Cytokine modulation

Abstract

To provide a cytokine modulation.SOLUTION: The present invention relates to the use of hemichannel blockers to modulate cytokine levels in a subject, including the angiogenic cytokine, VEGF, and their production, secretion and / or release, and to the use of hemichannel blockers to reduce or level cytokine activity, including in conditions characterized in whole or in part by angiogenesis and / or vessel leak. The invention provides e.g., a method for modulating cytokine activity in a subject, comprising administering an effective amount of a hemichannel blocker to the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 534,595, filed July 19, 2021, which is incorporated herein by reference. Field The present invention relates generally to connexin hemichannels, including connexin 43 hemichannels, and cytokines, including VEGF, IL-6, IL-8, MCP-1, and sICAM-1.

[0002] Incorporation by Reference All U.S. patents, U.S. patent application publications, foreign patents, foreign and PCT published applications, papers and other documents, references and publications mentioned herein, and all listed as references cited in any patent(s) originating herefrom, are incorporated herein by reference in their entirety. The incorporated information is effectively part of this application as if all of the text and other content were repeated in this application, and is treated as part of the text and content of this application as filed. [Background technology]

[0003] background The following contains information that may be useful in understanding the present invention. This is not an admission that any information, publication, or document specifically or implicitly referred to herein is prior art to or essential to the invention described or claimed herein.

[0004] Vascular endothelial growth factor (VEGF), an angiogenic cytokine, plays a central role in human growth and development and vascular maintenance. However, it is now well established that angiogenesis also plays an important role in the pathogenesis of various disorders. VEGF-mediated angiogenesis has been reported to be important for tumor growth, as well as exudative age-related macular degeneration (AMD), such as proliferative diabetic retinopathy and retinopathy of prematurity, all of which are characterized by abnormal neovascularization. In the case of solid tumors, neovascularization allows tumor cells to acquire a growth advantage and proliferative autonomy compared to normal cells. A correlation has been observed between the density of microvessels in tumor sections and patient survival in breast cancer and several other tumors. Weidner et al., Tumor Angiogenesis and Metastasis - Correlation in Invasive Breast Carcinoma. N Engl J Med 324:1-6 (1991); Horak et al., Angiogenesis, assessed by platelet / endothelial cell adhesion molecule antibodies, an indicator of node metastases and survival in breast cancer. Lancet 340:1120-1124 (1992); and Macchiarini et al., Relation of neovascularization to metastasis of non-small lung Cell cancer. Lancet 340:145-146 (1992). It has been reported that ischemia and inflammation result in VEGF-mediated breakdown of the blood-retinal barrier, which causes vision-reducing macular edema. Folkman et al. J. Biol. Chem. 267:10931-10934 (1992); Klagsbrun et al. Annu. Rev. Physiol. 53:217-239 (1991); and Garner A, Vascular diseases. In: Pathobiology of ocular disease. A dynamic approach. Garner A, Klintworth GK, Eds. 2nd Edition Marcel Dekker, NY, pp 1625-1710 (1994). To address these effects, anti-VEGF drugs (e.g., antibodies) , aptamers and tyrosine kinase inhibitors) have been developed for both systemic and topical (intraocular) use.

[0005] Despite the overall clinical success of anti-VEGF agents with regard to retinal damage, some AMD patients still require frequent injections to keep the disease under control. It has been stated that long-acting formulations or sustained-release technologies are needed for such cases. See Ferrara, N and Adamis AP, Ten years of anti-vascular endothelial growth factor therapy. Nature Reviews Drug Discovery 15:385-403 (2016). Furthermore, approximately 40% of patients with neovascular AMD have vision problems of less than 20 / 40. indicates a suboptimal treatment response, defined as visual acuity of 0.5 or less. Rosenfeld, PJ et al., Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355:1419-1431 (2006). Data from a phase 3 study demonstrate that the currently approved dose Higher doses may not be helpful, as studies have shown that 100% of patients with AMD and diabetic macular edema (DME) have a dose-response curve at or near the peak of the curve. BG, et al., Twelve-month efficacy and safety of 0.5 mg or 2.0 mg ranibizumab in patients with subfoveal neovascular age-related macular degeneration. Ophthalmology 120:1046-1056 (2013). With regard to cancer, the impact of VEGF inhibitors has not reached the impressive efficacy predicted in some early preclinical studies with other angiogenesis inhibitors. Boehm, T, et al. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404-407 (1997). Nevertheless, VEGF inhibitors are promising candidates for the treatment of advanced, difficult-to-treat malignancies. It has shown benefit in patients with advanced tumors and is currently the standard of care for the treatment of some metastatic cancers. However, heterogeneity in clinical response exists. Ferrara, N., Pathways Mediating VEGF-independent tumor angiogenesis. Cytokine Growth Factor Rev. 21:21-26 (2010).

[0006] Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role not only in the immune system but also in various biological processes. Dysregulated and persistent interleukin IL-6 production has been implicated in the development of various autoimmune and chronic inflammatory diseases and even cancer. It is a key regulator of both acute and chronic inflammation. Significantly elevated levels of IL-6 have been found in ocular fluids from patients with refractory / chronic uveitis, for example, and IL-6 has been shown to be required for inflammation induction in an experimental autoimmune uveitis model using IL-6 knockout mice. Actemra® (tocilizumab), a recombinant humanized anti-IL-6 receptor antibody, has been used in the treatment of several autoimmune diseases, including uveitis. For a summary of the efficacy and safety of tocilizumab treatment, see Mesquida, M, et al., Interleukin-6 blockade in ocular inflammatory diseases. Clin Exp Immunol. 176:301-309 (2-14). Anti-IL-6 receptor antibodies are a potential treatment for autoimmune disorders, including Castleman disease. It has also been used for sexual disorders.

[0007] Increased expression of interleukin-8 (IL-8) and / or its receptor has been characterized in many chronic inflammatory conditions, including COPD, as well as in many cancers, and its upregulation often correlates with disease activity. IL-8 is a pro-angiogenic cytokine that is overexpressed in many human cancers. Receptors for IL-8 are widely expressed on normal and various tumor cells, and IL-8 has been reported to induce pro-inflammatory, chemotactic, and matrix-degrading responses in many pathologies. As reviewed by Qazi et al., Recent Advances in Underlying Pathologies Provide Insight into Interleukin-8 Expression-Mediated Inflammation and Angiogenesis, International Journal of Inflammation Volume 2011 (2011), Article ID 908468. There has been hope for finding strategies to indirectly attenuate IL-8 signaling in cancer cells, with the goal of sensitizing cancer cells to conventional therapeutic interventions. Campbell, LM, et al., Rationale and Means to Target Pro-Inflammatory Interleukin-8 (CXCL8) Signaling in Cancer. Pharmaceuticals (Basel) 6:929-959 (2013).

[0008] Monocyte chemoattractant protein-1 (MCP-1 / CCL2) is one of the key chemokines that regulate monocyte / macrophage migration and infiltration. Both MCP-1 and its receptors have been reported to be induced and involved in various diseases and conditions, including multiple sclerosis (the correlation between MCP-1 and axonal injury), secondary multiple sclerosis and nociception (due to MCP-1-mediated neuronal depolarization), tumor angiogenesis (due to the influence of MCP-1 on macrophage infiltration), and insulin resistance (increased MCP-1). While there has been interest in discovering drugs that affect MCP-1 production, such as those targeting tissues experiencing chronic inflammation, the only possibility offered has been techniques such as silencing the MCP-1 gene using RNAi technology. See Deshmane, SL, et al., Monocyte Chemoattractant Protein-1 (MCP-1): An Overview, J Interferon Cytokine Res. 29:313-326 (2009) Id. at 321, stating that discovery of drugs that block upregulated chemokine receptors may prove effective if they are upstream of MCP-1 expression.

[0009] ICAM-1, a member of the immunoglobulin supergene family, is a single-chain cell surface glycoprotein constitutively expressed at low levels on various cell types. Plasma soluble ICAM-1 (sICAM-1) levels have been associated with coronary heart disease and other vascular diseases. Ridker PM, et al., Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men. Lancet 351:88-92 (1998). sICAM-1 Plasma concentrations have been reported to be significantly elevated in patients with acute myocardial infarction and unstable angina, but not in patients with stable angina (Pellegatta, F. et al., J. Cardiovasc. Pharmacol. 30:455-460 (1997);Miwa, K. et al., Cardiovasc. Res. 36:37-44, 1997; Ghaisas, NK et al., Am. J. Cardiol. 80:617-619 (1997); Ogawa, H. et al., Am. J. Cardiol. 83:38-42 (1999)). Elevated plasma concentrations of sICAM-1 have also been reported to be associated with cancer and multiple sclerosis (Kim, JS, J. Neurol. Sci. 137:69-78 (1996); Laskowitz, D. T. et al., J. Stroke Cerebrovasc. Dis. 7:234-241 (1998)). Gho et al. reported that sICAM-1 can apparently promote angiogenesis and stimulate tumor cell growth. Gho YS, et al. Angiogenic activity of human soluble intercellular adhesion molecule-1. Cancer Res 59:5128-32 (1999); Gho YS, et al., Stimulation of tumor growth by human soluble intercellular adhesion molecule-1. Cancer Res 61:4253-7 (2001). Elevated sICAM-1 levels have also been reported in patients with various malignancies and are thought to correlate with disease progression and tumor metastasis. Another paper reported that inflammatory factors (VEGF, IL-6, MCP-1, and sICAM-1) can induce increased vascular permeability and disrupt the blood-aqueous barrier in patients with macular edema. Noma, H, et al., Role of inflammation in previously untreated macular edema with branch retinal vein occlusion. BMC Ophthalmol. 14:67 (2014).

[0010] Connexin channels are ubiquitous, providing pathways for the movement of molecules between cells (gap junction channels) and for the release of molecular effectors into the extracellular environment (plasma membrane gap junction hemichannels). Gap junctions are specialized cell-cell connections found in most animal cell types. They are expressed in virtually all tissues of the body, except mature skeletal muscle and motile cell types such as sperm and red blood cells. Gap junctions directly connect the cytoplasm of two cells, allowing various molecules, ions, and electrical impulses to pass directly through regulated gates between the cells. A gap junction consists of two connexons (i.e., hemichannels) that connect across the intercellular space between adjacent cells, allowing the flow of intracellular molecules between them. Each connexon of a gap junction resides in the membrane of adjacent cells and is formed by the covalent oligomerization of six individual connexin ("Cx") proteins. A requirement for the formation of functional gap junctions is the assembly of connexin proteins into hemichannels and their insertion into the membrane. For intercellular communication, hemichannels from one cell must dock with their counterparts on the opposing membrane of adjacent cells to allow the transmission of signals through gap junctions from one cell to the other. Gap junctions and hemichannels are involved in the translocation of various small molecules with molecular masses up to approximately 1 kDa, such as ions, small metabolites, cAMP, ATP, IP3, and prostaglandins. Burra S and Jiang JX, Regulation of cellular function by connexin hemichannels, Int J Biochem Mol Biol. 2(2): 119-128 (2011). Under physiological conditions, most connexins form hemichannels in the plasma membrane, which are closed until they dock to form cell-cell channels during gap junction formation. With some exceptions, connexin hemichannel currents tend to be activated by strong depolarization or a reduction in extracellular calcium below 0.5 mM. Therefore, the activity of endogenous connexin hemichannels may not be significant under normal physiological conditions. However, it has been reported that, when surface-exposed, undocked hemichannels can mediate the exchange of molecules between the cytosol and the extracellular space. Thus, hemichannels are closed by default, but several cues, such as extracellular Ca, induce their opening. 2+ A decrease in ATP concentration (Evans et al., The gap junction cellular internet: connexin hemichannels enter the signaling limelight. Biochem J 397(1): 1-14 (2006)) or infection with enteric pathogens (Puhar et al., A Shigella Effector Dampens Inflammation by Regulating Epithelial Release of Danger Signal ATP through Production of the Lipid Mediator PtdIns5P, Immunity 39(6): 1121- 1131 (2013); Tran Van Nhieu et al., Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells, Nat Cell Biol 5(8): 720-726 (2003). See Puhar A and Sansonetti PJ, Dye-uptake Experiment through Connexin Hemichannels, Bio-protocol 4(17): e1221 (Sept. 2014). Gap junction, hemichannel, and connexin regulators are , has been proposed for a variety of therapeutic uses. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Weidner et al., Tumor Angiogenesis and Metastasis - Correlation in Invasive Breast Carcinoma. N Engl J Med 324:1-6 (1991) [Non-patent document 2] Horak et al., Angiogenesis, assessed by platelet / endothelial cell adhesion molecule antibodies, an indicator of node metastases and survival in breast cancer. Lancet 340:1120-1124 (1992) [Non-patent document 3] Macchiarini et al., Relation of neovascularization to metastasis of non-small lung cell cancer. Lancet 340:145-146 (1992)

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[0012] A brief summary The invention described and claimed herein has many aspects and embodiments, including but not limited to those shown or described or referred to in this Summary of the Invention. The invention is not intended to be comprehensive, and the invention described and claimed herein is not limited to or by the features or embodiments identified in this introduction, which are included for purposes of illustration only and not limitation.

[0013] This patent describes the use of hemichannel blockers to attenuate the production and release of cytokines. Cytokines are involved in several diseases, disorders, and conditions. Importantly, therefore, hemichannel blockers act upstream of current therapeutic approaches, including, for example, anti-VEGF antibodies, VEFG receptor blockers, IL-6 receptor blockers, and other regulators, as well as regulators of other cytokines and / or their receptors, including IL-8, MCP-1, and sICAM-1.

[0014] The inventors have now surprisingly discovered that, given the previously described properties of hemichannels, which have a molecular size passage limit of approximately 1 kDa, modulating or blocking hemichannels can reduce or stop the production, secretion, and / or release of proinflammatory cytokines, as evidenced by a reduction in IL-6, IL-8, MCP-1, and sICAM-1, as well as a reduction in the presence or amount of the angiogenic cytokine VEGF, following administration of a hemichannel blocker. These molecules range in size from approximately 11 kDa (MCP-1 and IL-8) to 90 kDa (sICAM-1). Hemichannels allow the transport of water, small molecules, and ions, such as Ca. 2+ (40 Da), as well as small signaling molecules such as ATP, cAMP, and NAD + , allowing only IP3, prostaglandins, and glutamate (140–700 Da) to pass through.

[0015] While not intending to be bound by any theory, the studies herein support the idea that connexin hemichannels play a critical role in disease processes by amplifying and perpetuating connexin hemichannel-mediated cytokine feedback loops, forming the basis for the conditions and diseases mentioned herein, as well as other conditions and diseases characterized at least in part by undesirable levels of VEGF and / or other cytokines, including, for example, IL-6, IL-8, MCP-1, and / or sICAM-1. Systemic and local release of pro-inflammatory cytokines is involved in the development and progression of, for example, diabetes mellitus and diabetic nephropathy. These results have implications for diseases including those mentioned in the background and elsewhere herein, as well as for example, heart failure (Butts, B., et al. Journal of Cardiac Failure, 21:586-593 (2015)); muscular dystrophy (Cea, L.A., et al. (2013). De novo expression of connexin hemichannels in denervated fast skeletal muscles leads to atrophy. Proceedings of the National Academy of Sciences, 110:16229-16234 (2013)); metabolic diseases (see de Torre-Minguela, C., et al. Frontiers in Immunology, Article 43 (January 27, 2017)); chronic respiratory diseases, e.g., chronic obstructive pulmonary disease, or COPD (Hosseinian, N, et al., Therapeutic advances in respiratory disease, 9:188-197 (2015); diabetes mellitus and diabetic nephropathy, and organ failure resulting in insulin resistance, impaired insulin secretion, and renal failure (Wada, J. and Makino, H. Innate immunity in diabetes and diabetic nephropathy. Nature Reviews Nephrology, 12:13-26 (2016)); and brain tumors (Zhou, K., et al. Journal of Immunology Research, 2016: 9238290).

[0016] In one aspect, the present invention relates to the use of hemichannel blockers to modulate cytokine levels, including the angiogenic cytokine VEGF, as well as their production, secretion and / or release, in a subject, for example, and to the use of hemichannel blockers to reduce or normalize cytokine activity, including in conditions characterized in whole or in part by angiogenesis and / or vascular leakage.

[0017] In one aspect, a method is provided for reducing the production, release and / or secretion of cytokines, including IL-6, IL-8, sICAM-1 and MCP-1.

[0018] In another aspect, a method is provided for reducing the production, release and / or secretion of VEGF. In one embodiment, the VEGF is VEGF-A.

[0019] In another aspect, a method is provided for modulating undesirably amplified and perpetuated connexin hemichannel-mediated cytokine feedback loops in a subject, including those in the diseases, disorders and conditions described or referred to herein, including in the Background Art.

[0020] In another aspect, a method is provided for modulating undesirably amplified and perpetuated connexin 43 hemichannel-mediated autocrine feedback loops in a subject, including those in the diseases, disorders and conditions described or referred to herein, including in the Background Art.

[0021] In another aspect, methods are provided for modulating connexin hemichannel-mediated autocrine cytokine feedback loops, including those mediated by connexin 43 hemichannels, to reduce cytokine production, secretion and / or release in diseases, disorders and conditions characterized at least in part by neoplastic or tumor growth associated with the angiogenic formation of new blood vessels.

[0022] This patent describes, in part, the use of compounds and methods for modulating connexin hemichannels, including connexin 43 hemichannels, to block or modulate cytokine release. Among other things, this patent describes compositions and methods that can be used to break the cycle of mediators of chronic disease.

[0023] The methods of the invention are useful in attenuating abnormal, elevated, dysregulated, and / or otherwise undesirable levels of cytokines, including, for example, IL-6, IL-8, sICAM-1, MCP-1, and VEGF, e.g., VEGF-A, in a subject by administering a connexin hemichannel blocker to the subject who would benefit therefrom.

[0024] The present invention is directed, in part, to methods for reducing cytokines and / or cytokine activity, comprising, consisting essentially of, or consisting of the administration of a hemichannel blocker, e.g., a peptidomimetic hemichannel blocker, such as Peptagon (Peptide 5), and / or a small molecule hemichannel blocker, such as Xiflam (Tonaversat). These methods are useful, for example, in treating VEGF (e.g., VEGF-A) levels associated with pathological or otherwise unwanted angiogenesis, conditions associated with type 2 (non-insulin-dependent) diabetes mellitus, and other diseases, disorders, and conditions described or referred to herein, including in the Background Art.

[0025] The methods are also useful in treating, for example, conditions associated with unwanted or pathological levels of interleukin-6 (IL-6), various human inflammatory diseases, e.g., Castleman's disease, and other diseases, disorders, and conditions described or referred to herein, including in the background art.

[0026] The methods are useful in treating, for example, conditions associated with a variety of diseases including unwanted or pathological levels of interleukin-8 (IL-8), peripheral arterial occlusive disease (PAOD), cystic fibrosis, ANCA-associated vasculitis (Wegener's granulomatosis), hematological malignancies, and other cancers, e.g., hepatocellular carcinoma, soft tissue sarcoma, and early-stage and metastatic breast cancer, as well as other diseases, disorders, and conditions described or referred to herein, including in the Background Art.

[0027] The methods are also useful in treating conditions associated with various acute and chronic inflammatory diseases, including, for example, unwanted or pathological levels of sICAM-1, lupus nephritis, neuromyelitis optica (NMO), systemic lupus erythematosus (SLE), and gingival support cells associated with plaque accumulation and inflammation, including in patients with gingivitis, adult periodontitis, and rapidly progressive periodontitis, and pathological processes associated with the metastatic behavior of tumor cells, including in patients with non-small cell lung cancer (NSCLC), as well as other diseases, disorders, and conditions described or referred to in this specification, including in the Background Art.

[0028] The methods are also useful in treating, for example, unwanted or pathological levels of MCP-1, autoimmune disorders (e.g., multiple sclerosis and secondary multiple sclerosis), pulmonary diseases (e.g., chronic obstructive pulmonary disease), cancer, and conditions involved in the pathology of several diseases including insulin resistance, tumor neovascularization, and other diseases, disorders, and conditions described or referred to herein, including in the background art.

[0029] In another aspect, this patent features a method of beneficially modulating cytokines, including IL-6, IL-8, sICAM-1, and MCP-1, in a subject by administering to the subject an effective amount of a hemichannel blocker. In one embodiment, the method of the invention is directed to reducing or modulating VEGF, including but not limited to VEGF-A. In another embodiment, the invention is directed to a method of reducing or modulating angiogenesis.

[0030] In another aspect, the present invention is directed to a method of reducing or modulating VEGF in a subject by administering a hemichannel blocker to the subject. In one embodiment, the VEGF is VEGF-A.

[0031] This patent describes the use of compositions and methods for reducing the production, secretion and / or release of VEGF to treat, for example, diseases, disorders and conditions characterized or mediated at least in part by angiogenesis and / or by VEGF, including but not limited to VEGF-A.

[0032] Thus, in one aspect, the invention relates to methods for blocking or reducing hemichannel opening to reduce or regulate VEGF, e.g., VEGF-A, and methods for the treatment of disorders in which modulation of VEGF and / or other cytokines may be beneficial.

[0033] In another aspect, the present invention provides a method for treating breast cancer, non-small cell lung cancer, or diabetes in a subject with breast cancer, non-small cell lung cancer, or diabetes by administering a hemichannel blocker to the subject. In one embodiment, the VEGF that is modulated or reduced is VEGF-A.

[0034] The hemichannel blocker compositions and methods can be used alone or in combination with one or more additional anti-VEGF therapeutic agents, including anti-VEGF antibodies, variant anti-VEGF antibodies, VEGF-traps, and other agents that inhibit the activity of VEGF and / or VEGF receptors (VEGFRs). In one embodiment, the VEGF therapeutic agent is a VEGF-A antagonist or blocker or a VEGF-A receptor antagonist or blocker.

[0035] It is an object of the present invention to provide compounds, compositions, formulations, kits and methods for their production and use for modulation of hemichannels to reduce the production, secretion and / or release or secretion of cytokines, e.g., VEGF, IL-6, IL-8, sICAM-1 and MCP-1, and their isoforms, including VEGF-A, in a subject in need thereof.

[0036] It is another object of the present invention to provide a method for attenuating abnormal, elevated, dysregulated and / or otherwise undesirable levels of cytokines in a subject by administering a connexin hemichannel blocker to a subject who would benefit therefrom.

[0037] It is another object of the present invention to provide compounds, compositions, formulations, kits and methods for the treatment of diseases, disorders and conditions that benefit from cytokine modulation.It is another object of the present invention to provide compounds, compositions, formulations, kits and methods for the treatment of diseases, disorders and conditions that benefit from reduced cytokines, reduced cytokine levels and / or reduced cytokine activity.

[0038] In some embodiments, the methods of treatment are applied to mammals, such as humans.

[0039] In another aspect, the present invention provides a gap junction hemichannel blocker, e.g., a small molecule, e.g., Xiflam and / or its analogs or prodrugs, or a peptidomimetic, e.g., Peptagon and / or its analogs or prodrugs, or another hemichannel blocker or prodrug thereof, for use in treating a disorder in which modulation of hemichannels may be beneficial. In some aspects, the hemichannel blocker is administered daily, weekly, monthly, bimonthly, or quarterly, or any combination of these periods. For example, treatment can be administered daily for a period of time, followed by weekly and / or monthly administration.

[0040] In another aspect, the present invention provides a hemichannel blocker for treating one or more diseases, disorders and conditions.In certain embodiments, one or more diseases, disorders or conditions are selected from the group consisting of, essentially consisting of, or consisting of, for example, Wegener's granulomatosis; Castleman's disease; angina pectoris, including unstable angina; renal failure; multiple sclerosis; muscular dystrophy; secondary multiple sclerosis; lupus nephritis; tumor neovascularization; COPD; PAOD; diabetes, including type 2 (non-insulin-dependent) diabetes mellitus; insulin resistance; diabetic nephropathy; heart failure; solid tumors, including brain tumors; cancers, including breast cancer, non-small cell lung cancer, hematologic malignancies; hepatocellular carcinoma, soft tissue sarcoma, early stage breast cancer and metastatic breast cancer.

[0041] The hemichannel blocker useful in the present invention can be administered alone or in combination with another therapeutic agent useful in treating target diseases, disorders or conditions.In some embodiments, the compound of Formula I, such as Xiflam and / or any analog or prodrug of the above-mentioned compounds, or a peptidomimetic, such as Peptagon or its analog or prodrug, or another hemichannel blocker, can be used together with a cytokine antagonist to treat disorders that may benefit from modulation of hemichannels.The administration of the hemichannel blocker can be simultaneous with, sequential to, or prior to the administration of the cytokine antagonist.

[0042] Thus, hemichannel blockers can be co-administered with, for example, VEGF or other cytokine antagonists. In methods comprising, consisting essentially of, or consisting of co-administration of a hemichannel blocker with a cytokine blocker or antagonist, such as a VEGF antagonist, for example, an anti-VEGF antibody or a VEGF receptor blocker, or an IL-6 receptor blocker, the co-administration of the hemichannel blocker can be simultaneous with, sequential to, or prior to the administration of the cytokine blocker or antagonist. VEGF-A and VEGF-A receptor antagonists are preferred in the present invention.

[0043] In still other aspects, various cytokine-related disorders can be treated by the compositions and methods of the present invention, including methods of treatment with hemichannel blockers alone or in combination with cytokine antagonists, including, but not limited to, those disorders described or referenced herein.

[0044] As noted, the present invention also features methods for treating a patient, comprising administering to the patient an anti-VEGF agent and / or an anti-cytokine agent as a therapeutic treatment in addition to administering a small molecule or peptide or peptidomimetic hemichannel blocker. In one aspect, the VEGF antagonist or another anti-cytokine agent is administered to the patient simultaneously with, or within about 1 to 5, 10, 30, 45, 60, 75, 90, or 100 to 180 days of, the administration of the hemichannel blocker in an amount sufficient to treat the patient. In certain embodiments of the methods of the present invention, the VEGF antagonist is administered simultaneously with the hemichannel blocker. In some aspects, by way of example, the VEGF antagonist can be a compound that inhibits and / or blocks VEGF or a compound that inhibits and / or blocks an upstream agonist of VEGF. In some aspects, VEGF antagonists include, for example, antagonists that bind and inhibit VEGF, compounds that inhibit VEGF expression, and / or viral vectors that contain VEGF inhibitors or encode proteins or antisense polynucleotides that block or inhibit VEGF. In some aspects, agents that inhibit VEGF and / or upstream agonists of VEGF can be, for example, antibodies or antibody fragments, nanobodies, peptides or peptidomimetics, receptor fragments, recombinant fusion proteins, aptamers, small molecules, or single-chain variable fragments (scFv). In one embodiment, the VEGF antagonist is a VEGF-A antagonist. In another exemplary embodiment, the VEGF antagonist is a nucleic acid molecule, an aptamer, an antisense RNA molecule, a ribozyme, an RNAi molecule, a protein, a peptide, a cyclic peptide, an antibody, a binding fragment of an antibody fragment, a sugar, a polymer, or a small molecule. In one embodiment, this method of the invention includes administering a VEGF antagonist that is an aptamer, e.g., the EYE001 aptamer. In another embodiment, the method of the invention comprises administration of a VEGF antagonist that is an antibody or binding fragment thereof, eg, Avastin® (bevacizumab) or Lucentis® (ranibizumab).In another embodiment, this method of the invention comprises administration of an IL-6 receptor antagonist, eg, Actemra® (tocilizumab).

[0045] Hemichannel blockers for modulating / reducing cytokine levels or activity, including the levels or activity of the angiogenic cytokine VEFG, include the hemichannel blocker compounds described or referenced herein or incorporated by reference herein.

[0046] Some preferred hemichannel blockers include small molecule hemichannel blockers (e.g., Xiflam (tonabersat)). In some embodiments, the hemichannel blocker is a small molecule other than Xiflam, such as a hemichannel blocker described in Formula I or Formula II in U.S. Patent Application Publication No. 20160177298, filed in the name of Colin Green et al., the disclosure of which is incorporated herein by reference in its entirety as noted above. Various preferred embodiments include the use of small molecules that block or improve or otherwise antagonize or inhibit hemichannel opening to treat diseases, disorders, and conditions characterized at least in part by abnormal, elevated, dysregulated, and / or otherwise undesirable, unwanted, or harmful levels or activity of cytokines, including those described or referenced herein. In various embodiments, the small molecule that blocks or improves or inhibits hemichannel opening is a prodrug of Xiflam or an analog thereof.

[0047] In other embodiments, hemichannel blockers include peptide and peptidomimetic hemichannel blockers (e.g., Peptagon, VDCFLSRPTEKT, peptidomimetics), as well as other peptidomimetic hemichannel blockers comprising, consisting essentially of, or consisting of the amino acid sequence SRPTEKT. In any of the aspects of the present invention, the hemichannel blocker is a connexin peptide or peptidomimetic, including a peptide or peptidomimetic comprising, consisting essentially of, or consisting of a connexin extracellular domain, a transmembrane region, and a connexin carboxy-terminal peptide. Connexin hemichannel-blocking peptides or peptidomimetics can be modified or unmodified. Connexin hemichannel-blocking peptides or peptidomimetics are chemically, synthetically, or otherwise manufactured. In some embodiments, the connexin hemichannel-blocking peptide or peptidomimetic is a Cx43 peptide or peptidomimetic. In some embodiments, the therapeutically effective modified or unmodified peptide or peptide mimetic comprises a portion of the extracellular or transmembrane domain of a connexin, e.g., Cx43 or Cx45, e.g., a portion of connexin extracellular loop 2, including a portion of Cx43 extracellular loop 2 and a portion of Cx45 extracellular loop 2.

[0048] In another aspect, the present invention provides use of a hemichannel blocker in the manufacture of a medicament for use in the treatment of one or more diseases, disorders, and conditions described or referenced herein. The medicament comprises, consists essentially of, or consists of a hemichannel blocker. In one embodiment, the medicament comprises, consists essentially of, or consists of a peptide hemichannel blocker. In one embodiment, the medicament comprises, consists essentially of, or consists of a peptidomimetic hemichannel blocker. In one embodiment, the medicament comprises, consists essentially of, or consists of a small molecule hemichannel blocker. In one embodiment, the medicament comprises, consists essentially of, or consists of a compound according to Formula I or Formula II in U.S. Patent Application Publication No. 20160177298. In one embodiment, the medicament comprises, consists essentially of, or consists of Xiflam (tonabersat). "Including," "containing," or "by" can be used interchangeably. The term "comprising," which is synonymous with "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or components (or steps, in the case of methods) from the medicament. The phrase "consisting of" excludes any element, step, or ingredient (or step, in the case of methods) not specified in the medicament. The phrase "consisting essentially of" refers to the specified materials and materials that do not substantially affect the basic and novel characteristics (or steps, in the case of methods) of the medicament. The basic and novel characteristics of the inventions are described throughout this specification, including the ability of the medicaments and methods of the invention to block or modulate connexin gap junction hemichannels and to attenuate the production, release, or activity of cytokines (including, for example, VEGF cytokines). Substantial variations in the basic and novel characteristics of the inventions, including the medicaments and methods described herein, include unwanted or clinically undesirable, harmful, adverse, or deleterious reduction in hemichannel modulation and / or cytokine attenuation. In one embodiment, the medicament comprises, consists essentially of, or consists of a connexin 43 hemichannel blocker, eg, a peptidomimetic or small molecule connexin 43 hemichannel blocker.

[0049] In another aspect, the present invention provides the use of a hemichannel blocker in the manufacture of a medicament (or a package or kit containing one or more medicaments and / or containers, with or without instructions for use) for modulating hemichannels and / or treating any of the diseases, disorders, and / or conditions described or referenced herein. In one aspect, for example, the present invention provides the use of a connexin hemichannel blocker, including, for example, Xiflam and / or analogs thereof, or Peptagon or analogs thereof, in the manufacture of a medicament or package or kit for treating a disorder in which hemichannel modulation may be beneficial. In one embodiment, the medicament comprises, consists essentially of, or consists of a connexin 43 hemichannel blocker, e.g., a peptidomimetic or small molecule connexin 43 hemichannel blocker. In one embodiment, the hemichannel blocker composition useful in the present invention may include a pharmaceutically acceptable carrier and may be formulated, for example, as a pill, solution, microsphere, nanoparticle, implant, matrix, or hydrogel formulation, or provided in lyophilized form.

[0050] In some embodiments, hemichannel blockers can be co-administered or used together with cytokine antagonists, e.g., VEGF antagonists, in the manufacture of a medicament for the treatment of disorders in which modulation of hemichannels and cytokines is beneficial.

[0051] In some embodiments, a hemichannel blocker may be used together with a cytokine antagonist, e.g., a VEGF antagonist, in the manufacture of separate or combined pharmaceuticals for the treatment of one or more diseases, disorders, and conditions mentioned herein.

[0052] In various embodiments, the hemichannel being modulated is connexin 23 (Cx23), connexin 25 (Cx25), connexin 26 (Cx26), connexin 30 (Cx30), connexin 30.2 (Cx30.2), connexin 30.3 (Cx30.3), connexin 31 (Cx31), connexin 31.1 (Cx31.1), connexin 31.9 (Cx31.9), connexin 32 (Cx32), In one embodiment, the hemichannels being modulated include one or more of connexin 36 (Cx36), connexin 37 (Cx37), connexin 40 (Cx40), connexin 40.1 (Cx40.1), connexin 43 (Cs43), connexin 45 (Cx45), connexin 46 (Cx46), connexin 47 (Cx47), connexin 50 (Cx50), connexin 57 (Cx57), connexin 59 (Cx59), and connexin 62 (Cx62). In one embodiment, the modulated hemichannels include one or more of Cx26, Cx30, Cx32, Cx36, Cx37, Cx40, Cx45, and / or Cx47 proteins. In a specific embodiment, the hemichannel and / or modulated hemichannel comprises one or more of Cx37, Cx40, and Cx43. In a specific embodiment, the hemichannel and / or modulated hemichannel comprises one or more of Cx30, Cx37, Cx40, Cx43, and Cx45. In some embodiments, the modulated hemichannel may include or exclude any of the above-mentioned connexins. In some aspects, the hemichannel blocker is a blocker of Cx37 hemichannel, Cx43 hemichannel, Cx40 hemichannel, and / or Cx45 hemichannel. In certain preferred embodiments, the hemichannel blocker is a connexin 43 hemichannel blocker. Pharmaceutical compositions of the invention for any of the uses featured herein may also include a hemichannel blocker capable of inhibiting or blocking Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50 or Cx57, or any other connexin or connexin hemichannel.In another embodiment, the pharmaceutical compositions for use in the methods and manufacture of the present invention for any of the uses and connexins featured herein may also include at least one cytokine antagonist, provided together or separately. In some embodiments, the blocking agent may include or exclude any of the connexins described above. In one embodiment, the hemichannel blocker blocks connexin hemichannels in blood vessels. In another embodiment, the hemichannel blocker blocks connexin hemichannels in microvessels. In another embodiment, the hemichannel blocker blocks connexin hemichannels in capillaries.

[0053] In one embodiment, the hemichannel blocker used in any of the administration, co-administration, compositions, kits, or treatment methods of the present invention is a Cx43 hemichannel blocker. Other embodiments include Cx45 hemichannel blockers, as well as blockers of hemichannels comprising, consisting essentially of, or consisting of Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50, and / or Cx57 hemichannels, or any other connexins mentioned above and herein. In some embodiments, the blockers may include or exclude any of the above-mentioned connexins, or other connexins mentioned in this patent. In some embodiments, the connexin hemichannels to be blocked are heteromeric hemichannels (i.e., hemichannels containing mixed, non-identical connexins).

[0054] Another embodiment of this aspect of the invention provides a pharmaceutical pack comprising a VEGF or other cytokine antagonist together with a small molecule or other hemichannel blocker. In one embodiment of this aspect, the pharmaceutical pack comprises a VEGF antagonist that is a VEGF-A antagonist. In another embodiment, the hemichannel blocker and the VEGF antagonist of the pharmaceutical pack are formulated separately and in individual dosage amounts. In yet another embodiment, the hemichannel blocker and the VEGF antagonist of the pharmaceutical pack are formulated together. In one embodiment, the hemichannel blocker is Xiflam. In another embodiment, the hemichannel blocker is Peptagon.

[0055] In another aspect of the present invention, the effect of hemichannel blocker treatment in a subject is assessed or monitored using a cytokine protein assay. Cytokine protein levels can be quantified by any conventional method that allows for the detection and quantification of proteins in a sample from a subject, including those described herein.

[0056] The present invention includes in vitro methods for predicting clinical outcomes in subjects treated with hemichannel blockers, and for initiating hemichannel blocker treatment, discontinuing hemichannel blocker treatment, modifying hemichannel blocker treatment, or further treating said subjects with hemichannel blockers and / or cytokine antagonists. Various embodiments of this aspect of the invention are described herein.

[0057] The activity of hemichannel blockers can be evaluated using certain biological assays. The effect of known or candidate hemichannel blockers on molecular mobility can be identified, evaluated, or screened for using the methods described in the Examples below, or other art-known or equivalent methods for determining the passage of compounds through connexin hemichannels. Various methods are known in the art, including dye translocation experiments, e.g., the translocation of molecules labeled with detectable markers, and the transmembrane passage of small fluorescently permeable tracers, which have been widely used to study the functional state of hemichannels. A method for use in identifying or evaluating the ability of a compound to block a hemichannel, comprising: (a) combining a test sample and a test system, wherein the test sample comprises one or more test compounds, and the test system comprises a system for assessing hemichannel blockade, wherein the system is activated by, for example, the introduction of hypoxia or ischemia into the system, a mediator of inflammation, or another compound or event that induces hemichannel opening, e.g., extracellular Ca. 2+ Various embodiments of this aspect of the invention are described herein, including methods comprising (a) determining the presence or amount of an increase in the dye or other labeled metabolite in the system; and (b) determining the presence or amount of an increase in the dye or other labeled metabolite in the system. Positive and / or negative controls may also be used. If desired, a predetermined amount of a hemichannel blocker (e.g., peptagon or xiflam) may be added to the test system. As mentioned herein, in one embodiment, hemichannel blockers, such as peptagon and xiflam, exhibit activity in in vitro assays at concentrations of less than about 1-5 nM, preferably less than about 10 nM, and more preferably less than about 50 pM. In in vivo assays, these compounds preferably exhibit hemichannel blockade at concentrations of less than about 10-100 micromolar (μM), more preferably less than about 50 μM. Other hemichannel blockers may be within these ranges and also within a range of less than about 200 pM. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method for modulating cytokine activity in a subject, comprising administering to the subject an effective amount of a hemichannel blocker. (Item 2) 2. The method of claim 1, wherein the presence or amount of the cytokine is reduced. (Item 3) 2. The method of claim 1, wherein an increase in the presence or amount of the cytokine is inhibited. (Item 4) 2. The method of claim 1, wherein the cytokine is selected from the group consisting of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), and soluble intercellular adhesion molecule-1 (sICAM-1). (Item 5) 2. The method of claim 1, wherein the cytokine is vascular endothelial growth factor. (Item 6) Item 6. The method according to item 5, wherein the vascular endothelial growth factor is vascular endothelial growth factor A. (Item 7) Item 10. The method of item 1, wherein the hemichannel blocker is a connexin 43 hemichannel blocker. (Item 8) Item 9. The method of item 1, wherein the hemichannel blocker is a small molecule hemichannel blocker. 9. The method of claim 8, wherein the small molecule hemichannel blocker is N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamide (Xiflam). (Item 10) 2. The method of claim 1, wherein the hemichannel blocker is a connexin peptidomimetic. (Item 11) 11. The method of claim 10, wherein the hemichannel blocker is VDCFLSRPTEKT (SEQ ID NO: 1). (Item 12) 11. The method of claim 10, wherein the hemichannel blocker consists essentially of SRPTEKT (SEQ ID NO: 2). (Item 13) 11. The method of claim 10, wherein the hemichannel blocker is selected from the group consisting of peptides consisting essentially of ADCFLSRPTEKT (SEQ ID NO: 3), VACFLSRPTEKT (SEQ ID NO: 4), VDCFLSRPTAKT (SEQ ID NO: 5), VDCFLSRPTEAT (SEQ ID NO: 6), CFLSRPTEKT (SEQ ID NO: 7), and LSRPTEKT (SEQ ID NO: 8). (Item 14) 10. The method of claim 1, further comprising administering a VEGF antagonist or a VEGF receptor antagonist. (Item 15) 15. The method of claim 14, wherein the VEGF is VEGF-A. (Item 16) The method of item 1, further comprising administering an IL-6 antagonist or an IL-6 receptor antagonist. (Item 17) 10. The method of claim 1, further comprising administering one or more of an IL-8 antagonist, an MCP-1 antagonist, or a sICAM-1 antagonist. (Item 18) Item 16. The method of item 15, wherein the hemichannel blocker is a connexin 43 hemichannel blocker. (Item 19) 19. The method of item 18, wherein angiogenesis is reduced or attenuated. (Item 20) Item 10. The method of item 1, wherein the hemichannel blocker is administered by injection. (Item 21) 2. The method of claim 1, wherein the hemichannel blocker is administered orally. (Item 22) 2. The method of claim 1, wherein the hemichannel blocker is administered PRN or on a predetermined schedule, or both. (Item 23) Item 10. The method of item 1, wherein the subject is a human. (Item 24) 11. The method of claim 10, wherein the hemichannel blocker is a modified peptidomimetic. (Item 25) 26. The method of claim 25, wherein the modification comprises C12-C12-VDCFLSRPTEKT (sequence number 171). (Item 26) 10. The method of claim 1, wherein the subject has a pathological, abnormal, unwanted, or undesirable amount of cytokine activity. [Brief explanation of the drawings]

[0058] [Figure 1] Figure 1 shows the secretion of IL-6, sICAM-1, MCP-1, and IL-8 under basal conditions and in response to high glucose (HG) conditions, cytokines, and co-application of HG and cytokines. Administration of cytokines alone, but not HG alone, induced IL-6 release. Neither cytokines nor HG alone induced sICAM-1 secretion. However, co-application of HG and cytokines resulted in increased IL-6 and sICAM-1 release compared to basal levels. Cytokines induced MCP-1 and IL-8 release. Co-administration with HG resulted in higher levels of MCP-1 and IL-8. Results are presented as mean ± SD; statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test; N = 3; t = 24 h; ns = not significant; ***p ≤ 0.001; ****p ≤ 0.0001.

[0059] [Figure 2]Figure 2 shows VEGF secretion in response to HG, cytokines, and co-application of HG and cytokines after treatment with a hemichannel blocker (Peptagon) and ATP addition under basal conditions. Co-administration of HG and cytokines induced VEGF release (p≦0.0001), whereas Peptagon treatment restored VEGF secretion to basal levels. Addition of exogenous ATP abolished hemichannel blocker-mediated protection against VEGF release. Results are expressed as mean ± SD; statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test; N=3; t=24 h; ns=not significant; **p≦0.01; ****p≦0.0001.

[0060] [Figure 3] Figure 3 shows Peptagon-mediated reduction in the expression of IL-6, IL-8, sICAM-1, and MCP-1 after co-application of HG and cytokines. Co-application of HG and cytokines induced IL-6, IL-8, sICAM-1, and MCP-1 release by ARPE-19 cells, and treatment with 5 μM, 10 μM, 25 μM, and 50 μM of a hemichannel blocker (Peptagon) reduced the levels of IL-6, IL-8, and MCP-1 in a concentration-dependent manner. Results are expressed as mean ± SD; statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparison test. All treatments were significantly different from co-application of high glucose and cytokines (p<0.0001 in all cases). N=3; t=24 h.

[0061] [Figure 4]Figure 4 shows the effect of a hemichannel blocker (Peptagon) in protecting against ATP release induced by HG and cytokines. Co-application of HG and cytokines results in increased ATP release compared to basal conditions. Peptide 5 prevented ATP release mediated by HG and cytokines. Importantly, there was no statistically significant difference between the Peptagon-treated group and basal conditions. Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test. N = 3; t = 24 h; ns = not significant; *p ≤ 0.05; ***p ≤ 0.001.

[0062] [Figure 5] Figure 5 shows that ATP reversed the hemichannel blocker-mediated reduction in IL-6, MCP-1, and IL-8 expression using Peptagon, but not sICAM-1. Coapplication of HG and cytokines induced IL-6, IL-8, sICAM-1, and MCP-1 release by ARPE-19 cells, whereas hemichannel blocker treatment with 25 μM connexin peptide mimetic (Peptagon) reduced cytokine secretion. Extracellular ATP (10 nM) reversed Peptagon-mediated protection against IL-6, MCP-1, and IL-8 release, but not sICAM-1. Results are expressed as mean ± SD; statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test. N = 3; t = 24 h; ns = not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p < 0.0001.

[0063] [Figure 6]Figure 6 shows immunohistochemical labeling of the NLRP3 complex. Inactive NLRP3 is normally dispersed within the cytoplasm, but upon inflammasome activation by high glucose and inflammatory cytokines, oligomerization concentrates multiple NLRP3 copies within the inflammasome complex, making them visible as small spots (arrows, A). Addition of a connexin peptide mimetic hemichannel blocker (peptagon) blocked inflammasome assembly, with only a few complex spots visible (B). High nuclear background labeling with this antibody was present under all conditions. Addition of exogenous 10 nM ATP reversed hemichannel blocker treatment, and inflammasome complexes re-formed within the cytoplasm (C). Results are expressed as mean ± SD; N = 3; t = 24 h; scale bar = 50 μm. DETAILED DESCRIPTION OF THE INVENTION

[0064] Detailed Description definition A "small molecule" is defined herein as having a molecular weight of less than about 600-900 daltons and is generally an organic compound. A small molecule can be the active agent of a hemichannel blocker prodrug. In one embodiment, the small molecule is less than 600 daltons. In another embodiment, the small molecule is less than 900 daltons.

[0065] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to a clinical intervention that alters the natural course of the individual, tissue, or cell being treated and can be performed either for prophylaxis or during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, disorder, or condition, alleviating signs or symptoms, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, remission or palliation of the disease state, and remission or improved prognosis. In some embodiments, the compounds, methods, and compositions of the invention can be used to delay the development of a disease, disorder, or condition or to slow the progression of a disease, disorder, or condition. The term does not necessarily imply that a subject is treated until complete recovery. Thus, "treatment" includes reducing, alleviating, or ameliorating the symptoms or severity of a particular disease, disorder, or condition, or preventing or otherwise reducing the risk of developing a particular disease, disorder, or condition. Treatment can also include maintaining or promoting a state of complete or partial remission of the condition. "Treatment," as used herein, also includes reducing, alleviating, or ameliorating cytokine levels or activity in a subject, e.g., the levels and / or activity of IL-6, IL-8, MCP-1, and sICAM-1, following administration of a hemichannel blocker, as well as reducing the presence or amount of the angiogenic cytokine VEGF.

[0066] Terms such as "treating a cytokine disorder," including diseases and conditions, can refer to preventing, slowing, reducing, diminishing, stopping and / or reversing the disorder, disease or condition and / or the level or activity of cytokines, including, for example, IL-6, IL-8, sICAM-1 and MCP-1 and / or VEGF.

[0067] The term "prevent" means to prevent, in whole or in part, or to ameliorate or control.

[0068] As used herein, "effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic or preventive result. For example, without limitation, "effective amount" can refer to the amount of a compound or composition disclosed herein that can treat the signs and / or symptoms of a disease, disorder, or condition in which cytokines are involved, or the amount of a hemichannel compound or composition that can advantageously modulate the production, secretion, and / or release of cytokines, including, for example, IL-6, IL-8, sICAM-1, and MCP-1 and / or VEGF.

[0069] As used herein, the "therapeutically effective amount" of a substance / molecule, agonist, or antagonist of the present invention can vary according to factors such as the individual's disease state, age, sex, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also preferably an amount in which any toxic or harmful effects of the substance / molecule, agonist, or antagonist can be outweighed by the therapeutically beneficial effects. A therapeutically effective amount of a hemichannel blocker reduces or inhibits an increase in cytokine levels or activity in a subject. A therapeutically effective amount of a hemichannel blocker modulates cytokine levels or activity in a subject.

[0070] As used herein, a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, although not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, disorder, or condition, the prophylactically effective amount will be less than the therapeutically effective amount.

[0071] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein, e.g., a hemichannel blocker, to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0072] As used herein, "pharmaceutically acceptable carrier" refers to an ingredient, other than the active ingredient, in a pharmaceutical formulation that can be safely administered to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, and preservatives.

[0073] As used herein, terms such as "subject," including "individual" and "patient," may all be used interchangeably herein and refer to any mammal, including humans, domestic and farm animals, and zoo, wildlife park, sport, or pet animals, such as dogs, horses, cats, sheep, pigs, cows, and the like. Preferred mammals are humans, including adults, children, and the elderly. Preferred sport animals are horses and dogs. Preferred pet animals are dogs and cats. The subject may be, for example, an aquarium animal, such as a dolphin, whale, sea lion, or walrus. In certain embodiments, the subject, individual, or patient is a human.

[0074] As used herein, the term "hemichannel" refers to a part of a gap junction (two hemichannels or connexons connect across the intercellular space between adjacent cells to form a gap junction) and is composed of several connexin proteins, typically hexamers of the same or different connexin proteins, i.e., homomeric or heteromeric, that form the gap junction pore between the cytoplasm of two adjacent cells. A hemichannel is provided by one cell on one side of the junction, and usually, two hemichannels from opposing cells join together to form a complete intercellular hemichannel. However, in some cells, and under certain circumstances, the hemichannel itself is active as a conduit between the cytoplasm and the extracellular space, allowing the passage of ions and small molecules.

[0075] Compounds of Formula I, such as Xiflam and / or analogs or prodrugs of any of the aforementioned compounds, can modulate the function and / or activity of hemichannels, preferably hemichannels containing any type of connexin protein. Thus, reference to a "hemichannel" should be interpreted broadly to include hemichannels that contain, consist essentially of, or consist of any one or more of several different connexin proteins, unless the context requires otherwise. However, by way of example, a hemichannel may contain one or more of connexin 23, 25, 26, 30, 30.2, 30.3, 31, 31.1, 31.9, 32, 36, 37, 40, 40.1, 43, 45, 46, 47, 50, 59, and 62 proteins. In one embodiment, a hemichannel consists of one of the aforementioned connexins. In one embodiment, the hemichannel comprises one or more of connexins 26, 30, 32, 36, 37, 40, 45, and 47. In one embodiment, the hemichannel consists of one of connexins 26, 30, 32, 36, 37, 40, 45, or 47. In one embodiment, the hemichannel consists of one of connexins 37, 40, or 43. In one embodiment, the hemichannel is a vascular hemichannel. In one embodiment, the hemichannel is a connexin hemichannel found in vascular endothelial cells. In one embodiment, the hemichannel is a connexin hemichannel found in vascular smooth muscle cells. In a particular embodiment, the hemichannel comprises one or more of connexins 30, 37, and connexin 43. In a particular embodiment, the hemichannel consists of connexin 30. In a particular embodiment, the hemichannel consists of connexin 37. In a particular embodiment, the hemichannel consists of connexin 43. In one particular embodiment, the hemichannel consists of one of connexin 45, connexin 46, or connexin 50. In one embodiment, the hemichannel comprises one or more connexins excluding connexin 26. In one embodiment, the composition may include or exclude hemichannel blockers of any connexin, including those mentioned above.

[0076] Hemichannels and hemichannels can be present in any type of cell. Thus, reference to a "hemichannel" or "hemichannel" should be interpreted as including reference to a hemichannel or hemichannel present in any cell type, unless the context requires otherwise. In one embodiment of the present invention, the hemichannel or hemichannel is present in a cell in an organ or in a cancer or tumor. In one embodiment, the hemichannel is a vascular hemichannel. In one embodiment, the hemichannel is a connexin hemichannel found in vascular endothelial cells and / or vascular smooth muscle cells.

[0077] As used herein, "modulation of a hemichannel" refers to modulation of one or more functions and / or activities of a hemichannel, typically the flow of molecules between cells through a hemichannel. Such functions and activities include, for example, the flow of molecules from the extracellular space or environment into a cell through a hemichannel, and / or the flow of molecules from the intracellular space or environment of a cell to the extracellular space or environment through a hemichannel. Compounds useful for modulating hemichannels may be referred to as "hemichannel modulators."

[0078] Modulation of hemichannel function can be achieved by any means. However, by way of example only, modulation can be achieved by one or more of inducing or promoting hemichannel closure; preventing, blocking, inhibiting, or reducing hemichannel opening; or triggering, inducing, or promoting cellular internalization of hemichannels and / or gap junctions. For example, the use of words such as "block," "inhibit," "prevent," "reduce," and "antagonize" may preferably be interpreted to imply complete blockage, inhibition, prevention, or antagonism, but need not be, and should be interpreted to include partial blockage, inhibition, prevention, or antagonism that at least reduces hemichannel function or activity and / or hemichannels. Similarly, "induce" or "promote" should not be interpreted to imply complete internalization of a hemichannel (or group of hemichannels), but should be interpreted to include partial internalization that at least reduces hemichannel function or activity.

[0079] As used herein, the term "hemichannel blocker" refers to a compound that interferes with the passage of molecules through connexin hemichannels. Hemichannel blockers can block or reduce hemichannel opening, block or reduce the release of molecules into the extracellular space through the hemichannel, and / or block or reduce the entry of molecules into the intracellular space through the hemichannel. Hemichannel blockers include compounds that completely or partially block hemichannel leakage or the passage of molecules into or from the extracellular space. Hemichannel blockers also include compounds that reduce the open probability of a hemichannel. Open probability is a measure of the percentage of time a channel remains open compared to its closed state (reviewed in Goldberg GS, et al., Selective permeability of gap junction channels Biochimica et Biophysica Acta 1662 (2004) 96-101). Examples of hemichannel blockers include peptides, small molecules, antibodies, and Hemichannel blockers include hemichannel modulators. Hemichannel blockers can directly or indirectly interfere with the passage of molecules through connexin hemichannels.

[0080] As used herein, the terms "cytokine modulation" and "modulating cytokine activity" refer to the reduction, decrease, leveling, or smoothing of the production, secretion, and / or release of cytokines, including the angiogenic cytokine VEGF. As used herein, "cytokine modulation" and "modulating cytokine activity" include the reduction, decrease, leveling, or smoothing of cytokine activity, including the activity of the angiogenic cytokine VEGF. Cytokine modulation is achieved using hemichannel blockers and is useful in the treatment of diseases, disorders, and conditions characterized in whole or in part by pathological, aberrant, or otherwise unwanted or undesirable cytokine activity, including those characterized in whole or in part by angiogenesis and / or vascular leakage. Compounds useful for cytokine modulation may be referred to as "cytokine modulators." The compounds of the invention can be used in methods of treatment to modulate cytokine activity, including in methods of treatment of diseases, disorders, or conditions characterized in whole or in part by pathological, aberrant, or otherwise unwanted or undesirable cytokine activity, where cytokine activity is modulated, e.g., where cytokine activity is reduced, diminished, leveled, and / or smoothed. Leveling or smoothing of cytokine activity includes leveling out and / or inhibiting a substantial increase in cytokine presence or amount or cytokine activity.

[0081] Inflammasomes are multiprotein complexes containing caspase-1, PYCARD, NALP, and optionally caspase-5 (also known as caspase-11 or ICH-3). The exact composition of inflammasomes depends on the activator that initiates inflammasome assembly. Inflammasomes promote the maturation of the inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). Hemichannel blockers according to the present invention can modulate or regulate inflammasome activity and the activation of the inflammasome pathway. Target inflammasomes for hemichannel blockers include the NLRP3 inflammasome.

[0082] The terms "peptide," "peptidomimetic," and "mimetics" include synthetic or genetically engineered chemical compounds that may have substantially the same structural and functional characteristics of the protein regions they mimic. In the case of connexin hemichannels, these may, for example, mimic the extracellular loops of hemichannel connexins.

[0083] As used herein, the term "peptide analog" refers to a compound that has properties similar to those of the template peptide and may be a non-peptide drug. "Peptidomimetics" (peptidomimetics) include peptides and peptide-based compounds. Peptide mimetics (also known as peptide mimetics) are compounds that mimic such non-peptide based compounds, e.g. For example, peptide analogs are also included. Peptide mimetics structurally similar to therapeutically useful peptides can be used to produce equivalent or enhanced therapeutic or prophylactic effects. Peptides and peptidomimetics can, in some embodiments, be modified or unmodified. Generally, a peptidomimetic is a structural or functional mimetic (e.g., identical or similar) to a reference polypeptide (i.e., a polypeptide having a biological or pharmacological function or activity), but can also have one or more peptide linkages optionally replaced by linkages selected from the group consisting of, for example, -CHNH-, -CHS-, -CH-CH-, -CH=CH- (cis and trans), -COCH-, -CH(OH)CH-, and -CHSO-. Mimetics can be composed entirely of either natural amino acids, synthetic chemical compounds, or non-natural analogs of amino acids, or are chimeric molecules of partially natural peptide amino acids and partially non-natural analogs of amino acids. Mimetics can also include any amount of conservative substitutions of natural amino acids, so long as such substitutions do not also substantially alter the mimetic's activity. In the case of connexin hemichannels, they can mimic, for example, the hemichannel extracellular loops involved in connexon-connexon docking and cell-cell channel formation. Peptide mimetics include those described herein as well as those that may be known in the art, whether currently known or later developed. Peptide and peptidomimetic hemichannel blockers can also be modified to increase stability, improve bioavailability, and / or increase cell membrane permeability.

[0084] This patent describes new methods for modulating cytokines, including IL-6, IL-8, sICAM-1, and MCP-1, and the angiogenic cytokine VEGF. The presence or amount of one or more of these cytokines is elevated, aberrant, dysregulated, impaired, or otherwise unwanted or undesirable in several diseases, disorders, or conditions, some of which are characterized by unwanted or pathological angiogenesis.

[0085] As used herein, the term "antagonist" refers to a compound whose presence results in a decrease in the magnitude of the biological activity of a protein. In certain embodiments, the presence of an antagonist blocks or attenuates the biological activity of a protein, i.e., results in its complete or partial inhibition. In certain descriptions, an antagonist may be referred to as an inhibitor or modulator. Thus, a cytokine "antagonist" refers to a compound or compounds that totally or partially inhibit the activity or function of a cytokine, for example, the activity or function of VEGF, preferably its angiogenic activity. A cytokine "receptor antagonist" refers to a compound or compounds that totally or partially inhibit the activation or function of a cytokine receptor, for example, a VEGF receptor.

[0086] Blockers of hemichannel opening, including hemichannel blockers, include small peptide and small molecule blockers. The blockers can be used alone or in combination with each other and / or other therapeutic agents to treat the diseases, disorders, or conditions described herein, including diseases, disorders, or conditions characterized by angiogenesis and / or chronic inflammation.

[0087] As described herein, the use of hemichannel blockers was evaluated in cell lines exposed to various mediators of inflammation, including the pro-inflammatory cytokines IL-1β and TNF-α, and the combination of IL-1β and TNF-α with high glucose, which synergistically increased inflammatory cytokine release. Assays used to measure the release of various cytokines showed increased secretion of VEGF, as well as IL-6, IL-8, MCP-1, and sICAM-1. Figure 1 shows cytokine release in the presence of inflammation (IL-1β and TNF-α) alone, along with a combination that included added glucose, which increased cytokine release. Application of hemichannel blockers reduced cytokine release and restored normal gap junction patterning. Exogenous ATP reversed hemichannel blocker protection, confirming that the cytokine effect was connexin hemichannel-mediated.

[0088] The present invention relates to the modulation of cytokine production, secretion and / or release, including the modulation of VEGF level or activity.As mentioned, hemichannel blockers include, for example, small peptide mimetics and small molecule blockers.Hemichannel blockers can be used alone or in combination with other drugs, such as cytokine antagonists, to treat the diseases, disorders or conditions described herein, including acute and chronic inflammatory diseases.

[0089] The present invention provides methods for treating cytokine disorders, including diseases and conditions characterized at least in part by elevated or undesirable levels and / or activity of cytokines, including VEGF, IL-6, IL-8, MCP-1, and sICAM-1, and for preventing, slowing, reducing, diminishing, halting, and / or reversing the production, secretion, and / or release of cytokines, including VEGF and IL-6, IL-8, MCP-1, and sICAM-1. A subject having a cytokine disorder is treated with a therapeutically effective amount of a hemichannel blocker.

[0090] The present invention provides, inter alia, methods for modulation of cytokine production, secretion and / or release by administration of hemichannel blockers, e.g., Peptagon and / or analogs thereof, compounds of formula I, e.g., Xiflam and / or analogs or prodrugs of any of the aforementioned compounds, for the treatment of diseases, disorders or conditions in which modulation to reduce cytokine production, secretion and / or release may be beneficial.

[0091] In certain embodiments, the inventors contemplate that the method of the present invention is used for treating the diseases, disorders or conditions described or mentioned herein, or that may benefit from reducing cytokine production, secretion and / or release.Diseases, disorders or conditions include, for example, Wegener's granulomatosis; Castleman's disease; renal failure; angina pectoris, including unstable angina; multiple sclerosis; muscular dystrophy; secondary multiple sclerosis; lupus nephritis; tumor neovascularization; COPD; PAOD; diabetes, including type 2 (non-insulin-dependent) diabetes mellitus; insulin resistance; diabetic nephropathy; heart failure; solid tumor; brain tumor; cancer, including breast cancer, non-small cell lung cancer, hematologic malignant tumor; hepatocellular carcinoma, soft tissue sarcoma, early stage breast cancer and metastatic breast cancer.

[0092] In some embodiments, the invention features the use of compounds of Formula I, e.g., Xiflam, and / or analogs or prodrugs of any of the above-described compounds, to directly and immediately block Cx43 hemichannels and to cause a concentration- and time-dependent reduction in cytokine production, secretion, and / or release. Connexins

[0093] In various embodiments, the modulated hemichannel is a connexin 23 (Cx23) hemichannel, a connexin 25 (Cx25) hemichannel, a connexin 26 (Cx26) hemichannel, a connexin 30 (Cx30) hemichannel, a connexin 30.2 (Cx30.2) hemichannel, a connexin 30.3 (Cx30.3) hemichannel, a connexin 31 (Cx31) hemichannel, a connexin 31.1 (Cx31.1) hemichannel, a connexin 31.9 (Cx31.9) hemichannel, a connexin 32 (Cx32) hemichannel, a connexin 33 (Cx33) hemichannel, a connexin 34 (Cx34) hemichannel, a connexin 35 (Cx35) hemichannel, a connexin 36 (Cx36) hemichannel, a connexin 37 (Cx37) hemichannel, a connexin 38 (Cx38) hemichannel, a connexin 39 (Cx39 ... These are connexin 36 (Cx36) hemichannels, connexin 37 (Cx37) hemichannels, connexin 40 (Cx40) hemichannels, connexin 40.1 (Cx40.1) hemichannels, connexin 43 (Cx43) hemichannels, connexin 45 (Cx45) hemichannels, connexin 46 (Cx46) hemichannels, connexin 47 (Cx47) hemichannels, connexin 50 (Cx50) hemichannels, connexin 57 (Cx57) hemichannels, connexin 59 (Cx59) hemichannels, and connexin 62 (Cx62) hemichannels. In one embodiment, the modulated hemichannel comprises one or more of Cx26, Cx30, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and / or Cx47 proteins. In a specific embodiment, the hemichannel and / or modulated hemichannel is Cx37 and / or Cx40 and / or Cx43 hemichannel. In a specific embodiment, the hemichannel and / or modulated hemichannel is Cx30 and / or Cx43 and / or Cx45 hemichannel. In some embodiments, the modulated hemichannel may include or exclude any of the connexin proteins described above. In some aspects, the hemichannel blocker is a blocker of Cx43 hemichannel, Cx40 hemichannel, and / or Cx45 hemichannel. In certain preferred embodiments, the hemichannel blocker is a connexin 43 blocker.Pharmaceutical compositions of the invention for any of the uses featured herein may also include hemichannel blockers capable of inhibiting or blocking Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, or Cx57 hemichannels, or any other connexin hemichannels (including homologous and heterologous hemichannels). In another embodiment, pharmaceutical compositions for use in the methods and manufacture of the invention for any of the uses and connexins featured herein may also include at least one cytokine antagonist, provided together or separately. In some embodiments, the modulated hemichannel may include or exclude any of the connexin hemichannels described above, or may be a heteromeric hemichannel.

[0094] In one embodiment, the hemichannel blocker used in any of the administration, co-administration, compositions, kits, or treatment methods of the present invention is a Cx43 hemichannel blocker. Other embodiments include Cx45 hemichannel blockers, Cx30 hemichannel blockers, Cx37 hemichannel blockers, Cx40 hemichannel blockers, and hemichannel blockers comprising, consisting essentially of, or consisting of Cx26, Cx31.1, Cx36, Cx50, and / or Cx57 hemichannels, or any other connexin mentioned above or herein. Some embodiments may include or exclude any of the above-mentioned connexins or hemichannels, or others mentioned in this patent. Hemichannel blockers

[0095] Hemichannel blockers are used in the methods of the invention to modulate cytokines, including, for example, VEGF and IL-6, IL-8, MCP-1, sICAM-1. Small molecule hemichannel blockers

[0096] Examples of hemichannel blockers include small molecule hemichannel blockers (e.g., Xiflam (tonabersat)). In some embodiments, the hemichannel blocker is a small molecule other than Xiflam, such as a hemichannel blocker described in Formula I in U.S. Patent Application Publication No. 20160177298, filed in the name of Colin Green et al., the disclosure of which, as noted above, is incorporated herein by reference in its entirety. Various preferred embodiments include the use of small molecules that block, improve, or otherwise antagonize or inhibit hemichannel opening to treat the diseases, disorders, and conditions described or referenced herein. In various embodiments, the small molecule that blocks, improves, or inhibits hemichannel opening is a prodrug of Xiflam or an analog thereof.

[0097] In some embodiments, the invention features the use of small molecule hemichannel blockers, including, for example, compounds of Formula I, e.g., Xiflam, and / or analogs or prodrugs of any of the above-mentioned compounds, to block Cx43 hemichannels and cause a concentration- and time-dependent reduction in cytokine production, secretion, and / or release.

[0098] As an example, the hemichannel blocker Xiflam may be known by the IUPAC name N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamide or (3S-cis)-N-(6-acetyl-3,4-dihydro-3-hydroxy-2,2-(dimethyl-d6)-2H-1-benzopyran-4-yl)-3-chloro-4-fluorobenzamide.

[0099] In one embodiment, Xiflam and / or its analogs or prodrugs have Formula I: [ka] [In the formula, Y is C-R1; R1 is acetyl; R2 is hydrogen, C 3~8 Cycloalkyl, optionally interrupted by oxygen or substituted by hydroxy 1~6 Alkyl, C 1~6 Alkoxy or substituted aminocarbonyl, C 1~6 Alkyl carbonyl, C 1~6 Alkoxycarbonyl, C 1~6 Alkylcarbonyloxy, C 1~6 Alkoxy, nitro, cyano, halo, trifluoromethyl or CF3S; or the group CF3-A- (wherein A is -CF2-, -CO-, -CH2-, CH(OH), SO2, SO, CH2-O- or CONH); or the group CF2H-A'- (wherein A' is oxygen, sulfur, SO, SO2, CF2 or CFH); trifluoromethoxy, C 1~6 Alkyl sulfinyl, perfluoro C 2~6 Alkyl sulfonyl, C 1~6 Alkyl sulfonyl, C 1~6 Alkoxysulfinyl, C 1~6 alkoxysulfonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, phosphono, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulfinyl, heteroarylsulfinyl, arylsulfonyl or heteroarylsulfonyl (wherein any aromatic moiety is optionally substituted), C 1~6 Alkylcarbonylamino, C 1~6 Alkoxycarbonylamino, C 1~6 Alkyl-thiocarbonyl, C 1~6 Alkoxy-thiocarbonyl, C 1~6 Alkyl-thiocarbonyloxy, 1-mercaptoC 2~7 alkyl, formyl, or aminosulfinyl, aminosulfonyl or aminocarbonyl (wherein any amino moiety may be one or two C 1~6 optionally substituted with alkyl groups), or C 1~6 Alkylsulfinylamino, C 1~6Alkyl sulfonyl amino, C 1~6 Alkoxysulfinylamino or C 1~6 Alkoxysulfonylamino, or C 1~6 alkylcarbonyl, ethylenyl terminally substituted by nitro or cyano, or -C(C 1~6 alkyl)NOH or -C(C 1~6 alkyl)NNH2; or one or two C 1~6 By alkyl or C 2~7 amino optionally substituted by alkanoyl; one of R and R is hydrogen or C 1~4 alkyl, and the other is C 1~4 Alkyl, CF3 or CH2X a Fluoro, chloro, bromo, iodo, C 1~4 Alkoxy, hydroxy, C 1~4 Alkylcarbonyloxy, -SC 1~4 Alkyl, nitro, one or two C 1~4 Amino, cyano or C optionally substituted with alkyl groups 1~4 or R3 and R4 together represent C 1~4 C optionally substituted with alkyl 2~5 is polymethylene; R5 is C 1~6 Alkylcarbonyloxy, benzoyloxy, ONO2, benzyloxy, phenyloxy or C 1~6 R6 and R9 are hydrogen, or R5 is hydroxy and R6 is hydrogen or C 1~2 alkyl and R9 is hydrogen; R7 is heteroaryl or phenyl, both of which are chloro, fluoro, bromo, iodo, nitro, C 1~4 Amino, cyano, azido, optionally substituted once or twice with alkyl, C 1~4 is optionally substituted one or more times independently with a group or atom selected from alkoxy, trifluoromethoxy, and trifluoromethyl; R8 is hydrogen, C 1~6 Alkyl, OR 11 or NHCOR 10 where R 11 is hydrogen, C 1~6 Alkyl, formyl, C 1~6 Alkanoyl, aroyl or aryl-C 1~6 alkyl, and R 10 is hydrogen, C 1~6 Alkyl, C 1~6 Alkoxy, mono or di C 1~6 Alkylamino, Amino, Amino-C 1~6 Alkyl, Hydroxy-C 1~6 Alkyl, Halo-C 1~6 Alkyl, C 1~6 Acyloxy-C 1~6 Alkyl, C 1~6 Alkoxycarbonyl-C 1~6 - alkyl, aryl, or heteroaryl; the R8-N-CO-R7 group is cis to the R5 group; X is oxygen or NR 12 where R 12 is hydrogen or C 1~6 alkyl] The compound is selected from the group of compounds having the formula:

[0100] For any of the Markush groups set forth above, the group may include or exclude any of the species listed for that group. Hemichannel blockers for use in the methods of the invention may include or exclude any of these compounds.

[0101] In another embodiment, the analog of Formula I is the compound carabersat (N-[(3R,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-4-fluorobenzamide) or trans-(+)-6-acetyl-4-(S)-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzo[b]pyran-3R-ol hemihydrate.

[0102] In certain embodiments, Xiflam and / or its analogs are in the form of a free base or a pharmaceutically acceptable salt. In other embodiments, one or more polymorphs, one or more isomers, and / or one or more solvates of Xiflam and / or its analogs may be used.

[0103] Various other small molecules have been reported to be useful in inhibiting hemichannel activity. Green et al., U.S. Patent Application Publication No. 20160177298, Formula II; Savory et al., U.S. Patent See U.S. Patent Application Publication No. 20160318891; and Savory et al., U.S. Patent Application Publication No. 20160318892, all of which, as noted above, are incorporated by reference in their entirety. Hemichannel blockers for use in the methods of the present invention may include or exclude any of these compounds. Peptide and peptidomimetic hemichannel blockers

[0104] In other embodiments, the present invention features the use of peptide hemichannel blockers, e.g., peptidomimetic compounds, such as Peptagon, to block connexin hemichannels and cause a concentration- and time-dependent reduction in cytokine production, secretion, and / or release. Hemichannel blockers may include peptides corresponding to specific sequences within the extracellular loops E1 and E2 and the cytoplasmic loop (Gap19 peptide), including the conserved QPG and SHVR motifs in E1 (Gap26 peptide) and the SRPTEK motif in E2 (Gap27 peptide). Hemichannel blockers for use in the methods of the present invention may include or exclude any of the "Gap" compounds. The most potent peptidomimetic is Peptagon (VDCFLSRPTEKT) (SEQ ID NO: 1). A preferred peptidomimetic compound contains the 7-mer motif SRPTEKT.

[0105] In some embodiments, the peptide and / or peptidomimetic hemichannel blocker (e.g., peptagon) comprises a connexin extracellular domain, a transmembrane region, and a connexin carboxy-terminal peptide. The connexin hemichannel-blocking peptide or peptidomimetic can be modified or unmodified. The connexin hemichannel-blocking peptide or peptidomimetic is chemically, synthetically, or otherwise manufactured. In some embodiments, the connexin hemichannel-blocking peptide or peptidomimetic is a Cx43 peptide or peptidomimetic. In some aspects, the therapeutically effective modified or unmodified peptide or peptidomimetic comprises a portion of the extracellular or transmembrane domain of a connexin, e.g., Cx43 or Cx45, e.g., a portion of connexin extracellular loop 2, including a portion of Cx43 extracellular loop 2 and a portion of Cx45 extracellular loop 2. In some embodiments, the peptide or peptidomimetic comprises a portion of the extracellular or transmembrane domain of connexin Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50, Cx57, or another connexin mentioned herein. Peptidomimetics corresponding to a portion of extracellular loop 2 of Cx43 are preferred herein.

[0106] Peptagon is a hemichannel blocker that can act in a dose-dependent manner, with lower doses blocking gap junction hemichannel opening and higher doses uncoupling gap junctions between cells. See, e.g., O'Carroll et al., 2008. With sustained low-dose application, there is also a gradual loss of gap junction coupling (parallel to the gradual removal of existing gap junctions during normal turnover), which is attributed to peptide interference with hemichannel docking. Peptagon has proven effective in several in vitro, ex vivo, and in vivo (animal) studies (e.g., Davidson et al., 2012; Danesh-Meyer et al., 2013). 2012; see O'Carroll et al, 2013).

[0107] In some embodiments, hemichannel blockers, e.g., Cx43 hemichannel blockers, may comprise peptides. The hemichannel blocker peptide sequence may comprise, consist essentially of, or consist of, for example, one or more of the following sequences: SRPTEKT "Mod3" (SEQ ID NO: 2), "Peptide 1" ADCFLSRPTEKT (SEQ ID NO: 3), "Peptide 2" VACFLSRPTEKT (SEQ ID NO: 4), "Peptide 11" VDCFLSRPTAKT (SEQ ID NO: 5), "Peptide 12" VDCFLSRPTEAT (SEQ ID NO: 6), "Peptide 5" VDCFLSRPTEKT (SEQ ID NO: 1), "Mod1" CFLSRPTEKT (SEQ ID NO: 7), "Mod2" LSRPTEKT (SEQ ID NO: 8). In some embodiments, the carboxy terminus may be modified. In some aspects, the carboxy terminus modification may include an n-alkyl chain, which may optionally be further linked to hydrogen or other moieties. In some embodiments, the hemichannel blocker peptide may include or exclude any of the peptides listed above or disclosed herein.

[0108] In one aspect, the present invention relates to the use of a pharmaceutical composition, alone or in a kit, package, or other article of manufacture, in a method for treating the diseases, disorders, or conditions referred to herein, as well as diseases, disorders, or conditions characterized by increased or impaired or otherwise unwanted or undesirable cytokines or angiogenesis, including IL-6, IL-8, sICAM-1, and MCP-1, and the angiogenic cytokine VEGF. The methods herein provide for treating a subject with a hemichannel blocker in an amount sufficient to reduce the production, secretion, and / or release of IL-6, IL-8, sICAM-1, and MCP-1, and / or VEGF. In some embodiments, the hemichannel blocker is a connexin 43 hemichannel blocker. Blockers of other connexin hemichannels, as noted, are within the scope of the present invention.

[0109] In some embodiments, a "promoiety" is a compound that masks a functional group within an active agent. "promoiety" refers to a species that acts as a protecting group for a drug, thereby converting the active drug into a prodrug. Typically, the promoiety is attached to the drug via a bond that is cleaved in vivo by enzymatic or non-enzymatic means, thereby converting the prodrug to its active form. In some embodiments, the promoiety can also be the active drug. In some embodiments, the promoiety can be attached to a hemichannel blocker. In some embodiments, the promoiety can be attached to, for example, either a peptide or peptidomimetic or a small molecule hemichannel blocker. In some embodiments, the promoiety can be attached to a compound of Formula I. In some embodiments, the prodrug is attached to another hemichannel compound, for example, the compound described in Green et al., U.S. Patent Application Publication No. No. 20160177298; Savory et al., U.S. Patent Application Publication No. 20160318891; or Savory et al., U.S. Patent Application Publication No. 20160318892.

[0110] In some aspects, hemichannel blockers include, for example, antibodies or antibody fragments, nanobodies, peptides or peptidomimetics, recombinant fusion proteins, aptamers, small molecules, or single-chain variable fragments (scFvs) that bind to connexin hemichannels, and others mentioned herein. In one preferred embodiment of the present invention, the connexin hemichannel is a Cx43 hemichannel.

[0111] In other embodiments, the hemichannel blocker is a connexin 43 peptide or peptidomimetic, sometimes referred to as a hemichannel-blocking peptide or peptidomimetic, including modified or unmodified Cx peptides or peptidomimetics comprising, consisting essentially of, or consisting of the connexin extracellular domain, transmembrane region, and connexin carboxy-terminal peptide. In some aspects, the therapeutically effective modified or unmodified peptide or peptidomimetic comprises a portion of the extracellular or transmembrane domain of connexin 43 or connexin 45. The protein sequence of connexin 43 is shown below. Connexin 43 (SEQ ID NO: 9) [ka] [ka]

[0112] Table 1 shows the extracellular loops for connexin 43 and connexin 45. In some embodiments, the therapeutically effective modified or unmodified peptide or peptidomimetic comprises a portion of the E2 extracellular domain (extracellular loop 2) of a connexin, e.g., connexin 43 or connexin 45, preferably connexin 43. In some embodiments, the therapeutically effective modified or unmodified peptide or peptidomimetic comprises a portion of the C-terminal domain of a connexin, e.g., connexin 43 or connexin 45, preferably connexin 43. When the peptide or peptidomimetic blocking agent comprises a portion of the intracellular domain of a connexin, the peptide may, in some embodiments, be conjugated to a cell-internalized transporter, and in some examples, may block junction of tight junctions (ZO-1) binding to connexin 43. [Table 1]

[0113] Shown are the sequences of the E2 domains of different connexin isotypes with amino acids homologous to peptide SEQ ID NO: 14 and peptide SEQ ID NO: 15 shown in bold in Table 2. Note that the last four amino acids of peptide SEQ ID NO: 15 are part of the fourth membrane domain.

[0114] Table 2 provides extracellular domains of connexin family members that can be used to prepare the peptide hemichannel blockers described herein. The peptides and fragments thereof provided in Table 2 are used as peptide hemichannel blockers in certain non-limiting embodiments. In other non-limiting embodiments, hemichannel blocker peptides comprising, consisting essentially of, or consisting of about 8 to about 15 or about 11 to about 13 contiguous amino acids of a peptide in this table are peptide hemichannel blockers of the invention. In other embodiments, conservative amino acid changes are made to the peptide or fragment thereof. [Table 2]

[0115] Other peptide hemichannel blockers are derived from the cytoplasmic loop (amino acids 119-144) L2 peptide of connexin 43 and subportions of the L2 peptide of connexin 43. In some embodiments, these peptides include, for example, the 9 amino acid sequence of Gap19, KQIEIKKFK (SEQ ID NO: 19); the native Gap19 sequence, DGVNVEMHLKQIEIKKFKYGIEEHGK (SEQ ID NO: 20); et al., Biophys. J. 91, 405404063, 2006) His144→Glu L2 derivative, DGVNVEMHLKQIEIKKFKYGIEEQGK (SEQ ID NO: 21); TAT-Gap19 sequence, YGRKKRRQRRRKQIEIKKFK (SEQ ID NO: 22); SH3 binding domain, CSSPTAPLSPMSPPGYK (SEQ ID NO: 23), or its subportion PTAPLSPMSPP (SEQ ID NO: 24); C-terminal sequence RPRDDEI (SEQ ID NO: 25), SRPRDDLEI (SEQ ID NO: 26), YGRKKRRQRRRSRPRDDEI (SEQ ID NO: 27), or YGRKKRRQRRRRPRDDEI (SEQ ID NO: 28) of CT9 or CT10 peptide with or without the TAT leader sequence for increased cell penetration. Other peptidomimetic sequences that may be included or excluded in compositions for use in the methods, kits, or articles of manufacture disclosed herein include those described in Dhein (Dhein, S., Naunyn-Schmiedeberg's Arch. Pharm., 350: 174-184, 1994); AAP10 peptide, H2N-Gly-Ala-Gly-4Hyp-Pro Tyr-CONH2 (SEQ ID NO: 29) and ZP123 peptide (rotigapeptide), Ac-DT yr-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2 (SEQ ID NO: 310) (Dhein, S., et al. Cell Commun. Adhes. 10, 371-378, 2013). Rotigaptide is composed of the D-form of the peptide for enhanced efficacy over the native L-form of the peptide.

[0116] Exemplary connexin 43 (Cx43) or Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40.1, Cx43, Cx46, Cx46.6 or Cx40 peptide blocking agents that may be included or excluded in certain embodiments of the present disclosure are provided in Table 3 below (E2 and T2 refer to the position of the peptide, e.g., in the second extracellular domain or second transmembrane domain). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0117] In some embodiments, connexin 43 blocking agents may include, for example, peptides or peptidomimetics comprising, consisting essentially of, or consisting of, for example, SEQ ID NO:2 (SRPTEKT). Peptides or peptidomimetics may also include, for example, SEQ ID NO:1 (VDCFLSRPTEKT). Peptides may contain one or more modified amino acids, amino acid analogs, or be otherwise modified to improve bioavailability or increase permeation across cell membranes. For example, SEQ ID NO:1 may be modified to obtain SEQ ID NOs:20-25 and 27. In some aspects, peptides or peptidomimetics comprising, consisting essentially of, or consisting of, for example, SEQ ID NO:2 (SRPTEKT) or SEQ ID NO:2 (VDCFLSRPTEKT), comprise 7-40 amino acids or amino acid analogs and do not include a C-terminal peptide. In some embodiments, peptides may also be used as promoieties.

[0118] In some embodiments, connexin 45 blocking agents can be peptides or peptidomimetics that comprise, consist essentially of, or consist of portions of the connexin 45 protein that antagonize, inhibit, or block connexin-connexin interactions. Exemplary peptide sequences for connexin 45 peptides and peptidomimetic blocking agents are provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

[0119] In some embodiments, a connexin 45 blocking agent may include, for example, a peptide or peptidomimetic comprising, consisting essentially of, or consisting of a portion of the E2 or C-terminal domain of connexin 45, e.g., a peptide or peptidomimetic comprising, consisting essentially of, or consisting of SEQ ID NO: 150 (SRPTEKT). The peptide or peptidomimetic may also include, for example, SEQ ID NO: 149 (DCFISRPTEKT). In some embodiments, the peptide may be as little as three amino acids in length, or longer, including SRL, PCH, LCP, CHP, IYY, SKF, QPC, VCY, APL, HVR.

[0120] In some aspects, connexin 40 hemichannel blockers can be peptides or peptidomimetics comprising, consisting essentially of, or consisting of portions of connexin 40 proteins. In some embodiments, connexin 43 blockers can comprise, consist essentially of, or consist of, for example, SEQ ID NO:2 (SRPTEKT), SEQ ID NO:1 (VDCFLSRPTEKT), or SEQ ID NO:1 conjugated to two dodecyl groups at the N-terminus via a linker. The peptides can contain one or more modified amino acids, amino acid analogs, or be otherwise modified, for example, conjugated or linked to a cell-internalized transporter.

[0121] In another non-limiting but preferred embodiment, the hemichannel blocker comprises a peptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to a portion of the transmembrane region of a connexin, e.g., Cx43 or Cx45, or Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx50, or Cx57. In certain non-limiting embodiments, the anti-connexin compound is a peptide having an amino acid sequence comprising about 3 to about 30 contiguous amino acids of a connexin, e.g., connexin 43 or 45 protein sequence, about 5 to about 20 contiguous amino acids of a connexin protein sequence, about 8 to about 15 contiguous amino acids of a connexin protein sequence, or about 11, 12, or 13 contiguous amino acids of a connexin protein sequence. Other non-limiting embodiments include anti-connexin compounds that are peptides having an amino acid sequence comprising at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 consecutive amino acids of a connexin protein sequence. In some aspects, hemichannel blockers may include or exclude any of the above.

[0122] In other anti-connexin compounds, mimetic peptides are based on the extracellular domain of connexin 43 corresponding to amino acids 37-76 and 178-208 of the connexin 43 protein sequence. Thus, certain peptides described herein have amino acid sequences corresponding to the regions of connexin 43 protein sequence positions 37-76 and 178-208. Peptides need not have identical amino acid sequences to these portions of the connexin 43 protein sequence; conservative amino acid changes can be made so that the peptide retains binding or functional activity in the assays described herein and other known in the art. In other embodiments, mimetic peptides are based on peptide target regions within the connexin protein other than the extracellular domain (e.g., portions of the connexin 43 protein sequence not corresponding to positions 37-76 and 178-208).

[0123] In preferred, though non-limiting, embodiments, the hemichannel blocker comprises, consists essentially of, or consists of a peptide comprising, consisting essentially of, or consisting of an amino acid sequence corresponding to a portion of the transmembrane region of connexin 45 or the C-terminal region of connexin 45. In certain non-limiting embodiments, for example, the anti-connexin compound is a peptide having an amino acid sequence comprising about 3 to about 30 consecutive amino acids of a known connexin 45 sequence, a peptide having an amino acid sequence comprising about 5 to about 20 consecutive amino acids of a known connexin 45 sequence, a peptide having an amino acid sequence comprising about 8 to about 15 consecutive amino acids of a known connexin 45 sequence, or a peptide having an amino acid sequence comprising about 11, 12, or 13 consecutive amino acids of a known connexin 45 sequence. Other non-limiting embodiments include anti-connexin compounds that are peptides having an amino acid sequence comprising at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 consecutive amino acids of a known connexin 45 sequence. In certain anti-connexin compounds provided herein, the mimetic peptide is based on the extracellular domain of connexin 45 corresponding to amino acids 46-75 and 199-228 of the known connexin 45 sequence. Accordingly, certain peptides described herein have amino acid sequences corresponding to the regions of positions 46-75 and 199-228 of the known connexin 45 sequence. The peptide need not have an identical amino acid sequence to these portions of the known connexin 45 sequence. Conservative amino acid changes can be made so that the peptide retains binding or functional activity in assays described herein and other known in the art. In other embodiments, the mimetic peptide is based on a peptide target region within the connexin protein other than the extracellular domain (e.g., a portion of the known connexin 45 sequence not corresponding to positions 46-75 and 199-228). WO 2006 / 134494, which discloses various connexin sequences, is incorporated by reference in its entirety. In some aspects, the hemichannel blocker may include or exclude any of the above. Other connexin hemichannel blockers

[0124] Hemichannel blockers, including peptides, peptidomimetics, antibodies, antibody fragments, and the like, e.g., connexin 43 or 45 blockers, are also suitable hemichannel blockers. Exemplary hemichannel blockers include, without limitation, polypeptides (e.g., antibodies, binding fragments thereof, and synthetic constructs), and other gap junction blocking agents, and Gap junction protein phosphorylation agents may be included. In some embodiments, the hemichannel blocker is a blocker of Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx50, or Cx57. Hemichannel blockers, for example, connexin 43 or 45 blockers, include recombinant antibodies and antibody fragments that can bind antigenic determinants (i.e., the portion of a molecule commonly referred to as an epitope) that contact a specific antibody or other binding molecule, such as monoclonal antibodies, polyclonal antibodies, antibody fragments (including, for example, Fab, F(ab')2, and Fv fragments); single-chain antibodies; single-chain Fv; and single-chain binding molecules, for example, those that comprise, consist essentially of, or consist of a binding domain, hinge, CH2, and CH3 domain. These binding proteins, including antibodies, antibody fragments, etc., can be chimeric, humanized, or otherwise rendered less immunogenic in the subject to which they are administered, and can be synthesized, recombinantly produced, or produced in an expression library. Any binding molecule known in the art or later discovered is contemplated, such as those mentioned herein and / or those further described in detail in the art. For example, binding proteins include not only antibodies, etc., but also ligands, receptors, peptidomimetics, or other binding fragments or molecules (e.g., produced by phage display) that bind to targets (e.g., connexins, hemichannels, or associated molecules).

[0125] Binding molecules generally have a desired specificity, including but not limited to binding specificity, and a desired affinity. For example, the affinity may be about 10 4 Higher than or equal to M-1, approximately 10 6Higher than or equal to M-1, approximately 10 7 Higher than or equal to M-1, approximately 10 8 Ka may be greater than or equal to M-1. 8 M-1, e.g., about 10 9 M-1, about 10 10 M-1, about 10 11 M-1 and about 10 12 Affinity equal to or greater than M-1 is suitable. The affinity of binding proteins according to the invention can be determined by conventional techniques, e.g., Scatchard et al. et al., (1949) Ann. NY Acad. Sci. 51: 660. , can be easily determined.

[0126] Exemplary compounds used to close gap junctions (e.g., phosphorylate tyrosine and / or serine residues of connexin 43) are reported in U.S. Patent Nos. 7,153,822 and 7,250,397. Exemplary peptides and peptidomimetics are reported in Green et al., WO2006134494. See also WO2006069181 and WO2003032964. Examples of other agents used to close gap junctions include anti-connexin agents, such as anti-connexin polynucleotides (e.g., connexin inhibitors, e.g., alpha-1 connexin oligodeoxynucleotides), anti-connexin peptides (e.g., antibodies and antibody-binding fragments) and peptidomimetics (e.g., alpha-1 anti-connexin peptides or peptidomimetics), gap junction-closing or blocking compounds, hemichannel-closing or blocking compounds, and connexin carboxy-terminal polypeptides, e.g., polypeptides that bind to ZO-1 or the ZO-1 binding site.

[0127] Other hemichannel blockers useful in the present invention may also include or be combined with compounds that block connexin hemichannels but maintain connexin gap junction function. For example, the linear peptide RRNYRRNY, the cyclic peptide CyRP-71, and the peptidomimetic molecule ZP2519 have been demonstrated to target the Cx43 carboxy-terminal domain and prevent Cx43-based gap junction closure under low pH conditions (Verma V, et al. Design and characterization of the first peptidomimetic molecule that prevents acidification-induced closure of cardiac gap junctions. Heart Rhythm 7:1491-1498 (2010); Verma V, et al. Novel pharmacophores of connexin43 based on the "RXP" series of Cx43-binding peptides. Circ. Res. 105:176-184 (2009)). These substances are of potential practical value for preventing gap junction closure. Furthermore, these molecules are potential hemichannel blockers and may therefore have dual actions aimed at preventing gap junction closure as well as inhibiting hemichannel opening.

[0128] Anti-connexin agents include peptides having an amino acid sequence comprising about 5-20 contiguous amino acids of a connexin protein, such as connexin 43 (SEQ ID NO: 19), about 8-15 contiguous amino acids of connexin 43, or about 11-13 contiguous amino acids of connexin 43. Other anti-connexin agents include peptides having an amino acid sequence comprising at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of connexin 43. Other anti-connexin 43 blocking agents include peptides or peptidomimetics comprising, consisting essentially of, or consisting of the extracellular domain of connexin 43, e.g., SRPTEKT or VDCFLSRPTEKT. Other anti-connexin 43 blocking agents include the C-terminal region of connexin 43 (see WO2006 / 069181), or modified versions thereof. Peptide chemical modification

[0129] In certain embodiments, the connexin 43 blocking peptides of the present invention can be linked to a cell-internalizing transporter at the amino or carboxy terminus. The cell-internalizing transporter linked to the connexin 43 blocking peptides of the present invention can be any internalizing sequence known in the art or newly discovered in the art, or a conservative variant thereof. Non-limiting examples of cell-internalizing transporters and sequences include the antennapedia sequence, TAT, HIV-Tat, penetratin, Antp-3A (Antp variant), buforin II, transportan, MAP (model amphipathic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidine-cholesterol), and BGTC (bisguanidinium-tren-cholesterol).

[0130] Other sequences of exemplary cell-internalizing peptides are provided in Table 5 below. [Table 5]

[0131] In one embodiment of the present invention, the amino acid sequence of the connexin 43 blocker peptide can be selected from the group consisting of any peptide SEQ ID NO: 30-90, or a conservative variant thereof. In a further embodiment of the present invention, the connexin 43 blocker peptide can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 30-90. In another embodiment of the present invention, the connexin 43 blocker peptide further comprises a cell-internalizing transporter. In a further embodiment, the connexin 43 hemichannel blocker peptide can be linked to the cell-internalizing transporter at the amino terminus.

[0132] When a specific protein is referred to herein, derivatives, variants, and fragments are contemplated. Protein derivatives and variants are well understood by those of skill in the art and may include amino acid sequence modifications. For example, amino acid sequence modifications may fall into one or more of three classes: insertion, substitution, or deletion variants. Insertions include amino- and / or carboxyl-terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions may be smaller than those of amino- or carboxyl-terminal fusions, for example, about 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at a final construct. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions are referred to as conservative substitutions. The replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. Conservative substitutions can replace one hydrophobic residue with another, or one polar residue with another. Conservatively substituted variations of each explicitly disclosed sequence are included within the scope of the peptides provided herein. Conservative substitutions typically have little to no effect on the biological activity of the resulting polypeptide. Conservative substitutions can be amino acid substitutions in a peptide that do not substantially affect the biological function of the peptide. A peptide can contain one or more amino acid substitutions, 2 to 10 conservative substitutions, 2 to 5 conservative substitutions, or 4 to 9 conservative substitutions. Chemical structure modification

[0133] In certain embodiments, the chemical structure of a hemichannel blocker peptide or peptidomimetic can be synthetically modified to increase activity or half-life. For example, the peptide or peptidomimetic can be modified by conjugating the peptide to a hydrophobic compound, in some embodiments via a linker moiety. The hydrophobic compound can be, for example, one or more n-alkyl groups, which can be, for example, C6-C14 alkyl groups. In some embodiments, the peptide can be modified by conjugating one or two n-alkyl groups, as described in Chen, YS et al., J.Pharm. Sci., 102: 2322-2331 (2013), which is incorporated herein by reference. It may be conjugated at the N-terminus to a dodecyl (C12) group. In one embodiment, the peptide sequence CFLSRPTEKT or VDCFLSRPTEKT may be conjugated to two dodecyl groups to create a modified peptide "C12-C12-Cxn43 MP" (SEQ ID NO: 171) that can modulate connexin 43. The resulting structure is shown below. [ka] Structure: Structure of C12-C12-Cxn43 MP (SEQ ID NO: 171). R1 and R2 can be hydrogen or alkyl groups. In some embodiments, R1 = R2 = n-dodecyl chain. Chemical Delivery Modification

[0134] Hemichannel blockers useful in the present invention for reducing or halting cytokine production, secretion, and / or release can also be formulated into microparticle (microsphere, MP) or nanoparticle (nanosphere, NP) formulations, or both. Particulate drug delivery systems include nanoparticles (1-1,000 nm) and microparticles (1-1,000 μm), which are further classified as nanospheres and microspheres and nanocapsules and microcapsules. In nanocapsules and microcapsules, drug particles or droplets are encapsulated in a polymeric membrane. Particulate systems have the advantage of delivery by injection, and their size and polymer composition significantly affect their biological behavior in vivo. Microspheres can remain in the vitreous for significantly longer periods than nanospheres; therefore, microparticles act like reservoirs after injection. Nanoparticles rapidly diffuse and are internalized in tissues and cells.

[0135] Evaluating hemichannel blocker activity Various methods can be used to evaluate the activity or effectiveness of hemichannel blockers. In one embodiment of the present invention, the effect of hemichannel blocker treatment in a subject is evaluated or monitored using a cytokine protein assay. Cytokine protein levels can be quantified by any conventional method that allows for the detection and quantification of proteins in a sample from a subject. As a non-limiting example, cytokine protein levels can be quantified, for example, by using an antibody (or a fragment thereof containing an antigenic determinant) with cytokine binding ability and subsequent quantification of the formed complex. The antibodies used in these assays can be labeled or unlabeled. Illustrative examples of markers that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There are a wide variety of known assays that can be used in the present invention using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies). These techniques include Western blot or Western transfer, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of protein microarrays or biochips containing specific antibodies, or colloidal precipitation-based assays in formats such as dipsticks. Other methods for detecting and quantifying cytokines include affinity chromatography techniques, ligand binding assays, etc. When immunological methods are used, any antibody or reagent known to bind to the cytokine protein with high affinity can be used to detect its amount. Nevertheless, the use of antibodies, such as polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, scFv, humanized diabodies, triabodies, tetrabodies, nanobodies, alphabodies, stapled peptides, cyclopeptides, and antibodies is preferred.There are commercially available anti-cytokine protein antibodies on the market provided by several commercial companies that can be used in accordance with the present invention.

[0136] The present invention includes an in vitro method for predicting clinical outcomes in a subject treated with a hemichannel blocker, and for initiating hemichannel blocker treatment, discontinuing hemichannel blocker treatment, modifying hemichannel blocker treatment, or further treating the subject with a hemichannel blocker and / or cytokine antagonist. In one embodiment, the method includes detecting one or more cytokines, such as VEGF-A, in a sample from the subject, where elevated cytokine levels, profile, or activity indicate a poor or inappropriate clinical outcome, and initiating treatment of the subject with a therapeutically effective amount of a hemichannel blocker, i.e., a therapy aimed at preventing and / or treating unwanted or undesirable cytokine levels and / or activity, selected from the group consisting of small molecule hemichannel blockers, peptidomimetic hemichannel blockers, and other compounds that function as hemichannel blocker agents capable of avoiding and / or preventing disease or disease progression in the subject. In another embodiment, hemichannel blocker treatment is discontinued or discontinued for a predetermined period of time. In another embodiment, hemichannel blocker treatment is modified, for example, by changing the dose or dose frequency, or by separate administration of another therapeutic agent, for example, a cytokine antagonist (e.g., an anti-VEGF antibody or a VEGFR blocker). In another embodiment, hemichannel blocker treatment is continued or continued for a predetermined period of time. Preferred small molecule hemichannel blockers of the present invention include Xiflam. Preferred peptidomimetic hemichannel blockers of the present invention include Peptagon.

[0137] The activity of hemichannel blockers can also be evaluated using certain biological assays. The effect of known or candidate hemichannel blockers on molecular mobility can be identified, evaluated, or screened using the methods described in the Examples below, or other art-known or equivalent methods for determining the passage of compounds through connexin hemichannels. Various methods are known in the art, including dye translocation experiments, e.g., the translocation of molecules labeled with detectable markers, and the transmembrane passage of small fluorescently permeable tracers, which have been widely used to study the functional status of hemichannels. See, for example, Schlaper, KA, et al. Currently Used Methods for Identification and Characterization of Hemichannels. Cell Communication and Adhesion 15:207-218 (2008). In vivo methods can also be used. See, for example, Danesh-Meyer, HV, et al. al. Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischemia. Brain, 135:506-520 (2012); Davidson, JO, et al. (2012). Connexin hemichannel blockade improves outcomes in a model of fetal ischemia. Annals of Neurology 71:121-132 (2012). See methods.

[0138] One method for use in identifying or assessing the ability of a compound to block hemichannels includes (a) combining a test sample and a test system, wherein the test sample comprises one or more test compounds, and the test system comprises a system for assessing hemichannel blockade, wherein the system is activated by, for example, the introduction of hypoxia or ischemia into the system, a mediator of inflammation, or other compound or event that induces hemichannel opening, e.g., extracellular Ca 2+ (b) determining the presence or amount of an increase in the dye or other labeled metabolite in the system, for example, in response to a decrease in the dye or other labeled metabolite. Positive and / or negative controls can also be used. Optionally, a predetermined amount of a hemichannel blocker (e.g., peptagon or xiflam) can be added to the test system.

[0139] Preferably, hemichannel blockers, such as peptagon and xiflam, exhibit activity in in vitro assays of less than about 1-5 nM, preferably less than about 10 nM, and more preferably less than about 50 pM. In in vivo assays, these compounds preferably exhibit hemichannel blockade at concentrations of less than about 10-100 micromolar (μM), more preferably less than about 50 μM. Other hemichannel blockers may be within these ranges and in the range of less than about 200 pM. Coadministration

[0140] Pharmaceutical compositions are also provided for co-administration in the form of combined preparations, for example, as admixtures of two or more hemichannel blockers, which may be modified or unmodified, or as one or more hemichannel blockers and one or more cytokine antagonists.

[0141] The term "combination preparation" includes a "kit of parts" or "article of manufacture" in the sense that the combination partners defined above, whether in pharmaceutical form or in dressing / matrix form or both, can be administered independently or by use of different fixed combinations with identified amounts of combination partners (a) and (b), i.e., simultaneously, separately or sequentially. The parts of the kit can then, for example, be administered simultaneously or chronologically staggered, i.e., at different time points and with equal or different time intervals for any part of the kit of parts.

[0142] In one embodiment, a combined preparation is administered, in which two or more separate blocking agent compositions are administered to a subject, wherein a first composition comprises a therapeutically effective amount of a blocking agent, e.g., a hemichannel blocking agent, e.g., an anti-connexin 43 peptide, peptidomimetic, or small molecule hemichannel-closing compound, and a second composition comprises a therapeutically effective amount of a second blocking agent, e.g., an anti-cytokine agent, e.g., an anti-VEGF antibody.

[0143] Pharmaceutical compositions for single, combined, simultaneous, separate, sequential or sustained administration can be used in the methods and kits described herein.In one embodiment, a composition comprising, consisting essentially of, or consisting of one or more hemichannel blockers is administered or provided at one or more desired doses for administration at one or more time points.In another embodiment, a composition comprising, consisting essentially of, or consisting of one or more hemichannel blockers is administered at approximately the same time as one or more cytokine antagonists.When the compositions are administered at different time points, they can be administered, for example, within 30 minutes, 1 hour, 1 day, 1 week, 1 month, or 3 months apart, or any time interval between any two of the listed periods.

[0144] In one embodiment, a composition comprising, consisting essentially of, or consisting of one or more hemichannel blockers is administered within at least about 30-60 minutes of one or more cytokine antagonists. The hemichannel blocker dose together with the antagonist dose (in a combined or separate formulation) can be administered QD, BID, TID, QID, or in weekly doses, e.g., QIW, BIW QW. They can also be administered PRN (i.e., as needed) and HS (before sleep, i.e., at bedtime). Dosage Forms and Formulations and Administration

[0145] Unless expressly stated otherwise, all statements regarding dosing apply to the hemichannel blockers of the present invention.

[0146] The hemichannel blockers may be dosed, administered or formulated as described herein.

[0147] The hemichannel blocker can be administered to a subject in need of treatment. Thus, the present invention provides a formulation that can modulate connexin hemichannels, such as connexin 43 or connexin 45 hemichannels, to reduce their opening probability in a transient or site-specific manner.

[0148] The hemichannel blocker can be present in the formulation in a substantially isolated form. It is understood that the product can be mixed with a carrier or diluent that does not interfere with the intended purpose of the product and still be considered substantially isolated. The product of the present invention can also be in a substantially purified form, in which case the product of the present invention generally contains about 80%, 85%, or 90%, for example, at least about 88%, at least about 90, 95%, or 98%, or at least about 99%, for example, a peptidomimetic or small molecule hemichannel blocker, or a preparation of dry mass.

[0149] The hemichannel blocker can be administered to a subject by any means capable of delivering the drug to a target site in the subject's body. For example, the hemichannel blocker and / or cytokine antagonist can be administered by one of the following routes: orally, topically, systemically (e.g., intravenously, intraarterially, intraperitoneally, transdermally, intranasally, or via suppository), parenterally (e.g., intramuscularly, subcutaneously, or intravenously or intraarterially), by implantation, and by injection via a device such as an osmotic pump, transdermal patch, etc. Exemplary administration routes are also outlined in Binghe, W. and B. Wang (2005). Drug delivery: principles and applications, Binghe Wang, Teruna Siahaan, Richard Soltero, Hoboken, NJ Wiley-Interscience, c2005. In one embodiment, the hemichannel blocker is administered systemically. In another embodiment, the hemichannel blocker is administered orally. In another embodiment, the hemichannel blocker is administered, for example, topically or directly to the organ, cancer, or tumor of interest.

[0150] In some aspects, the hemichannel blocker can be provided as or in conjunction with an implant. In some aspects, sustained delivery can be provided. In some embodiments, microneedles, needles, iontophoresis devices, or implants can be used to administer the hemichannel blocker. The implant can be, for example, a dissolvable disc material, such as that described in S. Pflugfelder et al., ACS Nano, 9 (2), pp. 1749-1758 (2015). In some embodiments, the hemichannel blockers of the present invention, e.g., connexin 43 hemichannel blockers, can be administered via intracerebroventricular and / or intrathecal and / or extradural and / or subdural and / or epidural routes.

[0151] The hemichannel blocker can be administered once, or more than once, or periodically. The hemichannel blocker can be administered PRN (as needed), or on a predetermined schedule, or both. In some embodiments, the hemichannel blocker is administered daily, weekly, monthly, bimonthly, or quarterly, or any combination of these periods. For example, treatment can be administered daily for a period of time, then weekly and / or monthly. Other methods of administering the blocker are featured herein. In one embodiment, the hemichannel blocker is administered to the patient at or between days 1-5, 10, 30, 45, 60, 75, 90, or 100-180 in an amount sufficient to treat the patient.

[0152] Hemichannel blockers, such as Peptagon and / or analogs or prodrugs thereof, compounds of Formula I, such as Xiflam and analogs or prodrugs of any of the above-mentioned compounds, may be administered alone or in combination with one or more additional ingredients and may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients, diluents and / or carriers.

[0153] "Pharmaceutically acceptable diluents, carriers, and / or excipients" are intended to include substances that are useful in preparing pharmaceutical compositions, that may be co-administered with compounds of Formula I, e.g., Xiflam, and any analogs of the compounds described above, while enabling them to perform their intended function, and that are generally safe, non-toxic, and not biologically or otherwise undesirable. Pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for veterinary and human pharmaceutical use. Suitable carriers and / or excipients will be readily apparent to those skilled in the art, taking into account the properties of the compounds of Formula I, e.g., Xiflam, and any analogs of the compounds described above. However, by way of example, diluents, carriers, and / or excipients include solutions, solvents, dispersion media, retardants, polymeric and lipid agents, emulsions, and the like. By way of further example, suitable liquid carriers, particularly for injectable solutions, include water, saline solution, aqueous dextrose, and the like, with isotonic solutions being preferred for intravenous, intrathecal, and intracisternal administration, and vehicles such as liposomes are also particularly suitable for administering drugs.

[0154] The composition can be in the form of any standard known dosage form, including tablets, pills, capsules, semisolids, powders, sustained-release formulations, solutions, suspensions, elixirs, aerosols, injectable solutions, gels, creams, transdermal delivery devices (e.g., transdermal patches), inserts, such as organ inserts, e.g., ophthalmic, or any other suitable compositions.Those skilled in the art to which the present invention pertains will easily understand the most appropriate dosage form without undue experimentation, taking into account the condition to be treated and the properties of the active agent used.It should be understood that one or more of the hemichannel blockers, such as Peptagon and / or its analogs, compounds of Formula I, such as Xiflam and analogs of any of the above-mentioned compounds, and / or cytokine antagonists, can be formulated into a single composition.In certain embodiments, preferred dosage forms include injectable solutions and oral formulations.

[0155] Compositions useful in the present invention may contain any appropriate level of hemichannel blocker, e.g., Peptagon and / or analogs thereof, compounds of Formula I, e.g., Xiflam and analogs of any of the above-mentioned compounds, and / or cytokine antagonists, taking into consideration the dosage form and mode of administration. However, by way of example, compositions for use in the present invention may contain from about 0.1% to about 99% by weight, preferably from about 1% to about 60%, of the hemichannel blocker, depending on the method of administration.

[0156] In addition to standard diluents, carriers, and / or excipients, the compositions of the present invention can be formulated with one or more additional components, or in a manner that enhances the activity or bioavailability of, for example, hemichannel blockers, such as Peptagon and / or its analogs, compounds of Formula I, such as Xiflam and analogs of any of the above-mentioned compounds, and / or cytokine antagonists, helps protect their integrity or increases their half-life or shelf life, allows for slow release upon administration to a subject, or provides other desirable benefits. For example, slow-release vehicles include macromers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone), or hydrogels. As further examples, the compositions can also include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, coating agents, buffers, etc. Those skilled in the art to which the present invention pertains can identify additives that may be desirable for a particular purpose.

[0157] Hemichannel blockers can be administered by sustained-release systems.Suitable examples of sustained-release compositions include semipermeable polymer matrices in the form of shaped articles, for example, films or microcapsules.Sustained-release matrices include polylactic acid (U.S. Pat. No. 3,773,919; EP58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate or poly-D-(-)-3-hydroxybutyric acid (EP133,988).Sustained-release compositions also include liposome-encapsulated compounds. Liposomes containing hemichannel blockers can be prepared by known methods, including those described in, for example, DE 3,218,121; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. Typically, liposomes are small (approximately 200-800 angstroms) unilamellar liposomes with a lipid content of more than about 30 mole percent cholesterol, with the selected ratio adjusted for the most effective treatment. For example, slow-release delivery using PGLA nanoparticles or microparticles or an in situ ion-activated gelation system can also be used.

[0158] Furthermore, it is contemplated that hemichannel blocker pharmaceutical compositions for use in accordance with the present invention may, in certain cases, be formulated with additional active ingredients or agents that may be therapeutically or otherwise beneficial to the subject. Those skilled in the art to which this invention pertains will recognize appropriate additional active ingredients in light of the description of the invention herein and the nature of the disorder being treated.

[0159] The compositions are described, for example, in Gennaro AR: Remington: The Science and Practice of Pharmacy, 20 thed., Lippincott, Williams & Wilkins, 2000. However, as a further example, the information provided in US2013 / 0281524 or US5948811 may be used.

[0160] In certain embodiments, the present invention provides a combination product comprising, consisting essentially of, or consisting of (a) a hemichannel blocker and (b) one or more additional active agents, e.g., cytokine antagonists, wherein components (a) and (b) are adapted for simultaneous or sequential administration.

[0161] In certain embodiments of the invention, the combination product according to the invention is used in such a way that at least one of the components is administered while the other component still has an effect on the subject being treated.

[0162] Any container suitable for storing and / or administering pharmaceutical compositions may be used for the hemichannel blocker product for use in the methods of the present invention.

[0163] In some embodiments, hemichannel blockers, such as connexin 43 hemichannel blockers, can be formulated to provide controlled and / or compartmentalized release to the site of administration. In some embodiments of the present invention, the formulation can be an immediate-release or extended- or sustained-release dosage form. In some embodiments, the dosage form can include both an immediate-release dosage form combined with an extended-release and / or sustained-release dosage form. In some embodiments, both immediate-release and sustained and / or extended-release of the hemichannel blocker can be achieved by combining an immediate-release form of, for example, a modified or unmodified peptide or peptidomimetic, or another hemichannel blocker. In some embodiments of the present invention, the hemichannel blocker is, for example, a connexin 43 blocker or other hemichannel blocker disclosed herein. In some embodiments of the present invention, the dosage form can be an implant, for example, a biodegradable or non-biodegradable implant.

[0164] In some embodiments of the present invention, hemichannel blockers, e.g., connexin 43 hemichannel blockers, can be formulated for compartmentalized release of the blockers, for example, by adjusting the particle size or coating. For example, in some embodiments, particle formulations of hemichannel blockers, e.g., connexin 43 blockers, can be administered for use in the methods of the present invention. In some embodiments, particle-containing drug delivery systems can include nanoparticles with an average diameter of less than 1,000 nm, e.g., 1-1,000 nm, and / or microparticles with an average diameter of 1-1,000 μm. The nanoparticles or microparticles can be, for example, nanospheres or microspheres, or encapsulated nanocapsules and microcapsules in which the hemichannel blockers are encapsulated in a polymeric coating. The particle formulations can also include liposomes. In some embodiments, the hemichannel blocker may or may not include a blocker of connexin 45, Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50, or Cx57 hemichannels or any other connexin hemichannel in blood vessels.

[0165] The present invention includes methods for modulating hemichannel function for the treatment of various disorders, which include administering a hemichannel blocker, optionally alone or in combination with one or more other agents (including, for example, active cytokine antagonist agents) or therapeutic agents.

[0166] In another embodiment, the hemichannel blocker, e.g., a compound of Formula I, e.g., Xiflam, or a peptide or peptidomimetic hemichannel blocker, can be administered systemically, e.g., by intravenous, intraarterial, or intraperitoneal administration, so that the final circulating concentration is from about 0.001 to about 150 micromolar, or higher concentrations up to 200, 300, 400, 500, 600, 700, 800, 900, or 1000 micromolar.The final circulating concentrations were 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, .4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 110, 120, 130, 140, or 150 micromolar concentrations, or any concentration between any of the two listed numbers, or any higher concentration and within the stated range as described above. As referred to herein, the present invention also includes combination therapies in which one or more additional active agents are also administered to a subject. Those skilled in the art will understand the desired dosage for one or more active agents, taking into account the properties of the agents and the principles previously discussed herein.

[0167] Administration of the hemichannel blocker, and optionally one or more other active agents, can occur at any time during the progression of the disorder, or before or after the development of the disorder or one or more symptoms of the disorder. In one embodiment, the hemichannel blocker is administered periodically for an extended period of time to assist in the ongoing management of symptoms. In another embodiment, the hemichannel blocker is administered periodically for an extended period of time or lifelong to prevent or delay the development of the disorder.

[0168] In some embodiments, hemichannel blockers, such as connexin 43 hemichannel blockers, can be administered as a pharmaceutical composition comprising one or more particles. In some aspects, the pharmaceutical composition can be, for example, an immediate-release formulation or a controlled-release formulation, such as delayed-release particles. In other aspects, the hemichannel blockers can be formulated in a granular formulation of one or more particles for selective delivery to the area to be treated. In some embodiments, the particles can be, for example, nanoparticles, nanospheres, nanocapsules, liposomes, polymeric micelles, or dendrimers. In some embodiments, the particles can be microparticles. The nanoparticles or microparticles can include biodegradable polymers. In other embodiments, the hemichannel blockers are prepared or administered as an implant or matrix, or formulated to provide compartmentalized release to the site of administration.

[0169] In some embodiments, the formulated hemichannel blocker is a connexin 43 or connexin 45 hemichannel blocker, preferably a connexin 43 hemichannel blocker. As used herein, "matrix" includes matrices, such as polymeric matrices, biodegradable or non-biodegradable matrices, and other carriers useful for creating implants or application structures for delivering hemichannel blockers. Implants include reservoir implants and biodegradable matrix implants.

[0170] In some embodiments, hemichannel blockers, e.g., connexin 43 hemichannel blockers, are administered to a subject using, for example, microneedles, microneedle arrays, needles, or implants to provide a therapeutically effective amount of the connexin 43 hemichannel blocker. In some embodiments, microneedles can be used to administer the hemichannel blockers. In some embodiments, microneedle penetration can be controlled to a desired depth within a tissue, organ, or organ compartment. In some embodiments, the microneedles can be coated with the hemichannel blockers, alone or in combination with other drug agents. In some embodiments, the volume of hemichannel blocker and / or drug agent administered by the microneedle is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 The volume may be from 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 295, or 300 μl, or any range of volumes between any two of the recited numbers, or any volume between any two of the recited numbers. For example, any suitable formulation of the present invention may be administered by microneedle injection, including nanoparticle or microparticle formulations, or other formulations injectable by microneedles. Combinations of connexin hemichannel blockers with cytokine inhibitors or other agents

[0171] In some embodiments, the VEGF antagonist for use in the present invention is a compound or composition that inhibits and / or blocks VEGF, or inhibits and / or blocks the upstream agonist of VEGF or its receptor.In some embodiments, the VEGF antagonist includes, for example, an antagonist that binds to and inhibits VEGF, a compound that inhibits the expression of VEGF, and / or a viral vector that contains a VEGF inhibitor or encodes a protein or antisense polynucleotide that blocks or inhibits VEGF or VEGFR.In some embodiments, the VEGF antagonist is, for example, an antibody or antibody fragment, a nanobody, a peptide or peptidomimetic, a receptor fragment, a recombinant fusion protein, an aptamer, a small molecule, or a single-chain variable fragment (scFv).In some embodiments, the VEGF antagonist antibody is, for example, Lucentis™ (ranibizumab) and / or Avastin™ (bevacizumab).

[0172] In some embodiments, the VEGF antagonist, which is antisense to an upstream agonist of a VEGF species that binds to and thus inhibits VEGF, may be an RTP801 inhibitor or a REDD1 blocker. In some embodiments, the RTP801 inhibitor or REDD1 blocker may be PF-655 (by Quark Pharmaceuticals and Pfizer), also known as REDD14NP or RTP801i. In some embodiments, the REDD1 blocker may have the mRNA sequence 5'-AGCUGCAUCAGGUUGGCAC-3' (SEQ ID NO: 172).

[0173] In some embodiments of the invention, the VEGF antagonist is, for example, a peptide or peptidomimetic, such as pegaptanib sodium (Macugen™), and AGN-150998. Macugen™ is a modified RNA sequence, ((2'-deoxy-2'-fluoro)C-Gm-Gm-AA-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-Am-Gm-(2'-deoxy-2'-fluoro)U-Gm-Am-Am-(2'-deoxy-2'-fluoro)U-Gm-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2'-fluoro)U-Am-(2'-deoxy-2'-fluoro)U-Am-(2' -deoxy-2'-fluoro)C-Am-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)C-Gm-(3'→3')-dT), 5'-ester with α,α'-[4,12-dioxo-6-[[[5-(phosphonooxy)pentyl]amino]carbonyl]-3,13-dioxa-5,11-diaza-1,15-pentadecanediyl]bis[ω-methoxypoly(oxy-1,2-ethanediyl)], sodium salt (SEQ ID NO: 173). AGN-150998 / MP0112 is an anti-VEGF DARPin, a small protein that binds to VEGF.

[0174] In some embodiments of the present invention, the VEGF antagonist is a recombinant fusion protein, such as aflibercept (Eyelea™) or conbercept. Aflibercept is a recombinant fusion protein consisting of portions of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Conbercept is a recombinant fusion protein consisting of the second Ig domain of VEGFR1 and the third and fourth Ig domains of VEGFR2 fused to the constant region (Fc) of human IgG1.

[0175] In some embodiments, the scFv VEGF antagonist is, for example, ESBA1008. ESBA1008 is a humanized monoclonal single-chain FV (scFv) antibody fragment that targets VEGFA.

[0176] In some embodiments, the viral vector VEGF antagonist may be AAV-sFLT01 (also known as "AVA-101"). AAV2-sFlt01 is an adeno-associated viral vector carrying a gene construct for a secreted chimeric protein, sFLT01, that binds to VEGF. sFLT01 is a VEGF-binding protein consisting of domain 2 of Flt-1 (sFlt01) linked to a human immunoglobulin G1 heavy chain Fc fragment, which is combined with an adeno-associated virus (AAV) to yield AAV2-sFlt01.

[0177] In some embodiments of the present invention, the VEGF antagonist is a small molecule such as vatalanib, cediranib, AL39324, pazopanib, TG100572, or TG100801. Vatalanib (N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine) is also known as PTK787, PTK / ZK, or CGP79787. Cediranib, also known as AZD2171, Recentin™, ZD2171, or CAS number 288383-20-0, is also known as 4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]-quinazoline. AL39324, also known as linifanib, CAS No. 796967-16-3, 1145655-58-8 (as the HCl salt), or 796967-17-4 (as the trifluoroacetic acid salt), is also known as 1-[4-(3-amino-1H-indazol-4-yl)phenyl]-3-(2-fluoro-5-methylphenyl)urea. Pazopanib, also known as Votrient™, Armala™, or Patorma™, is also known as 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzenesulfonamide monohydrochloride. TG100801 is the prodrug version of TG100572, also known as 4-chloro-3-(5-methyl-3-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-amino)benzo[e][1,2,4]triazin-7-yl)p; 4-chloro-3-[5-methyl-3-[[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]amino]-1,2,4-benzotriazin-7-yl]phenol 1-benzoic acid.

[0178] In some embodiments, the hemichannel blocker and the anti-cytokine treatment agent can be co-formulated for co-administration. In some aspects, the hemichannel blocker and anti-cytokine treatment agent formulations can be part of a pill, liquid, gel, pre-filled syringe, tablet, eye drops, or particle-based formulation.

[0179] When using hemichannel blockers, such as relatively short peptides and peptidomimetics, the methods of the present invention also contemplate an initial high dose / rapid onset dose (rapid release), followed by a sustained maintenance low dose. For separate or common administration, the formulation can be prepared to provide rapid or slow release; immediate release, delayed release, sustained release, or sustained release; or a combination thereof. The formulation can be in the form of a liquid, solution, suspension, emulsion, elixir, syrup, electuary, drops (including, but not limited to, eye drops), tablet, granule, powder, lozenge, troche, capsule, gel, ointment, cream, lotion, oil, foam, spray, mist, or aerosol. Combination products / kits of connexin hemichannel blockers with cytokine inhibitors or other agents

[0180] Another embodiment of the present invention provides an article of manufacture, or "kit," containing materials useful for treating the above-mentioned diseases and disorders. The kit includes a container containing, consisting essentially of, or consisting of a cytokine inhibitor and a connexin hemichannel blocker. The kit may further include a label or package insert on or associated with the container. The term "package insert" refers to instructions customarily included in commercial packaging of such therapeutic products, containing information regarding the indications, use, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container can hold a hemichannel blocker and / or cytokine antagonist, or a formulation thereof, that is effective for treating the condition and may have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a hemichannel blocker. The label or package insert indicates that the composition is used to treat the selected condition, such as any of the diseases, disorders, and / or conditions described or referenced herein. The label or package insert may also indicate that the composition can be used to treat other disorders. Alternatively, or in addition, the product may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fillers, needles, and syringes.

[0181] The kit may further include instructions for administering the hemichannel blocker and, if present, cytokine inhibitors or other agents that act on separate mechanisms derived from hemichannel modulation to treat a subject as described herein. For example, if the kit includes a first composition comprising, consisting essentially of, or consisting of a connexin hemichannel blocker, and a second pharmaceutical formulation, the kit may further include instructions for simultaneous, sequential, or separate administration of the first and second pharmaceutical compositions to a patient in need thereof.

[0182] In one embodiment, the kit may further comprise a third container comprising, consisting essentially of, or consisting of a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fillers, needles, and syringes.

[0183] In some embodiments, the first and second (and optionally third) compositions of the kit can be administered in combination, simultaneously, separately, sequentially, or in a continuous manner.

[0184] In this manner, other anti-connexin agents, such as one or more connexin hemichannel blocker peptides or peptidomimetics and / or small molecule hemichannel blockers, can also be used alone or in combination with any of the modulating agents in the manufacture of a medicament or kit. Suitable hemichannel blockers are, for example, blockers of Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, Cx57, or any other connexin described herein. As described, the kit may contain one or more pharmaceutical compositions in separate or divided containers, along with packaging and instructions for use. The kit may also include a pharmaceutically acceptable carrier. In some embodiments, the kit may also include components for administering the pharmaceutical composition, such as a syringe, needle, microneedle, loadable implant, or iontophoresis device. The connexin hemichannel blocker and the treatment partners described herein can be administered independently or by use of different fixed combinations with identified amounts of combination partners (a) and (b), i.e., simultaneously, separately, or sequentially, whether in pharmaceutical form, dressing / matrix form, or both. The parts of the kit can then be administered, for example, simultaneously or staggered in time, with different time points and equal or different time intervals for any part of the kit parts.

[0185] Also provided are articles of manufacture comprising, consisting essentially of, or consisting of a container containing a hemichannel blocker compound, composition, or formulation and instructions for use for treating a subject. For example, in another aspect, the invention includes an article of manufacture comprising, consisting essentially of, or consisting of a container containing a therapeutically effective amount of one or more connexin hemichannel blocker peptides or peptidomimetics and / or other hemichannel blockers, alone or in combination with an optional anti-cytokine agent, together with instructions for use, including use for treating a subject.

[0186] In some embodiments, the article of manufacture may include a matrix containing one or more connexin hemichannel blocker peptides or peptidomimetics or another hemichannel blocker, such as a small molecule hemichannel blocker, alone or in combination. Suitable connexin hemichannel blockers may be, for example, anti-connexin 43 or 45 hemichannel blockers. Dosage, volume and concentration

[0187] As will be appreciated, the dose of hemichannel blocker administered, the duration of administration, and the general administration regimen may vary from subject to subject depending on variables such as the target site to which it is delivered, the severity of any symptoms in the subject being treated, the type of disorder being treated, the size of the unit dose, the mode of administration selected, and the age, sex, and / or general health of the subject and other factors known to those skilled in the art.

[0188] Examples of effective doses that can be used to treat the diseases, disorders, or conditions described herein are described. In some embodiments, a therapeutically effective amount of a hemichannel blocker, e.g., a connexin 43 hemichannel blocker, is about 0.001 to about 1.0 micrograms / ml, or about 0.001 to about 0.01 mg / ml, or about 0.1 mg / ml to about 100 mg / ml or higher, or any range between any two of the described dosage amounts, or any dose between any two of the described numbers.The doses were: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg / ml, or any range between any two of the recited dosage amounts, or any dose between any two of the recited numbers. In some embodiments, the therapeutically effective amount of hemichannel blocker is present at a concentration ranging from about 0.5 to about 50 mg / mL. In some embodiments, the hemichannel blocker is present at a concentration ranging from about 0.3 to about 30 mg / mL. In some embodiments, the hemichannel blocker is present at a concentration ranging from about 0.1 or 1.0 to about 10 mg / mL.In some embodiments, the hemichannel blocker is present at a concentration ranging from about 0.1 or 1.0 to about 0.3 or 3.0 mg / mL, hi some embodiments, the hemichannel blocker is present at a concentration of about 3.0 mg / mL.

[0189] In some embodiments, the hemichannel blocker can be administered in a therapeutically effective amount of about 0.001 to about 100 mg / kg, about 0.001 to about 0.01 mg / kg, about 0.01 to about 0.1 mg / kg, 0.1 to about 1 mg / kg, about 1 to about 10 mg / kg, or about 10 to about 100 mg / kg, or any range between any two of the recited dosage amounts, or any dose between any two of the recited dosage amounts.In some embodiments, the dose is 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg / ml or any range between any two of the recited dosages or any dose between any two of the recited numbers.

[0190] It should be understood that administration may include a single daily dose, administration of several individual divided doses, or continuous administration, as appropriate. For example, the unit dose may be administered once or more than once per day, for example, 1, 2, 3, 4, 5, or 6 times per day, to achieve the desired total daily dose. By way of example, the unit dose of hemichannel blocker can be administered in a single daily dose or in several individual doses, or sequentially to achieve a daily dose of approximately 0.1 to 10 mg, 10 to 100 mg, 100 to 1000 mg, 1000 to 2000 mg, or 2000 mg to 5000 mg, 0.1 to approximately 2000 mg, approximately 0.1 to approximately 1000 mg, approximately 1 to approximately 500 mg, approximately 1 to approximately 200 mg, approximately 1 to approximately 100 mg, approximately 1 to approximately 50 mg, or approximately 1 to approximately 25 mg, or any range between any two of the recited dosage amounts, or any dose between any two of the recited dosage amounts.

[0191] By way of further example, a unit dose of hemichannel blocker can be administered once or more than once per day (e.g., 1, 2, 3, 4, 5, or 6 times per day, typically 1 to 4 times per day) so that the total daily dose is in the range of about 1 to about 1000 mg, e.g., about 1 to about 500 mg, or 500 mg to 1000 mg, 1000 to 2000 mg, or 2000 mg to 5000 mg, or any range between any two of the recited dosage amounts, or any dose between any two of the recited dosage amounts (for a 70 kg adult). For example, hemichannel blockers such as peptagon and / or analogs thereof, compounds of Formula I, e.g., xiflam, and analogs of any of the above compounds, can be administered to a subject at a dose range of about 0.01 to about 15 mg / kg / day, e.g., about 0.1 to about 6 mg / kg / day, e.g., about 1 to about 6 mg / kg / day, e.g., 6 mg / kg / day to 100 mg / kg / day, or any range or any dose between any two of the recited dosage amounts. In one embodiment, xiflam can be administered orally once daily at a dose of about 2 mg to about 40 mg.

[0192] In one embodiment, the dose of the hemichannel blocker is about 0.001 micromolar to 0.1 micromolar at the site of action, 0.1 micromolar to up to about 200 micromolar, or higher in the circulation to achieve these concentrations at the site of action. By way of example, and without limitation, the dose may be about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2 .,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3 .5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 1 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 micromolar final circulating concentration, or any range between any two of the recited concentrations, or any concentration between any two of the recited numbers. Further examples of doses expected to block hemichannels but not uncoupled gap junctions are described in O'Carroll et al., 2008, incorporated herein by reference. In some embodiments, Xiflam can be used at lower doses, e.g., 0.001 to 20 micromolar. Low doses include: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.9, 2.1, 2.2, 2.4, 2.6, 2.8, 2.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, 2.8, 2.9, 2.1, 2.2, 2.4, 7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.The concentration may be 0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 micromolar.

[0193] In one embodiment, the dose of hemichannel blocker, such as peptagon, and / or its analogs is from about 0.001 micromolar to a maximum of about 200 micromolar at the site of action, or from 200 to 2000 or 5000 micromolar, or higher in the circulation to achieve these concentrations at the site of action. By way of example, and without limitation, the dose may be about 1, 5, 10, 20, 50, 100, 200, 250, 500, 1000, 2000, 3000, 4000, or 5000 micromolar final circulating concentrations, or any range between any two of the listed doses, or any dose between any two of the listed doses. Doses of peptagon effective for blocking hemichannels, but not uncoupled gap junctions, are discussed in O'Carroll et al., 2008.

[0194] In some embodiments, Xiflam can be used at lower doses, such as 1-20 micromolar, 1-50 micromolar, 20-30, 30-40, or 40-50 micromolar. Low doses include: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, .9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7. The concentration may be 8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 micromolar.

[0195] In some embodiments, a suitable therapeutically effective dose of the hemichannel blocker may be at least about 1.0 mg / mL of the hemichannel blocker, hi some embodiments, the therapeutically effective dose of the hemichannel blocker may be about 0.001 mg / mL to 0.01 mg / mL, about 0.01 mg / mL to about 0.1 mg / mL, or about 0.1 mg / mL to about 100 mg / mL. In some embodiments, a suitable therapeutically effective dose of a hemichannel blocker is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0, 64.0, 65.0, 66.0, 67.0, 68.0, 69.0, 70.0, 71.0, 72.0, 73.0, 74.0, 75.0, 76.0, The dose may be 8.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5, 95.0, 97.5, or about 100.0 μg / mL, or any range or subrange between any two of the recited doses, or any dose within the range of about 0.1 to about 100 μg / mL.In some embodiments, a suitable therapeutically effective dose of a hemichannel blocker is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0, 64.0, 65.0, 66.0, 67.0, 68.0, 69.0, 70.0, 71.0, 72.0, 73.0, 74.0, 75.0, 76.0, In some embodiments, the hemichannel blocker may be present at a concentration ranging from about 0.5 to about 50 mg / mL. In other embodiments, the hemichannel blocker is present at a concentration ranging from about 0.3 to about 30 mg / mL. In other embodiments, the hemichannel blocker is present at a concentration ranging from about 0.1 or 1.0 to about 10 mg / mL. In other embodiments, the hemichannel blocker is present at a concentration ranging from about 0.1 or 1.0 to about 0.3 or 3.0 mg / mL. In other embodiments, the hemichannel blocker, such as a connexin 43 hemichannel blocker and / or a connexin 45 hemichannel blocker, is present at a concentration of about 3.0 mg / mL. In any of these embodiments, the hemichannel blocker may be a connexin 43 or connexin 45 hemichannel blocker. When the hemichannel blocker is a modified or unmodified peptide or peptidomimetic, its dosage may be reduced by 1-10, 25-50, 100-200, or 1000-fold.

[0196] In certain embodiments, hemichannel blockers, e.g., connexin 43 hemichannel blockers, can be administered at and / or adjacent to the treatment site at a final concentration of about 0.001 micromolar (μM), or 0.05 μM to about 200 μM, or up to 300 μM, or up to 1000 μM, or up to 2000 μM, or up to 3200 μM or higher, e.g., up to about 10 mM, 20 mM, or 30 mM, as well as any dose and dose range within these numerical values. In one embodiment, the hemichannel blocker composition is applied at a concentration greater than about 1000 μM. Preferably, the hemichannel blocker composition is applied at a final concentration of about 1000 μM to about 10 mM, more preferably, the anti-connexin agent composition is applied at a final concentration of about 3 mM to about 10 mM, and more preferably, the hemichannel blocker composition is applied at a final concentration of about 1-3 mM to about 5-10 mM. The hemichannel blocker concentrations were 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4 ,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6 , 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 ,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 micromolar; or 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0. 2., 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 millimolar concentrations, or any range between any two of the recited dosage amounts or any dose between any two of the recited numbers.

[0197] Additionally, the hemichannel blocker, e.g., a connexin 43 hemichannel blocker, may be present in the formulation at a final concentration of about 1 μM to about 50 μM; alternatively, the connexin 43 hemichannel blocker is present, for example, at a final concentration of about 5 μM to about 20 μM, or at a final concentration of about 10 μM to about 15 μM. In certain other embodiments, the hemichannel blocker is present at a final concentration of about 10 μM. In yet other embodiments, the hemichannel blocker is present at a final concentration of about 1 to 15 μM. In other embodiments, the hemichannel blocker is at about 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 10-200 μM, 200-300 μM, 300-400 μM, 400-500 μM, 500-600 μM, 600-700 μM, 700-800 μM, 800-900 μM, 900-1000 μM, 1000-1200 μM, 1200-1400 μM, 1400-1600 μM, 1600-1800 μM, 1800-2000 μM, 1800-2200 μM, 1800-2400 00μM, 800-900μM, 900-1000 or 1000-1500μM, or 1500μM-2000μM, 2000μM-3000μM, 3000μM-4000μM, 4000μM-5000μM, 5000μM-6000μM, 6000μM-7000μM, 7000μM up to 8000 μM, 8000 μM to 9000 μM, 9000 μM to 10,000 μM, 10,000 μM to 11,000 μM, 11,000 μM to 12,000 μM, 12,000 μM to 13,000 μM, 13,000 μM to 14,000 μM, 14,000 μM to 15,000 μM, 15,000 μM to 20,000 μM, 20,000 μM to 30,000 μM, 30,000 μM to 50,000 μM, or higher concentrations, or any range or subrange between any two recited doses, or any dose within the range of about 20 μM to about 50,000 μM.

[0198] Still other dosage levels are from about 1 nanogram (mg) / kg to about 1 mg / kg body weight / day of each hemichannel blocker described herein. In certain embodiments, the dosage of each of the target compounds is generally in the range of about 1 ng to about 1 microgram / kg body weight, about 1 ng to about 0.1 microgram / kg body weight, about 1 ng to about 10 ng / kg body weight, about 10 ng to about 0.1 microgram / kg body weight, about 0.1 microgram / kg body weight, about 20 ng to about 100 ng / kg body weight, about 0.001 mg to about 0.01 mg / kg body weight, about 0.01 mg to about 0.1 mg / kg body weight, or about 0.1 mg to about 1 mg / kg body weight. In certain embodiments, the dosage of each of the target compounds is generally in the range of about 0.001 mg to about 0.01 mg / kg body weight, about 0.01 mg to about 0.1 mg / kg body weight, or about 0.1 mg to about 1 mg / kg body weight. When more than one hemichannel blocker is used, the dosage of each hemichannel blocker need not be in the same range as the others. For example, the dosage of one connexin hemichannel blocker may be about 0.01 mg to about 10 mg / kg body weight, and the dosage of another connexin hemichannel blocker may be about 0.1 mg to about 1 mg / kg body weight, 0.1 to about 10 mg, 0.1 to about 20 mg, 0.1 to about 30 mg, 0.1 to about 40 mg, or about 0.1 to about 50 mg / kg body weight.Dosages may also be about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mg / kg body weight, or any range or subrange between any two of the recited doses, or any dose within the range of about 0.001 to about 100 mg / kg body weight.

[0199] As described above, the dose of the hemichannel blocker, for example, a connexin 43 or 45 hemichannel blocker, can be administered in a single application or in divided applications. The dose can be administered once or repeatedly. Typically, the application is repeated every week, every two weeks, every three weeks, every month, or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months, or more, as needed to prevent, slow, or treat any disease, disorder, or condition described herein. If cytokine levels or activity increase to an undesirable or undesirable level, the dose can be repeated and / or increased or decreased. The dose can also be administered every 12 hours to 7 days apart, or longer. For example, doses may be applied 12 hours apart, or 1, 2, 3, 4, 5, 6, or 7 days apart, or any time interval between any two of these times, or between 12 hours and 7 days. The connexin 43 hemichannel blocker may be administered for, for example, up to 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 weeks. For some indications, more frequent dosing may be used. Combination Products

[0200] In some embodiments, a hemichannel blocker can be used together with a cytokine antagonist in the manufacture of separate or combined medicaments for the treatment of one or more of the diseases, disorders, and conditions described herein.

[0201] The hemichannel blocker useful in the present invention can be administered alone or in combination with another therapeutic agent useful for treating target disease, disorder or condition.In some embodiments, the compound of Formula I, for example, Xiflam, and / or any analog or prodrug of the above-mentioned compound, or a peptidomimetic such as Peptagon or its analog or prodrug, or another hemichannel blocker can be used together with a cytokine antagonist to treat disorders that may benefit from modulation of hemichannel.The administration of the hemichannel blocker can be simultaneous with, followed by, or before the administration of the cytokine antagonist.

[0202] In certain embodiments, the present invention provides a combination product comprising, consisting essentially of, or consisting of (a) a hemichannel blocker and (b) one or more additional active agents, such as a cytokine antagonist, wherein components (a) and (b) are adapted for simultaneous or sequential administration. Any container suitable for storing and / or administering pharmaceutical compositions in which the hemichannel blocker and additional active agents are in the same or separate containers can be used for the hemichannel blocker combination product of the present invention.

[0203] In certain embodiments of the invention, the combination product according to the invention is used in such a way that at least one component is administered while the other component still has an effect on the subject being treated. Manufacturing and Purity

[0204] The method of synthesizing peptides and polypeptides, including antibodies and binding fragments, and peptidomimetics and peptide analogues, can be carried out using suitable methods.For example, see Lihu Yang et al., Proc. Natl. Acad. Sci. USA, 1; 95(18): 10836-10841 (September 1, 1998); Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Publications, New York; Harlow and Lane (1999) "Using Antibodies", A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0205] In some embodiments, the formulations of the invention are substantially pure. By substantially pure, it is meant that the formulation contains less than about 10%, 5%, or 1%, preferably less than about 0.1%, of any impurities. In some embodiments, the total impurities, including metabolites of the connexin 43 modulating agent, are 1-15% or less. In some embodiments, the total impurities, including metabolites of the connexin 43 modulating agent, are 2-12% or less. In some embodiments, the total impurities, including metabolites of the connexin 43 modulating agent, are 3-11% or less. In other embodiments, the total impurities, including metabolites of the connexin 43 modulating agent, are 4-10% or less. [Example]

[0206] The studies described in these Examples were conducted using an immortalized human retinal pigment epithelial cell line (ARPE-19) to assess the effects of high glucose and inflammation on cytokine release and the expression and localization of connexin 43. Inflammation was induced by exposing the cells to a combination of the potent inflammatory cytokines IL-1β and TNF-α. Zhou, J., et al. Role of intravitreal inflammatory cytokines and angiogenic factors in proliferative diabetic retinopathy. Current Eye Research, 37:416-420 (2012). The released cytokines tested were IL-6 (a pro-inflammatory cytokine), IL-8 (a neutrophil chemotactic factor), MCP-1 (a monocyte chemoattractant), and sICAM-1 (a leukocyte endothelial cell adhesion molecule), cytokines that have been widely studied in the literature (for review, see Tang, J, and Kern, TS. Inflammation in diabetic retinopathy. Progress in Retinal and Eye Research, 30(5), 343-358 (2011)). These include VEGF (a stimulator of angiogenesis), and VEGF (a stimulator of angiogenesis), all of which have been found to be elevated in the vitreous of patients with diabetic retinopathy (Zhou et al. supra; Abu El Asrar, AM, et al. (1992). Cytokines in the Vitreous of Patients With Proliferative Diabetic Retinopathy. American Journal of Ophthalmology, 114:731-736 (1972). The role of connexin hemichannels in the disease process was then evaluated using connexin 43 hemichannel blockers, which inhibit hemichannel opening. Example 1 method

[0207] Cell culture - Human adult retinal pigment epithelial cells (ARPE-19; American Type Culture Collection, Manassas, VA) were cultured in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 medium (DMEM-F12; Thermofisher Scientific Inc., USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1× antibiotic and antimycotic mixture (AA, 100× stock) at 37°C in a humidified 5% CO2 incubator. Cells were grown in T75 flasks and medium was changed twice a week until confluent and ready for experiments.

[0208] High glucose and / or cytokine challenge - At passages 6 to 12, 2.5 × 10 cells were cultured in 8-well chamber slides for immunohistochemistry or in 24-well plates for ATP release assays and cytometric bead array analysis. 5 Cells were seeded at 0.1 cells / mL. Once confluent, the culture medium was changed to serum-free DMEM-F12 containing 1x AA. Some cultures were challenged with 15 mM glucose (high glucose group), a combination of the inflammatory cytokines 10 ng / mL TNF-α (Peprotech, USA) and 10 ng / mL IL-1β (Peprotech, USA) (cytokine group), or a combination of high glucose, 10 ng / mL TNF-α, and 10 ng / mL IL-1β (high glucose + cytokine group). The untreated group received a medium change without further treatment (basal group). All evaluations were performed 24 h after treatment.

[0209] Hemichannel blocker treatment - peptide 5 (H-Val-As-Cys-Phe-Leu- Ser-Arg-Pro-Thr-Glu-Lys-Thr-OH; China Peptides, China) was administered to cells challenged with high glucose and inflammatory cytokines. Concentration-dependent effects were examined with 5, 10, 25, and 50 μM Peptagon 24 h later. These concentrations were shown to block connexin 43 hemichannels with minimal effects on gap junction intercellular coupling. O'Carroll, SJ, et al. Connexin 43 mimetic peptides reduce swelling, astrogliosis, and neuronal cell death after spinal cord injury. Cell communication & adhesion, 15:27-42 (2008).

[0210] Cytokine and chemokine measurement using cytometric bead arrays. Soluble cytokines and chemokines in ARPE-19 incubation medium were simultaneously measured using a multiplexed bead-based immunoassay, the cytometric bead array (CBA, BD Biosciences, USA). Triplicate 50 μL volumes were collected from duplicate cultures in 24-well plates after 24 h, transferred to 96-well plates, and used for the CBA assay. The analysis included six experiments per group. The assay was performed according to the manufacturer's instructions. Briefly, a 10-point standard curve ranging from 0 to 5000 pg / mL for each cytokine was prepared using the cytokine standards provided in each kit. The cytokines measured were human soluble CD54 (sICAM-1, Catalog No. 560269, BD Biosciences, USA), IL-6 (Cat. No. 558276), IL-8 (Cat. No. 558227), and MCP-1 (Cat. No. 558287). Samples and cytokine standards were incubated in the capture bead mixture for 1 h, and phycoerythrin (PE)-conjugated antibodies against each cytokine were added to the sample-bead mixture for a 2 h incubation at room temperature. All buffers used were from the CBA Human Soluble Protein Master Buffer Kit (Cat. No. 558265, BD Biosciences, USA). Beads were washed and analyzed using an Accuri C6 flow cytometer (BD Biosciences, USA). The mean fluorescence intensity for each bead cluster was calculated using the FCAP as previously described (O'Carroll et al., 2015). Conversion to cytokine concentrations was performed based on a 10-point standard curve using Array™ software (BD version 3.1).

[0211] ATP release assay - After 24 h incubation in treatment medium, ATP release was measured in triplicate using 50 μL of culture medium taken from duplicate wells of a 24-well plate. The sample size was 6 per group and repeated 3 times in separate experiments. ATP released into the culture medium was measured using the ATPlite Luminescence ATP Detection Assay System (PerkinElmer, USA) according to the manufacturer's instructions. ATP release (%) in cultures treated with peptide 5 (treatment group) was calculated using the formula: (OD of treatment group) 490 -OD in the injury group 490 ) / (OD of injury group 490 ) × 100% was used to calculate the percentage of cells treated with high glucose and cytokines (injured group).

[0212] Immunohistochemical analysis: After 24 h incubation in treatment medium, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min. Cells were then incubated overnight at 4 °C with mouse anti-NLRP3 (1:100; Abcam, USA) and then washed three times in PBS for 15 min. Goat anti-mouse Cy3 (1:500; Jackson Immuno) was used. Secondary antibodies (Agilent Research, USA) were applied to the slides and incubated for 3 h at room temperature. Secondary antibody-only controls showed no nonspecific labeling. Cell nuclei were stained with DAPI (1:1000; Sigma-Aldrich, USA). Cells were washed and mounted using Citifluor™ fluorescence retardant, and coverslips were sealed with nail polish. Labeling was repeated three times in separate experiments.

[0213] Image analysis—All images were captured on an Olympus FV1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan) and processed using the FV-10 ASW 3.0 Viewer and ImageJ software version 1.46r (National Institutes of Health, USA).

[0214] Quantification of NLRP3 immunolabeling—For NLRP3 immunohistochemical analysis, four images per well were analyzed, and all experiments were repeated three times.

[0215] Using ImageJ, each image was split into its RGB channels, with NLRP3 shown in the red channel and DAPI in the blue channel. Each NLRP3 image was converted to a binary image, and an equal threshold was applied to all images to reduce background. A sharpening filter was used to highlight only the NLRP3 complex, with upper and lower size thresholds set to allow inflammasome counting independent of noise speckling, and larger nuclei. The number of NLRP3 spots was counted for each image.

[0216] Statistical Analysis - Data are expressed as arithmetic mean ± standard deviation. Statistical comparisons between groups were performed using one-way analysis of variance. When each data point was compared to all other data points in series, a post-hoc Tukey's multiple comparison test was used. When each data point was compared to only one data point, a post-hoc Dunnett's multiple comparison test was used. The specific statistical method used for each data set is provided in the figure legends. P<0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 6 was used. Example 2 Co-application of high glucose plus cytokines increased IL-6, sICAM-1, MCP-1, IL-8 and VEGF secretion.

[0217] The effects of individual or combined application of high glucose and pro-inflammatory cytokines on the secretion of inflammatory pathway cytokines were evaluated. High glucose did not stimulate the release of IL-6, sICAM-1, MCP-1, or IL-8 compared to basal levels (Figure 1). Cytokines alone did not induce significant changes in sICAM-1 levels compared to basal conditions, but did induce higher levels of IL-6 (p ≤ 0.0001), MCP-1 (p = 0.0002), and IL-8 (p ≤ 0.0001). However, co-application of both high glucose and cytokines resulted in much higher IL-6, sICAM-1, MCP-1, and IL-8 release compared to basal, high glucose only, and cytokine only (p ≤ 0.0001 for all) (Figure 1). This increase was 2-3-fold higher than with inflammatory cytokines alone.

[0218] VEGF secretion was also assessed. When added separately, neither inflammatory cytokines nor high glucose had a significant effect on VEGF compared to basal levels (Figure 2). However, the combination of inflammatory cytokines and high glucose significantly increased VEGF release (p<0.0001), with VEGF concentrations more than double their baseline levels. Example 3 Connexin hemichannel blockade reduces IL-6, IL-8, sICAM-1, MCP-1, and VEGF expression after co-application of high glucose and cytokines

[0219] To assess the role of connexin 43 hemichannels in high glucose- and cytokine-mediated pathologies, cells were exposed to Peptagon, a well-established blocker of connexin hemichannels. Results show that Peptagon significantly reduced the secretion of IL-6, IL-8, sICAM-1, and MCP-1 (Figure 3, p<0.0001 for all). There was a slight trend toward a concentration-dependent decrease in IL-6, IL-8, and MCP-1 secretion with Peptagon treatment, but for sICAM-1, all peptide concentrations had the same effect. Furthermore, when connexin hemichannel blockers were added, VEGF release also completely abolished, and extracellular concentrations were reduced to nearly baseline levels, with no statistically significant difference between baseline and the high glucose + cytokine + peptide 5 treatment group (p=0.98) (Figure 2). Example 4 Connexin hemichannel blockade abolishes high glucose plus cytokine-induced ATP release

[0220] Cytokines are too large to pass through connexin hemichannels, and previous work in our laboratory suggested that open connexin 43 hemichannels release ATP. Increased ATP release triggers inflammasome activation and cytokine release. ATP release was assessed after high glucose and cytokine insults, as well as in response to additional peptide 5 treatment. Results showed that at 24 h, co-application of high glucose and cytokines resulted in high levels of ATP release (Figure 4), essentially double the levels released by ARPE-19 cells (p=0.0003). Hemichannel blocker treatment with peptagon significantly reduced ATP release compared with high glucose and cytokine treatment (p=0.0171), with no statistically significant difference between the peptagon-treated group and basal conditions (p=0.1119). Example 5 Exogenous extracellular ATP does not induce IL-6, sICAM-1, MCP-1, IL-8, or VEGF release, but reverses connexin hemichannel blockade protection against IL-6, IL-8, and VEGF release.

[0221] Released ATP was assessed after co-application of high glucose and cytokines to determine whether ATP alone was sufficient to induce cytokine release. As shown in Table A, the results showed that exposure of ARPE-19 cells to 10 nM exogenous ATP did not alter IL-6, sICAM-1, IL-8, or VEGF secretion, but did cause a decrease in MCP-1 release compared to basal conditions (p≦0.0001). [Table A]

[0222] Given that 10 nM exogenous ATP alone was not sufficient to induce cytokine release, further experiments were performed to determine whether adding the same concentration of ATP to the extracellular environment while exposing cells to high glucose, cytokines, and Peptagon could reverse Peptagon-mediated blockade of inflammatory cytokine secretion. Results were positive for IL-6, MCP1, and IL-8 release, with the presence of exogenous ATP resulting in cytokine secretion returning toward injurious levels (Figure 5). There was a trend toward increased sICAM-1 release in the presence of exogenous ATP, but it did not reach statistical significance. However, the addition of exogenous extracellular ATP completely reversed the effect of Peptagon hemichannel blockade on VEGF release, and VEGF levels again significantly increased (Figure 2).

[0223] We demonstrated that these results were due to the regulation of inflammasome complex assembly using immunohistochemical labeling of NLRP3 inflammasome complexes. Inactive NLRP3 is normally dispersed in the cytoplasm, but upon inflammasome activation, oligomerization concentrates multiple NLRP3 copies within inflammasome complexes, which can be visualized using immunohistochemical labeling. Upon the addition of high glucose and inflammatory cytokines, multiple complexes were labeled in ARPE-19 cells (small spots in Figure 6A). In turn, the addition of a peptagon hemichannel blocker blocked inflammasome assembly (Figure 6B), with faint labeling observed in the cytoplasm (note: this antibody gives high nuclear background under all conditions). However, the addition of exogenous ATP reversed the treatment, and inflammasome complexes were again seen to form in the cytoplasm (quantified in Figure 6C). Consideration

[0224] These examples demonstrate the surprising discovery of novel actions of hemichannel blockade, including direct and immediate effects on cytokine production and release or secretion via hemichannel modulation. Surprisingly, it has been discovered that connexin hemichannels mediate and play a key role in cytokine release, a discovery that has important implications in the treatment of a variety of diseases, disorders, and conditions characterized, in whole or in part, by cytokine activity, importantly, by angiogenic cytokine activity via VEGF.

[0225] Furthermore, it was surprisingly discovered that hemichannel blockers can reduce the release of inflammatory mediators IL-6, sICAM-1, MCP-1, and IL-8. The release of IL-6 and sICAM-1 indicates changes in the level of cellular stress and inflammation. IL-6, a proinflammatory cytokine, is a "death" signal, and its expression increases when cells are exposed to inflammatory stress (Planck et al., 1992). On the other hand, sICAM-1 can be cleaved from the cell surface and act as a regulatory molecule controlling leukocyte adhesion to the cell surface (Miyamoto et al., 2000). MCP-1 and IL-8 are involved in leukocyte recruitment and exacerbate the inflammatory response. Taken together, these examples and the findings herein support the idea that the use of hemichannel blockers at concentrations as low as 5 μM results in a statistically significant reduction in the secretion of inflammatory mediators. However, regulation of cytokine release by connexin hemichannels is not direct, as these molecules are too large to translocate through gap junction hemichannels, which have a size limit of approximately 1 kDa.

[0226] Finally, it is important to note that a single application of a hemichannel blocker reduced VEGF release to baseline levels, thus providing a new upstream approach to prevent excessive VEGF (and other inflammatory cytokine) release in the first instance, forming the basis for the treatment of, for example, chronic inflammatory diseases. References [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0227] The invention described and claimed herein includes many attributes and embodiments, including, but not limited to, those illustrated or described or referenced in this detailed disclosure. This is not intended to be comprehensive, and the invention described and claimed herein is not limited to or by the features or embodiments identified in this detailed disclosure; such features or embodiments are included merely for purposes of illustration and not limitation. Those skilled in the art will readily recognize that many components and parameters may be varied or modified to some extent or substituted with known equivalents without departing from the scope of the invention. It is to be understood that such modifications and equivalents are incorporated herein as if individually described. The invention also includes all steps, features, compositions, and compounds referred to or shown herein, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0228] All patents, publications, scientific articles, websites, and other documents and materials referenced or described in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it were individually incorporated by reference in its entirety or as if set forth in its entirety herein. Applicant reserves the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other referenced materials or documents. Reference herein to any applications, patents, and publications is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute legitimate prior art or form part of the common general knowledge in any country in the world.

[0229] The specific methods and compositions described herein represent preferred embodiments, are illustrative, and are not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Preferably, the invention illustratively described herein can be practiced in the absence of any element or elements, or limitation or limitations, not specifically disclosed herein as essential. Thus, for example, in each example herein and in embodiments or examples of the invention, any of the terms "comprising," "consisting essentially of," and "consisting of" can be substituted for either of the other two terms herein. Preferably, the methods and processes illustratively described herein can be practiced in different order of steps, and are not necessarily limited to the order of steps set forth in the specification or claims. Also, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Under no circumstances should the patent be construed as limited to the particular examples or embodiments or methods specifically disclosed herein. Under no circumstances should the patent be construed as limited by any statements made by any examiner or other officer or employee of the U.S. Patent and Trademark Office unless such statements are specifically, unrestrictedly, or unconditionally expressly adopted in applicant's responsive documents. Furthermore, titles, headings, and the like are provided to facilitate the reader's comprehension of this document and should not be read to limit the scope of the invention. Any examples of aspects, embodiments, or components of the invention described herein should be considered non-limiting.

[0230] The terms and expressions which have been employed are used as terms of description, not of limitation, and no intention is made in the use of such terms and expressions to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by preferred embodiments and necessary features, it will be understood that those skilled in the art may resort to modifications and variations of the concepts disclosed herein, and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0231] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a condition or negative limitation excluding any subject matter from a genus, regardless of whether the excised material is specifically described herein.

[0232] Other embodiments are within the scope of the following claims. Furthermore, when features or aspects of the invention are described in the form of a Markush group, those skilled in the art will recognize that the invention also is thereby described in the form of any individual member or subgroup of members of the Markush group.

Claims

[Claim 1] The invention as set forth in the drawings.

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