Treatment of Inflammatory Diseases

Abstract

A pharmaceutical composition comprising: a) at least one endothelin A receptor inhibitor or a pharma- ceutical acceptable salt thereof; and b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutical acceptable salt thereof. The pharmaceutical composition further comprises: c) at least one angiotensin type 1 receptor (AT1) inhibitor or a pharma- ceutical acceptable salt thereof. 1 R) blocking agent or a pharma- ceutical acceptable salt thereof. 1 The R blockers may be administered in the same dosage form or in separate dosage forms.

Description

[Technical field]

[0001] Technical Field The present invention relates to combination therapies comprising at least one chemokine receptor 2 pathway inhibitor and at least one endothelin A receptor inhibitor. [Background technology]

[0002] Background technology The pathology of inflammatory diseases, particularly those associated with the interaction between monocyte chemoattractant protein-1 (MCP-1) and CC chemokine receptor type 2 (CCR2) and with dysfunction of the endothelin system, is involved in uveitis, atherosclerosis, rheumatoid arthritis, multiple sclerosis, neuropathic pain, pruritus, Crohn's disease, nephritis, organ allograft rejection, fibrotic lung, renal failure, diabetes and diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic microangiopathy, tuberculosis, sarcoidosis, and glaucoma. It has been associated with diseases such as leucoidosis, invasive staphylococcal infections, inflammation after cataract surgery, allergic rhinitis, allergic conjunctivitis, chronic urticaria, allergic asthma, periodontal disease, periodontitis, gingivitis, gum disease, dilated cardiomyopathy, myocardial infarction, myocarditis, chronic heart failure, vascular stenosis, restenosis, reperfusion injury, glomerulonephritis, solid tumors and cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma, malignant myeloma, Hodgkin's disease, and carcinoma of the bladder, breast, cervix, colon, lung, prostate or stomach.

[0003] There is a need to provide new treatments for inflammatory disease conditions, including kidney, lung and heart disease, or at least to provide alternative treatments to complement previously known treatments for inflammatory disease conditions.

[0004] The preceding description of the background art is intended only to facilitate an understanding of the present invention. The discussion is not an admission or acknowledgement that any of the material referred to was or was part of the common general knowledge at the priority date of the application. Summary of the Invention

[0005] Summary of the Invention The present invention relates to a pharmaceutical composition comprising: a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof.

[0006] The present invention further comprises: a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; c) at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof.

[0007] Preferably, the endothelin A receptor inhibitor is selected from the group comprising endothelin A receptor antagonists such as sitaxsentan, ambrisentan, atrasentan, BQ-123 and zibotentan; endothelin A and B dual antagonists such as bosentan, macitentan and tezosentan; and heterologous receptor dual antagonists such as sparsentan.

[0008] Preferably, the chemokine receptor 2 (CCR2) inhibitor is selected from the group including: repagermanium; propagermanium; CCX140; cenicriviroc; PF-6309; PF-04178903; CCX872-B (CCX-872); MLN1202; and PF-04634817.

[0009] Preferably, the angiotensin type 1 receptor (AT1R) blocker is selected from the group including sparsentan, irbesartan, eprosartan, losartan, valsartan, telmisartan, candesartan, olmesartan, ZD-7115, fimasartan, azilsartan, DuP 753, EXP 3174, embusartan (BAY 10-6734), KRH-594, fonsartan (HR 720), and pratosartan (KT3-671).

[0010] The CCR2 pathway inhibitor, the endothelin A receptor inhibitor and / or the AT1R blocker may be administered in the same dosage form or in separate dosage forms.

[0011] The CCR2 pathway inhibitor, the endothelin A receptor inhibitor and / or the AT1R blocker may be administered simultaneously or sequentially.

[0012] The present invention also provides a method for treating, ameliorating or preventing a disease, comprising administering a therapeutically effective amount of a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) providing a method for treating, ameliorating or preventing a disease, comprising administering to a subject a combination of at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof.

[0013] The present invention further provides a method for treating, ameliorating or preventing a disease, comprising administering a therapeutically effective amount of a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; c) providing a method for treating, ameliorating or preventing a disease, comprising administering to a subject a combination of at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof.

[0014] Preferably, the disease is renal disease, pulmonary disease or cardiac disease.Renal disease can be selected from the list including focal segmental glomerulosclerosis (FSGS; including primary FSGS and secondary FSGS), fibrotic disorders in the kidney, chronic renal disease caused by diabetic nephropathy, renal failure (diabetic and non-diabetic), and renal failure symptoms including diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, proteinuria of primary renal disease and renal vascular hypertension.Renal disease can be infectious or non-infectious inflammatory lung disease, preferably selected from the list including community-acquired pneumonia, chronic obstructive pulmonary disease, asthma, bronchiectasis, bronchiolitis, bronchitis, emphysema, pleurisy or pulmonary fibrosis.Cardiac disease can be coronary artery disease, preferably selected from the list including arthrosclerosis, pulmonary hypertension, ischemic heart disease, cardiomyopathy and inflammatory heart disease.

[0015] The present invention also relates to a method for producing a dosage form for treating, ameliorating or preventing a disease. a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; The present invention contemplates the use of a pharmaceutical composition comprising:

[0016] The present invention further relates to a method for producing a dosage form for treating, ameliorating or preventing a disease. a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; c) at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof; The present invention contemplates the use of a pharmaceutical composition comprising:

[0017] The present invention provides at least one endothelin A receptor inhibitor, or a pharma- ceutically acceptable salt thereof, and at least one chemokine receptor 2 (CCR2) inhibitor, or a pharma- ceutically acceptable salt thereof, for use in a formulation for treating, ameliorating or preventing a disease.

[0018] The present invention provides at least one endothelin A receptor inhibitor, or a pharma- ceutically acceptable salt thereof, at least one chemokine receptor 2 (CCR2) inhibitor, or a pharma- ceutically acceptable salt thereof, and at least one angiotensin type 1 receptor (AT1R) blocker, or a pharma- ceutically acceptable salt thereof, for use in a formulation for the treatment, amelioration, or prevention of a disease.

[0019] The present invention provides the following: at least one endothelin A receptor inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with at least one CCR2 pathway inhibitor. at least one CCR2 pathway inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor. at least one endothelin A receptor inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with at least one CCR2 pathway inhibitor and at least one angiotensin type 1 receptor blocker. at least one CCR2 pathway inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor and at least one angiotensin type 1 receptor blocker. At least one angiotensin type 1 receptor blocker for use in a formulation for the treatment, amelioration or prevention of a disease, wherein at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor and at least one CCR2 pathway inhibitor.

[0020] The present invention relates to a kit for treating, ameliorating, or preventing a disease, the kit comprising: a) at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; c) providing a kit for treating, ameliorating or preventing a disease, comprising:

[0021] The present invention relates to a kit for treating, ameliorating, or preventing a disease, the kit comprising: a) at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; c) at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof; d) instructions for use. [Brief description of the drawings]

[0022] Further features of the present invention are described more fully in the following description of several non-limiting embodiments thereof. This description is included solely for the purpose of illustrating the present invention and should not be understood as a limitation on the broad summary, disclosure or description of the present invention set forth above. The following description is made with reference to the accompanying drawings. [Figure 1] Figure 1 is a graph of ETAR / Rluc8+Barr2 / Venus+CCR2, where ETAR is tagged with Rluc8 and β-arrestin is tagged with Venus in the presence of unlabeled CCR2, and shows that β-arrestin is recruited to ETAR not only in the presence of ET-1, as expected, but also in the presence of CCL2 / MCP-1, confirming the existence of a complex between the receptors. The presence of both ligands causes hyperactivation of β-arrestin recruitment, confirming this heteromeric interaction. [Diagram 2] Figure 2 is a graph of ETAR / Rluc8+Barr2 / Venus+CCR2, showing that β-arrestin recruitment to ETAR occurs when CCL2 is present, confirming the interaction of ETAR with unlabeled CCR2. Sparsentan is able to suppress hyperactivation when both CCL2 / CMP-1 and ET-1 ligands are present but does not abolish β-arrestin recruitment to ETAR. [Diagram 3] Figure 3 is a graph of ETAR / Rluc8+Barr2 / Venus+CCR2, showing that hyperactivation of ETAR and CCR2 heteromers is suppressed by the addition of either sparsentan inhibition or CCR2 inhibition. ETAR can recruit β-arrestin to a low extent when stimulated with ET-1, regardless of the presence of CCR2, and sparsentan inhibits this signaling independently of CCR2 activation. Complete inhibition of β-arrestin recruitment to ETAR occurs only in the presence of ET1 and CCL2 / MCP-1 when CCR2 and ETAR are simultaneously inhibited. [Figure 4]Figure 4 is a graph of CCR2 / Rluc8+Barr2 / Venus+HA-ETAR, showing the different experimental orientations in which CCR2 is tagged with Rluc8 and β-arrestin is tagged with Venus in the presence of unlabeled ETAR, and β-arrestin is recruited to CCR2 in the presence of CCL2 / MCP-1. Increased recruitment of β-arrestin is observed in the presence of both CCL2 / MCP-1 and ET-1 ligands, confirming the presence of an interaction between CCR2 and ETAR. [Diagram 5] FIG. 5 is a graph of CCR2 / Rluc8+Barr2 / Venus+HA-ETAR and shows that when ETAR activation is blocked with sparsetan, no increased recruitment of β-arrestin is observed in the presence of CCL2 / MCP-1 and ET-1. [Figure 6] FIG. 6 is a graph of CCR2 / Rluc8+Barr2 / Venus+HA-ETAR, which shows that when CCR2 is inhibited, with or without ETAR activation by ET-1, there is no recruitment of β-arrestin to CCR2 in the presence of CCL2 / MCP-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention Studies have shown that G protein-coupled receptors (GPCRs) can act not only as monomers, but also as homodimers and heterodimers, and / or homo- and hetero-oligomers (also known as homomers and heteromers), leading to changes in ligand binding, signal transduction, and endocytosis (Rios et al. (2000) Pharmacol. Ther. 92:71-87). Thus, the effect of a drug acting as an agonist or antagonist of a specific receptor may depend on the binding partner of this receptor. It may be desirable to limit the effect of a drug to a cellular response mediated by a particular receptor dimer or oligomer.

[0024] composition The present invention relates to a pharmaceutical composition comprising: a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof.

[0025] The present invention further comprises: a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; c) at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof.

[0026] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier or excipient.

[0027] Without being bound by any theory, it is believed that chemokine receptor 2 and endothelin A receptor form heterodimers / hetero-oligomers in some tissues. When CCR2 and ETAR are in their heterodimeric / hetero-oligomeric configuration, inhibition of both receptors simultaneously causes a greater effect than inhibition of either receptor alone or either receptor when the receptors are not in the heterodimeric configuration.

[0028] Chemokine receptor 2 and angiotensin type 1 receptor are further believed to form heterodimers / hetero-oligomers in some tissues. When CCR2 and AT1R are in their heterodimeric / hetero-oligomeric configuration, inhibition of both receptors simultaneously causes a greater effect than inhibition of either receptor alone or either receptor when the receptors are not in the heterodimeric configuration.

[0029] The above findings allow for specific and targeted intervention by simultaneous inhibition of both elements of the heterodimer / hetero-oligomer, which would result in a greater therapeutic effect in tissues where the heterodimer forms, but not in tissues where the receptor is not in heterodimeric form.

[0030] Chemokine Receptor 2 Pathway Inhibitors CC chemokine receptor type 2 (CCR2 or CD192) is a human G protein-coupled receptor. CCR2 is the primary receptor for monocyte chemoattractant protein-1 (MCP-1 / CCL2).

[0031] The phrase "chemokine receptor 2 pathway" should be understood to include any one of the pathways of chemokine receptor 2 that are activated by its native ligand, including, but not limited to, pathways that result in chemotactic migration, cell motility, extracellular regulated kinase (ERK) phosphorylation, cAMP production, actin recruitment, protein phosphorylation, nuclear protein localization, gene transcription or translation, G protein coupling, β-arrestin recruitment or mediated signal transduction.

[0032] The phrase "chemokine receptor 2 pathway inhibitor" is intended to include any compound or agent that inhibits or partially inhibits any one of the pathways associated with chemokine receptor 2. CCR2 pathway inhibitors include compounds or agents that inhibit or partially inhibit the activation of chemokine receptor 2. CCR2 pathway inhibitors can be peptides, polypeptides, or small chemical entities. For example, CCR2 pathway inhibitors can be proteins, binding proteins, or antibodies.

[0033] This may be due to binding to the CCR2 ligand, monocyte chemotactic protein-1 (MCP-1 / CCL2), such that it can no longer bind to chemokine receptor 2 (CCR2). In one embodiment, chemokine receptor 2 pathway inhibitors inhibit or partially inhibit the in vitro chemotactic migration of monocytes induced by MCP-1 by directly targeting MCP-1. Such compounds include agents that bind to MCP-1.

[0034] Alternatively, chemokine receptor 2 pathway inhibitors may bind to chemokine receptor 2 itself and block ligand access or binding. Such CCR2 pathway inhibitors may be direct CCR2 antagonists, direct CCR2 inverse agonists or direct CCR2 negative allosteric modulators. CCR2 antagonists may block CCR2 and prevent MCP-1 binding; CCR2 inverse agonists may bind to CCR2 and reduce its activity below basal levels; CCR2 negative allosteric modulators may bind to CCR2 and reduce its affinity and / or effectiveness for MCP-1 upon binding.

[0035] The phrase "chemokine receptor 2 pathway inhibitors" includes compounds or agents that inhibit or partially inhibit components of the chemokine receptor 2 pathway other than chemokine receptor 2 itself. For example, the inhibitor may inhibit or partially inhibit proteins associated with chemokine receptor 2, or may inhibit compounds or pathway steps before and / or after the chemokine receptor itself. In such cases, preferably, the chemokine receptor 2 pathway inhibitor is selected from an indirect CCR2 antagonist, an indirect CCR2 inverse agonist, or an indirect CCR2 negative allosteric modulator.

[0036] As used herein, the term "a component of the chemokine receptor 2 pathway other than chemokine receptor 2 itself" should be understood to refer to a component of the chemokine receptor 2 pathway that is not itself chemokine receptor 2. A component of the chemokine receptor 2 pathway may directly interact with chemokine receptor 2. Alternatively, a component of the chemokine receptor 2 pathway may indirectly interact with chemokine receptor 2 through a protein-protein interaction or complex formation. Alternatively, a component of the chemokine receptor 2 pathway may indirectly interact with chemokine receptor 2 through a signaling cascade. Preferably, the component is a protein, such as, but not limited to, a transduction or signaling protein.

[0037] In one aspect, when a chemokine receptor 2 pathway inhibitor inhibits or partially inhibits a component of the pathway other than chemokine receptor 2 itself, the inhibitor blocks MCP-1-induced monocyte migration and activation and chemotactic migration via targeting glycosylphosphatidylinositol (GPI)-linked proteins such as CD55, CD59 and CD16.

[0038] In another aspect, when the chemokine receptor 2 pathway inhibitor inhibits or partially inhibits a pathway component other than chemokine receptor 2, the inhibited component is selected from the list of CCR2 ligands including CCL7 (MCP-3), CCL8, CCL13 (MCP-4) and CCL16 (hemofiltrate CC chemokine (HCC)-4). In one embodiment, the chemokine receptor 2 pathway inhibitor inhibits or partially inhibits the in vitro chemotactic migration of monocytes induced by the above CCR2 ligands by directly targeting the ligand. Such compounds include agents that bind to CCL7, CCL8, CCL13 and CCL16.

[0039] In another embodiment, the chemokine receptor 2 pathway inhibitor inhibits or partially inhibits MCP-1-induced in vitro chemotactic migration of monocytes by targeting one or more GPI-linked proteins selected from the group including CD55, CD59 and CD16. In such a case, the chemokine receptor 2 pathway inhibitor may stabilize the complexes CCR2 / CD55 and / or CCR2 / CD59 and / or CCR2 / CD16.

[0040] Known inhibitors of the CCR2 pathway include repagermanium or propagermanium; RS504393; RS102895; MLN-1202 (Millennium Pharmaceuticals); INCB3344, INCB3284 and INCB8696 (Incyte Pharmaceuticals); MK-0812 (Merck); CCX140 and CCX872-B (CCX-872); (ChemoCentryx); BMS-741672 (Bristol-Myers Squibb); and PF-04136309 (also known as PF-6309), PF-04178903 and PF-04634817 (Pfizer); and cenicriviroc (TAK-652, TBR-652). Preferably, the chemokine receptor 2 pathway 2 inhibitor is selected from the list: repagermanium; propagermanium; CCX140; PF-6309 and PF-04634817.

[0041] In a preferred embodiment, the CCR2 pathway inhibitor is a direct antagonist of chemokine receptor 2, such as repagermanium, propagermanium or CCX140.

[0042] Propagermanium (3-oxygermylpropionic acid polymer), a molecule that has been used as a therapeutic agent against chronic hepatitis, has also been shown to specifically inhibit in vitro chemotactic migration of monocytes by MCP-1 through a mechanism that appears to require glycosylphosphatidylinositol (GPI)-linked proteins such as CD55, CD59 and CD16. Propagermanium is also known as 3-[(2-carboxyethyl-oxogermyl)oxy-oxogermyl]propanoic acid, proxygermanium, Ge-132, bis(2-carboxyethylgermanium) sesquioxide (CEGS), 2-carboxyethylgerma sesquioxane, SK-818, organogermanium, sesquioxide germanium, 3,3<'>-(1,3-dioxo-1,3-digermanoxanediyl)bispropionic acid, 3-oxygermylpropionic acid polymer, poly-fra / 7s-(2-carboxyethyl)germa sesquioxane, proxygermanium, repagermanium, and Serocion™.

[0043] Endothelin A receptor inhibitors Endothelin receptor type A (ETAR, ET A ET1 receptor, also known as EDNRA, ET1 receptor, ETA receptor, ETA, EDN1 and ET-AR, is a human G protein-coupled receptor.

[0044] The phrase "endothelin A receptor inhibitor" (also known as ETAR inhibitor) is intended to include any compound or agent that inhibits or partially inhibits any one of the pathways related to endothelin A receptor. ETAR inhibitors are understood to include any compound or agent that inhibits or partially inhibits the activation of ETAR. ETAR inhibitors can be peptides, polypeptides or small chemical entities. For example, ETAR inhibitors can be proteins, binding proteins or antibodies.

[0045] This may be due to binding to endothelin-1 (ET-1), an ETAR ligand, so that it can no longer bind to endothelin A receptor (ETAR). In one embodiment, ETAR inhibitors inhibit or partially inhibit ET-1-induced signal transduction by directly targeting ET-1. Such compounds include agents that bind to ET-1.

[0046] Alternatively, ETAR inhibitors can bind to ETAR itself and block access or binding of ligand. Such ETAR inhibitors can be direct ETAR antagonists, direct ETAR inverse agonists or direct ETAR negative allosteric modulators. ETAR antagonists can block ETAR and prevent ET-1 from binding; ETAR inverse agonists can bind to ETAR and reduce its activity below basal level; ETAR negative allosteric modulators can bind to ETAR and reduce its affinity and / or its effectiveness for ET-1 when ET-1 binds.

[0047] Known inhibitors of the endothelin A receptor include endothelin A receptor antagonists such as sitaxsentan, ambrisentan (Volibris®, GalaxoSmithKline), atrasentan (AbbVie), BQ-123, zibotentan (ZD4054, AstraZeneca); endothelin A and B dual antagonists such as bosentan, macitentan, tezosentan; and heterologous receptor dual antagonists such as sparsentan.

[0048] As an example, the endothelin A receptor inhibitor can be sparsentan. Sparsentan, also known as 2-[4-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N-(4,5-dimethyl-1,2-oxazol-3-yl)benzenesulfonamide, is a combined selective antagonist of the angiotensin II type 1 receptor (AT1R) and the ETA receptor.

[0049] Angiotensin type 1 receptor The angiotensin type 1 receptor (AT1R, AT1R, angiotensin II receptor type 1) is a G protein-coupled receptor.

[0050] The phrase "angiotensin type 1 receptor blocker" (also called AT1R blocker or ARB) is intended to include any compound or drug that inhibits or partially inhibits any one of the pathways related to the angiotensin type 1 receptor. AT1R blocker is understood to include any compound or drug that inhibits or partially inhibits the activation of AT1R. AT1R blocker can be a peptide, a polypeptide or a small chemical entity. For example, AT1R blocker can be a protein, a binding protein or an antibody.

[0051] This may be due to binding to AT1R ligand, such as angiotensin II (AngII), so that it can no longer bind to AT1R.In one embodiment, the AT1R blocker inhibits or partially inhibits the signal transduction induced by the binding of AT1R ligand to AT1R by directly targeting AT1R ligand.Such compounds include agents that bind to AT1R ligand, such as agents that bind to angiotensin II (AngII).

[0052] Alternatively, AT1R blockers can bind to AT1R itself and block the access or binding of ligand. Such AT1R blockers can be direct AT1R antagonists, direct AT1R inverse agonists or direct AT1R negative allosteric modulators. AT1R antagonists can block AT1R and prevent AT1R ligand binding; AT1R inverse agonists can bind to AT1R and reduce its activity below basal level; AT1R negative allosteric modulators can bind to AT1R and reduce its affinity to AT1R ligand and / or its effectiveness when AT1R ligand binds.

[0053] For example, the AT1R blocker may be selected from the group including sparsentan, irbesartan, eprosartan, losartan, valsartan, telmisartan, candesartan, olmesartan, ZD-7115, fimasartan, azilsartan, DuP753 (2-n-butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H)-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole potassium salt), EXP3174, embusartan (BAY10-6734), KRH-594, fonsartan (HR720) and pratosartan (KT3-671).

[0054] Taylor et al. (2011) reviewed AT1R blockers and noted that "there are no major differences in efficacy or other clinical characteristics between the older drugs in this class, but some of the newer agents may lower blood pressure more effectively than the older agents." The differences in properties appear to be related to interactions with other pathways and not to core angiotensin type 1 receptor blockade. However, they noted that "there are some pharmacological differences between ARBs, and some drugs in this class have special effects independent of angiotensin II receptor blockade, but there is currently no strong evidence that these translate into unique protection against target organ damage from hypertension." Thus, with regard to their effects on AT1R blockade, AT1R blockers are functionally interchangeable, despite their structural differences.

[0055] AT1R blockers were first marketed in 1990. Despite the long-term use of this class of drugs, physicians treat them as groups that are more or less interchangeable with each other. There are some specific subtleties in the exact pharmacology of some groups, but these do not seem to be related to their angiotensin type 1 receptor activity. Furthermore, all pharmacological products behave subtly differently in some patients, but the variability of some subgroups does not prevent them from being generally useful in therapy (Taylor et al., 2011).

[0056] As an example, the angiotensin receptor inhibitor can be irbesartan. Irbesartan is an angiotensin type 1 receptor antagonist also known as 2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1,3-diazaspiro[4.4]non-1-en-4-one. The angiotensin receptor inhibitor can be sparsentan. Sparsentan, also known as 2-[4-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N-(4,5-dimethyl-1,2-oxazol-3-yl)benzenesulfonamide, is a combined selective antagonist of the angiotensin II type 1 receptor (AT1R) and the ETA receptor.

[0057] Treatment Method The present invention also provides a method for treating, ameliorating or preventing a disease, comprising administering a therapeutically effective amount of a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) providing a method for treating, ameliorating or preventing a disease, comprising administering to a subject a combination of at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof.

[0058] The present invention further provides a method for treating, ameliorating or preventing a disease, comprising administering a therapeutically effective amount of a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; c) providing a method for treating, ameliorating or preventing a disease, comprising administering to a subject a combination of at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof.

[0059] The CCR2 pathway inhibitor and the endothelin A receptor inhibitor can be administered in the same dosage form or in separate dosage forms. The CCR2 pathway inhibitor and the endothelin A receptor inhibitor can be administered simultaneously or sequentially.

[0060] When the method of treatment, amelioration or prevention further comprises at least one angiotensin type 1 receptor (AT1R) blocker, the AT1R blocker can be administered in the same dosage form as the CCR2 pathway inhibitor and / or the ETAR inhibitor, or in separate dosage forms. The angiotensin type 1 receptor (AT1R) blocker, the CCR2 pathway inhibitor and the endothelin A receptor inhibitor can be administered simultaneously or sequentially.

[0061] The endothelin A receptor inhibitor, CCR2 pathway inhibitor and / or AT1R blocker may be a pharma- ceutically acceptable salt of the endothelin A receptor inhibitor, CCR2 pathway inhibitor and / or AT1R blocker.

[0062] One component of the treatment of the invention may already be administered to the subject, for example as a standard of care treatment, in which case the second (and third, if present) component of the treatment of the invention is administered as the second (and third) component in the treatment to provide the treatment of the invention.

[0063] Without intending to be limited to any particular mode of action, in a preferred embodiment, the CCR2 pathway inhibitor has greater affinity and / or potency and / or efficacy when interacting with CCR2 or regulating its downstream pathway when CCR2 associates with endothelin A receptor. For example, CCR2 and endothelin A receptor can associate as CCR2 / ETAR heterodimer / hetero-oligomer. In a further preferred embodiment, when the CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with an endothelin A receptor inhibitor, the combined affinity, potency and / or efficacy is greater than the affinity, potency and / or efficacy that would be achieved when the CCR2 pathway inhibitor is not administered (whether simultaneously or sequentially) with an endothelin A receptor inhibitor. In yet a further preferred embodiment, when the CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with an endothelin A receptor inhibitor, a synergistic effect (measured by affinity, potency and / or efficacy) is achieved.

[0064] Without intending to be limited to any particular mode of action, in a preferred embodiment, endothelin A receptor inhibitor has greater affinity and / or potency and / or efficacy when interacting with endothelin A receptor when endothelin A receptor is associated with CCR2. For example, CCR2 and endothelin A receptor can be associated as CCR2 / ETAR heterodimer / hetero-oligomer. In a further preferred embodiment, when endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with CCR2 pathway inhibitor, the combined affinity, potency and / or efficacy is greater than the affinity, potency and / or efficacy that can be achieved when endothelin A receptor inhibitor is not administered (whether simultaneously or sequentially) with CCR2 pathway inhibitor. In yet a further preferred embodiment, when endothelin A receptor inhibitor is administered to a subject together with CCR2 pathway inhibitor (whether simultaneously or sequentially), a synergistic effect (measured by affinity, potency and / or efficacy) is achieved.

[0065] Without intending to be limited to any particular mode of action, in a preferred embodiment, the CCR2 pathway inhibitor has greater affinity and / or potency and / or efficacy when interacting with CCR2 or regulating its downstream pathway when CCR2 associates with angiotensin receptor.For example, CCR2 and angiotensin receptor can associate as CCR2 / AT1R heterodimer / hetero-oligomer.In a further preferred embodiment, when the CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with an AT1R blocker, the combined affinity, potency and / or efficacy is greater than the affinity, potency and / or efficacy that would be achieved when the CCR2 pathway inhibitor is not administered (whether simultaneously or sequentially) with an AT1R blocker.In yet a further preferred embodiment, when the CCR2 pathway inhibitor is administered to a subject together with an AT1R blocker (whether simultaneously or sequentially), a synergistic effect (measured by affinity, potency and / or efficacy) is achieved.

[0066] Without intending to be limited to any particular mode of action, in a preferred embodiment, the AT1R blocker has greater affinity and / or potency and / or efficacy when interacting with angiotensin receptor when the angiotensin receptor is associated with CCR2. For example, CCR2 and angiotensin receptor can be associated as CCR2 / AT1R heterodimer / hetero-oligomer. In a further preferred embodiment, when the AT1R blocker is administered to a subject simultaneously or sequentially with a CCR2 pathway inhibitor, the combined affinity, potency and / or efficacy is greater than the affinity, potency and / or efficacy that would be achieved when the AT1R blocker is not administered (whether simultaneously or sequentially) with a CCR2 pathway inhibitor. In yet a further preferred embodiment, when the AT1R blocker is administered to a subject simultaneously or sequentially with a CCR2 pathway inhibitor, a synergistic effect (measured by affinity, potency and / or efficacy) is achieved.

[0067] Because CCR2 / ETAR heterodimers / hetero-oligomers, when activated simultaneously by their cognate ligands, have greater affinity, potency and / or efficacy than either component alone, and because CCR2 / AT1R heterodimers / hetero-oligomers, when activated simultaneously by their cognate ligands, have greater affinity, potency and / or efficacy than either component alone, simultaneous inhibition of the two sets of heterodimers / hetero-oligomers is expected to provide a high level of therapeutic benefit.

[0068] The subject to be treated is preferably a mammal, including a human mammal.

[0069] Symptoms or diseases Preferably, the CCR2 pathway inhibitors, endothelin A receptor inhibitors and / or AT1R blockers are used to treat pulmonary disease, renal disease, cardiovascular disease including congestive heart failure (acute and chronic), left ventricular dysfunction and hypertrophic cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, atrial fibrillation or atrial flutter, myocardial infarction and its sequelae, atherosclerosis, angina (unstable or stable), heart failure, angina pectoris, pectoris), diabetes, secondary aldosteronism, primary and secondary pulmonary hyperaldosteronism, primary and pulmonary hypertension, diabetic retinopathy, macular degeneration, eye disorders, insulin resistance, management of other vascular disorders, e.g., in the treatment, amelioration or prevention of a disease selected from the list including migraine, Raynaud's disease, luminal hyperplasia, cognitive impairment (such as Alzheimer's disease), stroke, hyperkalemia, pre-eclampsia, sarcoidosis, ischemia and reperfusion injury, atherogenesis, chronic obstructive pulmonary disease, asthma and allergic kidney disease, rheumatoid arthritis, neuropathic pain, and pruritus.

[0070] Endothelin A receptor inhibitors, CCR2 pathway inhibitors and / or AT1R blockers may be used in the treatment, amelioration or prevention of kidney diseases, more particularly diseases selected from the list including focal segmental glomerulosclerosis (FSGS; including primary FSGS and secondary FSGS), fibrotic disorders in the kidney, chronic kidney disease caused by diabetic nephropathy, kidney failure (diabetic and non-diabetic), and kidney failure symptoms including diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, proteinuria of primary kidney disease and renal vascular hypertension.

[0071] Endothelin A receptor inhibitors, CCR2 pathway inhibitors and / or AT1R blockers can be used to treat, ameliorate or prevent pulmonary diseases, preferably infectious or non-infectious inflammatory pulmonary diseases.Preferably, the infectious or non-infectious inflammatory pulmonary diseases are selected from the list including community-acquired pneumonia, chronic obstructive pulmonary disease, asthma, bronchiectasis, bronchiolitis, bronchitis, emphysema, pleurisy or pulmonary fibrosis.

[0072] Endothelin A receptor inhibitors, CCR2 pathway inhibitors and / or AT1R blockers may be used to treat, ameliorate or prevent cardiac disease. The cardiac disease may be selected from the list including coronary artery disease, including arthrosclerosis, pulmonary hypertension, ischemic heart disease, cardiomyopathy and inflammatory heart disease.

[0073] The term "prevention" includes administration of the compositions of the present invention to a subject having symptoms indicating that the subject is at risk of developing a disease, but may not be diagnosed with the disease at the time of treatment. For example, a subject may have diabetes, but may not yet be diagnosed with diabetes-related kidney disease.

[0074] The term "amelioration" includes administration of the compositions of the invention to a subject who has a disease and is at risk of developing post-disease symptoms, such as fibrosis and persistent inflammation. The post-disease symptoms may be due to the disease itself or to medical interventions, such as mechanical ventilation used during the acute phase of pulmonary disease.

[0075] Measurement of inhibition The phrase "endothelin A receptor inhibitor" is understood to include any compound or agent that inhibits or partially inhibits the activation of the endothelin A receptor.

[0076] The phrase "angiotensin type 1 receptor blocker" is understood to include any compound or agent that inhibits or partially inhibits the activation of the AT1R.

[0077] The phrase "chemokine receptor 2 pathway inhibitor" is intended to include any compound or agent that inhibits or partially inhibits any one of the pathways associated with chemokine receptor 2.

[0078] As used herein, the term "inhibit" refers to a decrease below detectable limits when compared to a reference. This phrase includes preventing, retarding, or impeding an action to prevent an undesirable outcome.

[0079] As used herein, the term "partially inhibit" refers to any detectable decrease when compared to a reference. This phrase includes preventing, retarding, or impeding an action to prevent an undesirable outcome.

[0080] Inhibition or partial inhibition of the CCR2 pathway, Endothelin A receptor and / or AT1R caused by (i) CCR2 pathway inhibitors and Endothelin A receptor inhibitors, or (ii) CCR2 pathway inhibitors, Endothelin A receptor inhibitors and AT1R blockers can be measured using in vitro methods described herein, including, but not limited to, biochemical or cellular assays to assess in vitro chemotactic migration of CCR2-expressing neutrophils and other cells known in the art, as well as the use of antibodies to assess inositol phosphate production, extracellular regulated kinase (ERK) phosphorylation, cAMP production, actin recruitment, protein phosphorylation, nuclear protein localization, gene transcription, label-free techniques (e.g., using impedance, light refraction or charge redistribution), G-protein coupling using proximity reporter systems or other approaches, β-arrestin recruitment or mediated signaling, transcription factor-based reporter systems, microscopy using fluorescent labels, receptor cellular localization (such as enzyme-linked immunosorbent assay) and fluorescence activated cell sorting.

[0081] The inhibition or partial inhibition of the CCR2 pathway Endothelin A receptor and / or AT1R caused by (i) a CCR2 pathway inhibitor and an Endothelin A receptor inhibitor, or (ii) a CCR2 pathway inhibitor, an Endothelin A receptor inhibitor and an AT1R blocker, can be measured using the in vivo methods described herein.

[0082] For example, when the disease is a renal disease, the measurements include, but are not limited to, serial measurements of renal function, such as by measuring plasma creatinine and urea, such as by an automated analyzer; measuring proteinuria, measuring albuminuria; and GFR or estimated GFR; evaluation of endpoints such as renal and / or cardiac and / or ocular structure, such as by light microscopy (LM) to evaluate glomerular and cardiac hypertrophy, glomerulosclerosis and / or fibrosis and / or glomerular epithelial cell (podocyte) changes, and / or; immunohistochemistry to measure the degree of matrix deposition and regulation of profibrotic growth factors and their activity; evaluation of systolic blood pressure, regulation of fasting plasma glucose with insulin, regulation of hemoglobin A1c; and molecular biology techniques to evaluate renal and cardiac and ocular structure according to conventional assays such as those known in the art. Inhibition or partial inhibition can be indicated by qualitative improvement of renal and / or cardiac and / or ocular structure measured by one or more of the above endpoints, or other patient benefit endpoints such as improved quality of life or renal or overall survival.

[0083] Alternatively, if the disease is a pulmonary disease, the measurements include, but are not limited to, measurements of pulmonary exudate cell and cytokine content, measurements of pulmonary function, including the physical capacity of lung function using spirometry-based tests, or pulmonary functional output measured using blood gas or other biochemical measurements, or improvements in functional benefit, including clinical benefit measured by quantitative methods such as survival or walking tests or qualitative methods such as patient-reported outcome assessments. Inhibition or partial inhibition can be indicated by qualitative improvements in lung structure measured by one or more of the above endpoints.

[0084] In another embodiment, the total effectiveness of the pharmaceutical preparation is higher when each component is administered without administration of any other component, compared with the effectiveness of endothelin A receptor inhibitor, AT1R blocker or CCR2 pathway inhibitor.Therefore, the combination preparation can be administered in a single dose that includes a subtherapeutic dose, or can be administered less frequently than any component can be administered as a single compound.

[0085] Preferably, the total efficacy of the pharmaceutical formulation is higher when compared to the sum of the efficacy of the endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor when any component is administered without administration of the other component. More preferably, a synergistic effect of efficacy is observed when the endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor are administered simultaneously or sequentially.

[0086] Alternatively, the total efficacy of the pharmaceutical formulation is equal to the sum of the efficacy of the endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor when any component is administered without administration of the other component. In a more preferred embodiment of this alternative, an additive effect of efficacy is observed when the endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor are administered simultaneously or sequentially.

[0087] In a further alternative, the total efficacy of the pharmaceutical formulation is less than the sum of the efficacy of the endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor when any component is administered without the administration of the other components. In a further embodiment, the combined efficacy is less than the sum of the efficacy of the endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor when each component is administered without the administration of the other components, but the treatment provides greater efficacy compared to a single treatment of the endothelin A receptor inhibitor, AT1R blocker or CCR2 pathway inhibitor administered alone.

[0088] Preferably, the components are co-administered simultaneously (e.g., taken as two or three tablets or taken as a single tablet, formulated with each component) or sequentially (e.g., one or two tablets taken after another). The dosing of each component may occur together (simultaneously) or sequentially, within seconds, minutes, days, weeks or months of each other.

[0089] delivery Dosage forms provided by the present invention may further comprise a vial, cartridge, container, tablet or capsule containing the pharmaceutical formulation of the present invention together with administration instructions for administration of the dosage form(s) to a subject for the treatment, amelioration or prevention of a disease.

[0090] The amount of each active ingredient that can be combined with a carrier material to produce a single dosage will vary depending on the host being treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to 1 g of each active compound with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95% w / w of the total formulation. Dosage unit forms generally contain about 0.5 mg to 500 mg of active ingredient.

[0091] Preferably, the CCR2 pathway inhibitor is provided at a dose between 0.5 mg and 2000 mg per day, provided in one or more doses. Even more preferably, the CCR2 pathway inhibitor is provided at a dose between 0.5 mg and 50 mg per day, provided in one or more doses.

[0092] Preferably, the endothelin A receptor inhibitor is provided at a dose of 0.5 mg to 2000 mg per day and provided in one or more doses. Even more preferably, the endothelin A receptor inhibitor is provided at a dose of 0.5 mg to 300 mg per day and provided in one or more doses. For example, the ETAR inhibitor is sparsentan, administered at a dose of 75, 150 or 300 mg per day and provided in one or more doses. Other exemplary doses include ambrisentan administered at 5 mg or 10 mg per day and provided in one or more doses; bosentan administered at a dose of 62.5 mg or 125 mg per day and provided in one or more doses; or macitentan administered at a dose of 10 mg per day and provided in one or more doses.

[0093] Preferably, the AT1R blocker is provided at 50 mg to 500 mg per day and is provided in one or more doses. Even more preferably, the AT1R blocker is provided at 75 mg to 300 mg per day. For example, the AT1R blocker is irbesartan, administered at a dose of 75, 150 or 300 mg per day and provided in one or more doses. For example, the AT1R blocker is sparsentan, administered at a dose of 75, 150 or 300 mg per day and provided in one or more doses.

[0094] The dose of each active agent may be provided in either a single dosage form, two separate dosage forms, or three separate dosage forms. The active agents may be provided as about 0.5 mg to 2 g of the CCR2 pathway inhibitor, about 0.5 mg to 2 g of the ETAR inhibitor, and / or about 50 mg to 500 mg of the AT1R blocker per day. The dose of the active agents may be provided in either a single dosage form, two separate dosage forms, or three separate dosage forms.

[0095] However, it will be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the particular compound used, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular condition or disease being treated.

[0096] The formulation of the present invention can be administered in various embodiments by injection, or can be prepared for oral, pulmonary, nasal or any other form of administration.Preferably, the formulation is administered by intravenous, subcutaneous, intramuscular, intraorbital, intraocular, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal or aerosol administration, for example, orally or via a gastric feeding tube.

[0097] The mode of administration is, in one aspect, at least suitable for the form in which the formulation is prepared. The mode of administration for the most effective response can be empirically determined, and the administration means described below are given as examples and in no way limit the method of delivery of the formulations of the present invention. All formulations provided are commonly used in the pharmaceutical industry and generally known to appropriately qualified physicians.

[0098] Injectable dosage form The formulation of the present invention in certain embodiments may contain pharmaceutically acceptable non-toxic excipients and carriers and may be administered by any parenteral technique, such as subcutaneous, intravenous and intraperitoneal injection.In addition, the formulation may contain one or more adjuvants as required.As used herein, a "pharmaceutical carrier" is a pharmaceutically acceptable solvent, suspending agent, excipient or vehicle for delivering a compound to a subject.The carrier may be liquid or solid, and is selected with the planned mode of administration in mind.

[0099] Pharmaceutical forms suitable for injection use include sterile aqueous solutions (if water soluble) or dispersions as necessary, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. Alternatively, the compounds of the present invention are in certain embodiments encapsulated in liposomes and delivered in injections to aid their transport across cell membranes. Alternatively, or in addition, such preparations contain components of self-assembling pore structures to facilitate transport across cell membranes. The carrier is, in various embodiments, a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by a coating such as, but not limited to, lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants.

[0100] The present invention also provides for the prolonged absorption of an injectable sustained release pharmaceutical formulation comprising a therapeutically effective pharmaceutical formulation according to the present invention and a release retardant, which can be, for example, aluminum monostearate and gelatin.

[0101] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent together with one or more other components as listed above as necessary, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and other necessary components from those listed above.For the preparation of sterile powder for sterile injectable solution, in certain embodiments, the preparation includes, but is not limited to, vacuum drying and freeze-drying techniques, which obtain the powder of active ingredient + any additional desired components from the solution that has been previously sterile filtered.

[0102] Oral dosage form Contemplated for use herein are oral dosage forms generally described in Chapter 89 of Martin, Remington's Pharmaceutical Sciences, 18th Ed. (1990 Mack Publishing Co. Easton PA 18042), which is incorporated herein by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, liquids, suspensions or pellets. Liposomal or proteinoid encapsulation can also be used to formulate the formulations of the present invention (e.g., as proteinoid microspheres, as reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation can also be used, and liposomes can be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms of therapeutic agents is described by Marshall in Modern Pharmaceutics, Chapter 10, Banker and Rhodes ed., (1979), which is incorporated herein by reference. In general, the formulations include a compound described as part of the present invention (or a chemically modified form thereof) and inert ingredients that allow for protection from the stomach environment and release of the biologically active material in the intestine.

[0103] The oral dosage form of the present invention may be a liquid, suspension or other suitable dosage form delivered directly to the feeding tube of a patient who is in a coma and / or on a ventilator and is unable to swallow tablets, etc.

[0104] For the CCR2 pathway inhibitor, endothelin A receptor inhibitor or AT1R blocker of the present invention, the location of release can be stomach, small intestine (duodenum, jejunum, or ileum), or large intestine.Those skilled in the art have available formulations that do not dissolve in the stomach but release material in the duodenum or other places in the intestine.In one embodiment, release avoids the harmful effects of the stomach environment by protection of the formulation or by release of compound beyond the stomach environment, such as the duodenum or other places in the intestine.

[0105] A coating or mixture of coatings not intended for protection against the stomach may also be used on tablets. This includes, but is not limited to, sugar coatings, or coatings that make the tablet easier to swallow. An exemplary capsule consists of a hard shell (such as gelatin) for delivery of dry therapeutics, i.e., powders, and for liquid forms, a soft gelatin shell may be used. The shell material for cachets in certain embodiments is thick starch or other edible paper. For pills, lozenges, molded tablets, or tablet triturates, moist massing techniques are also contemplated, but are not limited to these.

[0106] As used herein, the term "sustained release" refers to the gradual but continuous or sustained release of therapeutic compound content over a relatively long period of time after oral ingestion. The release may continue after the pharmaceutical formulation has passed from the stomach until and after the pharmaceutical formulation reaches the intestine. The phrase "sustained release" also refers to delayed release in which the release of the therapeutic compound does not begin immediately when the pharmaceutical formulation reaches the stomach, but is delayed for a period of time, for example, until the pharmaceutical formulation reaches the intestine. Once in the intestine, an increase in pH may trigger the release of the therapeutic compound from the pharmaceutical formulation.

[0107] The term "release retardant" as used herein means a substance that slows the release rate of a therapeutic compound from a pharmaceutical formulation when taken orally. The release retardant may be polymeric or non-polymeric. The release retardant may be used according to any one of several sustained release systems, including, for example, a diffusion system, a dissolution system, and / or an osmotic system.

[0108] In certain embodiments, the therapeutic agent is included in the formulation as fine multiparticulates in the form of granules or pellets with a particle size of about 1 mm. The formulation of the material for capsule administration is, in certain embodiments, a powder, lightly compressed plugs, or even tablets. In one embodiment, the therapeutic agent can be prepared by compression.

[0109] Colorants and flavoring agents are optionally included. For example, the compounds can be formulated (such as, but not limited to, liposomal or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage, that contains colorants and flavoring agents.

[0110] In one embodiment, the volume of the therapeutic agent is diluted or increased with an inert material. These diluents may include carbohydrates, particularly mannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans, and starch. Certain inorganic salts are also used as fillers, if necessary, including calcium triphosphate, magnesium carbonate, and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

[0111] In other embodiments, disintegrants are included in the formulation of the therapeutic agent into solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including the commercially available disintegrants based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge and bentonite are also contemplated. Another form of disintegrant is the insoluble cationic exchange resin. Powdered gums are also optionally used as disintegrants and binders, including, but not limited to, powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0112] Binders are intended to hold the therapeutic compounds together to form a hard tablet and include, but are not limited to, materials from natural products such as acacia, tragacanth, starch and gelatin. Other binders include, but are not limited to, methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) are intended for use in alcoholic solutions to granulate the therapeutic agent.

[0113] Antifriction agents may be optionally included in the formulation of the therapeutic agent to prevent sticking during the formulation process.Lubricants may be optionally used as a layer between the therapeutic agent and the die wall, and may include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes.Exemplary soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, and Carbowax 4000 and 6000 may also be used.

[0114] Glidants may be added as needed to improve the flow properties of the compound during formulation and potentially aid rearrangement during compression. Glidants may include, but are not limited to, starch, talc, pyrogenic silica, and hydrated silicoaluminate.

[0115] To aid in the dissolution of therapeutic agents in aqueous environments, in certain embodiments, surfactants may be added as wetting agents.Surfactants may include, for example, but are not limited to, anionic surfactants such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate.Cationic surfactants may be used as needed, and may include, but are not limited to, benzalkonium chloride or benzethonium chloride.A list of possible nonionic surfactants that may be included in the formulation as surfactants is lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose.When used, these surfactants may be present in the formulation of the compound alone or as a mixture of different ratios.

[0116] Excipients that potentially enhance uptake of the compound include, but are not limited to, the fatty acids oleic acid, linoleic acid, and linolenic acid.

[0117] Controlled release formulations may be desirable. These formulations are also contemplated. In certain embodiments, the compound can be incorporated into an inert matrix, i.e., gum, that allows release by either diffusion or leaching mechanisms. In some embodiments, a slowly degrading matrix may also be incorporated into the formulation. Another form of controlled release of this therapeutic agent is by a method based on the Oros Therapeutic System (Alza Corp.), i.e., the drug is encapsulated in a semipermeable membrane that allows water to enter a single small opening and push the drug out by osmotic effect. Some enteric coatings also have a delayed release effect.

[0118] In other embodiments, a mixture of materials may be used to provide the optimum film coating. Film coating can be performed, for example, but not limited to, in a pan coater, or in a fluidized bed, or by compression coating.

[0119] Pulmonary and nasal dosage forms Pulmonary delivery of the formulations of the present invention is also contemplated herein. In these embodiments, the CCR2 pathway inhibitor, endothelin A receptor inhibitor, or AT1R blocker can be delivered to the subject's lungs while inhaling and travel across the lung epithelial lining to the bloodstream.

[0120] Contemplated for use in the practice of the present invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including, but not limited to, nebulizers, metered dose inhalers and dry powder inhalers, all of which are well known to those skilled in the art.

[0121] Some specific examples of commercially available devices suitable for the practice of the present invention include, but are not limited to, the Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Massachusetts.

[0122] All such devices require the use of a formulation suitable for dispensing the compound. Typically, each formulation is specific to the type of device used, and may include the use of a suitable propellant material in addition to the usual diluents, adjuvants and / or carriers useful in therapy. Also contemplated is the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers.

[0123] The formulation suitable for use with either jet or ultrasonic nebulizer typically comprises a compound suspended in water.The formulation may also comprise, in one aspect, a buffer and a simple sugar (e.g., for protein stabilization and osmotic pressure regulation).In one embodiment, the nebulizer formulation may also contain a surfactant to reduce or prevent surface-induced aggregation of the compound caused by atomization of the solution when forming an aerosol.

[0124] Formulations for use with metered dose inhalation devices generally comprise a fine powder containing the compound suspended in a propellant, in one embodiment with the aid of a surfactant. The propellant may be any conventional material used for this purpose, such as, but not limited to, chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2 tetrafluoroethane, or combinations thereof. Suitable surfactants include, but are not limited to, sorbitan trioleate and soybean lecithin. Oleic acid may also be useful as a surfactant in certain embodiments.

[0125] Formulations for dispensing from powder inhalation devices include a dry fine powder containing the compound and may include a bulking agent such as, but not limited to, lactose, sorbitol, sucrose, or mannitol in an amount to facilitate dispersion of the powder from the device, e.g., 50-90% by weight of the formulation. In certain embodiments, the compound(s) are prepared in a particulate form having an average particle size of less than 10 microns, most preferably 0.5-5 microns, for most effective delivery to the distal lung.

[0126] Nasal delivery of compounds is also contemplated.Nasal delivery allows the passage of proteins directly into the bloodstream without the need to deposit the product in the lungs after administering therapeutic products to the nose.Formulations for nasal delivery include, but are not limited to, formulations that contain dextran or cyclodextran.

[0127] Dosage schedule It will be appreciated that in certain embodiments, the formulations of the present invention can be given as a single dose schedule, or preferably as a multiple dose schedule. A multiple dose schedule is one in which the primary course of delivery may be 1 to 10 separate doses, optionally followed by other doses given at subsequent time intervals as needed to maintain or enhance treatment. The dosing regimen will also be determined, at least in part, by the individual's needs and the physician's judgment.

[0128] Thus, the present invention provides a tablet comprising the pharmaceutical formulation of the present invention, a capsule comprising the pharmaceutical formulation of the present invention, an injectable suspension comprising the pharmaceutical formulation of the present invention, and a pulmonary formulation comprising the pharmaceutical formulation of the present invention. The endothelin A receptor inhibitor, the AT1R blocker and / or the CCR2 pathway inhibitor may be delivered in the same formulation or in separate formulations.

[0129] The endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor may be in the same dosage form or in separate dosage forms. The subject who is administered the endothelin A receptor inhibitor, AT1R blocker and / or CCR2 pathway inhibitor may already be receiving one or more active agents, and according to the present invention, may be administered other component(s) of the treatment of the present invention. The treatment of the present invention may include administration of only the CCR2 pathway inhibitor and the endothelin A receptor inhibitor, or may include administration of the CCR2 pathway inhibitor, the endothelin A receptor inhibitor and the AT1R blocker.

[0130] Excipients The CCR2 pathway inhibitor, ETAR inhibitor and AT1R blocker may be a pharma- ceutically acceptable salt of each active agent. Pharmaceutically and veterinarily acceptable salts include salts that retain the biological effectiveness and properties of the disclosed compounds and are not biologically or otherwise undesirable. In many cases, the compounds disclosed herein can form acid salts and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto. Acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines such as, by way of example only, alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkyl amines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkyl amines, disubstituted cycloalkyl amines, trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, )amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed diamines and triamines in which at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, etc. Also included are amines in which two or three of the substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group.

[0131] Pharmaceutically and veterinarily acceptable acid addition salts can be prepared from inorganic and organic acids. Inorganic acids that can be used include, by way of example only, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids that can be used include, by way of example only, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

[0132] The pharmaceutically or veterinarily acceptable salts of the compounds useful in the present disclosure can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods.In general, such salts can be prepared by reacting the free acid or free base form of these compounds with stoichiometric amounts of suitable base or acid in water or organic solvent, or in a mixture of the two, and generally non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.A list of suitable salts can be found in Remington's Pharmaceutical Sciences.17th ed., Mack Publishing Company, Easton, Pa.(1985), p.1418, the disclosure of which is incorporated herein by reference.Examples of such acceptable salts are iodide, acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, gamma-hydroxybutyrate, beta-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, hexyne-1,6-dioate, caprolate, caprate, chloride, cinnamate, citrate, decanoate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxymale ... eate), malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, propanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-I-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartrate, and the like.

[0133] use The present invention also relates to a method for producing a dosage form for treating, ameliorating or preventing a disease. a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; The present invention contemplates the use of a pharmaceutical composition comprising:

[0134] The present invention further relates to a method for producing a dosage form for treating, ameliorating or preventing a disease. a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) inhibitor or a pharma- ceutically acceptable salt thereof; c) at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof; The present invention contemplates the use of a pharmaceutical composition comprising:

[0135] The present invention provides at least one endothelin A receptor inhibitor, or a pharma- ceutically acceptable salt thereof, and at least one chemokine receptor 2 (CCR2) inhibitor, or a pharma- ceutically acceptable salt thereof, for use in a formulation for treating, ameliorating or preventing a disease.

[0136] The present invention provides at least one endothelin A receptor inhibitor, or a pharma- ceutically acceptable salt thereof, at least one chemokine receptor 2 (CCR2) inhibitor, or a pharma- ceutically acceptable salt thereof, and at least one angiotensin type 1 receptor (AT1R) blocker, or a pharma- ceutically acceptable salt thereof, for use in a formulation for the treatment, amelioration, or prevention of a disease.

[0137] The present invention provides the following: at least one endothelin A receptor inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with at least one CCR2 pathway inhibitor. at least one CCR2 pathway inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor. at least one endothelin A receptor inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with at least one CCR2 pathway inhibitor and at least one angiotensin type 1 receptor blocker. at least one CCR2 pathway inhibitor for use in a formulation for the treatment, amelioration or prevention of a disease, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor and at least one angiotensin type 1 receptor blocker. At least one angiotensin type 1 receptor blocker for use in a formulation for the treatment, amelioration or prevention of a disease, wherein at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor and at least one CCR2 pathway inhibitor.

[0138] Preferably, the disease is renal disease, pulmonary disease or cardiac disease.Renal disease can be selected from the list including focal segmental glomerulosclerosis (FSGS; including primary FSGS and secondary FSGS), fibrotic disorders in the kidney, chronic renal disease caused by diabetic nephropathy, renal failure (diabetic and non-diabetic), and renal failure symptoms including diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, proteinuria of primary renal disease and renal vascular hypertension.Renal disease can be infectious or non-infectious inflammatory lung disease, preferably selected from the list including community-acquired pneumonia, chronic obstructive pulmonary disease, asthma, bronchiectasis, bronchiolitis, bronchitis, emphysema, pleurisy or pulmonary fibrosis.Cardiac disease can be coronary artery disease, preferably selected from the list including arthrosclerosis, pulmonary hypertension, ischemic heart disease, cardiomyopathy and inflammatory heart disease.

[0139] The endothelin A receptor inhibitor, the CCR2 pathway inhibitor and / or the AT1R blocker can be administered in the same dosage form or in separate dosage forms. The endothelin A receptor inhibitor, the CCR2 pathway inhibitor and the AT1R blocker can be administered simultaneously or sequentially.

[0140] The endothelin A receptor inhibitor, CCR2 pathway inhibitor and / or AT1R blocker may be a pharma- ceutically acceptable salt of the endothelin A receptor inhibitor, CCR2 pathway inhibitor and / or AT1R blocker.

[0141] kit The present invention relates to a kit for treating, ameliorating, or preventing a disease, the kit comprising: a) at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; c) providing a kit for treating, ameliorating or preventing a disease, comprising:

[0142] The present invention relates to a kit for treating, ameliorating, or preventing a disease, the kit comprising: a) at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; c) at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof; d) instructions for use.

[0143] The contents of the kit can be lyophilized and the kit can further comprise a suitable solvent for reconstitution of the lyophilized components. The individual components of the kit can be packaged in separate containers and associated with such containers can be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which notice reflects approval by the agency of the manufacture, use, or sale for human administration.

[0144] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example a sterile aqueous solution. For in vivo use, the expression construct can be formulated into a pharma-ceutically acceptable injectable composition. In this case, the container means can itself be an inhalant, syringe, pipette, eye dropper, or other such device from which the formulation can be applied to the affected area of ​​the animal, such as the lungs, injected into the animal, or applied to and mixed with other components of the kit.

[0145] The components of the kit may also be provided in dry or lyophilized form. When the reagent or components are provided as dry form, reconstitution is generally by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. Regardless of the number or type of container, the kit of the present invention may also include or be packaged with an instrument to assist in the injection / administration or placement of the final complex composition into the animal's body. Such an instrument may be an inhalant, a syringe, a pipette, forceps, a measuring spoon, an eye dropper, or any such medically approved delivery vehicle.

[0146] general Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described.The invention includes all such variations and modifications.The invention also includes all of the steps, features, preparations and compounds mentioned or shown herein, individually or collectively, and includes any and all combinations of steps or features or any two or more of them.

[0147] Each document, reference, patent application or patent cited herein is expressly incorporated herein in its entirety by reference, meaning that it should be read and considered by the reader as part of this specification. It is for the sake of brevity only that documents, references, patent applications or patents cited herein are not repeated herein.

[0148] Any manufacturer's instructions, manuals, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein are incorporated by reference herein and may be used in the practice of the present invention.

[0149] The scope of the present invention is not limited by any of the specific embodiments described herein. These embodiments are for illustrative purposes only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0150] The invention described herein can include one or more ranges of values ​​(e.g., size, displacement, and field strength, etc.). A range of values ​​is understood to include all values ​​within the range, including the values ​​defining the range and the values ​​adjacent to the range that produce the same or substantially the same results as the values ​​immediately adjacent to the values ​​defining the boundaries for the range. Thus, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. Thus, "about 80%" means "about 80%" and also means "80%". At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0151] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of a recited integer or group of integers but not the exclusion of any other integer or group of integers. Also, in this disclosure, particularly in the claims and / or paragraphs, it is noted that terms such as "comprises," "comprised," "comprising," and the like can have the meaning ascribed to them in U.S. patent law. For example, they can mean "includes," "included," "including," and the like. Terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, for example, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention.

[0152] Other definitions of selected terms used herein can be found in the detailed description of the present invention and can be applied throughout.Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.The term "active agent" can mean one active agent or can include two or more active agents.

[0153] The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention, it being understood that these methods in no way serve to limit the true scope of the invention, but rather are presented for illustrative purposes. References · Taylor et al,(2011)J Clin Hypertens.13:677-686 · Rios et al.(2000)Pharmacol.Ther.92:71-87 · Pfleger et al.(2006)Cell Signal 18:1664-1670 · Pfleger et al. (2006) Nat Protoc 1:336-344 · Ayoub et al.(2015)PLoS One 10(3):e0119803 EXAMPLES

[0154] Further features of the present invention are more fully described in the following non-limiting examples, which are included solely for the purpose of illustrating the invention and should not be construed as a limitation on the broad description of the invention as set forth above.

[0155] Example 1 Assays for receptor functional interactions HEK293FT cells were seeded in 6-well plates at a density of approximately 700,000 cells / well and maintained at 37 °C and 5% CO2 in complete medium (DMEM (Thermo Fisher) containing 0.3 mg / ml glutamine, 100 IU / ml penicillin and 100 μg / ml streptomycin) supplemented with 10% fetal calf serum (FCS; Bovogen). 24 h after seeding, transient transfections were performed using FuGene6 (Promega) according to the manufacturer's instructions. 24 h after transfection, cells were washed with PBS, detached using 0.05% trypsin / 0.53 mM EDTA, resuspended in phenol red-free complete medium containing 5% FCS and added to poly-L-lysine-coated white 96-well microplates (Greiner Bio-One). 48 hours after transfection, cells were preincubated with EnduRen™ (Promega) at a final concentration of 30 μM for 2 hours at 37°C, 5% CO2, followed by a bioluminescence resonance energy transfer (BRET) assay. BRET measurements were performed at 37°C using a CLARIOstar (BMG Labtech). Filtered light emission was measured continuously for 1 second in each of the "donor wavelength window" (410-490 nm) and "acceptor wavelength window" (520-620 nm).

[0156] For antagonist assays, cells were pre-incubated in EnduRen for 2 hours. Antagonists (10 μM) were added and the assay was read for 30 minutes. Agonists (CXCL8 10 nM, AngII 100 nM) were then added and measurements continued for an additional hour.

[0157] The BRET signal observed between interacting proteins is normalized by subtracting the background BRET ratio. This can be done in one of two ways (see Pfleger et al. (2006) Cell Signal 18:1664-1670; Pfleger et al. (2006) Nat Protoc 1:336-344): 1) subtract the ratio of 520-550 nm emission to 460-490 nm emission for a cell sample containing only the donor construct from the same ratio for a sample containing the interacting acceptor and donor fusion proteins; 2) subtract the ratio of 520-550 nm emission to 460-490 nm emission for a vehicle-treated cell sample from the same ratio for a second aliquot of the same cell sample treated with ligand. In the following examples, the second calculation is used and the signal is described as "ligand-induced BRET".

[0158] Receptor-heteromer interrogation technology (receptor-HIT) is an assay configuration that provides insight into receptor complexes (Ayoub et al. (2015) PLoS One 10(3):e0119803). Receptor-HIT is also known as GPCR-HIT when evaluating GPCRs. It is an assay configuration in which one receptor (e.g., CCR2) is labeled with one component (e.g., Renilla luciferase variant Rluc8) of a proximity-based reporter system (e.g., BRET) and its complementary component (e.g., yellow fluorescent protein Venus) is fused to a receptor interaction partner (e.g., β-arrestin2). Treatment with a ligand (e.g., AngII) selective for the receptor that is not tagged for the BRET assay (e.g., hemagglutinin epitope-tagged AT1R; HA-AT1R) leads to modulation of the proximity of the BRET-tagged receptor to the interaction partner, resulting in a change in the BRET signal indicative of a functional interaction between the two receptors.

[0159] result Figure 1 is a graph of ETAR / Rluc8+Barr2 / Venus+CCR2, showing that when ETAR is tagged with Rluc8 and β-arrestin is tagged with Venus in the presence of unlabeled CCR2, β-arrestin is recruited to ETAR not only in the presence of ET-1, as expected, but also in the presence of CCL2 / MCP1, confirming the existence of a complex between the receptors. The presence of both ligands causes hyperactivation of β-arrestin recruitment, confirming this heteromeric interaction.

[0160] Figure 2 is a graph of ETAR / Rluc8+Barr2 / Venus+CCR2, showing that β-arrestin recruitment to ETAR occurs when CCL2 is present, confirming the interaction of ETAR with unlabeled CCR2. Sparsentan is able to suppress hyperactivation when both CCL2 / CMP-1 and ET-1 ligands are present but does not abolish β-arrestin recruitment to ETAR.

[0161] Figure 3 is a graph of ETAR / Rluc8+Barr2 / Venus+CCR2, showing that hyperactivation of ETAR and CCR2 heteromers is suppressed by the addition of either sparsentan inhibition or CCR2 inhibition. ETAR can recruit β-arrestin to a low extent when stimulated with ET-1, regardless of the presence of CCR2, and sparsentan inhibits this signaling independently of CCR2 activation. Complete inhibition of β-arrestin recruitment to ETAR occurs only in the presence of ET-1 and CCL2 / MCP-1 when CCR2 and ETAR are simultaneously inhibited.

[0162] Figure 4 is a graph of CCR2 / Rluc8+Barr2 / Venus+HA-ETAR, showing the different experimental orientations in which CCR2 is tagged with Rluc8 and β-arrestin is tagged with Venus in the presence of unlabeled ETAR, and β-arrestin is recruited to CCR2 in the presence of CCL2 / MCP-1. Increased recruitment of β-arrestin is observed in the presence of both CCL2 / MCP-1 and ET-1 ligands, confirming the presence of an interaction between CCR2 and ETAR.

[0163] FIG. 5 is a graph of CCR2 / Rluc8+Barr2 / Venus+HA-ETAR and shows that when ETAR activation is blocked with sparsetan, no increased recruitment of β-arrestin is observed in the presence of CCL2 / MCP-1 and ET-1.

[0164] FIG. 6 is a graph of CCR2 / Rluc8+Barr2 / Venus+HA-ETAR, which shows that when CCR2 is inhibited, with or without ETAR activation by ET-1, there is no recruitment of β-arrestin to CCR2 in the presence of CCL2 / MCP-1.

[0165] Example 2 formulation Exemplary formulations for use in the present invention. Table 1: Exemplary Formulations [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

Claims

1. 1. A pharmaceutical composition comprising: a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) pathway inhibitor, or a pharma- ceutically acceptable salt thereof.

2. At least one angiotensin type 1 receptor (AT １ 10. The pharmaceutical composition of claim 1, further comprising an R) blocking agent or a pharma- ceutically acceptable salt thereof.

3. 2. The pharmaceutical composition of claim 1, wherein the endothelin A receptor inhibitor is selected from the group consisting of endothelin A receptor antagonists; endothelin A and B dual antagonists; and heterologous receptor dual antagonists, and combinations thereof.

4. a) the endothelin A receptor antagonist is selected from the group comprising sitaxsentan, ambrisentan, atrasentan, BQ-123, zibotentan; b) the endothelin A and B dual antagonist is selected from the group comprising bosentan, macitentan, tezosentan, and / or c) the heterologous receptor dual antagonist is selected from the group comprising sparsentan; The pharmaceutical composition according to claim 3.

5. 2. The pharmaceutical composition of claim 1, wherein the CCR2 pathway inhibitor is selected from the group consisting of repagermanium; propagermanium; CCX140; cenicriviroc; PF-6309; PF-04178903; CCX872-B; MLN1202; and PF-04634817.

6. 3. The pharmaceutical composition of claim 2, wherein the AT 1 R blocker is selected from the group comprising sparsentan, irbesartan, eprosartan, losartan, valsartan, telmisartan, candesartan, olmesartan, ZD-7115, fimasartan, azilsartan, DuP 753, EXP 3174, embusartan, KRH-594, fonsartan, and pratosartan.

7. The CCR2 pathway inhibitor and the endothelin A receptor inhibitor are (i) in the same dosage form or in separate dosage forms; and / or (ii) Simultaneously or sequentially The pharmaceutical composition according to claim 1 or 2,

8. The CCR2 pathway inhibitor, the endothelin A receptor inhibitor and the AT １ R blockers are (i) in the same dosage form or in separate dosage forms; and / or (ii) Simultaneously or sequentially The pharmaceutical composition according to claim 2 .

9. For the manufacture of a dosage form for the treatment, amelioration or prevention of a disease, a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof; Use of.

10. The dosage form comprises at least one angiotensin type 1 receptor (AT １ 10. The use according to claim 9, further comprising an R) blocking agent or a pharma- ceutically acceptable salt thereof.

11. The present invention relates to a pharmaceutical composition for the manufacture of a dosage form for the treatment, amelioration or prevention of a disease, the pharmaceutical composition comprising at least one endothelin A receptor inhibitor or a pharma- ceutical acceptable salt thereof, at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutical acceptable salt thereof, and at least one angiotensin type 1 receptor (AT １ R) Use of a blocking agent or a pharma- ceutically acceptable salt thereof.

12. Use of at least one endothelin A receptor inhibitor or a pharma- ceutical acceptable salt thereof for the manufacture of a dosage form for the treatment, amelioration or prevention of a disease, wherein the at least one endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutical acceptable salt thereof.

13. Use of at least one chemokine receptor 2 (CCR2) pathway inhibitor, or a pharma- ceutical acceptable salt thereof, for the manufacture of a dosage form for the treatment, amelioration or prevention of a disease, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor, or a pharma- ceutical acceptable salt thereof.

14. Use of at least one endothelin A receptor inhibitor or a pharma- ceutical acceptable salt thereof for the manufacture of a dosage form for the treatment, amelioration or prevention of a disease, wherein the at least one endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutical acceptable salt thereof and at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutical acceptable salt thereof.

15. Use of at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutical acceptable salt thereof for the manufacture of a dosage form for the treatment, amelioration or prevention of a disease, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor or a pharma- ceutical acceptable salt thereof and at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutical acceptable salt thereof.

16. Use of at least one angiotensin type 1 receptor (AT 1 R) blocker or a pharma- ceutically acceptable salt thereof for the manufacture of a dosage form for the treatment, amelioration or prevention of a disease, wherein the at least one AT 1 R blocker is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof and at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof.

17. The use of any one of claims 9 to 16, wherein the endothelin A receptor inhibitor is selected from the group consisting of endothelin A receptor antagonists; endothelin A and B dual antagonists; and heterologous receptor dual antagonists, and combinations thereof.

18. a) the endothelin A receptor antagonist is selected from the group including sitaxsentan, ambrisentan, atrasentan, BQ-123, and zibotentan; b) the endothelin A and B dual antagonist is selected from the group comprising bosentan, macitentan, tezosentan, and / or c) the heterologous receptor dual antagonist is selected from the group comprising sparsentan; 18. The use according to claim 17.

19. The use of any one of claims 9 to 16, wherein the CCR2 pathway inhibitor is selected from the group including repagermanium; propagermanium; CCX140; cenicriviroc; PF-6309; PF-04178903; CCX872-B; MLN1202; and PF-04634817.

20. The use of any one of claims 10, 11, or 14-16, wherein the AT1R blocker is selected from the group including sparsentan, irbesartan, eprosartan, losartan, valsartan, telmisartan, candesartan, olmesartan, ZD-7115, fimasartan, azilsartan, DuP 753, EXP 3174, embusartan, KRH-594, fonsartan, and pratosartan.

21. The CCR2 pathway inhibitor and the endothelin A receptor inhibitor, (i) in the same dosage form or in separate dosage forms; and / or (ii) Simultaneously or sequentially The use according to any one of claims 9 to 16, wherein the drug is administered.

22. The CCR2 pathway inhibitor, the endothelin A receptor inhibitor and the AT1R blocker are (i) in the same dosage form or in separate dosage forms; and / or (ii) Simultaneously or sequentially The use according to any one of claims 10, 11 or 14 to 16, wherein the drug is administered

23. The use according to any one of claims 9 to 22, wherein the disease is a renal disease, a pulmonary disease or a cardiac disease.

24. The renal disease is selected from the list including focal segmental glomerulosclerosis (FSGS; including primary FSGS and secondary FSGS), fibrotic disorders in the kidney, chronic kidney disease caused by diabetic nephropathy, renal failure (diabetic and non-diabetic), and renal failure symptoms including diabetic nephropathy, glomerulonephritis, scleroderma, glomerulosclerosis, proteinuria of primary kidney disease, and renal vascular hypertension. said pulmonary disease is an infectious or non-infectious inflammatory pulmonary disease, optionally selected from the list comprising community-acquired pneumonia, chronic obstructive pulmonary disease, asthma, bronchiectasis, bronchiolitis, bronchitis, emphysema, pleurisy or pulmonary fibrosis; and / or The heart disease is a coronary artery disease, optionally selected from the list comprising arthrosclerosis, pulmonary hypertension, ischemic heart disease, cardiomyopathy, and inflammatory heart disease.

24. The use according to claim 23.

25. 1. A kit for use in the treatment, amelioration or prevention of a disease, said kit comprising: a) at least one chemokine receptor 2 (CCR2) pathway inhibitor, or a pharma- ceutically acceptable salt thereof; b) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; and c) instructions for use.

26. 26. The kit of claim 25, further comprising at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof.

27. ​​A combination for the treatment, amelioration or prevention of a disease, comprising: a) at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof; b) at least one chemokine receptor 2 (CCR2) pathway inhibitor, or a pharma- ceutically acceptable salt thereof; A combination comprising:

28. A combination for the treatment, amelioration or prevention of a disease, comprising at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof, at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof, and at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof.

29. A dosage form for the treatment, amelioration or prevention of a disease, comprising at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof, wherein said dosage form is administered to a subject simultaneously or sequentially with at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof.

30. A dosage form for the treatment, amelioration or prevention of a disease, comprising at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof.

31. A dosage form for the treatment, amelioration, or prevention of a disease, comprising at least one endothelin A receptor inhibitor or a pharma- ceutically acceptable salt thereof, wherein the at least one endothelin A receptor inhibitor is administered to a subject simultaneously or sequentially with at least one chemokine receptor 2 (CCR2) pathway inhibitor or a pharma- ceutically acceptable salt thereof and at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof.

32. A dosage form for the treatment, amelioration, or prevention of a disease, comprising at least one chemokine receptor 2 (CCR2) pathway inhibitor, or a pharma- ceutically acceptable salt thereof, wherein the at least one CCR2 pathway inhibitor is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor, or a pharma- ceutically acceptable salt thereof, and at least one angiotensin type 1 receptor (AT1R) blocker, or a pharma- ceutically acceptable salt thereof.

33. A dosage form for the treatment, amelioration, or prevention of a disease, comprising at least one angiotensin type 1 receptor (AT1R) blocker or a pharma- ceutically acceptable salt thereof, wherein the at least one AT1R blocker is administered to a subject simultaneously or sequentially with at least one endothelin A receptor inhibitor, or a pharma- ceutically acceptable salt thereof, and at least one chemokine receptor 2 (CCR2) pathway inhibitor, or a pharma- ceutically acceptable salt thereof.

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