Immunosuppressive drugs and methods of treatment

Abstract

Relaxin-2 is used as a broad spectrum substitute for glucocorticoids, mineralocorticoids, corticosteroids and analogs thereof for the treatment of tissue and endothelial injury without causing glycogen allogenesis, Cushing syndrome and Cushing-like adverse reactions. Particularly, under the condition of autoimmune diseases and organ transplantation, the medicine and treatment of tissue and endothelial injury repair are supported by promoting immunosuppressive regulatory T cells (Treg).

Description

Technical Field

[0001] This invention relates to pharmaceutical formulations containing low molecular weight peptide hormones of the human insulin superfamily and to pharmaceutical formulations containing selective glucocorticoid receptor modulators (SEGRMs) that inhibit the innate immune system (A61K38 / 1754; A61K38 / 1751; A61K38 / 2221). Background Technology

[0002] Glucocorticoids are steroid hormones secreted by the adrenal glands. They regulate a variety of physiological functions and are important in maintaining basal and stress-related homeostasis. At pharmacological doses, glucocorticoids and corticosteroids are effective immunosuppressants in the treatment of a variety of inflammatory, autoimmune, and lymphoproliferative disorders. At the cellular level, glucocorticoids and corticosteroids are functionally mediated by glucocorticoid receptors (GR), which, simply put, are receptors to which cortisol, cortisone, and glucocorticoids bind. GR belongs to a superfamily of receptors of nuclear transactivators with over 200 members and is ubiquitously expressed in almost all human tissues and organs. GR is a hormone and ligand-dependent transcription factor that regulates or influences the expression of GR-responsive genes, which may account for 3% to 10% of the human genome. For example, ligand-activated GR can upregulate the expression of anti-inflammatory proteins in the nucleus or inhibit the expression of pro-inflammatory proteins in the cytosol by preventing other transcription factors from translocating from the cytosol to the nucleus. It can also be inhibited by the ligand-activated GR complex binding to the same site on DNA where another transcription factor binds, thus rendering the other inactive. The activated GR complex functions pleiotropically and occurs in various parts of the body: controlling metabolism, physical development, and immune responses. This has led to steroids and glucocorticoids becoming the most commonly used drugs (see review by Nicolaides N et al., Glucocorticoid Receptor, edited by Feingold KR, Anawalt B, Boyce A et al., Endotext [Internet]. South Dartmouth (MA): 2021).

[0003] Relaxin was initially identified as a pregnancy hormone due to its activity. However, it functions not only at the maternal-fetal interface (Hisaw FL, Experimental relaxation of the public ligament of the guinea pig, Proc. Soc. Exp. Biol. Med. 1926; 23:661-663). Relaxin is a heterodimeric peptide of approximately 6 kDa, in which disulfide bonds, like in insulin, link the A and B chains. The insulin superfamily includes insulin, insulin-like growth factor I and II, relaxin-1, relaxin-2, and relaxin-3; and insulin-like factors 3, 4, 5, and 6. In humans, three distinct forms of relaxin have been identified, with relaxin-2 being the predominant storage form and the only form secreted into circulation. The biological role of relaxin-1 in humans is unclear, as is the biological role of relaxin-3, which is found only in the brain. Relaxin-2 has been shown to function as an endocrine and paracrine factor, dilating blood vessels and increasing blood flow in tissues (see reviews: Dschietzig T et al. Relaxin: a pregnancy hormone as a central player of body fluid and circulation homeostasis, CMLS 2003; 60:688-700; Dschietzig T et al. Relaxin-a pleiotropic hormone and its emerging role for experimental and clinical therapeutics, Pharmacol Ther 2006; 112:38e56).

[0004] Numerous clinical applications have been proposed for relaxin and relaxin agonists and antagonists: for the treatment of skin aging, androgenetic alopecia, atrophy, sclerosis, and miniaturization of hair and hair follicles (EP0793505); for controlling fetal growth (EP0991947); for improving fertility (EP1473034); as an adjuvant during stem cell differentiation (EP1696948); for increasing arterial compliance (EP1765149); and for diseases related to vasoconstriction (EP18544). 76); for tumor suppression (WO2007115414); for the treatment of diabetes and related complications (EP1909809); for the treatment of multiple sclerosis and other neurodegenerative disorders (EP2723366); for the treatment of symptoms of aging and neurodegenerative disorders (WO0048618); for the treatment of glucose toxicity and impaired glucose tolerance (EP2817026); for the treatment of dyspnea associated with acute heart failure (EP2829280); for the treatment of preserved ejection fraction. Heart failure (EP3145534); used to treat CNS, CNS trauma, demyelinating diseases and / or glial proliferation, multiple sclerosis (MS), Alzheimer's disease and Parkinson's disease, inflammatory conditions of the CNS, Schilder's diffuse cerebral sclerosis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, transverse myelitis, and neuromyelitis optica, concussion, traumatic brain injury, shaken baby syndrome, traumatic spinal cord injury, traumatic brain injury, ionizing radiation, Korsakoff syndrome, multiple sclerosis Systemic atrophy, prions, AIDS dementia syndrome, vasculitis, amyotrophic lateral sclerosis, Huntington's disease, autoimmune inflammatory diseases, retinal gliosis, encephalopathy, leukodystrophy, encephalitis, neuropathy (EP3347037); used to treat various inflammatory conditions, acidic airway hyperresponsiveness, asthma, rheumatoid arthritis, gout, ankylosing spondylitis, inflammatory bowel disease, myositis, systemic lupus erythematosus, sepsis, urticaria, psoriasis, allergic reactions (WO20220374669).However, a particular focus of attention is the use of relaxin in cardiac, renal, pulmonary, and hepatic infarction models for hemodynamic adaptation and systemic vascular resistance regulation (WO9303755, WO0240500; Dschietzig T et al. Plasma levels and myocardial expression of relaxin-2 are increased in human heart failure, CIRCULATION 2000, 102(18):594; Coulson CC et al. Central hemodynamic effects of recombinant human relaxin in the isolated, perfused rat heart model, Obstetrics & Gynecology 1996, 87(4):610-612; Masini E. et al. Relaxin counteracts myocardial damage induced by ischemia-reperfusion inisolated guinea pig hearts: evidence for an involvement of nitric oxide, Endocrinology 1997, 138:4713-4720; DiLascio G et al. Cellular retrograde cardiomyoplasty and relaxin therapy for postischemic myocardial repair in arat model, Tex Heart Inst J 2012, 39:488-499; Collino M et al. Acute treatment with relaxin protects the kidney against ischemia-reperfusion injury, J CellMol Med 2013, 17:1494-1505; Bausys A et al. supplemented with synthetic human relaxin decreases ischemia-reperfusion injury after porcine kidney transplantation,Int J Mol Sci.2021,22,11417; Alexiou K et al. Relaxin is acandidate drug for lung preservation:relaxin induced protection of rat lungs from ischemia-reperfusion injury,J Heart Lung Transplant 2010,29:454-460; Teichmann Relaxin by SL et al.: a review of the biology and potential role in treating heart failure, Curr Heart Fail Rep 2010;7:75-82). Furthermore, relaxin has been observed to reduce oxidative cell damage in orthotopic kidney and liver transplantation and in liver perfusion systems (DE102005040492; Boehnert MU, Relaxin as an additional protective model of isolate perfused liver, Ann NY Acad Sci 2005, 1041:434-440; Kageyama S et al., Relaxin in Liver Transplantation: A Personal Perspective Mol Cell Endocrinol. 2019, 487:75-79; Jakubauskiene L et al., Relaxin positively influences ischemia-reperfusion injury in solid organ transplantation: a comprehensive review, Int J Mol Sci. 2020, 21(2):631ff). These findings appear consistent with the observation that relaxin can function as a ligand for GR in somatic cells completely independently of the signaling cascade of G protein-coupled relaxin receptors RXFP1 and RXFP2 (formerly designated LGR7 and LGR8, respectively).Experiments conducted in HeLa cells have confirmed this, with HEK cells and Th1-activated macrophages showing that relaxin-2 activates the GR pathway, and that the relaxin-GR complex inhibits the stimulated secretion of cytokines IL-1, IL-6, and TNF-α (such as dexamethasone) (Dschietzig TB et al. Identification of the pregnancy hormone relaxin as a glucocorticoid receptor agonist, FASEB J 2004, 18:1536-1538; Dschietzig T et al. The pregnancy hormone relaxin binds to and activates the human glucocorticoid receptor, Ann NY Acad Sci. 2005, 1041:256-71; Dschietzig T et al. RXFP1-inactive relaxin activates human glucocorticoid receptor: further investigations into the relaxin-GR pathway, Regul Pept. 2009, 154:77-84; Dschietzig T et al.'s "Autoregulation of human relaxin-2 gene expression critically involves relaxin and glucocorticoid receptor binding to glucocorticoid response half-sites in the relaxin-2 promoter" (Regul Pept. 2009, 155:163-73). The presence of relaxin also stimulates gene expression in the NOTCH1 intracellular domain (NICD), and associated intercellular signaling appears to contribute to a reduction in ischemia-reperfusion injury. However, ischemia-reperfusion injury (IRI) is an unavoidable consequence of many clinical conditions, including trauma, sepsis, resection, and transplantation, and is an immune-driven inflammatory response leading to cell death and early graft dysfunction. However, human liver biopsies have shown that high NICD expression enhances resistance to IRI. The use of relaxin in organ preservation solutions from donor kidneys and livers appears to offer hope for improving IRI resistance. On the other hand, their side effects limit the clinical application of glucocorticoids and GR ligands.

[0005] Because almost all cells in the body express the same glucocorticoid receptors, the pharmacologically highly desirable effects of glucocorticoids and GR activation are proportionally associated with the specific adverse reactions they cause. Adverse metabolic and pharmacological effects of long-term glucocorticoid therapy include impaired wound healing, diabetic manifestations / dysregulation, adverse immunosuppression, increased risk of infection, osteoporosis, growth retardation in children, myopathy / muscle atrophy, skin atrophy, steroid acne, hirsutism, as well as typical symptoms of Cushing's syndrome (such as uncontrolled hypertension), saline imbalance, psychological and neurological disorders, depression, etc. Therefore, individual benefit-risk analyses must be performed for each glucocorticoid therapy (primarily when treating cancer patients and transplant recipients receiving such hormone therapy). This applies to patients with hormone-refractory tumors who will be treated with a combination of specific antibodies and steroids to inhibit cancer cell proliferation. This is especially true for patients with chronic inflammatory diseases who suffer from long-term glucocorticoid therapy for side effects that outweigh the suffering or debilitating effects of the underlying disease. The known prior art does not provide medically reasonable guidance on whether and when to use relaxin for treatment. Therefore, existing technologies in this field present problems. Summary of the Invention

[0006] This problem is addressed by a pharmaceutical composition for treating patients who require treatment to suppress the physiological inflammatory response caused by the innate immune system, the pharmaceutical composition comprising an effective amount of synthetic human relaxin-2, as well as a pharmacological solvent, a pharmacological diluent, or a pharmacological excipient.

[0007] In some implementations, patients suspected of needing to suppress the physiological inflammatory response caused by the innate immune system are tested for one or more of the following serum parameters: serum HMGB1 (high-mobility family box protein) greater than or equal to 4 ng / ml; serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.5 ng / ml; serum sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 2 ng / ml; or serum calprotectin greater than or equal to 10 μg / ml.

[0008] This problem is further addressed by a pharmaceutical composition for treating patients with inflammatory responses triggered by the innate immune system and / or requiring inhibition of glucocorticoid receptors activated by ligands, the pharmaceutical composition comprising an effective amount of synthetic human relaxin-2 as the active ingredient, as well as a pharmacological solvent, pharmacological diluent, or pharmacological excipient, thereby avoiding the manifestations or disorders of diabetes or symptoms of Cushing's syndrome.

[0009] In some implementations, patients suspected of having an inflammatory response triggered by the innate immune system meet the following clinical criteria: prediabetes (HbA1c > 5.7% and < 6.5%), obesity (BMI > 30 kg / m²). 2 High blood pressure (stage 1 or higher according to the 2017 ACC / AHA guidelines).

[0010] In some embodiments, a pharmaceutical composition comprising an effective amount of synthetic human relaxin-2 is used to treat patients who require alterations to the development of their body or organs, while also preventing the manifestations or disorders of diabetes or symptoms of Cushing's syndrome. The patients may already exhibit the following clinical characteristics: prediabetes (HbA1c > 5.7 and < 6.5%), obesity (BMI > 30 kg / m²). 2 High blood pressure (Stage 1 or higher).

[0011] In some embodiments, a pharmaceutical composition comprising an effective amount of synthetic human relaxin-2 is used to treat patients who have received allogeneic grafts and require suppression of the innate immune system and inflammatory response, while preventing impaired wound healing, manifestations or disorders of diabetes, or symptoms of Cushing's syndrome. In the case of transplant patients, the medical need for suppression of the innate immune system is met when one or more of the following four criteria are met: serum HMGB1 (high-mobility family box protein) ≥ 2 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) ≥ 0.25 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) ≥ 0.5 ng / ml, and / or serum calprotectin ≥ 4 μg / ml.

[0012] In some preferred embodiments, a pharmaceutical composition containing an effective amount of synthetic human relaxin-2 is used to treat patients who require chronic suppression of their innate immune system and inflammatory response, while preventing the manifestations or disorders of diabetes or symptoms of Cushing's syndrome.

[0013] In some implementations, the patient suspected of needing chronic suppression of the inflammatory response exhibits one or more of the following medical criteria: serum HMGB1 (high-mobility box cassette protein) greater than or equal to 4 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.5 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 2 ng / ml, and / or serum calprotectin greater than or equal to 10 μg / ml.

[0014] In some other embodiments, the pharmaceutical composition is used to treat patients with hormone-resistant cancers, wherein the hormone-resistant cancers include, but are not limited to, prostate cancer, breast cancer, primary cancers of glucocorticoid receptor-activated (GR) cells activated by ligands, and Kaposi's sarcoma; wherein the primary cancers of GR cells activated by ligands include, but are not limited to, multiple myeloma, Hodgkin's disease, and other lymphoid carcinomas; wherein the synthetic human relaxin-2 is used as a supplement and alternative to glucocorticoid receptor-activated hormones.

[0015] Another aspect of the invention relates to a method of treating a patient, the method comprising testing one or more of the following clinical parameters of the patient: serum HMGB1 (high-mobility family box protein) greater than or equal to 4 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.5 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 2 ng / ml, and / or serum calprotectin greater than or equal to 10 μg / ml; and, when administered, administering to the patient an effective amount of synthetic human relaxin-2 in a pharmacological solvent, pharmacological diluent, or pharmacological excipient to inhibit or suppress a physiological inflammatory response caused by the innate immune system.

[0016] In some embodiments, the method steps are used to treat patients suffering from an inflammatory response triggered by the innate immune system and / or requiring inhibition of glucocorticoid receptors activated by ligands, wherein the active pharmacological component is synthetic human relaxin-2 to avoid manifestations or disorders of diabetes or symptoms of Cushing's syndrome.

[0017] In some other embodiments, the treatment method includes administering an effective amount of synthetic human relaxin-2 to a patient who needs to alter the development of the body or organs, while preventing the manifestations or disorders of diabetes or symptoms of Cushing's syndrome.

[0018] In some embodiments, the method includes administering an effective amount of synthetic human relaxin-2 to a patient who has received an allogeneic graft and requires suppression of the innate immune system and inflammatory response without impairing wound healing, causing or disrupting diabetes, or inducing symptoms of Cushing's syndrome, after the patient has tested positive for one or more of the following medical criteria: serum HMGB1 (high-mobility family box protein) greater than or equal to 2 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.25 ng / ml, sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 0.5 ng / ml, and / or serum calprotectin greater than or equal to 4 μg / ml.

[0019] In some other embodiments, the treatment method includes administering an effective amount of synthetic human relaxin-2 to a patient diagnosed with hormone-resistant cancer requiring treatment, wherein the hormone-resistant cancer includes, but is not limited to, prostate cancer, breast cancer, primary cancers of ligand-activated GR, and Kaposi's sarcoma; wherein the primary cancers of ligand-activated GR include, but are not limited to, multiple myeloma, Hodgkin's disease, and other lymphoid carcinomas; wherein the synthetic human relaxin-2 is used to supplement and / or replace glucocorticoid receptor-activated hormone. Alternatively, in some embodiments, the method includes administering an effective amount of synthetic human relaxin-2 to a patient requiring additional glucocorticoid receptor-activated hormone or a substitute for glucocorticoid receptor-activated hormone.

[0020] In some embodiments, the treatment method includes administering an effective amount of synthetic human relaxin-2 to a patient diagnosed as requiring immunosuppressive therapy, primarily when the patient exhibits one or more of the following clinical features, wherein the synthetic relaxin-2 is used to supplement and / or replace glucocorticoid receptor-activating hormones to prevent or avoid manifestations or dysregulation of diabetes or symptoms of Cushing's syndrome: prediabetes (HbA1c > 5.7% and < 6.5%), obesity (BMI > 30 kg / m²). 2 High blood pressure (stage 1 or higher according to the 2017 ACC / AHA guidelines).

[0021] In some implementations of the treatment, the patient is initially diagnosed as requiring chronic doses of corticosteroids and / or glucocorticoids, and it includes the subcutaneous administration of an appropriate amount of synthetic human relaxin-2 to avoid manifestations or disorders of diabetes, impaired wound healing, or symptoms of Cushing's syndrome.

[0022] In some implementations of the above treatment methods, the patient suffers from various forms of autoimmune or rheumatic diseases; ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory types of arthritis, and rheumatoid arthritis (RA).

[0023] In some embodiments of the above-described treatment methods, the method includes testing one or more of the following clinical parameters of the patient: serum HMGB1 (high-mobility family box protein) greater than or equal to 4 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.5 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 2 ng / ml, and / or serum calprotectin greater than or equal to 10 μg / ml, and when administered, administering to the patient an effective amount of synthetic human relaxin-2 in a pharmacological solvent, pharmacological diluent, or pharmacological excipient to inhibit or suppress the physiological inflammatory response induced by the innate immune system.

[0024] In some embodiments of the above-described treatment methods, the methods include treating patients with SIRS (Systemic Inflammatory Response Syndrome), autoimmune or rheumatic diseases, thyroiditis, gastritis, pancreatitis, sialadenitis, adrenalitis, oophoritis, glomerulonephritis, polyarthritis, ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory types of arthritis, rheumatoid arthritis (RA), SARS-CoV-2, and SARS.

[0025] In some embodiments of the above treatment methods, the methods include treating patients with an immune response triggered by the innate immune system and / or exhibiting the following clinical criteria: prediabetes (HbA1c > 5.7% and < 6.5%), obesity (BMI > 30 kg / m²). 2 High blood pressure (stage 1 or higher according to the 2017 ACC / AHA guidelines).

[0026] In some embodiments of the above-described treatment methods, the methods include treating patients with inflammatory responses triggered by the innate immune system and / or requiring suppression of the inflammatory response via ligand-activated glucocorticoid receptors, wherein the active pharmacological component is synthetic human relaxin-2 to avoid manifestations or dysregulation of diabetes, impaired wound healing, and / or symptoms of Cushing's syndrome.

[0027] In some embodiments of the above treatment methods, the method includes treating patients who have received allogeneic grafts and require suppression of innate immune and inflammatory responses after testing a patient who is positive for one or more of the following medical criteria: serum HMGB1 (high-mobility family box protein) greater than or equal to 2 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.25 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 0.5 ng / ml, and / or serum calprotectin greater than or equal to 4 μg / ml.

[0028] In some embodiments of the above-described treatment methods, the method includes treating a patient with hormone-resistant cancer, wherein the hormone-resistant cancer includes, but is not limited to, prostate cancer, breast cancer, primary cancers of glucocorticoid receptor-activated (GR) cells activated by ligands, and Kaposi's sarcoma; wherein the primary cancers of GR cells activated by ligands include, but are not limited to, multiple myeloma, Hodgkin's disease, and other lymphoid carcinomas, wherein synthetic human relaxin-2 is used to supplement and / or replace glucocorticoid receptor-activated hormones.

[0029] In some embodiments of the above-described treatment methods, the method includes, particularly when a patient exhibits one or more of the following clinical features, treating a patient diagnosed as requiring immunosuppressive therapy, wherein synthetic relaxin-2 is used to supplement and / or replace glucocorticoid receptor-activating hormone to prevent or avoid manifestations or dysregulation of diabetes or symptoms of Cushing's syndrome: prediabetes (HbA1c > 5.7% and < 6.5%), obesity (BMI > 30 kg / m²). 2 High blood pressure (stage 1 or higher according to the 2017 ACC / AHA guidelines).

[0030] Further aspects and advantages of embodiments of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings, which illustrate the principles of the invention through examples. Attached Figure Description

[0031] In the accompanying figures:

[0032] Figure 1 This demonstrates the high affinity binding of H2 relaxin to the helical 12 of the ligand-binding domain of the human GC receptor in the absence of the coactivator TIF2 (K). D The image shows a microscale thermophoresis at a size of approximately 5 nM.

[0033] Figure 2 This demonstrates the low-affinity interaction (K) between H2 relaxin and the ligand-binding domain of the human GC receptor. D The image shows a microscale thermophoresis at a depth of approximately 500 nM.

[0034] Figure 3 This is a graph showing data on the release of lactate dehydrogenase (LDH) from cultured primary mouse hepatocytes after induced cell damage: Control (no cell damage); induced by H2O2; induced by H2O2 after treatment with relaxin-2 (Rlx) or dexamethasone (Dx); as a control after knockout of the GC receptor by added siRNA (GRsi) and disordered siRNA (scr) - all data are expressed as the percentage of maximum cytotoxicity induced by detergent.

[0035] Figure 4 The following is a graph showing data on the release of cleaved caspase-3 from cultured primary mouse hepatocytes after induced cell damage: Control (no cell damage); Cell damage induced by H2O2; Cells treated with relaxin-2 (Rlx) or dexamethasone (Dx); GC receptor knockout after addition of siRNA (GRsi) and out-of-order siRNA (scr) as a control - all data were normalized to β-actin and adjusted for the effect of H2O2.

[0036] Figure 5 This is a graph comparing the relative amounts of cytoplasmic GC receptors (GR) in cultured primary mouse hepatocytes after induced cell damage: Control (no cell damage); by addition of H2O2; after treatment with relaxin-2 (Rlx) or dexamethasone (Dx); relaxin-2 or dexamethasone (alone) - all data were normalized to control.

[0037] Figure 6 This is a graph comparing the relative levels of mitochondrial pyruvate dehydrogenase lipoamide kinase isoenzyme 4 (PDK-4) in cultured primary mouse hepatocytes after induced cell damage: control (no cell damage), cell damage by adding H2O2; cell damage after treatment with relaxin-2 (Rlx) or dexamethasone (Dx); addition of relaxin-2 or dexamethasone alone - all data were normalized to control.

[0038] Figure 7 This is a graph comparing the concentrations of tumor necrosis factor α (pg / mL), adipokines, and cytokines in the supernatant of activated Th1 macrophages: Control (no activation); Activated by lipopolysaccharide (endotoxin); After treatment of macrophages with relaxin-2 (Rlx), dexamethasone (Dx), mifepristone-RU486 (RU), LPS+Rlx+RU486, or LPS+Dx+RU486.

[0039] Figure 8 This is a graph comparing the concentrations of interleukin-6 (pg / mL), a pro-inflammatory cytokine, in the supernatant of activated Th1 macrophages: control (no activation); activated by lipopolysaccharide (endotoxin); after treatment of macrophages with relaxin-2 (Rlx), dexamethasone (Dx), mifepristone-RU486 (RU), LPS+Rlx+RU486, or LPS+Dx+RU486.

[0040] Figure 9This is a graph comparing the concentration (pg / mL) of circulating tumor necrosis factor α in rat blood 24 hours after E. coli endotoxin challenge: control (placebo - no endotoxin); challenged with E. coli endotoxin (125 μg LPS / kg body weight); 2 hours after continuous subcutaneous (sc.) infusion (4 μg / h) of synthetic relaxin-2 (Relaxera Pharmazeutische GmbH, Germany); intramuscular (im) injection of dexamethasone (10 mg / kg); orally administered RU-486 (single dose of 10 mg / kg body weight) or a combination thereof;

[0041] Figure 10 This is a graph comparing the concentrations of fasting blood glucose levels (24 hours) in rats after exposure to Escherichia coli endotoxin: control (placebo - no endotoxin); challenged with Escherichia coli endotoxin (125 μg LPS / kg body weight); challenged 2 hours after continuous subcutaneous infusion (4 μg / h) of synthetic relaxin-2 (Relaxera Pharmazeutische GmbH, Bensheim, Germany); intramuscular injection of dexamethasone (10 mg / kg); or oral administration of RU-486 (single dose of 10 mg / kg body weight) or a combination thereof.

[0042] Figure 11 This is a graph comparing the concentrations of fasting blood glucose levels (48 hours) in the blood of rats after challenge with E. coli endotoxin: control (placebo - no endotoxin); challenge with E. coli endotoxin (125 μg LPS / kg body weight); challenge 2 hours after continuous subcutaneous infusion (4 μg / h) of synthetic relaxin-2 (Relaxera, Bensheim, Germany); intramuscular injection of dexamethasone (10 mg / kg); oral administration of RU-486 (single dose of 10 mg / kg body weight) or a combination thereof.

[0043] Figure 12 This is a graph comparing the percentage of Treg macrophages (CD4+CD25+ regulatory T cells) in the blood of rats after continuous subcutaneous infusion (4 μg / h) of relaxin-2 or oral administration of RU-486 (single dose of 10 mg / kg body weight) or both - all data are percentages of total white blood cell count (WBC).

[0044] exist Figures 3 to 12 In the diagram, the box represents the interquartile range, with the inner line representing the median; the whisker represents 1.5 times the interquartile range; values ​​above 1.5 times the interquartile range (outliers) and 3 times the interquartile range (extreme range) are displayed as circles and stars, respectively. Detailed Implementation

[0045] Millions of patients take glucocorticoids to treat autoimmune and rheumatoid diseases, neurological disorders, lung diseases, cancer, and other conditions and causes. However, the chronic side effects and adverse consequences of glucocorticoids are concerning, particularly due to downregulation of the glucocorticoid receptor (GR), steroid-induced hyperglycemia or gluconeogenesis activation, and negative effects from Cushing's syndrome. The inventors have discovered that relaxin-2 binds to the ligand-binding domains of GR-like steroids and glucocorticoids, thereby forming an activated GR ligand complex. Unlike glucocorticoids or corticosteroids, the relaxin-GR complex activates the transcription of genes that suppress the innate immune system but does not activate genes that promote gluconeogenesis. This discovery expands the therapeutic application of relaxin-2 for patients requiring suppression of the innate immune system. This group of patients includes, for example, those who have received or have received allogeneic grafts and cancer patients. Another large group consists of patients with various forms of tissue / endothelial injury or tissue-damaging diseases, including autoimmune or rheumatic tissue-damaging diseases such as ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory types of arthritis, and rheumatoid arthritis (RA). Medications targeting these diseases include corticosteroids, oral and topical analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and COX-2 inhibitors, and disease-specific biologics. Particularly in allogeneic rejection, tissue damage, endothelial injury, and endothelial cell activation can be detected and monitored by increased concentrations of calprotectin and / or S100A12 in the extracellular fluid and bloodstream. This is because endothelial cells also play a crucial role in the recruitment and extravasation of immune cells. Calcium-binding S100 proteins, particularly calprotectin and S100A12, have broad intracellular and extracellular functions, including regulating calcium homeostasis, apoptosis, cell migration, differentiation, proliferation, energy metabolism, and inflammation. When triggered by tissue / cell damage, antibody stress, and endothelial stress, calcium-binding S100 proteins are released from the cytoplasm of endothelial cells. S100 proteins subsequently function as danger signals (DAMPs, or damage-associated molecular patterns) and are involved in regulating immune homeostasis (macrophage migration, invasion, and differentiation), post-traumatic injury, and inflammation. Therefore, they are biomarkers for certain diseases (such as inflammatory bowel disease), but their multiple functions must be allocated to cell migration, differentiation, tissue repair, immune homeostasis, and inflammation management. The lack of commonly used diagnostic tests for tissue damage, endothelial injury, endothelial stress, and anti-endothelial cell antibody binding undoubtedly leads to allogeneic graft dysfunction and allogeneic rejection, making calcium-binding S100A12 and calprotectin biomarkers for endothelial activation, immune cell recruitment, endothelial injury, endothelial antibody binding, and complement activation.

[0046] Furthermore, the inventors discovered that relaxin-2 not only binds to glucocorticoid receptors (such as...) Figure 1 (as shown), and Figure 12 The experimental results further indicate that administration of relaxin-2 leads to specific activation and promotion of regulatory T cells at both local and systemic levels, likely induced by the relaxin-GR complex. This allows for suppression of the immune response and significantly expands the use of relaxin-2 as the active ingredient in drugs for treating abnormal, excessive, and unwanted tissue-damaging immune responses to self and foreign antigens. While the promotion of peripherally induced Treg cells can also be achieved through the administration of glucocorticoids, such treatment is disadvantageous due to Cushing's-like adverse reactions of glucocorticoids, corticosteroids, and their synthetic analogues. Their adverse reactions are well-known and numerous (see 2022 ICD-10CM Code T38.0X5A).

[0047] Regulatory T cells (Treg cells) were initially defined as CD4+ T cells with high expression of CD25 (interleukin-2 receptor α chain). Regulatory T cells are further classified into thymic and peripheral induced Treg cells based on their developmental location. Based on the following findings, the Foxp3 gene, a member of the forkhead / winged helical family of transcriptional regulators, has been identified as a crucial regulator in Treg cell development: Scurfy mice with frameshift mutations in the Foxp3 gene exhibit T-cell inflammation and fatal autoimmune disease in multiple organs due to increased effector T-cell activation and cytokine production resulting from Treg cell deficiency. Furthermore, mutations in the human Foxp3 gene lead to IPEX syndrome (X-linked immune dysregulation, polyendocrine disorders, and enteropathy). Additionally, forced expression of Foxp3 in naive T cells results in immunosuppressive function. CD4 / CD25-naive T cells transfected with the Foxp3 gene can be converted into CD4+ T cells. + CD25 +Treg-like cells produce repressive cytokines and express typical Treg cell molecules such as CD25, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and glucocorticoid-induced tumor necrosis factor (TNF) receptor-associated protein (GITR). Therefore, FoxP3 is a lineage-specific marker and a crucial regulatory gene for the production, maintenance, and immunosuppressive function of Treg cells. Regulatory T cells require the suppression of aberrant or excessive immune responses and maintain homeostasis and self-tolerance by inhibiting T cell proliferation and cytokine production. Treg cells exert their immunosuppressive function primarily by consuming the cytokine interleukin-2 and by inducing apoptosis or killing effector cells or antigen-presenting cells (APCs) through interactions with repressive cytokines (TGF-β, IL-10, IL-35) and perforin, granzyme B, or Fas ligands. Other immunosuppressive mechanisms of Treg cells involve immune checkpoint molecules and include the suppression of effector T cells through lymphocyte activation of programmed cell death pathways or cytotoxic T-lymphocyte antigen-4 (CTLA-4). A third immunosuppressive mechanism could be the metabolic regulation of indoleamine 2,3-dioxygenase (IDO) expression, which affects the kynurenine-tryptophan pathway in dendritic cells. Therefore, Treg cells play a crucial role in suppressing autoimmunity and inflammation. Decreased Treg cell numbers and function are associated with human autoimmune diseases, and in clinical trials, Treg cell activation and enhancement have been shown to be beneficial in the treatment of autoimmune diseases (see review Margarita Dominquez-Vallar & David A. Hafler, Regulatory T cells in autoimmune disease, Nature Immunology 2018, 19, 665-673). Overall, all current results suggest that Treg cells contribute to maintaining self-tolerance by downregulating immune responses to both self and foreign antigens in an antigen-nonspecific manner. Therefore, it is reasonable to hypothesize that in post-transplant patients, relaxin 2-induced increases in Treg cells, in addition to preventing ischemic injury, also improve post-transplant outcomes. The same hypothesis can be applied when using relaxin-2 to treat autoimmune-induced tissue damage, endothelial cell damage, and diseases following endothelial cell activation. This hypothesis is also supported by the fact that a decrease in the proportion of Treg cells in peripheral blood is known to relieve general immunosuppression, thereby enhancing innate and adaptive immune responses to foreign and self antigens. Therefore, the current findings significantly expand the pharmaceutical toolbox.

[0048] Example

[0049] Example 1 - Relaxin binds to and activates glucocorticoid receptors

[0050] Figure 1 and Figure 2 This study investigated the in vitro binding and affinity of synthetic human relaxin-2 (shRlx) to the ligand-binding domain of the glucocorticoid receptor (GR-LBD) using microscale thermophoresis (MST). MST is based on measuring the directional movement of molecules within a localized temperature gradient generated by infrared laser radiation in a high-precision glass capillary containing the interacting couples—synthetic human relaxin-2 and recombinant GR-LBD. For this experiment, the GR-LBD ligand-binding domain was expressed in an *E. coli* expression system to obtain a large quantity of soluble protein stable for biophysical characterization. The recombinant GR-LBD showed minimal aggregation and was demonstrated to be fully functional. One of the interacting couples was labeled with a fluorescent dye and added to a serial series of 15 dilutions of the non-fluorescent couple. After incubation, the thermophoretic motion of the complex was detected. Conformational changes caused by ligand binding to the target or binding near the fluorophore elicited thermophoretic changes. The affinity of the interacting proteins was determined by analyzing changes in normalized fluorescence as a function of the titration concentration of the binding coupler. The next step was to determine the binding mode of relaxin and the activation mechanism of GR. Fluorescence polarization revealed two binding affinities in the picomolar and nanomolar ranges. Furthermore, human H2 relaxin can displace fluormone-labeled GS red from its binding pocket on the GR-LBD (see the method described by Hemmerling M et al. in Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases, J. Med. Chem. 2017, 60, 20, 8591-8605). Using this combination of biophysical and structural biology techniques, including microscale thermophoresis (MST), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and NMR, the relaxin-2 binding site of the glucocorticoid receptor, the steroid binding pocket of the GR-LBD, was identified.

[0051] Further investigation was conducted into the effect of relaxin binding on GR-LBD to determine whether relaxin binding activates the receptor as an agonist or inhibits transcriptional activity as an antagonist. GR-LBD possesses an activation-function-2 site, which recruits cofactors upon ligand binding. Cofactors (coactivators or corepressors) are cellular environment-specific. Therefore, the binding of relaxin to coactivator and corepressor motifs to form GR-LBD / relaxin complexes was tested.

[0052] Relaxin was found to bind to two cofactors, leading to different receptor conformational changes. Since thermophoresis is an intrinsic molecular phenomenon dependent on hydration shell and size, binding events can be identified by tracking relevant changes in thermophoresis within fluorescently labeled interacting couples. In summary, the results of microscale thermophoresis (see...) Figure 1 and Figure 2 This indicates that the synthesized human relaxin-2 exhibits both a high-affinity interaction and a low-affinity interaction (K) with GR-LBD. D (Approximately 500 nM).

[0053] According to hydrogen-deuterium exchange experiments (not shown), human relaxin-2 appears to bind to helical 12 of the LBD, but unlike typical glucocorticoids which lack the transcriptional co-regulatory factor NCoA-2 (nuclear receptor coactivator 2). NCoA-2 is also known as glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator-2 (SRC-2), or transcription mediator 2 (TIF2). NCoA-2 contains multiple nuclear receptor interacting domains and inherent histone acetyltransferase activity, and its role appears to be in acetylation of histones when GR recruits NCoA-2 to DNA facilitating sites, making downstream DNA more readily transcribed. The presence and quantity of NCoA-2 are cell type dependent. Therefore, NCoA2 (GRIP1, SRC-2, TIF2) supports the upregulation of DNA expression, which in turn leads to increased activation of genes responsible for gluconeogenesis. Since human relaxin-2 does not recruit NCoA-2 upon binding, this type of gene activation does not appear to be triggered when human relaxin-2 binds to the glucocorticoid receptor.

[0054] Example 2 - Relaxin-2 activation inhibits the transcription of genes that suppress peroxide-induced cytotoxicity, inflammation, and apoptosis.

[0055] refer to Figure 3 and Figure 4Mouse hepatocytes were isolated according to the description by Tamaki N et al. in Am J Physiol Gastrointest LiverPhysiol 2008 294, G499. In summary, the livers of mice anesthetized with pentobarbital were washed and perfused for 5 minutes with a buffer (pH 7.25) consisting of 8,000 NaCl, 400 KCl, 88.7 NaH2PO4·H2O, 120.45 Na2HPO4, 2,380 HEPES, 350 NaHCO3, 190 EGTA, and 900 glucose (all values ​​are mg / L). The livers were then treated for 15 minutes at 37°C with 0.03% collagenase in a digestion buffer (pH 7.25) containing 8,000 NaCl, 400 KCl, 88.7 NaH2PO4·H2O, 120.45 Na2HO4, 2,380 HEPES, 350 NaHCO3, and 560 CaCl2·2H2O (all values ​​are mg / L). Following collagenase perfusion, the liver capsule was separated, and cells were dispersed in Geys balanced salt solution (GBSS)-B, consisting of 8,000 mg / L NaCl, 370 mg / L KCl, 210 mg / L MgCl₂·6H₂O, 70 mg / L MgSO₄·7H₂O, 120 mg / L NaH₂PO₄, 30 mg / L KH₂PO₄, 991 mg / L glucose, 227 mg / L NaHCO₃, and 225 mg / L CaCl₂·2H₂O (pH 7.25). Cells were further separated by forcing the material through a steel mesh and collecting the cells by centrifugation at 50 G for 1 minute. The cell pellet was resuspended in GBSS-B solution and washed three times by intermittent centrifugation.

[0056] In a humidified atmosphere of 5% CO2-95% air at 37°C, with a cell density of 5×10⁻⁶ cells / day, the cells were subjected to 5×10⁻⁶ cells / day. 5Mouse hepatocytes isolated from cells / well were cultured in type I collagen-coated 6-well plates coated with Dulbecco modified Eagle medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. After plating, the medium was replaced with serum-free Dulbecco modified Eagle medium after 6 hours. The hepatocytes were then treated with hydrogen peroxide (2 mM H₂O₂ / L) for 5 hours, pretreated with either synthetic human relaxin-2 (10 nM / L, 24 hours) (Relaxera Pharmazeutische GmbH, Bensheim, Germany) or dexamethasone (0.5 mM, 24 hours, Sigma-Aldrich) or not. In addition, hepatocytes were transfected with GR siRNA or out-of-order siRNA using liposome reagents (Invitrogen) to test whether the release of LDH or caspase-3 into the culture medium in both experiments depended on ligand-activated GR reagents (n=5 per group). H2O2-induced cell damage (cytotoxicity) was immunologically determined by quantifying lactate dehydrogenase (LDH) released into the culture medium using enzyme-linked assays, according to the manufacturer's instructions (Goat LDH ELISA Kit, BiomolFeinchemikalien GmbH, Germany) and by Western blot analysis of cleaved caspase-3 (Caspase-3 Rabbit mAb #14220, Cell Signaling Technology, Danvers, Massachusetts, USA).

[0057] The results are summarized in Figure 3 and Figure 4 middle. Figure 3 The LDH release data shown are expressed as a percentage of the maximum detergent-induced cytotoxicity. Figure 4 The data on cleaved / activated caspase-3 shown are normalized to β-actin and adjusted for the effect of H2O2 (p < 0.05 relative to control *; #, p < 0.05 relative to H2O2; Kruskal-Wallis ANOVA with respect to rank was used for global testing, with pairwise comparisons performed using a post-hoc Mann-Whitney U test (Bonferroni-Holm adjustment for p). In summary, Figure 3 and Figure 4 It was shown that both relaxin-2 (Rlx) and dexamethasone (Dx) could significantly inhibit peroxide-induced cell damage (LDH release) and apoptosis (cleaved caspase-3), and this inhibition did not occur when GR expression was specifically knocked out by GR siRNA, while knockout using disordered siRNA (scr-siRNA) showed no effect.

[0058] Physiologically, LDH is an enzyme expressed in almost all living cells, including cardiomyocytes and blood cells, and it catalyzes the conversion of lactate to pyruvate and vice versa. Because it is released during tissue damage, it is a biomarker for common injuries, damaged tissues, and diseases involving tissue damage, such as heart failure. In contrast, the relative concentration of its substrates primarily regulates LDH activity. LDH is transcriptionally regulated in an estrogen-associated receptor α-dependent manner via peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α).

[0059] Caspase 3 (CASP3), or cysteine-dependent aspartate-directed protease 3, plays a crucial role in programmed cell death. Caspase-3 is synthesized as an inactive zymogen until it is cleaved following apoptotic signaling events. Caspase-3 is thought to ensure the controlled degradation of cellular components and that cell death has minimal impact on surrounding tissues. Caspase deficiency has been identified as a cause of tumor development, for example, by eliminating mutations in cell cycle genes that restrict cell growth, combined with mutations in apoptotic proteins (such as caspases) that trigger cell death in abnormally growing cells. Conversely, overactivation of caspase-3 can lead to excessive programmed cell death. This can be seen in various neurodegenerative diseases, such as Alzheimer's disease, in which nerve cells are lost. Caspases involved in processing inflammatory signals are also associated with disease. Insufficient caspase activation may increase an organism's susceptibility to infection because an appropriate immune response may not be triggered. For example, inflammatory caspase-1 is associated with the development of autoimmune diseases; drugs that block caspase activation have been used to improve patients' health.

[0060] In summary, synthetic relaxin-2, as a ligand for glucocorticoid receptors, appears to have similar cellular biological effects to dexamethasone in inhibiting inflammatory responses to tissue damage, as well as apoptosis and necrosis.

[0061] Example 3 - The relaxin-GR complex does not activate the transcription of genes involved in gluconeogenesis.

[0062] Mouse hepatocytes were obtained and glucocorticoid adverse effects were tested after treatment with synthetic human relaxin-2 or dexamethasone (500 nM, 24 h). The complex physiological adverse effects of glucocorticoid overdose are numerous and difficult to assess (Cushing's syndrome, diabetes, thinning of the skin, hypertension, osteoporosis, obesity, impaired wound healing, depression, etc.), but what can be measured in this cellular assay is the activation of genes involved in gluconeogenesis and diabetes, obesity, and impaired wound healing. Therefore, after treatment with 10 nM / L synthetic human relaxin-2 (Relaxera Pharma GmbH & Co. KG) and 500 nM / L dexamethasone (Sigma-Aldrich) for 24 hours, regulation of GR and PDK4 transcription was detected by qRT-PCR in normal primary mouse hepatocytes (for control) and in H2O2-stressed primary mouse hepatocytes from Example 2. Results are shown in… Figure 5 and Figure 6 In the block diagram.

[0063] Specifically, Figure 5 The results showed that relaxin-2 increased GR gene transcription by 100% to 200% in normal and H2O2-stressed primary mouse hepatocytes, while dexamethasone did not have this effect. Figure 6 It was shown that incubation with relaxin-2 had no effect on PDK-4 gene transcription in normal and H2O2-stressed primary mouse hepatocytes. However, when primary mouse hepatocytes were incubated with dexamethasone, PDK-4 gene transcription increased several-fold. Therefore, Figure 5 and Figure 6 The binding strongly suggests that the complex of relaxin and GR binds to genomic DNA loci that are different from steroid-activated GR.

[0064] Physiologically, PDK-4 (pyruvate dehydrogenase lipoamide kinase isoenzyme 4) is a mitochondrial protein that inhibits pyruvate dehydrogenase complex (PDH) by phosphorylating a subunit of PDH. An active PDH complex is required to convert pyruvate to acetyl-CoA, allowing glycolytic products to enter the citric acid cycle. Fasting leads to the induction of PDK-4 mRNA and the PDK-4 enzyme in both cardiac and skeletal muscle, thus inhibiting glucose oxidation and utilizing it for glucose maintenance as part of a complex response during starvation. Therefore, the PDK-4 enzyme is considered to play a crucial role in regulating glucose metabolism, and increased PDK-4 transcription indicates gluconeogenesis. PDK-4 expression is known to be physiologically regulated by glucocorticoids, retinoic acid, and insulin, which enhance transcription of the PDK-4 gene in white adipose tissue. Fatty acid oxidation also increases when PDK-4 levels are elevated. Insulin downregulates PDK-4 mRNA transcription. When cells are exposed to dexamethasone to increase PDK-4 mRNA expression, insulin blocks this effect and fatty acid oxidation. In type 2 diabetes, PDK-4 is overexpressed in skeletal muscle, leading to impaired glucose utilization. In obese patients, PDK-4 mRNA expression is also significantly reduced, which is associated with increased glucose uptake, possibly due to insulin downregulation of PDK-4. This is consistent with the hypothesis that fatty acid availability affects glucose metabolism by regulating the pyruvate dehydrogenase (PDH) complex. Indeed, in insulin-resistant individuals, insufficient downregulation of PDK-4 mRNA may lead to increased PDK-4 expression, resulting in impaired glucose oxidation and subsequent increased fatty acid oxidation. Conversely, downregulation of PDK-4 in myocardial tissue during heart failure is a physiological countermeasure (Razeghi P et al., Downregulation of metabolic gene expression in failing human heart before and after mechanical unloading, Cardiology 2002, 97(4):203–9).

[0065] Figures 3 to 6 The results indicate that relaxin-2 has anti-inflammatory effects similar to glucocorticoids, corticosteroids, and mineralocorticoids, but does not lead to impaired glucose oxidation, increased fatty acid oxidation, or gluconeogenesis. The generalized effects of PDK-4 further suggest that pharmaceutical compositions containing relaxin-2 and treatment with relaxin-2 can be an alternative to glucocorticoid therapy, as it leads to increased expression of glucocorticoid receptors (which suppresses immune and inflammatory responses) but does not lead to increased expression of PDK-4 (which is detrimental to glucose metabolism and homeostasis).

[0066] Glucocorticoid receptors (GRs) are evolutionarily conserved ligand-dependent transcription factors. Upon binding to steroid hormones or other ligands, the receptor migrates from the cytoplasm to the nucleus, where it binds to genomic DNA loci and actively or passively regulates the transcription rate of genes associated with those loci. Significant efforts have been made to elucidate the molecular signaling roles of GRs, including intracellular shuttle, transcriptional regulation, and interactions with other intracellular signaling pathways. In short, glucocorticoids are essential for maintaining resting states and stress responses; therefore, they are indispensable in the treatment of many diseases, including autoimmune, inflammatory, allergic, and lymphoproliferative disorders. The pathological and therapeutic significance of GRs cannot be overstated. This includes not only genetic alterations to human GR genes but also the development of disease-associated GR regulatory molecules and GR ligands with selective GR activity.

[0067] Figures 1 to 6 The presentation demonstrates that administration of synthetic relaxin-2 has therapeutic effects distinct from those of known corticosteroids and glucocorticoids. Experiments show that synthetic relaxin-2 induces GR-dependent glucocorticoid effects, including inhibition of apoptosis in mouse hepatocytes and inhibition of cytokine release in human macrophages (see Example 4 below), which avoids adverse effects such as GR downregulation or glucocorticoid- or steroid-induced hyperglycemia or gluconeogenesis activation, compared to typical corticosteroids and glucocorticoids.

[0068] Example 4 - Relaxin-2 inhibits the release of pro-inflammatory cytokines

[0069] THP-1 cells were differentiated into macrophages and cultured according to the description by Dschietzig T et al. in Identification of the pregnancy hormone relaxin as a glucocorticoid receptor agonist, FASEB J2004, 18:1536-1538. In short, THP-1 cells are derived from a cell line of human monocytic leukemia and are as well known in the art. When cells are treated with myristic acid-phorbol ester during passage, they differentiate into macrophages.

[0070] refer to Figure 7 and Figure 8Macrophages were then attacked for 24 hours with 10 ng / ml of Salmonella abortus equine endotoxin (Sigma-Aldrich) in the presence or absence of synthetic human relaxin-2 (10 nM / L) and dexamethasone (500 nM (L)) and / or the GR antagonist RU-486 (500 nM) (Sigma-Aldrich) or combinations thereof (n=5 per group). RU-486 binds to GR in the steroid pouch of the ligand-binding domain. Subsequently, the levels of TNF-α and interleukin-6 in the supernatant were determined by ELISA (R&D Systems).

[0071] The results are summarized in Figure 7 and Figure 8 The diagram shows that endotoxin induces these macrophages to produce and secrete pro-inflammatory cytokines tumor necrosis factor-α (TNF) and interleukin-6 (IL-6), and the inflammatory response is suppressed in the presence of synthesized human relaxin-2 and dexamethasone. This response can be classified as GR-dependent because RU-486 completely inhibits this effect.

[0072] As further background information, RU-486, also known as It is a steroidal antiprogesterone, antiglucocorticoid, and antiandrogen. It competitively antagonizes cortisol activity on the GR receptor. In humans, the antiglucocorticoid effect of RU-486 was observed through compensatory increases in adrenocorticotropic hormone (ACTH) and cortisol when the dose of RU-486 was greater than or equal to 4.5 mg / kg. In animals, prolonged administration of very high doses produced a weak antiandrogenic effect (Danco Laboratories, 2005, Mifeprex USprescribing information). Therefore, this example demonstrates that administration of synthetic human relaxin-2 can provide GR-mediated immune system regulation and has immunosuppressive properties, but without the side effects of inducing gluconeogenesis and insulin insensitivity.

[0073] Example 5 - Compared to the dexamethasone-GR complex, the relaxin-GR complex does not induce hyperglycemia.

[0074] Figure 9 , Figure 10 and Figure 11This is an animal study demonstrating the different effects of relaxin-2 and dexamethasone (Sigma-Aldrich) on blood glucose levels in rats 24 and 48 hours after administration. In short, male and female Sprague-Dawley rats (300–350 g) were treated with intraperitoneal injection of either E. coli endotoxin (125 μg / kg body weight) or a placebo (carrier). Blood was collected from the tail vein 24 hours later to measure circulating TNF-α (ELISA, R&D Systems) and fasting blood glucose, and fasting blood glucose was measured 48 hours later. Two hours prior to administration of endotoxin or placebo, animals were given dexamethasone (intramuscularly, 10 mg / kg body weight), synthetic relaxin-2 (Relaxera Pharmazeutische GmbH & Co. KG, Bensheim) (subcutaneously infused via an Alzet micropump (4 μg shRlx / h) over 12 hours), or oral RU-486 (single dose of 10 mg / kg body weight), or a combination thereof (n = 5 animals per group). Results are shown in Figure 10 and Figure 11 *, p < 0.05 compared to control; #, p < 0.05 compared to endotoxin + dexamethasone; Kruskal-Wallis ANOVA with respect to rank was used for global tests, and pairwise comparisons were performed using the post-hoc Mann-Whitney U test (with Bonferroni-Holm adjustment for p).

[0075] While dexamethasone significantly enhanced endotoxin-related hyperglycemia, reflecting the animals' disease and fever response to endotoxin, relaxin-2 reduced blood glucose levels compared to endotoxin alone. This was true at both 24 and 48 hours post-endotoxin exposure. Oral administration of RU-486 inhibited the effects of relaxin-2 and dexamethasone through its antagonistic effect on glucocorticoid receptors, also suppressing circulating TNF-α concentrations. In summary, human relaxin-2 alleviated the endotoxin-induced surge in circulating TNF-α in both sexes of rats. This effect can be identified as GR-dependent and comparable to that of dexamethasone (a typical glucocorticoid). However, unlike dexamethasone, relaxin-2 does not induce hyperglycemia, a medically significant finding.

[0076] Example 5 - Similar to glucocorticoids, relaxin-2 promotes the differentiation of naive T cells into regulatory T cells (Tregs) in mice.

[0077] In the following experimental groups, mice with a C57Bl / 6 background (n=5 per group) were administered intraperitoneal injections once daily for 3 consecutive days: synthetic human relaxin-2 (Relaxera) (10 μg / kg body weight), placebo (carrier, sodium acetate), RU-486 (2.5 mg / kg body weight), or relaxin-2 + RU-486. Subsequently, the mice were sacrificed, their spleens were processed using standard procedures, and splenic regulatory T cells (T cells) were analyzed by FACS. reg The percentage of T. reg The cell was initially defined as a CD4+FoxP3+ cell, where FoxP3 (forkhead transcription factor) is a T cell. reg Major regulatory factor. Regulatory T cells that express the transcription factor Forkhead Box P3 (FoxP3) are known to control immune responses and prevent autoimmunity.

[0078] like Figure 12 As shown, administration of relaxin-2 approximately doubled the percentage of regulatory T cells (CD4+FoxP3+ cells) compared to placebo. The GR antagonist RU-486 did not affect T cell differentiation, but its presence completely eliminated the relaxin-induced increase in Tregs when administered in conjunction with shRlx. #, p < 0.05 compared to control; Kruskal-Wallis ANOVA with respect to rank was used for global testing, and paired comparisons were performed using the post-hoc Mann-Whitney U test (Bonferroni-Holm adjustment for p).

[0079] Therefore, these findings confirm the stimulatory effect of synthetic human relaxin-2 on the differentiation of peripheral (spleen) regulatory T cells (Tregs) in mice and show that these effects are GR-dependent because they can be counteracted by GR antagonists such as RU-486.

[0080] Discussion and Summary

[0081] As mentioned above, glucocorticoids and corticosteroids are the main drugs for treating tissue-damaging autoimmune conditions such as rheumatoid arthritis, and they are also immunosuppressants after organ transplantation. A large body of literature addresses glucocorticoid-mediated regulation of the immune system, particularly glucocorticoid-mediated regulation of innate immunity and inflammation. Calcium-binding S100 proteins are universal markers of inflammation and the innate immune system, especially calprotectin (a complex of S100A8 and S100A9) and S100A12. Typically, the presence of S100A12 and calprotectin indicates tissue damage, endothelial cell activation, and an inflammation-mediated response. Cellular stress and / or inflammation induce the release of S100 proteins into non-cellular compartments, where they bind to cell surface receptors such as RAGE, TLR4, CD147, and GPCRs. The interaction between calcium-binding S100 proteins and their receptors activates intracellular signaling pathways such as AP1 and NFκB, which in turn initiates various cellular processes, including cell differentiation, migration, apoptosis, proliferation, and inflammation; activating protein 1 (AP1), extracellular signal-regulated protein kinase (ERK), G protein-coupled receptor (GPCR); interleukin-1 (IL-1); interleukin-7 (IL-7), nuclear factor repressor α (IκBα), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38), receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), and TNF receptor-associated factor 2 (Traf2). Mammalian cells secrete calprotectin during inflammatory responses. The exact mechanism by which mammalian cells secrete the S100A8 / S100A9 complex during inflammation is not yet known. When released into the extracellular space, S100 proteins are active in regulating immune homeostasis, post-traumatic injury, tissue damage, and inflammation. S100 proteins trigger inflammation by interacting with receptors RAGE and TLR4. Evidence suggests that calprotectin (S100A8 / S100A9) is an endogenous agonist of TLR4. Binding to TLR4 initiates a signaling cascade and regulates inflammation, cell proliferation, and differentiation in an NFκB-dependent manner. Besides TLR4, RAGE is also thought to bind to S100 proteins such as S100A7, S100A12, S100A8 / A9 (calprotectin), and S100B. Through interaction with RAGE, S100 proteins activate NFκB, inducing the production of pro-inflammatory cytokines, leading to the migration of neutrophils, monocytes, and macrophages. Therefore, extracellular S100 proteins are involved in regulating apoptosis and the migration of monocytes, macrophages, neutrophils, lymphocytes, myoblasts, epithelial cells, and endothelial cells. Therefore, the levels of the S100A8 / A9 complex (calprotectin) and S100A12 in extracellular fluid can be used as biomarkers to assess inflammation regulation and the extent of tissue damage.

[0082] Liver transplantation and cryopreservation were investigated in mice with the same genetic set. Therefore, in this syngeneic mouse model, all immune responses induced by surgical trauma, cryopreservation, and ischemic injury are mediated by the innate immune system, the function of which can be suppressed by corticosteroid administration. In this syngeneic mouse model, administration of relaxin-2 during cryopreservation and / or reperfusion demonstrated cytoprotective effects and significantly improved liver function and survival after transplantation (refer to Kageyama S et al., Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench-to-bedside, Hepatology 2018, 258-273). Although the authors hypothesized that the relaxin-2-GR complex plays a regulatory role in inflammatory damage in liver transplantation, they did not note the effects of relaxin on promoting peripheral Treg cells and activating and promoting immunosuppressive Treg cells. According to the knowledge of the present invention, continuous treatment of the receptor with relaxin-2 is reliable because activated peripheral Treg cells can suppress local immune responses as well as responses activated by any type of tissue damage and endothelial injury. Figure 10 and Figure 11 As shown, continued use of relaxin-2 is safe because relaxin-2 does not induce gluconeogenesis.

[0083] Regarding the anticancer activity of glucocorticoids, for example, De Bono et al. (2014, Clin Cancer Res 2014, 20:1925-1934, US2006018910) disclosed the treatment of hormone-refractory prostate cancer patients with a combination of docetaxel, anti-IGF-1R antibody, and dexamethasone. Since glucocorticoid receptors are upregulated in refractory prostate cancer cells, inhibition should be performed to weaken the proliferation of these cancer cells (Ruhr et al., 2018. Clin Cancer Res 24:927-938). The study found that activation of glucocorticoid receptors leads to cellular quiescence, cell cycle arrest via p57, and reprogramming of signals coordinated via insulin receptor substrate 2 (IRS2) / forkhead box O1 (FOXO1). Since synthetic relaxin-2 can be used as an alternative to dexamethasone, it can be advantageously used for such treatments without the adverse effects of glucocorticoids and their analogues.

[0084] Boehnert MU's Relaxin as an additional protective model of isolate-perfused rat liver, Ann NY Acad Sci 2005, 1041:434-440, and Bausys A et al. Supplemented with synthetic human relaxin decreases ischemia-reperfusion injury after porcine kidney transplantation. Int J Mol Sci. 2021, 22, 11417 describes how relaxin in perfusion solutions reduces ischemia-reperfusion injury (IRI) after kidney or liver transplantation, and that relaxin-2 (RLX) upregulates the expression of mitochondrial superoxide dismutase-2 (SOD2) and nuclear factor κB (NFκB), a regulator of innate immunity. Compared to controls, it downregulates the expression of receptor-interacting serine / threonine protein kinase 1 (RIPK1), which plays a role in apoptosis and necroptosis. It also downregulates the expression of mixed lineage kinase domain-like protein (MLKL), which plays a role in tumor necrosis factor (TNF)-induced necroptosis, and reduces the number of caspase-3 and MPO-positive cells in the graft after static refrigeration in a solution containing relaxin-2. Static cryopreservation in cardioplegic solution is the simplest, most convenient, and cheapest method of organ preservation in clinical practice. While relaxin-2 has been described for its anti-fibrotic, antioxidant, anti-inflammatory, and cytoprotective properties, there is no evidence that it can be beneficially used as a glucocorticoid alternative without inducing Cushing's-like adverse effects (particularly gluconeogenesis), nor has its promoting effect on immunosuppressive Treg cells been observed. However, this makes relaxin-2 a broad-spectrum drug for tissue and endothelial injury, rather than a vasodilator additive in perfusion solutions to prevent ischemia-reperfusion injury.

Claims

1. A pharmaceutical composition for treating a patient who needs long-term suppression of an inflammatory response caused by the innate immune system while avoiding the side effects of steroid therapy, the pharmaceutical composition comprising an effective amount of synthetic human relaxin-2, and a pharmacological solvent, a pharmacological diluent or a pharmacological excipient.

2. A pharmaceutical composition according to claim 1 for treating patients who need long-term suppression of inflammatory responses caused by the innate immune system when the side effects of glucocorticoid, corticosteroid, mineralocorticoid or other steroid treatment are to be avoided, wherein the active pharmacological ingredient is synthetic human relaxin-2, thereby avoiding the manifestations or disorders of diabetes, wound healing disorders and / or symptoms of Cushing's syndrome.

3. A pharmaceutical composition according to claim 1 or 2 for use in the treatment of a patient or an organ of a patient or an allograft of a patient infected with a tissue or endothelial damaging immune response and having one or more of the following increased clinical parameters compared to a healthy individual or organ: elevated serum HMGB1 (high mobility group box protein), elevated serum sTLR4 (soluble Toll-like receptor-4), elevated serum sRAGE (soluble receptor for advanced glycation end products), elevated serum calprotectin and / or serum S100A12 or elevated calprotectin and / or S100A12 in post-donation washout of the organ.

4. A pharmaceutical composition according to any one of the preceding claims 1 to 3 for use in the treatment of patients suffering from SIRS (Systemic Inflammatory Response Syndrome), autoimmune or rheumatic diseases, thyroiditis, gastritis, insulitis, sialadenitis, adrenalitis, oophoritis, glomerulonephritis, polyarthritis, ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory types of arthritis, rheumatoid arthritis (RA), SARS-Covid 19 and SARS.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the patient exhibits the following clinical criteria: prediabetes (HbA1C>5.7% and <6.5%), obesity (BMI>30kg / m 2 ), hypertension (stage 1 or higher according to the 2017 ACC / AHA guidelines).

6. The pharmaceutical composition according to claim 3, for treating allogeneic transplant recipients when one or more of the following four criteria are met: serum HMGB1 (high mobility group box protein) is greater than or equal to 2 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) is greater than or equal to 0.25 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) is greater than or equal to 0.5 ng / ml, and / or serum calprotectin is greater than or equal to 4 μg / ml.

7. The pharmaceutical composition according to claim 3, for treating an allogeneic transplant that has been subjected to an enhanced immune response caused by the innate immune system, and one or more of the following criteria is observed in the blood flushed out after organ donation: HMGB1 (high mobility group box protein)>2ng / ml, sTLR4 (soluble Toll-like receptor-4)>0.25ng / ml, serum sRAGE (soluble receptor for advanced glycation end products)>0.5ng / ml, or calprotectin>4μg / ml.

8. A pharmaceutical composition according to any one of claims 1 to 3, for treating a patient in need of hormone-refractory cancer treatment, wherein the hormone-refractory cancer includes but is not limited to prostate cancer, breast cancer, primary cancers of GR activated by ligands, and Kaposi's sarcoma; wherein the primary cancers of GR activated by ligands include but are not limited to multiple myeloma, Hodgkin's disease, and other lymphoid cancers; the synthetic human relaxin-2 is used as a supplement and substitute for glucocorticoids, cortisone, glucocorticoid receptor activating hormones or steroids.

9. A method of treating a patient comprising testing the patient for one or more of the following clinical parameters: serum HMGB1 (high mobility group box protein) greater than or equal to 4 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.5 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 2 ng / ml, and / or serum calprotectin greater than or equal to 10 μg / ml; and administering to the patient an effective amount of synthetic human relaxin-2 in a pharmacological solvent, pharmacological diluent or pharmacological excipient to inhibit or suppress the physiological inflammatory response caused by the innate immune system when administered.

10. A method of treating a patient according to claim 9, wherein the patient suffers from SIRS (Systemic Inflammatory Response Syndrome), autoimmune or rheumatic diseases, thyroiditis, gastritis, insulitis, sialadenitis, adrenalitis, oophoritis, glomerulonephritis, polyarthritis, ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory types of arthritis, rheumatoid arthritis (RA), SARS-Covid 19 and SARS.

11. The method of claim 9 for treating a patient who suffers from an immune response caused by the innate immune system and / or exhibits the following clinical criteria: prediabetes (HbA1C>5.7% and <6.5%), obesity (BMI>30 kg / m 2 ), hypertension (stage 1 or higher according to the 2017 ACC / AHA guidelines).

12. The method of claim 9 for treating a patient suffering from an inflammatory response triggered by the innate immune system and / or requiring inhibition of an inflammatory response by ligand activated glucocorticoid receptor, wherein the active pharmacological ingredient is synthetic human relaxin-2, for avoiding manifestations or disturbances of diabetes, wound healing disorders, and / or symptoms of Cushing's syndrome.

13. A method of treating a patient according to claim 9 who has received an allogeneic transplant and requires suppression of the innate immune system and inflammatory response, after testing the patient positive for one or more of the following medical criteria: serum HMGB1 (high mobility group box protein) greater than or equal to 2 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) greater than or equal to 0.25 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) greater than or equal to 0.5 ng / ml, and / or serum calprotectin greater than or equal to 4 μg / ml.

14. The method of claim 9 for treating a patient in need of hormone-refractory cancer treatment, wherein the hormone-refractory cancer includes but is not limited to prostate cancer, breast cancer, primary cancers that activate the GR via ligands, and Kaposi's sarcoma; wherein the primary cancers that activate the GR via ligands include but are not limited to multiple myeloma, Hodgkin's disease, and other lymphoid cancers; wherein the synthetic human relaxin-2 is used to supplement and / or replace the glucocorticoid receptor activating hormone.

15. A method of treating a patient diagnosed as requiring immunosuppressive therapy, wherein synthetic relaxin-2 is used to supplement and / or replace glucocorticoid receptor activating hormones to prevent or avoid manifestations or disorders of diabetes or symptoms of Cushing's syndrome, particularly when the patient exhibits one or more of the following clinical features: prediabetes (HbA1C>5.7% and <6.5%), obesity (BMI>30 kg / m 2 ), hypertension (stage 1 or higher according to the 2017 ACC / AHA guidelines).

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