Precision treatment of heart failure and cardiorenal syndrome

Abstract

This invention provides a method for treating diuretic resistance by administering an IL-6 antagonist to a patient requiring polyuria. This invention also provides a method for treating heart failure and, particularly, renal symptoms of heart failure, by administering an IL-6 antagonist to a patient with heart failure who has at least one copy of the major paired gene TMPRSS6 rs855791. Depending on the situation, the patient may have elevated urinary IL-6 levels, elevated plasma IL-6 levels, or both elevated urinary and plasma IL-6 levels. This invention also provides a method for detecting cardiorenal syndrome in a patient, comprising measuring the IL-6 level in the patient's urine or plasma.

Description

Technical Field

[0001] none Prior Technology

[0002] Kidney damage often occurs in heart failure, and heart failure often occurs in kidney disease. The term cardiorenal syndrome ("CRS") encompasses a variety of clinical conditions in which dysfunction of the heart, kidneys, or both leads to accelerated failure of both organs. This cross-infection of organs is a predictor of high morbidity and mortality. Despite its dire clinical impact, the mechanistic basis of CRS is only now beginning to be elucidated.

[0003] New methods are needed to treat kidney damage and injury in patients with heart failure. New methods are also needed to detect cardiorenal syndrome to determine which patients will respond to treatment and monitor its effectiveness. Summary of the Invention

[0004] As further described in Example 1 below, a prospective observational study will be conducted to enroll patients with continuous heart failure ("HF") receiving high-dose diuretic therapy at an outpatient treatment center. Plasma IL-6 levels will be measured to explore the systemic association between this pro-inflammatory cytokine and various disease parameters, and urinary IL-6 levels will be measured to explore the association between IL-6 and local inflammation and neurohormonal activation at the renal tissue level.

[0005] The study found little correlation between urinary IL-6 levels and plasma IL-6 levels.

[0006] In these patients with heart failure, elevated urinary IL-6 levels were significantly associated with measures of kidney damage, such as diuretic resistance, underestimation of glomerular filtration rate ("eGFR"), and increased tissue-level renin-angiotensin-aldosterone system ("RAAS") activation.

[0007] Although an inverse association was observed between diuretic efficiency and plasma IL-6, after adjusting for eGFR, only urinary IL-6 maintained a significant association with the risk of low diuretic efficiency in these patients. Furthermore, when both urinary and plasma IL-6 were input into the logistic regression model, only urinary IL-6 maintained an association with the risk of low diuretic efficiency, while plasma IL-6 showed no such association.

[0008] These data indicate that urinary IL-6 levels are a useful biomarker for kidney inflammation and can be used to measure renal dysfunction in patients with heart failure (cardiorenal syndrome). The data further indicate that continuous measurement of urinary IL-6 can be used to measure the renal efficacy of treatment administered to patients with heart failure, especially those with cardiorenal syndrome.

[0009] Urinary IL-6 data, and to some extent plasma IL-6 data, also predict that treatment with IL-6 antagonists should be effective in reducing kidney inflammation in patients with heart failure, i.e., in treating kidney symptoms of cardiorenal syndrome.

[0010] However, because infection is often a trigger for acute decompensation in patients with heart failure, it is important to limit anti-cytokine and other immunosuppressive therapies to these patients with potentially improved renal and / or cardiac function. The cost of long-term IL-6 antagonist therapy also limits treatment for these patients with potentially improved renal and / or cardiac function.

[0011] The analysis was expanded to 129 patients, and the genotype of the rs855791 single nucleotide polymorphism ("SNP") in transmembrane protease serine 6 ("TMPRSS6") was further evaluated in each patient.

[0012] Urinary IL-6 levels were inversely associated with diuretic efficiency only in patients with at least one copy of the major paired gene (AG and GG) of the TMPRSS6 rs855791 SNP; urinary IL-6 levels were not significantly associated with diuretic efficiency in patients with isotype conjugation of the minor paired gene (AA). Plasma IL-6 levels were inversely associated with diuretic efficiency only in patients with at least one copy of the major paired gene of the TMPRSS6 rs855791 SNP; plasma IL-6 levels were not associated with diuretic efficiency in patients with isotype conjugation of the minor paired gene.

[0013] In mouse M1 CCD cells, which are genotype-similar to human cells with the major pairing gene TMPRSS6 rs855791, the addition of IL-6 is associated with the expression of ion transport proteins NKCC2, ENaC-β, and NCC.

[0014] These data suggest that treatment of heart failure with IL-6 antagonists will improve renal symptoms in heart failure patients who have at least one copy of the TMPRSS6 rs855791 major paired gene.

[0015] The data described in the examples also demonstrate the association between IL-6 and diuretic response.

[0016] Accordingly, in the first instance, a method is provided for treating patients who require polyuria but are resistant to diuretics. The method involves administering a therapeutically effective dose of an IL-6 antagonist in combination with a diuretic to the patient.

[0017] In some embodiments, the patient has a high pre-treatment plasma IL-6 level. In some embodiments, the patient's pre-treatment plasma IL-6 level is greater than 2 pg / mL. In some embodiments, the patient's pre-treatment IL-6 level is greater than 3 pg / mL. In some embodiments, the patient's pre-treatment IL-6 level is greater than 5 pg / mL. In some embodiments, the patient's pre-treatment IL-6 level is greater than 10 pg / mL.

[0018] In some embodiments, the patient's diuretic efficiency is less than 500. In some embodiments, the patient's diuretic efficiency is less than 200. In some embodiments, the patient's diuretic efficiency is less than 150. In some embodiments, the patient's diuretic efficiency is less than 100.

[0019] In some embodiments, the patient suffers from antidiuretic heart failure. In some embodiments, the patient suffers from acute heart failure. In some embodiments, the patient suffers from chronic heart failure.

[0020] In some embodiments, the patient suffers from cardiorenal syndrome. In some of these embodiments, the patient suffers from type 4 cardiorenal syndrome.

[0021] In some embodiments, the patient has kidney disease. In other embodiments, the patient has hepatorenal syndrome.

[0022] In some embodiments, the IL-6 antagonist is an anti-IL-6 antibody or its antigen-binding fragment or derivative. In specific embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative binds to human IL-6 with a KD of less than 100 nM, less than 50 nM, less than 10 nM, or even less than 1 nM.

[0023] In some embodiments, the elimination half-life following intravenous administration of the anti-IL-6 antibody or antigen-binding fragment or derivative is at least 7 days, at least 14 days, at least 21 days, or at least 30 days.

[0024] In some embodiments, the IL-6 antagonist is a full-length monoclonal anti-IL-6 antibody. In specific embodiments, it is an anti-systemic IgG1 or IgG4 antibody. In some embodiments, it is an anti-systemic IgG1 antibody.

[0025] In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is wholly human. In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is humanized.

[0026] In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable regions (CDRs) of MEDI5117. In specific embodiments, the antibody comprises the VH and VL regions of MEDI5117. In particular embodiments, the anti-IL-6 antibody system comprises MEDI5117.

[0027] In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable regions (CDRs) of an antibody selected from the group consisting of: siltuximab, gerilimzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises a heavy chain V region and a light chain V region of an antibody selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymec, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0028] In some embodiments, the IL-6 antagonist is a single-domain antibody, a VHH nanoantibody, a Fab, or a scFv.

[0029] In some embodiments, the IL-6 antagonist is an anti-IL-6R antibody or its antigen-binding fragment or derivative. In some embodiments, the anti-IL-6R antibody, antigen-binding fragment, or derivative comprises six CDRs of tocilizumab or vobarilizumab.

[0030] In some embodiments, the IL-6 antagonist is a JAK inhibitor. In some embodiments, the JAK inhibitor is selected from the group consisting of: tofacitinib (Xeljanz), decernotinib, ruxolitinib, upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986, and AC-410.

[0031] In some embodiments, the IL-6 antagonist is a STAT3 inhibitor.

[0032] In some embodiments, the IL-6 antagonist is administered non-enterally. In certain embodiments, the IL-6 antagonist is administered subcutaneously.

[0033] In some embodiments, the IL-6 antagonist is administered orally.

[0034] In some embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to improve diuretic efficiency. In some embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to improve diuretic efficiency to normal levels. In some embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to improve eGFR. In specific embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to improve eGFR to normal levels.

[0035] In some embodiments, the method further includes the following subsequent steps: measuring IL-6 levels in urine, measuring IL-6 levels in plasma, or measuring IL-6 levels in both urine and plasma. In a particular embodiment, the method further includes a final step of adjusting the dose of the IL-6 antagonist for subsequent administration based on the IL-6 levels measured immediately preceding the previous step. Simple Explanation of the Diagram

[0036] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the exact configurations and means of the embodiments shown in the drawings.

[0037] [[] [picture] [1A] is a bar graph showing the diuretic efficiency ("DE") of urinary IL-6 in 129 patients with heart failure ("HF") who received high-dose diuretic therapy in the study described in Example 1 of this article. The bar extends from the 10th percentile to the 90th percentile.

[0038] [[] [picture] [1B] is a bar graph showing the diuretic efficiency (DE) of plasma IL-6 in 129 patients with heart failure ("HF"), as presented in Figure 1A. The bar extends from the 10th to the 90th percentile.

[0039] [[] [picture] [2A]] Depicting the urinary IL-6 group DE in patients reported in Figures 1A and 1B, which are further grouped according to genotype. The left panel shows the results of patients who are homotypic of the TMPRSS6 rs855791 SNP minor pair (2321G→A; A736V), and the right panel shows the results of patients who have at least one copy of the TMPRSS6 rs855791 SNP major pair (2321G; A736).

[0040] [[] [picture] [2B]] Depicting the plasma IL-6 groupings of patients reported in Figures 1A and 1B according to further genotype grouping, the left panel shows the results of patients with isotype conjugation of the TMPRSS6 rs855791 SNP minor pair (2321G→A; A736V), and the right panel shows the results of patients with at least one copy of the TMPRSS6 rs855791 SNP major pair (2321G; A736).

[0041] [[] [picture] [3A] [and] [[] [picture] [3B]] This figure depicts the association between (A) urinary IL-6 levels and (B) plasma IL-6 levels and the following clinical measurements in a subgroup of 98 out of 129 patients reported in Figures 1 and 2: decreased renal function, low diuretic efficiency ("DE"), increased neurohormonal activation, and risk of death. The whisker line represents the 95% confidence interval ("CI"). All analyses were adjusted for urinary and plasma IL-6 levels. Urinary IL-6 levels were measured using urinary creatinine. Due to the skewed distribution of urinary and plasma IL-6 variables, log transformation was applied before logarithmic and Cox regressions. [ ]

[0042] [Abbreviations]: OR = Odds ratio. HR = Hazard ratio. IL = Interleukin. SD = Standard deviation. eGFR = Estimated glomerular filtration rate. * = Adjusted for the use of angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin receptor blockers (ARBs). ** = Adjusted for baseline characteristics, including age, species, N-terminal B-type natriuretic peptide (NT-BNP), use of ACE-I or ARBs, home loop diuretic dosage, and eGFR.

[0043] [[] [picture] [4A]]、 [[] [picture] [4B] and [[] [picture] [4C] shows the expression of NKCC2, ENaC-β and NCC in M1 CCD cells after treatment with IL-6 and / or ruzotinib. [picture] [4A] Demonstrates the performance of NKCC2 after treatment with IL-6 and / or ruzotinib; [picture] [4B] Demonstrates the performance of ENaC-β after treatment with IL-6 and / or ruzoutinib; and [picture] [4C] Demonstrates the performance of NCC after treatment with IL-6 and / or ruzotinib.

[0044] [[] [picture] [5A]]、 [[] [picture] [5B] and [[] [picture] [5C] shows the expression of NKCC2, ENaC-β and NCC in M1 CCD cells after treatment with IL-6 and / or spironolactone. [picture] [5A] Demonstrates the performance of NKCC2 after treatment with IL-6 and / or spironolactone; [picture] [5B] Demonstrates the performance of ENaC-β after treatment with IL-6 and / or spironolactone; and [picture] [5C] Shows the performance of NCC after treatment with IL-6 and / or spironolactone.

[0045] [[] [picture] [6] []] This demonstrates the association between baseline characteristics and higher IL-6 levels in the PROJECT trial.

[0046] [[] [picture] [7A] and [[] [picture] [7B] shows the Kaplan-Meier survival curves for the IL-6 group. [picture] [7A] Show the all-cause mortality rate at 180 days, and [picture] [7B] Show all-cause mortality or cardiovascular-related rehospitalization rate at 60 days.

[0047] [[] [picture] [8A] and [[] [picture] [8B] shows the Carbon-Meyer survival curve illustrating the change in IL-6 between baseline and day 7. [picture] [8A] Show the all-cause mortality rate at 180 days, and [picture] [8B] Show all-cause mortality or cardiovascular-related rehospitalization rate at 60 days.

[0048] [[] [picture] [9] []] This demonstrates the association between baseline characteristics and higher IL-6 levels in the BIOSTAT-CHF study.

[0049] [[] [picture] [10A] and [[] [picture] [10B] shows the Carbon-Meyer survival curve for the IL-6 group. [picture] [10A] Show the all-cause mortality rate and / or readmission rate for heart failure at 2 years, and [picture] [10B] Shows the mortality rate from all causes at 2 years.

[0050] [[] [picture]

[11] []] This section presents an analysis of the relationship between ferritin levels and the IL-6 group. Implementation

[0051] [Cross-reference to related applications] [ ]

[0052] This application claims the benefit of U.S. Provisional Application No. 62 / 453,257, filed February 1, 2017, the entire contents of which are incorporated herein by reference. [Statement Regarding Federally Funded Research and Development] [ ]

[0053] This invention was carried out with government support under licenses 5R01HL128973 and 4K23HL114868 granted by the National Institutes of Health. The government holds certain rights to this invention. [Statement Regarding the Joint Research Agreement] [ ]

[0054] This invention was carried out under a joint research agreement with Yale University, MedImmune Ltd., AstraZeneca Pharmaceuticals LP, and Corvidia Therapeutics Ltd. [Sequence List] [ ]

[0055] This application contains a sequence list, which has been submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created in [Month] 2018 is named XXXXXUS_sequencelisting.txt and has a size of X,XXX,XXX bytes. [1.] [definition] [ ]

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0057] " [Transmembrane protease serine] [6 (, TMPRSS6 , )] "[Polypeptide]" refers to a polypeptide or fragment thereof that has at least about 85% or more of the amino acid sequence identical to the one provided by NCBI accession number NP_001275929 and possesses serine protease activity. The TMPRSS6 polypeptide, also known as interstitial protease-2 (MT2), is a protein that breaks down hepcidin and inhibits bone morphogenetic protein signaling. An illustrative TMPRSS6 amino acid sequence containing alanine (736A) at position 736 is provided below:

[0058] The following provides an illustrative TMPRSS6 amino acid sequence containing valine (736V) at position 736:

[0059] " [, TMPRSS6 , ] [, Nucleic acid molecules "," signifies a polynucleotide encoding the TMPRSS6 polypeptide (interstitial protease-2; MT2). ​​An illustrative TMPRSS6 nucleic acid sequence is provided under NCBI accession number NM_001289000. The following is a TMPRSS6 nucleic acid sequence containing a G ("G-pair"; "major pair") at nucleotide position 2321:

[0060] The following is the TMPRSS6 nucleic acid sequence containing the nucleotide A at position 2321:

[0061] " "[Variant]" refers to a polynucleotide or polypeptide sequence that differs from the reference sequence due to one or more nucleotides or one or more amino acids. An illustrative variant of TMPRSS6 is TMPRSS6 (A736V), caused by SNP rs855791 (G→A).

[0062] " [Single nucleotide polymorphism] or " [SNP] refers to a naturally occurring DNA sequence variant in which a single nucleotide differs between members of a biological species or between paired chromosomes in an individual. SNPs can be used as genetic markers for variant pairs. In one embodiment, the TMPRSS6 SNP is rs855791.

[0063] " [rs855791] refers to a single nucleotide polymorphism (SNP) in the human TMPRSS6 gene, 2321G→A, which causes a substitution from alanine to valine (A736V) in the catalytic region of interstitial protein 2 (MT2) encoded by the TMPRSS6 gene. The most frequent pair of genes in the human population (major pair) is 2321G, encoding 736A. The least frequent pair of genes in the human population (minor pair) is 2321A, encoding 736V.

[0064] " "[Heterosynthesis]" means that a chromosomal locus has two different paired genes. In one embodiment of the method described herein, heterosynthesis refers to the following genotype: one paired gene has a TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736 (e.g., G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 major paired gene), and the other paired gene has a variant TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide containing valine at amino acid position 736 (e.g., A or T at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 minor paired gene).

[0065] " [Isogamy] means that a chromosomal locus has two identical paired genes. In some embodiments of the methods described herein, isogamy refers to the genotype in which the two paired genes have a TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736 (e.g., having G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (the major paired gene of rs855791 isogamy). In some embodiments, isogamy refers to the genotype in which the two paired genes have a TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide containing valine at amino acid position 736 (e.g., having A or T at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (the minor paired gene of rs855791 isogamy).

[0066] " [Identify that the patient has] [, TMPRSS6 , ] [rs855791] "[At least one copy of the major paired gene]" includes, but is not limited to, performing analysis to determine that a patient has at least one copy of the TMPRSS6 rs855791 major paired gene; sequencing the analysis to determine that a patient has at least one copy of the TMPRSS6 rs855791 major paired gene; prescribing analysis to determine that a patient has at least one copy of the TMPRSS6 rs855791 major paired gene; otherwise guiding or controlling the analysis to determine that a patient has at least one copy of the TMPRSS6 rs855791 major paired gene; and reviewing TMRSS6 genotypic analysis data or protein or nucleic acid sequence data to determine that a patient has at least one copy of the TMPRSS6 rs855791 major paired gene.

[0067] " Interleukin [6]” or “ [IL-6] or " [IL-6] "[Polypeptide]" refers to a polypeptide or fragment thereof that shares at least approximately 85% or greater amino acid identity with the amino acid sequence provided under NCBI accession number NP_000591 and possesses IL-6 biological activity. IL-6 is a pleiotropic cytokine with multiple biological functions. Exemplary IL-6 biological activities include immunostimulatory and pro-inflammatory activities. Exemplary IL-6 amino acid sequences are provided below:

[0068] " Interleukin [6 (IL-6)] "[Nucleic Acid]" refers to a polynucleotide encoding the interleukin-6 (IL-6) polypeptide. An illustrative IL-6 nucleic acid sequence is provided with NCBI accession number NM_000600. An illustrative sequence with NCBI accession number NM_000600 is provided below.

[0069] " Interleukin [6] [Receptor] [(IL-6R)] The term "[complex]" refers to a protein complex containing the IL-6 receptor subunit α (IL-6Rα) and the interleukin-6 signal transducer glycoprotein 130, also known as the interleukin-6 receptor subunit β (IL-6Rβ).

[0070] " Interleukin [6] [Receptor Subunit] [α (IL-6Rα)] "[Polypeptide]" refers to a polypeptide or fragment thereof that shares at least approximately 85% or greater amino acid identity with the amino acid sequence provided under NCBI accession numbers NP_000556 or NP_852004 and possesses IL-6 receptor biological activity. Exemplary IL-6Rα biological activities include binding to IL-6, binding to glycoprotein 130 (gp130), and regulation of cell growth and differentiation. Exemplary IL-6R sequences are provided below:

[0071] " Interleukin [6] [Receptor Subunit] [β (IL-6Rβ)] "[Polypeptide]" refers to a polypeptide or fragment thereof that shares at least approximately 85% or greater amino acid identity with the amino acid sequence provided under NCBI accessions NP_002175, NP_786943, or NP_001177910 and possesses IL-6 receptor biological activity. Exemplary IL-6Rβ biological activities include binding to IL-6Rα, IL-6 receptor signaling activity, and regulation of cell growth, differentiation, and hepcidin expression. Exemplary IL-6Rβ sequences are provided below:

[0072] " [IL-6] "[Antagonist]" refers to an agent that can reduce the biological activity of IL-6. IL-6 antagonists include agents that reduce the level of IL-6 peptides in serum, including agents that reduce the expression of IL-6 peptides or nucleic acids; agents that reduce the ability of IL-6 to bind to IL-6R; agents that reduce the expression of IL-6R; and agents that reduce signal transduction via IL-6R receptors when IL-6 binds. In preferred embodiments, IL-6 antagonists reduce IL-6 biological activity by at least about 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. As further described in section 6.3.4 below, IL-6 antagonists include IL-6-binding peptides, such as anti-IL-6 antibodies and their antigen-binding fragments or derivatives; IL-6R-binding peptides, such as anti-IL-6R antibodies and their antigen-binding fragments or derivatives; and synthetic chemical molecules, such as JAK1 and JAK3 inhibitors.

[0073] " [IL-6] [Antibody] or " [anti] [IL-6] "[Antibody]" refers to an antibody that specifically binds to IL-6. Anti-IL-6 antibodies include monoclonal and polyclonal antibodies specific to IL-6, as well as their antigen-binding fragments or derivatives. IL-6 antibodies are described in more detail in section 3.6.1 below.

[0074] As used in this article, " "[Diuretic efficiency]" is calculated as mmol of urinary sodium (mmol Na / double the dose of Heinz diuretic) per doubling of the dose of Heinz diuretic, according to the method described in Hanberg et al., Circ. Heart Fail. 2016;9:e003180, the disclosure of which is incorporated herein by reference in its entirety.

[0075] " "[Antidiuretic heart failure]" means heart failure in patients with a diuretic efficiency of less than 100.

[0076] As used in this article, the term " [Biomarkers] or " "[Biomarker]" refers to a detectable molecule. Therefore, biomarkers according to the present invention include, but are not limited to, nucleic acids, polypeptides, carbohydrates, lipids, inorganic molecules, and organic molecules, each with widely varying sizes and properties. A "biomarker" can be a bodily substance related to a physical condition or disease. A "biomarker" can be detected using any means known in this art or by means previously unknown but becoming apparent only when considered by a person skilled in this art.

[0077] As used herein, in the context of this invention, " "[Biomarkers]" encompasses, but is not limited to, proteins, nucleic acids, and metabolites, as well as their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes and degradation products, elements, related metabolites, and other analyte or sample-derived measures. Biomarkers may also include mutant proteins or mutant nucleic acids. Biomarkers also encompass non-hematogenous factors or non-analyte physiological markers of health conditions, such as clinical parameters and traditional laboratory risk factors. As defined by the Food and Drug Administration (FDA), a biomarker is a characteristic (e.g., measurable DNA and / or RNA or protein) that is "objectively measured and assessed as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic or other intervention." Biomarkers also include any calculated index generated by mathematical methods or any combination of one or more of the aforementioned measurements, including temporal trends and variability. Biomarkers can be measured at any level of space or time location, including but not limited to within tumors, within cells, or on cell membranes.

[0078] " "[Pharmaceutical]" means any compound or composition suitable for administration in a therapeutic context, and explicitly includes chemical compounds; proteins, including antibodies or their antigen-binding fragments; peptides; and nucleic acid molecules.

[0079] " [individual]( [subject] / "[individual]" refers to individuals of humans or non-human mammals, including but not limited to cattle, horses, canines, sheep, felines, and rodents (including mice and house mice). "[Patient]" refers to a human individual.

[0080] As used in this article, the term " [treat]( "[treat / treating / treatment]" and similar terms refer to reducing or improving symptoms and / or signs or symptoms associated with the condition, or slowing or stopping its progression. It should be understood that, while not excluded, treating a condition does not require the complete elimination of the condition or its associated symptoms.

[0081] " "[Pre-treatment]" means before the first administration of an IL-6 antagonist, according to the methods described in this article. Pre-treatment does not exclude and often includes administration of treatments other than IL-6 antagonists, such as treatment with diuretics (such as heinz ring diuretics).

[0082] " "[Biological sample]" means any tissue, cell, body fluid, or other substance derived from a living organism (e.g., a human individual). In some embodiments, the biological sample is serum, plasma, urine, or whole blood.

[0083] As used in this article, " "[Guidance Material]" includes disclosures, records, diagrams, or any other medium of representation that can be used to convey the effectiveness of the components of the present invention in kits for detecting the biomarkers disclosed herein. The guidance material for the kits of the present invention may, for example, be affixed to or shipped together with a container containing the components. Alternatively, the guidance material may be shipped separately from the container, intended for use by a recipient in conjunction with the components.

[0084] One or more biomarkers "Level" refers to the absolute or relative amount or concentration of a biomarker in a sample, determined by measuring mRNA, cDNA, trace organic molecules, nucleotides, ions, or proteins, or any part thereof (such as oligonucleotides or peptides). In context, a biomarker level may refer to a global level, a level within a sub-part of an organism, or a level within a specific sample. By way of non-limiting examples, a level may refer to the amount or concentration of a biomarker in a urine sample, or the amount or concentration of the same biomarker in a plasma sample.

[0085] " [Measurement] [measuring] / [measurement]) or alternatively " [Detection] [detecting] / "[detection]" means determining the presence, absence, amount or quantity (which may be an effective amount) of a given substance in a clinical or subject-derived sample, including the derivation of the qualitative or quantitative concentration level of such substance, or otherwise determining the value or classification of a clinical parameter of a subject.

[0086] The " [reference level]" of a biomarker means the level of the biomarker that indicates the presence or absence of a particular phenotype or characteristic. When the level of the biomarker in a subject is higher than the reference level of the biomarker, it indicates the presence of a particular phenotype or characteristic or an increase in its level. When the level of the biomarker in a subject is lower than the reference level of the biomarker, it indicates the absence of a particular phenotype or characteristic or its relative lack. [1.] [Other rule interpretations] [ ]

[0087] Unless otherwise specified, the residue numbering of the antibody constant region is according to the EU index as in Kabat.

[0088] Range: Throughout this invention, various aspects of the invention may be presented in a range format. The range includes the recited endpoints. It should be understood that the description in range format is for convenience and brevity only and should not be construed as a fixed limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. and the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0089] Unless the context specifically dictates or is otherwise apparent, as used herein, the term " [or]" should be understood to be inclusive.

[0090] Unless the context specifically dictates or is otherwise apparent, as used herein, the term " [a]( [a] / [an])" and " [the]" should be understood to be singular or plural. That is, the use of the articles " [a]( [a] and The phrase "[an]") refers to one or more (i.e., at least one) grammatical objects of the article. By way of example, "one element" means one or more elements.

[0091] In this invention, " [Include]( [comprises] / [containing])”, “ [contain]"," [have]"," [include]( [includes] / [including]) and its linguistic variations have the meaning given to them under US patent law, and allow for the existence of additional components except as expressly stated therein.

[0092] Unless the context specifically specifies or otherwise makes it obvious, the term "as used herein" shall be used in accordance with the law. "[Approximately]" should be understood as being within the normal permissible range in this technique, such as within 2 standard deviations of the mean, and means covering a variation of ±20% or ±10%, preferably ±5%, even better ±1% and even better ±0.1% of the value.

[0093] The report describes the antibody equilibrium dissociation constant (KD), which is determined by surface plasma resonance, in which the antibody (or its antigen-binding fragment) is immobilized on the surface of a wafer on which ligands flow. [2.] [Overview of Experimental Observation Results] [ ]

[0094] As further described in Example 1 below, a prospective observational study will be conducted to enroll patients with continuous heart failure ("HF") receiving high-dose diuretic therapy at an outpatient treatment center. Plasma IL-6 levels will be measured to explore the systemic association between this pro-inflammatory cytokine and various disease parameters, and urinary IL-6 levels will be measured to explore the association between IL-6 and local inflammation and neurohormonal activation at the renal tissue level.

[0095] There is little correlation between plasma IL-6 levels and urine IL-6 levels.

[0096] In these patients with heart failure, elevated urinary IL-6 levels were significantly associated with measures of kidney damage, such as diuretic resistance, underestimation of glomerular filtration rate ("eGFR"), and increased tissue-level renin-angiotensin-aldosterone system ("RAAS") activation.

[0097] Although an inverse association was observed between diuretic efficiency and plasma IL-6, after adjusting for eGFR, only urinary IL-6 maintained a significant association with the risk of low diuretic efficiency in these patients. Furthermore, when both urinary and plasma IL-6 were input into the logistic regression model, only urinary IL-6 maintained an association with the risk of low diuretic efficiency, while plasma IL-6 showed no such association.

[0098] These data indicate that urinary IL-6 levels are a useful biomarker for kidney inflammation and can be used to measure renal dysfunction in patients with heart failure (cardiorenal syndrome). The data further indicate that continuous measurement of urinary IL-6 can be used to measure the renal efficacy of treatment administered to patients with heart failure, especially those with cardiorenal syndrome.

[0099] Urinary IL-6 data, and to some extent plasma IL-6 data, predict that treatment with IL-6 antagonists should be effective in reducing renal inflammation in patients with heart failure, i.e., treating the symptoms of cardiorenal syndrome. However, because infection is often a trigger for acute decompensation in patients with heart failure, it is important to limit the use of anti-cytokine and other immunosuppressive therapies in these patients with potentially improved renal and / or cardiac function. The cost of long-term IL-6 antagonist therapy also limits treatment in these patients with potentially improved renal and / or cardiac function.

[0100] As detailed in Example 2 below, the analysis in Example 1 was extended to 129 patients. Figure 1A is a bar graph showing the diuretic efficiency ("DE") of the urinary IL-6 group, confirming the inverse correlation between urinary IL-6 levels and diuretic efficiency observed in a subgroup of 98 patients. Figure 1B is a bar graph showing the diuretic efficiency ("DE") of the plasma IL-6 group of these 129 patients, confirming the inverse correlation between plasma IL-6 levels and diuretic efficiency.

[0101] The genotype of the rs855791 single nucleotide polymorphism (SNP) in the transmembrane protease serine 6 ("TMPRSS6") was assessed in each of the 129 patients. TMPRSS6 polypeptide, also known as interstitial protease-2 (MT2), is a protein that breaks down hepcidin and inhibits bone morphogenetic protein signaling. The rs855791 (G2321A) SNP alters the TMPRSS6 protein sequence: the most frequent pair in the population (major pair) is 2321G, encoding 736A; the least frequent pair in the human population (minor pair) is 2321A, encoding 736V.

[0102] As shown in Figure 2A, urinary IL-6 levels were inversely correlated with diuretic efficiency only in patients with at least one copy of the major pair gene of the TMPRSS6 rs855791 SNP (Figure 2A, right panel, "AG+GG"); urinary IL-6 levels were not significantly correlated with diuretic efficiency in patients with isotype conjugation of the minor pair gene (Figure 2A, left panel, "AA").

[0103] As shown in Figure 2B, plasma IL-6 levels were inversely correlated with diuretic efficiency only in patients with at least one copy of the major pair gene of the TMPRSS6 rs855791 SNP (Figure 2B, right panel, "AG+GG"); plasma IL-6 levels were not significantly correlated with diuretic efficiency in patients with isotype conjugation of the minor pair gene (Figure 2B, left panel, "AA").

[0104] These data suggest that treatment of heart failure with IL-6 antagonists will improve renal symptoms only in heart failure patients who have at least one copy of the TMPRSS6 rs855791 major paired gene.

[0105] In mouse M1 CCD cells, which are genotype-similar to human cells with the major pairing gene TMPRSS6 rs855791, the addition of IL-6 is associated with the expression of ion transport proteins NKCC2, ENaC-β, and NCC.

[0106] The data described in the examples also demonstrate the association between IL-6 and diuretic response. [3.] [Treatment of diuretic resistance] [ / ] [Methods to treat heart failure] [ ]

[0107] Accordingly, in the first embodiment, a method is provided for treating a patient who requires polyuria but is resistant to diuretics. The method comprises administering a therapeutically effective amount of an IL-6 antagonist in combination with a diuretic to the patient. In some embodiments, the patient has a higher pre-treatment plasma IL-6 level. In some embodiments, the patient has low diuretic efficiency.

[0108] In some embodiments, a method of treating heart failure is provided. The method includes administering a therapeutically effective amount of an IL-6 antagonist to a patient with heart failure, wherein the patient has been identified as having at least one copy of the TMPRSS6 rs855791 major paired gene. In some embodiments, the patient has elevated pre-treatment urinary IL-6 levels. In some embodiments, the patient has elevated pre-treatment plasma IL-6 levels. In some embodiments, the patient has elevated pre-treatment urinary and plasma IL-6 levels. In a typical embodiment, the patient has cardiorenal syndrome. In some embodiments, the patient has antidiuretic heart failure. [3.1. TMPRSS6 rs855791] [genotype] [ ]

[0109] In some embodiments, the patient has previously been identified as having at least one copy of the TMPRSS6 rs855791 major paired gene. In other embodiments, the method further includes an earlier step of identifying that the patient has at least one copy of the TMPRSS6 rs855791 major paired gene.

[0110] Preferably, the genotypes at the two pairs of genes are determined, thereby allowing the identification and differentiation of patients with isotyped TMPRSS6 rs855791 major pair of genes, patients with heterotyped major and minor TMPRSS6 rs855791 pairs of genes, and patients with isotyped TMPRSS6 rs855791 minor pair of genes.

[0111] Standard techniques were used to determine whether the SNP rs855791 (2321G→A) in the TMPRSS6 gene was absent (major pair) or present (minor pair).

[0112] Typically, biological samples obtained from patients are amplified using PCR.

[0113] In some embodiments, real-time PCR (RT-PCR) is used simultaneously to detect the absence or presence of polymorphism during amplification. In some embodiments, RT-PCR analysis employs 5' nucleases (TaqMan® probes), molecular beacons, and / or FRET hybridization probes. This was reviewed in Espy et al., Clin. Microbiol. Rev. 2006 January; 19(1): 165-256, which is incorporated herein by reference in its entirety. In typical embodiments, commercially available analyses are used. In selected embodiments, commercially available analyses are selected from the group consisting of: TaqMan™ SNP genotyping analysis (ThermoFisher); PCR SNP genotyping analysis (Qiagen); Novallele genotyping analysis (Canon); and SNP Type™ analysis (formerly SNPtype) (Fluidigm).

[0114] In some embodiments, the absence or presence of polymorphism is detected after amplification using hybridization with probes specific to SNP rs855791, restriction endonuclease digestion, nucleic acid sequencing, primer extension, microarray or gene chip analysis, mass spectrometry, and / or DNase protection assays. In some embodiments, paired gene variants are identified by sequencing. In some embodiments, Sanger sequencing is used. In some embodiments, one of various next-generation sequencing technologies is used, including, for example, sequencing technologies selected from the group consisting of: microarray sequencing, Solexa sequencing (Illumina), ion jet sequencing (Life Technologies), SOliD (Applied Biosystems), pyrosequencing, single-molecule real-time sequencing (Pacific Bio), nanopore sequencing, and tunneling current sequencing.

[0115] In some embodiments, the procedure described in Example 2 below is used to detect the absence or presence of polymorphism. [3.2.] [Before treatment] [IL-6] [level] [ ] [3.2.1.] [Urine before treatment] [IL-6] [level] [ ]

[0116] In some embodiments, the patient's pre-treatment urinary IL-6 level was greater than 5.0 pg IL-6 per gram of creatinine, 6.0 pg IL-6 per gram of creatinine, 7.0 pg IL-6 per gram of creatinine, 8.0 pg IL-6 per gram of creatinine, 9.0 pg IL-6 per gram of creatinine, or 10.0 pg IL-6 per gram of creatinine. In some embodiments, the patient's pre-treatment urinary IL-6 level was greater than 11.0 pg IL-6 per gram of creatinine, 12.0 pg IL-6 per gram of creatinine, 13.0 pg IL-6 per gram of creatinine, 14.0 pg IL-6 per gram of creatinine, or 15.0 pg IL-6 per gram of creatinine. In other embodiments, the patient's pretreatment urinary IL-6 level was greater than 16.0 pg IL-6 per gram of creatinine, 17.0 pg IL-6 per gram of creatinine, 18.0 pg IL-6 per gram of creatinine, 19.0 pg IL-6 per gram of creatinine, or 20.0 pg IL-6 per gram of creatinine. In certain embodiments, the patient's pre-treatment urinary IL-6 level was greater than 21.0 pg IL-6 per gram of creatinine, 22.0 pg IL-6 per gram of creatinine, 23.0 pg IL-6 per gram of creatinine, 24.0 pg IL-6 per gram of creatinine, 25.0 pg IL-6 per gram of creatinine, 26.0 pg IL-6 per gram of creatinine, 27.0 pg IL-6 per gram of creatinine, 28.0 pg IL-6 per gram of creatinine, 29.0 pg IL-6 per gram of creatinine, or even greater than 30.0 pg IL-6 per gram of creatinine. In some embodiments, the patient's pre-treatment urinary IL-6 level was greater than 35.0 pg IL-6 per gram of creatinine.

[0117] In some embodiments, the patient's pre-treatment urine level was greater than 14.2 pg IL-6 per gram of creatinine ("higher urine IL-6 level"). In other embodiments, the patient's pre-treatment urine level was less than 14.2 pg IL-6 per gram of creatinine.

[0118] In some embodiments, prior to treatment with an IL-6 antagonist and prior to treatment with Heinz cyclodiuretic, the patient's urinary IL-6 level was greater than 5.0 pg IL-6 per gram of creatinine, 6.0 pg IL-6 per gram of creatinine, 7.0 pg IL-6 per gram of creatinine, 8.0 pg IL-6 per gram of creatinine, 9.0 pg IL-6 per gram of creatinine, or 10.0 pg IL-6 per gram of creatinine. In some of these embodiments, the patient's urinary IL-6 level was greater than 11.0 pg IL-6 per gram of creatinine, 12.0 pg IL-6 per gram of creatinine, 13.0 pg IL-6 per gram of creatinine, 14.0 pg IL-6 per gram of creatinine, or 15.0 pg IL-6 per gram of creatinine. In other embodiments, prior to treatment with an IL antagonist and prior to treatment with a Heinz ring diuretic, the patient's urinary IL-6 level was greater than 16.0 pg IL-6 per gram of creatinine, 17.0 pg IL-6 per gram of creatinine, 18.0 pg IL-6 per gram of creatinine, 19.0 pg IL-6 per gram of creatinine, or 20.0 pg IL-6 per gram of creatinine. In certain embodiments, the patient's urinary IL-6 level was greater than 21.0 pg IL-6 per gram of creatinine, 22.0 pg IL-6 per gram of creatinine, 23.0 pg IL-6 per gram of creatinine, 24.0 pg IL-6 per gram of creatinine, 25.0 pg IL-6 per gram of creatinine, 26.0 pg IL-6 per gram of creatinine, 27.0 pg IL-6 per gram of creatinine, 28.0 pg IL-6 per gram of creatinine, 29.0 pg IL-6 per gram of creatinine, or even greater than 30.0 pg IL-6 per gram of creatinine. In some embodiments, the patient's urinary IL-6 level was greater than 35.0 pg per gram of creatinine before treatment with an IL antagonist and before treatment with a heinz cyclodiuretic.

[0119] In some embodiments, prior to treatment with an IL-6 antagonist and prior to treatment with a heinz diuretic, the patient's urinary IL-6 level was greater than 14.2 pg IL-6 per gram of creatinine. In other embodiments, prior to treatment with an IL antagonist and prior to treatment with a heinz diuretic, the patient's urinary IL-6 level was less than 14.2 pg IL-6 per gram of creatinine. [3.2.2.] [Pre-treatment plasma] [IL-6] [level] [ ]

[0120] In various embodiments, patients had high pre-treatment plasma IL-6 levels.

[0121] In some embodiments, the patient's pre-treatment plasma IL-6 level was greater than 2.0 pg / mL. In other embodiments, the patient's pre-treatment plasma IL-6 level was less than 2.0 pg / mL.

[0122] In some embodiments, the patient's pre-treatment plasma IL-6 level was greater than 1.0 pg / ml, 1.1 pg / ml, 1.2 pg / ml, 1.3 pg / ml, 1.4 pg / ml, 1.5 pg / ml, 1.6 pg / ml, 1.7 pg / ml, 1.8 pg / ml, 1.9 pg / ml, or 2.0 pg / ml. In some embodiments, the patient's pre-treatment plasma IL-6 level was greater than 2.1 pg / ml, 2.2 pg / ml, 2.3 pg / ml, 2.4 pg / ml, 2.5 pg / ml, 2.6 pg / ml, 2.7 pg / ml, 2.8 pg / ml, 2.9 pg / ml, or 3.0 pg / ml. In some embodiments, the patient's pretreatment plasma IL-6 level was greater than 3.1 pg / ml, 3.2 pg / ml, 3.3 pg / ml, 3.4 pg / ml, 3.5 pg / ml, 3.6 pg / ml, 3.7 pg / ml, 3.8 pg / ml, or 3.9 pg / ml.

[0123] In some embodiments, the patient's pre-treatment IL-6 level is greater than 2 pg / mL, such as greater than 3 pg / mL, 4 pg / mL, 5 pg / mL, 6 pg / mL, 8 pg / mL, 10 pg / mL, 15 pg / mL, or 20 pg / mL. In some embodiments, the patient's pre-treatment IL-6 level is greater than 3 pg / mL. In some embodiments, the patient's pre-treatment IL-6 level is greater than 5 pg / mL. In some embodiments, the patient's pre-treatment IL-6 level is greater than 10 pg / mL. [3.2.3.] [Before treatment] [IL-6] [level] [Measurement] [ ]

[0124] The concentration of IL-6 in urine, plasma, and serum can be determined using any standard analytical method known in this technique. When measuring IL-6 in urine, the IL-6 level can be indexed by another biomarker or normalized to another biomarker, in some embodiments of which is urinary creatinine.

[0125] In a particular embodiment, the MesoScale Discovery (MSD) platform (Meso Scale diagnostics, Gaithersburg, MD, USA) was used to measure the concentration. [3.3.] [Diuretic efficiency] [ ]

[0126] In some embodiments, patients requiring IL-6 antagonist treatment have a disease or condition requiring polyuria. In some embodiments, patients are already treated with diuretics. In some embodiments, patients are already treated with thiazide diuretics, such as chlorothiazide (Diuril®), chlorothiazide, hydrochlorothiazide (Microzide®), indapamide, or metolazone. In some embodiments, patients are already treated with heinz ring diuretics, such as bumetanide (Bumex®), ethacrynic acid (Edecrin®), furosemide (Lasix®), or torsemide (Demadex®). In some embodiments, patients are already treated with potassium-sparing diuretics, such as ampicillin, eplerenone (Inspra®), spironolactone (Aldactone®), or dyrenium®. In some embodiments, patients are already treated with more than one diuretic. In some embodiments, patients are already treated with different types of diuretics.

[0127] In various embodiments, patients requiring IL-6 antagonist treatment are resistant to diuretics. In some embodiments, patients resistant to diuretics require diuretic treatment with at least 40 mg of furosemide (or its equivalent) daily. In some of these embodiments, patients require diuretic treatment with at least 80 mg of furosemide (or its equivalent) daily. In some of these embodiments, patients require diuretic treatment with at least 120 mg of furosemide (or its equivalent) daily.

[0128] In various embodiments, patients resistant to diuretics exhibit low diuretic efficiency. Diuretic efficiency is calculated as the increase in sodium output corresponding to each doubling of the Heinz cyclodiuretic dose, centered at an intravenous dose of 40 mg furosemide equivalent: Diuretic efficiency = (Na output mmol) / (log2(administered dose of Heinz cyclodiuretic) - 4.32). See Hanberg et al., Circ. Heart Fail. 2016;9:e003180, the disclosure of which is incorporated herein by reference in its entirety.

[0129] In some embodiments, the patient's diuretic efficiency is less than 500, such as less than 450, 400, 350, 300, 250, or 200. In some embodiments, the patient's diuretic efficiency is less than 200, such as less than 195, 190, 185, 180, 175, 170, 165, 160, 155, or 150. In some embodiments, the patient's diuretic efficiency is less than 150, such as less than 145, 140, 135, 130, 125, 120, 110, 105, or 100. In some embodiments, the patient's diuretic efficiency is less than 100, such as less than 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50. In a particular embodiment, the patient's diuretic efficiency is less than 50, such as less than 45, 40, 35, 30, or even less than 25, 20, 15, or 10. [3.4.] [Heart failure] [ ]

[0130] In some embodiments of the methods described herein, the patient suffers from heart failure.

[0131] In some embodiments, the patient has NYHA functional class I heart failure. In some embodiments, the patient has NYHA functional class II heart failure. In some embodiments, the patient has NYHA functional class III heart failure. In some embodiments, the patient has NYHA functional class IV heart failure.

[0132] In some embodiments, the patient suffers from acute heart failure. In other embodiments, the patient suffers from chronic heart failure.

[0133] In some embodiments, the patient has a condition selected from the following [surface] [1] The type of heart failure. [Table 1] [ICD-10-CM] [describe] I50 Heart failure Heart failure complicated by miscarriage, ectopic pregnancy, or hydatidiform mole; postoperative heart failure; heart failure due to hypertension; heart failure due to hypertension associated with chronic kidney disease, obstetric surgery and procedures; rheumatic heart failure. I50.9 Heart failure, nonspecific Biventricular (cardiac) failure NOS NOS of heart, heart or heart failure congestive heart disease congestive heart failure Right ventricular failure (secondary to left heart failure) I50.1 Left ventricular failure Psychogenic asthma NOS with heart disease Pulmonary edema accompanied by heart failure Left heart failure NOS with heart disease Pulmonary edema accompanied by heart failure I50.20 Nonspecific systolic (congestive) heart failure I50.21 Acute systolic (congestive) heart failure I50.22 Chronic systolic (congestive) heart failure I50.23 Acute and chronic systolic (congestive) heart failure I50.30 Nonspecific diastolic (congestive) heart failure I50.31 Acute diastolic (congestive) heart failure I50.32 Chronic diastolic (congestive) heart failure I50.33 Acute and chronic diastolic (congestive) heart failure I50.40 Nonspecific systolic (congestive) and diastolic (congestive) combined heart failure I50.41 Acute systolic (congestive) and diastolic (congestive) combined heart failure I50.42 Chronic systolic (congestive) and diastolic (congestive) combined heart failure I50.43 Acute and chronic systolic (congestive) and diastolic (congestive) combined heart failure I50.1 Left ventricular failure Heart failure, nonspecific Biventricular (cardiac) failure NOS NOS of heart, heart or heart failure congestive heart disease congestive heart failure Right ventricular failure (secondary to left heart failure)

[0134] In some embodiments, the patient suffers from cardiorenal syndrome. In a particular embodiment, the patient suffers from type 1 cardiorenal syndrome. In a particular embodiment, the patient suffers from type 2 cardiorenal syndrome. In a particular embodiment, the patient suffers from type 3 cardiorenal syndrome. In a particular embodiment, the patient suffers from type 4 cardiorenal syndrome.

[0135] In some embodiments, the patient suffers from antidiuretic heart failure. In some of these embodiments, the diuretic efficiency of the heart failure patient is less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50. In certain embodiments, the patient's diuretic efficiency is less than 45, 40, 35, 30, or even less than 25, 20, 15, or 10. [3.5.] [Kidney disease] [ ]

[0136] In some embodiments of the methods described herein, the patient has kidney disease.

[0137] In some embodiments, the patient has antidiuretic nephropathy. In some of these embodiments, the diuretic efficiency of the nephropathy patient is less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50. In certain embodiments, the patient's diuretic efficiency is less than 45, 40, 35, 30, or even less than 25, 20, 15, or 10.

[0138] In a particular embodiment, the patient suffered from hepatorenal syndrome. [3.6. IL-6] [Antagonist] [ ]

[0139] The IL-6 antagonists used in the methods described herein can reduce the biological activity of IL-6. [3.6.1.] [anti] [IL-6] [Antibody] [ ]

[0140] In some embodiments, the IL-6 antagonist is an anti-IL-6 antibody or its antigen-binding fragment or derivative.

[0141] In some embodiments, the IL-6 antagonist is a full-length monoclonal anti-IL-6 antibody. In certain embodiments, the full-length monoclonal anti-IL-6 antibody is an IgG antibody. In some embodiments, the full-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the IL-6 antagonist is a multi-strain composition comprising a plurality of species of full-length anti-IL-6 antibodies, each of which has a unique CDR. In some embodiments, the IL-6 antagonist is an antibody fragment selected from Fab, Fab', and F(ab')2 fragments. In some embodiments, the IL-6 antagonist is scFv, disulfide-linked Fv (dsFv), or a single-domain antibody, such as a camel-derived VHH single-domain nanobody. In some embodiments, the IL-6 antagonist is an immunoconjugate or fusion comprising an IL-6 antigen-binding fragment. In some embodiments, the antibody is bispecific or multispecific, wherein at least one of the antigen-binding portions has specificity against IL-6.

[0142] In some embodiments, the antibody system is entirely human. In some embodiments, the antibody system is humanized. In some embodiments, the antibody system is chimeric and has a non-human V region and a human C region. In some embodiments, the antibody system is rodent-like.

[0143] In typical embodiments, the KD of the anti-IL-6 antibody binding to human IL-6 is less than 100 nM. In some embodiments, the KD of the anti-IL-6 antibody binding to human IL-6 is less than 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM. In specific embodiments, the KD of the anti-IL-6 antibody binding to human IL-6 is less than 5 nM, 4 nM, 3 nM, or 2 nM. In selected embodiments, the KD of the anti-IL-6 antibody binding to human IL-6 is less than 1 nM, 750 pM, or 500 pM. In particular embodiments, the KD of the anti-IL-6 antibody binding to human IL-6 is not greater than 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM.

[0144] In a typical embodiment, the anti-IL-6 antibody neutralizes the biological activity of IL-6. In some embodiments, the neutralizing antibody prevents IL-6 from binding to the IL-6 receptor.

[0145] In a typical embodiment, the elimination half-life of the anti-IL-6 antibody after intravenous administration is at least 7 days. In some embodiments, the elimination half-life of the anti-IL-6 antibody is at least 14 days, at least 21 days, or at least 30 days.

[0146] In some embodiments, the anti-IL-6 antibody has a human IgG constant region with at least one amino acid substitution, which prolongs the serum half-life compared to the unsubstituted human IgG constant region.

[0147] In some embodiments, the IgG constant domain comprises substitutions at residues 252, 254, and 256, wherein the amino acid substitution at residue 252 is a substitution of tyrosine, the amino acid substitution at residue 254 is a substitution of threonine, and the amino acid substitution at residue 256 is a substitution of glutamic acid (“YTE”). See U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety. In some extended half-life embodiments, the IgG constant domain contains a substitution selected from: T250Q / M428L (Hinton et al., J. Immunology 176:346-356 (2006)); N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)); or T307A / E380A / N434A (Petkova et al., International Immunology, 18: 1759-1769 (2006)).

[0148] In some embodiments, the elimination half-life of the anti-IL-6 antibody is enhanced by utilizing the FcRN binding property of human serum albumin. In some embodiments, the antibody binds to albumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001). In some embodiments, the anti-IL-6 antibody is fused to a bacterial albumin-binding domain (Stork et al., Prot. Eng. Design Science 20: 569-76 (2007)). In some embodiments, the anti-IL-6 antibody is fused to an albumin-binding peptide (Nguygen et al., Prot. Eng Design Sel 19: 291-297 (2006)). In some embodiments, the anti-IL antibody is bispecific, with one specificity against IL-6 and one specificity against human serum albumin (Ablynx, WO 2006 / 122825 (Bispecific Nanoantibody)).

[0149] In some embodiments, the elimination half-life of the anti-IL-6 antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)); by binding with HPMA copolymers (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)); by binding with polydextrose (Nuclear Medicine Communications, 16: 362-369 (1995)); by binding with homoamino acid polymers (HAP; HAP-ization) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)); or by polysialylation (Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)). [3.6.1.1. MEDI5117] [and derivatives] [ ]

[0150] In some embodiments, the anti-IL-6 antibody or its antigen-binding portion comprises all six CDRs of MEDI5117. In particular embodiments, the antibody or its antigen-binding portion comprises the heavy chain V region and the light chain V region of MEDI5117. In specific embodiments, the antibody is a full-length MEDI5117 antibody. The MEDI5117 antibody is described in WO 2010 / 088444 and US 2012 / 0034212, the disclosures of which are incorporated herein by reference in their entirety. The MEDI5117 antibody has the following CDRs and heavy and light chain sequences: [ ] [MEDI5117] [Rechain] [ ] [MEDI5117] [Light Chain] [ ]

[0151] In some embodiments, the anti-IL-6 antibody is a derivative of MEDI5117.

[0152] In some embodiments, the MEDI5117 derivative includes one or more amino acid substitutions in the V region of the MEDI5117 heavy chain and / or light chain.

[0153] In some embodiments, relative to the original VH and / or VL of the MEDI5117 anti-IL-6 antibody, the derivative contains fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, fewer than 2 amino acid substitutions, or 1 amino acid substitution, while retaining specificity for human IL-6.

[0154] In some embodiments, the MEDI5117 derivative comprises an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the VH and VL domains of MEDI5117. The percentage of sequence identity is determined using a BLAST algorithm with preset parameters.

[0155] In some embodiments, the MEDI5117 derivative comprises an amino acid sequence in which the CDR contains at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identical to the respective CDR of MEDI5117. The percentage of sequence identity is determined using a BLAST algorithm with preset parameters.

[0156] In some embodiments, the VH and / or VL CDR derivatives comprise one or more conserved amino acid substitutions on predicted non-essential amino acid residues (i.e., amino acid residues that are not essential for antibody-specific binding to human IL-6). [3.6.1.2.] [Other resistance] [IL-6] [Antibody] [ ]

[0157] In some embodiments, the anti-IL-6 antibody comprises six CDRs selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymezumab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In some embodiments, the anti-IL-6 antibody comprises the heavy chain V region and the light chain V region of an antibody selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymezumab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In a specific embodiment, the anti-IL-6 antibody is an antibody selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0158] In some embodiments, the anti-IL-6 antibody comprises six CDRs of antibodies selected from the antibodies described below: US 2016 / 0168243, US 2016 / 0130340, US 2015 / 0337036, US 2015 / 0203574, US 2015 / 0140011, US 2015 / 0125468, US 2014 / 0302058, US 2014 / 0141013, US 2013 / 0280266, US 2013 / 0017575, US 2010 / 0215654, US 2008 / 0075726, US Patent No. 5,856,135, US 2006 / 0240012, US The disclosures of Patent No. 2006 / 0257407 or U.S. Patent No. 7291721 are incorporated herein by reference in their entirety. [3.6.2.] [anti] [IL-6] [Receptor antibody] [ ]

[0159] In some embodiments, the IL-6 antagonist is an anti-IL-6 receptor antibody or its antigen-binding fragment or derivative.

[0160] In some embodiments, the IL-6 antagonist is a full-length anti-IL-6 receptor monoclonal antibody. In certain embodiments, the full-length monoclonal antibody is an IgG antibody. In some embodiments, the full-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the IL-6 antagonist is a multi-strain composition comprising a plurality of species of full-length anti-IL-6 receptor antibodies, each of which has a unique CDR. In some embodiments, the IL-6 antagonist is an antibody fragment selected from Fab and Fab' fragments. In some embodiments, the IL-6 antagonist is an scFv single-domain antibody, including a camel-derived VHH single-domain nanobody. In some embodiments, the antibody is bispecific or multispecific, wherein at least one of the antigen-binding portions has specificity against IL-6R.

[0161] In some embodiments, the antibody system is entirely human. In some embodiments, the antibody system is humanized. In some embodiments, the antibody system is chimeric and has a non-human V region and a human C region. In some embodiments, the antibody system is rodent-like.

[0162] In typical embodiments, the KD (knockout descent) of the anti-IL-6 receptor antibody binding to human IL-6R is less than 100 nM. In some embodiments, the KD of the anti-IL-6R antibody binding to human IL-6R is less than 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM. In specific embodiments, the KD of the anti-IL-6 receptor antibody binding to human IL-6R is less than 5 nM, 4 nM, 3 nM, or 2 nM. In selected embodiments, the KD of the anti-IL-6 receptor antibody binding to human IL-6R is less than 1 nM, 750 pM, or 500 pM. In particular embodiments, the KD of the anti-IL-6 receptor antibody binding to human IL-6R is not greater than 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM.

[0163] In a typical embodiment, anti-IL-6R reduces the biological activity of IL-6.

[0164] In a typical embodiment, the elimination half-life of the anti-IL-6R antibody after intravenous administration is at least 7 days. In some embodiments, the elimination half-life of the anti-IL-6R antibody is at least 14 days, at least 21 days, or at least 30 days.

[0165] In some embodiments, the anti-IL-6R antibody has a human IgG constant region with at least one amino acid substitution, which prolongs the serum half-life compared to the unsubstituted human IgG constant region.

[0166] In some embodiments, the IgG constant domain comprises substitutions at residues 252, 254, and 256, wherein the amino acid substitution at residue 252 is a substitution of tyrosine, the amino acid substitution at residue 254 is a substitution of threonine, and the amino acid substitution at residue 256 is a substitution of glutamic acid (“YTE”). See U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety. In some extended half-life embodiments, the IgG constant domain contains a substitution selected from: T250Q / M428L (Hinton et al., J. Immunology 176:346-356 (2006)); N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)); or T307A / E380A / N434A (Petkova et al., International Immunology, 18: 1759-1769 (2006)).

[0167] In some embodiments, the elimination half-life of the anti-IL-6R antibody is enhanced by utilizing the FcRN binding property of human serum albumin. In some embodiments, the antibody binds to albumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001). In some embodiments, the anti-IL-6R antibody is fused to a bacterial albumin-binding domain (Stork et al., Prot. Eng. Design Science 20: 569-76 (2007)). In some embodiments, the anti-IL-6 antibody is fused to an albumin-binding peptide (Nguygen et al., Prot. Eng Design Sel 19: 291-297 (2006)). In some embodiments, the anti-IL antibody is bispecific, with one specificity against IL-6R and one specificity against human serum albumin (Ablynx, WO 2006 / 122825 (Bispecific Nanoantibody)).

[0168] In some embodiments, the elimination half-life of the anti-IL-6R antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)); by binding with HPMA copolymers (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)); by binding with polydextrose (Nuclear Medicine Communications, 16: 362-369 (1995)); by binding with homoamino acid polymers (HAP; HAP-ization) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)); or by polysialylation (Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)).

[0169] In some embodiments, the anti-IL-6R antibody or its antigen-binding portion comprises all six CDRs of tocilizumab. In particular embodiments, the antibody or its antigen-binding portion comprises the heavy chain V region and the light chain V region of tocilizumab. In a specific embodiment, the antibody is a full-length tocilizumab antibody.

[0170] In some embodiments, the anti-IL-6R antibody or its antigen-binding portion comprises all six CDRs of sarilumab. In specific embodiments, the antibody or its antigen-binding portion comprises the V region of the sarilumab heavy chain and the V region of the light chain. In particular, the antibody is a full-length sarilumab antibody.

[0171] In some embodiments, the anti-IL-6R antibody or its antigen-binding portion comprises all six of the following CDRs: VX30 (Vaccinex), ARGX-109 (arGEN-X), FM101 (Formatech), SA237 (Roche), NI-1201 (NovImmune), or the antibody described in US 2012 / 0225060.

[0172] In some embodiments, the anti-IL-6R antibody or its antigen-binding portion is a single-domain antibody. In a particular embodiment, the single-domain antibody is a camel VHH single-domain antibody. In a specific embodiment, the antibody is vobarizumab (ALX-0061) (Ablynx NV). [3.6.3.] [anti] [IL-6:] [IL-6R] [Combined Antibody] [ ]

[0173] In some embodiments, the IL-6 antagonist is an antibody specific for the complex of IL-6 and IL-6R. In some embodiments, the antibody has six CDRs selected from the antibody described in US 2011 / 0002936, which is incorporated herein by reference in its entirety. [3.6.4. JAK] [and] [STAT] [Inhibitor] [ ]

[0174] It is known that IL-6 transmits information via the JAK-STAT path.

[0175] In some embodiments, the IL-6 antagonist is an inhibitor of the JAK signaling pathway. In some embodiments, the JAK antagonist is a JAK1-specific inhibitor. In some embodiments, the JAK antagonist is a JAK3-specific inhibitor. In some embodiments, the JAK inhibitor is a pan-JAK inhibitor.

[0176] In some embodiments, the JAK inhibitor is selected from the group consisting of: tofacitinib (Xeljanz), desensinib, ruzotinib, utpatatinib, baricitinib, finasteride, letotinib, paricitinib, pifiltinib, INCB-039110, ABT-494, INCB-047986, and AC-410.

[0177] In some embodiments, the IL-6 antagonist is a STAT3 inhibitor. In one specific embodiment, the inhibitor is AZD9150 (AstraZeneca, Isis Pharmaceuticals), a STAT3 antisense molecule. [3.6.5.] [additional] [IL-6] [Antagonist] [ ]

[0178] In some embodiments, the IL-6 antagonist is an antagonist peptide.

[0179] In some embodiments, the IL-6 antagonist is C326 (Avidia's IL-6 antagonist, also known as AMG220) or FE301, a recombinant protein inhibitor of IL-6 (Ferring International Center SA, Conaris Research Institute AG). In some embodiments, the anti-IL-6 antagonist comprises soluble gp130 or FE301 (Conaris / Ferring). [3.7.] [Dosage Regimen] [ ] [3.7.1.] [Antibody, antigen-binding fragment, peptide] [ ]

[0180] In a typical embodiment, the antibody, antigen-binding fragment, and peptide IL-6 antagonist are administered non-enterally.

[0181] In some non-enteral embodiments, the IL-6 antagonist is administered intravenously. In some intravenous embodiments, the IL-6 antagonist is administered as a bolus. In some intravenous embodiments, the IL-6 antagonist is administered by infusion. In some intravenous embodiments, the IL-6 antagonist is administered as a bolus followed by an infusion. In some non-enteral embodiments, the IL-6 antagonist is administered subcutaneously.

[0182] In some embodiments, antibodies, antigen-binding fragments, or peptide IL-6 antagonists are administered at a dose independent of the patient's weight or surface area (uniform dose).

[0183] In some embodiments, the uniform intravenous dose is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In some embodiments, the uniform intravenous dose is 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg. In some embodiments, the uniform intravenous dose is 25 mg, 30 mg, 40 mg, or 50 mg. In some embodiments, the uniform intravenous dose is 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. In some embodiments, the uniform intravenous dose is 1 to 10 mg, 10 to 15 mg, 15 to 20 mg, 20 to 30 mg, 30 to 40 mg, or 40 to 50 mg. In some embodiments, the uniform intravenous dose is 1 to 40 mg or 50 to 100 mg.

[0184] In some embodiments, the uniform subcutaneous dose is 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. In some embodiments, the uniform subcutaneous dose is 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg. In some embodiments, the uniform subcutaneous dose is 210 mg, 220 mg, 230 mg, 240 mg, or 250 mg. In some embodiments, the uniform subcutaneous dose is 10 to 100 mg, 100 to 200 mg, or 200 to 250 mg. In some embodiments, the uniform subcutaneous dose is 10 to 20 mg, 20 to 30 mg, 30 to 40 mg, 40 to 50 mg, 50 to 60 mg, 60 to 70 mg, 70 to 80 mg, 80 to 90 mg, or 90 to 100 mg. In some embodiments, the uniform subcutaneous dose is 100 to 125 mg, 125 to 150 mg, 150 to 175 mg, 175 to 200 mg, or 200 to 250 mg.

[0185] In some embodiments, antibodies, antigen-binding fragments, or peptide IL-6 antagonists are administered in doses based on the patient's weight.

[0186] In some embodiments, the antagonist is administered intravenously at doses of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1.0 mg / kg. In some embodiments, the antagonist is administered at doses of 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, or 5 mg / kg.

[0187] In some embodiments, the subcutaneous dose based on body weight is 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1.0 mg / kg. In some embodiments, the antagonist is administered at doses of 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, or 5 mg / kg.

[0188] In various intravenous embodiments, the IL-6 antagonist was administered once every 7 days, once every 14 days, once every 21 days, once every 28 days, or once a month. In various subcutaneous embodiments, the IL-6 antagonist was administered once every 14 days, once every 28 days, once a month, once every two months (every other month), or once every three months.

[0189] In some preferred embodiments, the IL-6 antagonist is a MEDI5117 antibody. In some embodiments, MEDI5117 is administered intravenously once weekly at a uniform dose of 1 to 30 mg. In some embodiments, the MEDI5117 antibody is administered intravenously once weekly at a uniform dose of 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 25, or 30 mg. In some embodiments, the MEDI5117 antibody is administered subcutaneously once monthly to every three months at a uniform dose of 25 to 250 mg. In specific embodiments, MEDI5117 is administered subcutaneously once monthly, every two months, or every three months at doses of 30 mg, 45 mg, 60 mg, 75 mg, 100 mg, 120 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, or 250 mg.

[0190] In some embodiments, the IL-6 antagonist is tocilizumab. In some embodiments, for patients ≥100 kg, tocilizumab is administered subcutaneously once weekly at an initial dose of 162 mg. In some embodiments, tocilizumab is administered intravenously at a dose of 4 mg / kg every 4 weeks, subsequently increased to 8 mg / kg every 4 weeks based on clinical response. [3.7.2. JAK] [and] [STAT] [Inhibitor] [ ]

[0191] In a typical embodiment, the small molecule JAK inhibitor and STAT inhibitor are administered orally.

[0192] In some embodiments, the inhibitor is administered orally once or twice daily at doses of 1 to 10 mg, 10 to 20 mg, 20 to 30 mg, 30 to 40 mg, or 40 to 50 mg. In some embodiments, the inhibitor is administered orally once or twice daily at doses of 50 to 60 mg, 60 to 70 mg, 70 to 80 mg, 80 to 90 mg, or 90 to 100 mg. In some embodiments, the inhibitor is administered orally once or twice daily at doses of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg. In some embodiments, the inhibitor is administered orally once or twice daily at a dose of 75 mg, or orally once daily at a dose of 10 ...

[0193] In some embodiments, the JAK inhibitor is tofacitinib, administered at a dose of 5 mg via post-oral administration (PO BID) or at a dose of 11 mg via post-oral administration once daily.

[0194] In some embodiments, the JAK inhibitor is desensinib, and it is administered PO BID at doses of 25 mg, 50 mg, 100 mg, or 150 mg.

[0195] In some embodiments, the inhibitor is ruzotinib, and it is administered at a dose of 25 mg PO BID, at a dose of 20 mg PO BID, at a dose of 15 mg PO BID, at a dose of 10 mg PO BID, or at a dose of 5 mg PO BID. [3.7.3.] [Treatment Goals] [ ]

[0196] In some embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to improve diuretic efficiency. In some of these embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to improve diuretic efficiency to normal levels.

[0197] In some embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to increase eGFR, and in some of these embodiments, the IL-6 antagonist is administered at a dose, duration, and duration sufficient to increase eGFR to normal levels. [3.7.4. IL-6] [Leveling Monitoring] [ ]

[0198] In some embodiments, the effects of IL-6 antagonist treatment on cardiorenal parameters can be monitored by measuring IL-6 levels in a patient's urine or plasma sample. This is particularly relevant considering that the method of the present invention can be used to monitor the efficacy of treatment for cardiorenal syndromes and can drive changes, for example, in dosage or therapeutics.

[0199] Accordingly, in some embodiments, the method further includes a subsequent step of determining IL-6 levels in urine, plasma, or both. In some of these embodiments, the method further includes a final step of adjusting at least one subsequent dose of the IL-6 antagonist based on the IL-6 levels determined in the preceding step. [3.8.] [Additional Healing Agent] [ ]

[0200] In some embodiments of the methods described herein, the method further includes administration of at least one therapeutic agent other than an IL-6 antagonist, wherein the additional therapeutic agent treats one or more cardiovascular or renal symptoms of heart failure. Specific treatment will be determined by the attending physician based on the specific circumstances. [3.8.1.] [Standard Heart Failure Medication] [ ]

[0201] In some embodiments, the additional therapeutic agent is a diuretic.

[0202] In certain embodiments, the diuretic is a Heinz ring diuretic. In selected embodiments, the Heinz ring diuretic is selected from the group consisting of furanylanilic acid, tosimimai, bumetanide, and ethacrynic acid. In certain embodiments, the Heinz ring diuretic is furanylanilic acid. In some embodiments, furanylanilic acid is administered orally. In some embodiments, furanylanilic acid is administered intravenously. In some embodiments, the diuretic is a thiazide diuretic. In certain embodiments, the thiazide diuretic is chlorothiazide, hydrochlorothiazide, chlorothiazide, indapamide, or metoprazone. In some embodiments, the diuretic is a potassium-retaining diuretic.

[0203] In some embodiments, the additional therapeutic agent is an ACE inhibitor. In some embodiments, the ACE inhibitor is selected from the group consisting of: benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril.

[0204] In some embodiments, the additional therapeutic agent is an angiotensin receptor blocker (“ARB”). In some embodiments, the ARB is eprosartan, olmesartan, valsartan, telmisartan, losartan, azilsartan medoxomil, candesartan, or irbesartan.

[0205] In some embodiments, the additional therapeutic agent is a beta-blocker, a calcium channel blocker, or a mineralocorticoid receptor antagonist.

[0206] In some embodiments, the additional therapeutic agent is a diuretic natriuretic peptide, such as B-type natriuretic peptide or N-terminal β-pro-B-type natriuretic peptide.

[0207] In some embodiments, the additional therapeutic agent is an adenosine antagonist, such as rolofylline.

[0208] The specific treatment will be determined by the attending physician based on the specific circumstances. [3.8.2.] [Nitro group donor] [ ]

[0209] In some embodiments, the additional therapeutic agent is a nitrogenous donor, and the method further includes administering a therapeutically effective amount of the nitrogenous donor.

[0210] In certain embodiments, the nitro group donor is selected from compounds described in one or more of the following patents: U.S. Patent Nos. 9,499,511, 9,487,498, 9,464,061, 9,458,127, 9,221,780, 9,181,213, 9,156,804, 9,115,064, 9,018,411, 8,987,326, RE45,314, 8,674,132, 8,227,639, and 8,030,356, the disclosures of which are incorporated herein by reference in their entirety.

[0211] In the selected embodiments, the nitro group donor is selected from the compounds described in U.S. Patent No. RE45,314. In a specific embodiment, the nitro group donor is selected from the compounds described in U.S. Patent No. 9,156,804. [3.8.3.] [Sodium-free chloride supplement] [ ]

[0212] In some embodiments, the additional therapeutic agent is a sodium-free chloride salt. In some embodiments, the agent is lysine chloride. [4.] [Methods for improving the treatment of heart failure] [ ]

[0213] In another embodiment, a method is provided to improve the treatment of heart failure by interrupting ineffective therapies, thereby reducing side effects and costs without losing therapeutic efficacy. The method involves interrupting the administration of an IL-6 antagonist to a patient with heart failure, wherein the patient has been identified as a TMPRSS6 rs855791 minor counterpart isotype conjugate. In one series of embodiments, the patient has previously been identified as a TMPRSS6 rs855791 minor counterpart isotype conjugate. In another series of embodiments, the method further includes an earlier step of identifying the patient as a TMPRSS6 rs855791 minor counterpart isotype conjugate.

[0214] In some embodiments, the patient had a higher pre-treatment urinary IL-6 level. In some embodiments, the patient had a higher pre-treatment plasma IL-6 level. In some embodiments, the patient had both a higher pre-treatment urinary IL-6 level and a higher pre-treatment plasma IL-6 level.

[0215] In a particular embodiment, the patient suffers from cardiorenal syndrome. [5.] [Diagnostic, Predictive, and Treatment Guidelines] [ ]

[0216] In another aspect, a method is provided for determining whether an individual will benefit from IL-6 antagonist treatment for heart failure. The method includes: measuring the level of IL-6 in a urine or plasma sample from the individual; comparing the measured IL-6 level with a predetermined reference level; and determining whether the measured IL-6 level is greater than the corresponding reference level, wherein IL-6 antagonist treatment is recommended when the measured IL-6 level is greater than the corresponding reference level.

[0217] IL-6 can be measured in plasma or urine using the methods described in section 3.2.3 above. Reference levels of IL-6 in urine and plasma can be determined by measuring IL-6 levels in a reference population. Those skilled in this technique can determine reference levels of biomarkers in a population based on clinical experience and the general levels of biomarkers from samples taken from that population.

[0218] In other cases, methods are provided for determining whether an individual requires IL-6 antagonist treatment for impaired glomerular filtration, low diuretic efficiency, high urinary angiotensin, high plasma renin, or whether the use of these methods places them at risk of death due to cardiorenal syndrome. As shown in Figure 3 and discussed in Example 1 below, high levels of IL-6 in urine or plasma are associated with these parameters. [6.] [Set] [ ]

[0219] In another embodiment, a kit is provided. Generally, the kit will contain a detection reagent suitable for detecting the presence of the biomarker of interest and instructions for use according to the method of the invention. The kit may contain an antibody or other immunohistochemical reagent capable of binding to IL-6. The kit may contain antibodies suitable for collecting and detecting IL-6 in urine or plasma for ELISA. In some embodiments, the kit may contain tools and reagents for preparing urine and plasma samples for ELISA or for indexing the concentration of IL-6 in urine using another biomarker, in some embodiments creatinine, as an index. [7.] [Other Embodiments] [ ]

[0220] Other embodiments are provided in the following numbered embodiments. 1. A method for treating heart failure, comprising: Administer a therapeutically effective dose of an IL-6 antagonist to a patient with heart failure. The patients have been identified as having at least one copy of the TMPRSS6 rs855791 major paired gene. 2. The method as in Example 1, wherein the patient has been previously identified as having at least one copy of the TMPRSS6 rs855791 major paired gene. 3. The method of Example 1, further comprising the following earlier steps: The patient was identified as having at least one copy of the TMPRSS6 rs855791 major paired gene. 4. The method of any one of Examples 1 to 3, wherein the patient has a higher pre-treatment urinary IL-6 level. 5. The method of any one of Examples 1 to 4, wherein the patient has a higher pre-treatment plasma IL-6 level. 6. The method of any one of Examples 1 to 5, wherein the patient suffers from acute heart failure. 7. The method of any one of Examples 1 to 5, wherein the patient suffers from chronic heart failure. 8. The method of any one of Examples 1 to 7, wherein the patient suffers from cardiorenal syndrome. 9. The method as described in Example 8, wherein the patient suffers from type 4 cardiorenal syndrome. 10. The method of any one of Examples 1 to 9, wherein the patient suffers from antidiuretic heart failure. 11. The method of Example 10, wherein the patient’s diuretic efficiency is less than 95%. 12. The method of Example 11, wherein the patient’s diuretic efficiency is less than 90%. 13. The method of Example 12, wherein the patient’s diuretic efficiency is less than 85%. 14. The method of Example 13, wherein the patient’s diuretic efficiency is less than 80%. 15. The method of Example 14, wherein the patient’s diuretic efficiency is less than 75%. 16. The method of Example 15, wherein the patient’s diuretic efficiency is less than 70%. 17. The method of Example 16, wherein the patient’s diuretic efficiency is less than 65%. 18. The method of any one of Examples 1 to 17, wherein the IL-6 antagonist is an anti-IL-6 antibody or its antigen-binding fragment or derivative. 19. The method of Example 18, wherein the KD of the anti-IL-6 antibody or antigen-binding fragment or derivative binding to human IL-6 is less than 100 nM. 20. The method of Example 19, wherein the KD of the antibody or antigen-binding fragment or derivative binding to human IL-6 is less than 50 nM. 21. The method of Example 20, wherein the KD of the antibody or antigen-binding fragment or derivative binding to human IL-6 is less than 10 nM. 22. The method of Example 21, wherein the KD of the antibody or antigen-binding fragment or derivative binding to human IL-6 is less than 1 nM. 23. The method of any one of Examples 18 to 22, wherein the elimination half-life after intravenous administration of the anti-IL-6 antibody or antigen-binding fragment or derivative is at least 7 days. 24. The method of Example 23, wherein the elimination half-life after intravenous administration of the anti-IL-6 antibody or antigen-binding fragment or derivative is at least 14 days. 25. The method of Example 24, wherein the elimination half-life after intravenous administration of the anti-IL-6 antibody or antigen-binding fragment or derivative is at least 21 days. 26. The method of Example 25, wherein the elimination half-life after intravenous administration of the anti-IL-6 antibody or antigen-binding fragment or derivative is at least 30 days. 27. The method of any one of Examples 18 to 26, wherein the IL-6 antagonist is a full-length monoclonal anti-IL-6 antibody. 28. The method of Example 27, wherein the anti-system IgG1 or IgG4 antibody is used. 29. The method of Example 28, wherein the anti-system IgG1 antibody is used. 30. The method of any one of Examples 18 to 29, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is completely human. 31. The method of any one of Examples 18 to 29, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is humanized. 32. The method of any one of Examples 18 to 31, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable regions (CDRs) of MEDI5117. 33. The method of Example 32, wherein the antibody comprises VH and VL of MEDI5117. 34. The method of Example 33, wherein the anti-system is MEDI5117. 35. The method of any one of Examples 18 to 31, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable regions (CDRs) of an antibody selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymec, esmomumab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). 36. The method of Example 35, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises a heavy chain V region and a light chain V region of an antibody selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymec, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). 37. The method of Example 36, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of: serotonin, grilinzumab, serotonin, clazazumab, onozymec, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). 38. The method of any one of Examples 18 to 26, wherein the IL-6 antagonist is a single-domain antibody, a VHH nanoantibody, a Fab or scFv. 39. The method of any one of Examples 1 to 17, wherein the IL-6 antagonist is an anti-IL-6R antibody or its antigen-binding fragment or derivative. 40. The method of Example 39, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative comprises six CDRs of tocilizumab. 41. The method of Example 39, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative comprises six CDRs of vobarbital. 42. The method of any one of Examples 1 to 17, wherein the IL-6 antagonist is a JAK inhibitor. 43. The method of Example 42, wherein the JAK inhibitor is selected from the group consisting of: tofacitinib (Xeljanz), desensinib, ruzotinib, eupatatinib, baricitinib, feicrotinib, letotinib, paricitinib, pifiltinib, INCB-039110, ABT-494, INCB-047986 and AC-410. 44. The method of any one of Examples 1 to 17, wherein the IL-6 antagonist is a STAT3 inhibitor. 45. The method of any one of Examples 18 to 41, wherein the IL-6 antagonist is not administered via the intestine. 46. ​​The method of Example 45, wherein the IL-6 antagonist is administered subcutaneously. 47. The method of any one of Examples 42 or 43, wherein the IL-6 antagonist is administered orally. 48. The method of any one of Examples 1 to 47, wherein the IL-6 antagonist is administered at a dose, duration and duration sufficient to improve diuretic efficiency. 49. The method of Example 48, wherein the IL-6 antagonist is administered at a dose, duration and duration sufficient to improve diuretic efficiency to a normal level. 50. The method of any one of Examples 1 to 49, wherein the IL-6 antagonist is administered at a dose, duration and duration sufficient to increase eGFR. 51. The method of Example 50, wherein the IL-6 antagonist is administered at a dose, duration and duration sufficient to increase eGFR to normal levels. 52. The method of any one of Examples 1 to 51, further comprising the subsequent step of determining the level of IL-6 in urine. 53. The method of any one of Examples 1 to 51, further comprising the subsequent step of determining the level of IL-6 in plasma. 54. The method of any one of Examples 1 to 51, further comprising the subsequent step of determining the levels of IL-6 in urine and plasma. 55. The method of any one of Examples 52 to 54, further comprising a final step of adjusting the dose of the IL-6 antagonist to be subsequently administered based on the IL-6 level determined in the preceding step. 56. A method for determining whether an individual needs treatment for cardiorenal syndrome, the method comprising: a) Measuring IL-6 levels in urine or plasma samples from individuals. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, the individual has cardiorenal syndrome and treatment is recommended. 57. A method for determining whether an individual needs treatment for impaired glomerular filtration, comprising: a) Measuring IL-6 levels in urine samples from individuals. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, the individual has impaired glomerular filtration and treatment is recommended. 58. A method for determining whether an individual needs treatment for low diuretic efficiency, comprising: a) Measuring IL-6 levels in urine samples from individuals. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, the individual has low diuretic efficiency and treatment is recommended. 59. A method for determining whether an individual needs treatment for high urinary angiotensin levels, comprising: a) Measuring IL-6 levels in urine samples from individuals. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, the individual has high urinary angiotensin levels, and treatment is recommended. 60. A method for determining whether an individual needs treatment for high plasma renin levels, comprising: a) Measuring IL-6 levels in plasma samples from patients. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, the individual has high plasma renin levels, and treatment is recommended. 61. A method for determining whether an individual is at risk of death due to cardiorenal syndrome and requires treatment, comprising: a) Measuring IL-6 levels in plasma samples from patients. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, the individual is at risk of death due to cardiorenal syndrome, and treatment is recommended. 62. The method of any one of Examples 56 to 61, wherein the patient is a patient with heart failure. 63. The method of any one of Examples 56 to 62, wherein enzyme-linked immunosorbent assay (ELISA) is used to measure IL-6 levels. 64. A method for treating cardiorenal syndrome in patients, comprising: a) Measure IL-6 levels in urine or plasma samples from the patient. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, it is used for the treatment of cardiorenal syndrome. 65. The method of Example 64, wherein the patient is a patient with heart failure. 66. The method of Example 64 or Example 65, wherein enzyme-linked immunosorbent assay (ELISA) is used to measure IL-6 levels. 67. The method of any one of Examples 64 to 66, wherein treatment for cardiorenal syndrome comprises administering one or more of the following: at least one diuretic, angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, natriuretic peptide, adenosine antagonist, IL-6 antagonist, or any combination thereof. 68. The method of Example 67, wherein treating cardiorenal syndrome comprises administering at least one anti-IL-6 antibody or anti-IL-6R antibody. 69. A kit comprising an analytical reagent for measuring IL-6 levels and written instructions, the written instructions comprising: a) Measuring IL-6 levels in urine or plasma samples from individuals. b) Compare the measured IL-6 level with the predetermined reference level. c) Determine whether the measured IL-6 level is greater than the corresponding reference level. When the measured IL-6 level is higher than the corresponding reference level, the individual has cardiorenal syndrome and treatment is recommended. 70. The kit as described in Example 69, wherein the instructions guide the use of the kit for patients with heart failure. 71. The kit as described in Examples 69 or 70, wherein the kit contains reagents for measuring IL-6 levels using enzyme-linked immunosorbent assay (ELISA). [8.] [Experimental Example] [ ]

[0221] The invention will be described in further detail with reference to the following experimental examples. Unless otherwise specified, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not be construed as limited to the following examples, but rather as encompassing any and all variations that become apparent from the teachings provided herein.

[0222] Without further description, it is believed that those skilled in the art will be able to practice the methods claimed in this invention using the above description and the following illustrative examples. Therefore, the following working examples specifically illustrate preferred embodiments of the invention and should not be construed as limiting the rest of the invention in any way.

[0223] The materials and methods used in the experiments described in this paper are now described. [8.1.] [Example] [1] [:] [Urine in patients with heart failure] [IL-6] [and plasma] [IL-6] [Related to] [ ]

[0224] [method] [.] Patients with continuous heart failure ("HF") receiving high-dose diuretic therapy at the Yale Transitional Care Clinic (YTCC) will be enrolled in a prospective observational study. [ ]

[0225] YTCC is an outpatient clinic dedicated to diuresis and fluid status management. Patients participate in 4 to 8 hours of treatment, during which they receive 1 to 3 doses of Heinz Cyclodiuretic. The dosing regimen is determined based on the patient's fluid status; oral (PO) tosimidazine or intravenous (IV) bumetanide is chosen with careful consideration by the treating physician. All urine produced during the treatment period is collected in a cumulative collection container and transported to the clinical laboratory for electrolyte measurement at the end of the consultation. Cumulative urine collection is performed during the treatment period. Additional single-point urine samples are collected one hour before and after diuretic administration. [ ]

[0226] [analyze] Urine and serum electrolytes were measured using an ion-selective electrode on the Randox RxDaytona™ Automated Clinical Chemicals Analyzer. Urea, creatinine, bicarbonate, and cystatin C were measured using Randox reagents (Randox Laboratories™, UK) according to the manufacturer's instructions. Interleukin-10 and IL-6 concentrations were measured using the MesoScale Discovery (MSD) platform (Meso Scale diagnostics™, Gaithersburg, MD, USA). N-terminal B-type natriuretic peptide (NT-BNP) levels were measured at the Yale Clinical Laboratory using a Roche Elecsys 120 analyzer (Roche Diagnostics, Indianapolis, USA). Plasma renin activity (PRA), proanginogen, and active renin were measured using a competing ELISA kit available from ALPCO™ (ALPCO™, Salem, NH, USA) according to the manufacturer's instructions. Total renin was also analyzed using a commercially available ELISA kit (R&D Systems™, Minneapolis, USA). The total renin immunoassay kit, purchased from R&D Systems™, identifies active renin and prorenin. The detection limit of the analytical method for total renin was 4.43 pg / ml, and the detection limit for active renin was 0.81 pg / ml. Urine bumetanide and tosimimai levels were measured using liquid chromatography-mass spectrometry. Ultra-high performance liquid chromatography (UPLC) was performed on an Agilent Infinity 1290 UPLC system™. Chromatographic separation was achieved at a flow rate of 0.6 mL / min on a Zorbax Bonus RP™ 2.1 × 50 mm 1.8 µ column. The mobile phase contained 0.1% formic acid (buffer A) and 80% acetonitrile in 0.1% formic acid (buffer B). Mass spectrometry analysis was performed in cation mode on an Agilent Q-TOF system™ (Agilent™, Santa Clara, CA, USA). [ ]

[0227] [Calculation and Definition] [.] eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. Heinz ring diuretics doses were converted to furosemide equivalents using the formula: 1 mg bumetanide = 20 mg tosimidazine = 40 mg intravenous furosemide = 80 mg oral furosemide. As previously published, diuretic efficiency was defined as the increase in urinary sodium output corresponding to each doubling of the Heinz ring diuretic dose centered at 40 mg intravenous furosemide equivalent during the treatment period; this ratio was chosen to interpret the S-shaped dose-response curves of these drugs. Urinary diuretic excretion was calculated by multiplying the volume of urine produced in the first 3 hours after diuretic administration by the concentration of the diuretic in the urine; this estimated amount of diuretic was then normalized to the dose of diuretic administered in furosemide equivalent form, taking into account the urinary clearance of the specific diuretic (bumetanide or tosimidazine) received by the patients in the previously published studies. The fractional excretion of sodium and potassium is calculated using the following formula: Fractional excretion of X (FEX) = [X]urine * [creatinine]serum / ([X]serum or plasma * [creatinine]urine). Urinary proteins, including renin, proangina pectoris, and IL-6, are measured using urinary creatinine as an indicator. Low urinary IL-6 is defined as less than the median value of 14.2 pg / g urinary creatinine. Low plasma IL-6 is defined as less than the median value of 2.0 pg / mL. High levels of urinary and plasma renin and proangina pectoris are defined as values ​​greater than or equal to the group median of these variables. [ ]

[0228] [Statistical Analysis] [.] The reported values ​​are mean + / - SD, median (1st quartile - 3rd quartile), and percentage. Correlation between continuous variables is Spearman's rho, except for adjusted correlations. Pearson's chi-squared test was used to compare categorical variables between groups. Studden's t-test or Wilcoxon Rank Sum test was used to compare continuous variables between groups. Log transformation was applied to skewed variables including plasma IL-6, urinary IL-6, and NT-promoting BNP before inputting them into the multivariate model for partial correlation analysis. Logistic regression was used to assess the association between low diuretic efficiency, eGFR < 60 ml / min / 1.73 m², or high levels of urinary or plasma neurohormonal parameters and plasma and urinary IL-6 levels, both at the univariate level and adjusted for plasma or urinary IL-6 and / or eGFR. Cox proportional hazards simulation was used to assess the time-event association of all-cause mortality rates. Statistical analyses were performed using IBM SPSS Statistical Edition 23 (IBM, Armonk, NY) and Stata Edition 13 (StataCorp™, College Station, TX). For all analyses, statistical significance was defined as a two-tailed test (p < 0.05).

[0229] The baseline characteristics of our group are as follows: [surface] [2] As described. In this subgroup, 98 patients underwent measurements of IL-6 levels in blood and urine. The median urinary IL-6 (IQR) level before diuresis was 14.2 pg / g creatinine (5.6 to 36.2 pg / g), while the median plasma IL-6 level was 2.0 pg / mL (1.2 to 3.9 pg / mL). There was little association between plasma IL-6 levels and urinary IL-6 levels (r=0.40, p<0.001). Notably, individuals with lower than median urinary IL-6 levels tended to be younger Caucasian, more commonly on angiotensin receptor blockers (ARBs) or angiotensin-converting enzyme (ACE-I) therapy, more likely to have heart failure, reduced ejection fraction (HFrEF), higher GFR, and substantially lower plasma NT-BNP levels. The distribution of patients defined by lower than median plasma IL-6 was slightly different but generally reflected the same trends. Table 2: Baseline Characteristics [Overall Group] [Low urine output] [IL-6] [High urine output] [IL-6] [, p , ] [-] [value] [Low plasma] [IL-6] [High plasma] [IL-6] [, p , ] [-] [value] (n=98) (n=49) (n=49) (n=49) (n=49) [Demographics] age 67.9 ± 13.5 62.1 ± 13.2 73.8 ± 11.2 <0.001* 65.8 ± 15.1 70.1 ± 11.5 0.12 African American, % 36 57 15 <0.001* 47 26 0.04* male,% 44 49 39 0.31 49 39 0.31 [Past medical history] [%] hypertension 91 94 88 0.29 96 86 0.08 diabetes 47 55 39 0.11 37 57 0.04* gout 13 10 16 0.37 10 16 0.37 Ischemic etiology 30 33 27 0.46 33 27 0.46 Follow-up consultation after discharge 63 71 55 0.09 61 65 0.68 [Baseline medication,] [%] ACEI or ARB 55 65 45 0.04* 67 43 0.02* β-blockers 77 82 71 0.23 82 71 0.23 Thiazide diuretics 14 12 16 0.56 6 twenty two 0.02* MRA 33 37 29 0.39 31 35 0.67 Digoxin 10 12 8 0.50 8 12 0.50 Home dosage of Heinz cyclodiuretic (furan phenylethanoid equivalent mg) 80 (60-160) 80 (60-160) 100 (80-160) 0.60 80 (40-120) 160 (80-200) 0.002* [Physical Examination] [ ] Weight(kg) 98.2 ± 33.4 106.0 ± 37.9 90.4 ± 26.4 0.02* 98.4 ± 31.5 98.0 ± 35.5 0.95 BMI 34.8 ± 10.8 36.7 ± 11.3 32.5 ± 9.9 0.09 33.5 ± 8.4 36.2 ± 13.0 0.28 Systolic blood pressure (mmHg) 122.6 ± 18.9 120.6 ± 19.1 124.5 ± 18.7 0.32 123.4 ± 18.9 121.7 ± 19.1 0.65 Heart rate (beats per minute): 75.9 ± 13.2 75.6 ± 13.2 76.2 ±13.4 0.82 74.9 ± 13.3 76.8 ±13.2 0.48 [Heartbeat Echo Image] [ ] Left ventricular ejection fraction (%) 45 (27-56) 35 (25-50) 51 (31-59) 0.03* 39 (29-54) 45 (26-57) 0.56 Ejection fraction ≥ 40% 53% 43% 63% 0.04* 49% 57% 0.42 [Laboratory Value] Sodium (mmol / L) 136 (134-140) 137 (134-138) 139 (134-141) 0.06 138 (135-140) 136 (132-140.0) 0.12 Chloride (mmol / L) 96 (95-102) 100 (96-102) 98 (95-102) 0.50 101 (98-103) 96 (91-101) <0.001* Low blood chloride, % 34 27 42 0.13 14 55 <0.001* Potassium (mmol / L) 4.1 (3.7-4.5) 4.2 (3.8-4.6) 4.1 (3.6-4.3) 0.053 4.1 (3.7-4.5) 4.1 (3.7-4.4) 0.64 Bicarbonate (mmol / L) 23.9 (21.7-26.4) 23.1 (21.6-25.3) 24.9 (22.2-27.9) 0.03* 23.1 (21.5-25.2) 25.0 (22.6-28.0) 0.03* Blood urea nitrogen (mg / dL) 29.0 (20.0-47.0) 27.0 (17.5-44.0) 32.0 (24.0-48.0) 0.10 27.0 (19.0-39.0) 36.0 (23.0-65.0) 0.04* Serum creatinine (mg / dL) 1.5 ± 0.8 1.4 ± 0.6 1.7 ± 0.9 0.046* 1.3 ± 0.5 1.8 ± 1.0 0.007* BUN / creatinine ratio 24.0 ± 8.3 24.1 ± 8.1 23.9 ± 8.5 0.92 23.6 ± 8.0 24.5 ± 8.5 0.58 Estimated glomerular filtration rate (eGFR) (mL / min / 1.73m2) 54.9 ± 28.2 64.3 ± 30.1 45.7 ± 22.8 <0.001* 61.4 ± 27.2 48.3 ± 27.8 0.02* eGFR < 60 mL / min / 1.73m2 61% 48% 73% 0.01* 55% 67% 0.24 Albumin (g / dL) 3.8 ± 0.4 3.9 ± 0.4 3.8 ± 0.4 0.045* 4.0 ± 0.4 3.7 ± 0.4 <0.001* Hemoglobin (g / dL) 12.2 ± 2.3 12.7 ± 2.2 11.7 ± 2.3 0.04* 12.8 ± 2.0 11.5 ± 2.4 0.004* [Diuretic parameters] Dosage of diuretic administered (Furfural phenylamine equivalent mg) 140 (40-240) 80 (40-160) 160 (80-280) 0.001* 80 (40-160) 160 (80-320) <0.001* The percentage of diuretics administered. Intravenous bumetanide: 45 27 63 <0.001* 33 57 0.02* Tosimai: 55 73 37 67 43 ACE-I = Angiotensin-converting enzyme inhibitor. ARB = Angiotensin receptor blocker. MRA = Mineralocorticoid receptor antagonist. IL = Interleukin. BUN = Blood urea nitrogen. BNP = Brain natriuretic peptide. BMI = Body mass index. [*] p< 0.05. Table 3: Plasma and urine biomarkers at baseline [Overall Group] [Low urine output] [IL-6] [High urine output] [IL-6] [, p , ] [-] [value] [Low plasma] [IL-6] [High plasma] [IL-6] [, p , ] [-] [value] (n=98) (n=49) (n=49) (n=49) (n=49) [Plasma biomarkers] IL-6 (pg / mL) 2.0 (1.2-3.9) 1.4 (0.9-2.9) 2.8 (1.5-6.1) <0.001* 1.2 (0.9-1.4) 3.9 (2.9-6.8) <0.001* IL-10 (pg / mL) 0.3 (0.2-0.7) 0.3 (0.2-0.4) 0.5 (0.3-0.8) 0.001* 0.3 (0.2-0.5) 0.5 (0.3-0.8) 0.01* IL-10 / IL-6 ratio 0.2 (0.1-0.4) 0.2 (0.1-0.3) 0.2 (0.1-0.4) 0.73 0.3 (0.2-0.6) 0.1 (0.1-0.2) <0.001* Total renin (pg / mL) 1167.7 (734.7-2759.7) 1169.2 (734.7-3013.5) 1166.3 (816.0-2390.9) 0.97 909.6 (595.2-1517.9) 1784.4 (921.6-3868.7) <0.001* Active renin (pg / mL) 36.8 (8.4-162.8) 79.0 (7.7-184.2) 28.1 (12.0-139.4) 0.38 13.0 (4.4-160.7) 49.1 (15.7-169.2) 0.07 Angiotensinogen (ng / mL) 167.9 (54.1, 445.3) 193.9 (48.2, 518.1) 160.3 (58.3, 430.2) 0.81 178.8 (31.6, 481.2) 167.2 (75.5, 445.3) 0.50 NT promotes BNP (pg / mL) 1825.0 (681.0-5030.0) 840.0 (256.0-3115.0) 3115.0 (1440.0-6070.0) <0.001* 1440.0 (412.0-3010.0) 3390.0 (792.0-6410.0) 0.01* [Urine biomarkers] IL-6 (picograms / gram of creatinine) 14.2 (5.6-36.2) 5.6 (2.5-7.7) 36.2 (20.7-68.9) <0.001* 7.0 (3.2-19.1) 21.2 (9.2-45.3) <0.001* IL-6 (pg / mL) 1.2 (0.5-2.6) 0.5 (0.2-0.7) 2.5 (1.6-4.0) <0.001* 0.8 (0.4-1.9) 1.7 (0.6-3.2) 0.03* Renin (picograms / mg creatinine) 0.3 (0.1-0.8) 0.2 (0.1-0.5) 0.6 (0.3-1.2) <0.001* 0.2 (0.1-0.6) 0.5 (0.2-1.5) <0.001* Renin (pg / mL) 28.2 (10.2-88.3) 19.9 (7.5-53.1) 37.0 (13.4-109.6) 0.01* 23.7 (7.5-53.1) 35.9 (12.3-109.6) 0.06 Angiotensinogen (picograms / mg creatinine) 18.7 (5.4-126.3) 6.2 (3.3-19.0) 123.5 (17.3-549.7) <0.001* 7.3 (3.7-39.7) 33.7 (12.3-318.9) 0.002* Angiotensinogen (pg / mL) 1530 (624-10941) 966 (231-1567) 5850 (1518-53865) <0.001* 1105 (463-5801) 2153 (966-23952) 0.02* FENa (%) 0.5 (0.2-1.3) 0.4 (0.2-1.2) 0.5 (0.2-1.4) 0.22 0.4 (0.1-1.0) 0.7 (0.3-1.8) 0.02* FEK (%) 18.4 (11.7-39.3) 15.5 (9.9-32.5) 21.6 (15.2-44.6) 0.02* 15.8 (9.6-24.5) 24.5 (13.9-50.3) 0.001* FE urea (%) 63.6 (44.8-81.3) 59.5 (44.4-80.5) 65.5 (44.9-81.6) 0.62 66.7 (47.5-82.8) 58.2 (44.6-76.0) 0.20 IL = Interleukin. NT-promoting BNP = N-terminal pro-beta-diuretic sodium peptide. FENa = Sodium fractionation. FEK = Potassium fractionation. FEurea = Urea fractionation. [8.1.1.] [Kidney function and] [IL-6]

[0230] As shown in Table 2, patients with high urinary or plasma IL-6 had lower eGFR, but this association was significant only between eGFR and urinary IL-6 (p=0.01). A correlation was observed between plasma IL-6 and eGFR (r=-0.26, p=0.01) and between urinary IL-6 and eGFR (r=-0.38, p<0.001). However, after adjusting for urinary IL-6, the association between plasma IL-6 and eGFR was no longer significant (p=0.20), while a significant association remained between urinary IL-6 and eGFR after adjusting for plasma IL-6 (partially r=-0.32, p=0.002). Similarly, the risk of decreased eGFR, as defined by eGFR < 60 ml / min / 1.73 m², increased with higher urinary IL-6 levels (OR = 1.9 per SD increment, 95% CI = 1.2, 3.1, p = 0.006) but not with higher plasma IL-6 levels (OR = 1.3 per SD increment, 95% CI = 0.8–2.0, p = 0.25). [8.1.2.] [Diuretic response and] [IL-6]

[0231] Diuretic efficiency was inversely associated with both urinary IL-6 (r = -0.43, p < 0.001) and plasma IL-6 (r = -0.31, p = 0.002; Figure 3). The probability of low diuretic efficiency increased with higher urinary IL-6 levels (OR = 2.3 per SD increment, 95% CI 1.4–3.8, p = 0.001) or plasma IL-6 levels (OR = 1.7 per SD increment, 95% CI 1.1–2.7, p = 0.02). After adjusting for eGFR, only urinary IL-6 remained significantly associated with the risk of low diuretic efficiency (adjusted OR = 1.8 per SD increment, 95% CI 1.1–3.1, p = 0.02; Figure 3). Furthermore, when both urinary IL-6 and plasma IL-6 were included in the logistic regression model, only urinary IL-6 remained associated with the risk of low diuretic efficiency (adjusted OR = 2.1 per SD increment, 95% CI 1.3–3.5, p = 0.004), while plasma IL-6 showed no such association (OR = 1.4, 95% CI 0.9–2.2, p = 0.17). [8.1.3.] [Neurohormone activation] [ ]

[0232] Plasma IL-6 was associated with higher plasma renin levels (Table 3 and Figure 3). Notably, higher plasma IL-6 levels carried an additional risk of elevated total plasma renin (OR = 1.9 per SD increment, 95% CI 1.2–3.0, p = 0.008), even after adjustments for ACE-I or ARB use and urinary IL-6 levels (adjusted OR = 2.3 per SD increment, 95% CI 1.3–3.9, p = 0.003). Urinary IL-6 was not associated with the risk of elevated plasma renin (OR = 1.0, 95% CI 0.7–1.5, p = 0.98).

[0233] Urinary IL-6 is largely associated with high levels of tissue-level RAAS activation, as measured by urinary angiotensinogen (OR = 4.2 per SD increment, 95% CI 2.2–7.9, p < 0.001) and urinary renin (OR = 2.1 per SD increment, 95% CI 1.3–3.4, p = 0.002; Figure 3). These associations remained after adjustments for plasma IL-6 levels and ACE-I or ARB use (adjusted OR = 4.2 per SD increment for high urinary angiotensinogen, 95% CI 2.2–8.3, p < 0.001; adjusted OR = 2.0 per SD increment for high urinary renin, 95% CI 1.2–3.3, p = 0.005). The association between plasma IL-6 and the risk of high urinary renin levels was not univariate (OR = 1.3 per SD increment, 95% CI 0.9–2.0, p = 0.16). Although a tendency was observed to be associated with urinary angiotensinogen (OR = 1.5 per SD increment, 95% CI 0.98–2.3, p = 0.06), this tendency was eliminated upon adjustment for urinary IL-6 (adjusted OR = 1.1, 95% CI 0.7–1.7, p = 0.76). [8.1.4.] [Relationship with survival rate] [ ]

[0234] After a median follow-up period of 713 days, 32 deaths occurred. Consistent with previous reports, elevated plasma IL-6 was associated with a higher risk of death (univariate HR = 2.8 per SD increment, 95% CI 2.0–4.0, p < 0.001). After multivariate adjustment for age, race, baseline NT-BNP levels, ACE-I or ARB use, Heinz cyclodiuretic dose, and baseline eGFR characteristics, plasma IL-6 maintained its association with mortality (adjusted HR = 2.3 per SD increment, 95% CI = 1.5–3.7, p < 0.001). In contrast, urinary IL-6 was not associated with the risk of death (univariate HR = 1.3 per SD increment, 95% CI 0.9–1.8, p = 0.15; adjusted HR = 1.02, 95% CI = 0.6–1.6, p = 0.93). [8.1.5.] [Overview] [ ]

[0235] Plasma and urinary IL-6 levels reveal a unique aspect of the role of IL-6 in the pathophysiology of cardiorenal diseases. Plasma IL-6 levels are associated with global measures of disease severity, such as the risk of death. In contrast, in patients with heart failure, urinary IL-6, a measure of possible kidney inflammation, is largely associated with multiple measures of cardiorenal syndrome (“CRS”), including diuretic resistance, renin-angiotensin-aldosterone system (RAAS) activation, and lower estimated glomerular filtration rate (eGFR).

[0236] These data indicate that urinary IL-6 levels are a useful biomarker for kidney inflammation, and are particularly suitable for assessing renal symptoms in patients with heart failure. Furthermore, it should be demonstrated that continuous urinary IL-6 levels are useful for monitoring and assessing the renal benefits of therapeutic interventions for heart failure. [8.2.] [Example] [2] [:] [TMPRSS6] [Genome grouping predicted kidney symptoms would be associated with...] [IL-6] [Patients with heart failure who react to antagonists] [ ]

[0237] Data obtained in Example 1 also predicted that treatment with IL-6 antagonists should be effective in reducing kidney inflammation in patients with heart failure.

[0238] However, because infection is often a trigger for acute decompensation in patients with heart failure, it is important to limit anti-cytokine and other immunosuppressive therapies to these patients with potentially improved renal and / or cardiac function. The cost of long-term IL-6 antagonist therapy also limits treatment for these patients with potentially improved renal and / or cardiac function.

[0239] The analysis in Example 1 was extended to 129 patients. Figure 1A shows the diuretic efficiency ("DE") of the urinary IL-6 group, confirming the inverse correlation between urinary IL-6 and diuretic efficiency observed in a subgroup of 98 patients. Figure 1B shows the diuretic efficiency ("DE") of the plasma IL-6 group of these 129 patients, confirming the inverse correlation between plasma IL-6 and diuretic efficiency (unadjusted for urinary IL-6 levels).

[0240] Additionally, the genotype of the rs855791 single nucleotide polymorphism (SNP) in the transmembrane protease serine 6 ("TMPRSS6") was assessed in each patient. TMPRSS6 polypeptide, also known as interstitial protease-2 (MT2), breaks down hepcidin-regulated protein and inhibits bone morphogenetic protein signaling. The rs855791 (G2321A) SNP alters the TMPRSS6 protein sequence: the most frequent pair gene (major pair gene) in the population is 2321G, encoding 736A; the least frequent pair gene (minor pair gene) in the human population is 2321A, encoding 736V.

[0241] Genomic DNA was isolated from leukocytes using the ReliaPrep large-volume HT gDNA isolation system (Promega, Madison, USA) on an HSM instrument. The purity of the isolated DNA was assessed using Nanodrop. Genotyping was performed at the Yale Center for genomic analysis. Complete genome genotyping was performed using the Illumina Infinium® Exome-24 v1.0 BeadChip kit according to the standard protocol provided by the manufacturer (Illumina, Inc., San Diego, CA). Amplification, fragmentation, precipitation, resuspension, and hybridization steps were performed manually. The array was scanned on an Illumina Hiscan instrument. The BeadChip was scanned using an Illumina HiScan or iScan system, using laser excitation of the fluorophores of the single-base extension products on the beads. The scanner recorded high-resolution images of the light emitted by the fluorophores. Genotyping data was extracted from intensity data archives (*.idat files) collected by the Illumina HiScan system using the Illumina GenomeStudio genotyping module included in the Illumina Infinium analysis system. Infinium Exome-24 v1.0 BeadChip contains variants and complete genome sequences of over 240,000 inferred functional exons selected from the exomes of over 12,000 individuals. These >240,000 markers represent diverse populations, including individuals from Europe, Africa, China, and Spain, and a range of common conditions such as type 2 diabetes, cancer, metabolic disorders, and mental health issues. Detailed Illumina genotyping protocols are available at http: / / support.illumina.com (infinium_hts_assay_protocol_user_guide_15045738_a.pdf). In addition, details of the SNPs in the phenotypic array chip can be found at http: / / support.illumina.com / downloads / infinium-exome-24-v1-0-product-files.html.

[0242] As shown in Figure 2A, urinary IL-6 levels were inversely correlated with diuretic efficiency only in patients with at least one copy of the major pair gene of the TMPRSS6 rs855791 SNP (Figure 2A, right panel, "AG+GG"); urinary IL-6 levels were not significantly correlated with diuretic efficiency in patients with isotype conjugation of the minor pair gene (Figure 2A, left panel, "AA").

[0243] As shown in Figure 2B, plasma IL-6 levels were inversely correlated with diuretic efficiency only in patients with at least one copy of the major pair gene of the TMPRSS6 rs855791 SNP (Figure 2B, right panel, "AG+GG"); plasma IL-6 levels were not significantly correlated with diuretic efficiency in patients with isotype conjugation of the minor pair gene (Figure 2B, left panel, "AA").

[0244] These data suggest that treatment of heart failure with IL-6 antagonists will improve renal symptoms only in heart failure patients who have at least one copy of the TMPRSS6 rs855791 major paired gene. [8.3.] [Example] [3] [:] [In the absence or presence of ruzoutinib] [IL-6] [and] [NKCC2] [、] [ENaC-] [β] [and] [NCC] [Correlation of performance] [ ] [method] [ ]

[0245] Mouse M1 CCD cell line (American Type Culture Collection, ATCC, catalog number CRL-2038) was kept in a cell culture medium containing equal amounts of DMEM (Sigma-Aldrich, catalog number D6046) and Han F12 (Sigma-Aldrich, catalog number 11765-047), supplemented with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin (Thermo Fisher Scientific, catalog number 15140-122), 1% insulin-transferrin-selenium (Thermo Fisher Scientific, catalog number 51500-056) and 100 nM dexamethasone (Sigma-Aldrich, catalog number D4902-100MG). [ ]

[0246] M1 CCD cells were seeded at 1 million cells / well in cell culture medium in 6-well plates and incubated overnight at 37°C with 5% CO2 on day 0. On day 1, the cell culture medium was changed to DMEM / F12 serum-free medium and the cells were incubated overnight at 37°C with 5% CO2. On day 2, the serum-free medium was removed and cell culture medium was added to each well. Luzotinib (Selleckchem, S1378) was added 1 µM or 100 µM 10 minutes before adding IL-6 (Sigma-Aldrich, catalog number SRP3096-20UG) at a final concentration of 10 ng / mL, 100 ng / mL, or 500 ng / mL. Control wells without luzotinib or IL-6 were included. Cells were treated with IL-6 and / or luzotinib for 24 hours. Cells from each well were washed once with 1×PBS and collected in 250 µL of 1×PIPA buffer (10×, Millipore, catalog 20-188) supplemented with 1% protease inhibitor mixture (100×, Thermo Fisher Scientific, catalog 78430). Protein expression was quantified by immunoblotting using anti-NKCC2 antibody (Millipore, catalog AB3562P), anti-ENaC-β antibody (Millipore, catalog AB3532P), or anti-NCC antibody (Millipore, catalog AB3553). Each experiment was performed in triplicate. [result] [ ]

[0247] Following IL-6 treatment in the absence or presence of the JAK inhibitor ruzotinib, we examined the expression of NKCC2 (Na-K-Cl cotransporter 2), ENaC-β (epithelial sodium channel, β subunit), and NCC (sodium-chloride cotransporter) proteins in mouse M1 CCD cell lines. Mouse M1 CCD cells are genotype-similar to human cells isotype-conjugated with the TMPRSS6 rs855791 major pair gene. As shown in Figures 4A, 4B, and 4C, IL-6 treatment enhanced the expression of NKCC2, ENaC-β, and NCC. Ruzotinib blocks the effect of IL-6 on ion transporter proteins. [8.4.] [Example] [4] [:] [In the absence or presence of spironolactone] [IL-6] [and] [NKCC2] [、] [ENaC-] [β] [and] [NCC] [Correlation of performance] [ ] [method] [ ]

[0248] Mouse M1 CCD cell line (American Type Culture Collection (ATCC), catalog number CRL-2038) was kept in a cell culture medium containing equal amounts of DMEM (Sigma-Aldrich, catalog number D6046) and Han F12 (Sigma-Aldrich, catalog number 11765-047), supplemented with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin (Thermo Fisher Scientific, catalog number 15140-122), 1% insulin-transferrin-selenium (Thermo Fisher Scientific, catalog number 51500-056) and 100 nM dexamethasone (Sigma-Aldrich, catalog number D4902-100MG).

[0249] M1 CCD cells were seeded at 1 million cells / well in cell culture medium in 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator on day 0. On day 1, the cell culture medium was changed to DMEM / F12 serum-free medium and the cells were cultured overnight at 37°C in a 5% CO2 incubator. On day 2, the serum-free medium was removed and cell culture medium was added to each well. Ten minutes before adding IL-6 (Sigma-Aldrich, catalog number SRP3096-20UG) at a final concentration of 10 ng / mL, 100 ng / mL, or 500 ng / mL, spironolactone (Selleckchem, catalog number S4054) was added at a final concentration of 1 µM or 100 µM. Control wells without spironolactone or IL-6 were included. Cells were treated with IL-6 and / or spironolactone for 24 hours. Cells from each well were washed once with 1×PBS and collected in 250 µL of 1×PIPA buffer (10×, Millipore, catalog 20-188) supplemented with 1% protease inhibitor mixture (100×, Thermo Fisher Scientific, catalog 78430). Protein expression was quantified by immunoblotting using anti-NKCC2 antibody (Millipore, catalog AB3562P), anti-ENaC-β antibody (Millipore, catalog AB3532P), or anti-NCC antibody (Millipore, catalog AB3553). Each experiment was performed in triplicate. [result] [ ]

[0250] Following IL-6 treatment in the absence or presence of the potassium-sparing diuretic spironolactone, we examined the expression of NKCC2 (Na-K-Cl cotransporter 2), ENaC-β (epithelial sodium channel, β subunit), and NCC (sodium-chloride cotransporter) proteins in mouse M1 CCD cell lines. Mouse M1 CCD cells are genotype-similar to human cells isotype-conjugated with the TMPRSS6 rs855791 major pair gene. As shown in Figures 5A, 5B, and 5C, IL-6 treatment enhanced the expression of NKCC2, ENaC-β, and NCC. Spironolactone blocks the effect of IL-6 on ion transport proteins. [8.5.] [Example] [5] [:] [Due to acute heart failure] [(HF)] [and] [Among hospitalized patients] [IL-6] [and] [Related to Diuretic Reactions] [ ]

[0251] [method] [.] Data from the PROTECT trial (Weatherley et al., 2010, J. Card. Fail. 16:25-35; Massie et al., 2010, N. Engl. J. Med. 363:1419-1428) were analyzed based on the IL-6 group. The PROTECT trial was a randomized placebo-controlled trial that tested the effects of the adenosine A1-receptor antagonist roxitholin on the relief of dyspnea, the risk of renal function deterioration, and clinical outcomes. The key inclusion and exclusion criteria for the trial are presented below. [Included] [exclude] >18 years old Acute contrast-induced nephropathy A history of HF (high heart disease) under diuretic therapy >14 days Temperature >38°C or sepsis requires intravenous antimicrobial treatment. Hospitalized due to ADHF*, requiring intravenous diuretic therapy. Serum potassium <3.5 mEq / L Within 24 hours of admission Intravenous therapy for ADHF with positive myocardial systolic agents, vasopressors, or vasodilators that is currently in use or planned. The expected intravenous furosemide requirement is >40 mg / day for at least 24 hours. BNP <500 pg / mL or NT-pro-BNP <2000 pg / mL; impaired renal function defined as creatinine clearance between 20 mL / min and 80 mL / min at admission (Cockcroft-Gault); currently undergoing or planned ultrafiltration therapy; systolic blood pressure >95 mm Hg. Severe lung disease Clinical signs of acute coronary syndrome within 2 weeks prior to screening Hgb <8 g / dL, or Hct <25%, or blood transfusion required. Randomized systolic blood pressure $160 mm Hg *ADHF: Dyspnea at rest or with minimal exertion and, on randomization, signs of fluid overload by at least one of the following: JVP > 8 cm, or rales ≥ 1 / 3 of the upper lung area that do not disappear with cough, or ≥ 2 cm of peripheral edema or presacral edema.

[0252] In total, the PROTECT study included 2033 patients with ADHF. Among these patients, IL-6 was measured using Singulex in 1445 newly admitted patients (baseline), 1462 day 2 patients (24 hours after baseline), and 1445 day 7 patients. Diuretic response was defined as the change in weight from baseline to day 3 per 40 mg furosemide (or equivalent dose) administered on day 4. The primary endpoint of this study was all-cause mortality at 180 days.

[0253] [Statistical Analysis] [.] Baseline characteristics were presented based on the IL6 group. Differences in baseline characteristics between groups were assessed using one-way ANOVA, with the Kruskal-Wallis or chi-square (chi2) test as appropriate. Univariate linear regression was performed using diuretic response as the dependent variable and baseline (log-transformed) IL6 as the independent variable, adjusted for clinically relevant variables associated with diuretic response. Survival analysis was performed using Cox regression analysis adjusted for clinically relevant variables and the PROTECT risk model (O'Connor et al., 2012, Eur. J. Heart Fail. 14:605-612). The PROTECT risk model included: prior hospitalization for HF, edema at admission, systolic blood pressure, sodium levels, BUN, creatinine, and albumin.

[0254] [result] The baseline characteristics of the population are described in Table 4 below. Higher IL6 levels at baseline were associated with higher BNP levels, anemia, eGFR <60, and older age (Figure 6). [surface] [4] [Baseline characteristics] [factor] [, No. , ] [, 1 , ] [, Group , ] [, No. , ] [, 2 , ] [, Group , ] [, No. , ] [, 3 , ] [, Group , ] [p-] [value] N 531 530 530 [Demographics] [0.66-7.8 pg / mL] [7.9-16.1 pg / mL] [16.2-274.2 pg / mL] [ ] age 69.2 (11.5) 70.8 (11.0) 72.5 (10.8) <0.001 gender 347 (65.3%) 349 (65.8%) 354 (66.8%) 0.88 BMI 28.3 (5.8) 28.8 (6.0) 29.1 (6.2) 0.093 eGFR 52.5 (20.2) 47.2 (19.3) 44.9 (17.4) <0.001 NYHA classification I / II 107 (21.0%) 71 (14.1%) 96 (19.2%) 0.008 III 267 (52.4%) 263 (52.2%) 237 (47.4%) IV 136 (26.7%) 170 (33.7%) 167 (33.4%) LVEF 30 (25, 40) 30 (20, 40) 30 (22, 40) 0.35 HFpEF 42 (15.6%) 39 (16.3%) 38 (14.9%) 0.92 contractile BP 126.2 (17.4) 124.5 (17.5) 123.6 (17.4) 0.043 diastolic blood pressure 75.3 (11.2) 73.5 (12.1) 72.3 (11.9) <0.001 Heart rate 79.2 (14.6) 79.4 (15.8) 81.3 (16.0) 0.052 respiratory rate 20.6 (4.3) 21.2 (4.1) 21.7 (4.9) <0.001 [Medical History] atrial fibrillation 95 (45.5%) 92 (43.6%) 78 (39.4%) 0.45 Valvular disease 196 (37.0%) 195 (36.8%) 208 (39.5%) 0.61 Mitral regurgitation 174 (32.8%) 178 (33.6%) 184 (34.8%) 0.78 Aortic stenosis 18 (3.4%) 16 (3.0%) 32 (6.0%) [0.027] aortic valve insufficiency 43 (8.1%) 18 (3.4%) 38 (7.2%) [0.004] Heart failure (HF) 510 (96.0%) 505 (95.3%) 501 (94.5%) 0.51 Hospitalization rate due to HF in the previous year 258 (48.6%) 266 (50.2%) 259 (48.9%) 0.86 Median number of hospitalizations for HF (Hospitalization with Fear of Irregular Flow) (IQR Version 1.0 (1.0, 2.0) Version 1.0 (1.0, 2.0) Version 1.0 (1.0, 2.0) 0.62 ischemic heart disease 362 (68.4%) 396 (74.9%) 365 (68.9%) [0.037] Myocardial infarction 255 (48.3%) 296 (56.0%) 242 (45.7%) [0.003] hypertension 424 (79.8%) 422 (79.6%) 428 (80.8%) 0.89 Stroke or PVD 78 (14.7%) 102 (19.3%) 117 (22.2%) [0.007] anemia 174 (36.3%) 216 (47.2%) 238 (50.6%) [<0.001] Thyroid diseases 60 (11.3%) 61 (11.5%) 57 (10.8%) 0.92 Depression 28 (5.3%) 41 (7.7%) 35 (6.6%) 0.27 hyperlipidemia 298 (56.1%) 267 (50.5%) 244 (46.0%) [0.004] Current smokers 92 (17.4%) 114 (21.6%) 105 (19.8%) 0.23 COPD or asthma 106 (20.0%) 102 (19.2%) 112 (21.2%) 0.73 diabetes 247 (46.5%) 229 (43.2%) 258 (48.7%) 0.2 History of atrial fibrillation / atrial flutter 260 (49.2%) 291 (55.0%) 310 (58.8%) [0.007] AICD 73 (13.7%) 106 (20.0%) 68 (12.8%) [0.002] Biventricular pacemaker 54 (10.2%) 60 (11.3%) 47 (8.9%) 0.41 pacemaker 50 (9.5%) 66 (12.5%) 65 (12.3%) 0.22 [drug] β-blockers 417 (78.7%) 396 (74.7%) 385 (72.6%) 0.068 ACEI 353 (66.6%) 312 (58.9%) 330 (62.3%) [0.033] ARB 85 (16.0%) 85 (16.0%) 71 (13.4%) 0.38 ACE-I / ARB 424 (80.0%) 390 (73.6%) 393 (74.2%) [0.026] MRA 264 (49.8%) 226 (42.6%) 229 (43.2%) [0.033] Digoxin 159 (30.0%) 153 (28.9%) 146 (27.5%) 0.68 nitrates 136 (25.7%) 146 (27.6%) 135 (25.5%) 0.68 hydrazine 17 (3.2%) 15 (2.8%) 13 (2.5%) 0.76 CCB 74 (14.0%) 71 (13.4%) 81 (15.3%) 0.67 [Signs and symptoms] Sit upright and breathe 445 (84.3%) 440 (84.5%) 435 (82.5%) 0.65 Dyspnea at rest (NYHA IV) 264 (50.6%) 304 (59.1%) 327 (64.8%) [<0.001] angina pectoris 134 (25.2%) 117 (22.1%) 111 (21.0%) 0.24 CCS Level III and IV 48 (36.4%) 38 (33.0%) 29 (26.6%) 0.27 edema 82 (15.4%) 151 (28.5%) 184 (34.8%) [<0.001] jugular vein dilation 190 (39.7%) 195 (40.5%) 199 (41.6%) 0.82 Edema and elevated JVP 127 (26.5%) 148 (30.7%) 150 (31.4%) 0.2 Luo Yin 37 (7.0%) 55 (10.4%) 62 (11.7%) 0.027

[0255] The association between IL-6 levels and diuretic response is shown in Table 5 below. Diuretic response was defined as the weight change from baseline to day 3 of each 40 mg furosemide (or equivalent dose) administered on day 4. [surface] [5] [Diuretic reaction] [β] [p-] [value] univariate 0.06 [0.027] Model 1 (Age, Gender) 0.06 [0.030] Model 2 (Model 1 + eGFR, BMI) 0.06 [0.035]

[0256] Table 6 and Figures 7A and 7B show the association between IL-6 levels and all-cause mortality at 180 days and the association between IL-6 levels and all-cause mortality and / or cardiovascular-related readmission rate (CV hosp) at 60 days. [surface] [6] [:] [Cox] [Regression Results] [At 180] [Timing] All-cause mortality rate [exist]

[60] [Morality rate due to all causes of weather conditions] [ / ] [or] [CV hosp] univariate 1.59 (1.43-1.76) <0.001 1.14 (1.04-1.26) 0.007 Model 1 1.57 (1.41-1.74) <0.001 1.14 (1.04-1.26) 0.007 Model 2 1.55 (1.39-1.72) <0.001 1.11 (1.00-1.22) 0.049 Model 3 1.50 (1.35-1.68) <0.001 1.09 (0.99-1.21) 0.087 PROTECT model 1.41 (1.26-1.58) <0.001 1.06 (0.95-1.17) 0.309 Model 1: Age, Gender Model 2: Model 1 + eGFR, BMI Model 3: Model 2 + BNP PROTECT model: age, previous hospitalization for HF, edema at admission, sodium, urinary tract infection (log), creatinine (log), and albumin. Table 7 and Figures 8A and 8B show unfavorable predictions for the incremental IL-6 level compared to the baseline on day 7. [surface] [7] [:] [Cox] [Regression Results] [exist]

[0180] [Morality rate due to all factors related to weather conditions] >1 pg / mL decrease <1.0 pg / mL increase / decrease >1.0 pg / mL increase univariate 2.13 (1.16-3.91) 0.014 refer to 3.01 (1.65-5.49) <0.001 Model 1 1.79 (0.96-3.32) 0.066 refer to 2.80 (1.53-5.11) 0.001 Model 2 1.80 (0.95-3.44) 0.073 refer to 2.65 (1.41-4.97) 0.002 Model 3 1.65 (0.87-3.16) 0.128 refer to 2.35 (1.25-4.44) 0.008 PROTECT 1.71 (0.90-3.26) 0.101 refer to 2.38 (1.30-4.47) 0.007 [exist]

[60] [Morality rate due to all causes of weather conditions] [ / ] [or] [CV hosp] >1 pg / mL decrease <1.0 pg / mL increase / decrease >1.0 pg / mL increase univariate 1.31 (0.91-1.92) 0.146 refer to 1.83 (1.26-2.65) 0.001 Model 1 1.31 (0.89-1.92) 0.002 refer to 1.81 (1.25-2.62) 0.002 Model 2 1.29 (0.88-1.92) 0.187 refer to 1.70 (1.16-2.48) 0.006 Model 3 1.28 (0.86-1.89) 0.223 refer to 1.67 (1.14-2.45) 0.008 PROTECT 1.20 (0.81-1.78) 0.353 refer to 1.57 (1.07-2.30) 0.020 Model 1: Age, Gender Model 2: Model 1 + eGFR, BMI Model 3: Model 2 + BNP PROTECT model: age, previous hospitalization for HF, edema at admission, sodium, urinary tract infection (log), creatinine (log), and albumin. [8.6.] [Example] [6] [:] [IL-6] [level] [Association with outcomes in groups experiencing worsening heart failure] [ ]

[0257] [method] [.] We analyzed data from the Biology Study to Tailored Treatment in Chronic Heart Failure (BIOSTAT-CHF) to investigate the association between IL6 and outcomes in patients with worsening heart failure. In short, BIOSTAT-CHF was a multicenter, multinational prospective observational study that included 2516 patients from 69 centers in 11 European countries (Voors et al., 2016, Eur. J. Heart Fail. 18:716-726). We performed a secondary analysis in the BIOSTAT-CHF study, excluding patients with ferritin <100 from subsequent analyses. Inclusion criteria for the index cohort included: age >18 years, symptoms of new-onset or worsening HF confirmed by LVEF ≤40% or BNP and / or NT-BNP plasma levels >400 pg / ml or >2,000 pg / ml, respectively. In addition, these patients were either not previously treated with ACEi / ARB and / or β-blockers or were receiving ≤50% of the target dose of these drugs at enrollment and were expected to begin or increase the titration of ACEi / ARB and β-blockers. All patients required treatment with henlex diuretics. [Inclusion Criteria] Age > 18 Diagnosed with HF The patient was previously admitted to the hospital with HF and required diuretic treatment. Treatment with furosemide >20 mg / day or equivalent Previously untreated with ACE inhibitors / ARBs and / or beta-blockers or receiving <50% of the target dose of ACE inhibitors / ARBs and / or beta-blockers according to 2008 ESC guidelines. Titration on ACE inhibitors / ARBs and / or β-blockers is expected.

[0258] In total, IL-6 was measured in 2,329 patients with worsening HF from the BIOSTAT-CHF study. The first outcome of this study was the composite result of all-cause mortality and hospitalization due to HF.

[0259] [Statistical Analysis] [.] Baseline characteristics were presented based on the IL-6 group. Differences in baseline characteristics between groups were tested using one-way ANOVA, with Kruskal-Wallis or chi-square tests as appropriate. Survival analyses were performed using Cox regression analysis adjusted for clinically relevant variables and a BIOSTAT-CHF risk model. The BIOSTAT-CHF risk model for all-cause mortality and / or hospitalization for heart failure included: age, N-terminal β-pro-BNP (NT-pro-BNP), hemoglobin (Hb), use of β-blockers at inclusion, hospitalization for HF in the previous year, peripheral edema, systolic blood pressure, high-density lipoprotein cholesterol, and sodium (Voors et al., 2017, Eur. J. Heart Fail. 19:627-634). We performed an interaction analysis between ferritin levels and IL-6 for the first outcome. This was to investigate the association between IL-6 and outcomes dependent on the location of the TMPRS6 SNP (rs855791).

[0260] [result] The baseline characteristics of the population are described in Table 8 below. Higher IL-6 levels at baseline are associated with higher NT-promoting BNP levels and anemia (Figure 9). [surface] [8] [Baseline characteristics] [factor] [No.] [1] [Group] [No.] [2] [Group] [No.] [3] [Group] [p-] [value] N 781 775 773 [Demographics] 0.3-3.4 pg / mL 3.5-7.8 pg / mL 7.9-260.7 pg / mL Age 66.3 (12.2) 69.3 (11.7) 70.9 (11.7) <0.001 Female 200 (25.6%) 200 (25.8%) 212 (27.4%) 0.67 HF status HFrEF 607 (84.1%) 568 (81.6%) 524 (78.3%) <0.001 HFmrEF 92 (12.7%) 74 (10.6%) 87 (13.0%) HFpEF 23 (3.2%) 54 (7.8%) 58 (8.7%) BMI 27.9 (5.1) 27.7 (5.6) 27.7 (5.6) 0.77 Ischemic etiology 329 (43.2%) 353 (46.4%)<000262​​​​​​​​​​​​​​​​​​​​​​​​​​199 (25.5%) 216 (27.9%) 251 (32.5%) IV 22 (2.8%) 25 (3.2%) 33 (4.3%) NA 90 (11.5%) 97 (12.5%) 107 (13.8%) contractile BP 126.4 (20.0) 124.3 (22.4) 123.7 (23.5) 0.034 diastolic blood pressure 76.7 (12.7) 74.8 (13.4) 73.5 (14.0) <0.001 LVEF 30.6 (8.9) 30.9 (11.1) 31.3 (11.7) 0.54 Heart rate 76.4 (18.2) 80.1 (18.9) 83.6 (21.0) <0.001 [Signs and Symptoms] Peripheral edema Does not exist 352 (57.2%) 253 (39.7%) 180 (26.4%) <0.001 ankle 170 (27.6%) 188 (29.5%) 221 (32.5%) Below the knee 83 (13.5%) 156 (24.5%) 194 (28.5%) above knee 10 (1.6%) 40 (6.3%) 86 (12.6%) Increased JVP no 420 (76.5%) 318 (58.9%) 289 (53.7%) <0.001 yes 106 (19.3%) 190 (35.2%) 216 (40.1%) uncertain 23 (4.2%) 32 (5.9%) 33 (6.1%) Hepatomegaly 75 (9.6%) 128 (16.6%) 132 (17.1%) <0.001 Sit upright and breathe 168 (21.6%) 273 (35.3%) 361 (46.8%) <0.001 [Medical History] anemia 158 (23.6%) 271 (38.1%) 348 (47.0%) <0.001 atrial fibrillation 293 (37.5%) 372 (48.0%) 387 (50.1%) <0.001 diabetes 208 (26.6%) 270 (34.8%) 276 (35.7%) <0.001 COPD 113 (14.5%) 126 (16.3%) 163 (21.1%) 0.002 Kidney disease 152 (19.5%) 229 (29.5%) 261 (33.8%) <0.001 hypertension 486 (62.2%) 496 (64.0%) 473 (61.2%) 0.51 Peripheral artery disease 60 (7.7%) 92 (11.9%) 105 (13.6%) <0.001 stroke 59 (7.6%) 84 (10.8%) 76 (9.8%) 0.075 PCI 163 (20.9%) 166 (21.4%) 173 (22.4%) 0.76 CABG 120 (15.4%) 122 (15.7%) 156 (20.2%) 0.02 [drug] Heinz Cyclodiuretics 779 (99.7%) 769 (99.2%) 769 (99.5%) 0.36 ACE / ARB 601 (77.0%) 564 (72.8%) 518 (67.0%) <0.001 β-blockers 675 (86.4%) 654 (84.4%) 603 (78.0%) <0.001 Aldosterone antagonists 446 (57.1%) 411 (53.0%) 382 (49.4%) 0.01 [laboratory] hemoglobin 13.7 (1.7) 13.2 (1.8) 12.7 (2.0) <0.001 Total cholesterol 4.5 (3.7, 5.5) 4.1 (3.4, 4.9) 3.7 (3.1, 4.5) <0.001 AST 24.0 (19.0, 32.0) 25.0 (19.0, 34.0) 27.0 (20.0, 39.0) <0.001 ALT 25.0 (18.0, 37.0) 25.0 (17.0, 37.0) 23.5 (15.0, 40.0) 0.12 sodium 140.0 (138.0, 142.0) 140.0 (137.0, 142.0) 139.0 (136.0, 141.0) <0.001 Potassium 4.3 (4.0, 4.6) 4.2 (3.9, 4.6) 4.2 (3.8, 4.6) <0.001 HbA1c 6.0 (5.6, 6.7) 6.5 (5.9, 7.5) 6.5 (5.9, 7.3) <0.001 NT-promotes BNP 2661.0 (1445.0, 4820.0) 4344.0 (2517.0, 7837.0) 5734.5 (3141.0, 11452.0) <0.001 Troponin I 0.0 (0.0, 0.1) 0.0 (0.0, 0.1) 0.0 (0.0, 0.1) <0.001

[0261] As shown in Table 9 and Figures 10A and 10B, the measured baseline IL-6 level was associated with the combined results of all-cause mortality and / or hospitalization rate due to HF over two years, and was also associated with all-cause mortality over two years. [surface] [9] [:] [Cox] [Regression Analysis] [, All-cause mortality rate within two years and , ] [, / , ] [, Or because , ] [, HF , ] [, Hospitalization rate , ] [, All-cause mortality rate within two years , ] HR (95% CI) p-value HR (95% CI) p-value univariate 1.38 (1.31-1.46) <0.001 1.42 (1.32-1.53) <0.001 Model 1 1.34 (1.26-1.42) <0.001 1.48 (1.38-1.58) <0.001 Model 2 1.25 (1.17-1.33) <0.001 1.34 (1.24-1.44) <0.001 Model 3 1.24 (1.16-1.32) <0.001 1.33 (1.23-1.44) <0.001 BIOSTAT model 1.13 (1.04-1.19) 0.001 1.20 (1.11-1.31) <0.001 Model 1: Age, Gender Model 2: Model 1 plus BMI, country of origin, history of hypertension, history of diabetes, and anemia. Model 3: Model 2 plus baseline β-blocker, baseline ACEi / ARB, and baseline MRA BIOSTAT model: age, N-terminal β-pro-BNP, hemoglobin (Hb), use of β-blockers at the time of inclusion, hospitalization for HF in the year prior to inclusion, peripheral edema, systolic blood pressure, high-density lipoprotein cholesterol, and sodium.

[0262] As shown in Table 10 and Figure 11, there is no differential association between IL-6 and ferritin-dependent results. [surface]

[10] [:] [Cox] [Regression Analysis] [, Ferritin , ] [, >100 μg / L , ] [, interaction, ] [, Ferritin*IL6 , ] HR (95% CI) p-value univariate 1.39 (1.27-1.52) <0.001 0.610 Model 1 1.35 (1.23-1.48) <0.001 Model 2 1.26 (1.14-1.40) <0.001 Model 3 1.26 (1.14-1.39) <0.001 BIOSTAT model 1.11 (0.99-1.22) 0.052 Model 1: Age, Gender Model 2: Model 1 plus BMI, country of origin, history of hypertension, history of diabetes, and anemia. Model 3: Model 2 plus baseline β-blocker, baseline ACEi / ARB, and baseline MRA BIOSTAT model: age, N-terminal β-pro-BNP, hemoglobin (Hb), use of β-blockers at the time of inclusion, hospitalization for HF in the year prior to inclusion, peripheral edema, systolic blood pressure, high-density lipoprotein cholesterol, and sodium.

[0263] As shown in Table 11, there is no differential association between IL-6 and TMPRSS6 genotype-dependent results. [surface]

[11] [:] [Based on TMPRSS6] [Double Gene] [IL6] TMPRSS6 HR (95% CI) p-value AA (n=191) 1.50 (1.19-1.89) 0.001 AG (n=579) 1.34 (1.17-1.54) <0.001 GG (n=409) 1.54 (1.32-1.79) <0.001 [Incorporation by reference] [ ]

[0264] Every patent, patent application, and publication disclosed herein is incorporated herein by reference in its entirety. [Equivalent] [ ]

[0265] Although the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention can be devised without departing from the true spirit and scope of the invention. The appended claims are intended to be understood to include all such embodiments and equivalent variations.

[0266] <![CDATA[ <110> Yale University NOVO NORDISK A / S, a Danish company <![CDATA[ <120> Precision treatment for heart failure and cardiorenal syndrome <![CDATA[ <150> US 62 / 453,257 <![CDATA[ <151> 2017-02-01 <![CDATA[ <160> 16 ]]> <![CDATA[ <170> PatentIn version 3.5]]> <![CDATA[<210> 1]]> <![CDATA[<211> 824]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 1]]> Met Pro Val Ala Glu Ala Pro Gln Val Ala Gly Gly Gln Gly Asp Gly 1 5 10 15 Gly Asp Gly Glu Glu Ala Glu Pro Glu Gly Met Phe Lys Ala Cys Glu 20 25 30 Asp Ser Lys Arg Lys Ala Arg Gly Tyr Leu Arg Leu Val Pro Leu Phe 35 40 45 Val Leu Leu Ala Leu Leu Val Leu Ala Ser Ala Gly Val Leu Leu Trp 50 55 60 Tyr Phe Leu Gly Tyr Lys Ala Glu Val Met Val Ser Gln Val Tyr Ser 65 70 75 80 Gly Ser Leu Arg Val Leu Asn Arg His Phe Ser Gln Asp Leu Thr Arg 85 90 95 Arg Glu Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gln Lys Met 100 105 110 Leu Lys Glu Leu Ile Thr Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser 115 120 125 Ser Ser Val Tyr Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp 130 135 140 Phe Ile Leu Gln Ile Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu 145 150 155 160 Val Val Gln Ala Leu Leu Val Glu Glu Leu Leu Ser Thr Val Asn Ser 165 170 175 Ser Ala Ala Val Pro Tyr Arg Ala Glu Tyr Glu Val Asp Pro Glu Gly 180 185 190 Leu Val Ile Leu Glu Ala Ser Val Lys Asp Ile Ala Ala Leu Asn Ser 195 200 205 Thr Leu Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gln Gly Gln Val Leu 210 215 220 Arg Leu Lys Gly Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu 225 230 235 240 Gln Gly Pro Lys Asp Leu Met Leu Lys Leu Arg Leu Glu Trp Thr Leu 245 250 255 Ala Glu Cys Arg Asp Arg Leu Ala Met Tyr Asp Val Ala Gly Pro Leu 260 265 270 Glu Lys Arg Leu Ile Thr Ser Val Tyr Gly Cys Ser Arg Gln Glu Pro 275 280 285 Val Val Glu Val Leu Ala Ser Gly Ala Ile Met Ala Val Val Trp Lys 290 295 300 Lys Gly Leu His Ser Tyr Tyr Asp Pro Phe Val Leu Ser Val Gln Pro 305 310 315 320 Val Val Phe Gln Ala Cys Glu Val Asn Leu Thr Leu Asp Asn Arg Leu 325 330 335 Asp Ser Gln Gly Val Leu Ser Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser 340 345 350 Pro Gln Thr His Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr 355 360 365 Gly Leu Ala Leu Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gln Lys Tyr 370 375 380 Asp Leu Pro Cys Thr Gln Gly Gln Trp Thr Ile Gln Asn Arg Arg Leu 385 390 395 400 Cys Gly Leu Arg Ile Leu Gln Pro Tyr Ala Glu Arg Ile Pro Val Val 405 410 415 Ala Thr Ala Gly Ile Thr Ile Asn Phe Thr Ser Gln Ile Ser Leu Thr 420 425 430 Gly Pro Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gln Ser Asp Pro 435 440 445 Cys Pro Gly Glu Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala 450 455 460 Cys Asp Gly Val Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys 465 470 475 480 Val Cys Arg Ala Thr Phe Gln Cys Lys Glu Asp Ser Thr Cys Ile Ser 485 490 495 Leu Pro Lys Val Cys Asp Gly Gln Pro Asp Cys Leu Asn Gly Ser Asp 500 505 510 Glu Glu Gln Cys Gln Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gln 515 520 525 Cys Glu Asp Arg Ser Cys Val Lys Lys Pro Asn Pro Gln Cys Asp Gly 530 535 540 Arg Pro Asp Cys Arg Asp Gly Ser Asp Glu Glu His Cys Asp Cys Gly 545 550 555 560 Leu Gln Gly Pro Ser Ser Arg Ile Val Gly Gly Ala Val Ser Ser Glu 565 570 575 Gly Glu Trp Pro Trp Gln Ala Ser Leu Gln Val Arg Gly Arg His Ile 580 585 590 Cys Gly Gly Ala Leu Ile Ala Asp Arg Trp Val Ile Thr Ala Ala His 595 600 605 Cys Phe Gln Glu Asp Ser Met Ala Ser Thr Val Leu Trp Thr Val Phe 610 615 620 Leu Gly Lys Val Trp Gln Asn Ser Arg Trp Pro Gly Glu Val Ser Phe 625 630 635 640 Lys Val Ser Arg Leu Leu Leu His Pro Tyr His Glu Glu Asp Ser His 645 650 655 Asp Tyr Asp Val Ala Leu Leu Gln Leu Asp His Pro Val Val Arg Ser 660 665 670 Ala Ala Val Arg Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu 675 680 685 Pro Gly Leu His Cys Trp Ile Thr Gly Trp Gly Ala Leu Arg Glu Gly 690 695 700 Ala Leu Arg Ala Asp Ala Val Ala Leu Phe Tyr Gly Trp Arg Asn Gln 705 710 715 720 Gly Ser Glu Thr Cys Cys Cys Pro Ile Ser Asn Ala Leu Gln Lys Ala 725 730 735 Asp Val Gln Leu Ile Pro Gln Asp Leu Cys Ser Glu Val Tyr Arg Tyr 740 745 750 Gln Val Thr Pro Arg Met Leu Cys Ala Gly Tyr Arg Lys Gly Lys Lys 755 760 765 Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Lys Ala Leu 770 775 780 Ser Gly Arg Trp Phe Leu Ala Gly Leu Val Ser Trp Gly Leu Gly Cys 785 790 795 800 Gly Arg Pro Asn Tyr Phe Gly Val Tyr Thr Arg Ile Thr Gly Val Ile 805 810 815 Ser Trp Ile Gln Gln Val Val Thr 820 <![CDATA[<210> 2]]> <![CDATA[<211> 824]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 2]]> Met Pro Val Ala Glu Ala Pro Gln Val Ala Gly Gly Gln Gly Asp Gly 1 5 10 15 Gly Asp Gly Glu Glu Ala Glu Pro Glu Gly Met Phe Lys Ala Cys Glu 20 25 30 Asp Ser Lys Arg Lys Ala Arg Gly Tyr Leu Arg Leu Val Pro Leu Phe 35 40 45 Val Leu Leu Ala Leu Leu Val Leu Ala Ser Ala Gly Val Leu Leu Trp 50 55 60 Tyr Phe Leu Gly Tyr Lys Ala Glu Val Met Val Ser Gln Val Tyr Ser 65 70 75 80 Gly Ser Leu Arg Val Leu Asn Arg His Phe Ser Gln Asp Leu Thr Arg 85 90 95 Arg Glu Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gln Lys Met 100 105 110 Leu Lys Glu Leu Ile Thr Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser 115 120 125 Ser Ser Val Tyr Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp 130 135 140 Phe Ile Leu Gln Ile Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu 145 150 155 160 Val Val Gln Ala Leu Leu Val Glu Glu Leu Leu Ser Thr Val Asn Ser 165 170 175 Ser Ala Ala Val Pro Tyr Arg Ala Glu Tyr Glu Val Asp Pro Glu Gly 180 185 190 Leu Val Ile Leu Glu Ala Ser Val Lys Asp Ile Ala Ala Leu Asn Ser 195 200 205 Thr Leu Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gln Gly Gln Val Leu 210 215 220 Arg Leu Lys Gly Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu 225 230 235 240 Gln Gly Pro Lys Asp Leu Met Leu Lys Leu Arg Leu Glu Trp Thr Leu 245 250 255 Ala Glu Cys Arg Asp Arg Leu Ala Met Tyr Asp Val Ala Gly Pro Leu 260 265 270 Glu Lys Arg Leu Ile Thr Ser Val Tyr Gly Cys Ser Arg Gln Glu Pro 275 280 285 Val Val Glu Val Leu Ala Ser Gly Ala Ile Met Ala Val Val Trp Lys 290 295 300 Lys Gly Leu His Ser Tyr Tyr Asp Pro Phe Val Leu Ser Val Gln Pro 305 310 315 320 Val Val Phe Gln Ala Cys Glu Val Asn Leu Thr Leu Asp Asn Arg Leu 325 330 335 Asp Ser Gln Gly Val Leu Ser Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser 340 345 350 Pro Gln Thr His Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr 355 360 365 Gly Leu Ala Leu Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gln Lys Tyr 370 375 380 Asp Leu Pro Cys Thr Gln Gly Gln Trp Thr Ile Gln Asn Arg Arg Leu 385 390 395 400 Cys Gly Leu Arg Ile Leu Gln Pro Tyr Ala Glu Arg Ile Pro Val Val 405 410 415 Ala Thr Ala Gly Ile Thr Ile Asn Phe Thr Ser Gln Ile Ser Leu Thr 420 425 430 Gly Pro Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gln Ser Asp Pro 435 440 445 Cys Pro Gly Glu Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala 450 455 460 Cys Asp Gly Val Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys 465 470 475 480 Val Cys Arg Ala Thr Phe Gln Cys Lys Glu Asp Ser Thr Cys Ile Ser 485 490 495 Leu Pro Lys Val Cys Asp Gly Gln Pro Asp Cys Leu Asn Gly Ser Asp 500 505 510 Glu Glu Gln Cys Gln Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gln 515 520 525 Cys Glu Asp Arg Ser Cys Val Lys Lys Pro Asn Pro Gln Cys Asp Gly 530 535 540 Arg Pro Asp Cys Arg Asp Gly Ser Asp Glu Glu His Cys Asp Cys Gly 545 550 555 560 Leu Gln Gly Pro Ser Ser Arg Ile Val Gly Gly Ala Val Ser Ser Glu 565 570 575 Gly Glu Trp Pro Trp Gln Ala Ser Leu Gln Val Arg Gly Arg His Ile 580 585 590 Cys Gly Gly Ala Leu Ile Ala Asp Arg Trp Val Ile Thr Ala Ala His 595 600 605 Cys Phe Gln Glu Asp Ser Met Ala Ser Thr Val Leu Trp Thr Val Phe 610 615 620 Leu Gly Lys Val Trp Gln Asn Ser Arg Trp Pro Gly Glu Val Ser Phe 625 630 635 640 Lys Val Ser Arg Leu Leu Leu His Pro Tyr His Glu Glu Asp Ser His 645 650 655 Asp Tyr Asp Val Ala Leu Leu Gln Leu Asp His Pro Val Val Arg Ser 660 665 670 Ala Ala Val Arg Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu 675 680 685 Pro Gly Leu His Cys Trp Ile Thr Gly Trp Gly Ala Leu Arg Glu Gly 690 695 700 Ala Leu Arg Ala Asp Ala Val Ala Leu Phe Tyr Gly Trp Arg Asn Gln 705 710 715 720 Gly Ser Glu Thr Cys Cys Cys Pro Ile Ser Asn Ala Leu Gln Lys Val 725 730 735 Asp Val Gln Leu Ile Pro Gln Asp Leu Cys Ser Glu Val Tyr Arg Tyr 740 745 750 Gln Val Thr Pro Arg Met Leu Cys Ala Gly Tyr Arg Lys Gly Lys Lys 755 760 765 Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Lys Ala Leu 770 775 780 Ser Gly Arg Trp Phe Leu Ala Gly Leu Val Ser Trp Gly Leu Gly Cys 785 790 795 800 Gly Arg Pro Asn Tyr Phe Gly Val Tyr Thr Arg Ile Thr Gly Val Ile 805 810 815 Ser Trp Ile Gln Gln Val Val Thr 820 <![CDATA[<210> 3]]> <![CDATA[<211> 3196]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 3]]> ggacaaacag aggctcctga ggcctgtgtg caggcccggc acctatctgc cgctcccaaa 60 ggatgcccgt ggccgaggcc ccccaggtgg ctggcgggca gggggacgga ggtgatggcg 120 aggaagcgga gccggagggg atgttcaagg cctgtgagga ctccaagaga aaagcccggg 180 gctacctccg cctggtgccc ctgtttgtgc tgctggccct gctcgtgctg gcttcggcgg 240 gggtgctact ctggtatttc ctagggtaca aggcggaggt gatggtcagc caggtgtact 300 caggcagtct gcgtgtactc aatcgccact tctcccagga tcttacccgc cgggaatcta 360 gtgccttccg cagtgaaacc gccaaagccc agaagatgct caaggagctc atcaccagca 420 cccgcctggg aacttactac aactccagct ccgtctattc ctttggggag ggacccctca 480 cctgcttctt ctggttcatt ctccaaatcc ccgagcaccg ccggctgatg ctgagccccg 540 aggtggtgca ggcactgctg gtggaggagc tgctgtccac agtcaacagc tcggctgccg 600 tcccctacag ggccgagtac gaagtggacc ccgagggcct agtgatcctg gaagccagtg 660 tgaaagacat agctgcattg aattccacgc tgggttgtta ccgctacagc tacgtgggcc 720 agggccaggt cctccggctg aaggggcctg accacctggc ctccagctgc ctgtggcacc 780 tgcagggccc caaggacctc atgctcaaac tccggctgga gtggacgctg gcagagtgcc 840 gggaccgact ggccatgtat gacgtggccg ggcccctgga gaagaggctc atcacctcgg 900 tgtacggctg cagccgccag gagcccgtgg tggaggttct ggcgtcgggg gccatcatgg 960 cggtcgtctg gaaagggc ctgcacagct actacgaccc cttcgtgctc tccgtgcagc 1020 cggtggtctt ccaggcctgt gaagtgaacc tgacgctgga caacaggctc gactcccagg 1080 gcgtcctcag caccccgtac ttccccagct actactcgcc ccaaacccac tgctcctggc 1140 acctcacggt gccctctctg gactacggct tggccctctg gtttgatgcc tatgcactga 1200 ggaggcagaa gtatgatttg ccgtgcaccc agggccagtg gacgatccag aaacaggaggc 1260 tgtgtggctt gcgcatcctg cagccctacg ccgagaggat ccccgtggtg gccacggccg 1320 ggatcaccat caacttcacc tcccagatct ccctcaccgg gcccggtgtg cgggtgcact 1380 atggcttgta caaccagtcg gacccctgcc ctggagagtt cctctgttct gtgaatggac 1440 tctgtgtccc tgcctgtgat ggggtcaagg actgccccaa cggcctggat gagagaaact 1500 gcgtttgcag agccacattc cagtgcaaag aggacagcac atgcatctca ctgcccaagg 1560 tctgtgatgg gcagcctgat tgtctcaacg gcagcgacga agagcagtgc caggaagggg 1620 tgccatgtgg gacattcacc ttccagtgtg aggaccggag ctgcgtgaag aagcccaacc 1680 cgcagtgtga tgggcggccc gactgcaggg acggctcgga tgaggagcac tgtgactgtg 1740 gcctccaggg cccctccagc cgcattgttg gtggagctgt gtcctccgag ggtgagtggc 1800 catggcaggc cagcctccag gttcggggtc gacacatctg tgggggggcc ctcatcgctg 1860 accgctgggt gataacagct gcccactgct tccaggagga cagcatggcc tccacggtgc 1920 tgtggaccgt gttcctgggc aaggtgtggc agaactcgcg ctggcctgga gaggtgtcct 1980 tcaaggtgag ccgcctgctc ctgcacccgt accacgaaga ggacagccat gactacgacg 2040 tggcgctgct gcagctcgac cacccggtgg tgcgctcggc cgccgtgcgc cccgtctgcc 2100 tgcccgcgcg ctcccacttc ttcgagcccg gcctgcactg ctggattacg ggctggggcg 2160 ccttgcgcga gggcgcccta cgggcggatg ctgtggccct attttatgga tggagaaacc 2220 aaggctcaga gacatgttgc tgccccatca gcaacgctct gcagaaagtg gatgtgcagt 2280 tgatcccaca ggacctgtgc agcgaggtct atcgctacca ggtgacgcca cgcatgctgt 2340 gtgccggcta ccgcaagggc aagaaggatg cctgtcaggg tgactcaggt ggtccgctgg 2400 tgtgcaaggc actcagtggc cgctggttcc tggcggggct ggtcagctgg ggcctgggct 2460 gtggccggcc taactacttc ggcgtctaca cccgcatcac aggtgtgatc agctggatcc 2520 agcaagtggt gacctgagga actgcccccc tgcaaagcag ggcccacctc ctggactcag 2580 agagcccagg gcaactgcca agcaggggga caagtattct ggcggggggt gggggagaga 2640 gcaggccctg tggtggcagg aggtggcatc ttgtctcgtc cctgatgtct gctccagtga 2700 tggcaggagg atggagaagt gccagcagct gggggtcaag acgtcccctg aggacccagg 2760 cccacaccca gcccttctgc ctcccaattc tctctcctcc gtccccttcc tccactgctg 2820 cctaatgcaa ggcagtggct cagcagcaag aatgctggtt ctacatcccg aggagtgtct 2880 gaggtgcgcc ccactctgta cagaggctgt ttgggcagcc ttgcctccag agagcagatt 2940 ccagcttcgg aagcccctgg tctaacttgg gatctgggaa tggaaggtgc tcccatcgga 3000 ggggaccctc agagccctgg agactgccag gtgggcctgc tgccactgta agccaaaagg 3060 tggggaagtc ctgactccag ggtccttgcc ccacccctgc ctgccacctg ggccctcaca 3120 gcccagaccc tcactgggag gtgagctcag ctgccctttg gaataaagct gcctgatcca 3180 aaaaaaaaaa aaaaaa 3196 <![CDATA[<210> 4]]> <![CDATA[<211> 3196]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 4]]> ggacaaacag aggctcctga ggcctgtgtg caggcccggc acctatctgc cgctcccaaa 60 [[ID=2�]]ggatgcccgt ggccgaggcc ccccaggtgg ctggcgggca gggggacgga ggtgatggcg 120 [[ID=۲۵]]aggaagcgga gccggagggg atgttcaagg cctgtgagga ctccaagaga aaagcccggg 180 [[ID=۳۰]]gctacctccg cctggtgccc ctgtttgtgc tgctggccct gctcgtgctg gcttcggcgg 240 [[ID=۳۵]]gggtgctact ctggtatttc ctagggtaca aggcggaggt gatggtcagc caggtgtact 300 [[ID=۴۰]]caggcagtct gcgtgtactc aatcgccact tctcccagga tcttacccgc cgggaatcta 360 请注意,原文中存在一些可能的错误或不规范之处,比如“[[ID=]]”中的数字序号有重复和不连续情况,以及部分标签编号似乎不太符合常见的规范格式,但我按照要求进行了准确翻译。如果这是特定系统或格式下的内容,建议进一步核对原始规范以确保准确理解和处理。gtgccttccg cagtgaaacc gccaaagccc agaagatgct caaggagctc atcaccagca 420 cccgcctggg aacttactac aactccagct ccgtctattc ctttggggag ggacccctca 480 cctgcttctt ctggttcatt ctccaaatcc ccgagcaccg ccggctgatg ctgagccccg 540 aggtggtgca ggcactgctg gtggaggagc tgctgtccac agtcaacagc tcggctgccg 600 tcccctacag ggccgagtac gaagtggacc ccgagggcct agtgatcctg gaagccagtg 660 tgaaagacat agctgcattg aattccacgc tgggttgtta ccgctacagc tacgtgggcc 720 agggccaggt cctccggctg aaggggcctg accacctggc ctccagctgc ctgtggcacc 780 tgcagggccc caaggacctc atgctcaaac tccggctgga gtggacgctg gcagagtgcc 840 gggaccgact ggccatgtat gacgtggccg ggcccctgga gaagaggctc atcacctcgg 900 tgtacggctg cagccgccag gagcccgtgg tggaggttct ggcgtcgggg gccatcatgg 960 cggtcgtctg gaaagggc ctgcacagct actacgaccc cttcgtgctc tccgtgcagc 1020 cggtggtctt ccaggcctgt gaagtgaacc tgacgctgga caacaggctc gactcccagg 1080 gcgtcctcag caccccgtac ttccccagct actactcgcc ccaaacccac tgctcctggc 1140 acctcacggt gccctctctg gactacggct tggccctctg gtttgatgcc tatgcactga 1200 ggaggcagaa gtatgatttg ccgtgcaccc agggccagtg gacgatccag aaacaggaggc 1260 tgtgtggctt gcgcatcctg cagccctacg ccgagaggat ccccgtggtg gccacggccg 1320 ggatcaccat caacttcacc tcccagatct ccctcaccgg gcccggtgtg cgggtgcact 1380 atggcttgta caaccagtcg gacccctgcc ctggagagtt cctctgttct gtgaatggac 1440 tctgtgtccc tgcctgtgat ggggtcaagg actgccccaa cggcctggat gagagaaact 1500 gcgtttgcag agccacattc cagtgcaaag aggacagcac atgcatctca ctgcccaagg 1560 tctgtgatgg gcagcctgat tgtctcaacg gcagcgacga agagcagtgc caggaagggg 1620 tgccatgtgg gacattcacc ttccagtgtg aggaccggag ctgcgtgaag aagcccaacc 1680 cgcagtgtga tgggcggccc gactgcaggg acggctcgga tgaggagcac tgtgactgtg 1740 gcctccaggg cccctccagc cgcattgttg gtggagctgt gtcctccgag ggtgagtggc 1800 catggcaggc cagcctccag gttcggggtc gacacatctg tgggggggcc ctcatcgctg 1860 accgctgggt gataacagct gcccactgct tccaggagga cagcatggcc tccacggtgc 1920 tgtggaccgt gttcctgggc aaggtgtggc agaactcgcg ctggcctgga gaggtgtcct 1980 tcaaggtgag ccgcctgctc ctgcacccgt accacgaaga ggacagccat gactacgacg 2040 tggcgctgct gcagctcgac cacccggtgg tgcgctcggc cgccgtgcgc cccgtctgcc 2100 tgcccgcgcg ctcccacttc ttcgagcccg gcctgcactg ctggattacg ggctggggcg 2160 ccttgcgcga gggcgcccta cgggcggatg ctgtggccct attttatgga tggagaaacc 2220 aaggctcaga gacatgttgc tgccccatca gcaacgctct gcagaaagtg gatgtgcagt 2280 tgatcccaca ggacctgtgc agcgaggtct atcgctacca agtgacgcca cgcatgctgt 2340 gtgccggcta ccgcaagggc aagaaggatg cctgtcaggg tgactcaggt ggtccgctgg 2400 tgtgcaaggc actcagtggc cgctggttcc tggcggggct ggtcagctgg ggcctgggct 2460 gtggccggcc taactacttc ggcgtctaca cccgcatcac aggtgtgatc agctggatcc 2520 agcaagtggt gacctgagga actgcccccc tgcaaagcag ggcccacctc ctggactcag 2580 agagcccagg gcaactgcca agcaggggga caagtattct ggcggggggt gggggagaga 2640 gcaggccctg tggtggcagg aggtggcatc ttgtctcgtc cctgatgtct gctccagtga 2700 tggcaggagg atggagaagt gccagcagct gggggtcaag acgtcccctg aggacccagg 2760 cccacaccca gcccttctgc ctcccaattc tctctcctcc gtccccttcc tccactgctg 2820 cctaatgcaa ggcagtggct cagcagcaag aatgctggtt ctacatcccg aggagtgtct 2880 gaggtgcgcc ccactctgta cagaggctgt ttgggcagcc ttgcctccag agagcagatt 2940 ccagcttcgg aagcccctgg tctaacttgg gatctgggaa tggaaggtgc tcccatcgga 3000 ggggaccctc agagccctgg agactgccag gtgggcctgc tgccactgta agccaaaagg 3060 tggggaagtc ctgactccag ggtccttgcc ccacccctgc ctgccacctg ggccctcaca 3120 gcccagaccc tcactgggag gtgagctcag ctgccctttg gaataaagct gcctgatcca 3180 aaaaaaaaaa aaaaaa 3196 <![CDATA[<210> 5]]> <![CDATA[<211> 274]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 5]]> Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys <![CDATA[ ]]> 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Ser Phe 245 250 255 Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala 260 265 270 Lys Ile <![CDATA[<210> 6]]> <![CDATA[<211> 1201]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400>]]> 6 aatattagag tctcaacccc caataaatat aggactggag atgtctgagg ctcattctgc 60 cctcgagccc accgggaacg aaagagaagc tctatctccc ctccaggagc ccagctatga 120 actccttctc caaagcgcc ttcggtccag ttgccttctc cctggggctg ctcctggtgt 180 tgcctgctgc cttccctgcc ccagtacccc your foot ttccaaagat gtagccgccc 240 cacagaca gccactcacc tcttcagaac gaattgacaa acaaattcgg tacatcctcg 300 acggcatctc agccctgaga aaggagacat gtaacaagag taacatgtgt gaagcagca 360 aagaggcact ggcagaaaac aacctgaacc ttccaaagat ggctgaaaaa gatggatgct 420 tccaatctgg attcaatgag gagacttgcc tggtgaaaat catcactggt cttttggagt 480 540 ctgtgcagat gagtacaaaa gtcctgatcc agttcctgca gaaaaaggca aagaatctag 600 atgcaataac cacccctgac ccaaccacaa atgccagcct gctgacgaag ctgcaggcac 660 agaaccagtg gctgcaggac atgacaactc atctcattct gcgcagcttt aaggagttcc 720 tgcagtccag cctgagggct cttcggcaaa tgtagcatgg gcacctcaga ttgttgttgt 780 taatgggcat tccttcttct ggtcagaaac ctgtccactg ggcacagaac ttatgttgtt 840 ctctatggag aactaaaagt atgagcgtta ggacactatt ttaattattt ttaatttatt 900 aatatttaaa tatgtgaagc tgagttaatt tatgtaagtc atatttatat ttttaagaag 960 taccacttga aacattttat gtattagttt tgaaataata atggaaagtg gctatgcagt 1020 ttgaatatcc ttgtttcag agccagatca tttcttggaa agtgtaggct tacctcaaat 1080 aaatggctaa cttatacata tttttaaaga aatatttata ttgtatttat ataatgtata 1140 aatggttttt ataccaataa atggcatttt aaaaaattca gcaaaaaaaa aaaaaaaaaa 1200 a 1201 <![CDATA[<210> 7]]> <![CDATA[<211> 468]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 7]]> Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro 1 5 10 15 Gly Ala Ala Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg 20 25 30 Gly Val Leu Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro 35 40 45 Gly Val Glu Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys 50 55 60 Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg 65 70 75 80 Leu Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys 85 90 95 Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val 100 105 110 Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser 115 120 125 Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr 130 135 140 Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp 145 150 155 160 Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys 165 170 175 Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met 180 185 190 Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe 195 200 205 Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val 210 215 220 Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp 225 230 235 240 Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg 245 250 255 Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp 260 265 270 Leu Gln His His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His 275 280 285 Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser 290 295 300 Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser 305 310 315 320 Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr 325 330 335 Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr 340 345 350 Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pro Leu Pro Thr Phe Leu 355 360 365 Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Leu Leu Cys Ile Ala Ile 370 375 380 Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Arg Ala Leu Lys Glu Gly 385 390 395 400 Lys Thr Ser Met His Pro Pro Tyr Ser Leu Gly Gln Leu Val Pro Glu 405 410 415 Arg Pro Arg Pro Thr Pro Val Leu Val Pro Leu Ile Ser Pro Pro Val 420 425 430 Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Ser Ser His Asn Arg Pro 435 440 445 Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp Ile Ser Asn Thr Asp Tyr 450 455 460 Phe Phe Pro Arg 465 <![CDATA[<210> 8]]> <![CDATA[<211> 918]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 8]]> Met Leu Thr Leu Gln Thr Trp Leu Val Gln Ala Leu Phe Ile Phe Leu 1 5 10 15 Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser 20 25 30 Pro Glu Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45 Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50 55 60 Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr Thr 65 70 75 80 Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser 85 90 95 Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu 100 105 110 Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys 115 120 125 Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Arg Cys 130 135 140 Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu 145 150 155 160 Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175 Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190 Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205 Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220 Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu 225 230 235 240 Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys 245 250 255 Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile 260 265 270 Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp 275 280 285 Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu 290 295 300 Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile 305 310 315 320 Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile 325 330 335 Asp Pro Ser His Thr Gln Gly Tyr Arg Thr Val Gln Leu Val Trp Lys 340 345 350 Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val 355 360 365 Thr Leu Thr Arg Trp Lys Ser His Leu Gln Asn Tyr Thr Val Asn Ala 370 375 380 Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu 385 390 395 400 Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala Ala Val Leu Thr Ile 405 410 415 Pro Ala Cys Asp Phe Gln Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425 430 Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445 Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460 Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr Val His Arg Thr 465 470 475 480 Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485 490 495 Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala 500 505 510 Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 515 520 525 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gln Leu Pro Val 530 535 540 Asp Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr 545 550 555 560 Ile Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr Glu 565 570 575 Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590 Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600 605 Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu Ala Ile Val Val Pro 610 615 620 Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys 625 630 635 640 Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro 645 650 655 Asp Pro Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro 660 665 670 Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe 675 680 685 Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700 Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn 705 710 715 720 Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 725 730 735 Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn 740 745 750 Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly Tyr Arg 755 760 765 His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln 770 775 780 Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp 785 790 795 800 His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys 805 810 815 Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His Phe Glu 820 825 830 Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845 Lys Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 850 855 860 Met Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly 865 870 875 880 Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895 Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln 900 905 910 Gly Gly Tyr Met Pro Gln 915 <![CDATA[ <210> 9]]> <![CDATA[ <211> 5]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 9]]> Ser Asn Tyr Met Ile 1 5 <![CDATA[ <210> 10]]> <![CDATA[ <211> 17]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 10]]> Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <![CDATA[ <210> 11]]> <![CDATA[ <211> 11]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 11]]> Trp Ala Asp Asp His Pro Pro Trp Ile Asp Leu 1 5 10 <![CDATA[ <210> 12]]> <![CDATA[ <211> ]]>11 <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 12]]> Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <![CDATA[ <210> 13]]> <![CDATA[ <211> 7]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 13]]> Lys Ala Ser Thr Leu Glu Ser 1 5 <![CDATA[ <210> 14]]> <![CDATA[ <211> 8]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 14]]> Gln Gln Ser Trp Leu Gly Gly Ser 1 5 <![CDATA[ <210> 15]]> <![CDATA[ <211> 450]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 15]]> Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Asn 20 25 30 Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Leu Tyr Tyr Tyr Ala Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Met Ser Arg Asp Ile Ser Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Ala Asp Asp His Pro Pro Trp Ile Asp Leu Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile 245 250 255 Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <![CDATA[ <210> 16]]> <![CDATA[ <211> 213]]> <![CDATA[ <212> PRT]]> <![CDATA[ <213> Artificial sequence <![CDATA[ <220> ]]> <![CDATA[ <223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 16]]> Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Leu Gly Gly Ser 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210

Claims

1. The use of an antibody for the preparation of a medicine that reduces the risk of hospitalization for patients due to heart failure, wherein the antibody is an anti-IL-6 antibody or an antigen-binding fragment or derivative thereof.

2. As claimed in claim 1, wherein the drug is prepared for subcutaneous administration.

3. As requested in item 1, wherein the patient has a high pre-treatment plasma IL-6 level.

4. As requested in item 3, wherein the patient’s pretreatment plasma IL-6 level is greater than 2 pg / mL.

5. As requested in item 1, where the patient’s diuretic efficiency is less than 500 mmol Na / Heinz cyclodiuretic dose is doubled.

6. As requested in item 5, where the patient’s diuretic efficiency is less than 200 mmol Na / Heinz diuretic dose is doubled.

7. For the purpose of claim 1, wherein the patient suffers from antidiuretic heart failure.

8. For the purpose of request item 1, wherein the patient suffers from acute heart failure.

9. For the purposes of claim 1, wherein the patient suffers from chronic heart failure.

10. The use of any of claims 1 to 9, wherein the antibody is MEDI5117.

11. As used in claim 10, wherein MEDI5117 comprises: (a) VH CDR1 having the amino acid sequence of SEQ ID NO: 9; (b) VH CDR2 having the amino acid sequence of SEQ ID NO: 10; (c) VH CDR3 having the amino acid sequence of SEQ ID NO: 11; (d) VL CDR1 having the amino acid sequence of SEQ ID NO: 12; (e) VL CDR2 having the amino acid sequence of SEQ ID NO: 13; and (f) VL CDR3 having the amino acid sequence of SEQ ID NO:

14.

12. As used in claim 10, wherein MEDI5117 comprises: (i) a heavy chain having the amino acid sequence of SEQ ID NO: 15; and (ii) a light chain having the amino acid sequence of SEQ ID NO:

16.

13. As claimed in claim 1 or 2, wherein the anti-IL-6 antibody is MEDI5117, and wherein MEDI5117 is administered subcutaneously at a uniform dose of 10 to 20 mg.

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