WO2019081456A1

WO2019081456A1 - Use of activators and stimulators of sgc comprising a beta2 subunit

Info

Publication number: WO2019081456A1
Application number: PCT/EP2018/078950
Authority: WO
Inventors: Peter Sandner; Tibor SCHOMBER; Elke Hartmann; Agnes Benardeau; Rene Hoet; Jan Robert KRÄHLING; Mira PAVKOVIC; Josef Johannes AUBURGER; Ilka MATHAR; Ana Lúcia FREITAS DE MESQUITA BARBA
Original assignee: Bayer Aktiengesellschaft
Priority date: 2017-10-24
Filing date: 2018-10-23
Publication date: 2019-05-02

Abstract

The present invention relates to the use of activators and stimulators of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit for use in the treatment and/or prevention of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit. The sGC activators and sGC stimulators according to the invention are antibodies, aptamers, or small molecules. The invention also provides assays and screening technologies to identify such sGC activators and sGC stimulators.

Description

Use of activators and stimulators of sGC comprising a beta2 subunit

Background of the invention The Nitric Oxide (NO), cyclic guanosine monophosphate (cGMP) pathway (NO/cGMP pathway) is of paramount importance for the regulation of cell, tissue and organ function and plays a major role in health and diseases. It is well established that the NO/cGMP pathway plays a critical role in diseases, including heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary diseases, such as heart failure, chronic and acute kidney disease, and pulmonary hypertension. This is confirmed by genetic evidence, e.g. from genome wide association studies (GWAS) which showed strong correlation of genetic alterations in this pathway with a variety of diseases.

In short, the pathway is as follows (see also Fig. 1):

1. NO is formed from L-Arginine (L-Arg), e.g. caused by endothelial shear stress, catalyzed by NO synthases

2. NO diffuses into the cell, and binds to the heme moiety of the beta subunit of the soluble

Guanylyl Cyclase (sGC)

3. NO-binding to the sGC activates the enzyme, which then catalyzes the formation of cGMP out of GTP

4. cGMP acts as 2^nd messenger on multiple downstream targets, like cGMP -regulated proteinkinases (PKGs, cGK-I/cGK-II), cGMP -regulated ion channel and cGMP -regulated phosphodiesterases (PDEs) and further downstream targets which are phosphorylated and/or dephosphorylated

5. cGMP is hydrolyzed to inactive GMP by phosphodiesterases (PDEs) terminating NO/cGMP signaling

Since NO/cGMP plays a critical role in cell, tissue and body homeostasis, a decrease of cGMP levels can have pathophysiological consequences. Therapeutic approaches addressing this condition encompass • administration of Nitrates or NO donors, e.g., in the treatment of angina pectoris. The respective agents release NO enzymatically or non-enzymatically, which binds to sGC and activates the latter, leading to an increased cGMP production. This approach has some shortcomings, like radical formation, development of tachyphylaxia, and kinetic limitations.

· administration of PDE inhibitors, like Sildenafil, Vardenafil or Tadalafil. These agents have been used in the treatment of erectile dysfunction (ED), pulmonary arterial hypertension (PAH) and to treat signs and symptoms of benign prostatic hyperplasia (BPH). This approach has some shortcomings, too, like the demand of a sufficiently high NO production and high endogenous cGMP levels, which frequently are low in patients suffering from ED, PAH, or BPH. To overcome said limitations, attempts have been made to directly stimulate or activate the sGC with a suitable agent. This approach has the advantages that it is NO-independent, that there is no radical formation, and that it is not dependent on a sufficiently high cGMP level in the patient. sGC is a heterodimer composed of one alpha and one heme-containing beta subunit. The beta subunit consists of four domains: an N-terminal HNOX domain, a PAS-like domain, a coiled-coil domain, and a C-terminal catalytic domain. The HNOX domain of the beta subunit contains a heme moiety with a Fe(II), which is the target of NO. Upon NO-binding, there is an increase in sGC activity, and cGMP is formed.

The HNOX (Heme Nitric oxide/OXygen binding) domain of the beta subunit of sGC contains the prosthetic heme group, and is part of a family of related sensor proteins found throughout a wide range of organisms. The HNOX domain uses the bound heme to sense gaseous ligands such as NO.

It is well accepted, that sGC stimulators act via direct stimulation of the sGC which does not require NO but requires the prosthetic heme-group. Therefore, this compound class of sGC stimulators is defined as NO-independent but heme-dependent sGC stimulators. sGC stimulators bind to the alpha subunit of the non-oxidized and heme-containing sGC (alphal/betal), also termed wild type sGC which leads to NO- independent formation and increase of intracellular cGMP [Stasch JP et al. 2001 ; Stasch and Hobbs 2009]. In addition, sGC stimulators enhance the NO-effect on cGMP when NO is bound to the sGC. Therefore, sGC stimulators also exhibit synergistic effects with NO on cGMP production. The indazole derivative YC-1 was the first NO-independent but heme-dependent sGC stimulator described [Evgenov et al., 2006]. Based on YC-1, further substances were discovered which are more potent than YC-1 and show no relevant inhibition of phosphodiesterases (PDE). This led to the identification of the pyrazolopyridine derivatives BAY 41-2272, BAY 41-8543 and Riociguat [Evgenov et al., 2006]. More recently other compound classes were discovered which show different pharmacokinetics and also different organ distribution which might have an impact on their treatment potential [Follmann M et al., 2017]. The exact binding site of the sGC stimulators at the wild type sGC is still being debated. If the heme group is removed from the sGC, the enzyme still has a detectable catalytic basal activity, i.e. cGMP is still being formed. The remaining catalytic basal activity of the heme-free enzyme cannot be stimulated by any of the stimulators mentioned above and can also not be stimulated by NO [Evgenov et al., 2006]. This observation is important since heme-free and oxidized forms of the sGC (alphal/betal), also termed apo sGC, are preferentially present at diseases which are linked to oxidative stress. The current understanding is that under oxidative stress conditions, the Fe²⁺ iron atom of the heme group in the beta- 1 subunit is oxidized to Fe³⁺ which destabilizes the binding of the heme group to the beta-1 subunit and renders the enzyme heme-free. With the discovery of Cinaciguat, a new chemical matter has found which is able to activate heme-free apo sGC. Therefore, Cinaciguat is the prototype of this class of sGC activators and this compound class is defined as NO-independent and heme-independent sGC activators. Common characteristics of these substances are that in combination with NO they only have an additive effect on enzyme activation, and that the activation of the oxidized or heme-free enzyme is markedly higher than that of the heme -containing enzyme [Evgenov et al., 2006; Stasch JP et al., 2002; Stasch JP et al., 2006]. Spectroscopic studies show that Cinaciguat displaces the oxidized heme group in the beta-1 subunit which, as a result of the weakening of the iron-histidine bond, is attached only weakly to the sGC. It has also been shown that the characteristic sGC heme binding motif Tyr-x-Ser-x-Arg is absolutely essential both for the interaction of the negatively charged propionic acids of the heme group and for the action of Cinaciguat. Therefore, it is assumed that the binding site of Cinaciguat at the sGC is identical to the binding site of the heme group in the beta-1 subunit. [Stasch JP et al., 2006]. More recently other classes of sGC activators have been discovered which are different in pharmacokinetics but also in organ distribution which might have an impact on their treatment potential.

Currently, sGC stimulators are investigated in preclinical research, are in clinical development, or even approved. The most prominent ones are the sGC stimulators Riociguat and Vericiguat. sGC activators have hence a huge potential for treating various diseases and disorders, especially related to NO/cGMP shortage.

It is hence an object of the present invention to provide therapeutic approaches that further extend the therapeutic potential of sGC activators.

It is another object of the invention to provide sGC activator-based therapies for indications that so far could not be treated adequately.

It is another object of the invention to provide assays and screening technologies to identify such sGC activators targeting the beta2 subunit of the sGC. Summary of the invention

These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.

Embodiments of the invention Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word "comprising" does not exclude other elements or steps. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

According to one aspect of the present invention, an activator or stimulator of soluble Guanylate Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment and/or prevention of a human or animal subject · suffering from,

• at risk of developing, and/or

• being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit.

According to one aspect of the present invention, an activator of soluble Guanylate Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment and/or prevention of a human or animal subject

• suffering from,

• at risk of developing, and/or

According to one aspect of the present invention, a stimulator of soluble Guanylate Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment and/or prevention of a human or animal subject

• suffering from,

• at risk of developing, and/or

According to one aspect of the present invention, the target tissue which comprises, expresses or overexpresses a sGC comprising a beta2 subunit, is at least one selected from the group consisting of

• cardiac tissue,

· cardiovascular tissue,

• lung tissue and/or

• renal tissue.

According to a further aspect of the invention, an activator or stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject · suffering from, • at risk of developing, and/or

• being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease or the prevention thereof, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue selected from the group consisting of cardiac tissue, cardiovascular tissue, lung tissue and renal tissue, a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer.

According to a further aspect of the invention, an activator or stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject · suffering from,

• at risk of developing, and/or

• being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease or the prevention thereof, wherein the subject overexpresses at least in a particular target tissue selected from the group consisting of cardiac tissue, cardiovascular tissue, lung tissue and renal tissue, a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer.

According to a further aspect of the invention, an activator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject · suffering from,

• at risk of developing, and/or

According to a further aspect of the invention, a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject • suffering from,

• at risk of developing, and/or

The terms Guanylate Cyclase and Guanylyl Cyclase are used synonymously herein. As used herein, the term "overexpress a sGC comprising a beta2 subunit" refers to the level of mRNA encoding for a sGC comprising a beta2 subunit and/or sGC protein comprising a beta2 subunit expressed in cells of a given tissue being elevated in comparison to the levels thereof as measured in normal cells (free from disease) of the same type of tissue, under analogous conditions. Said mRNA and/or protein expression level may be determined by a number of techniques known in the art including, but not limited to, quantitative RT-PCR, western blotting, immunohistochemistry, and suitable derivatives of the above.

The inventors of the present invention have shown that different cardiac, cardiovascular, lung or renal tissues comprise, or express, an sGC variant that comprises a beta2 subunit.

According to a further aspect of the invention, an activator or stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject

• suffering from,

• at risk of developing, and/or

• being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease or the prevention thereof, wherein the subject comprises, expresses or overexpresses a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer in renal tissue.

According to a further aspect of the invention, an activator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject · suffering from, • at risk of developing, and/or

According to a further aspect of the invention, a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject

• suffering from,

• at risk of developing, and/or

• suffering from,

• at risk of developing, and/or

• being diagnosed for a kidney disease or the prevention thereof, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue selected from the group consisting of cardiac tissue, cardiovascular tissue, lung tissue and renal tissue, a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer.

According to a further aspect of the invention, an activator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, for use in the treatment of a human or animal subject

• suffering from,

• at risk of developing, and/or • being diagnosed for a kidney disease or the prevention thereof, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue selected from the group consisting of cardiac tissue, cardiovascular tissue, lung tissue and renal tissue, a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer.

• suffering from,

• at risk of developing, and/or

• suffering from,

• at risk of developing, and/or

• being diagnosed for a kidney disease or the prevention thereof, wherein the subject comprises, expresses or overexpresses a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer in renal tissue.

• suffering from,

• at risk of developing, and/or • being diagnosed for a kidney disease or the prevention thereof, wherein the subject comprises, expresses or overexpresses a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer in renal tissue.

• suffering from,

• at risk of developing, and/or

· being diagnosed for a kidney disease or the prevention thereof, wherein the subject comprises, expresses or overexpresses a sGC comprising a beta2 subunit selected from (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, and (iv) a beta2 monomer in renal tissue.

As discussed above, the alpha and beta subunits of sGC are each comprised of four modular domains: the N-terminal heme-NO/0₂-binding (H-NOX), the Per/Arnt/Sim (PAS), the helical, and the C-terminal catalytic domain. The betal subunit comprises, in the H-NOX domain, a heme group. The beta2 subunit contains a frameshift, due to which the formation of an intact beta-subunit with a complete heme- binding domain is impaired. If such beta2 subunit dimerizes with an alphal or alpha2 subunit (or with another beta2 subunit forming a homodimer), the resulting sGC enzyme cannot adequately bind NO, resulting in reduced or impaired cGMP formation [Gupta et al., 1997; Behrends and Vehse 2000]. Without being bound to theory, the sGC activity in these tissues could be limited by the presence or expression of an sGC variant that comprises a beta2 subunit. The beta2 subunit can be upregulated during disease progression in cardiac, cardiovascular, lung, or renal or urological tissues but also in other tissues and organs. This can be seen in rat disease models and human patients. Upregulation of sGC beta2 subunit expression is detected in renal tissue in uninephrectomized rats compared to control rats (Table 2). Expression levels of sGC beta2 subunit in kidneys of ZSF-1 rats are higher compared to ZSF-1 lean/control (Table 3). Patients suffering from diabetic kidney disease (DKD) show a significant upregulation of beta2 subunit in the distal nephron and proximal tubules of the kidney (Fig. 2 and 3). As used herein, the heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease includes at least one of the indications discussed in the following.

The compounds according to the invention can therefore be used in medicaments for the treatment and/or prophylaxis of cardiovascular diseases, for example high blood pressure (hypertension), resistant hypertension, acute and chronic heart failure, coronary heart disease, stable and unstable angina pectoris, peripheral and cardiac vascular diseases, arrhythmias, disturbances of atrial and ventricular rhythm and conduction disturbances, for example atrioventricular blocks of degree I-III (AVB I-III), supraventricular tachyarrhythmia, atrial fibrillation, atrial flutter, ventricular fibrillation, ventricular flutter, ventricular tachyarrhythmia, torsade-de-pointes tachycardia, atrial and ventricular extrasystoles, AV-junction extrasystoles, sick-sinus syndrome, syncopes, AV-node reentry tachycardia, Wolff- Parkinson- White syndrome, acute coronary syndrome (ACS), autoimmune heart diseases (pericarditis, endocarditis, valvulitis, aortitis, cardiomyopathies), shock such as cardiogenic shock, septic shock and anaphylactic shock, aneurysms, Boxer cardiomyopathy (premature ventricular contraction (PVC)), for the treatment and/or prophylaxis of thromboembolic diseases and ischaemias such as myocardial ischaemia, myocardial infarction, stroke, cardiac hypertrophy, transient ischaemic attacks, preeclampsia, inflammatory cardiovascular diseases, spasms of the coronary arteries and peripheral arteries, development of oedema, for example pulmonary oedema, cerebral oedema, renal oedema or oedema due to heart failure, peripheral perfusion disturbances, reperfusion injury, arterial and venous thromboses, microalbuminuria, myocardial insufficiency, endothelial dysfunction, for preventing restenoses such as after thrombolysis therapies, percutaneous transluminal angioplasty (PTA), transluminal coronary angioplasty (PTCA), heart transplant and bypass operations, and micro- and macrovascular damage (vasculitis), increased level of fibrinogen and of low-density LDL and increased concentrations of plasminogen activator inhibitor 1 (PAI-1), and for the treatment and/or prophylaxis of erectile dysfunction and female sexual dysfunction. In the sense of the present invention, the term heart failure comprises both acute and chronic manifestations of heart failure, as well as more specific or related forms of disease such as acute decompensated heart failure, right ventricular failure, left ventricular failure, total heart failure, heart failure with reduced ejection fraction (HFrEF), heart failure with preserved ejection fraction (HFpEF) ischaemic cardiomyopathy, dilatated cardiomyopathy, hypertrophic cardiomyopathy, idiopathic cardiomyopathy, congenital heart defects, heart failure with valvular defects, mitral valve stenosis, mitral valve insufficiency, aortic valve stenosis, aortic valve insufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonary valve stenosis, pulmonary valve insufficiency, combined valvular defects, heart muscle inflammation (myocarditis), chronic myocarditis, acute myocarditis, viral myocarditis, diabetic heart failure, alcoholic cardiomyopathy, storage cardiomyopathies, diastolic heart failure and also systolic heart failure and acute phases of an existing chronic heart failure (worsening heart failure). In addition, the compounds according to the invention can also be used in medicaments for the treatment and/or prophylaxis of arteriosclerosis, disturbances of lipid metabolism, hypolipoproteinaemias, dyslipidaemias, hypertriglyceridaemias, hyperlipidaemias, hypercholesterolaemias, abetalipoproteinaemia, sitosterolaemia, xanthomatosis, Tangier disease, adiposity, obesity, and combined hyperlipidaemias and metabolic syndrome.

Moreover, the compounds according to the invention can be used in medicaments for the treatment and/or prophylaxis of primary and secondary Raynaud phenomenon, microcirculation disturbances, claudication, peripheral and autonomic neuropathies, diabetic microangiopathies, diabetic retinopathy, diabetic limb ulcers, gangrene, CREST syndrome, erythematous disorders, onychomycosis, rheumatic diseases and for promoting wound healing.

In addition, the compounds according to the invention can be used in medicaments for preventing ischaemia- and/or reperfusion-related damage to organs or tissues and also as additives for perfusion and preservation solutions of organs, organ parts, tissues or tissue parts of human or animal origin, in particular for surgical interventions or in the field of transplantation medicine. Furthermore, the compounds according to the invention can be used in medicaments for the treatment and/or prophylaxis of kidney diseases, in particular acute and chronic renal insufficiency, and acute and chronic renal failure. In the sense of the present invention, the term renal insufficiency comprises both acute and chronic manifestations of renal insufficiency, as well as underlying or related kidney diseases such as renal hypoperfusion, intradialytic hypotension, obstructive uropathy, glomerulopathies, glomerulonephritis, acute glomerulonephritis, glomerulosclerosis, tubulointerstitial diseases, nephropathic diseases such as primary and congenital kidney disease, nephritis, immunological kidney diseases such as kidney transplant rejection, immune complex-induced kidney diseases, nephropathy induced by toxic substances, contrast medium-induced nephropathy, diabetic and non-diabetic nephropathy, pyelonephritis, renal cysts, nephrosclerosis, hypertensive nephrosclerosis and nephrotic syndrome, which can be characterized diagnostically for example by abnormally reduced creatinine and/or water excretion, abnormally increased blood concentrations of urea, nitrogen, potassium and/or creatinine, altered activity of renal enzymes such as e.g. glutamylsynthetase, altered urine osmolarity or urine volume, increased microalbuminuria, macroalbuminuria, lesions of glomeruli and arterioles, tubular dilatation, hyperphosphataemia and/or need for dialysis. The present invention also comprises the use of the compounds according to the invention for the treatment and/or prophylaxis of sequelae of renal insufficiency, for example pulmonary oedema, heart failure, uraemia, anaemia, electrolyte disturbances (e.g. hyperkalaemia, hyponatraemia) and disturbances in bone and carbohydrate metabolism. Furthermore, the compounds according to the invention can be used in medicaments for the treatment and/or prophylaxis of asthmatic diseases, pulmonary arterial hypertension (PAH) and other forms of pulmonary hypertension (PH), comprising pulmonary hypertension associated with left ventricular disease, HIV, sickle cell anaemia, thromboembolism (CTEPH), sarcoidosis, COPD or pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI), alpha- 1 -antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonary emphysema (e.g. smoking-induced pulmonary emphysema) and cystic fibrosis (CF).

In one embodiment of the invention, said heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease is characterized by presence and/or upregulation of the expression of the beta2 subunit of sGC.

According to an embodiment of the present invention, preferred sGC stimulators for use according to the invention are:

• 2-[l-(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5-(4-morpholinyl)-4,6- pyrimidinediamine

• 2-[l-(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5-(4-pyridinyl)-4-pyrimidineamine

• methyl 4,6-diamino-2-[l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5- pyrimidinyl(methyl)carbamate (Riociguat)

• methyl 4,6-diamino-2-[l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5- pyrimidinylcarbamate (Nelociguat)

• methyl {4,6-diamino-2-[5-fluoro-l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridine-3- yl]pyrimidin-5-yl}carbamate (Vericiguat)

• methyl {4,6-diamino-2-[5-fluoro-l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridine-3- yl]pyrimidin-5 -yl} methylcarbamate

• methyl {4,6-diamino-2-[5-fluoro-l-(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridin-3- yl]pyrimidin-5-yl}(2,2,2-trifluoroethyl)carbamate

• 4-amino-2-[5-chloro-3(3,3,3-trifluoropropyl)-lH-indazol-l-yl]-5,5-dimethyl-5,7-dihydro- 6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-2[5-chloro-3-(2,3,6-trifluorobenzyl)-lH-indazol-l-yl]-5,5-dimethyl-5,7-dihydro- 6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-5,5-dimethyl-2-[3-(2,3,6-trifluorobenzyl)lH-thieno[3,4-c]pyrazol-l-yl]-5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-5,5-dimethyl-2-[3-(2,3,6-trifluorobenzyl)-lH-thieno[2,3-d]pyrazol-l-yl]-5,5- dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-5,5-dimethyl-2-[7-(2,3,6-trifluorobenzyl)imidazo[l,5-b]pyridazin-5-yl]-5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-2-[6-chloro-3-(2,3,6-trifluorobenzyl)imidazo[l,5-a]pyridin-l-yl]] -5,5-dimethyl- 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-2-[6-fluoro-3-(2,3,6-trifluorobenzyl)imidazo[l,5-a]pyridin-l-yl]] -5,5-dimethyl- 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one • 4-amino-2-[6-fluoro-3-(2,3,6 rifluorobenzyl)6-fluoroimidazo[l,5-a]pyridin-l-yl]-5,5- dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-5,5-dimethyl-2-[3-(2,4,6 rifluorobenzyl)imidazo[l,5-a]pyridin-l -yl]] -5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-2-[3-(2-cyclopentylethyl)imidazo[l,5-a]pyridin-l -yl]-5,5-dimethyl-5,7-dihydro- 6H-pyrrolo[2,3-d]pyrimidin-6-one

• 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-l -(2-fluorobenzyl)-lH-pyrazolo[3,4-^]pyridine

• 2-{5-fluoro-l-[(3-fluoropyridin-2-yl)methyl]-lH-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5- (trifluoromethyl)-4-[(3,3,3 rifluoropropyl)amino]-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin- 6-one

• en^N-[(2S)-amino-2-methylbutyl]-8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[l,2- a]pyridine-3-carboxamide (enantiomer A)

• en N-(2-amino-2-methylbutyl)-8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[l,2- a]pyridine-3-carboxamide (enantiomer B)

• eni-N-(2-amino-5,5,5-trifluoro-2-methylpentyl)-2,6-dimethyl-8-[(2,3,6- trifluorobenzyl)oxy]imidazo[l,2-a]pyridine-3-carboxamide (enantiomer B)

• eni-N-(2-amino-5,5,5-trifluoro-2-methylpentyl)-8-[(2,6-difluorobenzyl)oxy]-2,6- dimethylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer B)

• eni-N-(2-amino-5,5,5-trifluoro-2-methylpentyl)-8-[(2,6-difluorobenzyl)oxy]-2,6- dimethylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer A)

• eni-N-(2-amino-3-fluoro-2-methylpropyl)-2,6-dimethyl-8-[(2,3,6- trifluorobenzyl)oxy]imidazo[l ,2-a]pyridine-3-carboxamide (enantiomer B)

• eni-N-(2-amino-3-fluoro-2-methylpropyl)-8-[(2,6-difluorobenzyl)oxy]-2,6- dimethylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer B)

• eni-N-(2-amino-3-fluoro-2-methylpropyl)-8-[(2,6-difluorobenzyl)oxy]-2,6- dimethylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer A)

• rac-N-(2-amino-3-fluoro-2-methylpropyl)-8-[(2,6-difluorobenzyl)oxy]-2,6- dimethylimidazo[l ,2-a]pyridine-3-carboxamide formate

• eni-N-(2-amino-3-fluoro-2-methylpropyl)-2,6-dimethyl-8-[(2,3,6- trifluorobenzyl)oxy]imidazo[l,2-a]pyridine-3-carboxamide (enantiomer A)

• eni-N-(2-amino-3-fluoro-2-methylpropyl)-8-[(2,6-difluorobenzyl)oxy]-6-(difluoromethyl)- 2-methylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer B)

• eni-N-(2-amino-3-fluoro-2-methylpropyl)-8-[(2,6-difluorobenzyl)oxy]-6-(difluoromethyl)-

2- methylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer A)

• eni-N-(2-amino-3-fluoro-2-methylpropyl)-8-[(2,6-difluorobenzyl)oxy]-6-(fluoromethyl)-2- methylimidazo [ 1 ,2-a]pyridine-3 -carboxamide

• l,l,l,3,3,3-Hexafluoro-2-[( {5-fluoro-2-[l -(2-fluorobenzyl)-5-(l,2-oxazol-3-yl)-lH-pyrazol-

3- yl]-4-pyrimidinyl} amino)methyl]-2-propanol (Praliciguat)

• 5-fluoro-2-[l -(2-fluorobenzyl)-5-(l,2-oxazol-3-yl)-lH-pyrazol-3-yl]pyrimidin-4-ol (IWP- 051)

• IWP-121, IWP-427, IWP-953, IW-1701, IW-6463

According to a further embodiment of the present invention, preferred sGC stimulators for use according to the invention are: • 2-[l-(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5-(4-morpholinyl)-4,6- pyrimidinediamine

• 4-amino-5,5-dimethyl-2-[3-(2,3,6-trifluorobenzyl)lH-thieno[3,4-c]pyrazol-l -yl]-5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-2-[6-chloro-3-(2,3,6-trifluorobenzyl)imidazo[l,5-a]pyridin-l -yl]] -5,5-dimethyl- 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-2-[6-fluoro-3-(2,3,6-trifluorobenzyl)imidazo[l,5-a]pyridin-l-yl]] -5,5-dimethyl- 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-2-[6-fluoro-3-(2,3,6-trifluorobenzyl)6-fluoroimidazo[l,5-a]pyridin-l-yl]-5,5- dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 4-amino-5,5-dimethyl-2-[3-(2,4,6-trifluorobenzyl)imidazo[l,5-a]pyridin-l -yl]] -5,7- dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• eni-N-[(2S)-amino-2-methylbutyl]-8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[l,2- a]pyridine-3-carboxamide (enantiomer A)

• eni-N-(2-amino-2-methylbutyl)-8-[(2,6-difluorobenzyl)oxy]-2,6-dimethylimidazo[l,2- a]pyridine-3-carboxamide (enantiomer B)

• eni-N-(2-amino-5,5,5-trifluoro-2-methylpentyl)-8-[(2,6-difluorobenzyl)oxy]-2,6- dimethylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer B) • eni-N-(2-amino-5,5,5-trifluoro-2-methylpentyl)-8-[(2,6-difluorobenzyl)oxy]-2,6- dimethylimidazo[l,2-a]pyridine-3-carboxamide (enantiomer A)

• l,l,l,3,3,3-Hexafluoro-2-[( {5-fluoro-2-[l -(2-fluorobenzyl)-5-(l,2-oxazol-3-yl)-lH-pyrazol- 3-yl]-4-pyrimidinyl}amino)methyl]-2-propanol (Praliciguat)

According to a further embodiment of the present invention, preferred sGC stimulators for use according to the invention are:

• 4-amino-2-[6-fluoro-3-(2,3,6 rifluorobenzyl)6-fluoroimidazo[l,5-a]pyridin-l-yl]-5,5- dimethyl-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one

• 2-{5-fluoro-l-[(3-fluoropyridin-2-yl)methyl]-lH-pyrazolo[3,4-b]pyridin-3-yl}-5-methyl-5- (trifluoromethyl)-4-[(3,3,3-trifluoropropyl)amino]-5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin- 6-one

• l,l,l,3,3,3-Hexafluoro-2-[( {5-fluoro-2-[l -(2-fluorobenzyl)-5-(l,2-oxazol-3-yl)-lH-pyrazol- 3-yl]-4-pyrimidinyl} amino)methyl]-2-propanol (Praliciguat)

• methyl 4,6-diamino-2-[l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridin-3-yl]-5- pyrimidinylcarbamate (Nelociguat) • methyl {4,6-diamino-2-[5-fluoro-l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridine-3- yl]pyrimidin-5-yl}carbamate (Vericiguat)

• methyl {4,6-diamino-2-[5-fluoro-l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridine-3- yl]pyrimidin-5-yl}carbamate (Vericiguat) • methyl {4,6-diamino-2-[5-fluoro-l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridine-3- yl]pyrimidin-5 -yl} methylcarbamate

According to a further embodiment of the present invention, the preferred sGC stimulator for use according to the invention is:

It is hence an object of the present invention to provide therapeutic approaches that further extend the therapeutic potential of sGC stimulators.

It is another object of the invention to provide sGC stimulator-based therapies for indications that so far could not be treated adequately. It is another object of the invention to provide assays and screening technologies to identify such sGC stimulators targeting the beta2 subunit of the sGC.

According to an embodiment of the present invention, preferred sGC activators for use according to the invention are:

• 4-( {(4-carboxybutyl)[2-(2- {[4-(2- phenylethyl)benzyl] oxy } phenyl)ethyl] amino } methyl)benzoic acid

• 5-chloro-2-(5-chlorothiophene-2-sulfonylamino-N-(4-(morpholine-4- sulfonyl)phenyl)benzamide as sodium salt

• 2-(4-chlorophenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4- sulfonyl)phenyl)benzamide

• 1 - {6-[5-chloro-2-( {4-trans-4-}trifluoromethyl)cyclohexyl]benzyl} oxy)phenyl]pyridin-2- yl} -5-(trifluoromethyl)-lH-pyrazole-4-carboxylic acid

• l-[6-(2-(2-methyl-4-(4-trifluoromethoxyphenyl)benzyloxy)phenyl)pyridin-2-yl]-5- trifluoromethylpyrazole-4-carboxylic acid

• 1 [6-(3,4-dichlorophenyl)-2-pyridinyl-5-(trifluoromethyl)-lH-pyrazole-4-carboxylic acid

• l-( {2-[3-chloro-5-(trifluoromethyl)phenyl]-5-methyl-l ,3-thiazol-4-yl}methyl)-lH-pyrazole- 4-carboxylic acid

• 4-( {2- [3 -(trifluoromethyl)phenyl] - 1 ,3 -thiazol-4-yl} methyl)benzoic acid

• l-( {2-[2-fluoro-3-(trifluoromethyl)phenyl]-5-methyl-l ,3-thiazol-4-yl}methyl)-lH-pyrazole- 4-carboxylic acid

• 3-(4-chloro-3-{[(2S,3R)-2-(4-chlorophenyl)-4,4,4-trifluoro-3- methylbutanoyl] amino } phenyl)-3 -cyclopropylpropanoic acid

• 5- { [2-(4-carboxyphenyl)ethyl] [2-(2- { [3 -chloro-4'-(trifluoromethyl)biphenyl-4- yl]methoxy}phenyl)ethyl]amino} -5,6,7,8-tetrahydroquinoline-2-carboxylic acid

• 5-{(4-carboxybutyl)[2-(2- {[3-chloro-4'-(trifluoromethyl)biphenyl-4- yl]methoxy}phenyl)ethyl]amino} -5,6,7,8-tetrahydroquinoline-2-carboxylic acid

• (lR,5S)-3-[4-(5-methyl-2-{[2-methyl-4-(piperidin-l-ylcarbonyl)benzyl]oxy}phenyl)-l,3- thiazol-2-yl]-3-azabicyclo[3.2.1]octane-8-carboxylic acid

• l-[6-(5-methyl-2- {[2-(tetrahydro-2H-pyran-4-yl)-l,2,3,4-tetrahydroisoquinolin-6- yl]methoxy}phenyl)pyridin-2-yl]-5-(trifluoromethyl)-lH-pyrazole-4-carboxylic acid According to a further embodiment of the present invention, preferred sGC activators for use according to the invention are:

According to a further embodiment of the present invention, the preferred sGC activator for use according to the invention is:

• 3-(4-chloro-3-{[(2S,3R)-2-(4-chlorophenyl)-4,4,4-trifluoro-3- methylbutanoyl] amino } phenyl)-3 -cyclopropylpropanoic acid Further sGC activators in the context of the invention are known from the following publications:

WO2013/157528, WO2015/056663, WO2009/123316, WO2016/001875, WO2016/001876,

WO2016/001878, WO2000/02851, WO2012/122340, WO2013/025425, WO2014/039434,

WO2016/014463, WO2009/068652, WO2009/071504, WO2010/015652, WO2010/015653,

WO2015/033307, WO2016/042536, WO2009/032249, WO2010/099054, WO2012/058132,

US2010/0216764, WO2001/19776, WO2001/19780, WO2001/19778, WO2002/070459,

WO2002/070460, WO2002/070510, WO2002/070462, WO2007/045366, WO2007/045369,

WO2007/045433, WO2007/045370, WO2007/045367, WO2014/012935, WO2014/012934,

WO2011/141409, WO2008/119457, WO2008/119458, WO2009/127338, WO2010/102717,

WO2011/051165, WO2012/076466, WO2012/139888, WO2013/157528, WO2013/174736,

WO2014/012934, WO2015/056663, WO2017103888, WO2017112617, WO2016042536,

WO2016081668, WO2016191335, WO2016191334, WO2016001875, WO2016001876,

WO2016001878, WO2016014463, WO2016044447, WO2016044445, WO2016044446,

WO2015056663, WO2015033307, WO2015187470, WO2015088885, WO2015088886,

WO2015089182, WO2014084312, WO2014039434, WO2014144100, WO2014047111,

WO2014047325, WO2013025425, WO2013101830, WO2012165399, WO2012058132,

WO2012122340, WO2012003405, WO2012064559, WO2011149921, WO2011119518,

WO2011115804, WO2011056511, CN101670106, TW201028152, WO2010015653, WO2010015652, WO2010099054, WO2010065275, WO2009123316, WO2009068652, WO2009071504, WO2009032249, US2009209556.

As used herein, the term "stimulator" of soluble Guanylyl Cyclase (sGC) relates to an active compound that interacts with a reduced, heme containing sGC, to stimulate the latter to catalyze the formation of cGMP.

As used herein, the term "stimulation" is to be understood as increasing the measured production of cGMP by at least 5% as compared to a control, e.g., a non-treated control, preferably by at least 10%, more preferably by at least 15%, even more preferably by at least 20%, even more preferably by at least 25%, even more preferably by at least 30%> or by at least 40%> or by at least 50%>. Suitable controls are evident for the skilled person when considering the teaching of the present disclosure. Suitable assays to determine said stimulation are readily available to the skilled person from the pertinent literature. In one embodiment of the invention, the assays referred to herein below are being used to determine said stimulation.

As used herein, the term "activator" of soluble Guanylyl Cyclase (sGC) relates to an active compound that interacts with an oxidized or heme-free sGC (apo sGC) to activate the latter to catalyze the formation of cGMP.

As used herein, the term "activation" is to be understood as increasing the measured production of cGMP by at least 5%> as compared to a control, e.g., a non-treated control, preferably by at least 10%>, more preferably by at least 15%, even more preferably by at least 20%, even more preferably by at least 25%), even more preferably by at least 30%> or by at least 40%> or by at least 50%>. Suitable controls are evident for the skilled person when considering the teaching of the present disclosure. Suitable assays to determine said activation are readily available to the skilled person from the pertinent literature. In one embodiment of the invention, the assays referred to herein below are being used to determine said activation. This test distinguishes between sGC activators and sGC stimulators as follows: when the sGC was treated with the sGC inhibitor ODQ or when heme was removed by adding Tween, the sGC can still be activated by sGC activators. In contrast, sGC stimulators do not show any activity on sGC treated in such a way. For distinguishing between sGC activators and sGC stimulators test A-l can be used.

In one embodiment of the invention, the activator or stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is for use in the manufacture of a medicament for treatment and/or prevention of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a cardiac, cardiovascular, renal or cardiorenal disease, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit. In one embodiment of the invention, the activator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is for use in the manufacture of a medicament for treatment and/or prevention of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a cardiac, cardiovascular, renal or cardiorenal disease, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit.

In one embodiment of the invention, the stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is for use in the manufacture of a medicament for treatment and/or prevention of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a cardiac, cardiovascular, renal or cardiorenal disease, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit.

In another embodiment, a method of treatment and/or prevention of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease, is provided, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit. Said method of treatment and/or prevention comprises administration, in a sufficient amount or dosage, of an activator or a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit.

In another embodiment, a method of treatment and/or prevention of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease, is provided, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit. Said method of treatment and/or prevention comprises administration, in a sufficient amount or dosage, of an activator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit.

In another embodiment, a method of treatment and/or prevention of a human or animal subject suffering from, at risk of developing, and/or being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease, is provided, wherein the subject comprises, expresses or overexpresses at least in a particular target tissue, a sGC comprising a beta2 subunit. Said method of treatment and/or prevention comprises administration, in a sufficient amount or dosage, of a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit.

According to one embodiment of the invention, the sGC is (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, or (iv) a beta2 monomer.

According to one embodiment of the invention, the sGC is (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, or (iii) a beta2/beta2 homodimer. According to one further embodiment of the invention, the target tissue which comprises, expresses or overexpresses a sGC comprising a beta2 subunit is at least one selected from the group consisting of

• cardiac tissue,

• cardiovascular tissue,

· lung tissue and/or

• renal tissue.

According to one further embodiment of the invention, the sGC activator or stimulator is a monoclonal antibody, or a fragment or derivative of such antibody retaining target binding capacities.

As used herein, the term "monoclonal antibody", shall refer to an antibody composition having a homogenous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin, or an antigen binding fragment or derivative thereof. Particularly preferred, such antibody is selected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, or a fragment or derivative thereof.

As used herein, the term "fragment" shall refer to fragments of such antibody retaining target binding capacities, e.g.

• a CDR (complementarity determining region),

• a hypervariable region,

• a variable domain (Fv),

• an IgG heavy chain (consisting of VH, CHI, hinge, CH2 and CH3 regions),

· an IgG light chain (consisting of VL and CL regions), and/or

As used herein, the term "derivative" shall refer to protein constructs being structurally different from, but still having some structural relationship to the common antibody concept, e.g., scFv, Fab and/or F(ab)2, as well as bi-, tri- or higher specific antibody constructs or monovalent antibodies, and further retaining target binding capacities. All these items are explained below.

Other antibody derivatives known to the skilled person are Diabodies, Camelid Antibodies, Nanobodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerised constructs comprising CH3+VL+VH, and antibody conjugates (e.g. antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label). These types are well described in literature and can be used by the skilled person on the basis of the present disclosure. As discussed above, soluble Guanylyl Cyclase (sGC) is sufficiently specified to enable a skilled person to make an antibody, such as a monoclonal antibody there against. Routine methods encompass hybridoma, chimerization/humanization, phage display/transgenic mammals, and other antibody engineering technologies. Methods for the production of a hybridoma cell have been previously described (see Kohler and Milstein 1975, incorporated herein by reference). Essentially, e.g., a mouse is immunized with a human soluble Guanylyl Cyclase (sGC) protein, followed by B-cell isolation from said mouse and fusion of the isolated B-cell with a myeloma cell.

Methods for the production and/or selection of chimeric or humanized mAbs are known in the art. Essentially, e.g., the protein sequences from the murine anti sGC antibody which are not involved in target binding are replaced by corresponding human sequences. For example, US6331415 by Genentech describes the production of chimeric antibodies, while US6548640 by Medical Research Council describes CDR grafting techniques and US5859205 by Celltech describes the production of humanised antibodies. All of these disclosures are incorporated herein by reference. Methods for the production and/or selection of fully human mAbs are known in the art. These can involve the use of a transgenic animal which is immunized with human sGC, or the use of a suitable display technique, like yeast display, phage display, B-cell display or ribosome display, where antibodies from a library are screened against human sGC in a stationary phase.

In vitro antibody libraries are, among others, disclosed in US6300064 by MorphoSys and US6248516 by MRC/Scripps/Stratagene. Phage Display techniques are for example disclosed in US5223409 by Dyax. Transgenic mammal platforms are for example described in EP1480515A2 by TaconicArtemis. All of these disclosures are incorporated herein by reference.

IgG, scFv, Fab and/or F(ab)2 are antibody formats well known to the skilled person. Related enabling techniques are available from the respective textbooks. As used herein, the term "Fab" relates to an IgG fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody.

As used herein, the term "F(ab)2" relates to an IgG fragment consisting of two Fab fragments connected to one another by one or more disulfide bonds. As used herein, the term "scFv" relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide.

Modified antibody formats are for example bi- or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates and the like. These types are well described in literature and can be used by the skilled person on the basis of the present disclosure. Furthermore, also monovalent antibodies have been previously described in US 2004/0033561 Al (referred to therein as monobodies) or WO2007048037; both of which are incorporated herein by reference.

Finding a suitable antibody, or fragment or derivative, that is capable of acting as an activator or a stimulator of sGC comprising a beta2 subunit is hence a matter of routine for the skilled person, based on the public availability of the amino acid sequences of sGC. Polyclonal antibodies against the alphal, alpha2, betal, and beta2 subunits of sGC are commercially available for scientific research, e.g., from Abeam, Thermo Fisher, Genetex and the like.

Because sGC is an intracellular protein, the antibody or its fragment or derivative needs to be funneled or trafficked into the intracellular space. Routine technologies are available for this purpose, which are disclosed in Chen et al. (2003), Berguig et al. (2015) and Meunier (2014) incorporated herein by reference.

According to one embodiment of the invention, the sGC activator or sGC stimulator is an aptamer. Aptamers are oligonucleotides that have specific binding properties for a pre -determined target. They are obtained from a randomly synthesized library containing up to 10¹⁵ different sequences through a combinatorial process named SELEX ("Systematic Evolution of Ligands by Exponential enrichment"). Aptamer properties are dictated by their 3D shape, resulting from intramolecular folding, driven by their primary sequence. An aptamer3D structure is exquisitely adapted to the recognition of its cognate target through hydrogen bonding, electrostatic and stacking interactions. Aptamers generally display high affinity (K_d about micromolar (μΜ) for small molecules and picomolar (pM) for proteins). An overview on the technical repertoire to generate target specific aptamers is given, e.g., in Blind and Blank 2015, which is incorporated herein by reference. Aptamers can also be delivered into the intracellular space, as disclosed in Thiel and Giangrande (2010), incorporated herein by reference.

Finding a suitable aptamer that is capable of acting as an activator or a stimulator to sGC comprising a beta2 subunit is hence a matter of routine for the skilled person, based on the public availability of the amino acid sequences of the different sGC variants.

According to one embodiment of the invention, the sGC activator or sGC stimulator is a small molecule. Preferably, said small molecule is an organic molecule, and/or said small molecule has a molecular weight of < 550 Da, preferably < 500 Da, more preferably < 450 Da.

Preferably, the sGC beta2 subunit protein which is stimulated or activated by the antibody, aptamer or small molecule comprises an amino acid sequence as encoded by the nucleic acid according to SEQ ID NO: 1 (GenBank: AF038499.2), or has the amino acid sequence according to SEQ ID NO: 2 (UniProtKB - 075343 (GCYB2 HUMAN). Other potential sGC beta2 subunit proteins that can be used in the present invention, or are contemplated as targets for the stimulation according to the present invention, are disclosed, e.g., in Koglin et al, 2001.

According to one other aspect of the invention, an activator or a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit for use in the manufacture of a medicament in the treatment of a human or animal subject

• suffering from,

• at risk of developing, and/or

• being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease, or for the prevention of such condition, is provided. Therein, the subject exhibits, at least in a particular target tissue, a sGC comprising a beta2 subunit.

According to one other aspect of the invention, an activator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit for use in the manufacture of a medicament in the treatment of a human or animal subject

• suffering from,

• at risk of developing, and/or

According to one other aspect of the invention, a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit for use in the manufacture of a medicament in the treatment of a human or animal subject suffering from,

at risk of developing, and/or • being diagnosed for a heart, kidney, lung, cardiovascular, cardiorenal and cardiopulmonary disease, or for the prevention of such condition, is provided. Therein, the subject exhibits, at least in a particular target tissue, a sGC comprising a beta2 subunit. According to another aspect of the invention, a medicament for use according to the invention is provided comprising a sGC activator or sGC stimulator according to the above description.

According to another aspect of the invention, a combination of a medicament for use according to the invention and one or more other therapeutically active compounds is provided. Preferred examples of suitable active compound combinations include: · organic nitrates and NO donors, for example sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomine or SIN-1, and inhaled NO;

• compounds which inhibit the breakdown of cyclic guanosine monophosphate (cGMP), such as, for example, inhibitors of phosphodiesterases (PDE) 1, 2 and/or 5, in particular PDE 5 inhibitors such as sildenafil, vardenafil and tadalafil; · NO-independent, but heme-dependent stimulators of guanylate cyclase, such as, in particular, riociguat, nelociguat, vericiguat, and the compounds described in WO 00/06568, WO 00/06569, WO 02/42301 and WO 03/095451;

• NO- and heme-independent activators of soluble guanylate cyclase (sGC), such as in particular cinaciguat and also the compounds described in WO 01/19355, WO 01/19776, WO 01/19778, WO 01/19780, WO 02/070462, WO 02/070510, WO 2009/127338, WO 2010/102717, WO

2011/051165, WO 2012/076466, WO 2013/174736, WO 2012/139888, and WO 2014/012934;

• agents having an antithrombotic effect, for example and with preference from the group of platelet aggregation inhibitors, of anticoagulants or of profibrinolytic substances;

• active compounds which lower blood pressure, for example and preferably from the group of calcium antagonists, angiotensin All antagonists, ACE inhibitors, endothelin antagonists, renin inhibitors, alpha-receptor blockers, beta-receptor blockers, mineralocorticoid receptor antagonists and/or diuretics;

• active compounds which alter lipid metabolism, for example and with preference from the group of thyroid receptor agonists, cholesterol synthesis inhibitors such as, by way of example and preferably, HMG-CoA reductase inhibitors or squalene synthesis inhibitors, of ACAT inhibitors, CETP inhibitors, MTP inhibitors, PPAR-alpha, PPAR-gamma and/or PPAR-delta agonists, cholesterol absorption inhibitors, lipase inhibitors, polymeric bile acid adsorbents, bile acid reabsorption inhibitors and lipoprotein(a) antagonists.

Agents having antithrombotic activity preferably mean compounds from the group of platelet aggregation inhibitors, of anticoagulants or of profibrinolytic substances.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a platelet aggregation inhibitor such as, by way of example and preferably, aspirin, clopidogrel, ticlopidin or dipyridamol.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a thrombin inhibitor such as, by way of example and preferably, ximelagatran, melagatran, dabigatran, bivalirudin or clexane.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a GPIIb/IIIa antagonist such as, by way of example and preferably, tirofiban or abciximab. In another embodiment of the invention, the compounds according to the invention are administered in combination with a factor Xa inhibitor such as, by way of example and preferably, rivaroxaban, apixaban, fidexaban, razaxaban, fondaparinux, idraparinux, DU-176b, PMD-3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-1021, DX 9065a, DPC 906, JTV 803, SSR-126512 or SSR-128428.

In another embodiment of the invention, the compounds according to the invention are administered in combination with heparin or a low molecular weight (LMW) heparin derivative.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a vitamin K antagonist such as, by way of example and preferably, coumarin.

Agents which lower blood pressure are preferably understood to mean compounds from the group of calcium antagonists, angiotensin All antagonists, ACE inhibitors, endothelin antagonists, renin inhibitors, alpha-receptor blockers, beta-receptor blockers, mineralocorticoid receptor antagonists, and the diuretics.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a calcium antagonist such as, by way of example and preferably, nifedipine, amlodipine, verapamil or diltiazem.

In another embodiment of the invention, the compounds according to the invention are administered combination with an alpha- 1 receptor blocker such as, by way of example and preferably, prazosin. In another embodiment of the invention, the compounds according to the invention are administered in combination with a beta receptor blocker such as, by way of example and preferably, propranolol, atenolol, timolol, pindolol, alprenolol, oxprenolol, penbutolol, bupranolol, metipranolol, nadolol, mepindolol, carazalol, sotalol, metoprolol, betaxolol, celiprolol, bisoprolol, carteolol, esmolol, labetalol, carvedilol, adaprolol, landiolol, nebivolol, epanolol or bucindolol.

In another embodiment of the invention, the compounds according to the invention are administered in combination with an angiotensin All antagonist such as, by way of example and preferably, losartan, candesartan, valsartan, telmisartan or embusartan.

In another embodiment of the invention, the compounds according to the invention are administered in combination with an ACE inhibitor such as, by way of example and preferably, enalapril, captopril, lisinopril, ramipril, delapril, fosinopril, quinopril, perindopril or trandopril.

In another embodiment of the invention, the compounds according to the invention are administered in combination with an endothelin antagonist such as, by way of example and preferably, bosentan, darusentan, ambrisentan, macitentan or sitaxsentan.

In another embodiment of the invention, the compounds according to the invention are administered in combination with prostanoids and prostacyclin receptor agonists such as, for example Iloprost, Beraprost, Cicaprost, Epoprostenol, oder Treprostinil, Selexipag or Ralinepag.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a renin inhibitor such as, for example and preferably, aliskiren, SPP-600 or SPP-800.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a steroidal mineralocorticoid receptor antagonist such as, for example and preferably, spironolactone or eplerenone and/or administered in combination with a non-steroidal mineralocorticoid receptor antagonist such as, for example and preferably, finerenone, esaxerenone, apararenone, or PF- 03882845.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a diuretic, such as, for example and preferably, furosemide, bumetanide, torsemide, bendroflumethiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichlormethiazide, chlorthalidone, indapamide, metolazone, quinethazone, acetazolamide, dichlorphenamide, methazolamide, glycerol, isosorbide, mannitol, amiloride or triamterene and/or with aldosterone antagonists, for example spironolactone, potassium canrenoate, eplerenone and finerenone and thiazide diuretics, for example hydrochlorothiazide, chlorthalidone, xipamide, and indapamide In another embodiment of the invention, the compounds according to the invention are administered in combination with natriuretic peptides, such as, for example "atrial natriuretic peptide" (ANP, Anaritide), "B-type natriuretic peptide", "brain natriuretic peptide" (BNP, Nesiritide), "C-type natriuretic peptide" (CNP) or Urodilatin; In another embodiment of the invention, the compounds according to the invention are administered in combination with inhibitors of the endopeptidase (NEP -inhibitors) such as, for example Sacubitril, Omapatrilat or AVE-7688, or as dual combinations with Angiotensin receptor antagonists (for example Valsartan), such as, for example LCZ696.

In another embodiment of the invention, the compounds according to to the invention are administered in combination with type2 antidiabetic drugs, such as inhibitors of the Sodium-glucose co-transporter 2 (SGLT2 inhibitors), for example Empagliflozin, Canagliflozin, Dapagliflozin, Ipragliflozin, Tofogliflozin, Ertugliflozin, Remogliflozin, Sergliflozin and such as inhibitors of the Dipeptidyl peptidase 4 (DPP-4 inhibitors), for example sitagliptin, saxagliptin, linagliptin, alogliptin.

Agents which alter lipid metabolism are preferably understood to mean compounds from the group of CETP inhibitors, thyroid receptor agonists, cholesterol synthesis inhibitors such as HMG-CoA reductase inhibitors or squalene synthesis inhibitors, of ACAT inhibitors, MTP inhibitors, PPAR-alpha, PPAR- gamma and/or PPAR-delta agonists, cholesterol absorption inhibitors, polymeric bile acid adsorbents, bile acid reabsorption inhibitors, lipase inhibitors and lipoprotein(a) antagonists.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a CETP inhibitor such as, by way of example and preferably, torcetrapib (CP -529 414), JJT-705 or CETP vaccine (Avant).

In another embodiment of the invention, the compounds according to the invention are administered in combination with a CETP inhibitor, for example and preferably dalcetrapib, BAY 60-5521, anacetrapib or CETP-vaccine (CETi-1). In another embodiment of the invention, the compounds according to the invention are administered in combination with a thyroid receptor agonist such as, by way of example and preferably, D-thyroxin, 3,5,3'-triiodothyronin (T3), CGS 23425 or axitirome (CGS 26214).

In another embodiment of the invention, the compounds according to the invention are administered in combination with an HMG-CoA reductase inhibitor from the class of the statins such as, by way of example and preferably, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin or pitavastatin. In another embodiment of the invention, the compounds according to the invention are administered in combination with a squalene synthesis inhibitor such as, by way of example and preferably, BMS- 188494 or TAK-475.

In another embodiment of the invention, the compounds according to the invention are administered in combination with an ACAT inhibitor such as, by way of example and preferably, avasimibe, melinamide, pactimibe, eflucimibe or SMP-797.

In another embodiment of the invention, the compounds according to the invention are administered in combination with an MTP inhibitor such as, by way of example and preferably, implitapide, BMS- 201038, R-103757 or JTT-130. In another embodiment of the invention, the compounds according to the invention are administered in combination with a PPAR-gamma agonist such as, by way of example and preferably, pioglitazone or rosiglitazone.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a PPAR-delta agonist such as, for example and preferably, GW 501516 or BAY 68- 5042.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a cholesterol absorption inhibitor such as, by way of example and preferably, ezetimibe, tiqueside or pamaqueside.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a lipase inhibitor such as, by way of example and preferably, orlistat.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a polymeric bile acid adsorbent such as, by way of example and preferably, cholestyramine, colestipol, colesolvam, CholestaGel or colestimide.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a bile acid reabsorption inhibitor such as, by way of example and preferably, ASBT (= IBAT) inhibitors, for example AZD-7806, S-8921, AK-105, BARI-1741, SC-435 or SC-635.

In another embodiment of the invention, the compounds according to the invention are administered in combination with a lipoprotein(a) antagonist such as, by way of example and preferably, gemcabene calcium (CI- 1027) or nicotinic acid. The present invention further provides medicaments which comprise at least one compound according to the invention, typically together with one or more inert, nontoxic, pharmaceutically suitable auxiliaries, and the use thereof for the aforementioned purposes.

According to a further embodiment of the invention, a medicament comprising a sGC activator or sGC stimulator in combination with an inert, non-toxic, pharmaceutically suitable excipient for use according to the invention is provided.

The present invention further provides medicaments comprising an activator or a stimulator of sGC for use according to the invention in combination with a further active compound selected from the group consisting of organic nitrates, NO donors, cGMP-PDE inhibitors, antithrombotic agents, hypotensive agents and lipid metabolism modifiers.

The present invention further provides medicaments comprising a sGC activator for use according to the invention in combination with a further active compound selected from the group consisting of organic nitrates, NO donors, cGMP-PDE inhibitors, antithrombotic agents, hypotensive agents and lipid metabolism modifiers. The present invention further provides medicaments comprising a sGC stimulator for use according to the invention in combination with a further active compound selected from the group consisting of organic nitrates, NO donors, cGMP-PDE inhibitors, antithrombotic agents, hypotensive agents and lipid metabolism modifiers.

The compounds according to the invention may act systemically and/or locally. For this purpose, they can be administered in a suitable manner, for example by the oral, parenteral, pulmonal, nasal, sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctival, otic route, or as an implant or stent.

The compounds according to the invention can be administered in administration forms suitable for these administration routes.

Suitable administration forms for oral administration are those which work according to the prior art, which release the compounds according to the invention rapidly and/or in a modified manner and which contain the compounds according to the invention in crystalline and/or amorphized and/or dissolved form, for example tablets (uncoated or coated tablets, for example with gastric juice-resistant or retarded-dissolution or insoluble coatings which control the release of the compound according to the invention), tablets or films/wafers which disintegrate rapidly in the oral cavity, films/lyophilizates or capsules (for example hard or soft gelatin capsules), sugar-coated tablets, granules, pellets, powders, emulsions, suspensions, aerosols or solutions. Parenteral administration can bypass an absorption step (e.g. intravenously, intraarterially, intracardially, intraspinally or intralumbally) or include an absorption (e.g. intramuscularly, subcutaneously, intracutaneously, percutaneously or intraperitoneally). Administration forms suitable for parenteral administration include preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilizates or sterile powders.

For the other administration routes, suitable examples are inhalable medicament forms (including powder inhalers, nebulizers), nasal drops, solutions or sprays, tablets, films/wafers or capsules for lingual, sublingual or buccal administration, suppositories, ear or eye preparations, vaginal capsules, aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions, ointments, creams, transdermal therapeutic systems (e.g. patches), milk, pastes, foams, sprinkling powders, implants or stents.

Oral or parenteral administration is preferred, especially oral administration.

The compounds according to the invention can be converted to the administration forms mentioned. This can be done in a manner known per se, by mixing with inert, nontoxic, pharmaceutically suitable excipients. These excipients include carriers (for example microcrystalline cellulose, lactose, mannitol), solvents (e.g. liquid polyethylene glycols), emulsifiers and dispersing or wetting agents (for example sodium dodecylsulphate, polyoxysorbitan oleate), binders (for example polyvinylpyrrolidone), synthetic and natural polymers (for example albumin), stabilizers (e.g. antioxidants, for example ascorbic acid), dyes (e.g. inorganic pigments, for example iron oxides) and flavour and/or odour correctants.

In general, it has been found to be advantageous in the case of parenteral administration to administer amounts of about 0.001 to 1 mg/kg, preferably about 0.01 to 0.5 mg/kg, of body weight to achieve effective results. In the case of oral administration, the dosage is about 0.01 to 100 mg/kg, preferably about 0.01 to 20 mg/kg and most preferably 0.1 to 10 mg/kg of body weight.

It may nevertheless be necessary where appropriate to deviate from the stated amounts, specifically as a function of the body weight, route of administration, individual response to the active compound, nature of the preparation and time or interval over which administration takes place. For instance, in some cases, less than the aforementioned minimum amount may be sufficient, while in other cases the upper limit mentioned must be exceeded. In the case of administration of relatively large amounts, it may be advisable to divide these into several individual doses over the course of the day.

The working examples which follow illustrate the invention. The invention is not limited to the examples. The percentages in the tests and examples which follow are, unless indicated otherwise, percentages by weight; parts are parts by weight. Solvent ratios, dilution ratios and concentration data for liquid/liquid solutions are based in each case on volume.

According to another aspect of the invention, a method for identifying a compound for use as an activator or a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, which method comprises b) the screening of one or more test compounds against an assay system comprising

• an isolated beta2 subunit

• an isolated sGC enzyme containing the beta2 subunit

· transgenic or non-transgenic cells expressing sGC enzyme containing the beta2 subunit

• transgenic or non-transgenic isolated organs that are characterized by comprising cells that express sGC enzyme containing the beta2 subunit

• transgenic or non-transgenic animal models characterized by expressing the beta2 subunit in at least some of their tissues or cells, on whether or not the test compounds have a selective effect on said assay system, which effect is specific to activation or stimulation of the beta2 subunit, and c) isolating at least one test compound.

According to another aspect of the invention, a method for identifying a compound for use as an activator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, which method comprises b) the screening of one or more test compounds against an assay system comprising

• an isolated beta2 subunit

• an isolated sGC enzyme containing the beta2 subunit

• transgenic or non-transgenic cells expressing sGC enzyme containing the beta2 subunit · transgenic or non-transgenic isolated organs that are characterized by comprising cells that express sGC enzyme containing the beta2 subunit

According to another aspect of the invention, a method for identifying a compound for use as a stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit is provided, which method comprises b) the screening of one or more test compounds against an assay system comprising

• an isolated beta2 subunit

• an isolated sGC enzyme containing the beta2 subunit

• transgenic or non-transgenic cells expressing sGC enzyme containing the beta2 subunit

Functional sGC assays are well known to the skilled person. Some examples of such assays are disclosed in Corazza et al, 2006, and Sousa et al, 2006, incorporated herein by reference.

The art of creating transgenic cells, organs or animal models is entirely within the routine of the skilled person (see, e.g., Pray 2008). A cDNA/mRNA sequence of Homo sapiens Guanylyl Cyclase 1 soluble subunit beta2 is available under NCBI Reference Sequence: NR 003923.2, and provided as SEQ ID NO 1 herein, to allow the development of such transgenic cells, organs or animal models.

According to one embodiment, the method described above further comprises, prior to step b), a step a) of creation and/or provision of a library of test compounds.

According to another aspect of the invention, a method for determining whether a human or animal subject is suitable of being treated with a sGC activator or sGC stimulator according the above description is provided, said method comprising

• providing a tissue or liquid sample from said subject, and

• determining whether or not said sample is characterized by presence, expression or overexpression of an sGC comprising a beta2 subunit. According to another aspect of the invention, a method for determining whether a human or animal subject is suitable of being treated with a sGC activator according the above description is provided, said method comprising

• providing a tissue or liquid sample from said subject, and · determining whether or not said sample is characterized by presence, expression or overexpression of an sGC comprising a beta2 subunit.

According to another aspect of the invention, a method for determining whether a human or animal subject is suitable of being treated with a sGC stimulator according the above description is provided, said method comprising · providing a tissue or liquid sample from said subject, and

• determining whether or not said sample is characterized by presence, expression or overexpression of an sGC comprising a beta2 subunit.

Preferably, presence, expression or overexpression an sGC comprising a beta2 subunit is indicative of a patient being eligible to receive treatment with a sGC activator or a sGC stimulator according the above description.

Preferably, presence, expression or overexpression an sGC comprising a beta2 subunit is indicative of a patient being eligible to receive treatment with a sGC activator according the above description.

Preferably, presence, expression or overexpression an sGC comprising a beta2 subunit is indicative of a patient being eligible to receive treatment with a sGC stimulator according the above description. According to one embodiment of said method, the sample is a cardiac tissue sample, a cardiovascular tissue sample, a lung tissue sample and/or a renal tissue sample.

According to one embodiment of said method, the sample is a renal tissue sample.

Said sample can for example be a slice, biopsy, aspiration, smear, homogenized or liquid sample.

According to one further embodiment of said method, the presence, expression or overexpression of a sGC comprising a beta2 subunit is determined

• on an mRNA level (e.g., RT-PCR, in situ PCR and/or Fluorescence in situ hybridization (FISH) or • on a protein level (e.g., with Immunohistochemistry, Immunoblot, Western Blot, ELISA, and the like).

Methods for determination on an mRNA level are selected from RT-PCR, in situ PCR and Fluorescence in situ hybridization (FISH). Methods for determination on a protein level are selected from Immunohistochemistry, Immunoblot, Western Blot, and ELISA.

RT-PCR, in situ PCR and FISH are routine methods sufficiently established, and explained in the respective literature (see Bagasra 2007, Bayani & Squire 2004, all incorporated by reference herein). These methods rely on the use of suitable, sequence specific nucleic acid probes and primers. A cDNA/mRNA sequence of Homo sapiens Guanylyl Cyclase 1 soluble subunit beta2 is available under NCBI Reference Sequence: NR 003923.2, and provided as SEQ ID NO 1 herein, to allow the development of such suitable probes and primers, for the determination of the expression of the beta2 subunit on the mRNA level. The design of such probes and primers follows routine methods as discussed, e.g., in Rodriguez et al 2015, incorporated by reference herein.

As already discussed above, monoclonal polyclonal antibodies against the beta2 subunit of sGC are commercially available. Some examples are shown in the following table:

These and other antibodies are suitable to be used in immunodiagnostic methods for the determination of the presence, expression or overexpression of the sGC beta2 subunit in a sample. The said immunodiagnostic methods Immunohistochemistry, Immunoblot, Western Blot, ELISA, are routine methods, and enablingly disclosed in, e.g., Jeyapradha et al, 2012; Darwish 2006, Mahmood and Young 2012, all incorporated by reference herein. In one embodiment of the present invention, a companion diagnostic is provided for use in a method according to the above description, which companion diagnostic comprises at least one agent selected from the group consisting of

• a nucleic acid probe or primer capable of hybridizing to a nucleic acid (DNA or RNA) that encodes a sGC comprising a beta2 subunit

• an antibody that is capable of binding to sGC comprising a beta2 subunit and

• an aptamer that is capable of binding to sGC comprising a beta2 subunit.

Preferably, the probe, antibody or aptamer is labelled, or is detected by another probe, antibody or aptamer which is labeled. For the skilled person, the provision of such diagnostic is entirely within his routine. Based on technologies readily available, he is capable of making probes or primers which bind with sufficient selectivity and sensitivity to a nucleic acid (DNA or RNA) that encodes a sGC comprising a beta2 subunit. He is also capable of generating or buying antibodies (polyclonal or monoclonal) or aptamers that bind with sufficient selectivity and sensitivity to sGC comprising a beta2 subunit. A. Assessment of the pharmacological activity

Preferably, the determination of activity of a sGC comprising a beta2 subunit is performed by a sGC activity assay. Methods to determine the activity of a sGC in general are discussed in WO 2014/012934 as experiment B-l .

A-l . Activation and stimulation of recombinant soluble Guanylate Cyclase (sGC) in vitro Investigations on the activation and stimulation of recombinant soluble Guanylate Cyclase (sGC) being (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, or (iv) a beta2 monomer by the compounds according to the invention with and without sodium nitroprusside, and with and without the heme-dependent sGC inhibitor lH-l,2,4-oxadiazolo[4,3a]quinoxalin-l-one (ODQ), are carried out by the method described in detail in M. Hoenicka et al, 1999. The heme-free guanylate cyclase is obtained by adding Tween 20 to the sample buffer (0.5% in the final concentration).

The activation and stimulation of sGC being (i) an alphal/beta2 heterodimer (ii) an alpha2/beta2 heterodimer, (iii) a beta2/beta2 homodimer, or (iv) a beta2 monomer by a test substance is reported as x- fold stimulation of the basal activity.

A-2. Modulation of alphal/betal and beta2 homodimer soluble Guanylate Cyclase (sGC) in vitro Enzyme activity was measured by the formation of [32^p] cGMP from a[32^p]-GTP modified according to Gerzer et al., (1981) and Hoenicka et al. (1999). The modifications included using GTP, Mn²⁺ or Mg²⁺, and cGMP at concentrations of 200 μΜ, 3 mM, and 1 mM, respectively. Enzyme concentrations were chosen carefully to achieve a substrate turnover of less than 10%, thus avoiding substrate or cofactor depletion. The characterization of the purified enzymes, namely alphal/beta-1 heterodimers (controls) as well as beta-2 homodimers were performed at a protein concentration range of 0.1 - 0.8 μg/ml. The enzyme activity was tested in the absence and presence of NO-donors like DEANO or SIN-1. All measurements were performed in duplicate and were repeated six times unless otherwise indicated. For enzyme characterization the specific activity of sGC was expressed as x-fold stimulation vs. specific basal activity. The highest dimethyl sulfoxide (DMSO) concentration in the assay was 1% (v/v) and did not elicit any effect per se on cGMP production.

A-3. Gene expression of sGC beta2 subunit mRNA compared to sGC betal subunit mRNA in rat kidneys of a chronic kidney disease model All studies were carried out in accordance with local ethical regulations for the use of laboratory animals. The studies were conducted according to the German animal welfare law and permitted by the Landesamt fur Natur, Umwelt und Verbraucherschutz (LANUV) Nordrhein-Westfalen, Germany.

Male rats were obtained from Charles River Laboratories. They were housed in groups of two to three under controlled standard conditions (12 h light and 12 h dark, at 24 ± 1°C) and received food and water ad libitum.

Following groups of rats were analyzed:

• Normal Sprague Dawley (SD) rats which did not receive any treatment (SD control)

• Sprague Dawley (SD) rats which underwent unilateral nephrectomy (uninephrectomy, SD+UNx) At the end of all studies, rats were euthanized, kidneys removed and immediately snap frozen in liquid nitrogen. Samples were stored at -80°C until RNA isolation and further analysis.

RNA isolation and real time PCR:

Total RNA from kidney samples was isolated using TRlzol® (Invitrogen, Karlsruhe, Germany). Quality and quantity of isolated RNA was determined photometrically. 1 μg total RNA was reversely transcribed into cDNA with the ImProm II Reverse Transcription System (Promega, Mannheim, Germany) according to the manufacturer's instructions. Specific primers and TapMan® probes were designed for detecting the sGC betal and beta2 subunit transcripts (Table 1).

Table 1 Primer sequences (5 '-3')

Primer / Gucylbl Gucylb2 RpL32

Gene Forward TATACATACAGGTG CACAGAGCCCTGAAAAA GAAAGAGCAGCACA TCTCATGTC CAAAG GCTGGC

Reverse GGACACAGGACCTC GTGGTCATCTTTCCTTTC TCATTCTCTTCGCTG

TGTG CC CGTAGC

TaqMan CCAGAAAACTCGGA TTGAAATTGTCAGGAGA TCAGAGTCACCAAT probe TCCACAGTTCCACTT GGCGAGATCGAAGTG CCCAACGCCA qPCR was performed according to manufacturer's instructions with 4 μΐ cDNA in a total reaction volume of 20 μΐ with the qPCR Master Mix Plus (Eurogentech, Seraing, Belgium) on the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative gene expression was calculated using the ΔΔ-Ct-Method and ribosomal protein L32 (RpL32) as reference gene.

Table 2 Expression levels: sGC betal subunit vs. beta2 subunit in rat kidneys of uninephrectomized and control rats.

Table 2 shows the quantitative summary of expression levels of sGC betal subunit vs. beta2 subunit in rat kidneys. Upregulation of sGC beta2 subunit expression is detected in uninephrectomized rats compared to control rats. Data are relative gene expression levels and presented as means with standard deviation.

A-4. Expression of sGC beta2 subunit in kidneys of diseased ZSF-1 rats and their respective controls (lean ZSF-1 rats)

Kidney sections from either ZSF-1 or ZSF-l/lean rats were lysed in buffer: lOOmM Tris pH7.4, 150mM NaCl, ImM EGTA, ImM EDTA, 1% Triton X-100 substituted with protease inhibitor (cOmplete Mini EDTA-free; Roche, Ref: 11836170001) and phosphatase inhibitor (Phosphatase Inhibitor Cocktail 2; SIGMA, P5726-5ML). After lysis on ice, samples were centrifuged at 10000 rpm to remove debris. Supernatants were pipetted into fresh tubes and stored at -80°C. To analyse the protein expression in the lysed kidney samples, total protein content of the specific samples was analysed by the BCA method and 20μg per sample was loaded on a 4-20% SDS acrylamide gel (Mini-PROTEAN TGX Stain-Free Gels ; BioRad;Cat. #456-8093) for separation of the proteins. After 20 minutes of separation, proteins were transferred by a Turboblotter from BioRad for 10 minutes at 25V and 2.4A . After blotting, the membrane was transferred into blocking buffer ( 3% BSA in PBS/0.1%> Tween20) and incubated for 1 hour at room temperature. The membrane was then incubated at room temperature for one hour with the primary anti-sGC-beta2 antibody (Thermo Fisher; PA5-36523) 1 : 1000 diluted in PBS/0.1% Tween. After 5 washes in PBS/0.1%> Tween20 the membrane was incubated with the secondary anti -rabbit antibody from Jackson (#711 -035-152) 1 :5000 diluted in PBS/0.1% Tween20. After 5 washed for 5 minutes in PBS/0.1%> Tween20, the membrane was developed using the ECL reagent (Figure 1, Figure 2) and protein bands were detected using the BioRad ChemiDoc MP (Figure 3). The sGC beta2 subunit bands on the blot were normalized to the internal beta-actin bands, which served as loading control. Table 3 Semiquantitative sGC beta2 subunit expression levels in kidneys of ZSF-1 lean/control vs. ZSF-1 rats related to beta actin as sample-specific internal control

Table 3 shows higher sGC beta2 subunit expression levels in kidneys of ZSF-1 rats compared to ZSF-1 lean/control.

A-5. Staining of sGC beta2 subunit in kidney biopsies from patients with and without diabetic nephropathy (diabetic kidney disease. DKD)

For the evaluation of sGC beta2 subunit expression in human biopsies, sections from 10 normal kidney and 30 diabetic nephropathy kidney biopsies were stained by immunohistochemistry. The sections were first incubated for 20 minutes at 72°C in a heat chamber to solubilize the paraffin, followed by 2x 4 minutes deparaffmization in a BondMax from Leica. After blocking endogenous peroxidase, the epitope was demasked using the Epitope Retrieval Solution 1 (ER1/ Leica/ Menarini) for 20 min at 95/ 100°C.

The primary anti-GUCYlB2 antibody from Thermo Fisher (PA5-36523 /Lot RL2309481) was incubated on the sections for 60 minutes at room temperature. Detection of the primary antibody was done using the Bond Polymer Refine Detection (Leica/Menarini) for 10 minutes at room temperature.

The sections were then analyzed by a pathologist and scored for individual staining intensities.

Fig. 2 shows the staining of beta2 subunit in kidneys of non-DKD (left) and DKD (right) individuals, demonstrating a significant upregulation of beta2 subunit in the distal nephron of the kidney of DKD patients (p = 0.0151, two-tailed Mann-Whitney test).

Fig. 3 shows the staining of beta2 subunit in kidneys of non-DKD (left) and DKD (right) individuals, demonstrating a significant upregulation of beta2 subunit in the proximal tubules of the kidney of DKD patients (p = 0.0197, two-tailed Mann-Whitney test).

While the invention is illustrated and described in detail in the drawings and foregoing description, such illustration and description is to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Any reference signs should not be construed as limiting the scope. All amino acid sequences disclosed herein are shown from N-tenninus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

Fig. 1 gives an overview of the NO-cGMP pathway.

Fig. 2 shows upregulation of beta2 subunit in the distal tubules of the kidney of DKD patients. Fig. 3 shows upregulation of beta2 subunit in the proximal tubules of the kidney of DKD patients. References

Bagasra O, Nat Protoc. 2007;2(1 1):2782-95

Bayani J, Squire JA, Curr Protoc Cell Biol. 2004 Sep;Chapter 22:Unit 22.4

Behrends and Vehse, Biochem Biophys Res Commun 2000, 271 , 64-69

Berguig GY et al., Mol Ther 2015;23(5):907-17

Blind M, Blank M. Mol Ther Nucleic Acids 4, e223 (2015)

Chen BX, Erlanger BF, Immunol Lett 2003, 5;88(2): 87

Corazza S, Scarabottolo L, Lohmer S, Liberati C. Assay Drug Dev Technol. 2006 Apr;4(2): 165-73

Darwish IA, Int J Biomed Sci. 2006 Sep; 2(3): 217-235

Evgenov OV et al, Nat. Rev. Drug Discovery 2006, 5, 755-768

Follmann M et al. Angew Chem Int Ed Engl. 2013 Sep 2;52(36):9442-62

Follmann M et al., J. Med Chem 2017 Jun 22;60(12):5146-5161

Gupta G et al., J. Clin. Invest., 1997, Volume 100, Number 6, 1488-1492

Gerzer et al., Eur. J. Biochem. 1981 , 116: 479-486

Hoenicka, M et al, J. Mol. Med. 77 1999, 14-23

Jeyapradha D et al., J Pharm Bioallied Sci. 2012 4(Suppl 2): S307-S309

Koglin et al., J. Biol. Chem. 2001 ;276:30737-30743

Kohler and Milstein, Nature 256, August 1975, 495 - 497

Mahmood T and Yang P-C; N Am J Med Sci. 2012 Sep; 4(9): 429-434

Meunier, L. BioCellChallenge: Drug Dev. April (2014)

Pray, L, Nature Education 2008, 1 (1):51

Rodriguez A et al., Methods Mol Biol. 2015;1275:31 -56

Sousa EH et al., Anal Biochem., 2006 Jan l;348(l):57-63

Stasch JP et al., Nature 2001 , 410:212-215 Stasch JP et al., Br. J. Pharmacol. 2002, 136(5):773-783

Stasch JP and Hobbs AJ, Handb. Exp. Pharmacol. 2009, 191, 277 - 308

Stasch JP et al., J. Clin. Invest. 2006, 116(9):2552-61

Thiel KW, Giangrande, PH, Ther Deliv. 2010, 1(6): 849-61

Claims

What is claimed is:

1. An activator or stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit, for use in the treatment of a human or animal subject

• suffering from,

· at risk of developing, and/or

2. The sGC activator or stimulator for use according to claim 1, wherein the target tissue which comprises, expresses or overexpresses a sGC comprising a beta2 subunit is renal tissue.

3. The sGC activator or stimulator for use according to claim 1 or 2, wherein the disease is a kidney disease.

4. The sGC activator for use according to any of claims 1 to 3, wherein the sGC activator is selected from the group consisting of

5. The sGC activator for use according to any of claims 1 to 4, wherein the sGC activator is · 3-(4-chloro-3- {[(2S,3R)-2-(4-chlorophenyl)-4,4,4-trifluoro-3-methylbutanoyl]amino}phenyl)-3- cyclopropylpropanoic acid.

6. The sGC stimulator for use according to any of claims 1 to 3, wherein the sGC stimulator is selected from the group consisting of

• methyl {4,6-diamino-2-[5-fluoro-l -(2-fluorobenzyl)-lH-pyrazolo[3,4-b]pyridine-3 yl]pyrimidin-5-yl}carbamate (Vericiguat)

• 1,1,1, 3,3, 3-Hexafluoro-2-[( {5-fluoro-2-[l-(2-fluorobenzyl)-5-(l,2-oxazol-3-yl)-lH-pyrazol 3-yl]-4-pyrimidinyl}amino)methyl]-2-propanol (Praliciguat)

7. The sGC stimulator for use according to any of claims 1, 2, 3 or 6, wherein the sGC stimulator is selected from the group consisting of

8. Medicament comprising a sGC activator or stimulator for use according to any of claims 1 to 7 in combination with an inert, non-toxic, pharmaceutically suitable excipient.

9. Medicament comprising a sGC activator or stimulator for use according to any of claims 1 to 7 in combination with in combination with a further active compound selected from the group consisting of organic nitrates, NO donors, cGMP-PDE inhibitors, antithrombotic agents, hypotensive agents and lipid metabolism modifiers.

10. A method for identifying a compound for use as an activator or stimulator of soluble Guanylyl Cyclase (sGC) comprising a beta2 subunit, which method comprises: b) screening of one or more test compounds against an assay system comprising

• an isolated beta2 subunit

• an isolated sGC enzyme containing the beta2 subunit

• transgenic or non-transgenic isolated organs that are characterized by comprising cells that express sGC enzyme containing the beta2 subunit and/or

11. The method of claim 10, further comprising, prior to step b), a step a) of creation and/or provision of a library of test compounds.

12. A method for determining whether a human or animal subject is suitable of being treated with a sGC activator or stimulator according to any one of claims 1 to 8, said method comprising

• providing a tissue or liquid sample from said subject, and

13. The method according to claim 12, wherein the sample is a cardiac tissue sample, a cardiovascular tissue sample, a lung tissue sample and/or a renal tissue sample.

14. The method according to claim 12 or 13, wherein the tissue sample is a sample of renal tissue.

15. The method according to any one of claims 12 to 14, wherein the presence, expression or overexpression of a sGC comprising a beta2 subunit is determined

• on an mRNA level and/or

• on a protein level.

16. A companion diagnostic for use in a method according to any one of claims 12 to 15, which companion diagnostic comprises at least one agent selected from the group consisting of

• a nucleic acid probe or primer capable of hybridizing to a nucleic acid (DNA or RNA) that encodes an sGC comprising a beta2 subunit

• an aptamer that is capable of binding to sGC comprising a beta2 subunit.