated by several different causes. For example, liver inflammation can be caused by a bacterial or viral infection, injury, or attack by the body's own immune system. Although inflammation is generally a protective response and a necessary step in the healing process, prolonged or chronic inflammation can cause injury. Several embodiments described herein relate to RAR agonist-mediated modulation of innate immune mechanisms that lead to liver injury that occurs after inflammation, is associated with inflammation, or is caused by inflammation. Some embodiments relate to methods and compositions for RAR agonist-mediated suppression of type I NKT cell activity that modulate interactions between components of the innate immune system to promote tolerance to gut-derived or metabolite-derived antigens with little or no impact on the innate immune response to detectable foreign pathogens.

[0192] Because RAR agonists can directly suppress type I NKT cells, RAR agonists may be useful for treatment of any indication in which type I NKT cells act as a pathogen. Some examples of diseases that can be treated according to embodiments of the present invention include alcoholic hepatitis, non-alcoholic steatohepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemia and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0193] In some embodiments, various indications, including autoimmune or immune system-related diseases or disorders, are treated, prevented, or ameliorated by RAR agonist-mediated suppression of type I NKT cell activity. In particular, one aspect of the provided embodiment relates to a method of treating a patient suffering from symptoms of an autoimmune or immune system-related disease or disorder, such as, for example, multiple sclerosis, systemic lupus erythematosus, AIDS, Alzheimer's disease, rheumatoid arthritis, insulin-dependent diabetes mellitus, autoimmune hepatitis, asthma, celiac disease, primary sclerosing cholangitis, primary biliary cirrhosis, using an effective amount of a RAR agonist. In some embodiments, RAR agonist-mediated suppression of type I NKT cell activity provides for treatment, prevention, or amelioration of symptoms of asthma. In some embodiments, the patient is a human.

[0194] Some embodiments relate to a method for suppressing or preventing type I NKT cell-mediated inflammation following ischemic reperfusion by administering a RAR agonist. Type I NKT cells play a pathogenic role in conditions such as ischemic and reperfusion injury. Reperfusion injury can occur in a variety of tissues when blood flow is restored following ischemia. Examples include skeletal muscle tissue following a crush injury, cardiac muscle following myocardial infarction or cardiac surgery or ischemic heart disease, neural tissue following stroke or brain injury, and liver and kidney tissue following surgery or injury. Ischemia and reperfusion injury also has a key effect on the quality and function of graft tissue during organ transplantation. Ischemia and reperfusion injury are major contributors to increased length of hospital stay and decreased long-term graft survival. In some embodiments, RAR agonist-mediated suppression of type I NKT cell activity suppresses or prevents ischemia and reperfusion injury to the liver during surgery or trauma.

[0195] Embodiments described herein relate to the suppression of type I NKT cell activity using one or more retinoic acid receptor (RAR) agonists. Retinoic acid receptors include three major subtypes: RARα, RARβ, and RARγ. Some embodiments relate to the suppression of type I NKT cell activity using one or more universal RAR agonists, precursors of such universal RAR agonists, and mixtures thereof. As used herein, the term "universal RAR agonist" refers to a RAR agonist that acts on, e.g., activates, RARα, RARβ, and RARγ substantially equally or non-selectively. Some embodiments relate to the suppression of type I NKT cell activity using one or more active RAR agonists that are effective to selectively or even specifically affect, for example, the activation of at least one of RARβ and RARγ, and preferably both of said receptors, compared to RARα, precursors of said active RAR agonists, and mixtures thereof. In this context, the term "selectively" means that the RAR agonist, RAR agonist precursors, and mixtures thereof are more potent, preferably at least 10-fold, or about 100-fold, or about 1000-fold or more, on at least one of RARβ and RARγ, or both of said receptors, compared to RARα. Some embodiments relate to the suppression of type I NKT cell activity using one or more RAR agonists having subtype selectivity, precursors of said RAR agonists having subtype selectivity, and mixtures thereof. As used herein, the term "RAR agonist having subtype selectivity" refers to a RAR agonist that selectively acts on, e.g., activates, one of the RAR subtypes. Retinoid compounds having selectivity for RARα, RARβ, and RARγ are known in the art and are described, for example, in U.S. Patent Nos. 6,534,544 and 6,025,388, the disclosures of which are incorporated herein by reference in their entirety.

[0196] Several embodiments relate to a method of suppressing proinflammatory activity of type I NKT cells by administering one or more retinoic acid receptor (RAR) agonists. Examples of RAR agonists include, but are not limited to, the RAR agonists listed in Table 1.

[0197] In some embodiments, the proinflammatory activity of type I NKT cells is suppressed using one or more RAR agonists selected from the group consisting of ATRA, retinol, 9-cis-RA or 13-cis-RA, tretinoin, AM580, AC55649, CD1530, or tazarotene. In some embodiments, the proinflammatory activity of type I NKT cells is suppressed using one or more polyolefin retinoids, such as isotretinoin and acitretin. In some embodiments, the proinflammatory activity of type I NKT cells is suppressed using one or more RAR agonists selected from the group consisting of etretinate, acitretin, and isotretinoin.

[0198] Several embodiments relate to the suppression of proinflammatory activity of type I NKT cells using tazarotene, tazarotenic acid, or a mixture thereof. Tazarotene is an ethyl ester prodrug that is metabolized to the corresponding free acid, tazarotenic acid. Tazarotene has a rigid ring structure that has limited conformational flexibility compared to all-trans retinoic acid, the natural ligand of retinoic acid receptors (RAR). This structural change confers tazarotenic acid specificity for RAR and selectivity for RARβ and RARγ.

[0199] Examples of RAR agonists further include esters of cis- and trans-retinoic acids, such as alkyl esters of primary, secondary, or tertiary alcohols, including, but not limited to: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, hexyl, heptyl, ethylhexyl, octyl, nonyl, lauryl, oleyl, stearyl, hydroxyethyl, hydroxypropyl, benzyl, alpha-methylbenzyl, alpha-propylphenyl, amyl, isoamyl, anisyl, cetyl, menthyl, cinnamic, pinacol, furyl, or myristyl.

[0200] Pharmaceutically acceptable salts of RAR agonists can also be used to suppress the activity of type I NKT cells. The compounds described herein that contain sufficiently acidic, sufficiently basic groups, or both types of said functional groups can react with any of organic or inorganic bases and inorganic and organic acids to form a salt.

[0201] Examples of acids that can be used to prepare acid addition salts of RAR agonists containing basic groups include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of said salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenyl acetate, phenyl propionate, phenyl butyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc.

[0202] Examples of bases that can be used to prepare base addition salts of RAR agonists containing acidic groups include, but are not limited to, alkali metal hydroxides such as sodium, potassium, and lithium; alkaline earth metal hydroxides such as calcium and magnesium; other metal hydroxides such as aluminum and zinc; ammonium and other organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis- or tris-(2-hydroxy-lower alkyl)amines such as mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-lower alkyl)amines such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, etc.

[0203] As used herein, the term "patient" or "subject" refers to a recipient of therapeutic treatment and includes all organisms in the animal kingdom. In preferred embodiments, the animal is a member of the mammalian family, such as humans, cattle, sheep, pigs, cats, bison, dogs, goats, horses, donkeys, deer, and primates. In some embodiments, the animal includes a non-human mammal. The most preferred animal is a human. Accordingly, in some embodiments, the patient or subject is a human.

[0204] As used herein, the terms “treat,” “treatment,” and “method of treating” include “prevent,” “prevention,” and “method of preventing,” respectively.

[0205] As used herein, the term "effective amount" of an agent is an amount that is sufficient to treat, suppress, or prevent liver injury caused by proinflammatory activity of type I NKT cells.

[0206] Some embodiments relate to pre-treating patients with sulfatide and/or an RAR agonist prior to the onset of clinical manifestations of ALD. In some embodiments, patients are treated with an effective amount of sulfatide and/or an RAR agonist prior to alcohol abuse. In some other embodiments, patients are treated with an effective amount of sulfatide and/or an RAR agonist from about 0.5 hours to about 18 hours prior to alcohol abuse. In some other embodiments, patients are treated with an effective amount of sulfatide and/or an RAR agonist during a period of chronic alcohol use. In some embodiments, the patient is a human.

[0207] In some embodiments, the specific amount of RAR agonist administered to a patient may vary depending on the disease or condition being treated, as well as the age, weight, and gender of the patient being treated. In general, to provide a specified final concentration, for example in the intestine or blood, the amount of RAR agonist in a single dose of the composition of the present embodiments is generally from about 0.1 milligrams to about 100 milligrams, preferably from about 2.0 milligrams to about 60 milligrams, more preferably from about 20 milligrams to about 50 milligrams. Examples of suitable doses include: from about 0.1 to about 10 mg/day; from about 0.5 to about 2 mg/day; about 0.01-100 mg/day; from about 1 to about 50 mg/day; from about 0.1 mg/day to about 1.0 mg/day; from about 1.0 mg/day to about 5.0 mg/day; from about 5.0 mg/day to about 10.0 mg/day; from about 10.0 mg/day to about 15 mg/day; from about 15.0 mg/day to about 20.0 mg/day; from about 20.0 mg/day to about 25.0 mg/day; from about 30.0 mg/day to about 35.0 mg/day; from about 35.0 mg/day to about 40.0 mg/day; from about 40.0 mg/day to about 45.0 mg/day; from about 45.0 mg/day to about 50.0 mg/day; from about 50.0 mg/day to about 55.0 mg/day; from about 55.0 to about 60.0 mg/day; from about 60.0 mg/day to about 65.0 mg/day; from about 65.0 mg/day to about 70.0 mg/day; from about 70.0 mg/day to about 75.0 mg/day; from about 75.0 mg/day to about 80.0 mg/day; from about 80.0 mg/day to about 85.0 mg/day; from about 85.0 to about 90.0 mg/day; from about 85.0 mg/day to about 90.0 mg/day; from about 90.0 mg/day to about 95.0 mg/day; and from about 95.0 mg/day to about 100.0 mg/day. Obviously, the exact dosage may vary depending on the disease or disorder being treated, and preferred ranges can be readily determined. In some embodiments, the patient is a human.

[0208] When tazarotene is administered orally to reduce type I NKT cell activity, the daily dosage of tazarotene is preferably in the range of about 0.3 mg/day to about 7 mg/day or about 8 mg/day, more preferably in the range of about 0.6 mg/day to about 6.5 mg/day or about 7 mg/day. In some embodiments, tazarotene is administered orally with daily dosages of tazarotene including 0.4 mg/day, 0.75 mg/day, 1.5 mg/day, 2.8 mg/day, 3 mg/day, 4.5 mg/day, 6 mg/day, and 6.3 mg/day.

[0209] In some embodiments, the RAR agonist may be administered to alleviate symptoms in a patient or may be administered to counteract the mechanism of the disorder itself. In certain embodiments, the RAR agonist may be administered as a prophylactic measure. In some embodiments, multiple doses of the RAR agonist are administered. Those skilled in the art will appreciate that these treatment goals are often interrelated and that treatment methods may be adjusted for specific patients depending on various factors. These factors may include the age, gender, or health status of the patient and the progression of the autoimmune or immune-related disease or disorder. Accordingly, the treatment method for the patient may be adjusted with respect to dosage, timing of administration, route of administration, and whether other therapies are administered simultaneously or sequentially. In some embodiments, the patient is a human.

[0210] In some embodiments, one or more RAR agonist compounds can be administered alone or in combination with another therapeutic compound. For example, one or more RAR agonist compounds can be administered in combination with sulfatide. Any currently known therapeutic compound used to treat alcoholic liver disease, an inflammatory disease, an autoimmune disease, or reperfusion injury can be used. In some embodiments, the RAR agonist can be administered in combination with hydrogen sulfide (H2S). In some embodiments, the RAR agonist can be administered in combination with antioxidants. In some embodiments, the RAR agonist can be administered in combination with, for example, corticosteroids, biologics (e.g., anti-TNF-alpha and anti-IL-6 antibodies), immunomodulators (e.g., RU-486), disease-modifying antirheumatic drugs (DMARDs, such as leflunomide), COX-2 inhibitors (celecoxib), non-steroidal anti-inflammatory drugs (NSAIDs, such as naproxen), oral antidiabetic agents (OADs, such as metformin or sitaglipten), GLP-1 agonists, insulin, PPAR agonists/antagonists, EGF mediators (anti-cancer agents), other agents that are effective in the treatment of liver cancers, cell-based therapies for liver cancers; interferons (IFNs) for hepatitis C, multiple sclerosis, or systemic lupus; and LFA-1 antagonists.

[0211] The RAR agonist compounds and sulfatide compounds described herein can be used as an active ingredient included in a pharmaceutical composition. In some embodiments, the pharmaceutical composition can comprise one active ingredient. In some embodiments, the pharmaceutical composition can comprise two, three, four, five or more active ingredients. All routes of administration are contemplated, such as oral, rectal, parenteral, topical, intravenous, intramuscular, intrasternal or subcutaneous injection, or in a form suitable for inhalation. The formulations, if possible, can be advantageously included in separate dosage forms and prepared by any methods well known in the pharmaceutical art. The active ingredients are typically formulated together with one or more pharmaceutically acceptable excipients in accordance with known and established practice. Thus, the pharmaceutical composition can be prepared as a liquid, powder, elixir, injectable solution, suspension, suppository, etc.

[0212] In some embodiments, the proinflammatory activity of type I NKT cells is suppressed using one or more RAR agonists selected from the group consisting of ATRA, retinol, 9-cis-RA or 13-cis-RA, tretinoin, AM580, AC55649, CD1530, or tazarotene. In some embodiments, the proinflammatory activity of type I NKT cells is suppressed using one or more polyolefin retinoids, such as isotretinoin and acitretin. In some embodiments, the proinflammatory activity of type I NKT cells is suppressed using one or more RAR agonists selected from the group consisting of etretinate, acitretin, and isotretinoin.

[0213] Several embodiments relate to the suppression of proinflammatory activity of type I NKT cells using tazarotene, tazarotenic acid, or a mixture thereof. Tazarotene is an ethyl ester prodrug that is metabolized to the corresponding free acid, tazarotenic acid. Tazarotene has a rigid ring structure that has limited conformational flexibility compared to all-trans retinoic acid, the natural ligand of retinoic acid receptors (RAR). This structural change confers tazarotenic acid specificity for RAR and selectivity for RARβ and RARγ.

[0214] Examples of RAR agonists further include esters of cis- and trans-retinoic acids, such as alkyl esters of primary, secondary, or tertiary alcohols, including, but not limited to: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, hexyl, heptyl, ethylhexyl, octyl, nonyl, lauryl, oleyl, stearyl, hydroxyethyl, hydroxypropyl, benzyl, alpha-methylbenzyl, alpha-propylphenyl, amyl, isoamyl, anisyl, cetyl, menthyl, cinnamic, pinacol, furyl, or myristyl.

[0215] Pharmaceutically acceptable salts of RAR agonists can also be used to suppress the activity of type I NKT cells. The compounds described herein that contain sufficiently acidic, sufficiently basic groups, or both types of said functional groups can react with any of organic or inorganic bases and inorganic and organic acids to form a salt.

[0216] Examples of acids that can be used to prepare acid addition salts of RAR agonists containing basic groups include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of said salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenyl acetate, phenyl propionate, phenyl butyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc.

[0217] Examples of bases that can be used to prepare base addition salts of RAR agonists containing acidic groups include, but are not limited to, alkali metal hydroxides such as sodium, potassium, and lithium; alkaline earth metal hydroxides such as calcium and magnesium; other metal hydroxides such as aluminum and zinc; ammonium and other organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis- or tris-(2-hydroxy-lower alkyl)amines such as mono-, bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy-lower alkyl)amines such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, etc.

[0218] In some embodiments, the specific amount of RAR agonist administered to a patient may vary depending on the disease or condition being treated, as well as the age, weight, and gender of the patient being treated. In general, to provide a specified final concentration, for example in the intestine or blood, the amount of RAR agonist in a single dose of the composition of the present embodiments is generally from about 0.1 milligrams to about 100 milligrams, preferably from about 2.0 milligrams to about 60 milligrams, more preferably from about 20 milligrams to about 50 milligrams. Examples of suitable doses include: from about 0.1 to about 10 mg/day; from about 0.5 to about 2 mg/day; about 0.01-100 mg/day; from about 1 to about 50 mg/day; from about 0.1 mg/day to about 1.0 mg/day; from about 1.0 mg/day to about 5.0 mg/day; from about 5.0 mg/day to about 10.0 mg/day; from about 10.0 mg/day to about 15 mg/day; from about 15.0 mg/day to about 20.0 mg/day; from about 20.0 mg/day to about 25.0 mg/day; from about 30.0 mg/day to about 35.0 mg/day; from about 35.0 mg/day to about 40.0 mg/day; from about 40.0 mg/day to about 45.0 mg/day; from about 45.0 mg/day to about 50.0 mg/day; from about 50.0 mg/day to about 55.0 mg/day; from about 55.0 to about 60.0 mg/day; from about 60.0 mg/day to about 65.0 mg/day; from about 65.0 mg/day to about 70.0 mg/day; from about 70.0 mg/day to about 75.0 mg/day; from about 75.0 mg/day to about 80.0 mg/day; from about 80.0 mg/day to about 85.0 mg/day; from about 85.0 to about 90.0 mg/day; from about 85.0 mg/day to about 90.0 mg/day; from about 90.0 mg/day to about 95.0 mg/day; and from about 95.0 mg/day to about 100.0 mg/day. Obviously, the exact dosage may vary depending on the disease or disorder being treated, and preferred ranges can be readily determined. In some embodiments, the patient is a human.

[0219] When tazarotene is administered orally to reduce type I NKT cell activity, the daily dosage of tazarotene is preferably in the range of about 0.3 mg/day to about 7 mg/day or about 8 mg/day, more preferably in the range of about 0.6 mg/day to about 6.5 mg/day or about 7 mg/day. In some embodiments, tazarotene is administered orally with daily dosages of tazarotene including 0.4 mg/day, 0.75 mg/day, 1.5 mg/day, 2.8 mg/day, 3 mg/day, 4.5 mg/day, 6 mg/day, and 6.3 mg/day.

[0220] In some embodiments, the RAR agonist may be administered to alleviate symptoms in a patient or may be administered to counteract the mechanism of the disorder itself. In certain embodiments, the RAR agonist may be administered as a prophylactic measure. In some embodiments, multiple doses of the RAR agonist are administered. Those skilled in the art will appreciate that these treatment goals are often interrelated and treatment methods may be adjusted for specific patients depending on various factors. These factors may include the age, gender, or health status of the patient and the progression of the autoimmune or immune-related disease or disorder. Accordingly, the treatment method for the patient may be adjusted in terms of dosage, timing of administration, route of administration, and whether other therapies are administered concurrently or sequentially. In some embodiments, the patient is a human.

[0221] In some embodiments, one or more RAR agonist compounds can be administered alone or in combination with another therapeutic compound. For example, one or more RAR agonist compounds can be administered in combination with an NKT-2 activator (e.g., miltefosine or a miltefosine analog, or a phospholipid), sulfatide, or an NKT-2 activator (e.g., miltefosine, a miltefosine analog, or a phospholipid) and sulfatide.

NKT-2 activators

[0222] In some embodiments of the present invention, a variety of NKT-2 activators can be used. In some embodiments, the NKT-2 activator comprises a phospholipid, such as a lysophospholipid, such as lysophosphatidylcholine (LPC), miltefosine, or a miltefosine analog. The effects of certain LPC compounds on type II NKT cells are described in Maricic et al, "Recognition of Lysophosphatidylcholine by Type II NKT Cells and Protection from an Inflammatory Liver Disease," J. Immunology^ published online Sep. 26, 2014, 1400699, the contents of which are incorporated herein by reference in their entirety.

[0223] In some embodiments, the NKT-2 activator comprises an LPC having formula IV:

wherein R is selected from the group consisting of _C1 - _C40 alkyl, _C1 - _C40 substituted alkyl, _C1 - _C40 alkenyl, _C1 - _C40 substituted alkenyl, and _C1 - _C40 alkynyl. Exemplary LPCs include LPC(C18:0), the structure of which is depicted below:

LPC (CI6:0), the structure of which is shown below:

LPC (C18:1(9Z)), the structure of which is shown below:

LPC (C18:1(1-oleyl)), the structure of which is shown below:

and Lign A (LPC (C24:0)), the structure of which is shown below:

Additional exemplary phospholipids that have been considered as suitable NKT-2 activators include, but are not limited to, lyso-platelet activating factor (LPAF), such as LPAF (C18:0), the structure of which is depicted below:

and lysosphingomyelin (LSM), the structure of which is shown below:

[0224] In some embodiments, the NKT-2 activator comprises miltefosine. The structure of miltefosine is shown below:

[0225] In some embodiments, the NKT-2 activator comprises a miltefosine analog. As used herein, "miltefosine analog" refers broadly to structural analogs of miltefosine and functional analogs of miltefosine. Some miltefosine analogs according to some embodiments of the present invention include compounds of formula V:

where R is selected from the group consisting of _C1 - _C30 alkyl, _C1 - _C30 substituted alkyl, _C1 - _C30 alkenyl, _C1 - _C30 substituted alkenyl, and _C1 - _C30 alkynyl.

[0226] Exemplary miltefosine analogs that may be used in accordance with some embodiments of the present invention include, but are not limited to, the compounds listed below in Tables 2.1 and 2.2.

[0227] In some embodiments, the miltefosine analog comprises, consists of, or consists essentially of any of the compounds shown in Table 2.1.

In some embodiments, the miltefosine analog comprises, consists of, or consists essentially of a compound selected from Table 2.1. In some embodiments, the miltefosine analog comprises, consists of, or consists essentially of any of the compounds shown in Table 2.1 or Table 2.2. In some embodiments, the miltefosine analog comprises, consists of, or consists essentially of a compound selected from Table 2.1 or Table 2.2. In some embodiments, the miltefosine analog comprises, consists of, or consists essentially of any of the compounds shown in Table 2.2. In some embodiments, the miltefosine analog comprises, consists of, or consists essentially of a compound selected from Table 2.2. In some embodiments, the miltefosine analog comprises, consists of, or consists essentially of a compound of Formula V.

[0228] All routes of administration of miltefosine and miltefosine analogs are contemplated, for example, oral, rectal, parenteral, topical, or by intravenous, intramuscular, intrasternal or subcutaneous injection, or in a form suitable for inhalation. The formulations, if possible, may be advantageously presented in separate dosage forms and prepared by any of the methods well known in the pharmaceutical art. The active ingredients are typically formulated with one or more pharmaceutically acceptable excipients in accordance with known and customary practice. Thus, the pharmaceutical composition may be prepared as a liquid, powder, elixir, injectable solution, suspension, suppository, etc.

[0229] Miltefosine may be suitable for oral or topical administration. Thus, in some embodiments, miltefosine or a miltefosine analog is administered orally. In some embodiments, miltefosine or a miltefosine analog is administered topically. In some embodiments, miltefosine or a miltefosine analog is administered by intravenous, intramuscular, intrasternal, or subcutaneous injection, or in a form suitable for inhalation.

[0230] According to some embodiments of the present invention, the specific amount of miltefosine or a miltefosine analog that is administered to a subject may vary depending on the disease or condition being treated, as well as the age, weight, and gender of the patient being treated. In some embodiments, miltefosine or a miltefosine analog is administered at a dosage of about 0.1 to 10 mg/kg body weight. In some embodiments, miltefosine or a miltefosine analog is administered at a dosage of about 0.1 mg/kg body weight, 0.2 mg/kg body weight, 0.3 mg/kg body weight, 0.5 mg/kg body weight, 1 mg/kg body weight, 2 mg/kg body weight, 3 mg/kg body weight, 4 mg/kg body weight, 5 mg/kg body weight, 6 mg/kg body weight, 7 mg/kg body weight, 8 mg/kg body weight, 9 mg/kg body weight, including ranges between any two of the listed values, such as about 0.2-0.5 mg/kg body weight, 0.2-1 mg/kg body weight, 0.2-5 mg/kg body weight, 0.2-10 mg/kg body weight, 0.5-1 mg/kg body weight, 0.5-3 mg/kg body weight, 0.5-5 mg/kg body weight, 0.5-10 mg/kg body weight, 1-3 mg/kg body weight, 1-5 mg/kg body weight, 1-10 mg/kg body weight, 2-3 mg/kg body weight, 2-5 mg/kg body weight, 2-10 mg/kg body weight or 5-10 mg/kg body weight. Preferably, the said dosage is repeated if necessary. The dosage may optionally be repeated five times a day, four times a day, three times a day, twice a day, once a day, or less frequently, such as once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, including ranges between any two of these values, such as every 1-2 days, every 1-3 days, every 1-5 days, every 1-10 days, every 1-20 days, every 2-3 days, every 3-5 days, every 3-10 days, every 3-20 days, every 5-10 days, or every 5-20 days. In some embodiments, at least 2 dosages are administered, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 doses, including ranges between any two of the listed values, such as 2-10, 2-20, 2-50, 2-100, 5-10, 5-50, 5-100, 10-20, 10-50, 10-100, 20-50 or 20-100 doses. In some embodiments, the patient is a human.

[0231] In some embodiments, the subject to which miltefosine or a miltefosine analog is administered has an inflammatory condition, such as fatty liver disease, alcoholic hepatitis, nonalcoholic steatohepatitis, cirrhosis, nonalcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), autoimmune hepatitis, inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemia and reperfusion injury, solid organ transplant, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

Pharmaceutical compositions

[0232] NKT-2 activators, such as miltefosine or miltefosine analogs, or the phospholipids, RAR agonist compounds, and sulfatide compounds described herein can be used as an active ingredient included in a pharmaceutical composition. In some embodiments, the pharmaceutical composition can comprise one active ingredient. In some embodiments, the pharmaceutical composition can comprise two, three, four, five, or more active ingredients. Any of the following compositions containing an NKT-2 activator can be used to treat, alleviate, or prevent an inflammatory condition such as fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C, and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemia and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis, and inflammatory bowel disease (Crohn's disease and colitis).

[0233] In some embodiments, the pharmaceutical composition comprises an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) that is sufficient to activate type II NKT cells. The pharmaceutical composition optionally further comprises an amount of a RAR agonist, such as a retinoid, e.g., tazarotene, that is sufficient to suppress the activation of type I NKT cells.

[0234] In some embodiments, the pharmaceutical composition comprises an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) and an amount of sulfatide that, taken together, are sufficient to activate type II NKT cells.

[0235] In some embodiments, the pharmaceutical composition comprises an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) and an amount of sulfatide that, taken together, are sufficient to activate type II NKT cells, and further comprises an amount of a RAR agonist, such as a retinoid (e.g., tazarotene), that is sufficient to suppress the activation of type I NKT cells.

[0236] In some embodiments, the pharmaceutical composition comprises an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) that is sufficient to activate type II NKT cells and an amount of sulfatide that is sufficient to activate type II NKT cells.

[0237] In some embodiments, the pharmaceutical composition comprises an amount of an NKT-2 activator (e.g., miltefosine or a miltefosine analog or a phospholipid) that is sufficient to activate type II NKT cells, an amount of sulfatide that is sufficient to activate type II NKT cells, and an amount of a RAR agonist, such as a retinoid (e.g., tazarotene), that is sufficient to suppress type I NKT cell activation.

[0238] In some embodiments, the pharmaceutical composition comprises an amount of miltefosine or a miltefosine analog that is sufficient to activate type II NKT cells and an amount of tazarotene that is sufficient to suppress the activation of type I NKT cells.

[0239] All routes of administration are contemplated, for example, oral, rectal, parenteral, topical, or by intravenous, intramuscular, intrasternal or subcutaneous injection, or in a form suitable for inhalation. The formulations, if possible, may advantageously be presented in separate dosage forms and prepared by any of the methods well known in the pharmaceutical art. The active ingredients are typically formulated with one or more pharmaceutically acceptable excipients in accordance with known and customary practice. Thus, the pharmaceutical composition may be prepared as a liquid, powder, elixir, injectable solution, suspension, suppository, etc. Approaches to preparing pharmaceutical formulations are described in detail in Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, ed., Mack Publishing Company (1995), the contents of which are incorporated herein by reference in their entirety.

[0240] Active ingredients according to some embodiments can be included in a formulation intended for oral administration in the form of tablets, hard gelatin capsules, soft gelatin capsules containing the active substance in the form of a powder, a mixture with excipients, a liquid or solution, a suspension, a solid solution. Active ingredients according to some embodiments can be included in a formulation intended for administration inside the oral cavity (sublingual or buccal) in the form of a solid dosage form, fast-dissolving or effervescent tablets, thin films, buccal strips, or in the form of a liquid or semi-solid form such as a gel, solution, emulsion, suspension. Active ingredients according to some embodiments can be included in a formulation intended for administration by injection in the form of an aqueous or non-aqueous solution, suspension or emulsion. Oil solutions and suspensions contain mixtures of natural and/or synthetic oils, such as soybean oil, cottonseed oil, mineral oil, sesame oil, castor oil, hydrogenated vegetable oils, beeswax. According to some embodiments, the active ingredients can be included in a composition intended for transdermal administration in the form of a cream, gel, emulsion, aqueous solution, oil solution, suspension, film, patch, foam. According to some embodiments, the active ingredients can be included in a composition intended for intranasal administration in the form of a powder, suspension, solution, emulsion. According to some embodiments, the active ingredients can be included in a composition intended for administration by intrapulmonary delivery in the form of a milled powder. Oral administration is associated with the first-pass effect, as well as the induction of enzymes that carry out metabolism. Thus, the dosage amount and dosing regimen for oral administration of retinoic acids can be adjusted to provide an optimal effect. Alternative delivery methods, such as sublingual, buccal, injection, intrapulmonary and transdermal routes, may be preferred over oral administration. Alternative routes of administration, such as those described herein, prevent the first-pass effect and gastrointestinal absorption, provide reduced enzyme induction and stable pharmacokinetics upon repeated administration.

[0241] Pharmaceutical formulations according to embodiments of the present invention can provide immediate release or modified release (e.g., sustained release, pulsatile release, controlled release, delayed release, slow release). Due to the immediate onset of activity, pharmacological amounts of orally administered retinoid isomers can have side effects. These side effects impose serious limitations on the use of oral retinoids in therapy. Although retinoids have little teratogenicity when administered topically, this route of administration is associated with other toxicities, including skin irritation. The primary cause of toxicity with oral and topical administration is that the full amount of retinoids is immediately available after administration. A method in which the retinoid would become available in vivo at a slower rate and over a longer period of time would avoid peaks and valleys in the availability of the retinoid and thereby provide an effective level of the compound in vivo over a longer period of time, which would also prevent or substantially reduce toxicity that partly results from the immediate availability of excess amounts of the substance. An oil-based injectable formulation containing retinol, a retinol ester, in particular a fatty acid ester of retinol, retinoic acid or a retinoic acid ester can be administered intramuscularly once a week to provide systemic delivery with slow release in accordance with the above principles.

[0242] Some embodiments relate to a formulation in the form of an oral dosage form of one or more active ingredients according to embodiments of the present invention, such as those described herein. 10 g of retinoic acid are dissolved in a mixture of beeswax, butylated hydroxyanisole, disodium edetate, hydrogenated soybean oil flakes, hydrogenated vegetable oils, and an alcohol adduct of soybean oil at a slightly elevated temperature with high shear mixing. The mixture is enclosed within soft gelatin capsules in dosages of 2 mg, 5 mg, and 10 mg for oral administration.

[0243] Some embodiments relate to a formulation in the form of a bioadhesive buccal tablet comprising one or more active ingredients according to embodiments of the present invention. A mixture of excipients is prepared comprising 24% active ingredient, 21% HPMC, 18% corn starch, 24% lactose, 1% silicon dioxide, 2.5% polycarbophil (Noveon), 7.5% carbomer 974P, 1.2% talc and 0.7% magnesium stearate. The mixture is compressed into tablets with a diameter of about 1 cm.

[0244] Some embodiments relate to a sublingual film formulation comprising one or more active ingredients according to embodiments of the present invention. The following ingredients are mixed in 50 g of water: 3 rMethocelE5, 5 rMethocel E50, 1 rKlucel, 1 g maltodextrin, 1 g citric acid, 3 g sucralose, 5 g orange flavor, 0.2 g paraben, 0.1 g sodium edetate, and 5 g sorbitol. 1 g retinoic acid is added and the mixture is degassed with stirring. The composition is applied in a thin layer to a polyester film backing and allowed to air dry in the absence of light to prevent degradation. The film is then cut to provide doses of 2 to 10 mg.

[0245] Some embodiments relate to a kit that can comprise one or more sulfatides, RAR agonists, or any combination thereof, preferably in the form of a pharmaceutical composition. In some embodiments, cis-tetracosenoyl is provided in a pharmaceutically acceptable carrier. In some embodiments, ATRA is provided in a pharmaceutically acceptable carrier. In some embodiments, tazarotene is provided in a pharmaceutically acceptable carrier. In some embodiments, hydrogen sulfide (H2S) can optionally be provided. In some embodiments, antioxidants can optionally be provided. In some embodiments, the kits can further comprise suitable packaging and/or instructions for use. The kits can also comprise a means for delivering one or more sulfatides, RAR agonists, or any combination thereof, such as an inhaler, a nebulizer (e.g., for intranasal spraying), an injection syringe, a needle, an i.v. injection bag, or a sealed package for capsules, tablets, suppositories. One or more sulfatides, RAR agonists or any combination thereof may be in dry or lyophilized form or in solution, in particular in a sterile solution. In the case of dry form, the kit may contain a pharmaceutically acceptable diluent to obtain a liquid formulation. The kit may contain a device for administering or dispensing the compositions, including but not limited to a syringe, a pipette, a transdermal patch or an inhaler. Some embodiments relate to kits containing dosages of the compounds or composition that are sufficient to provide effective treatment to an individual for an extended period of time, such as a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks or 8 weeks or more.

CD8 regulatory T cells

[0246] It is contemplated that a combination comprising administering CD8 T _reg cells and administering an NKT-2 activator according to some embodiments of the present invention may be suitable for treating or preventing an inflammatory condition, such as an autoimmune disease. In some embodiments, CD8 T _reg cells are administered to a subject that is developing or is at risk of developing an inflammatory condition. The subject may also be administered an NKT-2 inhibitor. The NKT-2 inhibitor optionally comprises miltefosine. The inflammatory condition optionally includes at least one condition selected from: fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis (A, B, C and others), inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, ischemic and reperfusion injury, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis, amyotrophic lateral sclerosis and inflammatory bowel disease (Crohn's disease and colitis), or a combination of two or more of these conditions.

[0247] The immune system maintains a state of equilibrium in response to foreign antigens as well as self antigens. Control mechanisms to maintain homeostasis following an immune response to a foreign antigen or to prevent or interrupt a harmful response to self antigens include CTLA-4-mediated T cell suppression, activation-induced cell death (AICD), IL-2-mediated regulation, and regulatory T cells (T _reg ) (1 Abbas, A.K., Lohr, J., Knoechel, B. & Nagabhushanam, V. T cell tolerance and autoimmunity. Autoimmun Rev 3, 471-5 (2004)). The induction of CD8 lreg cells is also described in PCT Publication WO 2007/136518 and US Published Patent Application No. 2007/0286849, the contents of each of which are incorporated herein by reference in their entirety.

[0248] Experiments using depleting antibodies or animals with a specific gene knockout have shown an important regulatory role for CD8+ lymphocytes in autoimmune diseases (Koh, DR et al. Less mortality but more relapses in experimental allergic encephalomyelitis in CD8-/- mice. Science 256, 1210-3 (1992)), transplant tolerance (Seydel, K. et al. Anti-CD8 abrogates effect of anti-CD4-mediated islet allograft survival in rat model. Diabetes 40, 1430-4 (1991)), neonatal tolerance (Field, AC, Caccavelli, L., Bloch, MF & Bellon, B. Regulatory CD8 ⁺ T cells control neonatal tolerance to a Th2-mediated autoimmunity. J Immunol 170, 2508-15 (2003)) and homeostasis of normal cellular and humoral immune responses (Nanda, NK & Sercarz, E. A truncated T cell receptor repertoire reveals underlying immunogenicity of an antigenic determinant. J Exp Med 184, 1037-43 (1996)).

[0249] In multiple sclerosis, for example, the immune system abnormally recognizes some of the self-antigens of the myelin membranes as foreign and initiates an immune response directed against them. This results in demyelination, the destructive removal of myelin, which is an insulating and protective fatty protein that forms the sheath of nerve cells (neurons). Demyelination in multiple sclerosis is mediated by a T-cell-mediated immune response that is triggered by antigen-presenting events in the CNS or induced after peripheral activation by a systemic molecular mimicry response.

[0250] Without being limited by theory, it is believed that CD8αα ⁺ , TCRαβ ⁺ T _reg cells may be useful for treating and/or preventing indications including autoimmune and immune-related diseases and disorders. An NKT-2 inhibitor, such as miltefosine, can induce CD8 T _reg cells in the liver of a subject (see, for example, Figures 8A-B). Thus, in some embodiments, CD8 T _reg cells are administered to a subject having or at risk of developing an inflammatory condition, such as those described herein. Optionally, the subject is further administered an NKT-2 inhibitor. In some embodiments, the NKT-2 inhibitor comprises miltefosine. The CD8 T _reg cells administered are optionally isolated from the subject (i.e., they are "autologous" CD8 T _reg cells). The CD8 T _reg cells that are administered are not necessarily isolated from another individual (i.e., they are “allogeneic” CD8 T _reg cells). The CD8 T _reg cells that are administered are not necessarily expanded prior to administration to the subject, for example, the cells may be expanded in vitro. It is envisaged that pathological autoreactive immune responses that underlie inflammatory conditions such as autoimmune diseases or disorders may be treated and reduced or eliminated.

[0251] In general, to provide an effective final concentration, for example in the intestine or blood, the amount of expanded T _reg cell population in a single dose of the composition according to embodiments of the present invention is generally from about 10,000 to about 1 trillion cells, preferably from about 100,000 to about 100 million cells, more preferably from about 1 million to about 50 million cells, even more preferably from about 10 million to about 30 million cells, even more preferably from about 15 million to about 25 million cells, and even more preferably about 20 million cells. Similarly, the amount of secondary therapeutic compound in a single oral dose of the composition according to embodiments of the present invention is generally in the range of from about 0.01 milligrams to about 1000 milligrams, more preferably from about 0.1 milligrams to about 100 milligrams. While it is clear that the exact dosage may vary depending on the disease or disorder being treated, preferred ranges can be readily determined.

[0252] In some embodiments, the expanded population of T _reg cells can be combined with a pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients include, for example, phosphate buffered saline. In one embodiment, the patient is administered T _reg cells in an amount of from about 1,000 to about 3,000,000 cells/kg body weight. Preferably, the T _reg cells are administered in an amount of from about 100,000 to about 1 million cells/kg body weight. The dosage can be repeated as needed, hourly, daily, weekly, monthly, or irregularly. Exemplary dosages and administration schedules are discussed below.

[0253] In some embodiments, the administered T _reg cells comprise CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ cells. In some embodiments, the administered T _reg cells comprise CD8αα ⁺ , TCRαβ ⁺ , IL-2Rβ ⁺ (CD122 ⁺ ). In some embodiments, the administered T _reg cells comprise CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ , CD122 ⁺ . Without being limited by any theory, it is believed that each of the CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ T _reg cells, CD8αα ⁺ , TCRαβ ⁺ , CD122 ⁺ T _reg cells and CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ , CD122 ⁺ T _reg cells also control the activated Vβ8.2 ⁺ CD4 T cell population in vivo and can be used in a similar manner to the CD8αα ⁺ , TCRαβ ⁺ T _reg cells described herein.

[0254] In some embodiments, the regulatory T cells are administered to the same subject from which they were obtained. In other embodiments, the regulatory T cells may be administered to a subject other than the subject from which they were obtained. In other embodiments, the regulatory T cells may be obtained from a mammal that is not the subject. In some embodiments, the regulatory T cells administered comprise a mixture of cells obtained from at least two subjects, the subject to which the regulatory T cells are administered, a subject other than the subject to which the regulatory T cells are administered, and a mammal that is not the subject.

[0255] In some embodiments, anti-CD3 antibody coated plates containing growth factors such as IL-2, IL-7, and IL-15 are used to expand a population of T cells. In some embodiments, _Tregs can be expanded in vitro using recombinant TCR proteins or peptides, such as p42-50, derived from the TCR Vβ chain 8.2. Optionally, a TCR Vβ or Vα chain gene engaged by disease-specific pathogenic T cells can be determined. Proteins corresponding to these TCR Vβ or Vα chain genes can then be administered to the body to activate the corresponding population of _Tregs .

[0256] Various modes and methods of administration of a therapeutic population of T _reg cells are suitable for use in accordance with the embodiments described herein. In some embodiments, delivery methods include, for example, intravenous administration, inhalation, intramuscular, subcutaneous, intranasal, and oral administration, or any other delivery method available in the art. Depending on the particular mode of administration, the dosage form can be, for example, a solid, semi-solid, liquid, gaseous preparation, or aerosol. The dosage form can include, for example, additives, lubricants, stabilizers, buffers, coatings, and excipients available in the art of pharmaceutical formulation.

[0257] According to embodiments described herein, various pharmaceutical formulations are contemplated. In some embodiments, the pharmaceutical formulation comprises CD8 T _reg cells and an NKT-2 activator, as described herein. In some embodiments, the pharmaceutical formulation comprises CD8 T _reg cells and miltefosine. In some embodiments, the pharmaceutical formulation comprises at least one of the following types of CD8 T _reg cells: CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ ,TCRαβ ⁺ ,CD200 ⁺ CD122 ⁺ T cells, and further comprises an NKT-2 activator, as described herein. In some embodiments, the pharmaceutical composition comprises at least one of the following types of CD8 T _reg cells: CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ ,TCRαβ ⁺ ,CD200 ⁺ CD122 ⁺ T cells, and further comprises miltefosine. In some embodiments, the first pharmaceutical composition comprises CD8 T _reg cells and the second pharmaceutical composition comprises an NKT-2 activator, such as described herein.

[0258] In some embodiments, a first pharmaceutical formulation comprises CD8 T _reg cells and a second pharmaceutical formulation comprises miltefosine. In some embodiments, the first pharmaceutical formulation comprises at least one of the following types of CD8 T _reg cells: CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ ,TCRαβ ⁺ ,CD200 ⁺ CD122 ⁺ T cells, and the second pharmaceutical formulation comprises an NKT-2 activator. In some embodiments, the first pharmaceutical formulation comprises at least one of the following types of CD8 T _reg cells: CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ CD122 ⁺ T cells, and the second pharmaceutical formulation comprises miltefosine. In some embodiments, the first and second pharmaceutical formulations are administered simultaneously. In some embodiments, the first and second pharmaceutical formulations are administered at different times. In some embodiments, the first pharmaceutical formulation is administered before the second pharmaceutical formulation. In some embodiments, the second pharmaceutical formulation is administered before the first pharmaceutical formulation. Optionally, any of the pharmaceutical formulations described herein can be prepared by conventional methods using the following pharmaceutically acceptable excipients, etc.: auxiliary substances such as solvents (e.g. water, saline), bulking agents and fillers (e.g. lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, mild silicic anhydride and calcium carbonate); auxiliary substances such as solubilizing agents (e.g. ethanol and polysolvates), binding agents (e.g. starch, polyvinylpyrrolidine, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose and acacia), disintegrating agents (e.g. starch and calcium carboxymethyl cellulose), lubricating agents (e.g. magnesium stearate, talc and hydrogenated oil), stabilizers (e.g. lactose, mannitol, maltose, polysolvates, macrogol and polyoxyethylene hydrogenated castor oil), isotonic agents, wetting agents, lubricating agents, dispersing agents, buffering agents and solubility enhancing agents; and additives such as antioxidants, preservatives, flavorings and fragrances, analgesics, stabilizers, colorants and sweeteners.

[0259] The following examples are provided for illustrative purposes only and are not intended to limit in any way the embodiments described in this application.

EXAMPLE 1

[0260] In a plate-immobilized CD1d assay, miltefosine was able to stimulate the type II NKT cell hybridoma, Hu19.3. 96-well plates coated with CD1d protein were co-incubated with the indicated lipids for 6 hours, washed, and Hu19.3 type II NKT cells were added (50,000 cells/well) and IL-2 levels were measured in the supernatant after 16 hours. LSF, lysosulphatide; LPC, lysophosphatidylcholine.

[0261] The data obtained show that miltefosine, a lysophosphatidylcholine (LPC) analog, can bind to CD1d molecules and stimulate type II NKT cells.

EXAMPLE 2

[0262] Naïve B6 mice were administered DMSO (control) or 1 mg miltefosine (dissolved in DMSO and diluted in PBS) daily for 7 days by oral gavage. Splenocytes were grown in vitro for 90 hours with or without alpha-GalCer. Stimulation (stimulation ratio) and expansion of type I NKT cells (FACS staining for alpha-GalCer/CD1d ^- tetramer ⁺ cells) at 10 ug/mL alpha-GalCer are shown in Figure 2A and Figure 2B.

[0263] The data shown in Figure 2A and Figure 2B show that animals treated with miltefosine experienced significant suppression of type I NKT cell proliferation and growth in the presence of their recognized ligand, alpha-GalCer.

[0264] These data are consistent with the observation that other lipids, such as sulfatide (a glycolipid) or lysophosphatidylcholine (LPC, a phospholipid), activate type II NKT cells, resulting in the inactivation or suppression of type I NKT cells.

EXAMPLE

[0265] Group-specific BL/6 mice were administered DMSO/PBS or miltefosine 12 hours before ConA administration and liver injury was determined by H&E staining of liver sections, and serum liver enzymes such as ALT were measured 24 hours later (Figure 3A and Figure 3B). Data shown are representative of 2 independent experiments.

[0266] As shown in Figure 3B, staining consistent with necrosis (red H&E staining) 1 was observed in liver sections treated with Con A, but staining consistent with reduced necrosis (reduced red H&E staining) 2 was observed in liver sections treated with ConA and miltefosine.

[0267] Accordingly, suppression of ConA-induced hepatitis was observed following treatment with miltefosine. These data indicate that, similar to sulfatide or LPC, treatment of mice with miltefosine significantly suppresses liver injury following Con A injection.

EXAMPLE 4

[0268] C57BL/6 mice were randomly fed a liquid alcohol diet (LAD) or an isocaloric control diet (control) for 10 days that contained miltefosine (-500 micrograms per day) or DMSO/PBS. On day 11, 9 hours after alcohol ingestion (chronic and excess ingestion), the accumulation and activation of type I NKT cells (alpha-GalCer/CD1d ^- tetramer ⁺ cells) or neutrophils (Ly ^- 6G ⁺ CD11b ⁺ ) in the liver were examined. As shown in Figure 4A and Figure 4B, type I NKT cells accumulated in the livers of mice chronically fed the LAD diet and excess ingestion compared to the control diet. Furthermore, as shown in Figure 4C and Figure 4D, type I NKT cells were activated (as assessed by cell surface expression of CD69 on alpha-GalCer/CD1d tetramer ⁺ cells), which was accompanied by accumulation of neutrophils (Ly ^- 6G ⁺ CD11b ⁺ cells) in the liver. The accumulation of activated type I NKT cells and neutrophils drives the inflammatory cascade that causes hepatocyte injury in ALD. Notably, both these cell types do not accumulate in miltefosine-treated mice, providing protection against liver injury.

[0269] The obtained data indicate suppression of the accumulation of activated type I NKT cells and neutrophils in alcoholic liver disease (ALD) in mice administered with miltefosine. Thus, it is suggested that miltefosine can be used for the treatment of inflammatory liver diseases including ALD, NASH, PBC, PSC, viral hepatitis, etc.

EXAMPLE 5

[0270] Treatment with sulfatide was found to significantly suppress chronic and recurrent EAE in SJL/J mice. However, as shown in Figure 6, treatment with oral sulfatide, although initially suppressive of the disease when administered orally, was not very effective. Without wishing to be bound by theory, it is believed that sulfatide is not very stable in the intestine, consistent with the observed low efficacy. Referring to Figure 6, B6 mice were randomly administered sulfatide (200 micrograms in 200 microliters of PBS) orally for 7 days, beginning one day before disease induction. Clinical paralysis was monitored daily and scored on a scale of 1 to 5. Although protection was achieved during the first 4 to 5 days, disease severity then became similar in mice in both groups. Without being bound by any theory, it is believed that the observed similarity of results in both groups is consistent with the instability of sulfatide in the intestine.

[0271] As shown in Figure 5, the potential for suppression of chronic and relapsing EAE, which is the prototype of multiple sclerosis, by oral administration of miltefosine, which is believed to be relatively stable, was assessed. SJL mice were randomly assigned to receive miltefosine (1 mg in 200 microliters of 10% DMSO/PBS) or control (DMSO/PBS) by oral gavage twice weekly, as indicated by the arrows. Clinical disease severity was monitored daily by scoring paralysis in both groups of mice on a scale of 1 to 5, as shown in the figure.

[0272] The data shown in Figure 5 summarize the results of treatment of chronic and relapsing experimental autoimmune encephalomyelitis (EAE), which is the prototype of multiple sclerosis, with miltefosine. These experiments demonstrate that T-cell-mediated autoimmune diseases, including MS, T1DD, RA, lupus and IBD, etc., can be cured with miltefosine.

EXAMPLE 6

[0273] PBMCs (1 x 10 ⁶ /well) were stimulated in vitro with 100 ng/mL αGalCer, 100 ng/mL αGalCer + 5 μg/mL miltefosine, or no stimulation. After 12 days, cells were harvested and type I NKT cell percentage was assessed by flow cytometry. Stained cells were collected using BD FACS Canto. Data were analyzed using FlowJo software. Human type I NKT cells, identified as CD3+/Vα24Jα18 TCR+ cells (CD1d-restricted type I NKT cells), are shown as % NKT cells in the boxed area of Figure 7. The percentage of type I NKT cells in fresh PBMCs (not in culture) and in vitro cultured PBMCs is shown for each graph. The data shown in Figure 7 are representative of more than 5 independent experiments.

[0274] Accordingly, it has been shown that, in some embodiments of the present invention, treatment with miltefosine can result in an increase in human type I NKT cells.

[0275] The use of the singular forms "a" and "an" in this application may include references to multiple objects unless otherwise specifically stated or unless it would be apparent to those skilled in the art after reading this description that the singular form represents a single functional embodiment. Thus, for example, the singular form "an" may mean more than one, and "one embodiment" may mean that the description refers to multiple embodiments.

[0276] The foregoing description and examples describe in detail certain embodiments. It should be understood, however, that regardless of the detail of the description given in the text, the invention may be implemented in many ways and the invention should be considered in accordance with the appended claims and their equivalents.

EXAMPLE 7

[0277] Female SJL/J mice were randomly administered 1 mg miltefosine orally as shown in Figure 2 before and during disease. Mice were sacrificed on day 59. Flow cytometry profiles of liver mononuclear cells stained with CD4, CD8α, and CD8β mAbs are shown in Figures 8A-B. Interestingly, while regulatory CD8αα ⁺ T cells were significantly elevated in the liver, other CD4+ and CD8αβ ⁺ T cells were not significantly different between control and treatment groups.

[0278] Accordingly, administration of NKT-2 inhibitors such as miltefosine, according to some embodiments of the present invention, can enhance the induction of CD8αα ⁺ T cells in the liver.

EXAMPLE 8

[0279] To determine the in vivo regulatory potential of CD8 T _reg, 2D11 cells were adoptively transferred intravenously into syngeneic mice. Twenty-four hours later, recipients were immunized with MBPAc1-9/CFA/PT to induce EAE, clinical symptoms were monitored daily, and they were scored for 35-45 days using a 1-5 scale: where 1 corresponds to tail paralysis; 2 corresponds to hindlimb paralysis; 3 corresponds to hindlimb paralysis; 4 corresponds to hindlimb paralysis and partial body paralysis; and 5 corresponds to whole body paralysis or death. As shown in Figure 8A, mice injected with 1 million 2D11 cells recovered from attenuated disease more quickly compared to controls. To test the possibility that disease-fighting ability is determined by long-lived cultures of a particular clone, short-lived p42-50-reactive T cell lines were generated and used in similar adoptive transfer experiments. As shown in Figure 8B, mice injected with five million cells of the p42-50-reactive T cell line were completely protected from MBP-induced EAE, and transfer of only one million cells resulted in accelerated recovery from attenuated paralysis. Adoptive transfer of short-lived T cell lines to two minor TCR peptides had no effect on EAE (data not shown). See Figures 8A-B.

[0280] Accordingly, adoptive transfer of CD8 T _reg cell lines and clones according to some embodiments of the present invention provides rapid recovery and protection in EAE. Accordingly, it is believed that adoptive transfer of CD8 T _reg cell lines and clones according to some embodiments of the present invention can reduce and/or prevent at least one inflammatory condition.

EXAMPLE 9

[0281] The predominant use of the TCR Vβ6 chain by CD8 T _reg cells is demonstrated by assessing the potential impact of treating mice with anti-Vβ6 mAb on the CD8 T _reg repertoire and the course of MBPAc1-9-induced EAE. Following a single intravenous injection of anti-TCR Vβ6 mAb (RR4-7, 300 μg/mouse), splenocytes were assessed at various time points to determine the presence of Vβ6+ cells in vivo. As expected, injection of anti-TCR Vβ6 mAb resulted in early activation of TCR Vβ6 ⁺ T cells in vivo, as determined by expression of the early activation marker CD69 (Figure 9A, second column), and subsequent downregulation of Vβ6 cell surface expression (Figure 9A, first column). The effect of the anti-TCR Vβ6 mAb was TCR-specific, as there was no effect on Vβ8 ⁺ T cells under identical experimental conditions (Figure 9A, third and fourth columns). Real-time RT-PCR analysis of TCR Vβ6 mRNA expression in splenocytes from mice treated with the anti-TCR Vβ6 mAb showed that TCR Vβ6 ⁺ T cells were mostly, but not all, depleted after long-term treatment (Figure 9B and data not shown).

[0282] CD8αα T cells, primarily present in intraepithelial lymphocytes (IELs) in the intestine, are relatively resistant to killing by self-agonists and may differ from CD8αβ T cells in their response to T cell receptor activation. A study was conducted to examine whether anti-TCR Vβ6 mAb could kill or activate CD8αα T cells in peripheral organs using TL-tetramer staining. The data shown in Figure 9C indicate that TL-tetramer ⁺ CD8 ⁺ cells were not depleted following antibody administration. Indeed, antibody-treated mice showed a modest increase in the in vitro secondary response to p42-50 (data not shown). To further investigate the regulatory function following CD8αα ⁺ Vβ6 ⁺ T cell activation, mice were immunized with MBPAc 1-9/CFA/PT to induce EAE. One day and one week later, they were given a low-dose intravenous injection of anti-Vβ6 TCR mAb (100 μg/mouse), isotype control mAb (100 μg/mouse), or PBS. Clinical signs were monitored daily after the second injection. The data shown in Figure 9D and Table 4 show that in vivo activation of Vβ6 ⁺ T cells with anti-Vβ6 TCR mAb resulted in protection against EAE in B10.PL mice. In contrast, animals in the isotype control mAb or non-specific mAb injection groups were not protected from disease (data not shown).

[0283] Activation (CD69 expression) and subsequent downregulation/depletion of Vβ6+ but not Vβ8 ⁺ T cells following a single intravenous injection of anti-TCR Vβ6 mAb (300 μg/mouse). On days 0, 1, and 3, splenocytes were stained with TCR Vβ6-FITC/CD8a-PE/CD69-PerCP (first and second columns) or TCR Vβ8-FITC/CD8α-PE/CD69-PerCP (third and fourth columns). Cells were separated into TCR Vβ6/CD8a ⁺ T cells (second column) or TCR Vβ8/CD8a ⁺ T cells (fourth column) for analysis of expression of the early activation marker CD69. Data are representative of two independent experiments. (b) Significant depletion of Vβ6 ⁺ T cells following anti-V(36 antibody injection as determined by real-time GGTTR. mRNA expression is shown as relative amounts after normalization for two internal control genes (L32 and cyclophilin). Data are representative of two independent experiments, (c) TL-tetramer ⁺ CD8 T cells are not depleted following anti-V(36 TCR antibody injection. Splenocytes were stained with CD8α-FITC/TL-tetramer-PE on the indicated days following antibody injection. Data are representative of two independent experiments, (d) Mice injected with anti-V(36 TCR mAb are protected from EAE. One day and seven days after induction of EAE with MBPAc1-9/CFA/PT, mice were randomly injected with anti-Vβ6 TCR mAb (100 μg/mouse) or isotype control IgG or FBI. The paralyzing disease was monitored as described in the Methods section. Data were pooled for three independent experiments.

[0284] Accordingly, TCR activation of Vβ6 ⁺ T cells in vivo according to some embodiments of the present invention provides protection against EAE.

Claims (27)

1. The use of miltefosine in the manufacture of a medicament for alleviating or preventing at least one inflammatory condition in a subject, wherein the amount of miltefosine in the medicament is sufficient to activate type II NKT cells in the subject and wherein the inflammatory condition is selected from the group consisting of fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis A, B and C, inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, solid organ transplantation, systemic lupus erythematosus, rheumatoid arthritis and amyotrophic lateral sclerosis. 2. The use according to claim 1, wherein the medicinal product further comprises a RAR agonist and the amount of RAR agonist in the medicinal product is sufficient to suppress the activation of type I NKT cells in the subject, wherein the RAR agonist is selected from ATRA, isotretinoin, tazarotene or retinol or a retinol ester. 3. The use according to claim 1, wherein miltefosine is prepared in a form for oral administration. 4. The use according to claim 2, wherein the RAR agonist comprises ATRA or isotretinoin. 5. The use according to claim 2, wherein the RAR agonist comprises tazarotene. 6. The use according to claim 2, wherein the RAR agonist comprises retinol or a retinol ester. 7. The use according to claim 1, wherein the activation of type II NKT cells comprises at least one of: the production of IL-2 by type II NKT cells, the proliferation of type II NKT cells, or the expression of CD69 on the surface of type II NKT cells. 8. The use according to claim 2, wherein the suppression of activation of type I NKT cells comprises suppression of accumulation of alpha-GalCer/CD1d-tetramer ⁺ TCR-beta ⁺ cells. 9. The use according to claim 1, wherein the amount of NKT-2 activator in the medicinal product is sufficient to activate CD8αα ⁺ T cells in the liver of the subject. 10. The use according to claim 1, wherein the inflammatory condition is multiple sclerosis. 11. The use according to claim 1, wherein the inflammatory condition is systemic lupus erythematosus. 12. The use according to claim 1, wherein the inflammatory condition is non-alcoholic steatohepatitis. 13. The use of isolated CD8αα ⁺ , TCRαβ ⁺ T cells and miltefosine in the manufacture of a medicament for alleviating or preventing at least one inflammatory condition in a subject, wherein the amount of miltefosine in the medicament is sufficient to activate type II NKT cells in the subject and wherein the inflammatory condition is selected from the group consisting of fatty liver disease, alcoholic hepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, cirrhosis, non-alcoholic fatty liver disease, fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fulminant cirrhosis, idiopathic hepatitis, viral hepatitis A, B, and C, inflammatory hepatitis associated with hepatobiliary carcinoma, multiple sclerosis, type 1 diabetes, solid organ transplantation, systemic lupus erythematosus, and amyotrophic lateral sclerosis. 14. The use according to claim 13, wherein the amount of miltefosine is sufficient to activate CD8αα ⁺ T cells in the liver of the subject. 15. The use according to paragraph 13, additionally including: obtaining a cell sample from a mammal; isolation of CD8αα ⁺ , TCRαβ ⁺ T cells from a cell sample and cultivation of isolated T cells. 16. The use according to claim 15, wherein the mammal is the subject. 17. The use according to claim 16, wherein the mammal is an organism other than the subject. 18. The use according to claim 15, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ . 19. The use according to claim 15, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD122 ⁺ . 20. The use according to claim 15, wherein the isolated T cells are CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ , CD122 ⁺ . 21. The use according to claim 15, wherein the isolated T cells comprise a mixture of two or more cell types selected from CD8αα ⁺ TCRαβ ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD200 ⁺ T cells, CD8αα ⁺ TCRαβ ⁺ CD122 ⁺ T cells, and CD8αα ⁺ , TCRαβ ⁺ , CD200 ⁺ CD122 ⁺ T cells. 22. The use according to claim 13, wherein the inflammatory condition is multiple sclerosis. 23. The use according to claim 13, wherein the inflammatory condition is systemic lupus erythematosus. 24. The use according to claim 13, wherein the inflammatory condition is non-alcoholic steatohepatitis.