Cytokine modulation

Abstract

To provide cytokine modulation. [Solution] The present invention relates to the use of hemichannel blockers to modulate cytokine levels, including angiogenic cytokines VEGF, and their production, secretion, and / or release, in a subject, and to the use of hemichannel blockers to reduce or level out cytokine activity, including in a state characterized entirely or partially by angiogenesis and / or vascular leakage. The present invention provides, for example, a method for modulating cytokine activity in a subject, comprising the step of administering an effective amount of hemichannel blocker to the subject.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 534,595, filed Jul. 19, 2017, which is incorporated herein by reference. Field The present invention generally relates 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 publication publications, foreign patents, foreign and PCT published applications, papers, and other documents, references, and publications, as well as all those listed as references cited in any patent(s) derived from this specification, are hereby incorporated by reference in their entirety. The incorporated information is in effect 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 at the time of filing.

Background Art

[0003] Background The following includes information that may be useful in understanding the present invention. This is neither an admission that any information, publication, or document specifically or implicitly referred to herein is prior art to the invention described or claimed herein, nor an admission that any such information, publication, or document is essential to the invention described or claimed herein.

[0004] The angiogenic cytokine vascular endothelial growth factor (VEGF) plays a central role in human growth and development, as well as in vascular maintenance. However, it is now well established that angiogenesis also plays a crucial role in the pathogenesis of various disorders. VEGF-mediated angiogenesis has been reported to be important for tumor growth, as well as for exudative age-related macular degeneration (AMD), such as proliferative diabetic retinopathy and retinopathy of prematurity, all of which are characterized by abnormal neovascularization. In solid tumors, neovascularization allows tumor cells to acquire growth advantages 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). Ischemia and inflammation have been reported to lead to VEGF-mediated breakdown of the blood-retinal barrier, which causes macular edema that impairs vision. 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] Regarding retinal damage, despite the overall clinical success of anti-VEGF agents, some AMD patients still require frequent injections to keep the disease under control. It has been noted 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 are 20 / 40% under 40%. It exhibits a suboptimal treatment response, defined as full visual acuity. (Rosenfeld, PJ et al., Ranibizumab for neovascular age-related macular degeneration. N. Engl. J.) Med. 355:1419-1431 (2006). Data from the Phase 3 study are for the currently approved dose. However, since the dose-response curves for AMD and diabetic macular edema (DME) are at or near the peak, higher doses may not be beneficial. Busbee, 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). Regarding cancer, the effects of VEGF inhibitors have not reached the remarkable efficacy predicted in some early preclinical studies using 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 used in advanced, difficult-to-treat malignant tumors. It has shown benefits in patients with tumors and is currently the standard treatment for 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 a crucial role not only in the immune system but also in various biological processes. Dysregulated, persistent interleukin-IL-6 production has been linked to the development of various autoimmune chronic inflammatory diseases and even cancer. It is a major regulator of both acute and chronic inflammation. Significant elevations of IL-6 have been found, for example, in ocular fluid from patients with refractory / chronic uveitis, and IL-6 has been shown to be required to induce inflammation in experimental autoimmune uveitis models using IL-6 knockout mice. Actemra® (tocilizumab), a recombinant humanized anti-IL-6 receptor antibody, has been used to treat several autoimmune diseases, including uveitis. For an overview of the efficacy and safety of tocilizumab therapy, 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 used in autoimmune diseases, including Castleman disease. It has also been used for sexual disorders.

[0007] Increased expression of interleukin-8 (IL-8) and / or its receptor is 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. This is summarized in 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. While there has been hope for the discovery of strategies to indirectly attenuate IL-8 signaling in cancer cells, this hope has been aimed at 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 chemotactic protein-1 (MCP-1 / CCL2) is one of the key chemokines that regulate monocyte / macrophage migration and infiltration. Both MCP-1 and its receptor have been reported to be induced and involved in various diseases and conditions, including multiple sclerosis (correlation between MCP-1 and axonal injury), secondary multiple sclerosis and nociception (due to MCP-1-mediated depolarization of neurons), tumor neovascularization (due to the effect of MCP-1 on macrophage infiltration), and insulin resistance (increased MCP-1). While the discovery of drugs that affect MCP-1 production could potentially target tissues, such as those 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, which states that the discovery of drugs that block upregulated chemokine receptors may prove effective when 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 different cell types. Levels of soluble ICAM-1 (sICAM-1) in plasma 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)). 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. have reported that sICAM-1 can clearly 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 reports 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 and provide pathways for the movement of molecules between cells (gap junction channels) and pathways for the release of molecular effectors into the extracellular environment (plasma membrane gap junction hemichannels). Gap junctions are specialized intercellular connections found in most animal cell types. They are expressed in virtually all tissues of the body except for mature skeletal muscle and mobile cell types, such as sperm and erythrocytes. Gap junctions directly connect the cytoplasm of two cells, allowing various molecules, ions, and electrical impulses to pass directly through the regulated gate between cells. A single gap junction consists of two connexons (i.e., hemichannels) that connect across the intercellular space between adjacent cells, allowing intracellular molecules to flow between these cells. Each connexon of a gap junction is located in the adjacent cell membrane and is formed by the covalent oligomerization of six individual connexin ("Cx") proteins. The requirements for the formation of a functional gap junction are the assembly of connexin proteins into hemichannels and their insertion into the membrane. For intercellular communication, hemichannels from one cell need to dock with their counterparts on the opposing membrane of an adjacent cell to enable signal transmission from one cell to the other via the gap junction. Gap junctions and hemichannels are involved in the transport 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 remain closed until they dock during gap junction formation to form cell-cell channels. With some exceptions, connexin hemichannel currents tend to be activated by strong depolarization or a reduction of extracellular calcium below 0.5 mM. Thus, the activity of endogenous connexin hemichannels may not be prominent under normal physiological conditions. However, when surface-exposed, undocked hemichannels have been reported to be able to mediate molecular exchange between the cytosol and extracellular space. Thus, hemichannels are closed by default, but several cues can induce their opening, e.g., extracellular Ca 2+ Decreased concentration (Evans et al., The gap junction cellular internet: connexin hemichannels enter the signaling limelight. Biochem J 397(1): 1-14 (2006)) or infection by enteropathogenic 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., The study "Connexin-dependent intercellular communication increases invasion and dissemination of Shigella in epithelial cells, Nat Cell Biol 5(8): 720-726 (2003)" is mentioned. See Puhar A and Sansonetti PJ, Dye-uptake Experiment through Connexin Hemichannels, Bio-protocol 4(17): e1221 (Sept. 2014). Gap junctions, hemichannels, and connexin regulators are discussed. It has been proposed for various 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)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0012] Brief summary The inventions described and claimed herein have many features and embodiments, including but not limited to those shown, described or referenced in this summary of the invention. The invention is not intended to be comprehensive, and the inventions described and claimed herein are neither limited to nor limited by the features or embodiments identified in this introduction, which are included for illustrative purposes only, not limiting purposes.

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

[0014] The inventors hereby discover that, surprisingly, considering the previously described properties of hemichannels having a molecular size limit of approximately 1 kDa, modulating or blocking hemichannels can reduce or halt the production, secretion, and / or release of inflammatory cytokines, as evidenced by the reduction in IL-6, IL-8, MCP-1, and sICAM-1, as well as the reduction in the presence or amount of the angiogenic cytokine VEGF, after administration of hemichannel blockers. These molecules range in size from approximately 11 kDa (MCP-1 and IL-8) to 90 kDa (sICAM-1). Hemichannels are resistant to water, small molecules, and ions, such as Ca 2+ (40Da), as well as small signaling molecules, such as ATP, cAMP, and NAD + This allows only IP3, prostaglandins, and glutamates (140-700 Da) to pass through.

[0015] While not intended to be constrained by any particular theory, the research herein supports the idea that connexin hemichannels play a crucial role in disease processes by amplifying and perpetuating connexin hemichannel-mediated cytokine feedback loops, forming the basis for the conditions and diseases referred herein, as well as other conditions and diseases characterized at least in part by undesirable levels of VEGF and / or other cytokines, including 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 diabetes mellitus and diabetic nephropathy, for example. These results support the idea that connexin hemichannels play a vital role in disease processes, including those referred to elsewhere herein in the background technology, as well as, for example, heart failure (see Butts, B., et al. Journal of Cardiac Failure, 21:586-593 (2015)); muscular dystrophy (Cea, LA, et al. (2013)). See 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, i.e., COPD (Hosseinian, N, et al., Therapeutic See advances in respiratory disease, 9:188-197 (2015); diabetes mellitus and diabetic nephropathy, as well as organ failure resulting in insulin resistance, impaired insulin secretion and renal failure (Wada, J. and Makino, H. Innate immunity in diabetes and See diabetic nephropathy. Nature Reviews Nephrology, 12:13-26 (2016); and brain tumors (Zhou, K., et al. Journal of Immunology Research, This has an effect (see 2016: 9238290).

[0016] In one aspect, the present invention relates to the use of hemichannel blockers to modulate cytokine levels, including angiogenic cytokines VEGF, and their production, secretion, and / or release, in a subject, and to the use of hemichannel blockers to reduce or level out cytokine activity, including in a state characterized entirely or partially by angiogenesis and / or vascular leakage.

[0017] In one embodiment, 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 embodiment, a method is provided for reducing the production, release, and / or secretion of VEGF. In one embodiment, VEGF is VEGF-A.

[0019] In another embodiment, a method is provided for modulating a connexin hemichannel-mediated cytokine feedback loop that is undesirably amplified and persisted in a subject, including in diseases, disorders and conditions described or referenced herein, including in the background art.

[0020] In another embodiment, a method is provided for modulating a connexin 43 hemichannel-mediated autosecretion feedback loop that is undesirably amplified and persisted in a subject, including in diseases, disorders and conditions described or referenced herein, including in the background art.

[0021] In another embodiment, a method is provided for modulating a connexin hemichannel-mediated autosecretion cytokine feedback loop, including a connexin 43 hemichannel-mediated one, to reduce cytokine production, secretion and / or release in diseases, disorders and conditions characterized at least in part by neoplasm or tumor growth associated with the 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. In particular, this patent describes compositions and methods that may be used to disrupt the mediator cycle of chronic diseases.

[0023] The method of the present invention is useful in attenuating abnormal, elevated, dysregulated, and / or otherwise undesirable levels of cytokines in a subject by administering a connexin hemichannel blocker to the subject. Cytokines include, for example, IL-6, IL-8, sICAM-1, MCP-1, and VEGF, for example, VEGF-A.

[0024] The present invention relates in part to methods for reducing cytokines and / or cytokine activity, which include, or consist of, the administration of hemichannel blockers, such as peptide-mimicking hemichannel blockers, such as Peptagon (peptide 5), and / or small molecule hemichannel blockers, such as Xiflam (tonaversat). These methods are useful, for example, in the treatment of VEGF (e.g., VEGF-A) levels associated with pathological or otherwise unwanted angiogenesis, difficulties associated with type 2 (non-insulin-dependent) diabetes mellitus, and, for example, solid tumors and cancers, as well as other diseases, disorders and conditions described or mentioned herein, including in the background art.

[0025] The method is also useful in treating, for example, unwanted or pathological levels of interleukin-6 (IL-6), various human inflammatory diseases, such as conditions associated with Castleman disease, and other diseases, disorders, and conditions described or referred to herein, including in the background technology.

[0026] The method is useful, for example, in the treatment of various diseases and conditions associated with unwanted or pathological levels of interleukin-8 (IL-8), peripheral artery occlusive disease (PAOD), cystic fibrosis, ANCA-associated vasculitis (Wegener's granulomatosis), hematological malignancies, and other cancers, such as hepatocellular carcinoma, soft tissue sarcoma, and early and metastatic breast cancer, as well as other diseases, disorders and conditions described or referred to herein, including in the background art.

[0027] The method is also useful in the treatment of various acute and chronic inflammatory diseases and associated conditions, including, for example, in patients with unwanted or pathological levels of sICAM-1, lupus nephritis, neuromyelitis optica (NMO), systemic lupus erythematosus (SLE), gingival supporting cells associated with plaque buildup and inflammation, as well as in patients with gingivitis, adult periodontitis and rapidly progressive periodontitis, and pathological processes associated with the metastatic behavior of tumor cells, as well as in patients with non-small cell lung cancer (NSCLC), and other diseases, disorders and conditions described or mentioned herein, including in the background technology.

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

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

[0030] In another embodiment, the present invention relates to a method for reducing or regulating VEGF in a subject by administering a hemichannel blocker to the subject. In one embodiment, VEGF is VEGF-A.

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

[0032] Accordingly, in one embodiment, the present invention relates to a method for blocking or reducing hemichannel opening to reduce or modulate VEGF, for example VEGF-A, and a method for treating disorders in which modulation of VEGF and / or other cytokines may be beneficial.

[0033] In another embodiment, the present invention relates to the administration of a hemichannel blocker to subjects with breast cancer, non-small cell lung cancer, or diabetes. The present invention relates to methods for regulating or reducing VEGF. In one embodiment, the VEGF to be regulated or reduced is VEGF-A.

[0034] The hemichannel blocking compositions and methods may be used alone or in combination with one or more further 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] The object of the present invention is to provide compounds, compositions, formulations, kits and methods for the use and production of cytokines, such as VEGF, IL-6, IL-8, sICAM-1 and MCP-1, and their isoforms including VEGF-A, for the modulation of hemichannels to reduce the production, secretion and / or release or secretion, in subjects where such use is required.

[0036] Another object of the present invention is 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 the subject.

[0037] Another object of the present invention is to provide compounds, compositions, formulations, kits and methods for the treatment of diseases, disorders and conditions that benefit from cytokine modulation. Another object of the present invention is 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 treatment method is applicable to mammals, such as humans.

[0039] In another embodiment, the present invention provides gap junction hemichannel blockers, e.g., small molecules, e.g., Xiflam and / or its analogs or prodrugs, or peptide mimes, e.g., Peptagon and / or its analogs or prodrugs, or other hemichannel blockers or prodrugs, for use in the treatment of disorders in which hemichannel modulation may be beneficial. In some embodiments, the hemichannel blocker is administered daily, weekly, monthly, bimonthly, or quarterly, or any combination of these intervals. For example, the treatment may be administered daily for a period, and then weekly and / or monthly thereafter.

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

[0041] The hemichannel blockers useful in the present invention may be administered alone or in combination with other therapeutic agents useful in treating a target disease, disorder, or condition. In some embodiments, compounds of formula I, e.g., Xiflam and / or analogs or prodrugs of any of the compounds described above, or peptide mimes, e.g., Peptagon or its analogs or prodrugs, or other hemichannel blockers may be used together with cytokine antagonists for the treatment of disorders in which hemichannel modulation may be beneficial. The administration of the hemichannel blocker may be simultaneous with, sequentially with, or prior to the administration of the cytokine antagonist.

[0042] Therefore, hemichannel blockers can be co-administered, for example, with VEGF or other cytokine antagonists. In methods that are essentially the same as, or consist of, co-administration of a hemichannel blocker with a cytokine blocker or antagonist, such as a VEGF antagonist, such as an anti-VEGF antibody or VEGF receptor blocker, or an IL-6 receptor blocker, the co-administration of the hemichannel blocker may 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 other embodiments, various cytokine-related disorders may be treated by compositions and methods of the present invention, including treatment with hemichannel blockers alone or in combination with cytokine antagonists. These disorders include, but are not limited to, those described or referred to herein.

[0044] As noted, the present invention also features a method for treating a patient, comprising the step of administering an anti-VEGF agent and / or an anti-cytokine agent to the patient as a therapeutic treatment, in addition to the administration of a small molecule or peptide or peptide-mimicking hemichannel blocker. In one embodiment, the VEGF antagonist or another anti-cytokine agent is administered to the patient in an amount sufficient to treat the patient, either concurrently with the administration of the hemichannel blocker or within about 1–5, 10, 30, 45, 60, 75, 90 or 100–180 days thereafter. In certain embodiments of the method of the present invention, the VEGF antagonist is administered concurrently with the hemichannel blocker. In some embodiments, for example, the VEGF antagonist may be a compound that inhibits and / or blocks VEGF, or a compound that inhibits and / or blocks an upstream agonist of VEGF. In some embodiments, VEGF antagonists include, for example, antagonists that bind to and inhibit VEGF, compounds that inhibit VEGF expression, and / or VEGF inhibitors, or viral vectors encoding proteins or antisense polynucleotides that block or inhibit VEGF. In some embodiments, agents that inhibit VEGF and / or upstream agonists of VEGF may, for example, be antibodies or antibody fragments, nanobodies, peptides or peptide mimes, 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 present invention involves administration of a VEGF antagonist, which is an aptamer, for example, the EYE001 aptamer. In another embodiment, the method of the present invention comprises the administration of a VEGF antagonist, such as an antibody or a binding fragment thereof, for example, Avastin® (bevacizumab) or Lucentis® (ranibizumab).In another embodiment, this method of the present invention includes the administration of an IL-6 receptor antagonist, such as Actemra® (tocilizumab).

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

[0046] Some preferred hemichannel blockers include small molecule hemichannel blockers (e.g., Xiflam (Tonaversat)). In some embodiments, the hemichannel blocker is a small molecule other than Xiflam, e.g., a hemichannel blocker described by formula I or II in U.S. Patent Application Publication No. 20160177298 filed in the name of Colin Green et al., the disclosure of which, as mentioned above, is incorporated herein by this reference in its entirety. Various preferred embodiments include the use of small molecules that block or improve or otherwise antagonistize 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 mentioned herein. In various embodiments, the small molecule that blocks, improves or inhibits hemichannel opening is a prodrug of Xiflam or an analog thereof.

[0047] In other embodiments, hemichannel blockers include peptides and peptide-mimicking hemichannel blockers (e.g., Peptagon, VDCFLSRPTEKT, peptide mimetics), as well as other peptide-mimicking hemichannel blockers containing, essentially, or consisting of the amino acid sequence SRPTEKT. In any aspect of the present invention, the hemichannel blocker is a connexin peptide or peptide mime, including a peptide or peptide mime containing, essentially, or consisting of a connexin extracellular domain, transmembrane region, and connexin carboxy-terminal peptide. The connexin hemichannel-blocking peptide or peptide mime may be modified or unmodified. The connexin hemichannel-blocking peptide or peptide mime is produced chemically, by synthesis, or otherwise. In some embodiments, the connexin hemichannel-blocking peptide or peptide mime is a Cx43 peptide or peptide mime. In some embodiments, a therapeutically effective modified or unmodified peptide or peptide mime is a portion of the extracellular or transmembrane domain of a connexin, e.g., Cx43 or Cx45, e.g., a portion of the extracellular loop 2 of Cx43 and a portion of the extracellular loop 2 of Cx45, comprising a portion of the extracellular loop 2 of the connexin.

[0048] In another embodiment, the present invention provides the use of hemichannel blockers in the manufacture of pharmaceuticals for use in the treatment of one or more diseases, disorders and conditions described or referred to herein. The pharmaceutical comprises, essentially comprises, or consists of a hemichannel blocker. In one embodiment, the pharmaceutical comprises, essentially comprises, or consists of a peptide hemichannel blocker. In one embodiment, the pharmaceutical comprises, essentially comprises, or consists of a peptide mimetic hemichannel blocker. In one embodiment, the pharmaceutical comprises, essentially comprises, or consists of a small molecule hemichannel blocker. In one embodiment, the pharmaceutical comprises, essentially comprises, or consists of a compound according to Formula I or Formula II of U.S. Patent Application Publication No. 20160177298. In one embodiment, the pharmaceutical comprises, essentially comprises, or consists of Xiflam (Tonaversat). The term "comprising," which is synonymous with "characterized," is comprehensive or open-ended and does not exclude from the pharmaceutical any further unlisted elements or components (or steps, in the case of a method). The phrase "consisting of" excludes any elements, steps or components (or steps, in the case of a method) that are not specified in the pharmaceutical. The phrase "essentially consisting of" refers to the specified materials, as well as materials that do not substantially affect the basic and novel features (or steps, in the case of a method) of the pharmaceutical. The basic and novel features of the present invention are described throughout this specification and include the ability of the pharmaceuticals and methods of the present 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 changes in the basic and novel features of the present invention, including the pharmaceuticals and methods described herein, include unwanted, clinically undesirable, harmful, unfavorable or adverse reductions in hemichannel modulation and / or cytokine attenuation. In one embodiment, the pharmaceutical product comprises, or consists of, a connexin 43 hemichannel blocker, such as a peptide mimetic or a small molecule connexin 43 hemichannel blocker.

[0049] In another embodiment, the present invention provides the use of hemichannel blockers in the manufacture of a pharmaceutical (or package or kit containing one or more pharmaceuticals and / or containers, with or without instructions for use) for the modulation of hemichannels and / or the treatment of any of the diseases, disorders and / or conditions described or mentioned herein. In one embodiment, for example, the present invention provides the use of connexin hemichannel blockers, including, for example, Xiflam and / or its analogues or Peptagon or its analogues, in the manufacture of a pharmaceutical or package or kit for the treatment of a disorder in which hemichannel modulation may be beneficial. In one embodiment, the pharmaceutical is essentially or consists of a connexin 43 hemichannel blocker, for example, a peptide mimetic or a small molecule connexin 43 hemichannel blocker. In one embodiment, a 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 may be provided in lyophilized form.

[0050] In some embodiments, hemichannel blockers may be co-administered or used with cytokine antagonists, such as VEGF antagonists, in the manufacture of pharmaceuticals for the treatment of disorders in which modulation of hemichannels and cytokines is beneficial.

[0051] In some embodiments, hemichannel blockers may be used together with cytokine antagonists, such as VEGF antagonists, in the manufacture of separate or combination pharmaceuticals for the treatment of one or more diseases, disorders and conditions referred to herein.

[0052] In various embodiments, the modulated hemichannels are 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), 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 hemichannel includes one or more of the Cx26, Cx30, Cx32, Cx36, Cx37, Cx40, Cx45, and / or Cx47 proteins. In a particular embodiment, the hemichannel and / or modulated hemichannel includes one or more of Cx37, Cx40, and Cx43. In a particular embodiment, the hemichannel and / or modulated hemichannel includes one or more of Cx30, Cx37, Cx40, Cx43, and Cx45. In some embodiments, the modulated hemichannel may include or exclude any of the connexins described above. In some embodiments, the hemichannel blocker is a blocker of the Cx37 hemichannel, the Cx43 hemichannel, the Cx40 hemichannel, and / or the Cx45 hemichannel. In a particular preferred embodiment, the hemichannel blocker is a connexin 43 hemichannel blocker. The pharmaceutical compositions of the present invention for any of the uses featured herein may also include Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, or Cx57, or any other connexin, or hemichannel blockers capable of inhibiting or blocking connexin hemichannels.In another embodiment, the pharmaceutical compositions for use in the methods and manufactures of the present invention for any of the uses featured herein and for any of the connexins 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 yet 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, composition, kit, or treatment methods of the present invention is a Cx43 hemichannel blocker. Other embodiments include a Cx45 hemichannel blocker, and blockers of hemichannels comprising, essentially, 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 blocker may include or exclude any of the above-mentioned connexins or other connexins mentioned herein. In some embodiments, the connexin hemichannel to be blocked is a heteromeric hemichannel (i.e., a hemichannel containing a mixture of non-identical connexins).

[0054] Another embodiment of this aspect of the present invention provides a pharmaceutical pack containing a VEGF or other cytokine antagonist together with a small molecule or other hemichannel blocker. In one embodiment of this aspect, the pharmaceutical pack contains a VEGF antagonist, which is a VEGF-A antagonist. In another embodiment, the hemichannel blocker and the VEGF antagonist in the pharmaceutical pack are formulated separately and in individual dosages. In yet another embodiment, the hemichannel blocker and the VEGF antagonist in 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 on a subject is evaluated or monitored using a cytokine protein assay. Cytokine protein levels can be quantified by any conventional method that enables the detection and quantification of proteins in a sample derived from the subject, including those described herein.

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

[0057] The activity of hemichannel blockers can be evaluated using certain biological assays. The effects of known or candidate hemichannel blockers on molecular motility can be identified, evaluated, or screened using the methods described in the following examples, 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 transfer experiments, e.g., the transfer of molecules labeled with detectable markers, and 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 compounds to block hemichannels is provided, comprising the step of (a) putting together 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 evaluating hemichannel blockade, wherein the system includes, for example, the introduction of hypoxia or ischemia into the system, an inflammatory mediator, or other compounds or events that induce hemichannel opening, e.g., extracellular Ca 2+ Various embodiments of this aspect of the present invention are described herein, including a method comprising: (b) a step of determining the presence or amount of an increase in, for example, a dye or other labeled metabolite in the system, characterized in that it shows an increased migration in response to a decrease in; and (b) a step of determining the presence or amount of an increase in, for example, a dye or other labeled metabolite in the system. Positive and / or negative controls may also be used. If necessary, a predetermined amount of a hemichannel blocker (e.g., Peptagon or Xiflam) may be added to the test system. As referred to herein, in one embodiment, hemichannel blockers, e.g., Peptagon and Xiflam, exhibit activity of about 1 to less than 5 nM, preferably less than 10 nM, and more preferably less than 50 pM, in an in vitro assay, for example. In an in vivo assay, these compounds exhibit hemichannel blockade preferably at concentrations of less than 10 to 100 micromolar concentrations (μM), more preferably less than 50 μM. Other hemichannel blockers may be within these ranges and also within the range of less than 200 pM. In certain embodiments, for example, the following items are provided: (Item 1) A method for modulating cytokine activity in a subject, comprising the step of administering an effective amount of a hemichannel blocker to the subject. (Item 2) The method according to item 1, wherein the presence or amount of the cytokine is reduced. (Item 3) The method according to item 1, wherein the presence or increase in the amount of the cytokine is inhibited. (Item 4) The method according to item 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 intracellular adhesion molecule-1 (sICAM-1). (Item 5) The method according to item 1, wherein the cytokine is vascular endothelial growth factor. (Item 6) The method according to item 5, wherein the vascular endothelial growth factor is vascular endothelial growth factor A. (Item 7) The method according to item 1, wherein the hemichannel blocker is a connexin 43 hemichannel blocker. (Item 8) The method according to item 1, wherein the hemichannel blocker is a small molecule hemichannel blocker. (Item 9) The method according to item 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) The method according to item 1, wherein the hemichannel blocker is a connexin peptide mimetic. (Item 11) The method according to item 10, wherein the hemichannel blocker is VDCFLSRPTEKT (SEQ ID NO: 1). (Item 12) The method according to item 10, wherein the hemichannel blocker is essentially derived from SRPTEKT (SEQ ID NO: 2). (Item 13) The method according to item 10, wherein the hemichannel blocker is selected from the group consisting of peptides essentially comprising 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) The method according to item 1, further comprising the step of administering a VEGF antagonist or a VEGF receptor antagonist. (Item 15) The method according to item 14, wherein the VEGF is VEGF-A. (Item 16) The method according to item 1, further comprising the step of administering an IL-6 antagonist or an IL-6 receptor antagonist. (Item 17) The method according to item 1, further comprising the step of administering one or more IL-8 antagonists, MCP-1 antagonists, or sICAM-1 antagonists. (Item 18) The method according to item 15, wherein the hemichannel blocker is a connexin 43 hemichannel blocker. (Item 19) The method described in item 18, wherein angiogenesis is reduced or attenuated. (Item 20) The method according to item 1, wherein the hemichannel blocker is administered by injection. (Item 21) The method according to item 1, wherein the hemichannel blocker is administered orally. (Item 22) The method according to item 1, wherein the hemichannel blocker is administered via PRN, or according to a predetermined schedule, or both. (Item 23) The method described in item 1, wherein the subject is a human. (Item 24) The method according to item 10, wherein the hemichannel blocker is a modified peptide mimetic. (Item 25) The modification described above is the method described in item 25, which includes C12-C12-VDCFLSRPTEKT (sequence number 171). (Item 26) The method according to item 1, wherein the subject has pathological, abnormal, unwanted, or undesirable amounts of cytokine activity. [Brief explanation of the drawing]

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

[0059] [Figure 2]Figure 2 shows VEGF secretion in response to HG, cytokines, and co-administration of HG and cytokines, after treatment with a hemichannel blocker (Peptagon) and ATP supplementation, under basal conditions. Co-administration of HG and cytokines induced VEGF release (p ≤ 0.0001), but Peptagon treatment restored VEGF secretion to basal levels. Supplementation of exogenous ATP neutralized 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=24h; ns=not significant; **p ≤ 0.01; ****p ≤ 0.0001.

[0060] [Figure 3] Figure 3 shows the Peptagon-mediated reduction in IL-6, IL-8, sICAM-1, and MCP-1 expression after co-administration of HG and cytokines. Co-administration 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 hemichannel blockers (Peptagon) reduced IL-6, IL-8, and MCP-1 levels 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-administration of high glucose and cytokines (p<0.0001 in all cases). N=3; t=24h.

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

[0062] [Figure 5] Figure 5 shows that ATP reversed the hemichannel blocker-mediated reduction in the expression of IL-6, MCP-1, and IL-8, rather than sICAM-1. Co-administration of HG and cytokines induced IL-6, IL-8, sICAM-1, and MCP-1 release by ARPE-19 cells, while treatment with 25 μM connexin peptide mimetic (Peptagon) hemichannel blockers reduced cytokine secretion. Extracellular ATP (10 nM) reversed Peptagon-mediated protection against IL-6, MCP-1, and IL-8 release, rather than 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=24h; 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 in the cytoplasm, but upon inflammasome activation by high glucose and inflammatory cytokines, oligomerization enriched multiple NLRP3 copies within the inflammasome complex, which were visualized as small spots (arrows, A). Addition of the connexin peptide mimetic hemichannel blocker (Peptagon) blocked inflammasome assembly, resulting in only a few complex spots (B). High nuclear background labeling using this antibody was present under all conditions. Addition of exogenous 10 nM ATP reversed the hemichannel blocker treatment, and the inflammasome complex was re-formed in the cytoplasm (C). Results are expressed as mean ± SD; N=3; t=24h; scale bar=50 μm. [Modes for carrying out the invention]

[0064] Detailed explanation definition A “small molecule” is defined herein as having a molecular weight of approximately 600 to less than 900 daltons and is generally an organic compound. A small molecule may 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 its grammatical variations, e.g., “to treat” or “to treat”) refers to a clinical intervention that alters the natural course of the individual, tissue, or cell being treated, and may be performed either preventively or between clinicopathological conditions. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, disorder, or condition; reducing signs or symptoms; reducing any direct or indirect pathological consequences of the disease; slowing the rate of disease progression; achieving remission or mitigation of a disease state; and achieving a spleen or improved prognosis. In some embodiments, the compounds, methods, and compositions of the present invention may 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 the subject is treated until complete recovery. Accordingly, “treatment” includes reducing, mitigating, or relieving 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 may also include maintaining or promoting a state of complete or partial spleen of a condition. When used herein, “treatment” includes reducing, mitigating, or relieving cytokine levels or activity in a subject, such as IL-6, IL-8, MCP-1, and sICAM-1 levels and / or activity, as well as reducing the presence or amount of the angiogenic cytokine VEGF, after administration of a hemichannel blocker.

[0066] Terms such as “treating cytokine disorders,” including diseases and conditions, may refer to preventing, slowing, reducing, decreasing, stopping, and / or reversing the levels or activity of disorders, diseases or conditions, and / or cytokines, including, for example, IL-6, IL-8, sICAM-1, and MCP-1, and / or VEGF.

[0067] The term "prevent" means to prevent, either entirely or partially, or to achieve remission or control.

[0068] As used herein, “effective amount” means an effective amount in the dosage and duration required to achieve a desired therapeutic or preventive outcome. For example, “effective amount” may mean, but is not limited to, an amount of a compound or composition disclosed herein that can treat signs and / or symptoms of a disease, disorder or condition involving cytokines, or an amount of a hemichannel compound or composition that can favorably modulate the production, secretion and / or release of cytokines, such as IL-6, IL-8, sICAM-1 and MCP-1 and / or VEGF.

[0069] When used herein, the “therapeutic effective dose” of the substance / molecule, agonist, or antagonist of the present invention may 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. The therapeutic effective dose is preferably also the amount in which the therapeutically beneficial effects of the substance / molecule, agonist, or antagonist outweigh any toxic or adverse effects. A therapeutic effective dose of a hemichannel blocker reduces or inhibits an increase in cytokine levels or activity in a subject. A therapeutic effective dose of a hemichannel blocker modulates cytokine levels or activity in a subject.

[0070] As used herein, “prophylactic effective dose” refers to the effective amount of medication needed in the dosage and duration required to achieve the desired prophylactic outcome. Typically, but not always, the prophylactic effective dose is less than the therapeutic effective dose, as prophylactic doses are used in a subject before or in the early stages of a disease, disorder, or condition.

[0071] The term "pharmaceutical preparation" refers to a preparation that contains an active ingredient, such as a hemichannel blocker, in a form that enables its biological activity, and that does not contain any further components that are unacceptably toxic to the person to whom the preparation is administered.

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

[0073] Where used herein, terms such as “subject,” including “individual” and “patient,” are all interchangeable herein and refer to humans, domesticated animals and livestock, as well as any mammals, including zoo animals, wildlife park animals, sports animals or pet animals, such as dogs, horses, cats, sheep, pigs, and cattle. Preferred mammals are humans, including adults, children and the elderly. Preferred sports animals are horses and dogs. Preferred pet animals are dogs and cats. Subjects may be, for example, aquarium animals, such as dolphins, whales, sea lions or walruses. In certain embodiments, subject, individual or patient is human.

[0074] As used herein, the term “hemichannel” is a part of a gap junction (where 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 homomeric or heteromeric, connexin protein hexamers, that form a pore for the gap junction between the cytoplasm of two adjacent cells. A hemichannel is supplied to one side of the junction by one cell, and usually two hemichannels from the opposing cell come together to form a complete intercellular hemichannel. However, in some cells, and in cells under certain circumstances, the hemichannel itself is active as a conduit between the cytoplasm and the extracellular space, allowing for the movement of ions and small molecules.

[0075] Compounds of formula I, such as Xiflam and / or analogs or prodrugs of any of the compounds described above, can modulate the function and / or activity of hemichannels, preferably hemichannels comprising any type of connexin protein. Thus, references to “hemichannels” should be interpreted to broadly include hemichannels comprising, essentially comprising, or consisting of any one or more of several different connexin proteins, unless the context requires otherwise. However, as an example, a hemichannel may comprise one or more of the connexin proteins 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. In one embodiment, the hemichannel comprises one of the connexins described above. In one embodiment, the hemichannel comprises one or more 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 connexins 30, 37, and 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 comprises 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 a hemichannel blocker of any connexin, including those described above.

[0076] Hemichannels and hemichannels can be present in any type of cell. Therefore, a reference to or to "hemichannels" should be interpreted as including hemichannels or hemichannels present in any cell type unless the context requires otherwise. In one embodiment, hemichannels or hemichannels are present in cells in organs or in cancer or tumors. 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, “hemichannel modulation” refers to one or more functions and / or activities of hemichannels, typically the modulation of intercellular molecular flow mediated by hemichannels. Such functions and activities include, for example, the flow of molecules from extracellular space or the environment into the cell via hemichannels, and / or the flow of molecules from intracellular space or the environment into extracellular space or the environment via hemichannels. Compounds useful for hemichannel modulation may be called “hemichannel modulators.”

[0078] Modulation of hemichannel function can be performed by any means. However, only as an example, modulation can be performed by one or more of the following: inducing or promoting hemichannel closure; preventing, blocking, inhibiting or reducing hemichannel opening; or inducing, inducing or promoting the intracellular 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 as implying complete blockage, inhibition, prevention or antagonism, but may not be, and should be interpreted as including partial blockage, inhibition, prevention or antagonism that at least reduces the function or activity of hemichannels and / or hemichannels. Similarly, “inducing” or “promoting” should not be interpreted as implying complete internalization of hemichannels (or groups of hemichannels), but should be interpreted as including partial internalization that at least reduces the function or activity of hemichannels.

[0079] As used herein, the term “hemichannel blocker” refers to a compound that obstructs the passage of molecules through connexin hemichannels. Hemichannel blockers may block or reduce hemichannel opening, block or reduce the release of molecules into the extracellular space through hemichannels, and / or block or reduce the entry of molecules into the intracellular space through hemichannels. 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 probability of hemichannel opening. Probability of opening is a measure of the percentage of time a channel remains open compared to the closed state (as outlined 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 It contains antibody fragments. Hemichannel blockers contain 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 whole or partially by pathological, abnormal, or otherwise unwanted or undesirable cytokine activity, including in diseases, disorders, or conditions characterized whole or partially by angiogenesis and / or vascular leakage. Compounds useful for cytokine modulation may be called “cytokine modulators.” The compounds of the present invention may be used in methods for modulating cytokine activity, including in methods for treating diseases, disorders or conditions characterized whole or partially by pathological, abnormal, or otherwise unwanted or undesirable cytokine activity, where the cytokine activity is modulated, for example, by reducing, decreasing, leveling and / or smoothing. Leveling or smoothing of cytokine activity includes leveling and / or inhibiting a substantial increase in the presence or amount of cytokine or cytokine activity.

[0081] The inflammasome is a multiprotein complex comprising caspase 1, PYCARD, NALP, and optionally caspase 5 (also known as caspase 11 or ICH-3). The precise composition of the inflammasome depends on the activator that initiates inflammasome assembly. The inflammasome promotes 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 activation of the inflammasome pathway. Target inflammasomes for hemichannel blockers include the NLRP3 inflammasome.

[0082] The terms “peptide,” “peptide mime,” and “mimicking” include synthetic or genetically modified chemical compounds that may have substantially the same structural and functional characteristics as the protein region they mimic. In the case of connexin hemichannels, for example, these may mimic the extracellular loop of hemichannel connexin.

[0083] As used herein, the term "peptide analog" refers to a compound having properties similar to those of a template peptide, and may be a non-peptide drug. "Peptide mimes" (peptide mimes) include peptides and peptide-based compounds. (also known as peptide mimetics) such non-peptide-based compounds, e.g. For example, peptide analogs are also included. Peptide mimes that are structurally similar to therapeutically useful peptides may be used to produce equivalent or enhanced therapeutic or prophylactic effects. Peptides and peptide mimes may be modified or unmodified in some embodiments. Generally, peptide mimes are structural or functional mimes (e.g., identical or similar) to a reference polypeptide (i.e., a polypeptide having a biological or pharmacological function or activity), but may also have one or more peptide linkages that are optionally replaced by linkages selected from the group consisting of, for example, -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. Mimes may be composed entirely of natural amino acids, synthetic chemical compounds, or non-natural analogs of amino acids, or they may be chimeric molecules of a natural peptide amino acid and a partially non-natural analog of an amino acid. Mimes may also include any amount of conservative substitution of natural amino acids, provided that such substitutions do not substantially alter the mime activity. In the case of connexin hemichannels, these can, for example, mimic the extracellular hemichannel loops involved in connexon-connexon docking and cell-cell channel formation. Peptide mimes include those described herein, as well as those that may be known in the art, whether currently known or to be developed later. Peptides and peptide-mimicking hemichannel blockers may also be modified to increase stability, improve bioavailability, and / or increase cell membrane permeability.

[0084] This patent describes a novel method for modulating cytokines, including IL-6, IL-8, sICAM-1, and MCP-1, as well as the angiogenic cytokine VEGF. The presence or amount of one or more of these cytokines is elevated, abnormal, dysregulated, impaired, or otherwise unnecessary or undesirable in certain 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 reduction in the magnitude of the biological activity of a protein. In certain embodiments, the presence of an antagonist blocks or weakens the biological activity of a protein, i.e., results in its complete or partial inhibition. In certain descriptions, an antagonist may be called an inhibitor or modulator. Thus, a cytokine “antagonist” refers to a compound (single or multiple) that whole or partially inhibits the activity or function of a cytokine, e.g., the activity or function of VEGF, preferably angiogenesis. A cytokine “receptor antagonist” means a compound (single or multiple) that whole or partially inhibits the activation or function of a cytokine receptor, e.g., a VEGF receptor.

[0086] Hemichannel blockers, including small peptide and small molecule blockers, are used to treat diseases, disorders, or conditions described herein, including diseases, disorders, or conditions characterized by angiogenesis and / or chronic inflammation.

[0087] Where described herein, the use of hemichannel blockers was evaluated in cell lines exposed to various mediators of inflammation, pro-inflammatory cytokines IL-1β and TNF-α, and combinations of IL-1β and TNF-α with high glucose, which synergistically increases inflammatory cytokine release. Analysis 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, in combination with added glucose that increases it. Application of hemichannel blockers reduced cytokine release and restored normal gap junction pattern formation. Exogenous ATP reversed the hemichannel blocker protection, confirming that the cytokine effect is connexin hemichannel-mediated.

[0088] This invention relates to the modulation of cytokine production, secretion, and / or release, including the modulation of VEGF levels or activity. As mentioned, hemichannel blockers include, for example, small peptide mimes and small molecule blockers. Hemichannel blockers may be used alone or in combination with other agents, 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, decreasing, stopping, and / or reversing the production, secretion, and / or release of cytokines including VEGF and IL-6, IL-8, MCP-1, and sICAM-1. Subjects with cytokine disorders are treated with a therapeutically effective dose of a hemichannel blocker.

[0090] The present invention provides, in particular, a method for modulating cytokine production, secretion, and / or release by administering a hemichannel blocker, such as Peptagon and / or its analogues, a compound of formula I, such as Xiflam and / or an analogue or prodrug 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 intend to use the methods of the present invention for use in the treatment of diseases, disorders or conditions described or referred to herein, or in which attenuation of cytokine production, secretion and / or release may be beneficial. Diseases, disorders or conditions include, for example, Wegener's granulomatosis; Castleman disease; renal failure; angina pectoris including unstable angina; multiple sclerosis; muscular dystrophy; secondary multiple sclerosis; lupus nephritis; neovascularization; COPD; PAOD; diabetes mellitus including type 2 (non-insulin-dependent) diabetes mellitus; insulin resistance; diabetic nephropathy; heart failure; solid tumors; brain tumors; cancers including breast cancer, non-small cell lung cancer, and hematological malignancies; hepatocellular carcinoma, soft tissue sarcoma, early breast cancer, and metastatic breast cancer.

[0092] In some embodiments, the present invention features the use of a compound of formula I, e.g., Xiflam and / or any analog or prodrug of any of the compounds described above, to directly and immediately block the Cx43 hemichannel and to induce a concentration- and time-dependent reduction in cytokine production, secretion, and / or release. Connexin

[0093] In various embodiments, the modulated hemichannels include connexin 23 (Cx23) hemichannel, connexin 25 (Cx25) hemichannel, connexin 26 (Cx26) hemichannel, connexin 30 (Cx30) hemichannel, connexin 30.2 (Cx30.2) hemichannel, connexin 30.3 (Cx30.3) hemichannel, connexin 31 (Cx31) hemichannel, connexin 31.1 (Cx31.1) hemichannel, connexin 31.9 (Cx31.9) hemichannel, connexin 32 (Cx32) hemichannel, and connexin 31.9. These are the connexin 36 (Cx36) hemichannel, connexin 37 (Cx37) hemichannel, connexin 40 (Cx40) hemichannel, connexin 40.1 (Cx40.1) hemichannel, connexin 43 (Cx43) hemichannel, connexin 45 (Cx45) hemichannel, connexin 46 (Cx46) hemichannel, connexin 47 (Cx47) hemichannel, connexin 50 (Cx50) hemichannel, connexin 57 (Cx57) hemichannel, connexin 59 (Cx59) hemichannel, and connexin 62 (Cx62) hemichannel. In one embodiment, the modulated hemichannel includes one or more of the Cx26, Cx30, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and / or Cx47 proteins. In a particular embodiment, the hemichannel and / or modulated hemichannel is the Cx37 and / or Cx40 and / or Cx43 hemichannel. In a particular embodiment, the hemichannel and / or modulated hemichannel is the 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 embodiments, the hemichannel blocker is a blocker of the Cx43 hemichannel, the Cx40 hemichannel, and / or the Cx45 hemichannel. In a particular preferred embodiment, the hemichannel blocker is a connexin 43 blocker.The pharmaceutical compositions of the present invention for any of the uses featured herein may also include hemichannel blockers that can inhibit or block Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx45, Cx50, or Cx57 hemichannels, or any other connexin hemichannels (including homogeneous and heterogeneous hemichannels). In another embodiment, the pharmaceutical compositions for the uses featured herein and for use in the methods and manufactures of the present invention for any of the connexin uses 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 above-described connexin hemichannels, or may be heteromeric hemichannels.

[0094] In one embodiment, the hemichannel blocker used in any of the administration, co-administration, composition, kit, or treatment method 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, essentially, or consisting of Cx26, Cx31.1, Cx36, Cx50 and / or Cx57 hemichannels, or any other connexins mentioned above or herein. Some embodiments may include or exclude any of the connexins or hemichannels described above, or any others mentioned herein. Hemichannel blockers

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

[0096] Examples of hemichannel blockers include small molecule hemichannel blockers (e.g., Xiflam (Tonaversat)). In some embodiments, the hemichannel blocker is a small molecule other than Xiflam, e.g., the hemichannel blocker described by formula I in U.S. Patent Application Publication No. 20160177298 filed in the name of Colin Green et al., the disclosure of which, as mentioned above, is incorporated herein by this reference in its entirety. Various preferred embodiments involve the use of small molecules that block or improve hemichannel opening or otherwise antagonistize or inhibit it in order to treat the diseases, disorders and conditions described or mentioned 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 present invention features the use of small molecule hemichannel blockers, for example, compounds of formula I, such as Xiflam and / or analogs or prodrugs of any of the compounds described above, for blocking Cx43 hemichannels and causing a concentration- and time-dependent reduction in cytokine production, secretion, and / or release.

[0098] For example, the hemichannel blocker Xiflam may be known by the IUPAC names 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 analog or prodrug is formula I: [ka] [In the formula, Y is C-R1; R1 is acetyl; R2 is hydrogen, C 3~8 cycloalkyl, C interrupted by oxygen or substituted by hydroxy as required 1~6 alkyl, C 1~6 alkoxy or substituted aminocarbonyl, C 1~6 alkylcarbonyl, C 1~6 alkoxycarbonyl, C 1~6 alkylcarbonyloxy, C 1~6 alkoxy, nitro, cyano, halo, trifluoromethyl or CF3S; or the group CF3-A- (where A is -CF2-, -CO-, -CH2-, CH(OH), SO2, SO, CH2-O- or CONH); or the group CF2H-A’- (where A’ is oxygen, sulfur, SO, SO2, CF2 or CFH); trifluoromethoxy, C 1~6 alkylsulfinyl, perfluoro C 2~6 alkylsulfonyl, C 1~6 alkylsulfonyl, C 1~6 alkoxysulfinyl, C 1~6 alkoxysulfonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, phosphono, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulfinyl, heteroarylsulfinyl, arylsulfonyl or heteroarylsulfonyl (where any aromatic moiety is substituted as required), C 1~6 alkylcarbonylamino, C 1~6 alkoxycarbonylamino, C 1~6 alkyl-thiocarbonyl, C 1~6 alkoxy-thiocarbonyl, C 1~6 alkyl-thiocarbonyloxy, 1-mercapto C 2~7 alkyl, formyl, or aminosulfinyl, aminosulfonyl or aminocarbonyl (where any amino moiety is substituted by one or two C 1~6 alkyl groups as required), or C 1~6 alkylsulfinylamino, C 1~6Alkylsulfonylamino, C 1~6 Alkoxysulfinylamino or C 1~6 Alkoxysulfonylamino, or C 1~6 Ethyleneyl or -C(C) terminally substituted with alkylcarbonyl, nitro or cyano. 1~6 Alkyl)NOH or -C(C 1~6 Alkyl)NNH2; or one or two C 1~6 By alkyl or C 2~7 It is an amino that is optionally substituted with an alkanoyl; one of R3 and R4 is hydrogen or C 1~4 It is alkyl, and the other is C 1~4 Alkyl, CF3, or CH2X a It is fluoro, chloro, bromo, iodine, C 1~4 Alkoxy, hydroxy, C 1~4 Alkylcarbonyloxy, -SC 1~4 Alkyl, nitro, one or two C 1~4 Amino, cyano, or C atoms are substituted as needed with alkyl groups. 1~4 It is an alkoxycarbonyl; or R3 and R4 together are C 1~4 C is substituted with alkyl as needed. 2~5 It is a polymethylene compound; R5 is C 1~6 Alkylcarbonyloxy, benzoyloxy, ONO2, benzyloxy, phenyloxy or C 1~6 It is an alkoxy, and R6 and R9 are hydrogen, or R5 is hydroxyl and R6 is hydrogen or C 1~2 It is an alkyl group, and R9 is hydrogen; R7 is heteroaryl or phenyl, and together they are chloro, fluoro, bromo, iodine, nitro, and C. 1~4 Amino, cyano, azide, and C atoms that are substituted once or twice as needed with alkyl groups. 1~4 One or more groups or atoms are independently substituted as needed, selected from alkoxy, trifluoromethoxy, and trifluoromethyl; R8 is hydrogen, C 1~6 Alkyl, OR 11 or NHCOR 10 And in the formula, R 11 is hydrogen, C 1~6 Alkyl, formyl, C 1~6 Alkanoyl, aroyl, or aryl-C 1~6 It is alkyl, R 10 is hydrogen, C 1~6 Alkyl, C 1~6 Alkoxy, mono, or diC 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 - is alkyl, aryl, or heteroaryl; the R8-N-CO-R7 group is cis relative to the R5 group; X is oxygen or NR 12 And in the formula, R 12 is hydrogen or C 1~6 It is alkyl. It is selected from the group of compounds having the following properties.

[0100] For any of the Markush groups shown above, that group may include or exclude any of the species listed for that group. The hemichannel blockers for use in the methods of the present 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 analogues 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 analogues 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 these are incorporated by reference as a whole, as mentioned above. The hemichannel blockers for use in the methods of the present invention may include or exclude any of these compounds. Peptides and peptide-mimicking hemichannel blockers

[0104] In other embodiments, the present invention features the use of peptide hemichannel blockers, such as peptide mimetic compounds, e.g., Peptagon, to block connexin hemichannels and to induce concentration- and time-dependent reductions in cytokine production, secretion, and / or release. Hemichannel blockers may include peptides corresponding to specific sequences in 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 peptide mimetic is Peptagon (VDCFLSRPTEKT) (SEQ ID NO: 1). Preferred peptide mimetic compounds include SRPTEKT, which is a 7-mer motif.

[0105] In some embodiments, the peptide and / or peptide-mimicking 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 peptide mimetic may be modified or unmodified. The connexin hemichannel-blocking peptide or peptide mimetic may be produced chemically, by synthesis, or by other means. In some embodiments, the connexin hemichannel-blocking peptide or peptide mimetic is a Cx43 peptide or peptide mimetic. In some embodiments, the therapeutically effective modified or unmodified peptide or peptide mimetic comprises a portion of the connexin extracellular loop 2, including a portion of the extracellular or transmembrane domain of connexin, e.g., a portion of the Cx43 extracellular loop 2 and a portion of the Cx45 extracellular loop 2. In some embodiments, the peptide or peptide mimetic comprises a portion of the extracellular or transmembrane domain of connexin Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx50, Cx57, or another connexin referred to herein. A peptide mimetic corresponding to a portion of the extracellular loop 2 of Cx43 is preferred in the present invention.

[0106] Peptagon is a hemichannel blocker that can act in a dose-dependent manner; lower doses block gap junction hemichannel opening, while higher doses uncouple intercellular gap junctions. See, for example, O'Carroll et al., 2008. This refers to the fact that, with sustained low-dose application, there is also a gradual loss of gap junction coupling, which is considered peptide interference of hemichannel docking (parallel to the gradual removal of existing gap junctions during normal metabolic turnover). Peptagon has been proven effective in several in vitro, ex vivo, and in vivo (animal) studies (e.g., Davidson et al, 2012; Danesh-Meyer et al, (See 2012; O'Carroll et al, 2013).

[0107] In some embodiments, the hemichannel blocker, e.g., the Cx43 hemichannel blocker, may comprise a peptide. The hemichannel blocker peptide sequence may comprise, be essentially derived from, or be derived from, 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 carboxyl terminus may be modified. In some embodiments, the carboxyl terminus modification may comprise an n-alkyl chain, which may be further linked to hydrogen or other parts as needed. In some embodiments, the hemichannel blocker peptide may comprise or exclude any of the peptides listed above or disclosed herein.

[0108] In one embodiment, the present invention relates to the use of pharmaceutical compositions, alone or in kits, packages or other manufactured articles, in methods for treating diseases, disorders or conditions referred to herein, and diseases, disorders or conditions characterized by increased or impaired or otherwise unwanted cytokines or angiogenesis, including IL-6, IL-8, sICAM-1 and MCP-1 and the angiogenic cytokine VEGF. The methods herein provide treatment of a target with a sufficient amount of a hemichannel blocker 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. Other connexin hemichannel blockers are within the scope of the present invention, as referred to.

[0109] In some embodiments, the "promoiety" masks the functional groups within the active agent. This refers to a species that acts as a protecting group, thereby converting an active drug into a prodrug. Typically, the pro moiety is cleaved in vivo by enzymatic or non-enzymatic means, thereby attaching to the drug via a bond that converts the prodrug into its active form. In some embodiments, the pro moiety can also be an active drug. In some embodiments, the pro moiety can be bound to a hemichannel blocker. In some embodiments, the pro moiety can be bound to, for example, a peptide or peptide mime or a small molecule hemichannel blocker. In some embodiments, the pro moiety can be bound to a compound of formula I. In some embodiments, the prodrug is bound to another hemichannel compound, e.g., Green et al., U.S. Patent Application Publication No. It may be a compound described in Patent 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 embodiments, the hemichannel blocker includes, for example, antibodies or antibody fragments that bind to connexin hemichannels, nanobodies, peptides or peptide mimes, recombinant fusion proteins, aptamers, small molecules or single-chain variable fragments (scFv), and others as referred to 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 peptide mime, sometimes referred to as a hemichannel-blocking peptide or peptide mime, which includes modified or unmodified Cx peptides or peptide mimes that are essentially composed of, or comprise, the connexin extracellular domain, transmembrane region, and connexin carboxy-terminal peptide. In some embodiments, the therapeutically effective modified or unmodified peptide or peptide mime contains 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, a therapeutically effective modified or unmodified peptide or peptide mime is a connexin, e.g., connexin 43 or connexin 45, preferably a portion of the E2 extracellular domain (extracellular loop 2) of connexin 43. In some embodiments, a therapeutically effective modified or unmodified peptide or peptide mime is a connexin, e.g., connexin 43 or connexin 45, preferably a portion of the C-terminal domain of connexin 43. If the peptide or peptide mimetic blocker is a portion of the intracellular domain of connexin, the peptide may, in some embodiments, be conjugated to an intracellular transporter, and in some examples, block close-zone (ZO-1) binding to connexin 43. [Table 1]

[0113] Table 2 shows sequences of E2 domains of different connexin isotypes that have amino acids homologous to peptide sequence numbers 14 and 15, which are shown in bold. Note that the last four amino acids of peptide sequence number 15 are part of the fourth membrane domain.

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

[0115] Other peptide hemichannel blockers are derived from the cytoplasmic loop (amino acids 119-144) L2 peptide of connexin 43 and the sub-part of the L2 peptide of connexin 43. In some embodiments, these peptides are, for example, the 9-amino acid sequence of Gap19, KQIEIKKFK (SEQ ID NO: 19); the native Gap19 sequence, DGVNVEMHLKQIEIKKFKYGIEEHGK (SEQ ID NO: 20); Shibayama (Shibayama, J. Gap19 reported by 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 sub-part PTAPLSPMSPP (SEQ ID NO: 24); C-terminal sequences of CT9 or CT10 peptides with or without a TAT reader sequence to increase cell permeability, including or excluding RPRDDEI (SEQ ID NO: 25), SRPRDLEI (SEQ ID NO: 26), YGRKKRRQRRRSRPRDDEI (SEQ ID NO: 27), or YGRKKRRQRRRRPRDDEI (SEQ ID NO: 28). Other peptide mime sequences that may be included in or excluded from compositions for use in methods, kits, or manufactured articles disclosed herein include Dhein (Dhein, S., Naunyn-Schmiedeberg's As reported by 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 This is 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 a D-form peptide for enhanced efficacy beyond that of the native L-form 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 blockers that may be included in or excluded in certain embodiments of this disclosure are provided in Table 3 below (E2 and T2 refer, for example, to the position of the peptide in the second extracellular domain or the second transmembrane domain). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0117] In some embodiments, the connexin 43 blocker may include, for example, a peptide or peptide mime that is essentially or consists of, for example, SEQ ID NO: 2 (SRPTEKT). The peptide or peptide mime may also include, for example, SEQ ID NO: 1 (VDCFLSRPTEKT). The peptide may contain one or more modified amino acids, amino acid analogs, or be otherwise modified to improve bioavailability or to increase permeability across the cell membrane. For example, SEQ ID NO: 1 may be modified to obtain SEQ ID NOs: 20-25 and 27. In some embodiments, the peptide or peptide mime that includes, for example, SEQ ID NO: 2 (SRPTEKT) or SEQ ID NO: 2 (VDCFLSRPTEKT), essentially or consists of, for example, SEQ ID NO: 2 (SRPTEKT) or SEQ ID NO: 2 (VDCFLSRPTEKT) contains 7-40 amino acids or amino acid analogs and does not contain a C-terminal peptide. In some embodiments, the peptide may also be used as a pro-molecule.

[0118] In some embodiments, a connexin 45 blocker may be a peptide or peptide mime that contains, is essentially derived from, or comprises a portion of the connexin 45 protein that antagonistizes, inhibits, or blocks the connexin-connexin interaction. Exemplary peptide sequences of connexin 45 peptides and peptide mimetic blockers are provided in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

[0119] In some embodiments, the connexin 45 blocker may include, for example, a peptide or peptide mime that contains, is essentially, or consists of a portion of the E2 or C-terminal domain of connexin 45, such as, for example, SEQ ID NO: 150 (SRPTEKT). The peptide or peptide mime may also include, for example, SEQ ID NO: 149 (DCFISRPTEKT). In some embodiments, the peptide may be as few as three amino acids, including SRL, PCH, LCP, CHP, IYY, SKF, QPC, VCY, APL, and HVR, or longer.

[0120] In some embodiments, the connexin 40 hemichannel blocker may be a peptide or peptide mime that includes, is essentially derived from, or consists of a portion of the connexin 40 protein. In some embodiments, the connexin 43 blocker may include, be essentially derived from, 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 peptide may contain one or more modified amino acids, amino acid analogs, or be otherwise modified, for example, by being conjugated to or bound to an intracellular transporter.

[0121] In another non-limiting but preferred embodiment, the hemichannel blocker includes a peptide that is essentially derived from or comprises a connexin, for example, Cx43 or Cx45, or a portion of the transmembrane region of Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx50, or Cx57. In a particular non-limiting embodiment, the anti-connexin compound is a peptide having an amino acid sequence containing about 3 to about 30 consecutive amino acids of the connexin 43 or 45 protein sequence, a peptide having an amino acid sequence containing about 5 to about 20 consecutive amino acids of the connexin protein sequence, a peptide having an amino acid sequence containing about 8 to about 15 consecutive amino acids of the connexin protein sequence, or a peptide having an amino acid sequence containing about 11, 12, or 13 consecutive amino acids of the connexin protein sequence. Other non-limiting embodiments include anticonnexin compounds which 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 the connexin protein sequence. In some embodiments, the hemichannel blocker may include or exclude any of the above.

[0122] In other anti-connexin compounds, the mimetic peptide is based on the extracellular domain of connexin 43, corresponding to the amino acids at positions 37-76 and 178-208 of the connexin 43 protein sequence. Therefore, certain peptides described herein have amino acid sequences corresponding to the regions at positions 37-76 and 178-208 of the connexin 43 protein sequence. The peptide does not need to have the same amino acid sequence as these portions of the connexin 43 protein sequence; conserved amino acid changes may be made so that the peptide retains binding or functional activity in assays described herein and other known assays in the art. In other embodiments, the mimetic peptide is based on peptide target regions within the connexin protein other than the extracellular domain (e.g., portions of the connexin 43 protein sequence that do not correspond to positions 37-76 and 178-208).

[0123] In non-limiting but preferred embodiments, the hemichannel blocker includes, includes, or comprises a peptide that comprises, or comprises an amino acid sequence corresponding to a portion of the transmembrane region or the C-terminal region of connexin 45. In certain non-limiting embodiments, for example, the anticonnexin 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 an anticonnexin compound that is a peptide 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 the amino acids at positions 46-75 and 199-228 of a known connexin 45 sequence. Therefore, certain peptides described herein have amino acid sequences corresponding to the regions at positions 46-75 and 199-228 of a known connexin 45 sequence. The peptide does not need to have the same amino acid sequence as these portions of the known connexin 45 sequence. Conservative amino acid changes may be made such 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 a known connexin 45 sequence that does not correspond to positions 46-75 and 199-228). WO2006 / 134494, which discloses various connexin sequences, is incorporated in its entirety by reference. In some embodiments, the hemichannel blocker may include or exclude any of the above-described components. Other connexin hemichannel blockers

[0124] Hemichannel blockers including peptides, peptide mimes, antibodies, antibody fragments, etc., such as connexin 43 or 45 blockers, are also suitable hemichannel blockers. Exemplary hemichannel blockers include, without limitation, polypeptides (e.g., antibodies, their binding fragments, and synthetic constructs), and other gap junction blockers, and Gap junction protein phosphorylators may be included. In some embodiments, hemichannel blockers are blockers of Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx50, and Cx57. Hemichannel blockers, e.g., connexin 43 or 45 blockers, include, for example, monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., including Fab, F(ab')2, and Fv fragments); single-chain antibodies; single-chain Fv; and single-chain binding molecules, e.g., those essentially composed of or consisting of binding domains, hinges, CH2, and CH3 domains, etc., which are capable of binding antigenic determinants (i.e., parts of a molecule commonly called epitopes) to contact specific antibodies or other binding molecules. These binding proteins, including antibodies and antibody fragments, may be chimeric, humanized, or otherwise rendered less immunogenic in the target to which they are administered, and may be synthesized, recombinantly produced, or produced in expression libraries. Any binding molecules known or subsequently discovered in the art, e.g., those mentioned herein and / or described in more detail in the art, are envisioned. For example, binding proteins include not only antibodies but also ligands, receptors, peptide mimes, 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 possess desired specificity, including but not limited to binding specificity, and desired affinity. For example, affinity is about 10 4 Higher than or equal to M-1, about 10 6Higher than or equal to M-1, about 10 7 Higher than or equal to M-1, about 10 8 The Ka can be higher than or equal to M-1. Approximately 10 8 Even higher affinity than M-1, for example, about 10 9 M-1, about 10 10 M-1, about 10 11 M-1 and about 10 12 An affinity equal to or higher than that of M-1 is appropriate. The affinity of the binding protein according to the present invention is based on prior art, e.g., Scatchard Using the method described in et al., (1949) Ann. NY Acad. Sci. 51: 660 , can be easily determined.

[0126] Exemplary compounds used to close gap junctions (e.g., phosphorylating the tyrosine and / or serine residues of connexin 43) are reported in U.S. Patents 7,153,822 and 7,250,397. Exemplary peptides and peptide mimes are reported in Green et al., WO2006134494. See also WO2006069181 and WO2003032964. Other examples of agents used to close gap junctions include anticonnexin agents, e.g., anticonnexin polynucleotides (e.g., connexin inhibitors, e.g., alpha-l connexin oligodeoxynucleotides), anticonnexin peptides (e.g., antibodies and antibody-binding fragments) and peptide mimes (e.g., alpha-l anticonnexin peptides or peptide mimes), 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 ZO-1 binding sites.

[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 peptide mimetic 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 These molecules have potential practical value for preventing gap junction closure. Furthermore, these molecules are potential hemichannel blockers and therefore may have a dual effect, targeting not only the prevention of gap junction closure but also the inhibition of hemichannel opening.

[0128] Anticonnexin agents include peptides having an amino acid sequence containing approximately 5 to 20 consecutive amino acids of the connexin protein, e.g., connexin 43 (SEQ ID NO: 19), peptides having an amino acid sequence containing approximately 8 to 15 consecutive amino acids of connexin 43, or peptides having an amino acid sequence containing approximately 11 to 13 consecutive amino acids of connexin 43. Other anticonnexin agents include peptides having an amino acid sequence containing at least approximately 5, at least approximately 6, at least approximately 7, at least approximately 8, at least approximately 9, at least approximately 10, at least approximately 11, at least approximately 12, at least approximately 13, at least approximately 14, at least approximately 15, at least approximately 20, at least approximately 25, or at least approximately 30 consecutive amino acids of connexin 43. Other anticonnexin 43 blockers include peptides or peptide mimetics containing, essentially, or composed of the extracellular domain of connexin 43, e.g., SRPTEKT or VDCFLSRPTEKT. Other anticonnexin 43 inhibitors 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 peptide of the present invention may be ligated to an internalized transporter at its amino or carboxyl terminus. The internalized transporter ligated to the connexin 43 blocking peptide of the present invention may be any internalized sequence known in the art or newly discovered in the art, or a conserved variant thereof. Non-limiting examples of internalized transporters and sequences include Antennapedia sequences, TAT, HIV-Tat, penetratin, Antp-3A (Antp variant), buforin II, transportan, MAP (model amphiphilic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidium-spermidine-cholesterol), and BGTC (bis-guanidium-tren-cholesterol).

[0130] Other sequences of exemplary intracellularly located 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 blocking peptide may be selected from the group consisting of any peptide sequence numbers listed herein or their conserved variants. In a further embodiment of the present invention, the connexin 43 blocking peptide comprises, essentially comprises, or may comprise the amino acid sequences of Sequence Numbers 30 to 90. In another embodiment of the present invention, the connexin 43 blocking peptide further comprises an intracellular transporter. In a further embodiment, the connexin 43 hemichannel blocking peptide may be ligated to the intracellular transporter at its amino terminus.

[0132] Where a specific protein is referred to herein, derivatives, variants, and fragments are intended. Protein derivatives and variants are well understood by those skilled 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-terminated fusions as well as intrasequence insertions of one or more amino acid residues. Insertions may be smaller than amino or carboxyl-terminated fusion insertions, for example, of about 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final construct. A substitution variant is a variant in which at least one residue is removed and a different residue is inserted in its place. Such substitutions are called conservative substitutions. The replacement of one amino acid residue with another biologically and / or chemically similar amino acid residue is known to those skilled in the art as a conservative substitution. Conservative substitutions may involve replacing one hydrophobic residue with another hydrophobic residue, or replacing one polar residue with another polar residue. Each explicitly disclosed sequence with a conservative substitution falls within the range of peptides provided herein. Conservative substitutions typically have little to no effect on the biological activity of the resulting polypeptide. Conservative substitutions may also be amino acid substitutions in a peptide that substantially do not affect the biological function of the peptide. A peptide may contain one or more amino acid substitutions, two to ten conservative substitutions, two to five conservative substitutions, or four to nine conservative substitutions. Chemical structure modification

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

[0134] In the present invention, hemichannel blockers useful for reducing or stopping cytokine production, secretion, and / or release can also be formulated as microparticles (microspheres, Mp) or nanoparticles (nanospheres, Np), or both. Granular drug delivery systems include nanoparticles (1–1,000 nm) and microparticles (1–1,000 μm), which are further classified as nanospheres and microspheres, as well as nanocapsules and microcapsules. In nanocapsules and microcapsules, drug particles or droplets are encapsulated in a polymeric membrane. Granular systems have the advantage of delivery by injection, and their size and polymer composition significantly influence their biological behavior in vivo. Microspheres can remain in the vitreous humor for considerably longer periods than nanospheres, and therefore microparticles act like derva after injection. Nanoparticles diffuse rapidly and are internalized in tissues and cells.

[0135] Evaluate hemichannel blocking activity. Various methods may be used to evaluate the activity or efficacy of hemichannel blockers. In one aspect 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 may be quantified by any conventional method that allows for the detection and quantification of proteins in a sample derived from the subject. As a non-limiting example, cytokine protein levels may 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 may be labeled or unlabeled. Exemplary examples of markers that may be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There are a wide range of known assays that may be used in the present invention, using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies). These techniques include Western blotting 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 assays based on colloidal precipitation in the form of urine test strips. Other methods for detecting and quantifying cytokines include affinity chromatography techniques and ligand-binding assays. When immunological methods are used, any antibody or reagent known to bind to cytokine proteins with high affinity may be used to detect their quantity. Nevertheless, the use of antibodies, such as polyclonal serum, hybridoma supernatant 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 connection with the present invention.

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

[0137] The activity of hemichannel blockers can also be evaluated using certain biological assays. The effects of known or candidate hemichannel blockers on molecular motility can be identified, evaluated, or screened using the methods described in the following examples, 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 transfer experiments, e.g., the transfer of molecules labeled with detectable markers, and transmembrane passage of small fluorescently transparent tracers, which have been widely used to study the functional state 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 may also be used. See, for example, Danesh-Meyer, HV, et al. See the methods described in 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).

[0138] One method for use in identifying or evaluating the ability of compounds to block hemichannels is (a) a step of assembling 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 evaluating hemichannel blockade, wherein the system is subjected to, for example, the introduction of hypoxia or ischemia into the system, an inflammatory mediator, or other compounds or events that induce hemichannel opening, such as extracellular Ca 2+ (b) a step characterized in that it shows increased migration of, for example, a dye or labeled metabolite in response to a decrease in; (b) a step of determining, for example, 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 necessary, a predetermined amount of a hemichannel blocker (e.g., Peptagon or Xiflam) may be added to the test system.

[0139] Preferably, hemichannel blockers, such as Peptagon and Xiflam, exhibit activity of about 1 to less than 5 nM, preferably less than 10 nM, and more preferably less than 50 pM, in in vitro assays. In in vivo assays, these compounds preferably exhibit hemichannel blockade at concentrations of less than 10 to 100 micromolars (μM), more preferably less than 50 μM. Other hemichannel blockers may fall within these ranges and within a range of less than 200 pM. Co-administration

[0140] The pharmaceutical composition is also provided for co-administration in the form of a combination preparation, for example, a mixture of two or more hemichannel blockers, which may be modified or unmodified, or one or more hemichannel blockers and one or more cytokine antagonists.

[0141] The term “combination preparation” includes “kits of parts” or “manufactured articles” in the sense that the combination partners defined above, whether in pharmaceutical form, dressing / matrix form, or both, can be administered independently or by the use of different fixed combinations of identified amounts of combination partners (a) and (b), i.e., simultaneously, separately, or sequentially. The parts of the kit may then be administered, for example, simultaneously or alternately in a time series, i.e., at different times and at equal or different time intervals for any part of the kit of parts.

[0142] In one embodiment, a combination preparation is administered to a subject, in which case two or more separate blocking agent compositions are administered, wherein the first composition comprises a therapeutically effective amount of a blocking agent, e.g., a hemichannel blocker, e.g., an anticonnexin 43 peptide, a peptide mimetic, or a small molecule hemichannel occluding compound, and the second composition comprises a therapeutically effective amount of a second blocking agent, e.g., an anticytokine agent, e.g., an anti-VEGF antibody.

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

[0144] In one embodiment, a composition comprising, essentially, or consisting of one or more hemichannel blockers is administered within at least about 30 to 60 minutes of one or more cytokine antagonists. This can be administered alone or in combination with cytokines. Hemichannel blocker doses, in combination or separately, may be administered in QD, BID, TID, QID, or weekly doses, e.g., QIW, BIW, QW. These may also be administered in PRN (i.e., as needed) and HS (before sleep, i.e., at bedtime). Dosage form and formulation and administration

[0145] Unless otherwise explicitly stated, all descriptions relating to drug administration apply to the hemichannel blockers of the present invention.

[0146] Hemichannel blockers may be administered, given, or formulated as described herein.

[0147] Hemichannel blockers can be administered to subjects requiring treatment. Accordingly, the present invention provides formulations in which a connexin hemichannel, such as a connexin 43 hemichannel or a connexin 45 hemichannel, can be modulated to reduce its opening probability transiently or in a site-specific manner.

[0148] Hemichannel blockers may be present in the formulation in a substantially isolated form. It is understood that the product may 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 may 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%, of, for example, a peptide mimetic or small molecule hemichannel blocker, or a preparation by dry weight.

[0149] The administration of hemichannel blockers to a subject may be carried out by any means capable of delivering the drug to a target site within the subject's body. For example, hemichannel blockers and / or cytokine antagonists may be administered by one of the following routes: orally, topically, systemically (e.g., intravenously, intra-arterial, intraperitoneally, percutaneously, intranasally, or by suppositories), parenterally (e.g., intramuscularly, subcutaneously, or intravenously or intra-arterially by injection), by implantation, and by infusion via devices, e.g., osmotic pumps, percutaneous patches, etc. Exemplary routes of administration 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 target organ, cancer, or tumor.

[0150] In some embodiments, the hemichannel blocker may be provided as an implant or in conjunction with an implant. In some embodiments, sustained delivery may be provided. In some embodiments, a microneedle, needle, iontophoresis device, or implant may be used for administering the hemichannel blocker. The implant may be made of a dissolvable intervertebral 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, such as connexin 43 hemichannel blockers, may be administered via intraventricular and / or subarachnoid and / or extradural and / or epidural routes.

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

[0152] Hemichannel blockers, such as Peptagon and / or its analogs or prodrugs, compounds of formula I, such as Xiflam and any of the aforementioned compounds, may be administered alone or in combination with one or more further components, and may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients, diluents and / or carriers.

[0153] "Pharmacologically acceptable diluents, carriers, and / or excipients" are intended to include substances that are useful in preparing pharmaceutical compositions, can be co-administered with compounds of formula I, e.g., Xiflam and any analogues of the compounds described above, are generally safe and non-toxic, and are not biologically or otherwise undesirable, while enabling them to perform their intended function. Pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for veterinary and human medicinal use. Suitable carriers and / or excipients will be readily understood by those skilled in the art, taking into account the properties of compounds of formula I, e.g., Xiflam and any analogues of the compounds described above. However, examples of diluents, carriers, and / or excipients include solutions, solvents, dispersions, retarders, polymeric and lipid agents, emulsions, and the like. As further examples, suitable liquid carriers, particularly for injectable solutions, include water, saline solution, and dextrose aqueous solution, with isotonic solutions preferred for intravenous, intraspinal, and intracisional administration, and vehicles, such as liposomes, are also particularly suitable for drug administration.

[0154] The composition may take the form of any standard known dosage form, including tablets, pills, capsules, semi-solids, powders, sustained-release formulations, solutions, suspensions, elixirs, aerosols, injectable solutions, gels, creams, transdermal delivery devices (e.g., transdermal patches), implants (e.g., organ implants, e.g., eyes), or any other suitable composition. Those skilled in the art to which the present invention relates will readily understand, without any excessive experimentation, the most suitable dosage form, considering the condition being treated and the nature of the active agent used. It should be understood that hemichannel blockers, e.g., Peptagon and / or its analogues, compounds of formula I, e.g., Xiflam and any analogue of the compounds described above, and / or one or more cytokine antagonists may be formulated into a single composition. In certain embodiments, preferred dosage forms include injectable solutions and oral formulations.

[0155] A composition useful in the present invention may contain any appropriate level of hemichannel blockers, such as peptagon and / or its analogs, compounds of formula I, such as Xiflam and any of the aforementioned compounds, and / or cytokine antagonists, taking into consideration the dosage form and mode of administration. However, as an example, a composition for use in the present invention may contain approximately 0.1% to approximately 99% by weight, preferably approximately 1% to approximately 60%, of hemichannel blockers, depending on the method of administration.

[0156] In addition to standard diluents, carriers, and / or excipients, compositions according to the present invention may be formulated with one or more further components, or in a manner that enhances the activity or bioavailability of, for example, hemichannel blockers, e.g., Peptagon and / or its analogues, compounds of formula I, e.g., Xiflam and any analogue of any of the compounds described above, and / or cytokine antagonists, preserves their integrity, helps to increase their half-life or shelf life, enables 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. Further examples may 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 relates can identify additives that may be desirable for specific purposes.

[0157] Hemichannel blockers may be administered by sustained-release systems. Suitable examples of sustained-release compositions include semipermeable polymer matrices in the form of molded articles, e.g., films or microcapsules. Sustained-release matrices include polylactic acid (U.S. Patent 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-hydroxybutyrate (EP133,988). Sustained-release compositions also include liposome-encapsulated compounds. Liposomes containing hemichannel blockers can be prepared by known methods, including, for example, those described in DE3,218,121;EP52,322;EP36,676;EP88,046;EP143,949;EP142,641;Japanese Patent Application No. 83-118008;U.S. Patents No. 4,485,045 and No. 4,544,545; and EP102,324. Typically, the liposomes are small (or about 200-800 angstroms) monolayers with a lipid content of cholesterol greater than about 30 mole percent, and the selected proportions are adjusted for the most effective treatment. Slow-release delivery methods may also be used, for example, using PGLA nanoparticles or microparticles or in situ ion-activated gelation systems.

[0158] Furthermore, it is intended that hemichannel blocker pharmaceutical compositions for use according to the present invention may be formulated with additional active ingredients or agents that may, in particular, be therapeutically or otherwise beneficial to the subject. Those skilled in the art to which the present invention relates will understand appropriate additional active ingredients in consideration of the description of the invention herein and the nature of the disorder being treated.

[0159] The composition is, for example, Gennaro AR: Remington: The Science and Practice of Pharmacy, 20 thIt can be formulated according to standard techniques that can be found in standard references such as, ed., Lippincott, Williams & Wilkins, 2000. However, as further examples, the information provided in US2013 / 0281524 or US5948811 may be used.

[0160] In certain embodiments, the present invention provides a product comprising, or in combination thereof, (a) a hemichannel blocker and (b) one or more further active agents, such as cytokine antagonists, wherein components (a) and (b) are adapted for simultaneous or sequential administration.

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

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

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

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

[0165] The present invention includes a method for modulating the function of hemichannels to treat various disorders. The method of the present invention includes the step of administering a hemichannel blocker, either alone or in combination with one or more other agents (e.g., active cytokine antagonists) or therapeutic agents, as desired.

[0166] In another embodiment, a hemichannel blocker, such as a compound of formula I, such as Xiflam, or a peptide or peptide-mimicking hemichannel blocker, may be administered systemically, for example, by intravenous, intra-arterial, or intraperitoneal administration, such that the final circulating concentration is approximately 0.001 to approximately 150 micromoles, or higher concentrations of up to 200, 300, 400, 500, 600, 700, 800, 900, or 1000 micromoles.The final circulation 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 0.4, 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 either of the two enumerated numbers, or any concentration higher than those above and within the ranges mentioned. Where referred to herein, the present invention also includes combination therapies in which one or more further active agents are also administered to the target. 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] The administration of hemichannel blockers, and, if necessary, one or more other active agents, may be performed at any point 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 regularly for an extended period to help manage the progression of symptoms. In another embodiment, the hemichannel blocker is administered regularly for an extended period or for life to prevent the disorder or delay its development.

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

[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, for example, a polymeric matrix, a biodegradable or non-biodegradable matrix, and other carriers useful for fabricating an implant or application structure for delivering the hemichannel blocker. Implants include laser implants and biodegradable matrix implants.

[0170] In some embodiments, a hemichannel blocker, such as connexin 43 hemichannel blocker, may be administered to a subject using, for example, a microneedle, a microneedle array, or a needle to provide a therapeutically effective amount of connexin 43 hemichannel blocker, or an implant may be used for administering the hemichannel blocker. In some embodiments, a microneedle may be used to administer the hemichannel blocker. In some embodiments, the penetration of the microneedle may be controlled to a desired depth within the tissue or organ or organ compartment. In some embodiments, the microneedle may be coated with the hemichannel blocker, either alone or in combination with other drug agents. In some embodiments, the volume of hemichannel blockers and / or drugs administered by microneedles is approximately 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 listed numbers, or any volume between any two of the listed numbers. For example, any suitable formulation of the present invention, including nanoparticles or microparticle formulations, or other formulations that can be injected by microneedles, may be administered by microneedle injection. Connexin hemichannel blockers and combinations with cytokine inhibitors or other drugs

[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 an upstream agonist or receptor of VEGF. In some embodiments, VEGF antagonists include, for example, antagonists that bind to and inhibit VEGF, compounds that inhibit VEGF expression, and / or VEGF inhibitors, or viral vectors encoding proteins or antisense polynucleotides that block or inhibit VEGF or VEGFR. In some embodiments, VEGF antagonists are, for example, antibodies or antibody fragments, nanobodies, peptides or peptide mimes, receptor fragments, recombinant fusion proteins, aptamers, small molecules, or single-stranded variable fragments (scFv). In some embodiments, VEGF antagonist antibodies are, for example, Lucentis® (ranibizumab) and / or Avastin® (bevacizumab).

[0172] In some embodiments, the VEGF antagonist, which binds to VEGF and is therefore antisense against upstream agonists of VEGF species that inhibit it, 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 aspects of the present invention, the VEGF antagonist is, for example, a peptide or peptide mime, 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' It is the 5'-ester sodium salt (SEQ ID NO: 173) of -deoxy-2'-fluoro)C-Am-(2'-deoxy-2'-fluoro)U-(2'-deoxy-2'-fluoro)C-(2'-deoxy-2'-fluoro)C-Gm-(3'→3')-dT), 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)]. AGN-150998 / MP0112 is a small protein that binds to VEGF, and is an anti-VEGF DARPin.

[0174] In some aspects of the present invention, the VEGF antagonist is a recombinant fusion protein such as Aflibercept (Eyelea®) or Convercept. Aflibercept is a recombinant fusion protein consisting of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc region of human IgG1. Convercept 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-stranded 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 virus vector carrying a gene construct for the 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, when combined with adeno-associated virus (AAV), results in AAV2-sFlt01.

[0177] In some embodiments of the invention, the VEGF antagonist is a small molecule, for example, batatinib, cediranib, AL39324, pazopanib, TG100572, or TG100801. Batatinib (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 number 796967-16-3, 1145655-58-8 (as the HCl salt), or 796967-17-4 (as the trifluoroacetate 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 a prodrug version of TG100572 and is 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-benzoate.

[0178] In some embodiments, the hemichannel blocker and the anti-cytokine treatment agent can be co-formulated for co-administration. In some embodiments, the formulations of the hemichannel blocker and the anti-cytokine treatment agent may be part of a formulation based on pills, solutions, gels, pre-filled syringes, tablets, eye drops, or particles.

[0179] When using connexin hemichannel blockers, such as relatively short peptides and peptidomimetics, the methods of the present invention contemplate an initial high dose / rapid start dose (immediate release), followed by a sustained maintenance low dose. For separate or common administration, the formulations can be prepared to provide rapid or slow release; immediate release, delayed release, extended release, or sustained release; or combinations thereof. The formulations can be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, lozenges, troches, capsules, gels, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols (including, but not limited to, eye drops, etc.). Product / kit of combination of connexin hemichannel blocker with cytokine inhibitor or other agent

[0180] In another embodiment of the present invention, a product or “kit” containing materials useful for treating the above-mentioned diseases and disorders is provided. The kit comprises a container essentially consisting of, or comprising, a cytokine inhibitor and a connexin hemichannel blocker. The kit may further include a label or accompanying information sheet on or accompanying the container. The term “accompanying information sheet” is used to refer to instructions for use that are customarily included in the commercial packaging of such therapeutic products, containing information on 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, etc. Containers can be formed from a variety of materials, such as glass or plastic. Containers 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 (for example, the container may be a vial with a stopper that can be pierced by an intravenous solution bag or a subcutaneous injection needle). At least one activator in the composition is a hemichannel blocker. The label or accompanying information indicates that the composition is used to treat a selected condition, such as any of the diseases, disorders and / or conditions described or referenced herein. The label or accompanying information may also indicate that the composition can be used to treat other disorders. Alternatively, or further, the product may further include 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 a hemichannel blocker and, if present, other agents that treat the subjects described herein by acting on a separate mechanism derived from cytokine inhibitors or hemichannel modulation. For example, if the kit includes a first composition comprising, essentially comprising, 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.

[0182] In one embodiment, the kit may further include a third container which is essentially composed of or composed 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 which are desirable from a commercial and user standpoint, such as 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, one or more connexin hemichannel blocking peptides or peptide mimes and / or other anticonnexin agents, such as small molecule hemichannel blocking agents, can be used alone or in combination with any modulating agent in the manufacture of pharmaceuticals or in kits. Suitable hemichannel blocking agents are, for example, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, Cx57 or any other connexin blockers described herein. As described, the kit may contain one or more pharmaceutical compositions in separate containers or in divided containers, together with packaging and instructions for use. The kit may also contain a pharmaceutically acceptable carrier. In some embodiments, the kit may also contain components for administering the pharmaceutical composition, such as syringes, needles, microneedles, mountable implants, or iontophoresis devices. Connexin hemichannel blockers and the treatment partners described herein can be administered independently or in 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 alternately at different points in time and equal or different time intervals for any part of the kit.

[0185] Products comprising essentially the same, or products comprising essentially the same, are also provided, comprising a container containing a hemichannel blocking compound, composition or formulation and instructions for use for use in a treatment of a target. For example, in another embodiment, the present invention comprises essentially the same, or products comprising essentially the same, comprising a container containing, together with instructions for use for a treatment of a target, one or more connexin hemichannel blocking peptides or peptide mimes and / or other hemichannel blocking agents, either alone or in combination with any anti-cytokine agents.

[0186] In some embodiments, the product may include a matrix comprising one or more connexin hemichannel blocking peptides or peptide mimes or other hemichannel blocking agents, either alone or in combination. Suitable connexin hemichannel blocking agents may be, for example, anticonnexin 43 or 45 hemichannel blocking agents. Dosage, volume, and concentration

[0187] As should be understood, the dose, duration of administration, and general administration regimen of hemichannel blockers administered may vary among subjects depending on variables such as the target site to which it is delivered, the severity of any symptoms in the subject to be treated, the type of disorder to be treated, the size of the unit dose, the method of administration chosen, and the subject's age, sex, and / or general health and other factors known to those skilled in the art.

[0188] Examples of effective doses that may be used for the treatment of diseases, disorders, or conditions described herein are provided. In some embodiments, the therapeutically effective dose of a hemichannel blocker, for example, connexin 43 hemichannel blocker, is a concentration of 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 described doses or any number between any two described doses.The dosages are 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 listed dosages or any dose between any two of the listed numbers. In some embodiments, a therapeutically effective amount of hemichannel blocker is present at a concentration in the range of about 0.5 to about 50 mg / mL. In some embodiments, the hemichannel blocker is present at a concentration in the range of 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. In some embodiments, the hemichannel blocker is present at a concentration of about 3.0 mg / mL.

[0189] In some embodiments, the hemichannel blocker may be administered in a therapeutically effective dose of approximately 0.001 to approximately 100 mg / kg, approximately 0.001 to approximately 0.01 mg / kg, approximately 0.01 to approximately 0.1 mg / kg, 0.1 to approximately 1 mg / kg, approximately 1 to approximately 10 mg / kg, or approximately 10 to approximately 100 mg / kg, or in any range between any two of the listed doses or any dose between any two of the listed doses.In some embodiments, the dosages are 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 listed dosages or any dose between any two listed numbers.

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

[0191] As a further example, the unit dose of a hemichannel blocker may be administered once or more times a day (e.g., 1, 2, 3, 4, 5, or 6 times a day, typically 1 to 4 times) such that the total daily dose is approximately 1 to approximately 1000 mg, for example, approximately 1 to approximately 500 mg, or 500 mg to 1000 mg, 1000 to 2000 mg, or 2000 mg to 5000 mg, or any range between any two listed doses or any dose between any two listed doses (for a 70 kg adult). For example, hemichannel blockers such as peptagon and / or their analogues, compounds of formula I, such as Xiflam, and any analogue of any of the above compounds can be administered to a subject in a dose range of approximately 0.01 to approximately 15 mg / kg / day, for example, approximately 0.1 to approximately 6 mg / kg / day, for example, approximately 1 to approximately 6 mg / kg / day, for example, 6 mg / kg / day to 100 mg / kg / day, or any range between any two of the stated dosages, or any dose between any two of the stated dosages. In one embodiment, Xiflam can be administered orally once daily in a dose of approximately 2 mg to approximately 40 mg.

[0192] In one embodiment, the dose of the hemichannel blocker is approximately 0.001 micromolar to 0.1 micromolar at the site of action, 0.1 micromolar to a maximum of approximately 200 micromolar, or higher in circulation to achieve these concentrations at the site of action. For example, doses are (but are not limited to) approximately 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 0.5, 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, 5 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 The final circulating concentration may be 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 concentrations, or any range between any two listed concentrations, or any concentration between any two listed numbers. Further examples of doses expected to block hemichannels rather than non-conjugated gap junctions are described in O'Carroll et al, 2008, incorporated herein by reference. In some embodiments, Xiflam can be used at lower doses, for example, at concentrations of 0.001 to 20 micromoles. 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, 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 concentrations 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 micromolars.

[0193] In one embodiment, the dose of a hemichannel blocker such as Peptagon and / or its analogues ranges from approximately 0.001 micromolar to a maximum of approximately 200 micromolar at the site of action, or from 200 to 2000 or 5000 micromolar concentrations, or higher in circulation to achieve these concentrations at the site of action. For example, the dose may be (but not limited to) a final circulating concentration of approximately 1, 5, 10, 20, 50, 100, 200, 250, 500, 1000, 2000, 3000, 4000, or 5000 micromolar concentrations, or any range between any two of the listed doses, or any dose between any two of the listed doses. The dose of Peptagon effective in blocking hemichannels rather than unconjugated gap junctions is discussed in O'Carroll et al, 2008.

[0194] In some embodiments, Xiflam can be used at lower doses, for example, at concentrations of 1-20 micromoles, 1-50 micromoles, 20-30, 30-40, or 40-50 micromoles. 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. 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. The concentrations 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 micromolars.

[0195] In some embodiments, the preferred therapeutically effective dose of the hemichannel blocker may be at least about 1.0 mg / mL of hemichannel blocker. In 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, the preferred therapeutically effective dose of the hemichannel blocker is approximately 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, 3 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 approximately 100.0 μg / mL, or any range or subrange between any two of the listed doses, or any dose within the range of approximately 0.1 to approximately 100 μg / mL.In some embodiments, the preferred therapeutically effective dose of the hemichannel blocker is approximately 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, 3 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 mg / mL, or any range or subrange between any two of the listed doses, or any dose within the range of about 0.1 to about 100 mg / mL. In some embodiments, the hemichannel blocker is present at a concentration in the range of about 0.5 to about 50 mg / mL. In other embodiments, the hemichannel blocker is present at a concentration in the range of about 0.3 to about 30 mg / mL. In other embodiments, the hemichannel blocker is present at a concentration in the range of about 0.1 or 1.0 to about 10 mg / mL. In other embodiments, the hemichannel blocker is present at a concentration in the range of about 0.1 or 1.0 to about 0.3 or 3.0 mg / mL. In other embodiments, the hemichannel blocker, such as the connexin 43 hemichannel blocker and / or the 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 the connexin 43 or the connexin 45 hemichannel blocker. If the hemichannel blocker is a modified or unmodified peptide or peptide mime, its dose may be reduced by 1 to 10, 25 to 50, 100 to 200, or 1000 times.

[0196] In certain embodiments, a hemichannel blocker, such as a connexin 43 hemichannel blocker, may be administered at and / or adjacent to the treatment site at a final concentration of about 0.001 micromolars (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, for example, up to about 10 mM, 20 mM, or 30 mM, and at any dose and dose range within these numerical values. In one embodiment, the hemichannel blocker composition is applied at a concentration higher 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 anticonnexin 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 to 3 mM to about 5 to 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, and 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 concentrations; 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, 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 The doses may be 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 listed doses, or any dose between any two of the listed numbers.

[0197] Furthermore, a hemichannel blocker, such as a connexin 43 hemichannel blocker, may be present in the formulation at a final concentration of about 1 μM to about 50 μM, or the connexin 43 hemichannel blocker may be present at a final concentration of, for example, about 5 μM to about 20 μM, or about 10 to about 15 μM. In certain other embodiments, the hemichannel blocker may be present at a final concentration of about 10 μM. In yet another embodiment, the hemichannel blocker may be present at a final concentration of about 1 to 15 μM. In other embodiments, the hemichannel blocker is approximately 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, 700-8 00 μM, 800-900 μM, 900-1000 μM, or 1000-1500 μM, or 1500 μM-2000 μM, 2000 μM-3000 μM, 3000 μM-4000 μM, 4000 μM-5000 μM, 5000 μM-6000 μM, 6000 μM-7000 μM, 7000 μM It exists in concentrations of ~8000μM, 8000μM~9000μM, 9000μM~10,000μM, 10,000μM~11,000μM, 11,000μM~12,000μM, 12,000μM~13,000μM, 13,000μM~14,000μM, 14,000μM~15,000μM, 15,000μM~20,000μM, 20,000μM~30,000μM, 30,000μM~50,000μM, or higher, or in any range or subrange between any two listed doses, or in any dose within the range of approximately 20μM to approximately 50,000μM.

[0198] Further dose levels are approximately 1 nanogram (mg) / kg to approximately 1 mg / kg body weight / day of each hemichannel blocker described herein. In certain embodiments, the respective dosages of the compounds of interest are generally in the range of approximately 1 ng to approximately 1 microgram / kg body weight, approximately 1 ng to approximately 0.1 microgram / kg body weight, approximately 1 ng to approximately 10 ng / kg body weight, approximately 10 ng to approximately 0.1 microgram / kg body weight, approximately 0.1 microgram to approximately 1 microgram / kg body weight, approximately 20 ng to approximately 100 ng / kg body weight, approximately 0.001 mg to approximately 0.01 mg / kg body weight, approximately 0.01 mg to approximately 0.1 mg / kg body weight, or approximately 0.1 mg to approximately 1 mg / kg body weight. In certain embodiments, the respective dosages of the compounds of interest are generally in the range of approximately 0.001 mg to approximately 0.01 mg / kg body weight, approximately 0.01 mg to approximately 0.1 mg / kg body weight, or approximately 0.1 mg to approximately 1 mg / kg body weight. When more than one hemichannel blocker is used, the dosage of each hemichannel blocker does not need to be within the same range as the others. For example, the dosage of one connexin hemichannel blocker may be approximately 0.01 mg to approximately 10 mg / kg body weight, while the dosage of another connexin hemichannel blocker may be approximately 0.1 mg to approximately 1 mg / kg body weight, 0.1 to approximately 10, 0.1 to approximately 20, 0.1 to approximately 30, 0.1 to approximately 40, or approximately 0.1 to approximately 50 mg / kg body weight.The dosages are also approximately 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, 5 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 listed doses, or any dose within the range of approximately 0.001 to approximately 100 mg / kg body weight.

[0199] As described above, the dose of a hemichannel blocker, such as a connexin 43 or 45 hemichannel blocker, can be administered by single or divided administration. The dose can be administered once or the application can be repeated. Typically, the application is repeated weekly, every two weeks, every three weeks, monthly, 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 often, as required to prevent, slow or treat any of the diseases, disorders or conditions described herein. If cytokine levels or activity are not desired or increase to undesirable levels, the dose may be repeated and / or increased or decreased. The dose may also be applied every 12 hours to 7 days apart, or longer. For example, the dose may be applied 12 hours, or 1, 2, 3, 4, 5, 6 or 7 days apart, or at any time interval between any two of these times or between 12 hours and 7 days. A connexin 43 hemichannel blocker may be administered, for example, over a period of up to 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 weeks. For some indications, more frequent dosing can be used. Combination product

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

[0201] In the present invention, useful hemichannel blockers can be administered alone or in combination with other therapeutic agents useful for treating a target disease, disorder, or condition. In some embodiments, compounds of formula I, e.g., Xiflam, and / or analogs or prodrugs of any of the above compounds, or peptide mimes such as Peptagon or its analogs or prodrugs, or other hemichannel blockers can be used together with cytokine antagonists for treating disorders in which hemichannel modulation may be beneficial. The administration of the hemichannel blocker may be simultaneous with, after, or before the administration of the cytokine antagonist.

[0202] In certain embodiments, the present invention provides a combination product comprising essentially the hemichannel blocker and (b) one or more further activators 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 the pharmaceutical composition, in which the hemichannel blocker and the further activators 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 present invention, the combination product according to the present invention is used in such a manner that at least one component is administered, but the other components still have an effect on the object being treated. Manufacturing and Purity

[0204] Methods for synthesizing peptides and polypeptides, including antibodies and binding fragments, as well as peptide mimes and peptide analogs, can be carried out using preferred methods. For example, see Lihu Yang et al., Proc. Natl. Acad. Sci. USA, 1; 95(18): 10836-10841 (Sept 1 1998); Harlow and Lane (1988) "Antibodies: A Laboratory Manuel" Cold Spring Harbor Publications, New York; Harlow and Lane (1999) "Using Antibodies" A Laboratory Manuel, Cold Spring Harbor Publications, New York.

[0205] In some embodiments, the formulations of the present invention are substantially pure. Substantially pure means that the formulation contains any impurities in amounts less than about 10%, 5%, or 1%, preferably less than about 0.1%. 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. [Examples]

[0206] The studies described in these examples were conducted using immortalized human retinal pigment epithelial cell lines (ARPE-19) to evaluate the effects of high glucose and inflammation on cytokine release and connexin 43 expression and localization. Inflammation was induced by exposing 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 cytokines released and tested were IL-6 (inflammatory cytokine), IL-8 (neutrophil chemotactic factor), MCP-1 (monocyte chemoattractant), and sICAM-1 (leukocyte endothelial adhesion molecule), as well as other cytokines widely studied in the literature (for an overview, see Tang, J, and Kern, TS. Inflammation in diabetic retinopathy. Progress in Retinal and Eye Research, 30(5), 343-358 (2011)). (It is desired to be done), as well as VEGF (vascular growth factor), all of which have been found to be elevated in the vitreous humor 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). Next, the role of connexin hemichannels in the disease process was evaluated using connexin 43 hemichannel blockers that inhibit hemichannel opening. (Example 1) method

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

[0208] High glucose and / or cytokine attack - 6-12 passages, 2.5 × 10 in an 8-well chamber slide for immunohistochemical testing or in a 24-well plate for ATP release assay and cytometry bead array analysis. 5 Cells were seeded at a concentration of cells / mL. Once density was reached, the culture medium was replaced with serum-free DMEM-F12 containing 1×AA. Some cultures were treated with 15 mM glucose (high glucose group), a combination of inflammatory cytokines 10 ng / mL of TNF-α (Peprotech, USA) and 10 ng / mL of IL-1β (Peprotech, USA) (cytokine group), or a combination of high glucose, 10 ng / mL of TNF-α and 10 ng / mL of IL-1β (high glucose + cytokine group). The untreated group received a medium change without further treatment (basal group). All evaluations were performed 24 hours 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 attacked with high glucose and inflammatory cytokines. Concentration-dependent effects were tested after 24 hours with 5, 10, 25, and 50 μM Peptagon. These concentrations were shown to block connexin 43 hemichannels with minimal effect on gap junction cell-to-cell 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 Cytometry Bead Arrays - Soluble cytokines and chemokines in ARPE-19 incubation medium were simultaneously measured using a multiplexed bead immunoassay, cytometry bead array (CBA, BD Biosciences, USA). Three 50 μL volumes of test samples were collected from two sets of cultures in 24-well plates after 24 hours, transferred to 96-well plates, and used for the CBA assay. The analysis included six tests per group. The assays were performed according to the manufacturer's instructions. Briefly, a 10-point standard curve ranging from 0 to 5000 pg / mL was prepared for each cytokine using the cytokine standards provided in each kit. The cytokines measured were human soluble CD54 (sICAM-1, catalog number 560269, BD Biosciences, USA), IL-6 (catalog number 558276), IL-8 (catalog number 558227), and MCP-1 (catalog number 558287). Samples and cytokine standards were incubated in a capture bead mixture for 1 hour, and phycoerythrin (PE) conjugated antibodies against each cytokine were added to the sample-bead mixture for 2-hour incubation at room temperature. All buffers used were from the CBA Human Soluble Protein Master Buffer Kit (catalog number 558265, BD Biosciences, USA). The beads were washed and analyzed using an Accuri C6 flow cytometer (BD Biosciences, USA). The mean fluorescence intensity for each bead cluster was measured using FCAP as previously described (O'Carroll et al., 2015). Cytokine concentrations were converted based on a 10-point standard curve using Array™ software (BD version 3.1).

[0211] ATP Release Assay - After incubation in treatment medium for 24 hours, ATP release was measured in three sets of 50 μL culture medium taken from two wells of a 24-well plate. The sample size was 6 per group, and the experiment was repeated three 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 the culture treated with peptide 5 (treatment group) was expressed using the formula: (OD of the treatment group) 490 - OD in the injured group 490 ) / (OD in the injured group 490 The calculation was performed using ) × 100% for cells treated with high glucose and cytokines (injured group).

[0212] Immunohistochemical analysis - After incubation in treatment medium for 24 hours, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 minutes. The cells were then incubated overnight at 4°C with mouse anti-NLRP3 (1:100; Abcam, USA) and washed three times in PBS for 15 minutes each. Goat anti-mouse Cy3 (1:500; Jackson Immuno The secondary antibody (Research, USA) was applied to slides and incubated at room temperature for 3 hours. The control with secondary antibody alone did not show nonspecific labeling. Cell nuclei were stained with DAPI (1:1000; Sigma-Aldrich, USA). Cells were washed, mounted using Citifluor® fluorescence decay inhibitor, and the 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 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 were analyzed per well, and the entire experiment was repeated three times.

[0215] Using ImageJ, each image was split into its RGB channels, with NLRP3 represented by the red channel and DAPI by the blue channel. Each NLRP3 image was converted to a binary image, and the same 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 regardless of noise speckling, and to highlight 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 ANOVA. Post-hoc Tukey multiple comparison tests were used when comparing each data point in a series with all other data points. Post-hoc Dunnett multiple comparison tests were used when comparing each data point to only one data point. The specific statistical methods used for each dataset are shown in the legend of the figures. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism. This was done using 6. (Example 2) Simultaneous application of high glucose levels plus cytokines increased the secretion of IL-6, sICAM-1, MCP-1, IL-8, and VEGF.

[0217] The effects of individual or combined application of high glucose and inflammatory cytokines on cytokine secretion in the inflammatory pathway were evaluated. High glucose did not stimulate the release of IL-6 or sICAM-1, MCP-1 or IL-8 compared to basal levels (Figure 1). Cytokines alone did not induce a significant change in sICAM-1 levels compared to the basal condition, but did induce higher levels of IL-6 (p ≤ 0.0001), MCP-1 (p = 0.0002), and IL-8 (p ≤ 0.0001). However, simultaneous application of both high glucose and cytokines resulted in significantly higher release of IL-6, sICAM-1, MCP-1, and IL-8 compared to basal, high glucose alone, and cytokines alone (p ≤ 0.0001 for all) (Figure 1). This increase was 2–3 times higher than when inflammatory cytokines were used alone.

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

[0219] To evaluate the role of connexin 43 hemichannels in hyperglucose and cytokine-mediated pathology, cells were exposed to Peptagon, a well-established blocker of connexin hemichannels. Results showed that Peptagon significantly reduced the secretion of IL-6, IL-8, sICAM-1, and MCP-1 (Figure 3, p<0.0001 for all). While there was a slight tendency toward a concentration-dependent decrease in IL-6, IL-8, and MCP-1 secretion with respect to Peptagon treatment, all peptide concentrations had the same effect on sICAM-1. Furthermore, the addition of a connexin hemichannel blocker completely halted VEGF release, reducing extracellular concentrations to near baseline levels, with no statistical significance between baseline and the hyperglucose + cytokine + peptide 5 treatment group (p=0.98) (Figure 2). (Example 4) Blocking connexin hemichannels stops ATP release induced by high glucose and cytokines.

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

[0221] The released ATP was evaluated after the simultaneous 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 result in changes in IL-6, sICAM-1, IL-8, or VEGF secretion, but did cause a decrease in MCP-1 release compared to the basal condition (p ≤ 0.0001). [Table A]

[0222] Considering that 10 nM exogenous ATP was not sufficient to induce cytokine release on its own, further experiments were conducted 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. The results were positive for IL-6, MCP1, and IL-8 release, with the presence of exogenous ATP resulting in cytokine secretion returning to injury levels (Figure 5). There was a tendency toward increased sICAM-1 release in the presence of exogenous ATP, but this 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 increased significantly (Figure 2).

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

[0224] These examples demonstrate the remarkable discovery of novel effects of hemichannel blockade, including direct and immediate effects on cytokine production and release or secretion via hemichannel modulation. Remarkably, it was discovered that connexin hemichannels mediate and play a crucial role in cytokine release, and this discovery has significant relevance in the treatment of various diseases, disorders, and conditions characterized, either entirely or partially, by cytokine activity, and importantly, by VEGF-mediated angiogenic cytokine activity.

[0225] Furthermore, surprisingly, it was discovered that hemichannel blockers can also 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 levels of cellular stress and inflammation. IL-6, an inflammatory 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 that controls 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 of around 5 μM results in a statistically significant reduction in the secretion of inflammatory mediators. However, these molecules are too large to travel through gap junction hemichannels, which have a size limitation of approximately 1 kDa, so the regulation of cytokine release by connexin hemichannels is not direct.

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

[0227] The inventions described and claimed herein include, but are not limited to, many attributes and embodiments, including those described or mentioned or referenced in this detailed disclosure. It is not intended to be exhaustive, and the inventions described and claimed herein are not limited to, or thereby limited to, the features or embodiments identified in this detailed disclosure; such features or embodiments are included merely for illustrative purposes and are not limiting. 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. Such modifications and equivalents should be understood as being incorporated herein as if they were described separately. The invention also includes, individually or collectively, all steps, features, compositions and compounds mentioned or shown herein, and any and all combinations of any two or more of the aforementioned steps or features.

[0228] All patents, publications, scientific papers, websites, and other documents and materials referenced or mentioned herein represent the level of skill of those skilled in the art to which the present invention belongs, and each such referenced document and material is thus incorporated herein by reference as if it were individually incorporated in its entirety, or as if it were described herein in its entirety. The applicant reserves the right to physically incorporate into this specification any and all materials and information derived from any such patents, publications, scientific papers, websites, electronically available information, and other referenced materials or documents. No reference in this specification to any application, patent and publication constitutes, and should not be taken as, an acknowledgment of, or any form of suggestion that they constitute legitimate prior art or form part of common general knowledge in any country of the world.

[0229] The specific methods and compositions described herein are representative of preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. Those skilled in the art will readily realize that other purposes, aspects, and embodiments are conceivable and are included 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 inventions disclosed herein without departing from the scope and spirit of the invention. Preferably, the inventions described exemplary herein can be carried out in the absence of any or more elements, or limitations, that are not specifically disclosed as essential herein. Thus, for example, in each example herein, and in embodiments or examples of the invention, any of the terms “including,” “essentially consisting of,” and “consisting of” can be replaced with any of the other two terms herein. Preferably, the methods and processes described exemplary herein can be carried out in different order of steps, and are not necessarily limited to the order of steps shown herein or in the claims. Furthermore, where used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless otherwise explicitly stated in the text. In no event shall the patent be construed as being limited to any specific example, embodiment, or method specifically disclosed herein. In no event shall the patent be construed as being limited by any statement made by any examiner or other officer or employee of the United States Patent and Trademark Office unless such statement is specifically, unconditionally, or unconditionally expressed in the applicant's response. In addition, titles, headings, etc., are provided to facilitate the reader's understanding of this document and should not be read as limiting 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 used are intended to be descriptive, not restrictive, and there is no intention in using such terms and expressions to exclude any equivalent of the features or parts thereof shown and described, although it is recognized that various modifications are possible within the scope of the claimed invention. Thus, while the present invention has been specifically disclosed by preferred embodiments and features as necessary, it will be understood that those skilled in the art can rely on modifications and variations of the concepts disclosed herein, and that such modifications and variations are considered to fall within the scope of the present invention as defined by the appended claims.

[0231] The present invention has been described broadly and generally herein. Each of the narrower species and subgenera groups within the general disclosure also forms part of the present invention. This includes the general description of the present invention, conditionally or negatively, removing any subject matter derived from a genus, regardless of whether the cut material is specifically described herein.

[0232] Other embodiments are within the scope of the following claims. Furthermore, where any feature or aspect of the present invention is described in the form of a Markush group, a person skilled in the art will recognize that the present invention is also described therein in the form of any individual member or subgroup of a member of the Markush group.

Claims

[Claim 1] The invention as shown in the drawings.

Images