Immunosuppressant drugs and methods of treatment

Abstract

Relaxin-2 for use as a broad-spectrum alternative to glucocorticoids, mineralocorticoids, corticosteroids, and their analogues for treating tissue and endothelial damage without the induction of gluconeogenesis, Cushing's syndrome, and Cushingoid adverse effects.Medicines and treatments for supporting repair of tissue and endothelial damage by promoting immunosuppressive regulatory T cells (Tregs), particularly in cases of autoimmune pathologies and organ transplantation.

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical preparations containing small peptide hormones of the human insulin superfamily and selective glucocorticoid receptor modulators (SEGRMs) that attenuate the innate immune system (A61K 38 / 1754; A61K 38 / 1751; A61K 38 / 2221). [Background technology]

[0002] Glucocorticoids are steroid hormones secreted by the adrenal glands. They regulate various physiological functions and are important for maintaining basal and stress-related homeostasis. At pharmacological doses, glucocorticoids and corticosteroids are effective immunosuppressants for treating many inflammatory, autoimmune, and lymphoproliferative disorders. At the cellular level, glucocorticoid and corticosteroid activity is functionally mediated by the glucocorticoid receptor (GR), which is simply the receptor to which cortisol, cortisone, and glucocorticoids bind. GR belongs to the nuclear transactivator receptor superfamily, which has over 200 members and is widely expressed in almost all human tissues and organs. GR is a hormone- and ligand-dependent transcription factor that controls or influences the expression of GR-responsive genes, which likely represent 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 cytoplasm by preventing other transcription factors from translocating from the cytoplasm to the nucleus. Inhibition can also occur when the ligand-activated GR complex binds to the same DNA site where other transcription factors would bind, thereby rendering them ineffective. The functions of the activated GR complex are multifaceted and occur in various parts of the body: they play a role in the regulation of metabolism, body growth, and immune response. For this reason, steroids and glucocorticoids have become the most commonly used drugs (for a review, see Nicolaides N et al., Glucocorticoid Receptor in Feingold KR, Anawalt B, Boyce A, et al., eds. Endotext [Internet]. South Dartmouth (MA): 2021).

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

[0004] A number of clinical applications for relaxin and relaxin agonists and antagonists have been proposed, including: for the treatment of skin aging, male pattern baldness, atrophy, sclerosis, and miniaturization of hair and hair follicles (EP 0793505); for the control of fetal growth (EP 0991947); for increasing fertility (EP 1473034); as an adjuvant in stem cell differentiation (EP 1696948); for increasing arterial compliance (EP 1765149); for diseases associated with vasoconstriction (EP 1854476); for tumor suppression (WO2007115414); for the treatment of diabetes and related complications (EP 1909809); and for the treatment of multiple sclerosis and other neurodegenerative disorders (EP 2723366). ), for treating symptoms of aging and neurodegenerative dysfunction (WO0048618), for treating glucotoxicity and impaired glucose tolerance (EP2817026), in the treatment of dyspnea associated with acute heart failure (EP2829280), for the treatment of heart failure with preserved ejection fraction (EP3145534), for the treatment of diseases of the CNS, CNS trauma, demyelinating diseases and / or gliosis, 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's syndrome, multiple system atrophy, prion diseases, AIDS encephalopathy (AIDS) Relaxin has been used to treat dementia complex, vasculitis, amyotrophic lateral sclerosis, Huntington's disease, autoimmune inflammatory diseases, retinal gliosis, encephalopathy, leukodystrophies, encephalitis, and neuropathy (EP3347037), various inflammatory conditions, acid airway hyperresponsiveness, asthma, rheumatoid arthritis, gout, ankylosing spondylitis, inflammatory bowel disease, myositis, systemic lupus erythematosus, sepsis, urticaria, psoriasis, and allergic reactions (WO20220374669). However, of particular interest is the use of relaxin for hemodynamic adaptation and regulation of systemic vascular resistance in models of myocardial, renal, pulmonary, and hepatic infarction (WO9303755, WO0240500; Dschietzig T et al.在人类心力衰竭中,血浆中松弛素 - 2的水平及心肌表达增加(《循环》,2000年,第102卷,第18期:594页);库尔森·C·C等人在《重组人松弛素对离体灌注大鼠心脏模型的中心血流动力学影响》(《产科学与妇科学》,1996年,第87卷,第4期:610 - 612页);马西尼·E等人在《松弛素可对抗离体豚鼠心脏缺血再灌注诱导的心肌损伤:一氧化氮参与的证据》(《内分泌学》,1997年,第138卷:4713 - 4720页);迪拉斯西奥·G等人在《细胞逆行心肌成形术和松弛素治疗大鼠模型缺血后心肌修复》(《德克萨斯心脏研究所杂志》,2012年,第39卷:488 - 499页);科利诺·M等人在《松弛素急性治疗可保护肾脏免受缺血再灌注损伤》(《细胞与分子医学杂志》,2013年,第17卷:1494 - 1505页);鲍西斯·A等人在《添加合成人松弛素的 Custodiol(注册商标)可减少猪肾移植后的缺血再灌注损伤》(《国际分子科学杂志》,2021年,第22卷,第11417期);阿列克西乌·K等人in Relaxin is a candidate drug for lung preservation: relaxin induced protection of rat lungs from ischemia-reperfusion injury, J Heart Lung Transplant 2010, 29:454-460; Teichmann SL et al. in Relaxin: 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 attenuate oxidative cell injury occurring in orthotopic kidney and liver transplants and in liver perfusion systems (DE102005040492; Boehnert MU in Relaxin as an additional protective model of isolate perfused rat liver, Ann NY Acad Sci 2005, 1041:434-440; Kageyama S et al. in Relaxin in Liver Transplantation: A Personal Perspective Mol Cell Endocrinol. 2019, 487: 75-79; Jakubauskiene L et al. in Relaxin positively influences ischemia-reperfusion injury in solid organ transplantation: a comprehensive review, Int J Mol Sci.2020, 21(2):631ff). These findings appear to be consistent with the observation that relaxin can act as a GR ligand in somatic cells entirely independent of the signaling cascade of the G protein-coupled relaxin receptors RXFP1 and RXFP2 (previously named LGR7 and LGR8, respectively). Experiments in HeLa cells confirmed this, and HEK cells and Th1-activated macrophages showed that relaxin-2 activates GR, and that the relaxin-GR complex, like dexamethasone, suppresses the secretion of cytokines IL-1, IL-6, and TNF-α upon stimulation (Dschietzig TB et al. in Identification of the pregnancy hormone relaxin as a glucocorticoid receptor agonist, FASEB J 2004, 18:1536-1538; Dschietzig T et al. in The pregnancy hormone relaxin binds to and activates the human glucocorticoid receptor, Ann NY Acad Sci. 2005, 1041:256-71; Dschietzig T et al. in RXFP1-inactive relaxin activates human glucocorticoid receptor: further investigations into the relaxin-GR pathway, Regul Pept. 2009, 154:77-84; Dschietzig T et al. in 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 of the NOTCH1 intracellular domain (NICD), and the associated intercellular signaling appears to result in reduced ischemia-reperfusion injury. However, ischemia-reperfusion injury (IRI) is an inevitable outcome in many clinical situations, including trauma, sepsis, resection, and transplantation, and is an immune-driven inflammatory response that leads to cell death and early graft dysfunction. However, human liver biopsies suggest that high NICD expression enhances resistance to IRI. The use of relaxin in organ preservation solutions for donor kidneys and livers appears promising for improving IRI resistance. Meanwhile, the clinical use of glucocorticoids and GR ligands is limited due to their side effects.

[0005] Because nearly every cell in the body expresses the same glucocorticoid receptor, the highly desirable pharmacological effects of glucocorticoids and GR activation are equally associated with specific side effects. The adverse metabolic and pharmacological effects of long-term glucocorticoid treatment include impaired wound healing, development / deregulation of diabetes mellitus, adverse immunosuppression, increased risk of infection, osteoporosis, childhood growth retardation, myopathy / muscle atrophy, skin atrophy, steroid-induced acne, and hirsutism, in addition to the typical symptoms of Cushing's syndrome, such as uncontrolled hypertension, salt and water imbalance, psychiatric and neurological disorders, and depression. Therefore, an individualized benefit-risk analysis must be performed for each glucocorticoid therapy, primarily for cancer and transplant patients receiving such hormone therapy. This is especially true for patients with hormone-refractory tumors who are treated with specific antibody-steroid combinations to suppress cancer cell growth. This is particularly true for patients with chronic inflammatory diseases in whom the adverse side effects of long-term treatment with glucocorticoids may outweigh the painful and debilitating effects of the underlying disease. The known prior art provides no guidance as to whether and when treatment with relaxin is medically justified. Thus, the prior art in this area presents a challenge. Summary of the Invention

[0006] The above-mentioned problem is solved by a pharmaceutical composition for treating a patient in need of therapy that attenuates the physiological inflammatory response by the innate immune system, comprising an effective amount of synthetic human relaxin-2 and a pharmaceutical solvent, diluent, or formulation excipient.

[0007] In some embodiments, patients suspected of needing attenuation of the physiological inflammatory response by the innate immune system are tested for one or more of the following serum 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, or serum calprotectin greater than or equal to 10 micrograms / ml.

[0008] This problem is further solved by a pharmaceutical composition for treating a patient experiencing an inflammatory response triggered by the innate immune system and / or requiring suppression via ligand-activated glucocorticoid receptors, the pharmaceutical composition comprising an effective amount of synthetic human relaxin-2 as an active ingredient and a pharmaceutical solvent, diluent, or formulation excipient, thereby avoiding the onset or deregulation of symptoms of diabetes or Cushing's syndrome.

[0009] In some embodiments, patients suspected of having an inflammatory response caused by the innate immune system have the following clinical criteria: pre-diabetes (HbA1C > 5.7 and < 6.5%), obesity (BMI > 30 kg / m2), and hypertension (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 for treating a patient in need of altering bodily or organ development while preventing the onset or deregulation of symptoms of diabetes or Cushing's syndrome. The patient may already have the following clinical features: pre-diabetes (HbA1C >5.7 and <6.5%), obesity (BMI >30 kg / m2), hypertension (stage 1 or higher).

[0011] In some embodiments, the pharmaceutical composition comprising an effective amount of synthetic human relaxin-2 is for treating an allogeneic transplant patient in need of attenuated innate immune system and inflammatory response while preventing impaired wound healing, the onset or deregulation of symptoms of diabetes, or Cushing's syndrome. The medical need for attenuated innate immune system is met in transplant patients if one or more of the following four criteria are met: serum HMGB1 (high mobility group 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 micrograms / ml.

[0012] In some preferred embodiments, the pharmaceutical composition comprising an effective amount of synthetic human relaxin-2 is for treating patients requiring chronic suppression of the innate immune system and inflammatory response while preventing the onset or deregulation of symptoms of diabetes or Cushing's syndrome.

[0013] In some embodiments, the patient suspected of requiring chronic suppression of the inflammatory response exhibits one or more medical criteria selected from serum HMGB1 (high mobility group box protein) of 4 ng / ml or greater, serum sTLR4 (soluble Toll-like receptor-4) of 0.5 ng / ml or greater, serum sRAGE (soluble receptor for advanced glycation end products) of 2 ng / ml or greater, and / or serum calprotectin of 10 micrograms / ml or greater.

[0014] In some other embodiments, the pharmaceutical composition is for treating patients in need of treatment for hormone-refractory cancers, including but not limited to, prostate cancer, breast cancer, or treatment for primary cancers mediated by ligand-activated GR, including but not limited to, multiple myeloma, Hodgkin's disease, and other lymphoid cancers; Kaposi's sarcoma, wherein synthetic human relaxin-2 is used as a supplement or replacement for hormones that activate the glucocorticoid receptor.

[0015] Another aspect of the invention involves testing the patient for one or more of the following clinical parameters: serum HMGB1 (high mobility group box protein) of 4 ng / ml or more, serum sTLR4 (soluble Toll-like receptor-4) of 0.5 ng / ml or more, serum sRAGE (soluble receptor for advanced glycation end products) of 2 ng / ml or more, and / or serum calprotectin of 10 micrograms / ml or more; and when given, administering to said patient an effective amount of synthetic human relaxin-2 present in a pharmaceutical solvent, diluent, or excipient to attenuate or suppress a physiological inflammatory response by the innate immune system; The present invention relates to a method of treating a patient, comprising:

[0016] In some embodiments, the method steps are used to treat a patient experiencing an inflammatory response elicited by the innate immune system and / or in need of ligand-activated glucocorticoid receptor-mediated inhibition, wherein the pharmacologically active ingredient is synthetic human relaxin-2, to avoid the onset or deregulation of symptoms of diabetes or Cushing's syndrome.

[0017] In some other embodiments, the treatment method comprises administering an effective amount of synthetic human relaxin-2 to a patient in need of altering physical or organ development while preventing the onset or deregulation of symptoms of diabetes or Cushing's syndrome.

[0018] In some embodiments, the methods involve administering an effective amount of synthetic human relaxin-2 to a patient who has received an allogeneic transplant and who needs suppression of the innate immune response and inflammatory response without impairing wound healing, developing or deregulating diabetes, or inducing symptoms of Cushing's syndrome, after the transplant patient has tested 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 micrograms / ml.

[0019] In some other embodiments, the method of treatment comprises administering an effective amount of synthetic human relaxin-2 to a patient diagnosed with a need for treatment of a hormone-refractory cancer, including, but not limited to, prostate cancer, breast cancer, or a primary cancer mediated by a ligand-activated GR, including, but not limited to, multiple myeloma, Hodgkin's disease, and other lymphoid cancers, Kaposi's sarcoma, where the synthetic human relaxin-2 is used to supplement and / or replace hormones that activate the glucocorticoid receptor. Alternatively, in some embodiments, the method comprises administering an effective amount of synthetic human relaxin-2 to a patient in need of a supplement or replacement for hormones that activate the glucocorticoid receptor.

[0020] In some embodiments, the treatment method comprises administering an effective amount of synthetic human relaxin-2 to a patient diagnosed with the need for immunosuppressive therapy, wherein the synthetic relaxin-2 is used to supplement and / or replace hormones that activate the glucocorticoid receptor to prevent or avoid the onset or deregulation of symptoms of diabetes or Cushing's syndrome, primarily when the patient exhibits one or more of the following clinical features: pre-diabetes (HbA1C > 5.7 and < 6.5%), obesity (BMI > 30 kg / m2), or hypertension (Level 1 or higher according to the 2017 ACC / AHA guidelines).

[0021] In some embodiments of the treatment method, the patient is initially diagnosed as requiring chronic doses of corticosteroids and / or glucocorticoids, and the method comprises subcutaneously administering an appropriate amount of synthetic human relaxin-2, thereby avoiding the onset or deregulation of symptoms of diabetes, wound healing disorders, or Cushing's syndrome.

[0022] In some embodiments of the 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 the inflammatory form of arthritis, rheumatoid arthritis (RA).

[0023] In some embodiments of the treatment method, the method comprises: 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 micrograms / ml; and when given, administering to said patient an effective amount of synthetic human relaxin-2 present in a pharmaceutical solvent, diluent, or excipient to attenuate or suppress a physiological inflammatory response by the innate immune system; Includes.

[0024] In some embodiments of the methods of treatment, the methods include treating patients suffering from SIRS (systemic inflammatory response syndrome), autoimmune or rheumatic diseases, thyroiditis, gastritis, insulitis, sialadenitis, adrenitis, oophoritis, glomerulonephritis, polyarthritis, ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory forms of arthritis, rheumatoid arthritis (RA), SARS-Covid 19 and SARS.

[0025] In some embodiments of the methods of treatment, the methods include treating a patient who has generated an immunological response via the innate immune system and / or exhibits the following clinical criteria: pre-diabetes (HbA1C >5.7 and <6.5%), obesity (BMI >30 kg / m2), hypertension (stage 1 or higher according to the 2017 ACC / AHA guidelines).

[0026] In some embodiments of the treatment methods, the methods include treating a patient experiencing an inflammatory response elicited by the innate immune system and / or in need of suppression of an inflammatory response mediated by a ligand-activated glucocorticoid receptor, wherein the pharmacologically active ingredient is synthetic human relaxin-2, and the onset or deregulation of symptoms of diabetes, wound healing disorders, and / or Cushing's syndrome is avoided.

[0027] In some embodiments of the treatment methods, the methods include treating a patient who has received an allograft and who is in need of suppression of the innate immune system and inflammatory response after the patient has tested 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 micrograms / ml.

[0028] In some embodiments of the treatment methods, the methods include treating a patient in need of treatment for hormone-refractory cancers, including but not limited to prostate cancer, breast cancer, or primary cancers mediated by ligand-activated GR, including but not limited to multiple myeloma, Hodgkin's disease, and other lymphoid cancers, Kaposi's sarcoma, wherein synthetic human relaxin-2 is used to supplement and / or replace hormones that activate the glucocorticoid receptor.

[0029] In some embodiments of the above-described treatment methods, the methods include treating a patient diagnosed with a need for immunosuppressive therapy, wherein synthetic relaxin-2 is used to supplement and / or replace hormones that activate the glucocorticoid receptor to prevent or avoid the onset or deregulation of symptoms of diabetes or Cushing's syndrome, primarily when the patient exhibits one or more of the following clinical features: pre-diabetes (HbA1C > 5.7 and < 6.5%), obesity (BMI > 30 kg / m2), hypertension (Level 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, illustrating by way of example the principles of the invention. [Brief explanation of the drawings]

[0031] In the figures and drawings accompanying this specification [Figure 1] FIG. 1 shows microscale thermophoresis of the high affinity binding (KD ∼ 5 nM) of H2 relaxin to helix 12 of the ligand binding domain of the human GC receptor in the absence of the coactivator TIF2. [Figure 2] FIG. 2 is a microscale thermophoresis diagram showing the low affinity interaction (KD ∼ 500 nM) of H2 relaxin with the ligand binding domain of the human GC receptor. [Figure 3] Figure 3 shows data plots of lactate dehydrogenase (LDH) release from cultured primary mouse hepatocytes after induced cytotoxicity: control (no cytotoxicity); induction with HO; induction with HO after treating cells with relaxin-2 (Rlx) or dexamethasone (Dx); after knockdown of GC receptors with added siRNA (GRsi) and scrambled siRNA (scr) as a control - all data are percent of maximum detergent-induced cytotoxicity. [Figure 4] Figure 4 shows data plots of the release of cleaved caspase-3 from cultured primary mouse hepatocytes after induced cell injury: control (no cell injury); cell injury by HO; after treatment of cells with relaxin-2 (Rlx) or dexamethasone (Dx); after knockdown of GC receptors by addition of siRNA (GRsi) and scrambled siRNA (scr) as a control - all data were normalized to β-actin and corrected for the effect of HO. [Figure 5] Figure 5 shows data plots comparing the relative abundance of cytosolic GC receptors (GR) in cultured primary mouse hepatocytes after induced cell injury: control (no cell injury); with the addition of HO; after cells were treated with relaxin-2 (Rlx) or dexamethasone (Dx); and relaxin-2 or dexamethasone alone - all data were normalized to the control. [Figure 6]Figure 6 shows data plots comparing the relative abundance of mitochondrial pyruvate dehydrogenase-lipoamide kinase isoenzyme 4 (PDK-4) in cultured primary mouse hepatocytes after induced cell injury: control (no cell injury); cell injury due to the addition of HO; cell injury after treating cells with relaxin-2 (Rlx) or dexamethasone (Dx); and addition of relaxin-2 or dexamethasone alone - all data normalized to the control. [Figure 7] Figure 7 shows data plots comparing the concentrations of the adipokine and cytokine tumor necrosis factor alpha (pg / mL) in the supernatants of activated Th1 macrophages: control (no activation); activation with lipopolysaccharide (endotoxin); and after treatment of macrophages with relaxin-2 (Rlx) or dexamethasone (Dx) or mifepristone-RU486 (RU) or LPS+Rlx+RU486 or LPS+Dx+RU486. [Figure 8] Figure 8 is a data plot comparing the concentration (pg / mL) of the pro-inflammatory cytokine interleukin-6 in the supernatants of activated Th1-macrophages: control (no activation); activation with lipopolysaccharide (endotoxin); and after treatment of macrophages with relaxin-2 (Rlx) or dexamethasone (Dx) or mifepristone-RU486 (RU) or LPS+Rlx+RU486 or LPS+Dx+RU486. [Figure 9] Figure 9 is a data plot comparing circulating tumor necrosis factor alpha concentrations (pg / mL) in blood obtained from rats 24 hours after challenge with E. coli endotoxin: control (placebo - no endotoxin); challenge with E. coli endotoxin (125 μg LPS / kg body weight); 2 hours after continuous subcutaneous infusion (4 μg / h) of synthetic relaxin-2 (Relaxera Pharmazeutische GmbH, DE); intramuscular injection of dexamethasone (10 mg / kg); oral administration of RU-486 (single dose of 10 mg / kg body weight) or combinations thereof. [Figure 10]Figure 10 is a data plot comparing fasting blood glucose levels (24 hours) in rats after exposure to 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 Pharmazeutische GmbH, Bensheim, DE); 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 Pharmazeutische GmbH, Bensheim, DE); intramuscular injection of dexamethasone (10 mg / kg im.); oral RU-486 (single dose of 10 mg / kg body weight) or combinations thereof. [Figure 11] Figure 11 is a data plot comparing fasting blood glucose (48 hours) concentrations in blood obtained from rats after challenge with E. coli endotoxin: control (placebo - no endotoxin); challenge with E. coli endotoxin (125 μg LPS / kg body weight); continuous subcutaneous infusion (4 μg / h) of synthetic relaxin-2 (Relaxera, Bensheim, DE) 2 hours post-challenge; intramuscular injection of dexamethasone (10 mg / kg); oral RU-486 (single dose of 10 mg / kg body weight) or combinations thereof. [Figure 12] Figure 12 is a data plot comparing the percentage of Treg macrophages (CD4+CD25+ regulatory T cells) in the blood from rats after continuous subcutaneous infusion of relaxin-2 (4 μg / h) or oral RU-486 (single dose of 10 mg / kg body weight) or both - all data are percentages of total white blood cell count (WBC).

[0032] In Figures 3 to 12, boxes represent the interquartile range, inner lines represent the median, whiskers represent 1.5 times the interquartile range, and values ​​exceeding 1.5 times the interquartile range (outliers) and values ​​exceeding 3 times the interquartile range (extreme range) are indicated by circles and stars, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed description of the invention Millions of patients take glucocorticoids to treat autoimmune and rheumatic diseases, neurological disorders, pulmonary diseases, cancer, and other diseases and causes. However, the chronic side effects and adverse effects of glucocorticoids are highly feared, particularly due to downregulation of the glucocorticoid receptor (GR), steroid-induced hyperglycemia, activation of gluconeogenesis, and Cushing's syndrome. The present inventors have discovered that relaxin-2, like steroids and glucocorticoids, binds to the ligand-binding domain of the GR to form an activated GR-ligand complex. Unlike glucocorticoids and corticosteroids, the relaxin-GR complex activates the transcription of genes that attenuate the innate immune system but not genes that activate gluconeogenesis. This discovery expands the therapeutic application of relaxin-2 to patients requiring attenuation of the innate immune system. This patient group includes, for example, patients undergoing or who have undergone allogeneic transplants and cancer patients. Another major group of patients suffers from various forms of tissue / endothelial damage or tissue-damaging diseases, including autoimmune or rheumatic tissue-damaging diseases, including ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA), inflammatory forms of arthritis, and rheumatoid arthritis (RA). Pharmacological treatments for these diseases include corticosteroids, oral and topical analgesics, nonsteroidal anti-inflammatory drugs such as ibuprofen and COX-2 inhibitors, and disease-specific biologics. Tissue injury, endothelial injury, and endothelial cell activation, particularly in allograft rejection, can be detected and monitored by elevated levels of calprotectin and / or S100A12 in the extracellular fluid and bloodstream, as endothelial cells also play an important role in immune cell recruitment and extravasation. Calcium-binding S100 proteins, particularly calprotectin and S100A12, have a wide range of intracellular and extracellular functions, including the regulation of calcium balance, cell apoptosis, cell migration, differentiation, proliferation, energy metabolism, and inflammation.Calcium-binding S100 proteins are released from the cytoplasm of endothelial cells in response to tissue / cell injury, antibody stress, and endothelial stress. S100 proteins then function as danger signals and DAMP (damage-associated molecular pattern) molecules, and are involved in immune homeostasis (macrophage migration, infiltration, and differentiation), posttraumatic injury, and inflammation control. Therefore, they are biomarkers for several specific diseases, such as IBD (inflammatory bowel disease), although their multiple functions remain to be assigned to cell migration, differentiation, tissue repair, immune homeostasis, and inflammation management. Tissue injury, endothelial injury, endothelial stress, and anti-endothelial cell antibody binding undoubtedly contribute to allograft dysfunction and rejection, but there are no commonly available diagnostic tests for these. Therefore, calcium-binding S100A12 and calprotectin are biomarkers for endothelial activation, immune cell recruitment, endothelial injury, endothelial antibody binding, and complement activation.

[0034] Furthermore, the inventors have further discovered that not only does relaxin-2 bind to glucocorticoid receptors as shown in Figure 1, but also that the experimental results in Figure 12 indicate that administration of relaxin-2 results in the specific activation and promotion of regulatory T cells at both local and systemic levels, likely mediated by the relaxin-GR complex. This allows for the suppression of immune responses and significantly broadens the use of relaxin-2 as an active ingredient in pharmaceuticals 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 by administering glucocorticoids, such treatment is disadvantageous due to the Cushingoid side effects of glucocorticoids, corticosteroids, and their synthetic analogs. Their side effects are well known and numerous (see 2022 ICD-10CM code T38.0X5A).

[0035] Regulatory T cells (Treg cells) were originally defined as CD4+ T cells that highly express CD25 (interleukin-2 receptor α chain). Regulatory T cells are further classified into thymic-induced Treg cells and peripherally-induced Treg cells based on their location of origin. The Foxp3 gene, a member of the Forkhead / winged-helix family of transcription factors, was discovered to be a critical regulator of Treg cell development based on the following findings: Scurfy mice, which carry a frameshift mutation in the Foxp3 gene, have T cell inflammation in multiple organs and a fatal autoimmune disease due to increased effector T cell activation and cytokine production in the absence of Treg cells. Furthermore, mutations in the Foxp3 gene in humans lead to IPEX syndrome (X-linked immune dysregulation, polyendocrinopathy, and enteropathy). Furthermore, forced expression of Foxp3 in naive T cells leads to immunosuppressive functions. CD4 / CD25- naive T cells transfected with the Foxp3 gene can transform into CD4+ CD25+ Treg-like cells that produce suppressive cytokines and express typical Treg cell molecules, such as CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4), and glucocorticoid-inducible tumor necrosis factor (TNF) receptor-related protein (GITR). Thus, FoxP3 is a lineage-specific marker and a key regulatory gene for the generation, maintenance, and immunosuppressive function of Treg cells. Regulatory T cells are required to suppress aberrant or excessive immune responses and maintain homeostasis and self-tolerance by suppressing T cell proliferation and cytokine production. Treg cells exert their immunosuppressive function by dominantly consuming the cytokine interleukin-2, and by suppressive cytokines (TGF-β, IL-10, IL-35), and by inducing apoptosis or killing effector cells or antigen-presenting cells (APCs) through perforin, granzyme B, or Fas ligand interactions.Other immunosuppressive mechanisms of Treg cells involve immune checkpoint molecules, such as the lymphocyte activation programmed cell death pathway and the inhibition of effector T cells by the cytotoxic T lymphocyte antigen (CTLA-4). A third immunosuppressive mechanism may be metabolic regulation through the expression of indoleamine 2,3-dioxygenase (IDO), which affects the kynurenine-tryptophan pathway in dendritic cells. Thus, Treg cells play an important role in suppressing autoimmunity and inflammation. Decreased numbers and function of Treg cells are associated with human autoimmune diseases, and Treg cell activation and enhancement have been shown to be beneficial for the treatment of autoimmune diseases in clinical trials (see review by 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 the maintenance of self-tolerance by downregulating immune responses to self- and foreign antigens in an antigen-nonspecific manner. Therefore, it is reasonable to hypothesize that relaxin-2-induced increases in Treg cells in post-transplant patients will not only prevent ischemic injury but also improve post-transplant outcomes. The same holds true when relaxin-2 is used to treat autoimmune-induced tissue injury, endothelial cell injury, and diseases secondary to endothelial cell activation. This hypothesis is supported by the fact that a reduction in the proportion of Treg cells in peripheral blood is known to relieve general immunosuppression and enhance innate and adaptive immune responses to foreign and self-antigens. Thus, the present discovery significantly expands the pharmaceutical toolbox. <Example> Example 1 Relaxin binds to and activates the glucocorticoid receptor

[0036] Figures 1 and 2 refer to an in vitro binding and affinity study of synthetic human relaxin-2 (shRlx) for the glucocorticoid receptor ligand-binding domain (GR-LBD) using microscale thermophoresis (MST). Microscale thermophoresis is based on measuring the directed movement of molecules in a local temperature gradient generated by infrared laser irradiation within a precision glass capillary tube containing the interaction partners, synthetic human relaxin-2 and recombinant GR-LBD. For this experiment, the human glucocorticoid receptor ligand-binding domain (GR-LBD) was expressed in an E. coli expression system, yielding large amounts of soluble protein stable for biophysical characterization. The recombinantly produced GR-LBD showed little aggregation and was proven to be fully functional. One of the interaction partners was labeled with a fluorescent dye and added to a serial dilution series (15 dilutions each) of the non-fluorescent partner. After incubation, the thermophoretic motion of the complex was detected. Conformational changes due to ligand binding to the target or to the proximity of a fluorescent dye induce thermophoretic changes. The affinity of interacting proteins is determined by analyzing the change in normalized fluorescence as a function of titrated binding partner concentration. The next step was to determine the binding mode of relaxin and its mechanism of GR activation. Fluorescence polarization analysis revealed two binding affinities in the picomolar and nanomolar ranges. Furthermore, human H2 relaxin was able to displace fluorescein-labeled GS red from the binding pocket on the GR-LBD (see the method described in 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 in the glucocorticoid receptor was identified, which was the steroid-binding pocket of the GR-LBD.

[0037] To determine whether relaxin binding activates the receptor like an agonist or acts like an antagonist, suppressing transcriptional activity, we further investigated the effect of relaxin binding on the GR-LBD. The GR-LBD contains an activation function-2 site that recruits cofactors upon ligand binding. Cofactors (coactivators or corepressors) are specific to the cellular context. Therefore, we examined the binding of relaxin to the coactivator and corepressor motifs of the GR-LBD / relaxin complex.

[0038] Relaxin was found to bind to both cofactors but induce distinct receptor conformational changes. Because thermophoresis is an intrinsic molecular phenomenon influenced by hydration shell and size, binding events could be identified by tracking the associated changes in thermophoresis of fluorescently labeled interacting partners. In summary, the microscale thermophoresis results (see Figures 1 and 2) demonstrated a high-affinity interaction (KD ∼5 nM) and a separate low-affinity interaction (KD ∼500 nM) between synthetic human relaxin-2 and GR-LBD.

[0039] Hydrogen-deuterium exchange experiments (not shown) indicate that human relaxin-2 appears to bind to helix 12 of the LBD. However, in contrast to classical glucocorticoids, this binding occurs in the absence of the transcriptional coregulator 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 intermediary factor 2 (TIF2). NCoA-2 possesses several nuclear receptor-interacting domains and intrinsic histone acetyltransferase activity. When GR recruits NCoA-2 to DNA promoter sites, its role appears to be to acetylate histones, making downstream DNA more accessible for transcription. The presence and abundance of NCoA-2 are cell type-dependent. Thus, NCoA2 (GRIP1, SRC-2, TIF2) supports the upregulation of DNA expression, which also leads to increased activation of genes involved in gluconeogenesis. This type of gene activation does not appear to occur when human relaxin-2 binds to the glucocorticoid receptor, because human relaxin-2 does not recruit NCoA-2 upon binding. Example 2 Relaxin-2 activates the transcription of genes that attenuate peroxide-induced cytotoxicity, inflammatory responses, and apoptosis

[0040] Referring to Figures 3 and 4, mouse hepatocytes were isolated as described by Tamaki N et al. in Am J Physiol Gastrointest Liver Physiol 2008 294, G499. Briefly, livers obtained from pentobarbital-anesthetized mice were washed and perfused for 5 min with a pH 7.25 buffer consisting of 8,000 NaCl, 400 KCl, 88.7 NaH2PO4·H2O, 120.45 Na2HPO4, 2,380 HEPES, 350 NaHCO3, 190 EGTA, and 900 glucose, and then treated with 0.03% collagenase for 15 min at 37°C in a pH 7.25 digestion buffer containing 8,000 NaCl, 400 KCl, 88.7 NaH2PO4·H2O, 120.45 Na2HPO4, 2,380 HEPES, 350 NaHCO3, and 560 CaCl2·2H2O (all values ​​in mg / L). After collagenase perfusion, liver capsules were isolated and cells were dispersed in Geys' balanced salt solution (GBSS)-B (pH 7.25), consisting of 8,000 NaCl, 370 KCl, 210 MgCl₂·6H₂O, 70 MgSO₄·7H₂O, 120 NaH₂PO₄, 30 KH₂PO₄, 991 glucose, 227 NaHCO₃, and 225 CaCl₂·2H₂O (all values ​​in mg / L). Cells were further separated by passing the material through a steel mesh and centrifuging at 50 g for 1 minute to recover the cells. The cell pellet was resuspended in GBSS-B and washed three times with intermittent centrifugation.

[0041] Isolated mouse hepatocytes were collected at 5 x 10 5Hepatocytes were plated on type I collagen-coated 6-well plates at a cell density of 100 cells / well and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Six hours after plating, the medium was replaced with serum-free Dulbecco's modified Eagle's medium. Hepatocytes were then treated with hydrogen peroxide (2 mM H2O2 / L) for 5 hours, with or without pretreatment with synthetic human relaxin-2 (10 nM / L, 24 hours) (Relaxera Pharmazeutische GmbH, Bensheim, DE) or dexamethasone (0.5 mM, 24 hours) (Sigma-Aldrich). Hepatocytes were also transfected with GR-siRNA or scrambled siRNA using Lipofectamine reagent (Invitrogen) to test whether the release of LDH or caspase-3 into the medium in these two experiments depended on the ligand-activated GR reagent (n = 5 per group). HO-induced cytotoxicity was determined by quantifying lactate dehydrogenase (LDH) released into the culture medium using an enzyme-linked assay (Goat LDH ELISA Kit, Biomol Feinchemikalien GmbH, DE) according to the manufacturer's instructions, and immunologically by Western blot analysis of cleaved caspase-3 (Caspase-3 Rabbit Monoclonal Antibody #14220, Cell Signaling Technology, Danvers, MA, US).

[0042] The results are summarized in Figures 3 and 4. The LDH release data shown in Figure 3 are expressed as a percentage of maximum detergent-induced cytotoxicity. The cleaved / activated caspase-3 data shown in Figure 4 were normalized to β-actin and corrected for the effect of H2O2 (* indicates p<0.05 compared to control; # indicates p<0.05 compared to H2O2; Kruskal-Wallis ANOVA on ranks for global tests and post-hoc Mann-Whitney U tests for pairwise comparisons (Bonferroni-Holm p correction)). In summary, Figures 3 and 4 show that both relaxin-2 (Rlx) and dexamethasone (Dx) can significantly attenuate peroxide-induced cytotoxicity (LDH release) and apoptosis (cleaved caspase-3), and this attenuating effect did not occur when GR expression was specifically knocked out by GR-siRNA, whereas knockout using scrambled siRNA (scr-siRNA) had no effect.

[0043] Physiologically, LDH is an enzyme expressed in almost all living cells, including cardiac muscle and blood cells, where it catalyzes the conversion of lactate to pyruvate and vice versa. Because LDH is released upon tissue injury, it serves as a marker of general injury, damaged tissue, and diseases involving tissue damage, such as heart failure. Meanwhile, the relative concentrations of LDH substrates primarily regulate LDH activity. LDH is transcriptionally regulated by peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) in an estrogen-related receptor α-dependent manner.

[0044] Caspase-3 protein (CASP3), or cysteine-dependent aspartate-directed protease 3, plays an essential role in programmed cell death. Caspase-3 is synthesized as a zymogen that is inactive until cleavage following an apoptotic signaling event. Caspase-3 is thought to ensure that cellular components are degraded in a controlled manner and that cell death occurs with minimal impact on surrounding tissues. Caspase deficiency has been identified as a cause of tumorigenesis, for example, due to mutations in cell cycle genes that remove the limit on cell proliferation, combined with mutations in apoptotic proteins such as caspases that induce cell death in abnormally proliferating cells. Conversely, overactivation of caspase-3 leads to excessive programmed cell death, which is seen in several neurodegenerative diseases, such as Alzheimer's disease, resulting in neuronal loss. Caspases, involved in processing inflammatory signals, are also implicated in disease. Insufficient caspase activation may not evoke an appropriate immune response, making the organism more susceptible to infection. For example, the inflammatory caspase-1 has been implicated in the development of autoimmune diseases; drugs that inhibit caspase activation have been used to improve patient health.

[0045] In conclusion, synthetic relaxin-2, as a ligand for the glucocorticoid receptor, appears to have similar cell biological effects as dexamethasone in attenuating the inflammatory response to tissue injury and apoptosis and necrosis. Example 3: Relaxin-GR complex does not activate the transcription of genes involved in gluconeogenesis

[0046] Mouse hepatocytes were obtained and tested for glucocorticoid side effects after treatment with synthetic human relaxin-2 or dexamethasone (500 nM, 24 hours). While the complex physiological adverse effects of glucocorticoid excess are numerous and difficult to assess (e.g., Cushing's syndrome, diabetes, skin thinning, hypertension, osteoporosis, obesity, impaired wound healing, depression), this cellular assay measures the activation of genes involved in gluconeogenesis and in diabetes, obesity, and impaired wound healing. Therefore, qRT-PCR was used to examine the regulation of GR and PDK-4 transcription in control normal primary mouse hepatocytes and the HO-stressed primary mouse hepatocytes described in Example 2 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. The results are shown in block diagrams in Figures 5 and 6.

[0047] Specifically, Figure 5 shows that relaxin-2 increases GR gene transcription by 100% to 200% in normal and HO-stressed primary mouse hepatocytes, whereas dexamethasone has no such effect. Figure 6 shows that incubation with relaxin-2 does not affect PDK-4 gene transcription in normal and HO-stressed primary mouse hepatocytes. However, incubation of primary mouse hepatocytes with dexamethasone increases PDK-4 gene transcription several-fold. Therefore, the combination of Figures 5 and 6 provides strong evidence that the relaxin-GR complex binds to a genomic DNA locus distinct from the DNA locus of steroid-activated GR.

[0048] Physiologically, PDK-4 (pyruvate dehydrogenase lipoamide kinase isoenzyme 4) is a mitochondrial protein that inhibits the pyruvate dehydrogenase complex (PDH) by phosphorylating one of its subunits. An active PDH complex is required for the conversion of pyruvate to acetyl-CoA, allowing glycolytic products to enter the citric acid cycle. Fasting induces PDK-4 mRNA and the PDK-4 enzyme in both cardiac and skeletal muscle, suppressing glucose oxidation during starvation as part of an integrated response and for glucose maintenance. Therefore, the PDK-4 enzyme is thought to play an important role in the regulation of glucose metabolism, while increased PDK-4 transcription indicates gluconeogenesis. PDK-4 expression is well known to be physiologically regulated by glucocorticoids, retinoic acid, and insulin, which enhance PDK-4 gene transcription in white adipose tissue. Elevated PDK-4 levels also increase fatty acid oxidation. Insulin downregulates PDK-4 mRNA transcription. When cells are exposed to dexamethasone to increase PDK-4 mRNA expression, insulin inhibits this action and fatty acid oxidation. In type 2 diabetes, PDK-4 is overexpressed in skeletal muscle, resulting in impaired glucose utilization. In obese patients, increased glucose uptake is accompanied by a significant decrease in PDK-4 mRNA expression, likely due to insulin-induced downregulation of PDK-4. This is consistent with the hypothesis that fatty acid availability influences glucose metabolism by regulating the pyruvate dehydrogenase (PDH) complex. Indeed, in insulin-resistant individuals, insufficient PDK-4 mRNA downregulation can lead to increased PDK-4 expression, leading to impaired glucose oxidation and subsequently to increased fatty acid oxidation.Conversely, PDK-4 is downregulated in myocardial tissue during heart failure, which 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).

[0049] The results in Figures 3-6 indicate that relaxin-2, like glucocorticoids, corticosteroids, and mineralocorticoids, has anti-inflammatory effects, but does not lead to impaired glucose oxidation, increased fatty acid oxidation, or gluconeogenesis. The ubiquitous role of PDK-4 further suggests that pharmaceutical compositions containing and treatment with relaxin-2 may be an alternative to glucocorticoid treatment, because they increase the expression of glucocorticoid receptors, which suppress immune and inflammatory responses, but do not increase the expression of PDK-4, which is unfavorable for glucose metabolism and balance.

[0050] The glucocorticoid receptor (GR) is an evolutionarily conserved ligand-dependent transcription factor. Upon binding of steroid hormones or other ligands, the receptor translocates from the cytoplasm to the nucleus, where it binds to genomic DNA loci and positively or negatively regulates the transcription rate of genes associated with those loci. Extensive efforts have been made to clarify the molecular signaling functions of GR, including intracellular shuttling, transcriptional regulation, and interactions with other intracellular signaling pathways. In summary, glucocorticoids are essential for maintaining a resting state and stress response and are therefore crucial for the treatment of many diseases, including autoimmune, inflammatory, allergic, and lymphoproliferative disorders. The pathological and therapeutic significance of GR cannot be overemphasized. These include the discovery of disease-associated GR regulatory molecules and GR ligands with selective GR activity, excluding genetic alterations in the human GR gene.

[0051] Figures 1 through 6 demonstrate that administration of synthetic relaxin-2 has therapeutic effects distinct from those of known corticosteroids and glucocorticoids. The experiments presented demonstrate that synthetic relaxin-2 elicits GR-dependent glucocorticoid effects, including inhibition of apoptosis in mouse hepatocytes and inhibition of cytokine release in human macrophages (see Example 4 below), but in contrast to classical corticosteroids and glucocorticoids, this effect avoids undesirable effects such as GR downregulation or glucocorticoid- or steroid-induced hyperglycemia or activation of gluconeogenesis. Example 4 Relaxin-2 attenuates the release of inflammatory cytokines

[0052] THP-1 cells were differentiated into macrophages and cultured as described in Dschietzig T et al. in "Identification of the pregnancy hormone relaxin as a glucocorticoid receptor agonist," FASEB J 2004, 18:1536-1538. Briefly, THP-1 cells are derived from a cell line generated from human monocytic leukemia, and when these cells are serially treated with myristate-phorbol ester, they differentiate into macrophages as is well known in the art.

[0053] Referring to Figures 7 and 8, macrophages were then challenged with 10 ng / ml Salmonella abortus equii 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 a combination thereof, for 24 hours (n = 5 for each group). RU-486 binds to GR in the steroid pocket of the ligand-binding domain. Supernatant levels of TNF-α and interleukin-6 were then measured by ELISA (R&D Systems).

[0054] The results, summarized in the block diagrams of Figures 7 and 8, demonstrate that the endotoxin induced these macrophages to produce and secrete the proinflammatory cytokines tumor necrosis factor alpha (TNF) and interleukin-6 (IL-6), and that the inflammatory response was suppressed both in the presence of synthetic human relaxin-2 and in the presence of dexamethasone. This response can be classified as GR-dependent, since RU-486 completely inhibited this effect.

[0055] For further background, RU-486, also known as Mifepristone®, is a steroidal antiprogesterone, as well as an antiglucocorticoid and antiandrogen. RU-486 competitively antagonizes the action of cortisol at the GR receptor. In humans, the antiglucocorticoid effect of RU-486 is observed at doses of 4.5 mg / kg or higher through a compensatory increase in adrenocorticotropic hormone (ACTH) and cortisol. In animals, a weak antiandrogenic effect is observed with chronic administration of very high doses (Danco Laboratories, 2005, Mifeprex US prescribing information). Thus, this example demonstrates that administration of synthetic human relaxin-2 can confer GR-mediated control of the immune system and has immunosuppressive properties without the side effects of inducing gluconeogenesis and insulin insensitivity. Example 5: Relaxin-GR complex does not induce hyperglycemia, in contrast to dexamethasone-GR complex

[0056] Figures 9, 10, and 11 refer to animal experiments and show the differential effects of administration of relaxin-2 and dexamethasone (Sigma Aldrich) on rat blood glucose levels 24 and 48 hours after administration. Briefly, male and female Sprague-Dawley rats (300-350 g body weight) were treated with intraperitoneal injection of E. coli endotoxin (125 μg / kg body weight) or placebo (vehicle). Blood was collected from the tail vein 24 hours later for determination of circulating TNF-α (ELISA, R&D Systems) and fasting blood glucose, and 48 hours later for measurement of fasting blood glucose. Two hours before endotoxin or placebo administration, animals received dexamethasone (10 mg / kg body weight intramuscularly), synthetic relaxin-2 (Relaxera Pharmazeutische GmbH & Co. KG, Bensheim) as a subcutaneous infusion over 12 hours (4 μg shRlx / h) via an osmotic Alzet minipump, RU-486 (a single dose of 10 mg / kg body weight) or a combination thereof (n = 5 per group). The results are shown in Figures 10 and 11. Here, * indicates p < 0.05 compared to control; # indicates p < 0.05 compared to endotoxin + dexamethasone; Kruskal-Wallis ANOVA on ranks for global tests and post-hoc Mann-Whitney U tests for pairwise comparisons (Bonferroni-Holm p correction).

[0057] Dexamethasone significantly enhanced the endotoxin-associated rise in blood glucose levels, reflecting the animals' sickness and hyperthermic response to endotoxin, whereas relaxin-2 reduced glucose levels compared with endotoxin alone. This was true both 24 and 48 hours after endotoxin administration. Oral administration of RU-486 blocked the effects of relaxin-2 and dexamethasone by antagonizing glucocorticoid receptors, as well as circulating TNF-α levels. In conclusion, human relaxin-2 attenuated the endotoxin-induced surge in circulating TNF-α in both sexes of rats. This effect was identified as GR-dependent and comparable to that of the classical glucocorticoid dexamethasone. However, unlike dexamethasone, relaxin-2 did not induce hyperglycemia. This is a finding of great medical importance. Example 5 Relaxin-2, like glucocorticoids, promotes the differentiation of naive T cells into regulatory T cells (Treg) in mice.

[0058] Mice (n = 5 per group) on a C57Bl / 6 background were intraperitoneally injected once daily for 3 consecutive days with one of the following experimental groups: synthetic human relaxin-2 (Relaxera) (10 micrograms / kg body weight), placebo (vehicle, sodium acetate), RU-486 (2.5 mg / kg body weight), or relaxin-2 + RU-486. The mice were then sacrificed, and their spleens were processed using standard procedures to isolate splenic regulatory T cells (T). reg The percentage of T was analyzed by FACS. reg CD4+FoxP3+ cells were originally defined as cells expressing FoxP3 (a forkhead transcription factor) and reg Regulatory T cells expressing the transcription factor Forkhead box P3 (FoxP3) are known to control immune responses and prevent autoimmunity.

[0059] As shown in Figure 12, 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 when administered together with synthetic human relaxin-2 (shRlx), the presence of RU-486 significantly reduced relaxin-induced T cell differentiation. reg The increase was completely abrogated. # denotes p<0.05 compared to control; Kruskal-Wallis ANOVA on ranks for global tests and post-hoc Mann-Whitney U tests for pairwise comparisons (Bonferroni-Holm p correction).

[0060] Thus, these findings demonstrate the stimulatory effects of synthetic human relaxin-2 on the differentiation of peripheral (splenic) regulatory T cells (Tregs) in mice and indicate that these effects are GR-dependent, as they are abolished by GR antagonists such as RU-486. ●Considerations and Overview

[0061] As mentioned above, glucocorticoids and corticosteroids are the mainstay of treatment for tissue-damaging autoimmune pathologies such as rheumatoid arthritis and are also used as immunosuppressants after organ transplantation. An overwhelming amount of literature exists on glucocorticoid-mediated immune system regulation, particularly glucocorticoid-mediated regulation of innate immunity and inflammation. Calcium-binding S100 proteins are universal markers of inflammation and the innate immune system, including calprotectin (a complex of S100A8 and S100A9) and S100A12. Generally, the presence of S100A12 and calprotectin indicates tissue injury, endothelial cell activation, and inflammation-mediated responses. Cellular stress and / or inflammation induce the release of S100 proteins into cell-free compartments, where they bind to cell surface receptors such as RAGE, TLR4, CD147, and GPCRs. Interaction between calcium-binding S100 proteins and their receptors activates intracellular signaling pathways, such as AP1 and NFκB, initiating multiple cellular processes, including cell differentiation, migration, apoptosis, proliferation, and inflammation; activator protein 1 (AP1), extracellular signal-regulated protein kinase (ERK), G protein-coupled receptor (GPCR); interleukin-1 (IL-1), interleukin-7 (IL-7), nuclear factor kappa light polypeptide gene enhancer in B-cells inhibitor alpha (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 tumor necrosis factor receptor-associated factor 2 (Traf2). Mammalian cells secrete calprotectin during inflammatory responses. The exact mechanism by which the S100A8 / S100A9 complex is secreted from mammalian cells during inflammation remains unclear. When released into the extracellular space, S100 proteins have activity in the regulation of immune homeostasis, post-traumatic injury, tissue damage, and inflammation.S100 proteins mediate inflammation through interaction with their receptors, RAGE and TLR4, and there is evidence that calprotectin (S100A8 / S100A9) is an endogenous agonist of TLR4. Binding to TLR4 initiates a signaling cascade that regulates inflammation, cell proliferation, and differentiation in an NFκB-dependent manner. In addition to TLR4, RAGE has been suggested to bind to other S100 proteins, including S100A7, S100A12, S100A8 / A9 (calprotectin), and S100B. By interacting with RAGE, S100 proteins activate NFκB and induce the production of proinflammatory cytokines, which lead to the migration of neutrophils, monocytes, and macrophages. Therefore, extracellular S100 proteins are involved in the regulation of cell apoptosis and migration of monocytes, macrophages, neutrophils, lymphocytes, myoblasts, epithelial cells, and endothelial cells. Consequently, the levels of the S100A8 / A9 complex (calprotectin) and S100A12 in the extracellular fluid can be used as biomarkers to assess the degree of inflammatory control and tissue injury.

[0062] Liver transplantation and liver hypothermic preservation were studied in mice with the same genetic makeup. In this syngenic mouse model, all immunological responses to surgical trauma, hypothermic preservation, and ischemic injury were mediated by the innate immune system, and their effects were attenuated by corticosteroid administration. In this syngenic mouse model, administration of relaxin-2 during hypothermic preservation and / or reperfusion proved cytoprotective and significantly improved post-transplant liver function and survival (see Kageyama S et al. in Recombinant relaxin protects liver transplants from ischemia damage by hepatocyte glucocorticoid receptor: From bench-to-bedside, Hepatology 2018, 258-273). The authors hypothesized a regulatory role for relaxin-2-GR complex in inflammatory injury in liver transplantation, but the effects of relaxin on peripheral Treg cell promotion and immunosuppressive Treg cell activation and promotion were not described. The findings of the present invention justify continuous treatment of recipients with relaxin-2, since activated peripheral Treg cells can suppress local immune responses and responses activated by any type of tissue injury and endothelial injury. As shown in Figures 10 and 11, relaxin-2 does not lead to gluconeogenesis, so continuous administration of relaxin-2 is also justified.

[0063] Regarding the anti-cancer activity of glucocorticoids, for example, De Bono et al. (2014) (Clin Cancer Res 2014, 20:1925-1934, US2006018910) disclosed that glucocorticoid receptors are upregulated in refractory prostate cancer cells, and therefore should be inhibited to impair the proliferation of these cancer cells. They therefore reported treating hormone-refractory prostate cancer patients with a combination of docetaxel, anti-IGF-IR antibody, and dexamethasone (Ruhr et al., 2018. Clin Cancer Res 24: 927-938). Activation of the glucocorticoid receptor was found to lead to p57-mediated cell cycle arrest and acquisition of quiescence, which was assisted by reprogramming of signal transduction orchestrated through insulin receptor substrate 2 (IRS2) / forkhead box 01 (FOXO1). Synthetic relaxin-2 can be used as an alternative to dexamethasone and is therefore preferable for such treatments without the adverse effects of glucocorticoids and their analogs.

[0064] Boehnert MU in Relaxin as an additional protective model of isolate perfused rat liver, Ann NY Acad Sci 2005, 1041:434-440, and Bausys A et al. in Custodiol® supplemented with synthetic human relaxin decreases ischemia-reperfusion injury after porcine kidney transplantation, Int J Mol Sci. 2021, 22, 11417, reported that relaxin in the perfusate 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. The expression of receptor-interacting serine / threonine protein kinase 1 (RIPK1), which plays a role in apoptosis and necroptosis, was downregulated compared to controls. Furthermore, the expression of mixed lineage kinase domain-like protein (MLKL), which plays a role in tumor necrosis factor (TNF)-induced necroptosis, was downregulated, and the number of caspase-3- and MPO-positive cells was reduced in grafts after static hypothermia storage in a solution containing relaxin-2. Static hypothermia storage in a cardioplegic solution is the simplest, most convenient, and least expensive method of organ preservation in clinical practice. Although relaxin-2 has been described for its antifibrotic, antioxidant, anti-inflammatory, and cytoprotective properties, there has been no evidence that it can be beneficially used as a glucocorticoid substitute without inducing its Cushingoid-like adverse effects, particularly on gluconeogenesis, nor has relaxin-2 been observed to promote immunosuppressive Treg cell activity.However, from the above, relaxin-2 is not merely a vasodilator additive in the perfusate to prevent ischemia-reperfusion injury, but rather a broad-spectrum drug against tissue and endothelial injury.

Claims

1. A pharmaceutical composition for the treatment of patients requiring long-term attenuation of inflammatory responses caused by the innate immune system when it is desirable to avoid the side effects of steroid treatment, comprising an effective amount of synthetic human relaxin-2 and a pharmaceutical solvent, diluent, or excipient. , the aforementioned pharmaceutical composition.

2. A pharmaceutical composition according to claim 1 for the treatment of patients requiring long-term suppression of inflammatory responses caused by the innate immune system when it is desired to avoid the side effects of glucocorticoids, corticosteroids, mineralocorticoids, or other steroid therapies, wherein the pharmaceutical composition has synthetic human relaxin-2 as its pharmacologically active ingredient in order to avoid the manifestation or discontrollation of symptoms of diabetes, impaired wound healing, and / or Cushing's syndrome.

3. The patient has undergone an immune response that damages tissue or endothelium, and the following clinical parameters are elevated compared to a healthy individual or organ: Elevated serum HMGB1 (high mobility 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 during the post-organ donation flush. The pharmaceutical composition according to claim 1 for treating a patient having one or more of the following, or a patient's organ, or a patient's allograft.

4. A pharmaceutical composition according to any one of claims 1 to 3 for the treatment of patients suffering from SIRS (systemic inflammatory response syndrome), autoimmune or rheumatic diseases, thyroiditis, gastritis, pancreatitis, sialadenitis, adrenal nephritis, oophoritis, glomerulonephritis, polyarthritis, ankylosing spondylitis (AS) and spondyloarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune diseases, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory arthritis, rheumatoid arthritis (RA), SARS-Covid 19, and SARS.

5. The patient meets the following clinical criteria: Prediabetes (HbA1c > 5.7 and < 6.5%), obesity (BMI > 30 kg / m2), hypertension (stage 1 or higher according to the 2017 ACC / AHA guidelines) A pharmaceutical composition according to any one of claims 1 to 3, which shows the above.

6. The following four criteria: Serum HMGB1 (high mobility box protein) is ≥ 2 ng / ml, serum sTLR4 (soluble Toll-like receptor-4) is ≥ 0.25 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) is ≥ 0.5 ng / ml, and / or serum calprotectin is ≥ 4 micrograms / ml. The pharmaceutical composition according to claim 3 for treating an allograft recipient when one or more of the following conditions are met.

7. Allogeneic grafts that have undergone an elevated immunological response by the innate immune system should be subjected to the following criteria in the blood washed away after organ donation: HMGB1 (high mobility box protein) > 2 ng / ml, sTLR4 (soluble Toll-like receptor-4) > 0.25 ng / ml, serum sRAGE (soluble receptor for advanced glycation end products) > 0.5 ng / ml, or calprotectin > 4 μg / ml The pharmaceutical composition according to claim 3, for treatment when one or more of the following are observed.

8. A pharmaceutical composition according to any one of claims 1 to 3, for the treatment of a patient requiring treatment of hormone-refractory cancer, including but not limited to prostate cancer and breast cancer, or primary cancer via ligand-activated GR, including but not limited to multiple myeloma, Hodgkin's disease, and other lymphoid cancers, Kaposi's sarcoma, wherein the synthetic human relaxin-2 is used as a supplement and substitute for a glucocorticoid, cortisone, glucocorticoid receptor-activating hormone, or steroid.

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