EDVI: end-diastolic volume index; Ejection Fraction: left ventricular ejection fraction; E‘: maximum velocity of diastolic left ventricular myocardial relaxation in m/s); AET: Aortic Ejection Time; MV E/E‘: a dimensionless measure of left ventricular compliance (E, maximum velocity of mitral diastolic inflow in m/s; E‘, maximum velocity of diastolic left ventricular myocardial relaxation in m/s); GLS: Global Longitudinal Strain. [0047] Diminished Global Longitudinal Strain (GLS) is a well-established myocardial deformation analysis that predominantly reflects the function of subendocardial longitudinally oriented fibres, which are most susceptible to ischaemic damage and wall stress. Therefore, they may exhibit abnormal contraction patterns in the setting of an apparently normal left ventricular ejection fraction (LVEF). The E/E’ criterion also defines diastolic dysfunction in patients; its increase in absolute terms is mainly due to an impaired rate of myocardial relaxation (E’). In addition, the left atrial enlargement (see left atrial area) is a key feature of structural remodelling due to reduced left ventricular compliance. In combination with general clinical signs of heart failure as pulmonary oedema (the Lung Score), this detection of diastolic dysfunction completes the diagnostic criteria for HFpEF.

[0048] Using this animal model, ZSF1 rats (n = 8-12 per group) with experimental

HFpEF then received synthetic human relaxin-2 to assess the effects of relaxin-2. In brief, nine-week old ZSF1 lean rats were obtained from Charles River and fed with Purina Diet (#5008). After a 1-week laboratory adaptation period, animals underwent phenotypic evaluation and echocardiographic evaluation. From this point onward, the group of ZSF1 obese rats was allocated to receive HFD (Research Diet Inc, #D12468). To assess diastolic function, peak velocity of early (E) and late (A) mitral inflow signals and the ratio of E over e'

(peak velocity of early diastolic lateral mitral annular motion) were measured as an indication of LV filling pressure. More precisely, E/E‘ is a dimensionless measure of left ventricular compliance (E, maximum velocity of mitral diastolic inflow in m/s; e‘, maximum velocity of diastolic left ventricular myocardial relaxation in m/s). Furthermore, the Left Atrial Area was determined which reflects left atrial remodeling (enlargement) attributable to impaired left ventricular filling characteristics (impaired compliance). At 20 weeks, animals further underwent hemodynamic evaluation and biomechanical studies under anesthesia.

Investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 85-23, revised 1996); cf. Figs. 3A-D. [0049] Treatment with relaxin started at week 20, and relaxin-2 was compared to placebo to see whether relaxin-2 can significantly improve in rat the above echocardiography parameters of diastolic dysfunction, which are also important in the clinical assessment of human HFpEF.

[0050] Results: The effect of the continuous infusion of 400pg per kg rat body weight per day over two weeks of human relaxin-2 using an osmotic minipump in these rats with experimental HFpEF have been summarized in Fig. 4A and Fig. 4B The data support that the continuous administration of relaxin-2 has a substantial effect on the echocardiographic parameter (E/e’) of diastolic dysfunction as well as on the Left Atrial Area, which - most importantly - are also crucial for the clinical assessment of human HFpEF. Statistical analyses were performed using a two-way nonparametric ANOVA followed by Mann-Whitney U test for ranks. *, P < 0.05; **, P < 0.01 ; ***, P < 0.001 ; ****, P < 0.0001 .

[0051] The same animal model was used as in Example 3, but in this 2-week comparative experiment, the human relaxin-2 was administered as a daily subcutaneous bolus of 100 micrograms/kg bodyweight, starting at week 20. Figs. 4C-4F summarise the effect of

2 weeks treatment of ZSF1 rats (n = 5-6 per group) with daily subcutaneous boluses of 100 micrograms/kg body weight of synthetic human relaxin-2. As with continuous infusion, this relaxin drug significantly improves diastolic dysfunction in this HFpEF animal model compared to placebo injections. This includes E and E/e‘ as a echocardiographic parameter of left ventricular compliance and left atrial length measured in PSLAX (parasternal long axis, a specific echo view) as measure of atrial remodeling and also the Lung Score. Statistical analyses were performed using a two-way non-parametric ANOVA followed by Mann-Whitney

U test for ranks. *, P < 0.05; **, P < 0.01 ; ***, P < 0.001 ; ****, P < 0.0001 .

[0052] It is noteworthy, that the total amount of relaxin-2 drug administered by the daily subcutaneous bolus was only a quarter of the amount of relaxin-2 administered by the comparative continuous infusion as prescribed in the prior art. However, the essential therapeutic effect was the same, if not statistically greater. Consequently, this suggests that the therapeutic effects of relaxin-2 do not depend on a momentary serum concentration, but that the relaxin activity appears to affect mechanisms at the DNA level. In this context, it should not been overlooked that relaxin-2 appears to act on the glucocorticoid receptor and on the canonical Wnt signaling, where relaxin-2 seems to able to by-pass the effects of Dickkopf proteins in serum. Without wishing to be bound by theory, the present inventors assume that the reversal of myocardial ageing by relaxin-2 is mediated by the reactivation of canonical Wnt signaling, which primarily requires initiation rather than a constant trigger.

[0053] The use of zinc complexes of relaxin-2 further result in a flattened and prolonged pharmacokinetic curve with delayed peak levels. Their use may prolong individual bolus persistence in circulation and thereby confer another degree of freedom to optimally adjust the described bolus administration for clinical studies or therapeutic interventions. EXAMPLE 5 - Effects of Relaxin-2 on the Glucocorticoid Receptor

[0054] The new dosis regimen and mode of application for HFpEF patients affect also the treatment other diseases. Millions of patients take glucocorticoids to treat autoimmune and rheumatoid diseases, neurological disorders, pulmonary diseases, cancer, and other diseases and causes. However, the chronic side effects of glucocorticoids and their adverse consequences are much feared, particularly the negative impact due to glucocorticoid receptor (GR) down-regulation, steroid-induced hyperglycemia, or activation of gluconeogenesis as well

1 as Cushing’s syndrome. The present inventors have discovered that relaxin-2 binds the ligand- binding domain of the GR like steroids and glucocorticoids, forming an activated GR-ligand complex. The relaxin-GR complex activates the transcription of genes dampening the innate immune system but no genes activating gluconeogenesis, unlike the glucocorticoids or corticosteroids. This discovery enlarges the therapeutic applications of relaxin-2 for patients in need of a dampened innate immune system. This group of patients comprises for example patients who receive or have received an allotransplant, and cancer patients. Another large group are patients suffering from forms of tissue/endothelial injury or tissue-damaging diseases, including autoimmune or rheumatic tissue-damaging diseases comprising ankylosing spondylitis (AS) and spondylarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune disease, osteoarthritis (OA), psoriatic arthritis (PsA), and inflammatory types of arthritis, rheumatoid arthritis (RA). Medications for these diseases include corticosteroids, oral and topical analgesics, non-steroidal anti-inflammatory drugs such as ibuprofen and COX-2 inhibitors, and disease-specific biologies. Tissue injury, endothelial injury, and endothelial cell activation, particularly in allograft rejection, can be detected and monitored by increased concentrations of calprotectin and/or S100A12 in extracellular fluids and the bloodstream. This is because endothelial cells also play a critical role in immune cell recruitment and extravasation. The calcium-binding S100 proteins, particularly calprotectin and S100A12, have a wide range of intracellular and extracellular functions, including regulation of calcium balance, cell apoptosis, cell migration, differentiation, proliferation, energy metabolism, and inflammation. The calcium-binding S100 proteins are released from the cytoplasm of the endothelial cell when triggered by tissue/cell damage, antibody stress, and endothelial stress.

The S100 proteins then serve as danger signals, DAMP (damage-associated molecular pattern) molecules, and are involved in the regulation of immune homeostasis (macrophage migration, invasion, and differentiation), post-traumatic injury, and inflammation. They are therefore biomarkers in some specific diseases such as IBD (inflammatory bowel disease), although their multiple functions must be assigned to cell migration, differentiation, tissue repair, immune homeostasis, and inflammation management. The lack of commonly available diagnostic tests for tissue injury, endothelial injury, endothelial stress, and anti-endothelial cell antibody binding, which undoubtedly lead to allograft dysfunction and allograft rejection, makes calcium-binding S100A12 and calprotectin biomarkers for endothelial activation, immune cell recruitment, endothelial injury, endothelial antibody binding, and complement activation.

[0055] The present inventors discovered that relaxin-2 not only binds to the glucocorticoid receptor but that the experimental results further indicate that the administration of relaxin-2 leads to a specific activation and promotion of regulatory T cells at local and systemic levels, likely induced by the relaxin-GR complex. This allows suppression of immune responses and significantly widens the use of relaxin-2 as an active ingredient in pharmaceuticals for treating abnormal, excessive, and undesired tissue-damaging immune responses to self- and foreign antigens. While the promotion of peripherally induced Treg cells could also be achieved by the administration of glucocorticoids, such treatment is disadvantageous due to the Cushingoid adverse effects of glucocorticoids, corticosteroids, and synthetic analogs thereof. Their adverse effects are well-known and numerous (cf. 2022 ICD-

10CM Code T38.0X5A).

[0056] Regulatory T cells (Treg cells) were originally defined as CD4+ T cells with a high expression of CD25 (interleukin-2 receptor a-chain). The regulatory T cells are further classified into thymic and peripherally induced Treg cells based on where they develop. The

Foxp3 gene, a member of the Forkhead/winged-helix family of transcriptional regulators, was discovered to be an important regulator in the development of Treg cells based on the following findings: Scurfy mice with a frameshift mutation in the Foxp3 gene have T cell inflammation in multiple organs and a lethal autoimmune disease due to effectorT cell activation and increased cytokine production caused by the absence of Treg cells. In addition, mutation of the Foxp3 gene in humans leads to IPEX syndrome (X-linked immune dysregulation, polyendocrinopathy, and enteropathy). In addition, forced expression of FoxP3 in naive T cells leads to immune suppressive function. CD4/CD25-naive T cells transfected with the Foxp3 gene can transform into CD4⁺CD25⁺ Treg-like cells that produce inhibitory 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-related protein (GITR). Thus, FoxP3 is a lineage- specific marker and an important regulatory gene for the generation, maintenance, and immune suppressive functions of Treg cells. Regulatory T cells are required to suppress abnormal or excessive immune responses and to maintain homeostasis and self-tolerance by inhibiting T cell proliferation and cytokine production. The Treg cells exert their immunosuppressive function through dominant consumption of the cytokine interleukin-2 and by inhibitory cytokines (TGF-B, IL-10, IL-35) as well as induction of apoptosis or a killing of effector or antigen-presenting cells (APC) by perforin, granzyme B or Fas ligand interaction.

The other immunosuppressive mechanisms of Treg cells involve immune checkpoint molecules and include inhibition of effector T cells by the lymphocyte activation programmed cell death pathways or the cytotoxic T-lymphocyte antigen (CTLA-4). A third immunosuppressive mechanism may be metabolic modulation by indoleamine 2,3- dioxygenase (IDO) expression, which affects the kynurenine-tryptophan pathway in dendritic cells. The Treg cells thereby play a critical role in suppressing autoimmunity and inflammation.

Reduced number and function of Treg cells are associated with human autoimmune disease, and activation and augmentation of Treg cells have been shown to be beneficial in treating autoimmune diseases in clinical trials (see the review of 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 the immune response to self and foreign antigens in an antigen- nonspecific manner Therefore, it is reasonable to hypothesize that a relaxin-2-induced increase in Treg cells in a posttransplant patient also improves post-transplant outcomes beyond the prevention of ischemic injury. The very same can also be assumed when relaxin-2 is used in treating autoimmune induced tissue injury, endothelial cell injury and diseases following endothelial cell activation. The hypothesis is also supported by the fact that a

1 i reduction in the proportion of Treg cells in the peripheral blood is known to lift general immune suppression, thereby enhancing the innate and acquired immune response to foreign and self- antigens. Therefore, the present discovery significantly expands the pharmaceutical toolbox.

EXAMPLE 5 Relaxin binds and activates the glucocorticoid receptor

[0057] Fig.5a and Fig. 5b refer to in vitro binding and affinity studies of synthetic human relaxin-2 (shRIx) to the ligand-binding domain of the glucocorticoid receptor (GR-LBD) using microscale thermophoresis (MST). The microscale thermophoresis is based on measuring the directed movement of molecules in localized temperature gradients created by IR laser radiation in high-precision glass capillary tubes containing the interacting partners - synthetic human relaxin-2 and recombinant GR-LBD. For this experiment, the human glucocorticoid receptor (GR-LBD) ligand-binding domain was expressed in an E. coli expression system to obtain a large amount of soluble protein stable for biophysical characterization. The recombinantly produced GR-LBD showed little aggregation and proved to be fully functional. One of the interacting 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 movement of the complex is detected. Conformational changes due to ligand binding to the target or binding near the fluorophore induce thermophoretic changes. The affinity of interacting protein is determined by analyzing the change in normalized fluorescence as a function of the concentration of titrated binding partner. The next step was to determine the binding mode of relaxin and the activation mechanism of GR. Fluorescence polarisation revealed two binding affinities in the pico- and nanomolar range. Moreover, human

H2 relaxin could displace fluormone labeled GS red out of its binding pocket on GR-LBD (see 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 was identified, which was the steroid-binding pocket of the GR-LBD.

[0058] The effects of relaxin binding on GR-LBD have further been investigated to determine whether relaxin binding activates the receptor like an agonist or represses transcriptional activity by acting like an antagonist. GR-LBD has an activation function-2 site that will recruit cofactors upon ligand binding. The cofactors (coactivators or corepressors) are specific to the cellular environment. Therefore, the binding of relaxin with coactivator and corepressor motifs to the GR-LBD/relaxin complex was tested. [0059] It was found that relaxin binds to both cofactors, resulting in different receptor conformational changes. Since thermophoresis is an intrinsic phenomenon of a molecule that depends on the hydration shell and the size, the binding events could be identified by tracking the associated changes to thermophoresis in a fluorescently labeled interacting partner. In summary, the results of the microscale thermophoresis (see Figs. 5a and 5b) indicate a high- affinity interaction (KD ~ 5 nM) and another lower-affinity interaction (KD ~ 500 nM) of synthetic human relaxin-2 with the GR-LBD.

[0060] According to hydrogen-deuterium exchange experiments (not shown), human relaxin-2 appears to bind to helix 12 of the LBD, but in contrast to classical glucocorticoids in absence of the transcription co-regulator 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 contains several nuclear receptor interacting domains and intrinsic histone acetyltransferase activities, and when GR recruits NCoA-2 to a DNA promotion site, the role of NCoA-2 also appears to be in acetylating histones so that the downstream DNA becomes more accessible for transcription.

The presence and amount of NCoA-2 are cell-type dependent. NCoA2 (GRIP1 , SRC-2, TIF2) therefore supports up-regulation of DNA expression which also 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. Relaxin-2 activates transcription of genes dampening peroxide-induced cytotoxicity, inflammatory responses, and apoptosis

[0061] With reference to Fig. 5c and Fig. 5d, mouse hepatocytes were isolated as described by Tamaki N et al. in Am J Physiol Gastrointest Liver Physiol 2008294, G499. Briefly, livers from mice anesthetized with pentobarbital were washed and perfused for 5 min with buffer consisting of (all values are mg/L) 8,000 NaCI, 400 KCI, 88.7 NaH2PO₄ H2O, 120.45

Na₂HPO₄, 2,380 HEPES, 350 NaHCO₃, 190 EGTA, and 900 glucose, pH 7.25, then treated with 0.03% collagenase at 37°C for 15 min in digestion buffer containing (all values are mg/L)

8,000 NaCI, 400 KCI, 88.7 NaH₂PO₄ H₂O, 120.45 Na₂HPO₄, 2,380 HEPES, 350 NaHCO₃, and

560 CaCl2'2H2O , pH 7.25. After collagenase perfusion, the liver capsule was isolated, and the cells dispersed in Geys balanced salt solution (GBSS)-B consisting of (all values in mg/L) 8,000

NaCI, 370 KCI, 210 MgCI₂'6H₂O, 70 MgSO₄-7H₂O, 120 NaH₂PO₄, 30 KH₂PO₄, 991 glucose,

227 NaHCO₃, and 225 CaCl2'2H2O (pH 7.25). Cells were additionally separated by forcing the material through a steel mesh and collecting the cells by centrifugation at 50 G for 1 min. The cell pellet was resuspended in GBSS-B solution and washed three times with intermittent centrifugations.

[0062] Isolated mouse hepatocytes were cultured on type I collagen-coated 6-well plates coated at a cell density of 5 x 10⁵ cells/well with Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 U/ml penicillin, and 100 pg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2-95% air. After plating, the medium was replaced with serum- free Dulbecco's modified Eagle medium after 6 hours. The hepatocytes were then subjected to hydrogen peroxide treatment (2 mM H2O2/L) for 5 hours with or without pretreatment with synthetic human relaxin-2 (10 nM/L, 24 h) (Relaxera Pharmazeutische GmbH, Bensheim, DE) or dexamethasone (0.5 mM, 24 h) (Sigma Aldrich). In addition, 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 was dependent on the ligand-activated GR (n = 5 for each group). H2O2-induced cell injury

(cytotoxicity) was determined immunologically by quantification of lactate dehydrogenase (LDH) released into the culture medium using an enzyme-linked assay according to the manufacturer’s instructions (Goat LDH ELISA kit, Biomol Feinchemikalien GmbH, DE) and by

Western Blot analysis of cleaved caspase-3 (Caspase-3 Rabbit mAb #14220, Cell Signaling

Technology, Danvers, MA, US). [0063] The results are summarized in Figs. 5c and 5d. LDH release data shown in

Fig. 5c are expressed as a percentage of detergent-induced maximum cytotoxicity. Data on cleaved/activated caspase-3 shown in Fig. 5d were normalized to p-actin and adjusted for the effect of H2O2 (* p < 0.05 vs. control; #, p < 0.05 vs. H2O2; Kruskal-Wallis ANOVA on ranks for global testing with post hoc Mann-Whitney U-tests for pairwise comparisons (Bonferroni-Holm adjustment of p). In summary, Figs. 5c and 5d show that both relaxin-2 (Rix) and dexamethasone (Dx) can markedly dampen peroxide-induced cell injury (LDH release) and apoptosis (cleaved caspase-3) and that this attenuating effect does not occur when the expression of GR is specifically knocked out by GR-siRNA, whereas a knock-out using scrambled siRNA (scr-siRNA) showed no effect. [0064] Physiologically, LDH is an enzyme expressed in nearly all living cells, including heart muscles and blood cells, and it catalyzes the conversion of lactate to pyruvate and back.

Because it is released during tissue injury, it is a marker of common injuries, damaged tissues, and diseases involving tissue damage such as heart failure. In contrast, the relative concentrations of its substrates primarily regulate LDH activity. LDH is subject to transcriptional regulation by peroxisome proliferator-activated receptor-y coactivator 1a (PGC-1a) in an estrogen-related receptor-a-dependent manner. [0065] Caspase 3 protein (CASP3) or cysteine-dependent aspartate-directed protease 3 plays an essential role in programmed cell death. Caspase-3 is synthesized as an inactive zymogen until cleaved after apoptotic signaling events. 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 tumor development, for example, by a mutation in a cell cycle gene that removes cell growth restrictions in combination with mutations in apoptotic proteins such as caspases that would trigger cell death in abnormally growing cells. Conversely, over-activation of caspase-3 can lead to excessive programmed cell death. This is seen in several neurodegenerative diseases in which neural cells are lost, such as Alzheimer's disease. Caspases involved in processing inflammatory signals are also implicated in disease. Insufficient caspase activation can increase an organism's susceptibility to infection because an appropriate immune response may not be triggered. For example, inflammatory caspase-1 has been linked to the development of autoimmune diseases; drugs that block caspase activation have been used to improve patients’ health.

[0066] In conclusion, synthetic relaxin-2 as a ligand of the glucocorticoid receptor appears to have cell biological effects like dexamethasone in attenuating an inflammatory response to tissue injury as well as apoptosis and necrosis.

Relaxin-GR complex does not activate transcription of genes involved in gluconeogenesis [0067] Mouse hepatocytes were obtained and tested for glucocorticoid-adverse effects after treatment with synthetic human relaxin-2 or dexamethasone (500 nM, 24 hours). The complex physiological adverse effects of glucocorticoid excess are numerous and difficult to assess (Cushing’s syndrome, diabetes, skin thinning, hypertension, osteoporosis, obesity, impaired wound healing, depression, etc.), but measurable in this cell assay is the activation of genes involved in gluconeogenesis and in diabetes, obesity, and impaired wound healing. Consequently, the regulation of GR- and PDK-4 transcription was examined by qRT-PCR in normal primary mouse hepatocytes for control and in H2O2-stressed primary mouse hepatocytes from example 2 after 24 hours of treatment with 10 nM/L synthetic human relaxin-

2 (Relaxera Pharma. GmbH&Co.KG) and 500nM/L dexamethasone (Sigma Aldrich). Results are shown in the block diagrams of Figs. 5e and 5f.

[0068] Specifically, Fig. 5e shows that relaxin-2 increases GR gene transcription in normal and H2O2-stressed primary mouse hepatocytes by 100% to 200 %, whereas dexamethasone has no such effect. Fig. 5f shows that the incubation with relaxin-2 has no effect on PDK-4 gene transcription in normal and H2O2-stressed primary mouse hepatocytes. However, when primary mouse hepatocytes are incubated with dexamethasone, PDK-4 gene transcription increases severalfold. The combination of Figs. 5e and 5f is therefore strong evidence that the complex of relaxin and GR binds to a genomic DNA locus that is different from the DNA locus of steroid-activated GR.

[0069] Physiologically, PDK-4 (pyruvate dehydrogenase lipoamide kinase isozyme 4) is a mitochondrial protein that inhibits the pyruvate dehydrogenase complex (PDH) by phosphorylating one of its subunits. An active PDH complex is required to convert pyruvate to acetyl-CoA for the glycolytic products to enter the citric acid cycle. Fasting results in an induction of PDK-4 mRNA and PDK-4 enzyme in both cardiac and skeletal muscle, suppressing glucose oxidation during starvation as part of an integrative response and for glucose maintenance. The PDK-4 enzyme is therefore thought to play a critical role in regulating glucose metabolism, whereas an increased PDK-4 transcription indicates gluconeogenesis. It is well known that PDK-4 expression is physiologically regulated by glucocorticoids, retinoic acid, and insulin which enhance the transcription of the PDK-4 gene in white adipose tissue. Oxidation of fatty acids is also increased when the PDK-4 level is elevated. Insulin downregulates PDK-4 mRNA transcription. When cells are exposed to dexamethasone to increase PDK-4 mRNA expression, insulin blocks this effect and the oxidation of fatty acids. In type 2 diabetes, PDK-4 is overexpressed in skeletal muscle, resulting in impaired glucose utilization. In patients suffering from obesity, PDK-4 mRNA expression is also markedly decreased in association with increased glucose uptake, likely due to the downregulation of PDK-4 by insulin. 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, an inadequate downregulation of PDK-4 mRNA may cause increased PDK-4 expression, leading to impaired glucose oxidation followed by increased fatty acid oxidation. Conversely, PDK-4 is downregulated in cardiac muscle tissue during heart failure, which is a physiological countermeasure (Razeghi P et al., in

Downregulation of metabolic gene expression in failing human heart before and after mechanical unloading, Cardiology 2002, 97(4):203-9).

[0070] The results in Figs. 5c through 5f show that relaxin-2 like glucocorticoids, corticosteroids, and mineralocorticoids has anti-inflammatory effects, but without leading to impaired glucose oxidation, increased fatty acid oxidation, and gluconeogenesis. The ubiquitous role of PDK-4 further suggests that a pharmaceutical composition containing relaxin-2 and treatment with relaxin-2 may be an alternative to glucocorticoid treatment because it results in increased expression of the glucocorticoid receptor, which suppresses immune and inflammatory responses, but not in increased expression of PDK-4, which is unfavorable for glucose metabolism and balance.

[0071] The glucocorticoid receptor (GR) is an evolutionarily conserved ligand- dependent transcription factor. Upon binding of a steroid hormone or other ligand, the receptor migrates from the cytoplasm to the nucleus where it binds to a genomic DNA locus and positively or negatively modulates the transcription rates of the locus-associated genes.

Tremendous efforts have been made to uncover the molecular signaling actions of the GR, including intracellular shuttling, transcriptional regulation, and interaction with other intracellular signaling pathways. In brief, glucocorticoids are essential for the maintenance of resting-state and stress response and, therefore, they are essential in the treatment of numerous diseases, including autoimmune, inflammatory, allergic, and lymphoproliferative disorders. The pathological or therapeutic implications of the GR cannot be overstated. These include, except for genetic alterations in the human GR gene, disease-associated GR regulatory molecules and the development of GR ligands with selective GR action. [0072] Figs. 5a to 5f show that the administration of synthetic relaxin-2 has therapeutic effects that differ from 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), which, in contrast to classical corticosteroids and glucocorticoids, avoid unwanted effects such as GR down-regulation or glucocorticoid- or steroid-induced hyperglycemia or activation of gluconeogenesis. Relaxin-2 dampens the release of pro-inflammatory cytokines [0073] THP-1 cells were differentiated into macrophages and cultured as described by Dschietzig T et al. in Identification of the pregnancy hormone relaxin as a glucocorticoid receptor agonist, FASEB J 2004, 18:1536-1538. Briefly, the THP-1 cells are derived from a cell line generated from a human monocyte leukemia, and when the cells are treated in the passage with myristate-phorbol ester, they differentiate into macrophages as is well known in the art. [0074] Referring to Figs. 5g and 5h, the macrophages were then challenged for 24 hours 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 combinations thereof (n = 5 for each group). RU-486 binds to the GR in the steroid pocket of the ligand-binding domain.

Subsequently, the supernatant levels of TNF-a and interleukin-6 were determined by ELISA

(R&D Systems). [0075] The results are summarized in the block diagrams for Figs. 5g + h, and demonstrate that the endotoxin induced these macrophages to produce and excrete the proinflammatory cytokines tumor necrose factor alpha (TNF) and interleukin-6 (IL-6), which inflammatory response was both depressed in the presence of synthetic human relaxin-2 and dexamethasone. This response can be classified as GR-dependent, as RU-486 completely inhibited this effect. [0076] As further background, RU-486, also known as Mifepristone®, is a steroidal antiprogesterone as well as an antiglucocorticoid and antiandrogen. It competitively antagonizes cortisol action at the GR receptor. In humans, an antiglucocorticoid effect of RU-

486 is observed at doses greater or equal to 4.5 mg/kg through a compensatory increase in adrenocorticotropic hormone (ACTH) and cortisol. In animals, a weak antiandrogenic effect is seen with prolonged administration of very high doses (Danco Laboratories, 2005, Mifeprex

U.S. prescribing information). Thus, this example demonstrates that the administration of synthetic human relaxin-2 can provide GR-mediated regulation of the immune system, and has immune-suppressive properties, but without the side effects of inducing gluconeogenesis and insulin insensitivity.

Relaxin-GR complex does not induce hyperglycemia other than to dexamethasone-GR [0077] Figs. 5i, 5k, and 5L refer to animal experiments and show the different effects of relaxin-2 and dexamethasone (Sigma Aldrich) administration on blood glucose levels in rats after 24 and 48 hours. In brief, male and female Sprague-Dawley rats (body weight 300 - 350 g) were treated with an intraperitoneal injection of E. coli endotoxin (125 pg/kg body weight) or placebo (vehicle). Blood from the tail vein was taken after 24 hours for the determination of circulating TNF-a (ELISA, R&D Systems) and fasting blood glucose and at 48 hours for measurement of fasting blood glucose. Two hours before administration of endotoxin or placebo, animals received dexamethasone (intramuscular 10 mg/kg body weight), synthetic relaxin-2 (Relaxera Pharmazeutische GmbH&Co.KG, Bensheim) as a subcutaneous infusion (4 pg shRIx/h) over 12 hours via osmotic Alzet minipumps, oral RU-486 (single dose of 10 mg/kg body weight), or combinations thereof (n = 5 animals per group). The results are shown in Figs. 5k and 5L; *, p < 0.05 vs. control; #, p < 0.05 vs. endotoxin plus dexamethasone;

Kruskal-Wallis ANOVA on ranks for global testing with posthoc Mann-Whitney U-tests for pairwise comparisons (Bonferroni-Holm adjustment of p).

[0078] While dexamethasone markedly enhanced the endotoxin-related increase in blood glucose, reflecting the animal’s disease and hyperthermia response to endotoxin, relaxin-2 decreased glucose levels compared with endotoxin alone. This was true at both 24 and 48 hours after endotoxin. Oral administration of RU-486 inhibited the effects of relaxin-2 and dexamethasone by its antagonistic actions at the glucocorticoid receptor, as also the concentration of TNF-a in the circulation. In conclusion, human relaxin-2 attenuated the endotoxin-induced surge of circulating TNF-a in rats of both sexes. The effect could be identified as GR-dependent and comparable to that of dexamethasone, a classic glucocorticoid. However, unlike dexamethasone, relaxin-2 did not induce hyperglycemia which is a medically extremely important finding.

Relaxin-2 furthers differentiation of naive T cells into regulatory T cells (Treg) in mice

[0079] Mice of C57BI/6 background (each groups n=5) were intraperitoneally injected once daily for 3 consecutive days in 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), relaxin-2 plus RU-486. Thereafter, mice were sacrificed, their spleens were processed using standard procedures, and the percentage of spleen regulatory T cells (Treg) was analyzed by FACS. T_reg cells were originally defined as characterized as CD4+FoxP3+ cells wherein FoxP3 (a Forkhead transcription factor) is the T_reg master regulator. Regulatory T cells expressing the transcription factor Forkhead box P3 (FoxP3) are known to control immune responses and prevent autoimmunity.

[0080] As shown in Fig. 5m the administration of relaxin-2 approximately doubled the percentage of regulatory T cells (CD4+FoxP3+ cells) as compared with placebo. The GR antagonist RU-486 did not affect the differentiation of T cell but when administered together with relaxin-2, its presence completely abrogated the relaxin-induced T_reg increase. #, p < 0.05 vs. control; Kruskal-Wallis ANOVA on ranks for global testing with posthoc Mann-Whitney U- tests for pairwise comparisons (Bonferroni-Holm adjustment of p).

[0081] These findings, therefore, substantiate a stimulatory effect of synthetic human relaxin-2 on the differentiation of peripheral (spleen) regulatory T cells (Treg) in mice and show that these effects are GR-dependent because they could be nullified by a GR-antagonist such as RU-486. DISCUSSION AND SYNOPSIS

[0082] Conventionally, the administration of relaxin-2 in clinical trials and studies has been based on the assumption of a direct and immediate relationship between serum levels and relaxin activity, as is known from insulin. However, as shown in Examples 3 and 4, relaxin-

2 can reverse maladaptive remodeling of the aged heart, but the mechanisms of action are still unknown. In this regard, relaxin-2, although structurally an insulin-like hormone of the insulin superfamily, has been shown to suppress atrial fibrillation, inflammation, and fibrosis in aged rats. However, the administration of relaxin-2 in clinical trials and strudies has still been based i on the assumption of a continuous relation of circulating relaxin levels and relaxin activity. The present application shows that, although the mechanisms of action remain to become elucidated, there is no such direct relationship and that relaxin-2 probably acts by specifically activating genes in tissue development. This is a mechanism of action common to steroids and glucocorticoids as well as to the canonical wnt-signaling pathway. The wnt-signalling pathway is known to play a critical role in embryonic and tissue development which can be inhibited by the circulating proteins of the Dickkopf family.

[0083] In addition, glucocorticoids and corticosteroids are mainstays in the treatment of tissue-damaging autoimmune pathologies such as rheumatoid arthritis, and they are immunosuppressants following organ transplantation. An overwhelming amount of literature exists on the glucocorticoid-mediated regulation of the immune system and in particular on the glucocorticoid-mediated regulation of innate immunity and inflammation. The calcium-binding S100 proteins are universal markers for inflammation and of the innate immune system, notably calprotectin (the complex of S100A8 and S100A9) as well as S100A12. Generally, the presence of S100A12 and calprotectin indicate tissue injury, endothelial cell activation, and inflammation-mediated responses. Cell stress and/or inflammation induce the release of S100 proteins to acellular compartments where they bind cell surface receptors such as RAGE, TLR4, CD147, and GPCR. The interactions between the calcium-binding S100 proteins and their receptors activate intracellular signaling pathways such as AP1 and NFKB, which further initiates multiple cellular processes such as 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 of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IKBO), c-Jun

N-terminal kinase (JNK), p38 mitogen-activated protein kinase (P38), the receptor for advanced glycation end products (RAGE), toll-like receptor 4 (TLR4), TNF receptor-associated factor 2 (Traf2). Mammalian cells secrete calprotectin during the inflammatory response. The exact mechanism by which the complex of S100A8/S100A9 is secreted by mammalian cells during inflammation remains unknown. When released to the extracellular space, S100 proteins have activities in the regulation of immune homeostasis, post-traumatic injury, tissue damage, and inflammation. S100 proteins trigger inflammation through interacting with 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 and regulates inflammation, cell proliferation, and differentiation in an NFKB-dependent manner Apart from

TLR4, RAGE has also been suggested to bind S100 proteins such as S100A7, S100A12, S100A8/A9 (calprotectin), and SWOB. By interacting with RAGE, S100 proteins activate NFKB, inducing the production of pro-inflammatory cytokines leading to the migration of neutrophils, monocytes, and macrophages. Extracellular S100 proteins are therefore involved in the regulation of cell apoptosis, migration of monocytes, macrophages, neutrophils, lymphocytes, myoblasts, epithelial cells, and endothelial cells. Consequently, the levels of S100A8/A9 complex (calprotectin) and S100A12 in extracellular fluids can be used as biomarkers to assess the degree of inflammatory regulation and tissue injury.

[0084] Liver transplantation and cold storage of livers were studied in mice having the same genetic setup. In this syngeneic mouse model, all immunological responses due to surgical trauma, cold storage, and ischemia injury are therefore mediated by the innate immune system whose effects can be dampened by the administration of corticosteroids. In this syngeneic mouse model, the administration of relaxin-2 at cold storage and/or at reperfusion proved cell-protective and markedly improved posttransplant liver function and survival (cf. 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). Although the authors hypothesize a regulatory role of the relaxin-2-GR complex in the inflammatory injury in liver transplants, the impact of relaxin on the promotion of peripheral

Treg cells and the activation and promotion of immune-suppressive Treg cells was not noted.

With the knowledge of the present invention, continuous treatment of the recipient with relaxin-

2 is warranted as the activated peripheral Treg cells can suppress local immune responses and responses activated by any kind of tissue- and endothelial injury. A continuous administration of relaxin-2 is also warranted as relaxin-2 does not lead to gluconeogenesis as shown in Figs. 5k and 5I. [0085] Concerning the anti-cancer activity of glucocorticoids, for example, De Bono et al. 2014 (Clin Cancer Res 2014, 20:1925-1934, US2006018910) disclose treating hormone- refractory prostate cancer patients with a combination of docetaxel, anti-IGF-IR antibodies, and dexamethasone, since the glucocorticoid receptor is upregulated in refractory prostate cancer cells, and therefore should be inhibited to impair the proliferation of these cancer cells (Ruhr et al., 2018. Clin Cancer Res 24: 927-938). It was found that activation of the glucocorticoid receptor leads to the acquisition of quiescence, subserved by cell cycle arrest through p57 and reprogramming of signaling orchestrated via Insulin Receptor Substrate 2 (IRS2)/Forkhead Box 01 (FOXOI). As synthetic relaxin-2 can be used as a substitute for dexamethasone, it may be favorably used in such therapy without incurring the adverse effects of glucocorticoids and their analogs.

[0086] Boehnert MU in Relaxin as an additional protective model of isolate perfused rat liver, Ann N Y 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 describe that relaxin in the perfusion solution reduces ischemia-reperfusion injury (IRI) after kidney or liver

1 transplantation, and that relaxin-2 (RLX) upregulates the expressions of mitochondrial superoxide dismutase-2 (SOD2) and nuclear factor kappa B (NFKB), 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.

Also downregulated was the expression of mixed lineage kinase domain-like protein (MLKL), which plays a role in tumor necrosis factor (TNF)-induced necroptosis, and the number of caspase 3- and MPO-positive cells was also decreased in the grafts after static cold storage in a solution containing relaxin-2. Static cold storage in a cardioplegic solution is the simplest, most convenient, and least expensive method of organ preservation in clinical practice. While relaxin-2 has been described for its antifibrotic, antioxidant, anti-inflammatory, and cytoprotective properties, there was no evidence that relaxin-2 can be used beneficially as a substitute for glucocorticoids without inducing their Cushingoid adverse effects, particularly gluconeogenesis, and its promotion of immune suppressive Treg cells was also not observed, which, however, makes relaxin-2 a broad-spectrum drug fortissue and endothelial injury, rather than a vasodilator additive in a perfusion solution to prevent ischemia-reperfusion injury.

- SYNOPSIS [0087] In a model of HFpEF, ZSF1 -obese, where both male and female rats on high- fat diet develop this specific type of heart failure, synthetic human relaxin-2 significantly improves diastolic dysfunction, the echocardiographic measure that reflects left ventricular stiffening and subsequent left atrial remodelling (enlargement). These beneficial effects may be attributed to relaxin’s pleiotropic actions, including anti-inflammation, anti-fibrosis, up- regulation of anti-oxidant enzymes, protection of the functionality of the nitric oxide - cGMP- protein kinase G axis responsible for myocardial relaxation (titin phosphorylation), stimulation of active myocardial relaxation via phospholamban and SERCA-2a, as previously reviewed the article by Dschietzig TB, Relaxin-2 for heart failure with preserved ejection fraction (HFpEF): Rationale for future clinical trials, Molecular and Cellular Endocrinology 2019, 487: 54-8.

[0088] The present application discloses a novel mode of drug administration, namely subcutaneous injection of a bolus of relaxin-2 at daily intervals, which not only simplifies chronic clinical use of relaxin-2 and thus increases patient compliance, but also points to an unknown mechanism of its physiological actions and roles. The equipotentiality of a daily bolus of relaxin-

2 compared to continuous subcutaneous infusion could be explained by a slow dissociation of bound relaxin-2 from its cognate receptor, RXFP1 (Valkovic AL et al. in Real-time examination of cAMP activity at relaxin family peptide receptors using a BRET-based biosensor, Pharmacological Research and Perspectives 2018, e00422) and a variety of intracellular signaling cascades in response, and/or by the genomic effects via different pathways including cAMP response elements (CRE), the glucocorticoid receptor as described in this application, protein kinase G-mediated effects, as well as by Wnt-1 -mediated myocardial effects (Martin B et al. in Relaxin reverses maladaptive remodeling of the aged heart through Wnt-signaling,

Scientific Reports 2019, 9: 18545). However, these publications cannot suggest a daily relaxin bolus for the treatment of HFpEF, even when genomic effects may be longer-lasting in nature, since a reversal of the cardiac ageing as proven in Figures 4D to F is very multifactorial which escapes the usual explanations.

SEQUENCE LISTING

<110> Relaxera Pharmazeutis che Gesells chaft mbH

<120> RELAXIN MEDICATION

<130> RLX00257 PC

<160> 10

<170> Patentin version 3 . 5

<210> 1

<211> 29

<212> PRT

<213> Homo sapiens

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Asp Ser Trp Met Glu Glu Vai lie Lys Leu Cys Gly Arg Glu Leu Vai 1 5 10 15

Arg Ala Gin lie Ala lie Cys Gly Met Ser Thr Trp Ser

20 25

<210> 2

<211> 24

<212> PRT

<213> Homo sapiens

<400> 2

Glx Leu Tyr Ser Ala Leu Ala Asn Lys Cys Cys His Vai Gly Cys Thr 1 5 10 15

Lys Arg Ser Leu Ala Arg Phe Cys 20

<210> 3

<211> 27

<212> PRT

<213> Homo sapiens

<400> 3

Arg Ala Ala Pro Tyr Gly Vai Arg Leu Cys Gly Arg Glu Phe lie Arg 1 5 10 15

Ala Vai lie Phe Thr Cys Gly Gly Gly Arg Trp

20 25

<210> 4

<211> 24

<212> PRT

<213> Homo sapiens

<400> 4

Asp Vai Leu Ala Gly Leu Ser Ser Ser Cys Cys Lys Trp Gly Cys Ser

1 5 10 15 Lys Ser Glu lie Ser Ser Leu Cys

20

<210> 5

<211> 31

<212> PRT

<213> homo sapiens

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Pro Thr Pro Glu Met Arg Glu Lys Leu Cys Gly His His Phe Vai Arg 1 5 10 15

Ala Leu Vai Arg Vai Asp Gly Gly Pro Arg Trp Ser Thr Glu Ala 20 25 30

<210> 6

<211> 26

<212> PRT

<213> Homo sapiens

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Ala Ala Ala Thr Asn Pro Ala Arg Tyr Cys Cys Leu Ser Gly Cys Thr 1 5 10 15

Gin Gin Asp Leu Leu Thr Leu Cys Pro Tyr

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<212> PRT

<213> Homo sapiens

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Lys Glu Ser Vai Arg Leu Cys Gly Leu Glu Tyr lie Arg Thr Vai lie 1 5 10 15

Tyr lie Cys Ala Ser Ser Arg Trp

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<213> Homo sapiens

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Glx Asp Leu Gin Thr Leu Cys Cys Thr Asp Gly Cys Ser Met Thr Asp 1 5 10 15

Leu Ser Ala Leu Cys

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<210> 9

<211> 30 <212> PRT

<213> Homo sapiens

<400> 9

Phe Vai Asn Gin His Leu Cys Gly Ser His Leu Vai Glu Ala Leu Tyr 1 5 10 15

Leu Vai Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30

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<212> PRT

<213> Homo sapiens

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Gly lie Vai Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu 1 5 10 15

Glu Asn Tyr Cys Asn

20

Claims

1. A medicament for daily (q.d.) or twice daily (b.i.d.) subcutaneous injection over a prolonged period comprising as bolus a solution of synthetic human relaxin-2 from 75 pg to 2250 micrograms and a pharmacologically acceptable adjuvant, excipient, or diluent.

2. The medicament of claim 1 , wherein synthetic human relaxin-2 is dissolved in 0.5 to 1 .5 ml aqua bidest.

3. The medicament of claim 1 , wherein synthetic human relaxin-2 is dissolved in 0.5 to 1 .5 ml aqueous sodium acetate pH 5.0.

4. The medicament of any preceding claim, wherein the bolus comprises a complex of zinc and human relaxin-2.

5. The medicament of any preceding claim, wherein the bolus comprises a hexamer complex composed of Zn²⁺ ions and human relaxin-2.

6. The medicament of any preceding claim, wherein the bolus comprises as adjuvant a carbohydrate, preferably selected from mannitol, dextrin, B-dextrin, to adapt osmolality and solubility.

7. The medicament of any preceding claim, wherein the bolus is contained in an injection pen designed for ready-to-use subcutaneous injection.

8. The medicament of any preceding claim, which is contained in a set of pens intended for daily (q.d.) or twice daily (b.i.d.) subcutaneous injections for a period of at least one week, preferably three to twelve months.

9. The medicament of any preceding claim wherein the set of pens is intended for treating heart failure with preserved ejection fraction for a period of at least three months.

10. The medicament of any preceding claim wherein the set of pens is intended for the treatment of a patient suffering from heart failure with preserved ejection fraction for a period of six months or more. 38

11. The medicament of any preceding claim wherein the set of pens is intended for the treatment of a patient suffering from diabetes and heart failure with preserved injection fraction.

12. The medicament of any preceding claim wherein the set of pens is intended for the treatment of a patient suffering from atrial fibrillation and heart failure with preserved injection fraction.

13. The medicament of any preceding claim wherein the set of pens is intended for the treatment of a patient suffering from ischemic heart disease.

14. The medicament of any preceding claim wherein the set of pens is intended for adjunctive medical therapy to coronary angioplasty.

15. The medicament of any preceding claim intended for the protective effects of synthetic human relaxin-2 associated with ischemic heart disease, including the reduction of inflammatory leukocyte and platelet responses, inhibition of the release of proinflammatory and arrhythmogenic mediators by leukocytes and mast cells and other harmful substances generated locally at reperfusion by inflammation, oxidative stress, and necrosis.

16. The medicament of any preceding claim intended for treating a patient requiring a chronic dampening of the innate immune system, of inflammatory responses triggered by the innate immune system and/or suppression through a ligand-activated glucocorticoid receptor.

17. The medicament of any preceding claim intended for the treatment of a patient suspected of having inflammatory responses triggered by the innate immune system and having 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).

18. The medicament of any preceding claim wherein the set of pens is intended for the treatment of a patient who has received an allotransplant and needs a chronic dampening of the innate immune system and inflammatory responses while preventing compromised wound healing, manifestation, or deregulation of diabetes or symptoms of a Cushing’s syndrome.

19. The medicament of any preceding claim intended for the treatment of a transplant patient when one or more of the following four criteria are fulfilled: serum HMGB1 (high- mobility group box protein) greater or equal to 2 ng/ml, serum sTLR4 (soluble Toll-like receptor-4) greater or equal to 0.25 ng/ml, serum sRAGE (soluble receptor of advanced glycation end-products) greater or equal to 0.5 ng/ml, and/or serum calprotectin greater or equal to 4 micrograms/ml.

20. The medicament of any preceding claim intended for the treatment of a patient in need of hormone-refractory cancer therapy, including, but not limited to prostate cancer, breast cancer, or primary cancer therapy through a ligand-activated GR, including, but not limited to Multiple Myeloma, Hodgkin’s Disease, and other Lymphoid Cancers; Kaposi Sarcoma, the synthetic human relaxin-2 being used as supplement and substitute of the glucocorticoid receptor activating hormone.

21 . The medicament of any preceding claim intended for treating a patient suffering from forms of autoimmune or rheumatic disease; ankylosing spondylitis (AS) and spondylarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune disease, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory types of arthritis, rheumatoid arthritis (RA).

22. The medicament of any preceding claim intended for treating a patient suffering from SIRS (systemic inflammatory response syndrome), autoimmune or rheumatic diseases, thyroiditis, gastritis, insulitis, sialoadenitis, adrenalitis, oophoritis, glomerulonephritis, polyarthritis, ankylosing spondylitis (AS) and spondylarthritis, fibromyalgia, gout, infectious arthritis, lupus, systemic autoimmune disease, osteoarthritis (OA), psoriatic arthritis (PsA) and inflammatory types of arthritis, rheumatoid arthritis (RA), SARS-Covid 19 und SARS.

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