Diagnosis and / or treatment of heart failure with preserved ejection fraction

Abstract

The present invention relates to compositions and methods for treating and / or preventing heart failure with preserved ejection fraction (HFpEF), particularly in subjects with NO-cGMP-PKG signaling axis disorders. The inventors have established that not all clinically defined HFpEF patients suffer from NO-cGMP-PKG signaling deficiency. According to the present invention, patients diagnosed with HFpEF can be selected / stratified based on biomarkers such as NADPH oxidase type 5 (Nox5) plasma levels, nitration of tyrosine residues on plasma proteins, and / or cell-based assays. Furthermore, the inventors have found that treating this signaling network at two or more nodes, particularly sGC and NO synthase, achieves a causally related, highly precise treatment based on a first-in-class mechanism of action for the HFpEF endotype defined by NO-cGMP-PKG signaling deficiency.

Description

Technical Field

[0001] The present invention relates to compositions and methods for treating and / or preventing heart failure with preserved ejection fraction (HFpEF) in subjects with NO-cGMP-PKG signaling axis disorders. The present invention provides a method for treating such subjects, the method comprising the step of evaluating whether the subject is eligible for treatment, and optionally comprising a composition for use in said treatment.

Background Art

[0002] Measured in disability-adjusted life years (DALYs), cardiovascular diseases cause the most significant disease burden worldwide. The majority of these are due to chronic heart failure, which is a general term for a complex set of symptoms that permanently reduce the efficiency of the heart muscle to pump or fill with blood. The diagnosis rate of heart failure in developed countries is generally estimated to be 1-2% of the adult population; however, about half of all cases may not be detected. In developed countries, the actual prevalence of all types in people aged 65 and over is estimated to be 11.8%. Worldwide, more than 65 million people may be affected at any given time.

[0003] There are two major subgroups of chronic heart failure. One was originally known as systolic heart failure and is now called heart failure with reduced ejection fraction (HFrEF), which is characterized by a significantly lower ratio of the amount of blood ejected from the left ventricle per heartbeat to the maximum filling volume compared to the standard value. In the other form, HFpEF (formerly called diastolic heart failure), the ejection fraction is relatively preserved, but the ventricles are stiff and unable to relax properly during diastole, and thus are not filled properly. This ventricular stiffness usually develops against a background of old age, diabetes, and fibrotic diseases. Myocardial remodeling in HFpEF is different from that in HFrEF, which is caused by the loss of cardiomyocytes. The cellular, molecular, and metabolic differences between HFrEF and HFpEF are more prominent than the advanced clinical symptoms.

[0004] HFpEF was once considered a relatively rare disease, but it is now known to affect at least half of heart failure patients and accounts for the majority of heart failure cases in elderly diabetic patients. Looking at the temporal trend, it can be seen that the increase in the incidence of HFpEF and the decrease in the incidence of HFrEF have been balanced over the past 20 years. HFpEF patients do not show significant symptoms at rest in the early stages of the disease, showing only an increase in exercise intolerance and may remain undiagnosed for a long time, so the epidemiological trend may be even worse. In HFrEF, treatment with angiotensin-converting enzyme inhibitors and β-blockers can achieve reversal of cardiac remodeling and improve cardiac function, but in HFpEF, despite many clinical trials conducted using very different strategies, there are still no specific pharmacological options other than treatment for the underlying risk factors.

[0005] The mechanisms that drive the onset and maintenance of HFpEF have not yet been elucidated, which is a major reason for the lack of effective drug treatment. Furthermore, as the inventors have discovered, clinical HFpEF is not a molecularly single disease, but rather a common final endpoint of different endotypes that develop along different pathways.

[0006] In 2013, Paulus and Tschoepe (J Am Coll Cardiol. 2013;62(4):263 - 71) proposed a new paradigm for HFpEF. According to these authors, HFpEF develops based on a systemic inflammation - inducing state that causes inflammation of the coronary microvascular endothelial cells; this reduces the bioavailability of nitric oxide (NO), the cyclic guanosine monophosphate (cGMP) content, and ultimately decreases the activity of protein kinase G (PKG) within cardiomyocytes. As a result, hypophosphorylation of titin (the "molecular spring" responsible for the passive elasticity of muscle) occurs, the cardiomyocytes become stiff, and interstitial fibrosis develops. In this scenario of NO - cGMP - PKG signaling impairment, which has been confirmed to exist in heart disease (J Cardiovasc Pharmacol. 2020;75(5):370 - 84), HFpEF should, at least theoretically, be treatable by restoring the activity of this axis. However, to date, all attempts to achieve this have ended in failure. Since the INDIE - HFpEF trial, which investigated inorganic nitrite (JAMA 2018;320(17):1764 - 73), and the NEAT - HFpEF trial, which used organic nitrates (Am J Cardiol. 2019;123(10):1660 - 66), both failed to provide benefits to patients, a new class of soluble guanylate cyclase (sGC) modulators has come into focus. These agents act as stimulators and activators, enhancing the enzymatic activity of sGC independently of exogenous NO donors to generate cGMP and showing excellent efficacy in HFrEF. However, praliciguat failed in the CAPACITY - HFpEF trial (JAMA. 2020;324(15):1522 - 1531), and vericiguat also failed in the VITALITY - HFpEF phase III trial (JAMA. 2020;324(15):1512 - 21), despite high expectations in the phase II SOCRATES - PRESERVED trial (Eur J Heart Fail. 2017;19(6):782 - 91).These all reproduce the failure of the initial RELAX study that pursued variations of this strategy using the phosphodiesterase 5 (PDE5) inhibitor sildenafil (JAMA. 2013;309(12):1268 - 77). SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] From the above, HFpEF is still a large area with unmet medical needs and is expected to deteriorate further due to population dynamics and the increasing prevalence of diabetes. The object of the present invention is to provide an effective pharmacological treatment for HFpEF. MEANS FOR SOLVING THE PROBLEMS

[0008] The inventors surprisingly discovered that not all clinically defined HFpEF patients suffer from impairments in NO - cGMP - PKG signaling, a feature widely seen not only in heart failure as a whole but also in HFpEF. This hitherto unrecognized pathway heterogeneity, if not considered, will inevitably lead to a diminished therapeutic effect and provides at least a partial explanation for the failure of clinical trials when it is assumed that the NO - cGMP - PKG signaling axis is consistently impaired. The inventors also found that patients diagnosed with HFpEF according to current clinical criteria and suffering from NO - cGMP - PKG signaling axis disorders can be selected based on biomarkers such as plasma levels of NADPH oxidase type 5 (Nox5), nitration of tyrosine residues on plasma proteins, or cell - based assays. Furthermore, the inventors found that by treating this signaling network at two or more nodes, particularly sGC and NO synthase, a causally related, highly precise treatment based on a first - in - class mechanism of action can be achieved for the HFpEF endotype defined by impairments in NO - cGMP - PKG signaling.

[0009] Although not bound by a particular theory, the inventors believe that the Paulus & Tschoeppe model is very accurate in the basic assumption of NO-cGMP-PKG axis impairment, but the amount of reactive oxygen species (ROS) generated by the pro-inflammatory state induced by metabolic syndrome or its components (obesity, diabetes, and hypertension) is often not sufficient to disrupt the NO-cGMP-PKG axis to the extent necessary for the development of HFpEF. Rather, this only applies when NADPH oxidase type 5 (Nox5), a strong ROS-generating enzyme, is overexpressed or overactivated in endothelial cells, as seen in the endotype of age-related systolic hypertension (PLoS Biol. 2020;18(11):e3000885). Initially mainly generated by Nox5, ROS depletes tetrahydrobiopterin to dissociate cardiac NOS, generating superoxide anion instead of NO; this is a unique feature of this class of enzymes and has been suggested as a cause of diastolic dysfunction (Circulation. 2010;121(4):519-28), but the extremely important role of Nox5 has not been mentioned. At the same time, superoxide and other ROS directly deplete NO further by converting it to peroxynitrite, which generates even more ROS (see Figure 1), causing a vicious cycle leading to the development of HFpEF. Figure 8 shows the NOX5 plasma levels of HFpEF patients according to two HFpEF classification scores, namely the H2FPEF score and the HFAPEFF score. In HFpEF patients determined by the HFAPEFF score, a subgroup or endotype of patients with high NOX5 values (110 ng / mL or higher) can be distinguished. This endotype corresponds to approximately 25% of all HFpEF patients according to the HFAPEFF score. Figure 9 shows the 3-NT plasma levels of HFpEF patients according to two HFpEF classification scores, namely the H 2Display according to the FPEF score and the HFAPEFF score. Five out of seven patients with high NOX5 levels also had high 3-NT levels. Based on these findings and the hypothesis described above, in one aspect, the present invention is directed to the treatment of a specific subgroup of subjects who have or are at risk of having HFpEF, particularly subjects with a disorder in the NO-cGMP-PKG axis. In a further aspect, in the treatment of HFpEF, particularly in said subgroup of patients with a NO-cGMP-PKG axis disorder, a combination of active agents that target different nodes of the NO-cGMP-PKG axis is used. More specifically, according to a preferred embodiment of the present invention, patients preselected by a clinical HFpEF score are endotyped as having a disorder in the NO-cGMP-PKG axis. According to the present invention, HFpEF patients, particularly those endotyped as having a disorder in the NO-cGMP-PKG axis, are preferably treated as follows: i) administering an sGC modulator to reactivate and / or stimulate sGC so that a normal amount of cGMP can be synthesized from the NO that has become available again; ii) administering one or more agents that reverse NOS uncoupling so that NOS can resume NO production instead of generating additional ROS; iii) administering an excess of NOS substrate or precursor to avoid further uncoupling of the newly re-coupled NOS.

[0010] Accordingly, a first aspect of the present invention relates to a method for prophylactic and / or therapeutic treatment of a subject having or at risk of having HFpEF, preferably a subject having a disorder in the NO-cGMP-PKG axis, said method typically comprising administering to said subject a pharmaceutical composition comprising an sGC (positive) modulator, in combination with a NOS re-coupler and / or a NOS substrate (precursor).

[0011] A further aspect of the present invention relates to a pharmaceutical composition comprising an sGC (positive) regulator for use in a method for the prophylactic and / or therapeutic treatment of a subject suffering from or at risk of developing HFpEF, preferably a subject having a disorder in the NO-cGMP-PKG axis, said method typically comprising administering to said subject a pharmaceutical composition comprising an sGC (positive) regulator, in combination with a NOS rebinder and / or a NOS substrate (precursor).

[0012] Furthermore, a further aspect of the present invention relates to the use of an sGC (positive) regulator in the manufacture of a pharmaceutical composition for use in a method for the prophylactic and / or therapeutic treatment of a subject suffering from or at risk of developing HFpEF, preferably a subject having a disorder in the NO-cGMP-PKG axis, said method typically comprising administering to said subject a pharmaceutical composition comprising an sGC (positive) regulator, in combination with a NOS rebinder and / or a NOS substrate (precursor).

[0013] Another aspect of the present invention relates to a diagnostic method for determining whether a subject is suffering from the HFpEF endotype of the present invention, preferably a pharmaceutical composition comprising an sGC (positive) regulator in combination with a NOS rebinder and / or a NOS substrate (precursor), preferably in unit dosage form; and a package containing a plurality of such pharmaceutical unit dosage forms, and a leaflet comprising printed instructions for repeated self-administration of said unit dosage form for the treatment and / or prevention of HFpEF in a subject preferably having a disorder in the NO-cGMP-PKG axis.

[0014] Specific details and preferred embodiments of the foregoing methods, as well as the compositions and pharmaceutical kits used therein, will become apparent to those skilled in the art based on the following detailed description and the attached experimental part. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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Mode for Carrying Out the Invention

[0016] Pharmaceutical Composition The pharmaceutical composition according to the present invention comprises, as a first active ingredient ("API1"), a positive regulator of soluble guanylate cyclase ("sGC"), typically a stimulator or activator. In the usage herein, the term "sGC regulator" or "sGC positive regulator" refers to an agent that enhances the enzymatic activity of soluble guanylate cyclase independently of NO to produce cGMP by acting on either the holoenzyme (referred to in the art as "sGC stimulator" and / or "sGCs") or the apoenzyme that has lost its heme moiety (referred to in the art as "sGC reactivator", "sGC activator" and / or "sGCa"). Suitable examples of compounds that (positively) regulate sGC include riociguat, vericiguat, ataciguat, neliciguat, etoriciguat, lifitegrast, IW-1701, IW-1973, IWP-051, IWP-121, IWP-427, IWP-953, BAY-60-2770, A-344905, A-350619, A-778935, BI-684067, BI-703704, BAY-41-2272, BAY-41-8543, BAY60-4552, CF-1571, cinaciguat, and HMR-1766.

[0017] According to a particularly preferred embodiment of the present invention, the sGC (positive) regulator is riociguat or vericiguat, or a pharmaceutically acceptable salt, hydrate or solvate thereof. Riociguat has the IUPAC name methyl N-[4,6-diamino-2-[1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-pyrimidinyl]-N-methylcarbamate and is the international nonproprietary name for the compound having the following structural formula (I). Vericiguat has the IUPAC name methyl N-[4,6-diamino-2-[5-fluoro-1-[(2-fluorophenyl)methyl]pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl]carbamate and is the international nonproprietary name for the compound having the following structural formula (II). [Chemical formula]

[0018] As used herein, the term "pharmaceutically acceptable" has its conventional meaning and is considered appropriate for contact with mammalian, particularly human, tissue without causing undue toxicity, irritation, allergic response, and other complications by sound medical judgment, and refers to compounds, materials, compositions, and / or dosage forms that are commensurate with a reasonable benefit / risk ratio.

[0019] As used herein, "pharmaceutically acceptable salts" include any salts that retain the activity of the active agent and are acceptable for pharmaceutical use. Pharmaceutically acceptable salts of the disclosed compounds may be prepared by methods well known to those skilled in the art.

[0020] Furthermore, the composition may contain the active ingredient in the form of a solvated compound comprising a pharmaceutically acceptable solvent such as water ("hydrate"), ethanol, etc. In general, for the purposes of the present invention, the solvated form is considered equivalent to the unsolvated form.

[0021] As used herein, the term "pharmaceutical composition" refers to a composition comprising an sGC (positive) regulator and, optionally, one or more additional non-toxic ingredients, which composition is in a form suitable for administration to a (human) subject by any route of administration and is a physiologically acceptable composition for such administration.

[0022] In preferred embodiments, the composition comprises one or more carriers and / or excipients. As is well known to those skilled in the art, the appropriate selection of excipients depends on a number of factors, such as the physicochemical properties of the API, the preferred pharmaceutical form, the preferred route of administration, the desired release rate, etc. The compositions of the present invention can be formulated for various routes of administration, although oral administration is particularly preferred. Pharmaceutical compositions adapted for oral administration can be provided as capsules or tablets, as powders or granules to be dissolved before use, as solutions, syrups or suspensions, or as edible foams or whips; or as individual units such as emulsions. Excipients suitable for tablets or hard gelatin capsules include lactose, corn starch or its derivatives, stearic acid or its salts. Excipients suitable for use in soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols, etc. Excipients that may be used for the preparation of solutions and syrups include, for example, water, polyols, sugars, etc. It is within the knowledge of an average skilled person in the art to devise and develop appropriate formulations based on general knowledge described in textbooks such as Remington’s Pharmaceutical Sciences (Meade Publishing Co., Easton, Pa., 20th Ed., 2000) (the entire disclosure of which is incorporated herein by reference) and routine development efforts.

[0023] According to various aspects of the present invention, the pharmaceutical composition is preferably provided in a unit dosage form. The term "unit dosage form" refers to physically discrete units suitable for administration to a human subject, and each unit contains a predetermined amount of the active substance calculated to produce the desired therapeutic effect in combination with any suitable pharmaceutical carrier and / or excipient. Exemplary and non-limiting unit dosage forms include tablets, caplets, capsules (e.g., hard capsules or soft capsules), lozenges, films, strips, gel caps, and measured amounts of solutions, suspensions, syrups or elixirs, etc., which can be contained in, for example, vials, syringes, applicator devices, sachets, sprays, micropumps, etc. According to a particularly preferred embodiment of the present invention, the unit dosage form is a unit dosage form suitable for oral administration. Most preferably, it is a unit dosage form such as a tablet for oral ingestion.

[0024] According to various aspects of the present invention, the pharmaceutical composition is provided in a unit dosage form comprising an sGC (positive) regulator in a dose of at least 0.01 mg, preferably at least 0.05 mg, at least 0.1 mg, at least 0.2 mg, at least 0.25 mg, at least 0.3 mg, at least 0.4 mg, or at least 0.5 mg. According to various aspects of the present invention, the composition is typically provided in a unit dosage form comprising an sGC (positive) regulator in a dose of 100 mg or less, more preferably 75 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, 12.5 mg or less, 12 mg or less, or 11 mg or less. According to various aspects of the present invention, the composition is preferably provided in a unit dosage form comprising an sGC (positive) regulator in a dose within the range of 0.1 to 100 mg, 0.2 to 50 mg, 0.3 to 50 mg, 0.4 to 25 mg, or 0.5 to 10 mg.

[0025] According to various aspects of the present invention, the pharmaceutical composition is provided in a unit dosage form comprising at least 0.1 mg, preferably at least 0.25 mg, at least 0.5 mg, at least 1 mg, at least 1.25 mg, at least 1.5 mg, at least 2 mg, or at least 2.5 mg of velisiguate; or a salt, solvate or hydrate of velisiguate in an equipotent dosage. According to various aspects of the present invention, the composition is typically provided in a unit dosage form comprising at most 100 mg, more preferably at most 75 mg, 50 mg, 40 mg, 30 mg, 20 mg, 15 mg, 12.5 mg, 12 mg, or 11 mg of velisiguate; or a salt, solvate or hydrate of velisiguate in an equipotent dosage. According to various aspects of the present invention, the composition is preferably provided in a unit dosage form comprising velisiguate in a dosage within the range of 0.5 to 100 mg, 1 to 50 mg, 1.5 to 50 mg, 2 to 25 mg, or 2.5 to 10 mg; or a salt, solvate or hydrate of velisiguate in an equipotent dosage.

[0026] According to various aspects of the present invention, the composition is provided in a unit dosage form comprising at least 0.01 mg, preferably at least 0.05 mg, at least 0.1 mg, at least 0.2 mg, at least 0.25 mg, at least 0.3 mg, at least 0.4 mg, or at least 0.5 mg of riociguat; or a salt, solvate or hydrate of riociguat in an equipotent dosage. According to various aspects of the present invention, the composition is typically provided in a unit dosage form comprising at most 50 mg, more preferably at most 25 mg, 20 mg, 15 mg, 10 mg, 7.5 mg, 5 mg, 4 mg, 3 mg, or 2.5 mg of riociguat; or a salt, solvate or hydrate of riociguat in an equipotent dosage. According to various aspects of the present invention, the composition is preferably provided in a unit dosage form comprising riociguat in a dosage within the range of 0.1 to 50 mg, 0.2 to 25 mg, 0.3 to 10 mg, 0.4 to 5 mg, 4 mg or 0.5 to 2.5 mg; or a salt, solvate or hydrate of riociguat in an equipotent dosage.

[0027] As used herein, the term "equipotent" means having equal efficacy or the ability to produce a pharmacological effect of a particular strength equivalently. In the art, it is also common to refer to a particular amount of a compound as "equivalent" to a particular amount of a reference compound. For example, when a composition comprises a salt of an sGC modulator, the amount of the salt administered and / or incorporated into a unit dosage form needs to be adjusted taking into account the difference in molecular weight between the free base and the salt form. When representing the dosage in the label and / or product information of an approved pharmaceutical product comprising a salt form of an active compound that can also be used in the free base form, it is customary to specify the dosage of the free base that is equivalent to the dosage of the salt used. In this context, the term "equipotent" is considered synonymous with the term "equivalent".

[0028] According to various aspects of the present invention, a method of treatment comprises the administration of a NOS rebinder, as previously described / defined herein. As used herein, the term "NOS rebinding" refers to the phenomenon in which nitric oxide synthase enzyme (NOS) "dissociates from the binding" to generate superoxide anion (O(2)(-)) instead of nitric oxide (NO), and NOS rebinding can be brought about by supplying a level of tetrahydrobiopterin sufficient to replenish the depleted stores that caused the dissociation from the binding (Free Radic Biol Med. 2013;65:234-43). In one embodiment of the present invention, NOS rebinding is achieved by the administration of folate. As described elsewhere herein, the NOS rebinder may be administered as a separate formulation or in a "fixed-dose combination product" in combination with an sGC(+) modulator.

[0029] Accordingly, in a preferred embodiment of the present invention, the pharmaceutical composition as defined herein comprises, as a further active ingredient ("API2"), a NOS rebinder, preferably a NOS rebinder selected from the group consisting of folic acid and pharmaceutically acceptable salts thereof. As used herein, the term "folate" refers to folate (active form), and more specifically, may be used to refer to folic acid (pteroylmonoglutamic acid or pteroylglutamic acid), which is a prodrug thereof in any pharmaceutically acceptable form.

[0030] According to various aspects of the present invention, such pharmaceutical compositions are provided in unit dosage forms comprising a NOS rebinder, preferably folate, in a dose of at least 0.01 mg, preferably at least 0.05 mg, at least 0.1 mg, at least 0.2 mg, at least 0.25 mg, at least 0.3 mg, at least 0.4 mg, or at least 0.5 mg. According to various aspects of the present invention, the composition is typically provided in a unit dosage form comprising a NOS rebinder, preferably folate, in a dose of 100 mg or less, more preferably 75 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, 12.5 mg or less, 12 mg or less, or 11 mg or less. According to various aspects of the present invention, the composition is preferably provided in a unit dosage form comprising a NOS rebinder, preferably folate, in a dose within the range of 0.1 to 100 mg, 0.2 to 50 mg, 0.3 to 50 mg, 0.4 to 25 mg, or 0.5 to 10 mg.

[0031] According to various aspects of the present invention, a method or treatment comprises administration of a NOS substrate or a precursor thereof, as previously described / defined herein. In one embodiment, the NOS substrate is L-arginine. In another embodiment, the NOS substrate is L-citrulline, which is a natural precursor of L-arginine and has higher bioavailability than L-arginine because it avoids first-pass metabolism in the liver and has a longer residence time in the systemic circulation. In yet another embodiment, L-arginine and L-citrulline are administered together. According to the present invention, L-citrulline, as well as L-arginine, may be administered in any pharmaceutically and / or nutritionally acceptable form, particularly in the form of a pharmaceutically and / or nutritionally acceptable salt, hydrate or solvate compound. As described herein, the NOS substrate (precursor) may be administered as a separate formulation or a "fixed-dose combination product" may be provided in combination with an sGC (positive) modulator and / or a NOS rebinder (two-component or three-component).

[0032] Thus, in an embodiment of the present invention, a pharmaceutical composition as defined herein comprises, as a further active ingredient ("API3"), a NOS substrate or a NOS substrate precursor, preferably a NOS substrate (precursor) selected from the group consisting of L-citrulline, L-arginine, and pharmaceutically acceptable salts, hydrates, solvate compounds thereof, and combinations thereof.

[0033] According to various aspects of the present invention, such pharmaceutical compositions are provided in unit dosage forms comprising a NOS substrate (precursor), preferably L-citrulline or a salt thereof, in a dosage of at least 0.1 mg, preferably at least 0.25 mg, at least 0.5 mg, at least 1 mg, at least 1.25 mg, at least 1.5 mg, at least 2 mg, or at least 2.5 mg. According to various aspects of the present invention, the compositions are typically provided in unit dosage forms comprising a NOS substrate, preferably L-citrulline or a salt thereof, in a dosage of 100 mg or less, more preferably 75 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, 14 mg or less, 13 mg or less, 12.5 mg or less, or 12 mg or less. According to various aspects of the present invention, the compositions are preferably provided in unit dosage forms comprising a NOS substrate, preferably L-citrulline or a salt thereof, in a dosage within the range of 0.1 - 50 mg, 0.5 - 25 mg, 1 - 20 mg, 2 - 15 mg, or 3 - 12 mg.

[0034] Therapeutic indications As described above herein, the present invention provides a method for treating and / or preventing a subject in need of treatment, wherein the treatment preferably comprises administering an sGC modulator in combination with a NOS rebinder and / or a NOS substrate (precursor). More specifically, the present invention provides a method for treating and / or preventing heart failure with preserved ejection fraction (HFpEF) in such a subject. The present invention further provides a method for treating and / or preventing one or more symptoms associated with HFpEF in such a subject, preferably by administering an sGC modulator in combination with a NOS rebinder and / or a NOS substrate (precursor). The present invention further provides a method for treating and / or preventing one or more pathological conditions associated with and / or caused by HFpEF in such a subject, preferably by administering an sGC modulator in combination with a NOS rebinder and / or a NOS substrate (precursor).

[0035] As used herein, the terms "heart failure with preserved ejection fraction" and its abbreviation "HFpEF" mean, for example, a type of heart failure diagnosed according to current clinical and blood chemistry consensus criteria and having an ejection fraction of at least 50% of the nominal value. Non-limiting examples of HFpEF consensus criteria include the European Society of Cardiology HFA-PEFF algorithm (Eur Heart J. 2019;40(40):3297-317); the H2FPEF score published by Reddy et al. (Circulation. 2018;138(9):861-70); or other diagnostic algorithms discussed by Kaplon-Cieslicka et al. (Cardiol J. 2020;27(5):449-68).

[0036] The terms "treat", "treating", or "treatment", when used in conjunction with a particular disease or condition (e.g., "a method of treating a disease..."), refer to curing, alleviating, or suppressing the disease and / or attendant symptoms, reducing the degree of the disease, stabilizing the disease state (i.e., not worsening), retarding or moderating the progression of the disease, improving the disease state, and extending the survival period (compared to the predicted survival period without treatment). As used herein, the terms "prevent", "preventing", or "prevention" refer to reducing the risk that a subject will develop a disease and / or attendant symptoms and delaying the time at which a subject develops a disease. The terms "treat", "treating", or "treatment", when used in relation to a patient or subject (e.g., "a method of treating a subject"), typically refer to the act of administering a therapeutic compound to the patient or subject, regardless of whether the purpose is therapeutic and / or prophylactic.

[0037] Subjects to be treated As described above in this specification, the method of the present invention is directed to the treatment and / or prevention of a subject suffering from or at risk of suffering from HFpEF.

[0038] The term "subject" refers to an organism, typically a mammal, particularly a human subject. In one embodiment of the present invention, the subject is male. In another embodiment of the present invention, the subject is female.

[0039] In a further preferred embodiment of the present invention, the subject is at increased risk based on age, e.g., the subject is 35 years or older, 40 years or older, 45 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, or 70 years or older; typically in combination with one or more other risk factors as defined herein.

[0040] In a particularly preferred embodiment of the present invention, the subject is a subject suffering from and / or diagnosed with HFpEF.

[0041] In a particular embodiment of the present invention, the subject is a subject having a HFPEF score of 3 or more, 4 or more, 5 or more, or 6 or more, where the 2 HFPEF score refers to the score of the subject in the scoring system developed and published by Reddy et al. (Circulation. 2018;138(9):861-70). 2 The HFPEF score refers to the score of the subject in the scoring system developed and published by Reddy et al. (Circulation. 2018;138(9):861-70).

[0042] In a particular preferred embodiment of the present invention, the subject is a subject having a HFAPEFF score of 4 or more, preferably 5 or more, where the HFAPEFF score refers to the score of the subject in the scoring system developed and published by the Heart Failure Association (HFA) of the European Society of Cardiology (ESC) (Eur. Heart J. 2019;40:3297-3317).

[0043] In a further preferred embodiment of the present invention, the subject is a subject who is at risk of developing or having HFpEF, typically considered to be at above-average risk. In a preferred embodiment of the present invention, the subject is a subject suffering from one or more conditions known to have a causal and / or epidemiological correlation with the development of HFpEF, such as anemia, atrial fibrillation, chronic kidney disease, chronic obstructive pulmonary disease (COPD), coronary artery disease, diabetes, hypertension (high blood pressure), inflammatory or autoimmune diseases, obesity, sleep apnea, etc. In a further preferred embodiment of the present invention, the subject is a subject who genetically has a predisposition to develop HFpEF. In a further preferred embodiment of the present invention, the subject is a subject who has a tendency to develop HFpEF as a result of lifestyle / habitual factors.

[0044] In some embodiments of the present invention, the subject to be treated has normal blood pressure. As used herein, the term "normal blood pressure" refers to blood pressure within the range considered normal according to the recommendations of the American College of Cardiology (ACC) / American Heart Association (AHA), or according to the recommendations of the European Society of Cardiology (ESC) / European Society of Hypertension (ESH). According to the present invention, normal blood pressure typically refers to a subject having a systolic blood pressure of 129 mmHg or less, preferably 120 mmHg or less, and / or a diastolic blood pressure of 84 mmHg or less, preferably 80 mmHg or less.

[0045] In some embodiments of the present invention, the subject to be treated has hypertension. As used herein, the term "hypertension" refers to elevated blood pressure according to the recommendations of the American College of Cardiology (ACC) / American Heart Association (AHA), or according to the recommendations of the European Society of Cardiology (ESC) / European Society of Hypertension (ESH). According to an embodiment of the present invention, "hypertension" typically refers to a subject having a systolic blood pressure of 130 mmHg or more, such as 139 mmHg or more or 140 mmHg or more, such as in the range of 130 - 180 mmHg or 130 - 179 mmHg, and / or a diastolic blood pressure of 80 mmHg or more, 85 mmHg or more or 90 mmHg or more, such as in the range of 80 - 110 mmHg, 85 - 110 mmHg, 89 - 110 mmHg or 90 - 110 mmHg.

[0046] According to an embodiment of the present invention, "hypertension" refers to a subject suffering from blood pressure elevation, hypertension stage 1, or hypertension stage 2 in accordance with the recommendations of the ACC / AHA, as defined in the following table. In another embodiment of the present invention, "hypertension" refers to a subject having blood pressure classified as "normal" (above the optimal value), "high normal", grade 1 hypertension, grade 2 hypertension, or grade 3 hypertension, preferably "high normal", grade 1 hypertension, grade 2 hypertension, or grade 3 hypertension, in accordance with the recommendations of the ESC / ESH, as defined in the following table. According to an embodiment of the present invention, "normal blood pressure" refers to a subject whose blood pressure is classified as "normal" in accordance with the recommendations of the ACC / AHA, as defined in the following table. In another embodiment of the present invention, "normal blood pressure" refers to a subject whose blood pressure is classified as "optimal" or "normal", preferably "optimal", in accordance with the recommendations of the ESC / ESH, as defined in the following table.

[0047]

Table 1

[0048] Furthermore, as is apparent from the foregoing, a subject to be treated according to the present invention typically has a NO-cGMP-PKG signaling axis disorder. As will be understood by those skilled in the art, based on this teaching, a dysfunction of the NO-cGMP-PKG axis can be established based on biomarker-based criteria, but the present invention is not particularly limited in this regard. In an exemplary embodiment, a dysfunction of the NO-cGMP-PKG axis is established by collecting a blood sample from a subject; processing the sample to obtain a plasma / serum / exosome sample and / or a cell / exosome sample; and determining one or more of the following biomarker-based criteria: A) The expression level of NADPH oxidase type 5 (Nox5) in the plasma / serum / exosome sample; B) The level of nitrotyrosine in the plasma / serum / exosome sample;

[0049] The methodology for determining A) and / or B) in a non-limiting manner is as follows: ● Collect whole blood into a tube by venipuncture, with or without an anticoagulant. Immediately centrifuge the blood at 800×g for 10 minutes. ● Plasma / serum / exosomes may be collected and stored at -80 °C until biomarker measurement. ● Next, the plasma / serum / exosome sample is appropriately diluted, and the biomarker level is measured using a standardized commercially available antibody or ELISA kit.

[0050] According to a preferred embodiment of the present invention, the subject to be treated has an increased NOX5 level compared to the average NOX5 level of healthy subjects, for example, a NOX5 plasma level of at least 90 ng / mL, at least 95 ng / mL, at least 100 ng / mL, at least 102.5 ng / mL, at least 105 ng / mL, at least 107.5 ng / mL, at least 110 ng / mL, at least 112.5 ng / mL, at least 115 ng / mL, at least 117.5 ng / mL, or at least 120 ng / mL. According to the present invention, the NOX5 concentration can be measured by ELISA.

[0051] According to a preferred embodiment of the present invention, the subject to be treated has a nitrotyrosine level of at least 500 nM, more preferably at least 600 nM, at least 800 nM, at least 1000 nM, or at least 1200 nM. According to the present invention, the nitrotyrosine concentration can be measured by ELISA.

[0052] In a further exemplary embodiment, the dysfunction of the NO-cGMP-PKG axis is established by a step of collecting a blood sample from a subject; and a step of processing the sample to obtain a cell and / or exosome sample and quantitatively determining the degree to which the cGMP synthesis and / or downstream signaling phosphorylation response is stimulated in the cell and / or exosome sample. The methodology for making this determination in a non-limiting manner is as follows: ● After collecting whole blood by venous puncture, the blood is centrifuged to isolate different fractions (e.g., buffy coat) containing cells and / or exosomes. ● These fractions may be appropriately cryopreserved by adding a cryopreservation reagent and storing at -80°C. ● After a specific thawing procedure, these cell and / or exosome samples are exposed to a drug compound that modulates cGMP, and cGMP signaling is evaluated by measuring the production and / or phosphorylation response of cGMP.

[0053] In certain embodiments of the present invention, this methodology involves determining the induction of cGMP synthesis as a response to an NO donor and / or the induction of PKG-dependent protein phosphorylation as a response to an NO donor. As is known to those skilled in the art, cGMP synthesis can be determined (quantitatively) by ELISA. As is known to those skilled in the art, PKG-dependent protein phosphorylation can be determined (quantitatively) by Western blot.

[0054] According to a preferred embodiment of the present invention, a response disorder of a subject to a stimulation of cGMP synthesis is reflected by the level of cGMP production in a cell and / or exosome-containing sample as a response to an NO donor being less than a predetermined reference value, e.g., less than 90% of the predetermined reference value, preferably less than 80%, 70%, 60%, 50%.

[0055] In the usage of this specification, the term "predetermined reference value", which is used in the context of establishing a change capable of stimulating cGMP synthesis, refers to a threshold or cut-off value that distinguishes a normal non-pathological state from a pathological state. A value above the threshold indicates a normal non-pathological endotype, and a value below the threshold indicates a pathological endotype (or vice versa). Typically, such a "threshold" or "cut-off value" can be determined experimentally, empirically, or theoretically. In embodiments of the present invention, a pathological state is characterized by a decrease in response to a specific stimulus (compared to a healthy non-pathological state), and the predetermined reference value may be the 10th percentile cut-off point established in a normal non-pathological reference population of sufficient size, more preferably, the 5th percentile cut-off point, 4th percentile cut-off point, 3rd percentile cut-off point, 2nd percentile cut-off point, or 1st percentile cut-off point established in a normal non-pathological reference population of sufficient size using the same test procedure. In embodiments of the present invention, a pathological state is characterized by an increase in response to a specific stimulus (compared to a healthy non-pathological state), and the predetermined reference value may be the 90th percentile cut-off point established in a normal non-pathological reference population of sufficient size, more preferably, the 95th percentile cut-off point, 96th percentile cut-off point, 97th percentile cut-off point, 98th percentile cut-off point, or 99th percentile cut-off point established in a normal non-pathological reference population of sufficient size using the same test procedure.

[0056] According to another preferred embodiment of the present invention, the response disorder of a subject to the stimulation of cGMP synthesis, as a response to an NO donor, is reflected by the level of PKG-dependent protein phosphorylation in a cell and / or exosome-containing sample being less than a predetermined reference value, for example, less than 90%, 80%, 70%, 60%, or 50% of the predetermined reference value.

[0057] In certain embodiments of the present invention, this methodology involves determining the induction of cGMP synthesis as a response to an sGC stimulator and / or the induction of PKG-dependent protein phosphorylation as a response to an sGC stimulator. According to a preferred embodiment of the present invention, an impaired response of a subject to stimulation of cGMP synthesis, as a response to an sGC stimulator, is reflected by the level of cGMP production in a cell and / or exosome-containing sample being less than a predetermined reference value, for example, less than 90%, 80%, 70%, 60%, or 50% of the predetermined reference value. According to another preferred embodiment of the present invention, an impaired response of a subject to stimulation of cGMP synthesis, as a response to an sGC stimulator, is reflected by the level of PKG-dependent protein phosphorylation in a cell and / or exosome-containing sample being less than a predetermined reference value, for example, less than 90%, 80%, 70%, 60%, or 50% of the predetermined reference value.

[0058] In certain embodiments of the present invention, this methodology involves determining the induction of cGMP synthesis as a response to an sGC activator and / or the induction of PKG-dependent protein phosphorylation as a response to an sGC activator. According to a preferred embodiment of the present invention, an impaired response of a subject to stimulation of cGMP synthesis, as a response to an sGC activator, is reflected by the level of cGMP production in a cell and / or exosome-containing sample exceeding a predetermined reference value, for example, exceeding 10%, 20%, 30%, 40%, or 50% of the predetermined reference value. According to another preferred embodiment of the present invention, an impaired response of a subject to stimulation of cGMP synthesis, as a response to an sGC activator, is reflected by the level of PKG-dependent protein phosphorylation in a cell and / or exosome-containing sample exceeding a predetermined reference value, for example, exceeding 10%, 20%, 30%, 40%, or 50% of the predetermined reference value.

[0059] According to a preferred embodiment of the present invention, the response disorder of a subject to cGMP synthesis stimulation is, for example, less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of a predetermined reference value, i.e., below the predetermined reference value, and is reflected in the ratio between i) the level of cGMP production in a cell and / or exosome-containing sample in response to an sGC stimulator and ii) the level of cGMP production in a cell and / or exosome-containing sample in response to an sGC activator. According to another preferred embodiment of the present invention, the response disorder of a subject to cGMP synthesis stimulation is, for example, less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of a predetermined reference value, i.e., below the predetermined reference value, and is reflected in the ratio between i) the level of PKG-dependent protein phosphorylation in a cell and / or exosome-containing sample in response to an sGC stimulator and ii) the level of PKG-dependent protein phosphorylation in a cell and / or exosome-containing sample in response to an sGC activator. According to a preferred embodiment of the present invention, this ratio is determined in a fresh sample (cell preparation), and the predetermined reference value determined in a sample from a healthy / non-pathological subject is about 8, about 8.25, about 8.5, about 8.75, about 9, about 9.25, about 9.75, or about 10. According to another preferred embodiment of the present invention, this ratio is determined in a cryopreserved and thawed sample, and the predetermined reference value determined in a sample from a healthy / non-pathological subject is about 5.75, about 6, about 6.25, about 6.5, about 6.75, about 7, about 7.25, or about 7.5.

[0060] According to another preferred embodiment of the present invention, the NO-cGMP-PKG axis dysfunction of the subject is reflected by the oxidation state of the subject's sGC. The oxidation state can be evaluated by comparing cGMP-dependent protein phosphorylation in vitro in response to sGC stimulators and sGC activators. In a particularly preferred embodiment, this ratio, herein referred to as the sGCa / sGCs ratio, is measured as the pVASP response (pVASP / VASP) to 1 μM lancyguat (sGCa) in the presence of 500 μM IBMX (3-isobutyl-1-methylxanthine) and the pVASP response (pVASP / VASP) to 100 μM riociguat (sGCs) in the presence of 500 μM IBMX in a P-WBC (platelet-concentrated white blood cell) sample obtained from the subject, and the sGCa / sGCs ratio is determined by dividing each of these responses. As those skilled in the art will understand based on the teachings of the present invention, in this assay, cGMP is generated in response to stimulation, PKG is activated, and VASP is phosphorylated. IBMX inhibits PDE, which causes the degradation of cGMP, and maintains the generated cGMP levels. The setup will be described in detail in the examples. According to a preferred embodiment of the present invention, it involves the following steps: (a) Centrifuge whole blood at 800 g for 10 minutes to collect P-WBC, then isolate the P-WBC and cryopreserve it at -80 °C with 6% DMSO; (b) Thaw the P-WBC at 37 °C for 5 minutes and dilute it 1:100 with JNL buffer; (c) Pretreat the cells with IBMX (500 μM, 10 minutes); (d) Treat the cells with lancyguat (1 μM) or riociguat (100 μM) at 37 °C for 15 minutes; (e) Centrifuge the sample at 750 g for 10 minutes; (f) Lyse the cell pellet with ROTI® Load Laemmli supplemented with phosphatase inhibitors and protease inhibitors; (g) Boil the boiling lysate at 95 °C for 5 minutes; (h) Separate the cell lysate by SDS-PAGE; (i) Analysis by Western blot using anti-phospho-VASP Ser239 (Nanotools) and total VASP (Enzo) as primary antibodies, where ECL detection was performed for visualization of signals and densitometric analysis reported the response to stimulation as relative intensity (a.u.) reflecting phosphorylation of VASP.

[0061] In a non-limiting manner, the methodology for obtaining P-WBC samples used to determine the sGCa / sGCs ratio is as follows. ● Collect whole blood, with or without anticoagulant, for example by venipuncture, and subsequently centrifuge the blood at a speed of 200 - 800×g, preferably 400 - 800×g, more preferably 600 - 800×g, most preferably about 800×g for 5 - 20 minutes, more preferably 7.5 - 15 minutes, for example about 10 minutes; ● Collect plasma and isolate P-WBC; ● Cryopreserve P-WBC by storing at a temperature of -60 to -90°C, preferably -70 to -85°C, most preferably about -80°C, using a cryoprotectant, preferably DMSO, for example 6% DMSO; and ● Thaw cryopreserved P-WBC at a temperature of about 37°C for, for example, about 5 minutes before measuring the sGCa / sGCs ratio using the method defined above.

[0062] As shown in the following examples, a subgroup of patients with an sGCa / sGC value exceeding 1.05 can be identified, where the apo-sGC form of the enzyme is more prominent. Thus, according to a preferred embodiment of the present invention, a subject to be treated is characterized by an sGCa / sGCs ratio exceeding a specific predetermined threshold, such as at least 1.05, at least 1.06, at least 1.07, at least 1.08, at least 1.09, at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14, or at least 1.15, in the assay defined above.

[0063] In a particularly preferred embodiment of the present invention, the subject to be treated meets a combination of criteria based on the biomarkers defined herein. ● The NOX5 plasma / serum / exosome concentration defined herein, in combination with the following; ● Decrease in the production and / or phosphorylation of cGMP in response to the stimulus defined above.

[0064] In a particularly preferred embodiment of the present invention, the subject to be treated meets the following criteria: ● The NOX5 plasma / serum / exosome concentration defined herein, in combination with the following; ● Decrease in cGMP synthesis and / or decrease in PKG-dependent protein phosphorylation in response to the NO donor defined above.

[0065] In a particularly preferred embodiment of the present invention, the subject to be treated meets the following criteria: ● The NOX5 plasma / serum / exosome concentration defined herein, in combination with the following; ● Decrease in cGMP synthesis and / or decrease in PKG-dependent protein phosphorylation in response to the sGC stimulator defined above.

[0066] In a particularly preferred embodiment of the present invention, the subject to be treated meets the following criteria: ● The NOX5 plasma / serum / exosome concentration defined herein, in combination with the following; ● Increase in cGMP synthesis and / or increase in PKG-dependent protein phosphorylation in response to the sGC activator agent defined above.

[0067] In a particularly preferred embodiment of the present invention, the subject to be treated meets the following criteria: ● The NOX5 plasma / serum / exosome concentration defined herein, in combination with the following; ● i) The decrease in the ratio between the cGMP production level in response to the divided sGC stimulator and ii) the cGMP production level in response to the sGC activator, and / or the decrease in pvas between i) the level of KG-dependent protein phosphorylation in response to the sGC stimulator and ii) the level of PKG-dependent protein phosphorylation in response to the sGC activator as defined above.

[0068] In a particularly preferred embodiment of the invention, the subject to be treated meets one or both of the following criteria based on the biomarkers previously defined herein: ● A NOX5 plasma / serum / exosome concentration of at least 105 ng / mL; and / or ● An sGCa / sGCs ratio of at least 1.05, which can be determined using the assay defined above.

[0069] Method of treatment All aspects of the invention defined herein relate to a method of treatment comprising the administration, typically in a therapeutically effective dose, of a typically sGC (positive) regulator, typically in repeated doses, in combination with a NO rebinder and / or a NO substrate (precursor) as defined herein.

[0070] As used herein, the terms "therapeutically acceptable amount" or "therapeutically effective dose" refer to an amount of a drug or combination of drugs sufficient to reduce the clinical symptoms of HFpEF, or test parameters that are surrogate markers for HFpEF, or the risk of developing HFpEF. In some embodiments, a "therapeutically acceptable amount" does not induce or elicit undesirable side effects. The amount can be determined by first administering a low dose and then gradually increasing the dose until the desired result is obtained. In a preferred embodiment of the invention, the method involves repeatedly administering one or more pharmaceutical compositions or unit dosage forms as defined elsewhere herein at an intensity and frequency sufficient to result in a reduction in the clinical symptoms of HFpEF or test parameters that are surrogate markers for HFpEF or the risk of developing HFpEF.

[0071] The agent according to the present invention can be administered orally or parenterally; the oral route is particularly preferred.

[0072] Thus, in a particularly preferred embodiment of the present invention, this method comprises administration of an sGC regulator at a daily dose of at least 0.01 mg, preferably at least 0.05 mg, at least 0.1 mg, at least 0.2 mg, at least 0.25 mg, at least 0.3 mg, at least 0.4 mg, or at least 0.5 mg, preferably oral administration. According to various aspects of the present invention, this method comprises administration of an sGC regulator at a daily dose of 100 mg or less, preferably 75 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, 12.5 mg or less, 12 mg or less, or 11 mg or less, preferably oral administration. According to various aspects of the present invention, this method comprises administration of an sGC regulator at a daily dose within the range of 0.1 to 100 mg, 0.2 to 50 mg, 0.3 to 50 mg, 0.4 to 25 mg, or 0.5 to 10 mg.

[0073] According to various aspects of the present invention, this method comprises administration of vericiguat or a salt, solvate, or hydrate of vericiguat at an equivalent potency at a daily dose of at least 0.1 mg, preferably at least 0.25 mg, at least 0.5 mg, at least 1 mg, at least 1.25 mg, at least 1.5 mg, at least 2 mg, or at least 2.5 mg, preferably oral administration. According to various aspects of the present invention, this method comprises administration of vericiguat or a salt, solvate, or hydrate of vericiguat at an equivalent potency at a daily dose of 100 mg or less, more preferably 75 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, 12.5 mg or less, 12 mg or less, or 11 mg, preferably oral administration. According to various aspects of the present invention, this method comprises administration of vericiguat or a salt, solvate, or hydrate of vericiguat at an equivalent potency at a daily dose within the range of 0.5 to 100 mg, 1 to 50 mg, 1.5 to 50 mg, 2 to 25 mg, or 2.5 to 10 mg, preferably oral administration.

[0074] According to various aspects of the present invention, the method comprises administering an equipotent dose of riociguat, or a salt, solvate, or hydrate of riociguat, preferably orally, at a daily dose of at least 0.01 mg, preferably at least 0.05 mg, at least 0.1 mg, at least 0.2 mg, at least 0.25 mg, at least 0.3 mg, at least 0.4 mg, or at least 0.5 mg. According to various aspects of the present invention, the method comprises administering riociguat, or a salt, solvate, or hydrate of riociguat at an equipotent dose, preferably orally, at a daily dose of 50 mg or less, more preferably 25 mg or less, 20 mg or less, 15 mg or less, 10 mg or less, 7.5 mg or less, 5 mg or less, 4 mg or less, 3 mg or less, or 2.5 mg. According to various aspects of the present invention, the method comprises administering riociguat, or a salt, solvate, or hydrate of riociguat at an equipotent dose, preferably orally, at a daily dose within the range of 0.1 to 50 mg, 0.2 to 25 mg, 0.3 to 10 mg, 0.4 to 5 mg, 4 mg or 0.5 to 2.5 mg.

[0075] According to various aspects of the present invention, the method comprises administering a NOS rebinder as defined herein, preferably folate, at a daily dose of at least 0.01 mg, preferably at least 0.05 mg, at least 0.1 mg, at least 0.2 mg, at least 0.25 mg, at least 0.3 mg, at least 0.4 mg, or at least 0.5 mg, preferably orally. According to various aspects of the present invention, the method comprises administering a NOS rebinder at a daily dose of 100 mg or less, preferably 75 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, 12.5 mg or less, 12 mg or less, or 11 mg or less, more preferably orally. According to various aspects of the present invention, the method comprises administering a NOS rebinder at a daily dose within the range of 0.1 to 100 mg, 0.2 to 50 mg, 0.3 to 50 mg, 0.4 to 25 mg, 4 mg or 0.5 to 10 mg, preferably orally.

[0076] According to various aspects of the present invention, the method comprises administration, preferably oral administration, of a NOS substrate (precursor) as defined herein at a daily dose of at least 0.1 mg, preferably at least 0.25 mg, at least 0.5 mg, at least 1 mg, at least 1.25 mg, at least 1.5 mg, at least 2 mg, or at least 2.5 mg, preferably L-citrulline and / or a salt thereof. According to various aspects of the present invention, the method comprises administration, preferably oral administration, of a NOS substrate, preferably L-citrulline and / or a salt thereof, at a daily dose of 100 mg or less, more preferably 75 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, 15 mg or less, 14 mg or less, 13 mg or less, 12.5 mg or less, or 12 mg or less. According to various aspects of the present invention, the method comprises administration, preferably oral administration, of a NOS substrate, preferably L-citrulline and / or a salt thereof, at a daily dose within the range of 0.1 to 50 mg, 0.5 to 25 mg, 1 to 20 mg, 2 to 15 mg, or 3 to 12 mg.

[0077] As will be understood by those skilled in the art, based on the present teachings, it is not particularly important whether the sGC modulator, NOS rebinder, and / or NOS substrate (precursor) are administered simultaneously or sequentially, and in the case of sequential administration, it is not particularly important whether each agent is administered one after another in a short time / directly or at different times of the day. Further, based on the teachings herein, it will be apparent to those skilled in the art that the daily dose of each active agent shown herein may be contained in a single unit dosage form or divided into multiple unit dosage forms and administered at specific time intervals throughout the day.

[0078] In certain embodiments of the present invention, the method comprises administering the sGC regulator once or twice a day, most preferably once a day, at a total daily dose within the range defined above. In a further embodiment of the present invention, the method comprises administering the NOS rebinder once or twice a day, most preferably once a day, at a total daily dose within the range defined above. In a further embodiment of the present invention, the method comprises administering the NOS substrate (precursor) once, twice or three times a day, most preferably once or twice a day, at a total daily dose within the range defined above.

[0079] It will be understood that, from the perspective of patient convenience and / or patient compliance with taking the medicine, it is preferred that the therapeutic agent be administered simultaneously with, or immediately after, other therapeutic agents. For this purpose, the use of a fixed-dose combination product comprising at least two or all three of the active agents defined elsewhere in this specification is particularly preferred.

[0080] In a particularly preferred embodiment of the present invention, the treatment comprises administering, once, twice, three times or four times a day, preferably once, twice or three times a day, more preferably once or twice a day, most preferably once a day, a single unit dosage form comprising the sGC regulator, the NOS rebinder and the NOS substrate (precursor) at the total daily dose of each active agent defined herein.

[0081] In another particularly preferred embodiment of the present invention, the treatment comprises administering, once or twice a day, preferably once a day, a single unit dosage form comprising the sGC regulator and the NOS rebinder at the total daily dose of each active agent defined herein; and administering, once, twice or three times a day, preferably twice a day, a single unit dosage form comprising the NOS substrate (precursor) at the total daily dose defined herein.

[0082] Embodiments are also envisaged in which a unit dosage form comprising an amount of the active agent higher than the daily dose indicated herein is used. This may include, for example, the use of a sustained-release dosage form that remains in the body and continues to release the active ingredient for a sufficient period of time.

[0083] According to the present invention, the treatment comprises administering a combined sGC regulator of a NOS recombinase and / or a NOS substrate (precursor) for a period of at least 1 month, at least 3 months, at least 4 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, preferably, according to the regimen defined above. There is no particular upper limit; the treatment may be continued, for example, throughout the life of the subject, as long as it is determined to be beneficial to the overall health and well-being of the subject (as determined by a medical professional with appropriate qualifications).

[0084] As is apparent from the teachings of the present invention, the methods of the present invention are particularly suitable for the treatment of subjects suffering from HFpEF with impairments in the NO-cGMP-PKG axis. Thus, in certain embodiments of the present invention, provided are methods as defined herein that comprise, in a subject such as a subject suffering from or at risk of suffering from HFpEF, a preceding or initial step of determining NO-cGMP-PKG axis function and / or diagnosing an impairment of NO-cGMP-PKG axis function. In embodiments of the present invention, the step of determining NO-cGMP-PKG axis function and / or diagnosing an impairment of NO-cGMP-PKG axis function comprises obtaining a blood sample from the subject; processing the blood sample to obtain a plasma / serum / exosome sample and / or a cell and / or exosome-containing sample (a "cell / exosome-containing sample"); A) the expression level of NADPH oxidase type 5 (Nox5) in the plasma / serum / exosome sample; B) the level of nitrotyrosine in the plasma / serum / exosome sample; C) the degree to which cGMP synthesis can be stimulated in the cell and / or exosome-containing sample and determining one or more of the criteria based on the biomarker.

[0085] The methodology for determining A) and B) in a non-limiting manner is as follows: ● Collect whole blood into a tube by venipuncture with or without an anticoagulant. Without delay, centrifuge the blood at 800×g for 10 minutes. ● Collect plasma / serum / exosomes and then store them at -80°C until biomarker measurement. ● Next, appropriately dilute the plasma / serum / exosome sample and measure the biomarker level using a standardized commercially available antibody or ELISA kit.

[0086] The methodology for determining C), namely the change in response to the stimulation of cGMP synthesis in a cell and / or exosome-containing sample, in a non-limiting manner is as follows. ● After collecting whole blood by venipuncture, centrifuge the blood and isolate different fractions (e.g., buffy coat) containing cells and / or exosomes. These fractions may be appropriately cryopreserved by adding a cryopreservation reagent and storing at -80°C. After a specific thawing procedure, expose these cell and / or exosome fractions to a drug that regulates cGMP and evaluate cGMP signaling by measuring the production and / or phosphorylation response of cGMP.

[0087] In a particularly preferred embodiment of the present invention, this method comprises ● the NOX5 plasma / serum / exosome concentration of the subject; and / or ● the nitrotyrosine plasma / serum / exosome level of the subject; and / or ● the response to the stimulation of cGMP synthesis in the cells and / or exosomes of the subject a step of determining, and subsequently comparing the value with one or more predetermined reference values as defined elsewhere in this specification.

[0088] As will be understood by those skilled in the art, following the determination that a subject suffering from HFpEF and / or at risk of developing HFpEF has a dysfunction in the function of the NO-cGMP-PKG axis, typically, in accordance with the teachings of the present invention, preferably, the step of treating the subject by continuously / repeatedly administering an sGC regulator in combination with a NOS recombinase and / or a NOS substrate (precursor) follows.

[0089] Diagnostic method In a further aspect of the present invention, a method for diagnosing the HFpEF endotype of the present invention itself is provided. In a preferred embodiment of the present invention, such a method comprises: i) collecting a blood or plasma sample from a patient suffering from HFpEF; ii) analyzing a biomarker characteristic of the function of the NO-cGMP-PKG axis; and iii) determining that the patient is suffering from HFpEF due to a dysfunction of the NO-cGMP-PKG axis if the biomarker is above or below a predetermined threshold level.

[0090] In a preferred embodiment of the present invention, the biomarker is NOX5, and the method comprises: ● collecting whole blood, with or without an anticoagulant, for example by venipuncture, and subsequently (immediately) centrifuging the blood; ● collecting the plasma and storing it at -80°C until biomarker measurement if necessary; ● measuring the NOX5 level of the plasma sample using a standardized commercially available ELISA kit; ● determining that the patient is suffering from HFpEF due to NO-cGMP-PKG axis dysfunction if the NOX5 plasma level is at least 90 ng / mL, at least 95 ng / mL, at least 100 ng / mL, at least 102.5 ng / mL, at least 105 ng / mL, at least 107.5 ng / mL, at least 110 ng / mL, at least 112.5 ng / mL, at least 115 ng / mL, at least 117.5 ng / mL, or at least 120 ng / mL and including.

[0091] In a preferred embodiment of the present invention, the biomarker is damaged sGC, and the method is as follows: ● Collect whole blood with or without an anticoagulant, for example, by venipuncture, and then centrifuge the blood immediately at a speed of 200-800×g, preferably 400-800×g, more preferably 600-800×g, most preferably about 800×g for 5-20 minutes, more preferably 7.5-15 minutes, for example, about 10 minutes; ● Collect plasma and isolate P-WBC; ● Cryopreserve P-WBC by storing it at a temperature of -60 to -90°C, preferably -70 to -85°C, most preferably about -80°C using a cryoprotectant, preferably DMSO, for example, 6% DMSO; ● Thaw the cryopreserved P-WBC at a temperature of about 37°C, for example, for about 5 minutes; ● Using the P-WBC sample, typically, using the methodology described above, determine the pVASP response (pVASP / VASP) to 1 μM lancyguat (sGCa) in the presence of 500 μM IBMX (3-isobutyl-1-methylxanthine), and the pVASP response (pVASP / VASP) to 100 μM riociguat (sGCs) in the presence of 500 μM IBMX; ● When the ratio of the response to each stimulus, referred to herein as the GCa / sGCs ratio, is at least 1.05, at least 1.06, at least 1.07, at least 1.08, at least 1.09, at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14, or at least 1.15, determine that the patient is suffering from HFpEF due to dysfunction of the NO-cGMP-PKG axis including.

[0092] One aspect of the present invention relates to a method for preparing a P-WBC sample suitable for determining the sGCa / sGCs ratio. Therefore, ●Collect whole blood, with or without anticoagulant, for example by venipuncture, and subsequently centrifuge the blood immediately at a speed of 200 - 800×g, preferably 400 - 800×g, more preferably 600 - 800×g, most preferably about 800×g for 5 - 20 minutes, more preferably 7.5 - 15 minutes, for example about 10 minutes; ●Collect plasma and isolate P-WBC; ●Cryopreserve by storing P-WBC at a temperature of -60 to -90°C, preferably -70 to -85°C, most preferably about -80°C using a cryoprotectant, preferably DMSO, for example 6% DMSO; A method for preparing a P-WBC sample is provided, which comprises the above steps.

[0093] The cryopreserved sample is suitable for shipment to a central laboratory equipped with facilities for determining any biomarker, such as damaged sGC (based on the sGCa / sGC ratio defined herein), in a highly standardized and reliable manner. The cryopreserved P-WBC can be properly stored for at least 3 months before the biomarker is determined. In a preferred embodiment of the present invention, before the determination of the biomarker, the cryopreserved P-WBC is thawed at a temperature of about 37°C for about 5 minutes.

[0094] Pharmaceutical kit Another aspect of the present invention relates to a pharmaceutical kit comprising a package containing a plurality of unit dosage forms and a leaflet, wherein the unit dosage form contains the pharmaceutical composition according to the present invention, and the leaflet contains printed instructions for repeatedly self-administering the unit dosage form to achieve any of the treatment purposes defined herein.

[0095] According to an embodiment of the present invention, a pharmaceutical kit comprises a container such as a box for accommodating one or more blister packs, and the one or more blister packs contain a plurality of solid unit dosage forms described above in this specification. In a particularly preferred embodiment of the present invention, the pharmaceutical kit comprises at least 5, at least 8, at least 10, at least 12 or at least 15 of said unit dosage forms, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of said unit dosage forms.

[0096] According to the present invention, a pharmaceutical kit comprises a leaflet inserted into the container, typically a patient information leaflet containing printed information, and the information may include an explanation of the shape and composition of the unit dosage form contained in the kit, an indication of the therapeutic indication intended for the product, an explanation of how to use the product, and information and warnings regarding adverse effects and contraindications associated with use. The leaflet, which is part of the kit according to the present invention, will typically include information regarding the therapeutic indication, method of use, treatment regimen, etc. related to the treatment method of the present invention as described above, which will be understood by those skilled in the art based on the information presented herein. In a particularly preferred embodiment of the present invention, the leaflet specifically includes printed instructions for repeated (self-)administration of the unit dosage form for treating and / or preventing HFpEF in a subject in whom the NO-cGMP-PKG signaling axis is impaired, as defined elsewhere in this specification.

[0097] In one embodiment of the present invention, a pharmaceutical kit comprises a plurality of unit dosage forms as defined herein, preferably containing an sGC (positive) regulator, an NO rebinder and an NO substrate (precursor) in amounts defined elsewhere in this specification.

[0098] In one embodiment of the present invention, the pharmaceutical kit comprises a plurality of unit dosage forms as defined herein, preferably containing an sGC (positive) regulator and an NO rebinder in amounts defined elsewhere herein, and a plurality of unit dosage forms as defined herein, preferably containing an NO substrate (precursor) in amounts defined elsewhere herein.

[0099] In one embodiment of the present invention, the pharmaceutical kit comprises a plurality of unit dosage forms as defined herein, preferably containing an sGC (positive) regulator and an NO rebinder in amounts defined elsewhere herein, and a leaflet containing printed instructions for repeated (self-) administration of the unit dosage forms in combination with treatment with an NO substrate (precursor).

[0100] In one embodiment of the present invention, the pharmaceutical kit comprises a plurality of unit dosage forms as defined herein, preferably containing an sGC (positive) regulator in amounts defined elsewhere herein, and a leaflet containing printed instructions for repeated (self-) administration of the unit dosage forms in combination with treatment with an NO rebinder and / or an NO substrate (precursor).

[0101] Others It should be understood that many or most of the pharmacologically and / or physiologically active ingredients contained in the compositions defined herein may exist in the form of pharmaceutically acceptable salts or solvates. Such forms are typically equally suitable for use in the present invention.

[0102] Unless otherwise defined, all terms, including technical and scientific terms used in the disclosure of the present invention, have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Further guidance includes definitions of terms for better understanding of the teachings of the present invention.

[0103] As used herein, "a", "an", and "the" refer to both the singular and plural forms unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more compartments.

[0104] As used herein, "about" refers to measurable values such as parameters, amounts, durations, etc., and is intended to include variations of ±10% or less, more preferably ±5% or less, still more preferably ±1% or less from the specified value, as long as such variations are appropriate for the disclosed invention. However, it should be understood that the value itself to which the modifier "about" refers is also specifically disclosed.

[0105] As used herein, "Comprise", "comprising", "comprises", and "comprised of" are synonymous with "include", "including", or "contain", "containing", "contains", and are inclusive or open-ended terms that specify the presence of subsequent content such as components, and do not exclude or preclude the presence of additional components, features, elements, members, steps that are known or disclosed in the art.

[0106] The recitation of numerical ranges by endpoints includes not only the recited endpoints but also all numbers and fractions subsumed within that range. One of ordinary skill in the art will understand that the present invention can incorporate any number of the specific features described above.

Examples

[0107] Example 1: Identification of an HFpEF endotype characterized by elevated ROS-related biomarkers and impaired NO-cGMP-PKG signaling Plasma samples from HFpEF patients were collected from Biobank Maastricht UMC+. Patients were classified as HFpEF if the HFA-PEFF score returned by the HFA-PEFF diagnostic algorithm was 5 or higher. The HFA-PEFF diagnostic algorithm is a multi-step process that diagnoses by examining the patient's echocardiogram and natriuretic peptide levels.

[0108] The NOX5 plasma levels in these samples were measured using a commercially available NOX5 ELISA kit (MyBiosource, #MBS2512643). The 3-nitrotyrosine plasma levels were also measured using a commercially available 3-nitrotyrosine ELISA kit (Abcam, #ab210603). The results are presented in Figures 8 and 9.

[0109] As can be seen from Figure 8, the patient population of the biobank under study was divided into two groups based on the plasma NOX5 concentration of approximately 110 ng / mL (Figure 8a). Patients with NOX5 plasma levels above this cut-off value (i.e., 110 ng / mL) are candidates who may benefit from the above triple combination therapy including vericiguat, L-citrulline, and folic acid, and may be further endotyped based on cGMP production and / or phosphorylation in samples containing cells and / or exosomes responsive to stimulation.

[0110] Example 2: Triple combination therapy including vericiguat, L-citrulline, and folic acid in a mouse model of heart failure with preserved ejection fraction (HFpEF) In a mouse model of heart failure with preserved ejection fraction, the therapeutic efficacy of a triple combination therapy including vericiguat, L-citrulline, and folic acid was investigated. In this model, low-dose angiotensin II was chronically infused into the mice. The infusion of angiotensin II used in this model affects the production and signaling of ROS. Therefore, this model is considered to represent an HFpEF endotype characterized by impaired NO-cGMP-PKG signaling.

[0111] An osmotic micropump (Alzet, model 1004, DURECT Corporation) was subcutaneously implanted via a small incision into the flanks of male C57BL / 6J wild-type mice aged 8 - 12 weeks. Low-dose angiotensin II (AT-II, 0.2 mg / kg / day, A9525, Sigma-Aldrich) or saline was chronically infused via the micropump for 28 days to induce HFpEF or used as a control, respectively. After 28 days, the physical parameters of the mice were evaluated and the hearts were examined by echocardiography and histology (Figure 3). The left atrial area (Figure 4a) and isovolumic relaxation time (Figure 4b) were significantly increased, while the ejection fraction basically remained unchanged (Figure 4c), indicating the onset of HFpEF. The micropump was replaced to maintain chronic infusion treatment with AT-II or saline.

[0112] At the same time, mice injected with AT-II were randomly assigned to the treatment group and orally administered low-dose (3 mg / kg / day) or high-dose (10 mg / kg / day) vericiguat together with a combination of L-citrulline (500 mg / kg / day) and folic acid (15 mg / kg / day) for 28 days after HFpEF induction. Systolic and diastolic arterial blood pressures were non-invasively measured in a conscious state using a tail-cuff sphygmomanometer (CODA System, Kent Scientific, Torrington, CT) at baseline, day 28, and day 56.

[0113] On day 50, mice were individually placed in a cage equipped with a rotating wheel connected to a data acquisition device. The health status of the mice and the functionality of the rotating wheel were monitored daily. On the last day, the mice were anesthetized with isoflurane and echocardiography was performed in the supine position. Parameters of systolic and diastolic function (e.g., LVEF, PW-Doppler blood flow profile, left atrial area, isovolumic relaxation time, and early filling deceleration time) were obtained. The evaluation of the diastolic function of the mice was performed using a published algorithm (Schnelle et al., J. Mol. Cellular Cardiol. 114 (2018) 20 - 28). After echocardiographic evaluation, the mice were sacrificed and tissues were collected for histological analysis.

[0114] Both triple therapies significantly reduced left atrial area and isovolumic relaxation time, indicating a substantial improvement in left ventricular diastolic function, while significant impairment of diastolic function was shown in the control treatment (Figs. 4a - c). Importantly, neither treatment significantly affected the left ventricular ejection fraction, an indicator of systolic function (Fig. 4c). The triple therapy did not induce significant systolic blood pressure, diastolic blood pressure, or mean blood pressure (n = 6 - 8; n.s. not significant) (Figs. 6a - c). The wheel - turning activity was significantly improved after both triple therapies (n = 6 - 8; p < 0.05).

[0115] Example 3: Clinical trial of triple therapy (vericiguat, L - citrulline, and folic acid) in HFpEF patients with Nox5 - overexpressing endotype of HFpEF Approximately 100 HFpEF patients over 45 years old diagnosed according to the HFA - PEFF algorithm (NYHA class II - IV, left ventricular ejection fraction of 50% or more, elevated NT - proBNP value, evidence of structural heart disease by echocardiogram) were screened for Nox5 overexpression by ELISA. A Nox5 level of ≧110 ng / mL (the "Nox5 - overexpressing endotype") is considered characteristic of damage to the NO - cGMP - PKG axis.

[0116] Approximately 20 Nox5 - overexpressing endotype patients were randomly assigned to a group receiving triple therapy with folic acid, L - citrulline, and vericiguat added to standard therapy for 12 weeks and a group receiving standard therapy only. The safety parameters of the triple therapy measured by drug - adverse reactions that may be related or definitely related to the investigational drug constitute the primary evaluation items. The secondary evaluation items are the change in KCCQ score, NYHA functional class, change from baseline in echocardiogram examination parameters, 6 - minute walk distance, and N - terminal proBNP.

[0117] Interim analysis is performed for the primary and secondary parameters when 6 - 10 patients have completed the trial.

[0118] Example 4: Diagnosis of cGMPopathy for Intervention Based on the Mechanism of Action in Subtypes of Heart Failure with Preserved Ejection Fraction I. Introduction / Background If there were a diagnostic assay based on the mechanism of action, such failures could be prevented, and endotype / subtype classification and patient stratification based on the mechanism of action would be possible. At present, such an assay is not available, and it has been found that most of them cannot measure the drug response related to sGC even by measuring the plasma cGMP level derived from the natriuretic peptide signaling. Other cGMP-related biomarkers include mainly the natriuretic peptide level for HF diagnosis and phosphorylated vasodilator-stimulated phosphoprotein (P-VASP), which is the target of cGMP-dependent protein kinase proposed as guidance for antiplatelet therapy. However, due to the high rate of dephosphorylation, the most reliable method for measuring P-VASP is to induce phosphorylation in vitro, which is not a practical approach in the clinical setting or point-of-care environment.

[0119] Therefore, the development of a simple and point-of-care compatible workflow was aimed at, including cell-based analysis of cGMP signaling. First, in healthy individuals, platelet-enriched leukocyte fraction (P-WBC) was isolated from whole blood samples, followed by cryopreservation and transportation to a specialized testing facility to verify whether cGMP signaling could be detected with minimal effort at the clinical site. The best indicator of ROS affecting sGC is an increase in the ratio of the oxidatively damaged heme-free apotype of sGC to the healthy heme-containing type. Both states can be investigated by using the immunoblotting of P-VASP protein as a readout with sGC activator and sGC stimulator, respectively. Regarding the possible sources of ROS that can cause diseases, NOX5 has been recently identified as the closest protein. Therefore, alternative or additional relationships with subtypes of HFpEF related to cGMP were investigated in parallel. Next, detection of NOX5 levels in plasma by ELISA was applied to biobank samples from HFpEF patients (Maastricht HFpEF cohort) selected based on the definition of either the European Society of Cardiology or the American Heart Association. Next, samples from patients considered for the REPO-HFpEF II trial based on mechanism of action, which investigated the effect of sGC stimulator combined with NO synthase recombination in ROCG-positive HFpEF patients using both assays, were analyzed.

[0120] II. Materials and Methods Chemicals The ACD-A tube was obtained from Fisher Scientific (BD 366645). Riosiguat (BAY 63-2521) and Lanciguat (BAY 110-1042) were from MedChemExpress, and IBMX was from Enzo. BAY 58-2667 was manufactured by Merck, and BAY 41-2772 was manufactured by Enzo. The Pierce phosphatase inhibitor cocktail was manufactured by ThermoFischer Scientific, and the cOmplete™ mini protease inhibitor cocktail was manufactured by Merck. The ROTI® Load 1 buffer was obtained from Carl Roth. The anti-phospho VASP Ser 239 (clone 16C2) antibody was purchased from Nanotools (0047-100), and the total VASP polyclonal antibody (ALX-210-898) was from Enzo. The HRP-conjugated goat anti-rabbit antibody and rabbit anti-mouse antibody were obtained from Agilent Dako. DMSO and trehalose were obtained from Sigma. The NOX5 Elisa was obtained from MyBioSource (MBS2512643).

[0121] Protein-Protein Interaction Module Using the NeDRex Cytoscape plugin for network medicine, the neighboring proteins of soluble guanylate cyclase (sGC) were explored, and related disease modules were extracted. All sGC subunits (GUCY1A1, GUCY1A1, and GUCY1B1) in UniProt were considered and used as seeds to start constructing the network. GUCY1B2 was not considered because there is no experimentally known human protein interaction in IID. Next, the first neighboring interacting proteins were added to generate the final protein-protein interaction network. To explore the potential link between this network and the recently reported TCP1 subunit 7 (CCT7)-containing chaperonin, the shortest path to the network was calculated with a Steiner tree.

[0122] P-WBC Preparation for cGMP Assay Optimization Human blood was collected from healthy volunteers after obtaining informed consent in accordance with the Helsinki Declaration. Ethical approval was given by the FHML Research Ethics Committee of Maastricht University (FHML-REC / 2022 / 055). Whole blood (WB) was collected into ACD-A tubes (sodium citrate: 22.0 g / L, dextrose 24.5 g / L, citric acid: 8.0 g / L) by venipuncture. Centrifugation was performed at various speeds (200 g, 400 g, 600 g, 800 g) for 10 minutes at 18 °C (acceleration 1, deceleration 0). Plasma was collected and P-WBC was carefully separated (500 μl). Cell counting was performed using an automated hematology analyzer Sysmex XP-300. Subsequently, P-WBC was diluted with an equal volume of JNL buffer (130 mM NaCl, 3 mM KCl, 9 mM NaHCO3, 0.81 mM KH2PO4, 0.9 mM MgCl2, 10 mM sodium citrate, 6 mM dextrose, 10 mM Tris base, 2 mM HEPES) at pH 7.4. Then, 20 μl of the diluted P-WBC was further diluted 1:5 with JNL buffer and used for cell treatment. The cell suspension was treated with BAY 41-2272 (30 μM) and BAY 58-2667 (10 μM) for 10 minutes at 37 °C, with riociguat and lanciguat for 15 minutes; in one case, pretreated with IBMX for 10 minutes. The samples were centrifuged at 750 g for 10 minutes. The cell pellet was lysed with ROTI® Load Laemmli containing SDS (about 2% w / v), β-mercaptoethanol (about 5% v / v), glycerol (about 10% v / v), with phosphatase inhibitors and protease inhibitors added. The lysate was boiled at 95 °C for 5 minutes and stored at -20 °C.

[0123] Cryopreservation for cGMP assay optimization As described in the results, P-WBCs were cryopreserved at -80°C using cryopreservation reagents (DMSO, trehalose) (Figure 13). After thawing, the samples were diluted 1:100 with JNL-buffer to bring the cryoprotectant concentration below 0.1% and treated with an sGC stimulator (BAY 41-2272, 30 μM) or an sGC activator (BAY 58-2667, 10 μM). Cell lysates were prepared as described above, and the inducible phosphorylation response of VASP was measured by protein immunoblotting.

[0124] Western blot analysis Cell lysates were separated by SDS-PAGE using an 8% or 10% Bis-Tris gel and transferred to a PVDF membrane. The membrane was stained with Ponceau-S and blocked with 3% non-fat dry milk in TBS-Tween (0.1%) for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C (anti-phospho VASP Ser 239 1:400, total VASP 1:2000). The membrane was washed 3 times for 10 minutes each with TBS-Tween (0.1%). Appropriate rabbit anti-mouse or goat anti-rabbit secondary antibodies were used for 1 hour at room temperature. Membrane stripping for reprobing with the total antibody was performed 3 times for 7 minutes each with a boiling solution of 100 mM glycine, pH 2. ECL detection (Amersham) was performed for signal visualization, and densitometric analysis was performed using iBright analysis software.

[0125] HFpEF samples - Maastricht cohort After obtaining approval from the Medical Research Ethics Committee of Maastricht University Medical Center and Maastricht University (METC azM / UM) (NL67997.068.18 and NL76585.068.21), informed consent was obtained, and blood for biobanking was collected from HFpEF patients. After venipuncture, the blood tubes were centrifuged at 2000 g for 10 minutes, and plasma was collected and stored at -80°C. Subsequently, NOX5 was measured in the plasma samples.

[0126] HFpEF samples - Valencia cohort Once the principle of the cGMP-assay was established, after obtaining approval from the Ethics Committee for Pharmaceutical Research of the Hospital Clinico Universitario de Valencia in Valencia (2022 / 248), informed consent was obtained, and blood was collected from 20 HFpEF patients. The HFpEF patients were selected based on the inclusion criteria of the planned REPO-HFpEF II trial. After centrifugation at 800 g for 10 minutes, 1.5 ml of plasma was collected, aliquoted, and frozen at -80°C. P-WBCs were isolated, cryopreserved with 6% DMSO, and stored at -80°C. NOX5 measurements were performed on plasma samples. One aliquot (125 μl) of the cryopreserved P-WBCs was thawed at 37°C for 5 minutes, diluted 1:100 with JNL buffer, and divided into 9 conditions. Cell treatment was performed by pretreatment with IBMX (500 μM, 10 minutes), followed by treatment with ranolazine (1 μM) and riociguat (100 μM) at 37°C for 15 minutes. Subsequently, the cell lysates were separated by SDS-PAGE and analyzed by Western blot as described above.

[0127] NADPH Oxidase 5 Measurement The levels of NADPH oxidase 5 (NOX5), an enzyme that forms reactive oxygen species (ROS), were measured in the plasma of HFpEF patients by ELISA according to the manufacturer's guidelines (MyBioSource, MBS2512643, sensitivity: 18.75 pg / mL, detection range: 31.25 - 2000 pg / mL, coefficient of variation less than 10%). Plasma samples were thawed on ice and diluted according to the pre-measured values. The enzyme-substrate reaction was terminated by the addition of the stop solution, and the color changed to yellow. The optical density (OD) was measured by a spectrophotometer at a wavelength of 450 nm. The OD value is proportional to the concentration of human NOX5.

[0128] Statistical Analysis All data are represented as the mean ± standard error of the mean (SEM) of at least three independent experiments. Analyses and curve fitting were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, USA) or RStudio (Posit team (2022) RStudio: Integrated Development Environment for R. Posit Software, PBC, Boston, Massachusetts. URL http: / / www.posit.co / ). A non-linear least squares regression model was used for dose-response curves. For comparison, an independent Student's t-test was performed and a p-value was determined to be significant.

[0129] III. Results Disease network based on the interactome of soluble guanylate cyclase To define the signaling modules of the proposed underlying causal mechanisms, the first neighborhood protein-protein interaction (PPI) network was constructed using the NeDRex platform for network medicine from all subunits of the soluble guanylate cyclase (sGC) enzyme, namely GUCY1A1, GUCY1A2, and GUCY1B1. These subunits were selected as seeds and mapped to the protein interactome to extract the first adjacent interactions, resulting in a PPI network with 31 proteins and 81 interactions (Figure 11). Notably, the reactive oxygen species (ROS)-forming enzyme, NADPH oxidase 5 (NOX5), and endothelial NO synthase (NOS3) were found to bind directly to sGC, specifically to subunit GUCYB1. To investigate the recently observed association between chaperonin-containing TCP1 subunit 7 (CCT7) and cardiovascular disease, the shortest path between CCT7 and the sGC network was calculated. Thus, CCT7 was only indirectly connected to the network via STIP1 homology and U-box-containing protein 1 (STUB1).

[0130] Development of a cGMPopathy diagnostic assay To address the current challenges in the diagnostic field of cGMP endotyping, a diagnostic assay has been developed that enables simple and rapid patient testing for abnormal cGMP signaling. The established protocol includes blood collection from patients suspected of having cGMPopathy, one-step rapid centrifugation, isolation of P-WBC, and cryopreservation of the samples. Subsequently, the samples are sent to a specialized laboratory, thawed immediately upon arrival, and analyzed to detect the cGMP endotype of the relevant patient population. To establish the diagnostic protocol, optimizations were performed to evaluate different conditions regarding i) centrifugation, ii) cryopreservation, thawing, and storage period, and iii) drugs targeting the cGMP pathway (sGC stimulators and activators).

[0131] i) Optimization of centrifugal force: WB was centrifuged at different centrifugal forces (200 g, 400 g, 600 g, 800 g) for 10 minutes. Centrifugation speeds exceeding 800 g were not selected to ensure platelet integrity, and centrifugation exceeding 10 minutes was not tested to achieve time efficiency. The samples were separated into three fractions: plasma, P-WBC, and red blood cells (Figure 12B). Cell counts were performed on P-WBC. The cell recovery rate was calculated as cell recovery rate (%) = [number of cells in P-WBC / number of cells in whole blood] × 100. The average cell recovery rates of platelets and white blood cells collected at 800 g were 16% and 32% respectively (Figure 12C). The sGC / stimulated sGC ratio was determined via the downstream phospho-VASP (pVASP) response using an sGC stimulator (sGCs) and an sGC activator (sGCa) targeting damaged-form sGC. Treatments with sGC or sGCa were performed on P-WBC. The ratio obtained by dividing the pVASP response to sGC by the response to sGCa was similar at 200 g and 400 g, but increased at 600 g and 800 g (Figure 12D). When comparing only the responses to sGC, surprisingly, the pVASP signal was detected at 800 g (Figure 12E). Therefore, 800 g was selected as the optimal centrifugation speed thereafter.

[0132] ii) Cryopreservation, thawing, and optimization of storage conditions: Next, different cryopreservation conditions were evaluated, namely, using DMSO alone or in combination with the cryoprotectant trehalose at different concentrations, with and without using a freezing container (Mr. Frosty™, cooling rate around -1°C / min). Samples cryopreserved for one week were thawed, and the ratio of pVASP responses induced by sGCs and sGCa was tested. The inducible response using 6% DMSO without a freezing container showed the highest signal among the tested conditions, indicating the most protective effect on cryopreservation (Figures 13B, C). In addition to the conditions described in the figure, 2.5% DMSO + 30 mM trehalose with and without controlling the freezing rate was also tested, but a very weak signal intensity was obtained, suggesting a very low cell viability in this case (non-quantifiable data, not shown). Therefore, 6% DMSO without a controlled freezing container was selected as the cryopreservation condition. Next, different thawing methods were tested to obtain the best recovery after cryopreservation. Using the ratio of pVASP reactions induced by sGCs- and sGCa-, 5 minutes at 37°C showed the highest reaction, and this was subsequently selected as the preferred thawing method (Figure 13D). Subsequently, the period for which the samples could be cryopreserved at -80°C was evaluated. When the pVASP response induced by sGC was divided by the response induced by sGCa, no statistically significant difference was observed between 1 month and 3 months (Figure 13E). Therefore, cryopreserved samples can be analyzed within at least 3 months after storage, providing sufficient time for collection, shipping, and analysis. Furthermore, when used for endotype selection in clinical trials, it is highly unrealistic and unlikely to store samples for more than 3 months.

[0133] iii) Verification and optimization of sGC target compounds: To verify the sGC oxidation state in patient samples, it was necessary to select the optimal concentrations for both sGC and sGCa. Since this assay is considered an in vitro pre-test of the patient's response to treatment, it focused on clinically relevant agents. Riociguat was selected because it is an sGC already available clinically. Since there is currently no approved sGCa, lanciguat, which is currently being investigated for diabetic retinopathy (NCT04722991) and chronic kidney disease (NCT04507061), was tested. The pVASP concentration-response curve for riociguat did not reach maximum effect even when tested at a high dose of 100 μM, resulting in an incomplete curve. However, lanciguat showed maximum effect at 100 μM (Figure 14B). Therefore, to inhibit the activity of phosphodiesterase (PDE), the samples were pre-incubated with IBMX. The pVASP responses to different IBMX concentrations in the presence of sub-threshold doses of riociguat or lanciguat showed that an IBMX dose between 500 - 700 μM was optimal for enhancing the signal induced by the test compound without duplication (Figure 14C). Subsequently, the concentration-response curves of riociguat and lanciguat were repeated in the presence of 500 μM IBMX. The response of riociguat peaked at 100 μM and the response of lanciguat peaked at 1 μM, which means these are the optimal doses to fully activate sGC and apo-sGC and evaluate their oxidation state (Figure 14D).

[0134] Identification of ROCG subtypes in HFpEF NOX5 was found to be directly connected to sGC within the network (Figure 11) and has previously been associated with an endotype of hypertension. Therefore, plasma NOX5 levels in HFpEF patients from the Maastricht UMC biobank were classified and compared according to either the HFA-PEFF (Figure 15A) or the H2FPEF score (Figure 17). Using the HFA-PEFF score, a subgroup of patients (33%; 8 out of 24) was identified with higher NOX5 levels (>105 ng / ml) than the other patients, suggesting that NOX5 is a biomarker based on potential mechanisms of action (Figure 15A). Furthermore, in the HFpEF cohort from Valencia (selected based on the REPO-HFpEF inclusion criteria), the state of damaged and intact sGC, i.e., the apo-sGC / sGC ratio obtained by dividing the pVASP response induced by sGCa by the response induced by sGC, was evaluated. In a subgroup of patients (30%; 6 out of 20), higher sGCa / sGCs values (above 1.05) were observed, meaning that the apo-form of the enzyme was more prominent in these patients (Figure 15C). In parallel, measuring the plasma levels of NOX5 in the Valencia cohort, a subgroup (25%, 5 out of 20) reappeared based on the aforementioned NOX5 cut-off value, suggesting its importance for the pathology of the disease (Figure 15D). In summary, these two cut-off values, i.e., (i) NOX5 plasma levels above 105 ng / ml and (ii) an sGCa / sGCs ratio above 1.05, which rarely overlaps in the same patients (only 1 out of 20) (Figure 15E), indicate that both biomarkers are required to identify the pathophysiological mechanisms of all ROCG modules.

[0135] IV Discussion A simple, point-of-care-compatible diagnostic workflow was developed to detect abnormal regulation of the ROCG signaling module in patients with HFpEF. This includes a cell-based analysis of cGMP signaling, namely the ratio of apo-sGC and sGC in P-WBC, combined with a more straightforward measurement of NOX5 plasma levels. Applying this to HFpEF patients selected based on the major definitions of two societies, the ESC and the AHA, the ESC's HFA-PEFF score appeared to be excellent. This is presumably because the AHA H2FPEF score is strongly biased towards atrial fibrillation. Most notably, clinical trials do not follow any of these definitions and use independent scores that, although overlapping, are distinct. In the preparation of the REPO-HFpEF II trial, a more clinically feasible score that does not require extensive invasive diagnostics was applied. In any case, the European Union's Horizon Europe REPO-TRIAL project (repo-trial.eu) and the REPO4EU platform (repo4.eu) aim to overcome such symptomatological-phenotypical disease definitions and define patients based on underlying causal mechanisms. Applying the ROCG module diagnosis to the patient cohort selected as screening targets according to the REPO-HFpEF II protocol, approximately half were ROCG positive; one-quarter were NOX5; another quarter had sGC dysfunction; and 1 in 20 patients met both criteria.

[0136] Based on this mechanism of action, the stratum-specific REPO-HFpEF II trial, led by the principal investigator, was designed as a safety, Phase IIa, proof-of-concept, single-center, prospective randomized, standard-of-care-controlled, open-label clinical trial (Figure 16). ROCG-positive (NOX5, sGC signaling) HFpEF patients received, in addition to best standard-of-care, combination therapy with the sGC stimulator vericiguat (2.5, 5–10 mg), the NO synthase rebounder L-citrulline (3 g), and folate (5 mg), where NOS3 bound directly to sGC through protein–protein interactions. Twenty-one patients were randomly assigned 2:1 and received, in an open-label manner, triple combination therapy or standard-of-care daily for 12 weeks. Clinical trial endpoints (see supplement) were evaluated at the end of the treatment period. Additionally, blood was drawn and a comprehensive panel of 630 metabolites was evaluated (MxP Quant500, Biocrates). The primary objective of this study was to evaluate the safety profile of the triple combination therapy based on this mechanism of action. Secondary objectives were to investigate the potential benefits of treatment for patients, reported outcomes, maximal functional capacity, and echocardiogram and clinical findings. Based on strict inclusion and exclusion criteria, all HFpEF patients recruited were expected to benefit from the cardioprotective effects of this approach to achieve better outcomes. Along with an adequate safety profile, significant improvements in hemodynamics measured by echocardiogram, exercise tolerance measured by peak oxygen consumption (peak VO2) evaluated by CPET, and quality of life (QOL) evaluated by the Kansas City Cardiomyopathy Questionnaire (KCCQ) were expected.

[0137] This stratification and revised treatment scheme may effectively make HFpEF therapy using sGC stimulators such as vericiguat for at least the following two reasons: (1) sGC stimulation is combined with NO synthase recombination, thereby indirectly curing the dissociation of NOX5-induced NO synthase within the ROCG disease module; (2) Only ROCG-positive HFpEF patients are treated. In previous clinical trials using GC stimulators, it was highly likely that half of the patient group, who were ROCG-negative, were being treated, and the binding deficiency of NOX5-dependent NO synthase remained untreated with sGC stimulators alone. Therefore, only one-fourth of the patients had the opportunity to respond, and the effectiveness may have been significantly diluted.

Claims

1. A pharmaceutical composition comprising an sGC positive regulator for use in the treatment of a subject suffering from or at risk of suffering from HFpEF, said method comprising the step of administering said composition to said subject, wherein said subject has a NO-cGMP-PKG signaling axis disorder.

2. A pharmaceutical composition for use according to claim 1, wherein said sGC (positive) regulator is an sGC stimulator or an sGC activator, preferably riociguat, vericiguat, ataciguat, neliciguat, etoriciguat, reficiguat, IW-1701, IW-1973, IWP-051, IWP-121, IWP-427, IWP-953, BAY-60-2770, A-344905, A-350619, A-778935, BI-684067, BI-703704, BAY-41-2272, BAY-41-8543, BAY60-4552, CF-1571, cinaciguat, and HMR-1766.

3. A pharmaceutical composition for use according to claim 1 or 2, wherein said treatment method further comprises the step of administering to said subject a NO rebinder, preferably a NO rebinder selected from the group consisting of folic acid and folates.

4. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein said treatment method further comprises the step of administering to said subject a NO substrate or a NO substrate precursor, preferably a NO substrate or a NO substrate precursor selected from the group consisting of L-arginine, L-citrulline, their salts, their hydrates, their solvates, and combinations thereof.

5. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein said sGC positive regulator is vericiguat, and said treatment comprises administration of vericiguat at a daily dose in the range of 1 mg to 50 mg.

6. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein said sGC positive regulator is riociguat, and said treatment comprises administration of riociguat at a daily dose in the range of 0.2 mg to 25 mg.

7. A pharmaceutical composition for use according to any one of claims 4 to 6, wherein the NO substrate (precursor) is L-citrulline, and the treatment comprises administration of L-citrulline at a daily dose in the range of 0.5 mg to 25 mg.

8. A pharmaceutical composition for use according to any one of claims 1 to 7, wherein the plasma NOX5 level of the subject is at least 102.5 ng / mL, more preferably at least 105 ng / mL.

9. A pharmaceutical composition for use according to any one of claims 1 to 7, wherein the subject has an sGCa / sG Cs ratio of at least 1.05, the sGCa / sG Cs ratio is a value determined by performing an assay, and in a P-WBC sample obtained from the subject, the pVASP response (pVASP / VASP) to 1 μM sGCa rank siguat in the presence of 500 μM IBMX (3-isobutyl-1-methylxanthine), and the pVASP response (pVASP / VASP) to 100 μM sG Cs riociguat (sG Cs) in the presence of 500 μM IBMX are determined, and the sGCa / sG Cs ratio is calculated by dividing the response to sGCa by the response to sG Cs.

10. A pharmaceutical composition for use according to claim 9, wherein the subject further has a NOX5 plasma level of at least 102.5 ng / mL, more preferably at least 105 ng / mL.

11. A pharmaceutical composition for use according to any one of claims 1 to 10, wherein the subject has an altered response to stimulation of cGMP synthesis, as established in a sample containing cells and / or exosomes obtained from the subject's blood.

12. A pharmaceutical composition for use according to claim 11, wherein the altered response to stimulation of cGMP synthesis is - a low level of cGMP synthesis and / or a low level of PKG-dependent protein phosphorylation in response to an NO donor in a cell and / or exosome-containing sample, as compared to a predetermined reference value; - a low level of cGMP synthesis and / or a low level of PKG-dependent protein phosphorylation in response to an sG C stimulator in a cell and / or exosome-containing sample, as compared to a predetermined reference value; - High-level cGMP synthesis and / or high-level PKG-dependent protein phosphorylation in response to an sGC activator in a cell and / or exosome-containing sample, as compared to a predetermined reference value; - A decrease in the ratio of: i) the level of cGMP synthesis in response to an sGC stimulator to ii) the level of cGMP synthesis in response to an sGC activator, in a cell and / or exosome-containing sample, as compared to a predetermined reference value; and / or - A decrease in the ratio of: i) the level of PKG-dependent phosphorylation in response to an sGC stimulator to ii) the level of PKG-dependent phosphorylation in response to an sGC activator, in a cell and / or exosome-containing sample, as compared to a predetermined reference value A pharmaceutical composition established thereby.

13. A pharmaceutical composition for use according to any one of claims 1 to 12, wherein the pharmaceutical composition further comprises an NO rebinder and / or an NO substrate (precursor).

14. i) An sGC (positive) regulator, preferably an sGC stimulator or an sGC activator, more preferably riociguat, vericiguat, ataciguat, neliciguat, etoriciguat, lifitegrast, IW-1701, IW-1973, IWP-051, IWP-121, IWP-427, IWP-953, BAY-60-2770, A-344905, A-350619, A-778935, BI-684067, BI-703704, BAY-41-2272, BAY-41-8543, BAY60-4552, CF-1571, cinaciguat, and HMR-1766; an sGC stimulator or an sGC activator selected from the group consisting of; ii) An NO rebinder, preferably an NO rebinder selected from the group consisting of folic acid and folates; and iii) An NO substrate or an NO substrate precursor, preferably an NO substrate or an NO substrate precursor selected from the group consisting of L-arginine, L-citrulline, their salts, their hydrates, their solvates, and combinations thereof A pharmaceutical composition comprising.

15. A method for preventive and / or therapeutic treatment of a subject suffering from or at risk of suffering from HFpEF, said method comprising administering to said subject an sGC (positive) regulator, preferably an sGC stimulator or sGC activator, more preferably riociguat, vericiguat, ataciguat, neliciguat, etoriciguat, reficiguat, IW-1701, IW-1973, IWP-051, IWP-121, IWP-427, IWP-953, BAY-60-2770, A-344905, A-350619, A-778935, BI-684067, BI-703704, BAY-41-2272, BAY-41-8543, BAY60-4552, CF-1571, cinaciguat, and HMR-1766, wherein said subject has a NO-cGMP-PKG signaling axis disorder.

16. A diagnostic method for the HFpEF endotype characterized by a dysfunction of the NO-cGMP-PKG axis in a subject, said method comprising: ● collecting whole blood from said subject with or without an anticoagulant and subsequently immediately centrifuging the blood; ● collecting plasma; ● measuring the NOX5 level in said plasma sample; ● when said NOX5 plasma level is at least 105 ng / mL, determining that said patient is suffering from HFpEF due to a dysfunction of the NO-cGMP-PKG axis comprising the steps of.

17. A diagnostic method for the HFpEF endotype characterized by a dysfunction of the NO-cGMP-PKG axis in a subject, said method comprising: ● collecting whole blood with or without an anticoagulant and subsequently immediately centrifuging the blood at a speed of 600-800×g for 7.5-15 minutes; ● collecting plasma and isolating P-WBC; ● cryopreserving P-WBC by storing at a temperature of -60 to -90°C using a cryoprotectant, preferably 6% DMSO; ● thawing the cryopreserved P-WBC; ● Using the P-WBC, determining the pVASP response (pVASP / VASP) to 1 μM lanaciguat (sGCa) in the presence of 500 μM IBMX (3-isobutyl-1-methylxanthine), and the pVASP response (pVASP / VASP) to 100 μM riociguat (sGCs) in the presence of 500 μM IBMX; ● Determining that the patient has HFpEF due to dysfunction of the NO-cGMP-PKG axis when the sGCa / sGCs ratio, defined as the response to sGCa divided by the response to sGCs, is at least 1.05; A method comprising the above steps.

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