Methods for treating inflammation and related diseases and disorders by inhibiting alpha-protein kinase 1

Abstract

The present disclosure provides methods related to inhibiting alpha-kinase 1 (ALPK1) for treating inflammation and inflammatory diseases, disorders, and conditions, as well as for treating autoimmune diseases, diseases or disorders caused by bacterial infection, or cancer.

Description

[Technical Field]

[0001]

[01] The present invention relates to methods for treating inflammation and related diseases and disorders by inhibiting alpha-protein kinase 1 (ALPK1). [Background technology]

[0002]

[02] Research into the mechanisms of the inflammatory response has identified various protein kinases that act as essential signaling components, and defects in protein kinases are often associated with the pathogenesis of human inflammatory diseases, cancer, and diabetes.

[0003]

[03] Alpha-kinases represent a novel protein kinase superfamily that shows little sequence similarity to conventional protein kinases. A total of six alpha-kinase members have been identified, including alpha-protein kinase 1 (ALPK1), ALPK2, ALPK3, elongation factor 2 kinase (eEF2K), and transient receptor potential cation channels M6 and M7 (TRPM6 and TRPM7). Ryazanov AG et al., Curr Biol 1999, 9(2):R43-45; Ryazanov AG et al., Proc Natl Acad Sci USA 1997, 94(10):4884-4889.

[0004]

[04] ALPK1 was identified as a novel component of raft-containing sucrose isomerase (SI) vesicles in epithelial cells. Heinet M et al., J. Biol. Chem. (2005) 280(27):25637-25643. ALPK1 phosphorylates myosin 1 and has been shown to play an important role in exocytotic transport to the apical plasma membrane. Homozygous transposon insertional inactivation of ALPK1 in mice resulted in motor coordination defects that could be rescued by overexpressing full-length ALPK1. Chen M et al., (2011) BMC Neurosci. 12:1.

[0005]

[05] Several genetic association studies have linked ALPK1 to gout risk, although not all identified polymorphisms are replicated in all populations. Wang SJ et al., (2011) J. Mol. Med. 89:1241-1251; Ko AM et al., (2013) J. Intl. Epidemiol. 42:466-474; Chiba T et al., (2015) Human Cell 28:1-4. Other genetic association studies have linked ALPK1 as a risk factor for chronic kidney disease, myocardial infarction, and diabetes. Yamada Y et al., (2013) J Med Genet 50:410-418; Fujimaki T et al., (2014) Biomed Report. 2:127-131; Shimotaka S et al., (2013) Biomed Report. 1:940-944; Yamada Y et al., (2015) Biomed.Report DOI:10.3892 / br.2015.439.

[0006]

[06] Additional functional studies have implicated ALPK1 as a regulator of innate immunity to bacteria. For example, ALPK1 has been suggested to be a regulator of innate immunity to bacteria through its promotion of TIFA oligomerization and IL-8 expression in response to infections with S. flexneri, S. typhimurium, and Neisseria meningitides. Milivojevic M et al. (2017) PLoS Pathog 13(2):e1006224. Overexpression of ALPK1 in mice resulted in decreased testosterone levels and increased production of the pro-inflammatory cytokines IL-1β and TGF-β, suggesting that the balance between ALPK1 and testosterone may play a role in testosterone-mediated inhibition of pro-inflammatory cytokines. Kuo TM et al. (2015) J Steroid Biochem Mol Biol (2015) 154:150–158. Recently, myosin IIA has been shown to interact with ALPK1-regulated TNF transport in gouty plaques. Lee CP et al. (2016) Sci. Report 6:25740.

[0007]

[07] ALPK1 expression and mutations have also been found in certain cancers, including lung, colorectal, and breast cancer. Liao HF et al. (2016) Scientific Reports. 6:27350; Strietz J et al. (2016) Oncotarget 1-16.

[0008]

[08] Overexpression of ALPK1 accelerated multiple early renal damage in a mouse model of hyperglycemia. Kuo TM et al. (2016) Biochimka Biophysika Acta 1862:2034-2042. Summary of the Invention [Problem to be solved by the invention]

[0009]

[09] There are many diseases, disorders, and conditions whose clinical symptoms result from excessive and / or chronic inflammation. There is a need for new methods for inhibiting and / or reducing inflammation in target tissues to treat such diseases, disorders, and conditions. The present disclosure addresses this need. [Means for solving the problem]

[0010]

[10] The present invention is based, in part, on the discovery that inhibition of ALPK1 reduces inflammation and its clinical effects in several different animal models of inflammation, and is also effective in reducing the production of pro-inflammatory cytokines in various human and mouse cell lines. Accordingly, the present disclosure provides a method for treating inflammation in a subject in need of such treatment, comprising administering an ALPK1 inhibitor to the subject. The present disclosure also provides a method for treating an inflammatory disease, disorder, or condition, comprising administering an ALPK1 inhibitor to a subject in need of such treatment. In an embodiment, the inflammatory disease, disorder, or condition is characterized by chronic or excessive inflammation.

[0011]

[11] In embodiments, the inflammatory disease or disorder is selected from inflammatory bowel disease, arthritis, obesity, gout, radiation-induced inflammation, psoriasis, cardiovascular disease, diabetes, epithelial cancers including lung, colon, and breast cancer, T-cell mediated hypersensitivity diseases, allergic diseases, atopic dermatitis, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, systemic lupus erythematosus (SLE), autoimmune thyroiditis (Graves' disease), multiple sclerosis, ankylosing spondylitis, and bullous diseases caused by overproduction of pro-inflammatory cytokines.

[0012]

[12] In embodiments, the inflammatory bowel disease or disorder is selected from inflammatory bowel disease, arthritis, obesity, and radiation-induced inflammation. In embodiments, the inflammatory bowel disease is selected from Crohn's disease and ulcerative colitis.

[0013]

[13] In an embodiment, the ALPK1 inhibitor is a kinase inhibitor. In an embodiment, the kinase inhibitor is 1-benzyl-3-hexadecyl-2-methyl-1H-imidazol-3-ium iodide salt or other halogen salts thereof.

[0014]

[14] In an embodiment, the ALPK1 inhibitor is an antibody directed against ALPK1 or an anti-ALPK1-Fc fusion protein.

[15] In an embodiment, the ALPK1 inhibitor is an ALPK1 antisense polynucleotide. In an embodiment, the ALPK1 inhibitor is an interfering RNA directed against ALPK1 selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA).

[0015]

[16] In embodiments, the present disclosure also provides a pharmaceutical composition comprising an ALPK1 inhibitor and a carrier or excipient for use in the methods described herein. In embodiments, the ALPK1 inhibitor is a kinase inhibitor. In embodiments, the kinase inhibitor is 1-benzyl-3-hexadecyl-2-methyl-1H-imidazol-3-ium iodide salt or other halogen salts thereof. In embodiments, the pharmaceutical composition is formulated for oral or rectal delivery. In embodiments, the pharmaceutical composition is formulated as an oral dosage form in the form of a tablet or capsule. In embodiments, the pharmaceutical composition is formulated as a rectal dosage form in the form of an ointment, suppository, or enema. In embodiments, the pharmaceutical composition is formulated as a parenteral dosage form. In embodiments, the parenteral dosage form is suitable for administration via intravenous, intraarterial, or intramuscular routes, for example, by injection of an aqueous liquid.

[0016]

[17] The present disclosure also provides a method for treating an autoimmune disease, a disease or disorder caused by a bacterial infection, or cancer in a subject in need of such treatment, comprising administering to the subject an alpha-kinase 1 (ALPK1) inhibitor.

[0017]

[18] In embodiments where the method is a method of treating cancer, the cancer may be selected from lung cancer, colorectal cancer, and breast cancer.

[19] In embodiments where the method is for treating an autoimmune disease, the autoimmune disease is selected from the group consisting of systemic vasculitis, glomerulonephritis (poststreptococcal glomerulonephritis), Sjogren's syndrome, psoriatic arthritis, gout, gouty arthritis, reactive arthritis, septic shock, Graves' disease, Goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, autoimmune myositis, pernicious anemia, celiac disease, eczema, autoimmune thyroiditis, autoimmune myocarditis, celiac disease, juvenile idiopathic arthritis, Graves' disease, Goodpasture's syndrome, psoriatic arthritis, gout, gouty arthritis, reactive arthritis, septic shock, Graves' disease, Goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, autoimmune myositis, pernicious anemia, celiac disease, eczema, autoimmune thyroiditis, autoimmune myocarditis, celiac disease, juvenile idiopathic arthritis, Graves' disease, idiopathic thrombocytopenic purpura, autoimmune myositis, pernicious anemia, celiac disease, eczema, autoimmune thyroiditis, autoimmune myocarditis, juvenile idiopathic arthritis, Graves' disease, idiopathic myocard ... The condition may be selected from Reb's ophthalmopathy, polymyalgia rheumatica, autoimmune uveitis, alopecia areata, Wegener's disease, vitiligo, primary sclerosing cholangitis, primary biliary cirrhosis, autoimmune hepatitis, Guillain-Barré syndrome, antiphospholipid syndrome, sarcoidosis pain, alopecia areata, Lambert-Eaton myasthenic syndrome, autoimmune hemolytic anemia, cold agglutinin disease, warm autoimmune hemolytic anemia, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), and Behcet's disease.

[0018]

[20] In embodiments where the method is for treating a disease or disorder caused by a bacterial infection, the disease or disorder may be selected from chronic infection, sepsis, and cytokine storm. In embodiments, the disease or disorder may be caused by a bacterium selected from Neisseria, Escherichia, Klebsiella, Salmonella, Shigella, Vibrio, Helicobacter, Pseudomonas, Burkholderia, Haemophilus, Moraxella, Bordetella, Francisella, Pasteurella, Borrelia, Campylobacter, Yersinia, Rickettsia, Treponema, Chlamydia, and Brucella. [Brief explanation of the drawings]

[0019] [Figure 1]

[21] Figure showing that inactivating ALPK1 mutations confer resistance to DSS-induced colitis. Percentage change in body weight (%). Triangles, wild type (WT); diamonds, heterozygous mutants (HE); squares, homozygous mutants (HO). [Figure 2]

[22] Figure 2A shows that inactivating ALPK1 mutations confer resistance to radiation-induced weight loss. The y-axis shows percentage weight change. Figure 2B shows that inactivating ALPK1 mutations confer resistance to radiation-induced weight loss. The y-axis shows survival rate. [Figure 3]

[23] Figure 2 shows that an inactivating ALPK1 mutation confers resistance to high-fat diet-induced obesity. "HFD" refers to "high-fat diet treatment," and the y-axis shows percentage body weight change. [Figure 4]

[24] Figure 4A shows that inactivating ALPK1 mutations confer resistance to collagen-induced arthritis. ALPK1 mutant males. Footpad RA score. Figure 4B shows that inactivating ALPK1 mutations confer resistance to collagen-induced arthritis. ALPK1 mutant males. Difference in left and right hind footpad thickness. [Figure 5]

[25] Figure 1 shows that ALPK1 knockdown by siRNA results in decreased cytokine expression in HEK293T cells. [Figure 6]

[26] Figure 6A shows that knockdown of ALPK1 in HEK293T cells results in decreased cytokine expression induced by S. flexneri cell lysates and overexpression of human and mouse ALPK1. Figure 6B shows that IL8 was measured and demonstrated efficient IL8 expression by Flag-ALPK1, mouse ALPK1, 6*His-ALPK1-3*Flag, and 6*His-ALPK1, but not by the 6*His-ALPK1-E mutant. ALPK1 can rescue the impaired cytokine expression, but not the kinase-dead mutant. [Figure 7]

[27] Figure 7A shows that CRISPR / Cas9 knockout of ALPK1 in HEK293T cells results in decreased S. flexneri cell lysate-induced cytokine expression and secretion. Relative expression normalized to wild-type HEK293T control. Figure 7B shows that CRISPR / Cas9 knockout of ALPK1 in HEK293T cells results in decreased S. flexneri cell lysate-induced cytokine expression and secretion. IL8 concentration in conditioned medium of HEK293T 4 hours after S. flexneri cell lysate induction. [Figure 8]

[28] Figure 1 shows that knockdown of ALPK1 in HEK293 cells results in a decrease in S. flexneri and S. typhimurium cell lysate-induced cytokine expression. [Figure 9]

[29] Figure 9A shows that shRNA-mediated ALPK1 knockdown in THP-1 macrophage cells results in reduced expression of pro-inflammatory cytokines in the presence of LPS induction. Figure 9B shows that shRNA-mediated ALPK1 knockdown in THP-1 macrophage cells results in reduced expression of pro-inflammatory cytokines in the absence of LPS induction. [Figure 10]

[30] Figure 10A shows that shRNA-mediated knockdown of ALPK1 in THP-1 macrophage cells results in inhibition of TNFα secretion. Figure 10B shows that shRNA-mediated knockdown of ALPK1 in THP-1 macrophage cells results in inhibition of IL1β secretion. [Figure 11]

[31] Figure 3 shows that ALPK1 knockdown by siRNA in MDA-MB-468 human breast cancer cell line macrophages resulted in decreased expression of TNFα, IL1β, and IL8. [Figure 12]

[32] Figure 12A shows that shRNA-mediated ALPK1 knockdown in the RAW264.7 mouse macrophage-like cell line results in decreased cytokine expression in the presence of LPS. Figure 12B shows that shRNA-mediated ALPK1 knockdown in the RAW264.7 mouse macrophage-like cell line results in decreased cytokine expression in the absence of LPS. [Figure 13]

[33] Figure 1 shows that pro-inflammatory cytokine expression was reduced in bone marrow-derived macrophage cells isolated from mice carrying an inactivating ALPK1 mutation. [Figure 14]

[34] Figure 3 shows that the ALPK1 inhibitor MI6C improves recovery in a DSS-induced colitis model. Mice were treated with 3% DSS from day 0 to day 5, then switched to drinking water and given daily IP injections of either MI6C (1 mg / kg) dissolved in DMSO (n=6) or DMSO alone (n=5). The Y-axis shows the change in body weight relative to day 0, and the X-axis shows time (days). [Figure 15A]

[35] Figure 15A shows that ALPK1 mutation reduces tumor growth and metastasis in the MMTV-PyVT breast cancer model. In each panel, mice with ALPK1 mutations are represented by dark bars, and mock transgenic controls are represented by light bars. A, age at breast tumor onset; B, breast tumor burden 10 weeks after breast tumor onset; C, lung tumor burden (metastasis from breast tumors) 10 weeks after breast tumor onset; D, lung weight (metastasis from breast tumors) 10 weeks after breast tumor onset. [Figure 15B]

[35] Figure 15B shows that ALPK1 mutation reduces tumor growth and metastasis in the MMTV-PyVT breast cancer model. In each panel, mice with ALPK1 mutations are represented by dark bars, and mock transgenic controls are represented by light bars. A, age at breast tumor onset; B, breast tumor burden 10 weeks after breast tumor onset; C, lung tumor burden (metastasis from breast tumors) 10 weeks after breast tumor onset; D, lung weight (metastasis from breast tumors) 10 weeks after breast tumor onset. [Figure 15C]

[35] Figure 15C shows that ALPK1 mutation reduces tumor growth and metastasis in the MMTV-PyVT breast cancer model. In each panel, mice with ALPK1 mutations are represented by dark bars, and mock transgenic controls are represented by light bars. A, age at breast tumor onset; B, breast tumor burden 10 weeks after breast tumor onset; C, lung tumor burden (metastasis from breast tumors) 10 weeks after breast tumor onset; D, lung weight (metastasis from breast tumors) 10 weeks after breast tumor onset. [Figure 15D]

[35] Figure 15D shows that ALPK1 mutation reduces tumor growth and metastasis in the MMTV-PyVT breast cancer model. In each panel, mice with ALPK1 mutations are represented by dark bars, and mock transgenic controls are represented by light bars. A, age at breast tumor onset; B, breast tumor burden 10 weeks after breast tumor onset; C, lung tumor burden (metastasis from breast tumors) 10 weeks after breast tumor onset; D, lung weight (metastasis from breast tumors) 10 weeks after breast tumor onset. [Figure 16]

[36] Figure 1 shows the amino acid sequence of human ALPK1 isoform 1. The sequence of isoform 2 differs from isoform 1 as follows: 1-92: MNNQKVVAVL...VIGAGLQQLL → MCRKRTRARTSAAE DETAILED DESCRIPTION OF THE INVENTION

[0020]

[37] The present invention is based, in part, on the discovery that ALPK1 is a potent inducer of inflammatory responses in a variety of cellular and animal models of inflammation, and therefore its inhibition is effective in inhibiting inflammation and ameliorating its harmful effects. Accordingly, the present disclosure provides methods for treating inflammation and diseases, disorders, and conditions characterized by excessive and / or chronic inflammation, as well as for treating autoimmune diseases, diseases and disorders caused by bacterial infection, and cancer, by administering an ALPK1 inhibitor to a subject in need of such treatment, or by inhibiting ALPK1 in the subject, for example, using gene therapy approaches and related compositions.

[0021]

[38] The term "ALPK1" is used herein interchangeably to refer to isoform 1 (Q96QP1-1) of the human sequence - Q96QP1 (ALPK1_HUMAN) identified by UniProtKB or its alternative splice variant isoform 2 (Q96QP1-2), unless the context clearly refers to a specific isoform. See Figure 16 and SEQ ID NO: 1. Isoform 2 differs from isoform 1 as follows: 1~92:MNNQKVVAVL...VIGAGLQQLL → MCRKRTRARTSAAE

[39] In embodiments, the inflammatory disease or disorder is selected from inflammatory bowel disease, arthritis, obesity, gout, radiation-induced inflammation, psoriasis, cardiovascular disease, diabetes, epithelial cancers including lung, colon, and breast cancer, T-cell mediated hypersensitivity disorders, allergic disorders, and atopic dermatitis due to the overproduction of pro-inflammatory cytokines.

[0022]

[40] In embodiments, the inflammatory disease or disorder is selected from inflammatory bowel disease, arthritis, obesity, and radiation-induced inflammation. In embodiments, the inflammatory bowel disease or disorder is selected from Crohn's disease and ulcerative colitis.

[0023]

[41] In embodiments, the autoimmune disease is systemic vasculitis, glomerulonephritis (poststreptococcal glomerulonephritis), Sjogren's syndrome, psoriatic arthritis, gout, gouty arthritis, reactive arthritis, septic shock, Graves' disease, Goodpasture's syndrome, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, autoimmune myositis, pernicious anemia, celiac disease, eczema, autoimmune thyroiditis, autoimmune myocarditis, celiac disease, juvenile idiopathic arthritis, Graves' ophthalmopathy, rheumatoid arthritis ... The disease is selected from polymyalgia pemphigoid, autoimmune uveitis, alopecia areata, Wegener's disease, vitiligo, primary sclerosing cholangitis, primary biliary cirrhosis, autoimmune hepatitis, Guillain-Barré syndrome, antiphospholipid syndrome, sarcoidosis pain, alopecia areata, Lambert-Eaton myasthenic syndrome, autoimmune hemolytic anemia, cold agglutinin disease, warm autoimmune hemolytic anemia, eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), and Behcet's disease.

[0024]

[42] In embodiments, the disease or disorder caused by bacterial infection is selected from chronic infection, sepsis, and cytokine storm. In embodiments, the disease or disorder is caused by a bacterium selected from Neisseria, Escherichia, Klebsiella, Salmonella, Shigella, Vibrio, Helicobacter, Pseudomonas, Burkholderia, Haemophilus, Moraxella, Bordetella, Francisella, Pasteurella, Borrelia, Campylobacter, Yersinia, Rickettsia, Treponema, Chlamydia, and Brucella.

[0025]

[43] In embodiments, the cancer is selected from soft tissue sarcoma, breast cancer, head and neck cancer, melanoma, cervical cancer, bladder cancer, hematological malignancies, glioblastoma, pancreatic cancer, prostate cancer, colon cancer, breast cancer, renal cancer, lung cancer, Merkel cell carcinoma, small intestine cancer, thyroid cancer, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), gastric cancer, gastrointestinal stromal tumor, non-Hodgkin's lymphoma, Hodgkin's lymphoma, liver cancer, leukemia, lymphoma, T-cell lymphoma, brain cancer, and multiple myeloma.

[0026]

[44] In an embodiment, the ALPK1 inhibitor is a kinase inhibitor. In one embodiment, the kinase inhibitor is 1-benzyl-3-hexadecyl-2-methyl-1H-imidazol-3-ium (NH-125, also known as CAS 278603-08-0, which may be referred to herein as MI6 or MI6C):

[0027] [ka]

[0028] In some embodiments, the ALPK1 inhibitor is the halogen salt of 1-benzyl-3-hexadecyl-2-methyl-1H-imidazol-3-ium.In some embodiments, the halogen is selected from fluorine, chlorine, bromine, iodine and astatine.In some embodiments, the halogen is iodine.

[0029]

[45] In embodiments, the ALPK1 inhibitor is an antibody directed against ALPK1 or an anti-ALPK1-Fc fusion protein. In embodiments, the antibody is a fully human antibody, a humanized antibody, a camelid antibody, a chimeric antibody, a CDR-grafted antibody, a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a Fab fragment, or an antigen-binding fragment of any of the foregoing. The generic term "antibody" includes immunoglobulin molecules and their antigen-binding active fragments, i.e., molecules containing an antigen-binding site. Such fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, the Fc region or a fragment thereof. Antigen-binding fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments. These fragments lack the heavy-chain constant fragment (Fc) of intact antibodies and may be preferred because they tend to be cleared more rapidly from the circulation and less prone to nonspecific binding than intact antibodies. Such fragments are produced from intact antibodies using methods known in the art, for example, by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). Preferably, the antigen-binding fragment is a heavy chain dimer (camelid antibodies), a single-chain Fv (scFv), a disulfide-linked Fv (sdFv), a Fab fragment, or a F(ab')2 fragment. Those skilled in the art will recognize that other fusion products can be produced, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions. Immunoglobulin molecules can be of any type, including IgG, IgE, IgM, IgD, IgA, and IgY, and of any class, or subclass, including IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0030]

[46] Preferably, the therapeutic antibody in the methods described herein is a monoclonal antibody, preferably an IgG antibody. Monoclonal antibodies are derived from a substantially homogeneous population of antibodies specific for a particular antigen, which population contains substantially similar epitope-binding sites. Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and any subclass thereof. Methods for producing monoclonal antibodies are known in the art, such as hybridoma technology. In embodiments, the antibody is a chimeric, human, or humanized antibody, or an antigen-binding fragment thereof, preferably an antigen-binding fragment that exhibits low toxicity when administered to a subject, preferably a human subject.

[0031]

[47] In an embodiment, the ALPK1 inhibitor is an ALPK1 antisense polynucleotide. In an embodiment, the ALPK1 inhibitor is an interfering RNA directed against ALPK1 selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA).

[0032]

[48] ​​In embodiments, gene therapy approaches can be used to inhibit ALPK1. In embodiments, gene therapy approaches can include introducing inactivating mutations into ALPK1 using gene editing techniques. In embodiments, gene editing techniques can include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas-9-based techniques.

[0033]

[49] In the context of the methods described herein, the term "treating" can refer to the amelioration or stabilization of one or more symptoms associated with the disease, disorder, or condition being treated. The term "treating" can also encompass the management of a disease, disorder, or condition, and refers to the beneficial effects a subject derives from treatment that do not result in a cure of the underlying disease, disorder, or condition. In the context of this disclosure, the term "prevention" refers to preventing the recurrence, occurrence, progression, or onset of one or more symptoms of a disease, disorder, or condition.

[0034]

[50] In embodiments in which a therapeutically effective amount of a composition is administered to a subject, the therapeutically effective amount is an amount sufficient to achieve the desired therapeutic result, e.g., an improvement or stabilization of one or more symptoms of the disease, disorder, or condition being treated, or, in the context of prevention, an amount sufficient to prevent the recurrence, occurrence, progression, or onset of one or more symptoms of the disease, disorder, or condition.

[0035]

[51] In embodiments, a therapeutically effective amount is the amount required to achieve at least an equivalent therapeutic effect compared to the standard of care, an example of which is an FDA-approved drug indicated to treat the same disease, disorder, or condition.

[0036]

[52] In the context of any of the methods described herein, the subject is preferably a human, but can also be a non-human mammal, preferably a non-human primate. In other embodiments, the non-human mammal can be, for example, a dog, cat, rodent (e.g., mouse, rat, rabbit), horse, cow, sheep, goat, or any other non-human mammal.

[0037]

[53] In embodiments, the human subject is selected from an adult human, a pediatric human, or a geriatric human, as those terms are understood by physicians and as defined, for example, by the U.S. Food and Drug Administration.

[0038]

[54] In embodiments, the present disclosure provides ALPK1 inhibitors in the form of small organic molecules (e.g., 1-benzyl-3-hexadecyl-2-methyl-1H-imidazol-3-ium iodide salt or other halogen salts thereof), or in the form of large biomolecules such as proteins (e.g., antibodies directed against ALPK1 or Fc fragments thereof) or nucleic acids (e.g., antisense polynucleotides directed against ALPK1, or interfering RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), or short hairpin RNAs (shRNAs)). In the context of this disclosure, the generic term "compound" is intended to encompass both small organic molecules and large biomolecules.

[0039]

[55] In embodiments, the present disclosure provides compositions comprising an ALPK1 inhibitor and one or more excipients or carriers, preferably pharmaceutically acceptable excipients or carriers. As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, carriers, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Excipients for preparing pharmaceutical compositions are generally known to be safe and non-toxic when administered to the human or animal body. Examples of pharmaceutically acceptable excipients include, but are not limited to, sterile liquids, water, buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), oils, surfactants, suspending agents, carbohydrates (e.g., glucose, lactose, sucrose, or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, and suitable mixtures of any of the foregoing. The particular excipient utilized in the composition will depend upon various factors, including the chemical stability and solubility of the compound being formulated and the intended route of administration.

[0040]

[56] Pharmaceutical compositions can be provided in bulk or in unit dosage form. It is particularly advantageous to formulate pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The unit dosage form can be an ampoule, vial, suppository, dragee, tablet, capsule, IV bag, or a single pump in an aerosol inhaler.

[0041]

[57] In therapeutic applications, dosages may vary depending on the chemical and physical properties of the active compound, as well as the clinical characteristics of the subject, including, for example, age, weight, and comorbidities. Generally, dosages should be therapeutically effective. An effective amount of a pharmaceutical composition is an amount that provides an objectively identifiable improvement as recognized by a physician or other qualified observer, e.g., alleviating the symptoms of a disorder, disease, or condition.

[0042]

[58] Pharmaceutical compositions can take any suitable form (e.g., liquid, aerosol, solution, inhalant, mists, sprays; or solids, powders, ointments, pastes, creams, lotions, gels, patches, etc.) for administration by any desired route (e.g., pulmonary, inhalation, intranasal, oral, buccal, sublingual, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal, transdermal, transmucosal, rectal, etc.). In embodiments, pharmaceutical compositions take the form of orally acceptable dosage forms, such as, but not limited to, capsules, tablets, buccal tablets, troches, lozenges, and oral liquids in the form of emulsions, aqueous suspensions, dispersions, or solutions. Capsules may contain excipients, such as inert fillers and / or diluents, for example, starches (e.g., corn, potato, or tapioca starch), sugars, artificial sweeteners, powdered cellulose such as crystalline and microcrystalline cellulose, flour, gelatin, gums, etc. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch, Lubricating agents such as magnesium stearate can also be added.

[0043]

[59] In embodiments, the pharmaceutical composition is in the form of a tablet. The tablet may contain a unit dose of the compound described herein together with an inert diluent or carrier, such as a sugar or sugar alcohol, e.g., lactose, sucrose, sorbitol, or mannitol. The tablet may further contain a non-sugar-derived diluent, such as sodium carbonate, calcium phosphate, or calcium carbonate, or a starch, such as cellulose or its derivatives, e.g., methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and corn starch. The tablet may further contain a binder and granulating agent, such as polyvinylpyrrolidone, a disintegrant (e.g., a swellable cross-linked polymer, such as cross-linked carboxymethylcellulose), a lubricant (e.g., a stearate salt), a preservative (e.g., paraben), an antioxidant (e.g., butylated hydroxytoluene), a buffer (e.g., a phosphate or citrate buffer), and an effervescent agent, such as a citric acid / bicarbonate mixture. The tablet may be coated. The coating may be a protective film coating (e.g., a wax or varnish) or a coating designed to modify the release of the active compound, e.g., for delayed release (release of the active substance after a predetermined lag time after ingestion) or release at a specific location in the gastrointestinal tract. The latter can be achieved, for example, using enteric film coatings such as those sold under the brand name Eudragit®.

[0044]

[60] Tablet formulations can be made by conventional compression, wet granulation, or dry granulation methods and can utilize pharmaceutically acceptable diluents, binders, lubricants, disintegrants, surface modifiers (including surfactants), suspending agents, or stabilizers, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starch, and powdered sugar. Preferred surface modifiers include nonionic and anionic surface modifiers. Representative examples of surface modifiers include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphate salts, sodium dodecyl sulfate, magnesium aluminum silicate, and triethanolamine.

[0045]

[61] In an embodiment, the pharmaceutical composition is in the form of a hard or soft gelatin capsule. Depending on the formulation, the compound of the present invention may be in solid, semi-solid, or liquid form.

[0046]

[62] In embodiments, the pharmaceutical compositions are in the form of sterile aqueous solutions or dispersions suitable for parenteral administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0047]

[63] In embodiments, the pharmaceutical compositions are in the form of sterile aqueous solutions or dispersions suitable for administration either by direct injection or by addition to sterile infusion fluids for intravenous infusion, and include solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, or one or more vegetable oils. Solutions or suspensions can be prepared in water with the aid of cosolvents or surfactants. Examples of suitable surfactants include polyethylene glycol (PEG)-fatty acids and PEG-fatty acid monoesters and diesters, PEG glycerol esters, alcohol-oil transesterification products, polyglyceryl fatty acids, propylene glycol fatty acid esters, sterols and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugars and their derivatives, polyethylene glycol alkylphenols, polyoxyethylene-polyoxypropylene (POE-POP) block copolymers, sorbitan fatty acid esters, ionic surfactants, fat-soluble vitamins and their salts, water-soluble vitamins and their amphiphilic derivatives, amino acids and their salts, and organic acids and their esters and anhydrides. Dispersions can also be prepared in, for example, glycerol, liquid polyethylene glycols, and mixtures thereof in oils.

[0048]

[64] In embodiments, the compounds or compositions described herein can be administered as monotherapy or adjunctive therapy. In embodiments, the compounds or compositions described herein can be administered alone or in combination with one or more additional therapeutic agents (i.e., additional APIs) or treatments, for example, as part of a treatment regimen that includes aspects of diet and exercise. In embodiments, the methods described herein involve administration of an ALPK1 inhibitor as a primary therapy. In other embodiments, administration of an ALPK1 inhibitor is an adjunctive therapy. In either case, the methods of the present invention contemplate administration of an ALPK1 inhibitor in combination with one or more additional therapeutic agents and / or treatments for the treatment or prevention of a disease, disorder, or condition as described herein. The term "treatment" refers to any method, protocol, and / or agent that can be used to prevent, treat, manage, or ameliorate a disease, disorder, or condition, or one or more symptoms thereof.

[0049]

[65] The present disclosure also provides kits containing and packaging pharmaceutical compositions for use in the methods described herein. The kits may include one or more containers selected from the group consisting of bottles, vials, ampoules, blister packs, and syringes. The kits may further include one or more instructions for use, one or more syringes, one or more applicators, or a sterile solution suitable for reconstituting the compounds or compositions described herein.

[0050]

[66] All percentages and ratios used herein are by weight unless otherwise indicated.

[67] The present invention is further described and illustrated by the following non-limiting examples. [Example]

[0051]

[68] The following examples demonstrate that inhibiting ALPK1 is effective in treating inflammation directly or indirectly. Example 1 Inactivating ALPK1 mutations confer resistance to DSS-induced colitis

[69] Using a mouse model of dextran sulfate sodium (DSS)-induced colitis, we evaluated whether inactivating mutational inhibition of ALPK1 (CRISPR vectors targeting two sites in the intron, one before exon 13 and one after exon 13, were injected into fertilized mouse embryos to generate an ALPK1 mouse mutant lacking exon 13. Deletion of exon 13, which encodes part of the kinase domain of ALPK1, abolishes the kinase activity of ALPK) can ameliorate inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. DSS treatment causes destruction of colonic epithelial cells, inducing colitis, which in turn causes weight loss. Weight loss was used as an indicator of disease progression. This model system has been previously described, for example, by Okayasu H et al., 1990. Briefly, 8-9 week-old female mice were treated with 2% DSS (MW 40K-50K, MP Bioanalytical) dissolved in autoclaved drinking water for 7 days. Mice were weighed daily and at the end of the study period. All data are expressed as mean ± standard error of the mean (SEM). Experimental groups were ALPK1 wild-type (WT) (n = 3); heterozygous (HE) inactivating ALPK1 mutant (n = 2); and homozygous (HO) inactivating ALPK1 mutant (n = 3).

[0052]

[70] Results: Inactivating ALPK1 mutations were protective in all three measures of disease progression / severity and weight loss in the DSS-induced colitis model (Figure 1). Example 2 Inactivating ALPK1 mutations confer resistance to radiation-induced inflammation

[71] A mouse model of radiation-induced inflammation was used to evaluate whether inhibition of ALPK1 by an inactivating mutation could ameliorate radiation-induced inflammation. Radiation stimulates the immune system and activates inflammatory responses. This model system has been previously described, for example, by Biju G et al., 2012. Briefly, 8- to 9-week-old female mice were irradiated with 9 Gray (Gy) total body irradiation (gamma rays from Co60) per mouse on day 1. Animal survival and body weight were measured daily. Radiation caused damage to the gastrointestinal and hematopoietic systems. Shifts in gut flora combined with immune system dysfunction can lead to sepsis and death.

[0053]

[72] Experimental groups included ALPK1 wild-type (WT) (n = 4); heterozygous (HE) inactivating ALPK1 mutation (n = 6); and homozygous (HO) inactivating ALPK1 mutation (n = 5). In this study, only the heterozygous mutation demonstrated a protective effect against radiation-induced weight loss (Figure 2A) and lethality (Figure 2B).

[0054] Example 3 Inactivating ALPK1 mutation confers resistance to high-fat diet-induced obesity

[73] Using a mouse model of high-fat diet-induced obesity, we evaluated whether inhibition of ALPK1 by an inactivating mutation could ameliorate inflammation in this model system. Obesity shares with many chronic diseases the presence of an inflammatory component, which contributes to the development of metabolic disease. This inflammatory state is reflected by increased circulating levels of pro-inflammatory proteins. High-fat diet (HFD) feeding can induce obesity and metabolic disorders in rodents, resembling human metabolic syndrome. This model system has been previously described, e.g., Bourgeois A et al., 1983; Takahashi I et al., 1999. Briefly, 8- to 9-week-old male mice were treated with an HFD (60% fat, Research Diets Inc., New Brunswick, NJ) from day 0. Body weight was measured weekly from day 1 to day 84 after the HFD. Percentage weight gain (%g) was expressed as the mean ± standard deviation (SD).

[0055]

[74] Experimental groups were ALPK1 wild-type (WT) (n = 6); heterozygous (HE) inactivating ALPK1 mutation (n = 5); and homozygous (HO) inactivating ALPK1 mutation (n = 4). In this study, both heterozygous and homozygous inactivating mutations in ALPK1 conferred protection against HFD-induced weight gain (Figure 3).

[0056] Example 4 Inactivating ALPK1 mutations confer resistance to collagen-induced arthritis

[75] Using a mouse model of collagen-induced arthritis, we evaluated whether inhibition of ALPK1 by an inactivating mutation could ameliorate inflammation in this model system. Rheumatoid arthritis is recognized as an inflammatory immune process in which the body's overactive immune system attacks its own tissues, such as joint linings and cartilage. Inflammation can cause the pain and swelling commonly seen in rheumatoid arthritis. This model system has been previously described, for example, in Campbell H et al., 2000. Briefly, 8-week-old male mice were treated with 20 μl bovine type II collagen (immunization grade bovine type II collagen, solution (Chondrex, catalog no. 20022) and complete Freund's adjuvant (CFA, inactivated Mycobacterium tuberculosis H37Ra (Chondrex, catalog no. 7001) at 4 mg / ml) (1 mg / ml final concentration for both) by subcutaneous injection in the footpad of one hind paw; the other paw was injected with 20 μl PBS as a control. Bovine type II collagen induces paw swelling due to inflammation. Mice were evaluated for rheumatoid arthritis score, and paw thickness for paw swelling was measured every other day until 25 days after collagen injection. The rheumatoid arthritis (RA) score is the sum of all four paw scores on a scale of 0 to 16, with each paw scored as follows: Score 0 - normal foot; Score 1 - one toe is inflamed and swollen; Score 2 - more than one toe is inflamed and swollen, but not the entire foot, or mild swelling of the entire foot; Score 3 - the entire foot is inflamed and swollen; and Score 4 - Very inflamed and swollen or ankylosed (stiff or immobile) paw.

[0057]

[76] Data are presented as the mean ± standard deviation of the number of experimental groups as indicated. The experimental groups were ALPK1 wild-type (WT) (n = 8); heterozygous (HE) inactivating ALPK1 mutation (n = 7); and homozygous (HO) inactivating ALPK1 mutation (n = 7). In this study, both indicators of inflammation, footpad RA score (Figure 4A) and footpad thickness difference (Figure 4B), were lower in mice with heterozygous or homozygous inactivating mutations in ALPK1.

[0058] Example 5 ALPK1 knockdown inhibits the expression of pro-inflammatory cytokines in various human and mouse cells

[77] The results are summarized in Table 1 and described in more detail below.

[0059] [Table 1]

[0060] 5A. ALPK1 knockdown by siRNA in human embryonic kidney 293T (HEK293T) cells results in decreased cytokine expression

[78] HEK293T cells were transfected with siRNA (ALPK1-siRNA-1, ALPK1-siRNA-2, scrambled siRNA) using Lipofectamine® RNAiMAX Reagent (Life Technologies Corporation, Grand Island, NY). At 42 hours posttransfection, HEK293T cells were harvested for RNA extraction using Trizol Reagent (Life Technologies Corporation). RNA was reverse transcribed to cDNA using the PrimeScript™ RT Reagent Kit (Takara Bio Inc., #RR037A) and measured by qPCR using SYBR Green I reagent on an Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System (Life Technologies Corporation). Gene expression of ALPK1, IL-10, IL-1β, IL-6, IL-8, and TNF-α was normalized to GAPDH. Expression of all five of these pro-inflammatory cytokines was reduced by ALPK1 knockdown (Figure 5).

[0061] 5B. Knockdown of ALPK1 in HEK293T cells results in reduced S. flexneri cell lysate-induced cytokine expression, and overexpression of human and mouse ALPK1, but not kinase-dead mutants, can rescue the impaired cytokine expression.

[0062]

[79] HEK293T cells were transfected with ALPK1-siRNA-2 or scrambled siRNA using Lipofectamine® RNAiMAX Reagent (Life Technologies Corporation). Two days later, cells were transfected with no cDNA (empty) or an overexpression construct (pCDNA3.1-) containing the following: ALPK1 tagged with 1*Flag at the N-terminus (Flag-ALPK1) Mouse C-terminal HA-tagged ALPK1 (mouse ALPK1) ALPK1 tagged with a 6*His tag at the N-terminus and 3*Flag at the C-terminus (6*His-ALPK1-3*Flag) ALPK1 tagged with a 6*His tag at the N-terminus (6*His-ALPK1) · E1190A mutant of 6*His-ALPK1 (6*His-ALPK1-E mutant).

[0063]

[80] To confirm both efficient knockdown of ALPK1 by siRNA and overexpression by the overexpression vector, we measured ALPK1 gene expression (Figure 6A). IL8 expression was also measured (Figure 6B), and we found that Flag-ALPK1, mouse ALPK1, 6*His-ALPK1-3*Flag, and 6*His-ALPK1 induced efficient IL8 expression, but not the 6*His-ALPK1-E mutant, suggesting that the kinase activity of ALPK1 is required for ALPK1-induced IL8 expression.

[0064] CRISPR / Cas9 knockout of ALPK1 in HEK293T cells results in decreased S. flexneri cell lysate-induced cytokine expression and secretion

[81] CRISPR HEK293T cells were generated by transfection of CRISPRv1.0 containing ALPK1 exon 3 targeting sequences and ALPK1 exon 14 targeting sequences. One day after transfection, HEK293T cells were selected on 2.5 μg / ml puromycin for 3 days. Single colonies were expanded to generate stable transfectants. NF-κB signaling was activated by treatment of HEK293T cells with S. flexneri cell lysate. After 4 hours of treatment with 1% S. flexneri cell lysate, 293T cells were harvested using Trizol reagent (LIFE) for analysis of mRNA expression. ALPK1, IL-10, IL-1β, IL-6, IL-8, and TNF-α mRNA levels were normalized to GAPDH expression (Figure 7A). IL-8 secretion in the supernatant HEK293T cell cultures was measured using a human IL-8 ELISA kit (BD Biosciences) (Fig. 7B).

[0065] 5D. Knockdown of ALPK1 in HEK293 cells results in reduced cytokine expression induced by S. flexneri and S. typhimurium cell lysates.

[82] S. flexneri cell lysate (SFL) and S. typhimurium cell lysate (STL) can induce the expression of the pro-inflammatory cytokines IL6, IL8, and TNFα in HEK293 cells. Knockdown of ALPK1 by siRNA reduces the expression of all three of these cytokines (Figure 8). The siRNAs were as described in 5A above.

[0066] 5E. Knockdown of ALPK1 by shRNA in THP-1 macrophage cells results in decreased cytokine expression, regardless of whether LPS induction is induced.

[83] THP-1 cells were infected with lentivirus carrying shRNA-1190 directed against ALPK1, shRNA-2027 directed against ALPK1, or a null vector (Neg) for 5 days and selected using puromycin. PMA (50 ng / ml) was added to induce macrophage differentiation for 2 days, and LPS (10 ng / ml) was added to induce the NFκβ pathway. Expression of IL1β, IL-8, and TNFα was reduced by shRNA knockdown of ALPK1 in both LPS-stimulated (Figure 9A) and unstimulated (Figure 9B) cells. Expression of actin (actb) and GADPH (gadph) is also shown.

[0067] 5F. Knockdown of ALPK1 by shRNA in THP-1 macrophage cells results in inhibition of TNFα and IL-1β secretion.

[84] When THP-1 cells were induced to differentiate into macrophages by PMA (50 ng / ml) for 2–6 days, shRNA knockdown of ALPK1 in THP-1 macrophages resulted in inhibition of LPS (10 ng / ml)-induced secretion of TNFα (Figure 10A) and IL1β (Figure 10B).

[0068] ALPK1 knockdown in 5G.MDA-MB-468 cells results in decreased cytokine expression

[85] ALPK1 knockdown by siRNA in the breast cancer cell line MDA-MB-468 resulted in decreased expression of IL1β, IL8, and TNFα (Figure 11).

[0069] 5H. ALPK1 knockdown in RAW 264.7 cells results in decreased cytokine expression

[86] Knockdown of ALPK1 by shRNA in the murine macrophage cell line RAW264.7 resulted in decreased cytokine expression, both with (Figure 12A) and without (Figure 12B) LPS (100 ng / ml) stimulation.

[0070] 5I. Primary mouse macrophage cells from mice carrying an inactivating ALPK1 mutation

[87] Mouse bone marrow-derived macrophage cells (BMDMs) were collected from wild-type (WT) mice and homozygous (HO) and heterozygous (HE) ALPK1 mice as described in Example 1 above. Mature BMDMs were treated with 100 ng / ml LPS to induce cytokine expression. mRNA expression of the cytokines IL1β, TNFα, TGFβ1, IL6, IFNα, IL15, IL10, and TNFβ was reduced in cells from both homozygous (HO) and heterozygous (HE) ALPK1 knockout mice (Figure 13).

[0071] Example 6 The small molecule kinase inhibitor, MI6, improves recovery in a DSS-induced colitis model.

[88] identified ALPK1 inhibitors by screening a library of small molecules for inhibitory activity against ALPK1. Various assays, including binding assays, kinase assays, and cell-based assays for cytokine secretion, were used to screen the library. MI6 was identified as an ALPK1 inhibitor based on its performance in the binding and kinase assays, as well as at least two cell-based assays.

[0072]

[89] The ability of MI6 to ameliorate inflammation in a DSS-induced colitis model was evaluated as follows. Eight-week-old female wild-type FVC mice were treated with 3% DSS (sodium salt, MW 40K-50K, Affymetrix) from day 0 to day 5. Afterward, mice were switched to drinking water. Mice were injected intraperitoneally (IP) daily with either MI6 (1 mg / kg) dissolved in DMSO (n = 6) or DMSO alone (n = 5). Animals were weighed daily. Within 3 days of treatment with MI6, treated animals began to show improved weight gain compared to untreated animals (Figure 14).

[0073] Example 7 ALPK1 mutations reduce tumor growth and metastasis in breast cancer models In the MMTV-PyVT transgenic tumor model, polyomavirus middle T antigen (PyVT) is expressed under the mouse mammary tumor virus (MMTV) promoter, which drives mammary tissue-specific expression of PyVT. PyVT is an oncogene that activates multiple oncogenic pathways, including src and phosphatidylinositol-3-kinase, resulting in an invasive tumor phenotype. Virgin females carrying the transgene develop multifocal, poorly differentiated, highly invasive ductal carcinomas by 10–12 weeks of age and have a high incidence of pulmonary metastases derived from their primary mammary tumors. At 5 weeks of age, females develop noninvasive focal lesions that fall into four groups: simple, solid, cystic, and mixed (solid and cystic). Solid lesions consist of large foci with dense masses of atypical cells in nodular sheets. The cystic lesions vary in size and complexity and are lined by multilayered epithelium containing significant amounts of clear fluid.

[0074]

[91] Metastasis of primary tumors to distant sites remains a significant cause of mortality in many cancer types, highlighting the importance of metastasis models. The MMTV-PyVT transgenic tumor model of spontaneous mammary carcinogenesis is a powerful tool for studying mechanisms associated with tumor progression and for the development of novel chemotherapeutics.

[0075]

[92] We investigated the protective effect of the ALPK1-inactivating mutation in the MMTV-PyVT model. As shown in Figures 15A-D, both the tumor burden in mammary tissue (Figure 15B) and metastatic lung tumor burden (Figure 15D) were statistically significantly lower than those in mock MMTV-PyVT transgenic mice that did not contain the ALPK1 mutation, indicating the protective effect of this mutation.

[0076] equivalent

[93] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0077]

[94] All references cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0078]

[95] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising an alpha-kinase 1 (ALPK1) inhibitor for use in a method for treating inflammation in a subject in need thereof, wherein the ALPK1 inhibitor is an ALPK1 antisense polynucleotide or an interfering RNA directed against ALPK1, and the subject has inflammatory bowel disease or radiation-induced inflammation.

2. A pharmaceutical composition comprising an alpha-kinase 1 (ALPK1) inhibitor for use in a method for treating an inflammatory disease, disorder, or condition in a subject in need thereof, wherein the ALPK1 inhibitor is an ALPK1 antisense polynucleotide or an interfering RNA directed against ALPK1, and the inflammatory disease or disorder includes or is associated with inflammatory bowel disease, radiation-induced inflammation, or systemic lupus erythematosus (SLE).

3. The pharmaceutical composition of claim 2 , wherein the inflammatory disease or disorder comprises radiation-induced inflammation.

4. The pharmaceutical composition of claim 2, wherein the inflammatory disease or disorder comprises inflammatory bowel disease, including colitis.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the ALPK1 inhibitor is an ALPK1 antisense polynucleotide.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the ALPK1 inhibitor is an interfering RNA directed against ALPK1 selected from the group consisting of microRNA (miRNA), small interfering RNA (siRNA), and small hairpin RNA (shRNA).

7. A pharmaceutical composition comprising an alpha-kinase 1 (ALPK1) inhibitor for use in a method for treating sepsis in a subject in need thereof, wherein the ALPK1 inhibitor is an ALPK1 antisense polynucleotide or an interfering RNA directed against ALPK1.

8. The pharmaceutical composition of claim 7, wherein the sepsis is caused by S. flexneri or S. typhimurium infection.

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