Combination of zipotentan and dapagliflozin for treatment of chronic kidney disease

Abstract

The present disclosure relates to a combination of zipotentan and dapagliflozin, i.e., the endothelin receptor antagonist (ERA) zipotentan in combination with the sodium-dependent glucose cotransporter 2 (SGLT-2) inhibitor dapagliflozin, for use in the treatment of certain endothelin-related diseases.

Description

[0001] This application is a divisional application of the invention patent application with application date of July 9, 2021, application number 202180049043.7, and name “Combination of Zipotentan and Dapagliflozin for the treatment of chronic kidney disease”. Technical Field

[0002] The present disclosure relates to the combination of the endothelin receptor antagonist (ERA) zilpotentan and the sodium-dependent glucose cotransporter 2 (SGLT-2) inhibitor dapagliflozin for use in the treatment of certain endothelin-related diseases. Background Art

[0003] Endothelin-1 (ET-1) is a potent vasoconstrictor peptide. ET-1 (composed of endothelin A and B (ET A and E.T. B ) receptor regulation) is a highly potent systemic vasoconstrictor and a driver of renal disease progression. In chronic kidney disease (CKD), ET-1 levels increase with the urine albumin-creatinine ratio (UACR) and the severity of renal impairment (Grenda et al., Nephrol Dial Transplant. 2007; 22(12):3487-3494; Kohan Am J Kidney Dis. 1997; 29(1):2-26). The pathological effects of ET-1 (including proteinuria, vasoconstriction, and inflammation) are thought to be primarily mediated by endothelial A (ET A ) receptor drive (Goddard et al., Circulation. 2004; 109(9): 1186-1193). A Antagonists have demonstrated renal protection. In diabetic kidney disease (DKD), a 30% decrease in urine albumin-creatinine ratio (UACR) has been demonstrated (Heerspink et al., Diabetes Obes Metab. 2018; 20(8): 1829-1835; Heerspink et al., Lancet 2019; 393(10184): 1937-1947). However, clinical development using ETA receptor antagonists has been limited by problems with fluid retention and hospitalization for heart failure (Heerspink et al., Lancet 2019; 393(10184): 1937-1947). Zipotentan is an effective ET inhibitor developed for the treatment of prostate cancer. A receptor antagonist, but was discontinued in 2011 due to insufficient efficacy in Phase 3 and a 17% increase in the incidence of peripheral edema compared with placebo.

[0004] SGLT-2 inhibitors can act as osmotic diuretics due to glycosuria, thereby increasing urine volume and reducing volume overload largely independent of changes in systemic sodium load. SGLT-2 inhibitors block the reabsorption of glucose by the kidneys, increase glucose excretion, and reduce blood glucose concentrations. In addition to this well-characterized mode of action, SGLT-2 inhibitors also lower blood pressure, reduce vascular stiffness, improve endothelial function, and have anti-inflammatory and anti-fibrotic properties similar to ERA (HJHeerspink et al., Circulation [Circulation] (2016), 134 (10): 752-772). SGLT2 inhibitors have been shown to be effective for DKD (Stephens et al., Diabetes Obes Metab. [Diabetes Obes Metab. [Diabetes Obesity and Metabolism] 2020; 22 Supplement 1: 32-45). The DAPA-CKD trial is studying the efficacy of dapagliflozin for CKD, which was recently terminated early due to overwhelming efficacy. A posthoc analysis of the SONAR trial presented at the 2019 American Society of Nephrology annual Kidney Week meeting in Washington, D.C., showed that patients taking ET A In the subset of DKD patients (n=14) taking both the antagonist atrasentan and an SGLT2 inhibitor, the decrease in UACR was increased compared with atrasentan alone, and the weight gain caused by atrasentan, an alternative to fluid retention, was reduced.

[0005] Zipotentan (N-(3-methoxy-5-methylpyrazin-2-yl)-2-[4-(1,3,4-oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide) has the chemical structure of Formula I

[0006]

[0007] Zipotentan (also known as ZD4054) has been disclosed as an endothelin receptor antagonist together with details of the chemical synthesis of zilpotentan in WO 1996040681. Specific inhibition of endothelin A receptors by zilpotentan has been reported by Morris et al., British Journal of Cancer (2005), 92, 2148-2152.

[0008] Dapagliflozin ((1S)-1,5-anhydride-1-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-glucitol) has the chemical structure of Formula II

[0009]

[0010] Dapagliflozin is a potent, highly selective and orally active human renal sodium-dependent glucose transporter 2 (SGLT2) inhibitor (SGLT2i) approved to improve glycemic control in adults with type 2 diabetes (as an adjunct to diet and exercise) and to reduce the risk of hospitalization for heart failure in adults with type 2 diabetes and established cardiovascular disease or multiple cardiovascular risk factors. Dapagliflozin has been disclosed in WO 2003099836 together with details of its chemical synthesis.

[0011] When the endothelin A receptor specific inhibitor zilpotentan is combined with the SGLT-2 inhibitor dapagliflozin, the diuretic effect of dapagliflozin may be mitigated by the fluid retention side effect associated with the endothelin receptor antagonist zilpotentan, thereby mitigating the risks associated with each of the two individual compounds. Summary of the invention

[0012] In the present specification, dapagliflozin has been shown to mitigate zilpotentan-driven hemodilution, where hemodilution is defined as a decrease in hematocrit concentration, and thus highlights the potential for the combination of zilpotentan and dapagliflozin to demonstrate efficacy in treating certain endothelin-related diseases.

[0013] Endothelin-1 (composed of endothelin A and B (ET A and E.T. B ) receptor regulation) is a highly potent systemic vasoconstrictor and driver of renal disease progression. A Antagonist blockade improves renal function, while ET B Blocking is not preferred because ET B Also clears circulating endothelin-1.

[0014] Because endothelin appears in many cardio-renal metabolic diseases, endothelin-related diseases are associated with the increase of vasoconstriction, proliferation or inflammation. Examples of such endothelin-related diseases are hypertension, coronary artery disease, heart failure, renal ischemia and myocardial ischemia, chronic kidney disease (CKD). In particular, CKD is associated with hypertension or diabetes (diabetic kidney disease, DKD). ERA may be useful in treating peripheral arterial occlusive diseases, including diabetic arterial disease, by having acute (peripheral vasodilation) and chronic (vasodilation, improvement of vascular structure and regulation of sympathetic nerve activity, antithrombotic, anti-inflammatory) effects.

[0015] The mode of action of SGLT-2 results in the simultaneous inhibition of glucose and sodium uptake in the proximal tubule of the nephron, which is thought to result in a resetting of tubular-glomerular feedback, presumably leading to the phenomenon of glomerular hyperfiltration. The efficacy of SGLT-2 inhibitors is thought to decrease with decreasing plasma glucose levels or with decreasing glomerular filtration rate (GFR), and therefore, SGLT-2 inhibitors have an inherently low risk of developing hypoglycemia. Therefore, the properties of SGLT-2 inhibitors may open up an avenue for the treatment of HF, including HFpEF, even in non-diabetic patients (P. Martens et al., Curr Treat Options Cardio Med [Current Treatment Options in Cardiovascular Medicine] (2017), 19:23).

[0016] Side effects associated with the pharmacological actions of SGLT-2 inhibitors are volume depletion / intravascular volume contraction, which may lead to dehydration, hypovolemia, orthostatic hypotension or hypotension. Thus, SGLT-2 inhibitors generally induce an increase in the hematocrit (Hot, a marker of blood concentration) and increased blood viscosity, which is a putative cause of vascular damage in the context of peripheral vascular disease.

[0017] Through the pharmacological action of SGLT-2 inhibitors, serum creatinine increases and eGFR decreases. AWhen the prescription of ERA (ERA of receptor) combined with dapagliflozin is combined, zilpotentan may be suitable for treating endothelin-related diseases. When ERA is combined with SGLT-2 inhibitor, the diuretic effect of this SGLT-2 inhibitor and its potential pharmacological effect in reducing the risk of heart failure may be suitable for alleviating the most prominent side effects usually associated with ERA, such as fluid retention and the risk of increased congestive heart failure associated with potential. This combination therapy can produce a pharmacological effect on the disclosed endothelin-related diseases, while maintaining a favorable side effect curve even at the best effective dose of zilpotentan, and even when compared with the maximum tolerated dose of zilpotentan alone, a favorable side effect curve can be maintained at an increased dose of zilpotentan. When zilpotentan is used in combination with dapagliflozin, the increased dose of zilpotentan that can be obtained (for example, due to the reduced side effects) can allow for an expansion of the effect on diseases caused by the harmful effects of the endothelin paracrine system widely distributed in the organism. This combination therapy can improve the benefit / risk ratio. ERAs have been described as reducing hematocrit (Hot) via hemodilution. Therefore, zilpotentan, when used in combination with dapagliflozin, can antagonize the most prominent side effects commonly associated with SGLT-2 inhibitors, such as hemoconcentration due to the volume depletion effect. ERAs have been described as improving blood glucose levels by various mechanisms (increasing blood flow, improving insulin signaling). Therefore, zilpotentan, when used in combination with dapagliflozin, can have an additive or even synergistic effect on blood glucose reduction.

[0018] In a first aspect, zilpotentan for use in treating chronic kidney disease (CKD) in a human patient is provided, wherein zilpotentan is administered in combination with dapagliflozin.

[0019] In another aspect, a method of treating chronic kidney disease (CKD) in a human patient in need of such treatment is provided, the method comprising administering to the human patient a therapeutically effective amount of zilpotentan, wherein zilpotentan is administered in combination with a therapeutically effective amount of dapagliflozin.

[0020] In another aspect, there is provided a use of zilpotentan in the preparation of a medicament for treating chronic kidney disease (CKD) in a human patient, wherein zilpotentan is administered in combination with dapagliflozin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Individual zilpotentan blood concentrations following oral administration of zilpotentan in Example 1, Experiment 1 are shown.

[0022] Figure 2 Individual zilpotentan blood concentrations following oral administration of zilpotentan alone or in combination with dapagliflozin in Example 1 Experiment 2 are shown.

[0023] Figure 3 Individual dapagliflozin blood concentrations after oral administration of dapagliflozin alone or in combination with zilpotentan in Experiment 2 of Example 1 are shown.

[0024] Figure 4 The effect of Zipotentan on Hct concentration in Example 1 Experiment 1 is shown.

[0025] Figure 5 The effect of zilpotentan on body weight in Example 1, Experiment 1 is shown.

[0026] FIG6 shows the effects of zilpotentan, dapagliflozin and the combination on Hct in Experiment 2 of Example 1.

[0027] Figure 7 The effects of zilpotentan, dapagliflozin and the combination on body weight in Experiment 2 of Example 1 are shown.

[0028] FIG8 shows the effects of dapagliflozin and zilpotentan on glucose, water and food intake in Experiment 2 of Example 1.

[0029] Fig. 9 The UACR-lowering effect of dapagliflozin in CKD patients (with or without type 2 diabetes) from the DAPA CKD Phase 3 trial is shown.

[0030] Fig.10 Shown are the regression and progression of UACR in CKD patients (stratified by diabetes status) from the DAPA CKD Phase 3 trial. DETAILED DESCRIPTION

[0031] In a first aspect, zilpotentan for use in treating chronic kidney disease (CKD) in a human patient is provided, wherein zilpotentan is administered in combination with dapagliflozin.

[0032] In another aspect, a method of treating chronic kidney disease (CKD) in a human patient in need of such treatment is provided, the method comprising administering to the human patient a therapeutically effective amount of zilpotentan, wherein zilpotentan is administered in combination with a therapeutically effective amount of dapagliflozin.

[0033] In another aspect, there is provided a use of zilpotentan in the preparation of a medicament for treating chronic kidney disease (CKD) in a human patient, wherein zilpotentan is administered in combination with dapagliflozin.

[0034] In certain embodiments, chronic kidney disease (CKD) is stage 1 to 4 CKD as defined by the Kidney Disease Improving Global Outcomes (KDIGO) guidelines.

[0035] In certain embodiments, the CKD is stage 2-3 CKD.

[0036] In certain embodiments, the CKD is stage 3-4 CKD.

[0037] In certain embodiments, the CKD is stage 4 CKD.

[0038] In certain embodiments, the CKD is stage 3a or 3b CKD.

[0039] Zipotentan (N-(3-methoxy-5-methylpyrazin-2-yl)-2-[4-(1,3,4-oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide) has the chemical structure of Formula I

[0040]

[0041] Zipotentan is also known as ZD4054.

[0042] In embodiments, zilpotentan or a pharmaceutically acceptable salt thereof is administered once daily.

[0043] In an embodiment, the total daily dose of zilpotentan is about 10 mg.

[0044] In an embodiment, the total daily dose of zilpotentan is about 5 mg.

[0045] In an embodiment, the total daily dose of zilpotentan is about 1.5 mg.

[0046] In an embodiment, the total daily dose of zilpotentan is about 0.5 mg.

[0047] In an embodiment, the total daily dose of zilpotentan is about 0.25 mg.

[0048] In an embodiment, zilpotentan or a pharmaceutically acceptable salt thereof is in tablet form.

[0049] In an embodiment, zilpotentan or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. In further embodiments, the composition comprises one or more pharmaceutical diluents, one or more pharmaceutical disintegrants, or one or more pharmaceutical lubricants.

[0050] Dapagliflozin ((1S)-1,5-anhydride-1-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-glucitol) has the chemical structure of Formula II

[0051]

[0052] In an embodiment, dapagliflozin or a pharmaceutically acceptable salt thereof is administered once daily.

[0053] In at least one embodiment, dapagliflozin is in the form of a pharmaceutically acceptable solvate, mixed solvate, or complex. In some aspects provided herein, dapagliflozin is in the form of a non-crystalline solid. In some aspects provided herein, dapagliflozin is in the form of a crystalline solid. In some aspects provided herein, dapagliflozin is in the form of a (S)-propylene glycol ((S)-PG) solvate having the following structure:

[0054]

[0055] In at least one embodiment, dapagliflozin is in the form of a pharmaceutically acceptable solvate, mixed solvate, or complex. In some aspects provided herein, dapagliflozin is in the form of a non-crystalline solid. In some aspects provided herein, dapagliflozin is in the form of a crystalline solid.

[0056] In some aspects provided herein, the pharmaceutical composition is administered to the patient orally. In some aspects provided herein, the pharmaceutical composition is administered to the patient in tablet form.

[0057] In some aspects provided herein, a pharmaceutical composition containing dapagliflozin comprises a dose equivalent of about 2.5 mg / day to about 10 mg / day of dapagliflozin, which is administered to a patient. In some aspects provided herein, a pharmaceutical composition containing dapagliflozin comprises a dose equivalent of about 2.5 mg / day, about 5 mg / day, or about 10 mg / day of dapagliflozin, which is administered to a patient. In some aspects provided herein, a pharmaceutical composition containing dapagliflozin comprises a dose equivalent of about 5 mg / day of dapagliflozin, which is administered to a patient once daily. In some aspects provided herein, a pharmaceutical composition containing dapagliflozin comprises a dose equivalent of about 10 mg / day of dapagliflozin, which is administered to a patient once daily.

[0058] In one aspect, zilpotentan for use in treating CKD in a human patient is provided, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient.

[0059] In one aspect, a method for treating CKD in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient.

[0060] In one aspect, there is provided the use of zilpotentan in the preparation of a medicament for treating CKD in a human patient, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient.

[0061] On the one hand, there is provided a zipotentan for reducing UACR in a human patient, comprising administering to the human patient separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0062] In one aspect, a method for reducing UACR in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0063] In one aspect, there is provided the use of zilpotentan in the preparation of a medicine for reducing the UACR of a human patient, wherein the treatment includes administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0064] On the one hand, there is provided a zipotentan for reducing UACR in a human patient to <300 mg / g, comprising administering to a human patient separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0065] In one aspect, a method is provided for reducing UACR to <300 mg / g in a human patient in need of such treatment, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0066] In one aspect, there is provided the use of zilpotentan in the preparation of a medicine for reducing the UACR of a human patient to <300 mg / g, wherein the treatment includes administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0067] On the one hand, there is provided a zipotentan for reducing the risk of progression in a human patient to UACR≥3000mg / g, comprising administering to a human patient: i) zipotentan, and ii) dapagliflozin, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0068] In one aspect, a method for reducing the risk of progression to UACR ≥ 3000 mg / g in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0069] In one aspect, there is provided the use of zilpotentan in the preparation of a drug for reducing the risk of progression of a human patient to UACR≥3000mg / g, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0070] On the one hand, there is provided a zipotentan for reducing the risk of eGFR decline in human patients, comprising administering to human patients separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In another embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In another embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In another embodiment, the human patient is a CKD human patient with type 2 diabetes. In other embodiments, the human patient is a CKD human patient without type 2 diabetes. In some embodiments, the use reduces the risk of eGFR decline of ≥30%. In some embodiments, the use reduces the incidence of eGFR decline of ≥40%.

[0071] In one aspect, a method for reducing the risk of eGFR decline in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient separately, sequentially or simultaneously. In another embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In another embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In another embodiment, the human patient is a CKD human patient with type 2 diabetes. In other embodiments, the human patient is a CKD human patient without type 2 diabetes. In some embodiments, the method reduces the risk of eGFR decline of ≥ 30%. In some embodiments, the method reduces the incidence of a decrease in eGFR of ≥ 40%.

[0072] In one aspect, there is provided the use of zilpotentan in the preparation of a medicament for reducing the risk of eGFR decline in a human patient, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient with type 2 diabetes. In other embodiments, the human patient is a CKD human patient without type 2 diabetes. In some embodiments, the use reduces the risk of eGFR decline of ≥30%. In some embodiments, the use reduces the incidence of eGFR decline of ≥40%.

[0073] On the one hand, there is provided a zilpotentan for reducing the risk of fluid retention (edema) in a human patient, comprising administering to a human patient separately, sequentially or simultaneously: i) zilpotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes. In some embodiments, the reduction in the risk of fluid retention (edema) can be measured by, for example, a reduction in Hct or an increase in body weight.

[0074] In one aspect, a method for reducing the risk of fluid retention (edema) in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes. In some embodiments, the reduction in risk of fluid retention (edema) can be measured by, for example, a reduction in Hct or an increase in body weight.

[0075] In one aspect, there is provided the use of zilpotentan in the preparation of a medicine for reducing the risk of fluid retention (edema) in a human patient, wherein the treatment includes administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes. In some embodiments, the reduction in the risk of fluid retention (edema) can be measured by, for example, a reduction in Hct or an increase in body weight.

[0076] On the one hand, there is provided a zipotentan for reducing the total water content in a human patient, comprising administering to the human patient separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0077] In one aspect, a method for reducing total body water in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0078] In one aspect, there is provided the use of zilpotentan in the preparation of a medicament for reducing the total body water of a human patient, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0079] On the one hand, there is provided a zipotentan for reducing blood pressure in a human patient, comprising administering to the human patient separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0080] In one aspect, a method for lowering blood pressure in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0081] In one aspect, there is provided the use of zilpotentan in the preparation of a medicament for reducing the blood pressure of a human patient, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0082] On the one hand, there is provided a zipotentan for reducing the risk of elevated blood pressure in a human patient, comprising administering to a human patient separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0083] In one aspect, a method for reducing the risk of elevated blood pressure in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0084] In one aspect, there is provided the use of zilpotentan in the preparation of a medicament for reducing the risk of elevated blood pressure in a human patient, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0085] On the one hand, there is provided a method for reducing the incidence of a composite endpoint of end-stage renal disease (ESKD) and cardiovascular or renal death in a human patient by decreasing eGFR by ≥40%, which includes administering to a human patient separately, sequentially or simultaneously: i) zilpotentan, and ii) dapagliflozin. In another embodiment, the human patient is a CKD human patient. In another embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In another embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In another embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0086] In one aspect, a method is provided for reducing the composite endpoint of eGFR decline>40%, reaching end-stage renal disease (ESKD), and the incidence of cardiovascular or renal death in a human patient in need of such treatment, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0087] In one aspect, there is provided a use of zilpotentan in the preparation of a drug for reducing the composite endpoint of eGFR decline>40%, reaching end-stage renal disease (ESKD), and the incidence of cardiovascular or renal death in a human patient, wherein the treatment includes administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In another embodiment, the human patient is a CKD human patient. In another embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In another embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In another embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In another embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In another embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0088] On the one hand, there is provided a zipotentan for reducing the incidence of end-stage renal disease (ESKD) in human patients, comprising administering to human patients separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0089] In one aspect, a method for reducing the incidence of end-stage renal disease (ESKD) in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0090] In one aspect, there is provided the use of zilpotentan in the preparation of a medicine for reducing the incidence of end-stage renal disease (ESKD) in a human patient, wherein the treatment includes administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b phase patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 phase patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0091] On the one hand, there is provided a zipotentan for reducing the incidence of cardiovascular or renal death in a human patient, comprising administering to a human patient separately, sequentially or simultaneously: i) zipotentan, and ii) dapagliflozin. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient who does not suffer from type 2 diabetes.

[0092] In one aspect, a method for reducing the incidence of cardiovascular or renal death in a human patient in need of such treatment is provided, the method comprising administering a therapeutically effective amount of zilpotentan to the human patient, wherein the treatment comprises administering i) a therapeutically effective amount of zilpotentan and ii) a therapeutically effective amount of dapagliflozin to the human patient, respectively, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0093] In one aspect, there is provided the use of zilpotentan in the preparation of a medicament for reducing the incidence of cardiovascular or renal death in a human patient, wherein the treatment comprises administering i) zilpotentan and ii) dapagliflozin to the human patient separately, sequentially or simultaneously. In a further embodiment, the human patient is a CKD human patient. In a further embodiment, the human patient is a CKD human patient classified as a 1-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3-4 stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 4-stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 3a or 3b stage patient. In a further embodiment, the human patient is a CKD human patient classified as a 2-3 stage patient. In a further embodiment, the human patient is a CKD human patient suffering from type 2 diabetes. In other embodiments, the human patient is a CKD human patient not suffering from type 2 diabetes.

[0094] In any of the above aspects, the methods and uses thereof can also be related to human patients who receive at least one single SGLT2-I (e.g., dapagliflozin, empagliflozin, canagliflozin, etc.) or a combination thereof with at least one standard CKD care agent. In these aspects, the standard CKD care agent can be ACE-I (e.g., captopril, enalapril and lisinopril) and / or ARB (valsartan, losartan and irbesartan).

[0095] In any of the above aspects, the methods and uses thereof can be related to baseline patients. In some embodiments, the methods and uses thereof can be related to baseline patients compared to patients receiving at least one single SGLT2-I or a combination thereof with standard CKD care.

[0096] In one aspect, a kit is provided, the kit comprising:

[0097] a first pharmaceutical composition comprising zilpotentan and a pharmaceutically acceptable carrier; and

[0098] A second pharmaceutical composition comprising dapagliflozin and a pharmaceutically acceptable carrier.

[0099] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, relieve the symptoms of, and / or stop the progression of a diagnosed pathological condition or disorder. Thus, those patients in need of treatment include those who have been diagnosed with or suspected of having the condition. Patients or subjects in need of treatment may include those diagnosed with CKD.

[0100] "Therapeutically effective amount" or "effective amount" refers to the amount of at least one compound of the present disclosure or a pharmaceutical composition comprising at least one such compound that is effective to produce at least one therapeutic effect when administered to a patient as a single dose or as part of a series of doses. The optimal dose can generally be determined using experimental models and / or clinical trials. The design and implementation of preclinical and clinical studies of each therapeutic agent described herein (including when administered for preventive benefit) are within the skill of those of ordinary skill in the relevant art. The optimal dose of the therapeutic agent may depend on the patient's constitution, weight, and / or blood volume. The effectiveness of the treatment of a patient can generally be monitored using assays suitable for the disease, disorder, and / or condition being treated or prevented, which assays are familiar to those of ordinary skill in the art and are described herein. The level of the compound administered to the patient can be monitored by determining the level of the compound (or metabolite of the compound) in a biological fluid such as blood, a blood fraction (e.g., serum), and / or urine, and / or other biological samples from the patient. During the course of the treatment regimen, the level of the compound can be measured using any method practiced in the art for detecting a compound or its metabolite. Alternatively, the pharmacological and / or physiological effect can be preventive, that is, the effect completely or partially prevents a disease or its symptoms. In this regard, the disclosed methods include administering a "prophylactically effective amount" of a drug (e.g., zilpotentan or a pharmaceutically acceptable salt, solvate, mixed solvate, complex or prodrug thereof, and dapagliflozin or a pharmaceutically acceptable salt, solvate, mixed solvate, complex or prodrug thereof). A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive outcome (e.g., preventing CKD or disease onset) at the desired dosage and time period.

[0101] As used herein, the terms "subject" and "patient" are used interchangeably. In some aspects, the subject is a human.

[0102] As used herein, the term "end-stage renal disease (EKSD)" refers to (i) having an eGFR that is persistently <15 mL / min / 1.73 m2, (ii) receiving chronic dialysis treatment, or (iii) receiving a kidney transplant. In some embodiments, "persistent" means that a second eGFR test 3 months apart confirms a similar eGFR measurement.

[0103] As used herein, the terms "administer, administering, administration," and the like refer to methods that can be used to enable delivery of a drug (e.g., zilpotentan or a pharmaceutically acceptable salt, solvate, mixed solvate, complex, or prodrug thereof, and dapagliflozin or a pharmaceutically acceptable salt, solvate, mixed solvate, complex, or prodrug thereof, as described herein). Administration techniques that can be used with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa. In some aspects, zilpotentan and dapagliflozin are administered orally.

[0104] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to preparations that are in such form that the biological activity of one or more active ingredients is effective and that do not contain additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such preparations may be sterile.

[0105] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier commonly used in the art for use with therapeutic agents and together constituting a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0106] A "sterile" formulation is aseptic or substantially free of viable microorganisms and their spores.

[0107] As used herein, the term "prodrug" refers to esters and carbonates that can be converted to zilpotentan or dapagliflozin, for example, under physiological conditions or by solvolysis. Therefore, the term prodrug includes pharmaceutically acceptable metabolic precursors of zilpotentan or dapagliflozin. The term prodrug also includes covalently bound carriers that release zilpotentan or dapagliflozin in vivo when such prodrugs are administered to patients. Non-limiting examples of prodrugs include esters and carbonates.

[0108] Various forms of prodrugs are known in the art. For examples of such prodrug derivatives, see: (1) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, pp. 309-396, edited by K. Widder et al. (Academic Press, 1985); (2) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 "Design and Application of Pro-drugs", H. Bundgaard, pp. 113-191 (1991); (3) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); (4) H. Bundgaard et al., Journal of Pharmaceutical Design and Development, vol. 42, pp. 309-396, edited by K. Widder et al. (Academic Press, 1985); Sciences, 77, 285 (1988); and (5) N. Kakeya et al., Chem Pharm Bull, 32, 692 (1984).

[0109] It should be understood that wherever aspects are described herein with the language "comprising", other similar aspects described with respect to "consisting of" and / or "consisting essentially of" are also provided. In the present disclosure, "comprises or comprising", "containing", and "having" and the like may mean "includes or including" and the like; "consisting essentially of or consists essentially of" is open-ended, allowing for existence beyond what is recited, as long as the basic or novel features recited are not altered by existence beyond what is recited, but excluding prior art aspects.

[0110] Unless explicitly stated or obvious from the context, as used herein, the term "or" is to be understood as being inclusive.

[0111] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range indicate that deviations of 10% above and 10% below the numerical value or range are still within the intended meaning of the recited value or range. It should be understood that wherever aspects are described herein using the language "about" or "approximately" numerical values ​​or ranges, other similar aspects that refer to specific numerical values ​​or ranges (without "about") are also provided.

[0112] Example

[0113] Experimental Procedure

[0114] Example 1

[0115] The effects of dapagliflozin and zilpotentan on hematocrit (Hct) concentration in male Wistar rats fed a 4% salt diet were studied.

[0116] In Experiment 1, zilpotentan was orally administered once daily at 30, 100 or 300 mg / kg for 14 days. On Day 7, all three doses of zilpotentan resulted in a significant decrease in Hct concentration compared to vehicle (p<0.05). On Day 14, a significant decrease in Hct concentration was found at higher doses of zilpotentan (100 mg / kg and 300 mg / kg) compared to vehicle (p<0.05), but not at a lower dose of zilpotentan (30 mg / kg).

[0117] In Experiment 2, Hct concentrations were measured 7 days after administration of zilpotentan (30 or 100 mg / kg) or dapagliflozin (3.0 mg / kg) alone or in combination in male Wistar rats fed a 4% salt diet. Zilpotentan (30 or 100 mg / kg) significantly reduced Hct concentrations. Co-administration of dapagliflozin (3.0 mg / kg) with zilpotentan (30 mg / kg) resulted in Hct concentrations that were not significantly different from vehicle-treated animals. As revealed by one-way ANOVA, post hoc Tukey's test, co-administration of dapagliflozin (3.0 mg / kg) with zilpotentan (100 mg / kg) attenuated the effect of zilpotentan on Hct concentration (zilpotentan -4.00±0.87 vs. zilpotentan+dapagliflozin -1.72±0.68, # p<0.05). Zipotentan did not affect the effect of dapagliflozin on urinary glucose excretion.

[0118] Materials and methods

[0119] Formulation for Zipotentan alone: ​​Suspension in 30% (w / w) TEG, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000 cps, 0.1% (w / w) Tween 80, 67.4% purified water

[0120] Formulation for dapagliflozin alone: ​​solution in 30% (w / w) TEG, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000 cps, 0.1% (w / w) Tween 80, 67.4% purified water

[0121] Formulation for the combination of zilpotentan and dapagliflozin: Suspension / solution in 30% (w / w) TEG, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000cps, 0.1% (w / w) Tween80, 67.4% purified water

[0122] The study was divided into two experiments.

[0123] In the first experiment (Experiment 1), the effect of zilpotentan on hematocrit (Hct) concentration was determined when zilpotentan was administered QD orally (po) for 14 days. Three days before the first administration of the study drug, animals were randomized into the following groups based on body weight (BW); 1) vehicle, 2) zilpotentan (30 mg / kg), 3) zilpotentan (100 mg / kg), 4) zilpotentan (300 mg / kg). Throughout the experiment, animals were weighed every day before dosing. Blood samples (20 μL) were taken from awake animals via the tail vein at 4 hr and 24 hr after the first dose, and from anesthetized animals via the retro-orbital route 24 hr after the last dose for bioanalysis. Blood samples (100 μL) were collected via the tail vein 1-2 hr after dosing on the 7th and 14th days for Hct concentration and hemoglobin (Hb) measurements. Twenty-four-hour urine was collected between doses nine and ten for measurement of urine glucose, urine volume, and electrolytes. Animals were terminated 24 hr after the last dose. On the day of termination, animals were anesthetized with 5% isoflurane and blood was collected via the retro-orbital route (placed in a heparin-coated microvette, Sarsted) and the blood was centrifuged at 3,500 rpm for 10 minutes at 4°C, and plasma was collected and stored at -80°C before analysis. The animals were then euthanized by removing the heart. The heart and kidneys were removed and weighed. 4 mm thick tissue sections were collected from the right kidney and fixed in 4% formaldehyde for subsequent histological analysis, and a piece of cortical kidney tissue and heart tissue were collected for gene expression analysis.

[0124] In the second experiment (Experiment 2), the effects of zilpotentan and dapagliflozin (alone or in combination) on Hct concentration and Hb were determined. 2 to 3 days before the study drug was administered, 72 animals were randomly divided into the following groups based on BW and Hct concentration; 1) vehicle, 2) zilpotentan (30 mg / kg), 3) zilpotentan (100 mg / kg), 4) dapagliflozin (3 mg / kg), 5) zilpotentan (30 mg / kg) + dapagliflozin (3 mg / kg) and 6) zilpotentan (100 mg / kg) + dapagliflozin (3 mg / kg). For practical reasons, the study was conducted in two consecutive tanks, with 36 animals (6 / group) in each tank. Compound poQD was administered for seven days. On the third and seventh days, blood samples for bioanalysis were collected at 4hr and 24hr after the first dose and 24hr after the last dose for Hct concentration and Hb analysis, as described in Experiment 1. Twenty-four-hour urine was collected between the third and fourth doses. Twenty-four-hour food and water intake was measured between days 2-3 and between days 6-7. There were two animals in each cage, and the food and water intake of each animal in the cage was estimated by dividing the food and water intake of the cage by two.

[0125] The experimenters were blinded to the treatment assignment of the animals during termination, blood sampling, and sample analysis.

[0126] The blood and urine concentrations of zilpotentan and dapagliflozin were determined by a bioanalytical method using liquid chromatography with mass spectrometry detection (LC-MS / MS).

[0127] Whole blood samples for bioanalysis of dapagliflozin and zilpotentan followed the same procedure. A volume of 20 μL of whole blood was precipitated with 150 μL of acetonitrile containing internal standard and quickly vortexed, followed by centrifugation at 3220 g and 4 °C for 20 min. The supernatant was transferred to a fresh deep-well plate and diluted 1:1 with water prior to LC-MS analysis. Matrix-matched calibration samples and blanks were processed in the same manner as the study samples. The zilpotentan samples were further diluted two and three times with 33% acetonitrile at a ratio of 1:10 to ensure that all diluted samples were detectable within the calibration range.

[0128] Using Waters TargetLynx software version XS V4.2 SCN986, weighted linear regression (1 / X 2 ), sample concentrations were determined relative to matrix-spiked calibration standards in the range of 0.010 μM-10.0 μM (dapagliflozin) and 0.75 μM-376 μM (zipotentan). Calibration sample residuals showed a homoscedastic distribution. Analytical bias was <15% over the concentration range for both analytes.

[0129] The samples were analyzed by reversed phase high pressure liquid chromatography with rapid gradient elution. The compounds were detected by Waters Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA). Chromatographic separation was performed by ACQUITY BEH C18 1.8 μM, 2.1x50 mm column at a temperature of 40 ° C. Mobile phase A was 2% acetonitrile and 0.2% formic acid in water (A), and mobile phase B was 0.2% formic acid in acetonitrile (B). The separation was completed using the following elution gradient: 0 min to 1.5 min, 4% B increased to 95% B; 1.5 min to 2.3 min, maintained at 95% B; 2.3 min to 2.4 min, 95% B to 4% B and maintained at 4% B until 2.7 min. The flow rate was 0.7 mL / min. Zipotentan eluted after 0.82 min and was detected using multiple reaction transition monitoring (425.3 m / z > 139.25 m / z) in positive electrospray mode.

[0130] The samples were analyzed by reversed-phase high pressure liquid chromatography with rapid gradient elution. Compounds were detected using a Waters XevoTQ-S triple quadrupole mass spectrometer (Waters, Milford, Massachusetts, USA). Mobile phase A was 1M ammonium acetate / acetonitrile / water (0.1 / 2 / 97.5, v / v / v), and mobile phase B was 1M ammonium acetate / acetonitrile (0.1 / 99.5, v / v). Separation was performed using the following gradient on an ACQUITY BEH C18 1.8 μM, 2.1 × 50 mm column from Waters: 0 min to 2.5 min, 4% B increased to 60% B; 2.5 min to 2.9 min, 60% B to 95% B; 2.9 min to 3.4 min remained at 95% B, and from 3.4 min to 3.5 min mobile phase B was returned to 4% B and remained at 4% B until 4 min. The flow rate was 0.6 mL / min and the column temperature was 40° C. Dapagliflozin eluted after 2.19 min and was detected in negative electrospray mode, with a transition of 407.1>329.07 for dapagliflozin and 1.390.7 for 13 C 6 -Dapagliflozin internal standard, conversion 413.0>335.13.

[0131] Bioanalysis of Zipotentan in Rat Urine: 50 μL of PBS containing 5% BSA was added to 50 μL of urine sample and then added to 500 μL of acetonitrile containing nifedipine as internal standard. The sample was vortexed for 1 min and centrifuged at 2400 g for 5 min at 4 °C. 400 μL of supernatant was transferred to a new plate and dried under heated nitrogen. The sample was reconstituted in 100 μL of deionized water / formic acid (100 / 0.2, v / v), vortexed and centrifuged before analysis. Matrix-matched calibration samples, quality control samples and blanks were processed in the same manner as the study samples. The samples were analyzed on an Acquity i-class UPLC system (Waters, Milford, MA, USA) coupled to an API 4500 (Sciex LLC, Framingham, MA, USA). Mobile phase A was acetonitrile containing 0.2% formic acid, and mobile phase B was water containing 0.2% formic acid. Separation was performed by Acquity UPLC BEH C18 column (50 mm×2.1 mm, 1.7 um particle size, from Waters) using the following gradient: 0 min to 1.5 min, 5% A to 100% A; 1.5 min to 2.0 min, 100% A to 100% A; 2.0 min to 2.5 min, 100% A to 5% A. The flow rate was 0.75 mL / min and the column temperature was 60° C. Zipotentan (425.1 m / z>361.2 m / z) was analyzed in positive ionization mode, the source temperature was 700° C., and the ion spray voltage was 5000 V.

[0132] The lower limit of quantitation (LLOQ) was 0.010 μM with an analytical bias of <15%, except for the lowest QC standard, where the CV% was 13.6% and the bias was 21.0%.

[0133] Bioanalysis of dapagliflozin in rat urine: 50 μL of PBS containing 5% BSA was added to 50 μL of urine sample and then added to 500 μL of acetonitrile / formic acid (100 / 0.5, v / v) containing nifedipine as internal standard. The sample was vortexed for 1 min and centrifuged at 2400 g for 5 min at 4 ° C. 450 μL of supernatant was transferred to a new plate and dried under heated nitrogen. The sample was reconstituted in 400 μL of 1M ammonium acetate / water / formic acid (20 / 75 / 5, v / v / v) and vortexed. The sample was further purified by SPE using SOLA HRP (10 mg) solid phase extraction (SPE) plates (ThermoFisher Scientific, Waltham, MA, USA) on a liquid handling robot. The plate is adjusted with 450 μL of methanol followed by 450 μL of 1M ammonium acetate / water / formic acid (20 / 75 / 5, v / v / v). The sample is loaded onto the SPE plate and washed with 450 μL of 1M ammonium acetate / water / formic acid (20 / 75 / 5, v / v / v) and 450 μL of acetonitrile / water (95 / 5, v / v). The sample is eluted twice with 250 μL of acetonitrile / deionized water / acetic acid (80 / 20 / 1, v / v / v) and dried under a stream of heated nitrogen. The sample is reconstructed, vortexed and centrifuged in 100 μL of deionization, and then analyzed. The calibration sample, quality control sample and blank of matrix matching are processed in the same manner as the research sample.

[0134] Samples were analyzed on an Acquity i-class UPLC system (Milford Waters, Massachusetts, USA) coupled to an API 4500 (Sciex, Inc., Framingham, Massachusetts, USA). Mobile phase A was 1M ammonium acetate / acetonitrile / water (0.5 / 95 / 4.5, v / v / v), and mobile phase B was 1M ammonium acetate / acetonitrile / water (0.5 / 25 / 74.5, v / v / v). Separation was performed using the following gradient by an Acquity UPLC HSS T3 column (50mm×2.1mm, 1.8μm particle size, from Waters): 0min to 3.0min, 5% A to 50% A; 3.0min to 3.5min, 50% A to 100% A; 3.5min to 4min, 100% A to 5% A. Flow rate was 0.45mL / min and column temperature was 25°C. The acetate adduct of dapagliflozin (467.2 m / z > 329.0 m / z) was used for quantification and analyzed in negative ionization mode with a source temperature of 750 °C and an ion spray voltage of -4500 V. The LLOQ was 0.010 μM, and the CV and bias were typically <5% (at the lowest QC standard, 0.03 μM, the CV was 10.3%).

[0135] Plasma and urine albumin, creatinine, glucose, urea, potassium, and sodium levels were analyzed using an ABX Pentra 400 instrument (Horiba Medical, Irvine, California, USA) according to the manufacturer's protocol.

[0136] For Hct and Hb analysis, 100 μl of blood was collected from the rat tail vein and measured using a CG8 cartridge and analyzed using an iSTAT instrument (Abbott Point of Care Inc, Abbot Park, IL, USA).

[0137] The data of each measured feature were checked for normal distribution, and R was used to logarithmically transform the non-normally distributed data when appropriate. Since the data were grouped by single-factor zilpotentan dose or combination treatment, one-way ANOVA was used to analyze the traits measured at a single time point (GraphPad version 8 or R statistical package emmeans), and multiple measurements of body weight were analyzed using R's linear mixed effects model (nlme package). When comparing the treatment group with the vehicle group, the differences between the groups were tested using Dunnett's test (R's emmeans package or GraphPad v8) or Tukey's comparison (R's emmeans package or GraphPad v8) for specific pairwise comparisons of the treatment group. All model assumptions were checked using the model diagnostic diagram in R statistics. The results are expressed as the mean ± standard error of the mean (SEM) of the pharmacodynamic parameters and the mean ± standard deviation (SD) of the compound concentrations in blood and urine. Significance was set at p<0.05.

[0138] result

[0139] Exposure to zilpotentan was confirmed by measuring zilpotentan concentrations above the LLOQ in all blood samples collected from animals receiving zilpotentan at 4 hr or 24 hr after the first dose or 24 hr after the last dose (Tables 1 and 2, Figure 1 and Figure 2 ).

[0140] Table 1 Zibo blood concentration in Experiment 1 (μmol / L)

[0141]

[0142] Data presented are mean ± SD (number of samples).

[0143] LLOQ Lower limit of quantification

[0144] SD Standard Deviation

[0145] Table 2 Blood concentrations of Zibo and Dapa in Experiment 2

[0146]

[0147] Data presented are mean ± SD (number of samples).

[0148] LLOQ Lower limit of quantification

[0149] SD Standard Deviation

[0150] - Not detected, all concentrations were below LLOQ.

[0151] Exposure to dapagliflozin was confirmed by measuring dapagliflozin concentrations above the LLOQ in all blood samples collected from animals receiving dapagliflozin at 4 hr or 24 hr after the first dose or 24 hr after the last dose. Figure 3 , Table 2). In Experiment 2, concentrations of zilpotentan above the LLOQ were measured in all urine samples collected overnight between Days 3 and 4 from animals receiving zilpotentan (Table 3), and concentrations of dapagliflozin above the LLOQ were measured in all urine samples from animals receiving dapagliflozin (Table 3).

[0152] Table 3 Amounts of Dapa and Zibo excreted in urine in Experiment 2

[0153]

[0154] Data presented are mean ± SD (number of samples).

[0155] LLOQ Lower limit of quantitation

[0156] SD Standard deviation

[0157] - Not detected, all concentrations were below LLOQ

[0158] In Experiment 1, quantifiable concentrations of zilpotentan were detected in 6 blood samples from control animals on Day 14. The apparent concentrations of zilpotentan in these positive samples were at least 13-fold lower than the mean blood concentrations in the low-dose group (30 mg / kg) (using the highest observed (0.54 μM) vehicle animal, relative to the mean (7.39 μM) in the 30 mg / kg group). In Experiment 2, there were no concentrations of zilpotentan or dapagliflozin above the LLOQ in blood samples from control animals, but zilpotentan concentrations above the LLOQ were detected in all 5 urine samples from control animals. The amount of zilpotentan excreted in urine in these samples was at least 190-fold lower than the mean amount excreted in urine in the zilpotentan-treated group (showing the lowest amount) (30 mg / kg zilpotentan + 3 mg / kg dapagliflozin).

[0159] In Experiment 2, zilpotentan blood concentrations 24 hr after the last dose were 3- to 4-fold lower in the group receiving zilpotentan in combination with dapagliflozin compared to the group receiving zilpotentan alone.

[0160] Zipotentan significantly reduced Hct concentrations compared to vehicle at all doses (30 mg / kg, 100 mg / kg, and 300 mg / kg) on ​​day 7 and at 100 mg / kg and 300 mg / kg on day 14 (p<0.05). Figure 4 ).

[0161] For each dose of zilpotentan (30 mg / kg, 100 mg / kg and 300 mg / kg), body weight was measured daily and compared to vehicle. There were no differences in body weight change for any of the zilpotentan treated groups compared to vehicle (Table 4, Figure 5 ). On day 10, the animals lost weight due to being placed in metabolic cages.

[0162] Table 4 Body weight comparison between Zipotentan (30 mg / kg, 100 mg / kg and 300 mg / kg) and vehicle

[0163]

[0164] *Differences in means are expressed on a log scale and estimated using linear mixed-effects models with Dunnett's post hoc test.

[0165] Zipotentan resulted in significant reductions in Hb compared to vehicle at all three doses on day 7 (30 mg / kg, 100 mg / kg, and 300 mg / kg) and at the two higher doses on day 14 (100 mg / kg and 300 mg / kg) (Table 5).

[0166] Table 5 Effect of Zibo on Hb in Experiment 1

[0167]

[0168] Effects of 30 mg / kg, 100 mg / kg and 300 mg / kg of Zipotentan on Hb. N=8-10 / group, *p<0.05 vs. vehicle. Data were analyzed using one-way ANOVA and Dunnett's test for multiple comparisons, and data are presented as mean±SEM.

[0169] As shown in Table 6, there were no changes in the levels of Na, K, glucose, and urea in plasma in response to different doses of zilpotentan compared to vehicle.

[0170] Table 6 Effects of Zibo on plasma Na, K, glucose and urea in Experiment 1

[0171]

[0172] Effects of different doses of zilpotentan on plasma Na, K, glucose and urea. N = 8-10 / group. Data were analyzed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM.

[0173] As shown in Table 7, there were no changes in urine volume, Na, K, urea, creatinine, and glucose levels in response to different doses of zilpotentan compared to vehicle.

[0174] Table 7 Effects of Zibo on urine volume and Na, K, urea, creatinine, and glucose in urine in Experiment 1

[0175]

[0176] Effects of different doses of zilpotentan on urine volume, Na, K, glucose, creatinine, and urea. N = 8-10 / group. Data were analyzed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM.

[0177] After 14 days of zilpotentan treatment, kidney weights were increased for the 300 mg / kg dose, both normalized and not normalized to tibia length, and heart weights were increased for 100 mg / kg zilpotentan compared to vehicle, but this increase was not statistically significant after normalization to tibia length (Table 8).

[0178] Table 8 Effects of Zibo on kidney weight and heart weight in Experiment 1

[0179]

[0180] Effects of 30 mg / kg, 100 mg / kg and 300 mg / kg of Zipotentan on kidney weight and heart weight. N=8-10 / group. Data were analyzed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM, *p<0.05 vs. vehicle.

[0181] Hct data were analyzed using a linear mixed effects model to estimate marginal means and standard deviations within the vehicle and treatment groups estimated at Day 3, Day 7, and throughout the study for post hoc comparisons. Treatment groups were compared to vehicle (Dunnett's test) to determine if there was an effect on Hct compared to the vehicle-treated control group, and the zilpotentan-treated group was compared to the zilpotentan plus dapagliflozin group (using the Tukey test) to determine if the combination therapy mitigated the effect of zilpotentan on Hct.

[0182] There was no difference in the absolute level of Hct at baseline among the groups ( Fig. 6A On day 3, zilpotentan (30 mg / kg and 100 mg / kg) administered with or without dapagliflozin (3 mg / kg) reduced Hct ( Figure 6B , Table 9A). On day 7, zilpotentan (30 mg / kg and 100 mg / kg) reduced Hct compared to vehicle, and when co-administered with dapagliflozin (3 mg / kg), both doses resulted in Hct similar to vehicle ( Figure 6C , Table 9B). Overall, zilpotentan 100 mg / kg plus dapagliflozin had higher Hct levels than zilpotentan 100 mg / kg alone (Table 9C).

[0183] Table 9A: Comparison of Hct levels between paired treatment groups and between treatment groups and normal controls on day 3 of treatment

[0184]

[0185] *Differences in means are expressed on a logarithmic scale and estimated by linear mixed effects models with Dunnett's post hoc test for comparisons with vehicle and Tukey's comparisons for pairwise comparisons.

[0186] On day 7, 30 mg / kg and 100 mg / kg of zilpotentan alone induced a decrease in Hct, but this effect was abolished when zilpotentan was combined with dapagliflozin ( Figure 6C , Table 9B).

[0187] Table 9B: Comparison of Hct levels between paired treatment groups and between treatment groups and normal controls on day 7 of treatment

[0188]

[0189] Table 9C: Differences in mean Hct values ​​for treatment groups compared to vehicle for all time points

[0190]

[0191] Dapagliflozin is known to increase weight loss (Hansen et al., Endocr Pract. 2014; 20(11): 1187-1197). To evaluate the effect of the combination therapy on weight loss, body weight was measured daily and compared using a linear mixed effects model. As expected, vehicle-treated animals gained weight during administration, and dapagliflozin alone prevented this BW increase compared to vehicle (p<0.05), while zilpotentan alone or in combination with dapagliflozin did not prevent ( Figure 7 , Table 10).

[0192] Table 10: Intergroup comparison of body weight changes among treatment groups throughout the study.

[0193]

[0194] As expected, vehicle-treated animals gained weight during the dosing period, and dapagliflozin monotherapy prevented this BW increase compared to vehicle (p<0.05), whereas zilpotentan alone or in combination with dapagliflozin did not ( Figure 7 ).

[0195] Dapagliflozin significantly increased urinary glucose excretion, whereas zilpotentan had no effect ( Fig. 8A Co-administration of zilpotentan did not alter the dapagliflozin-mediated increase in urinary glucose ( Fig. 8A ). Zipotentan did not alter water and food intake compared with vehicle. Dapagliflozin treatment increased water and food intake compared with vehicle, as Figure 8B and Figure 8C Animals co-administered with dapagliflozin and zilpotentan had significantly increased water and food intake (1.3 and 2.5, respectively) when compared to vehicle and to similar levels observed in animals administered dapagliflozin alone. Figure 8B and Figure 8C ).

[0196] Table 11 Effects of Zibo and Dapa on Hb in Experiment 2

[0197]

[0198] Effects of 30 mg / kg and 100 mg / kg zilpotentan with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and vehicle on Hb, N=8-12 / group, *p<0.05 vs. vehicle, # p<0.05 vs. the single-treatment group of zilpotentan. Data were analyzed using one-way ANOVA and Tukey's test for multiple comparisons. Data are presented as mean ± SEM.

[0199] Plasma Na, K, glucose, urea concentrations were not altered by treatment with zilpotentan, dapagliflozin, or their combination compared to vehicle, as shown in Table 12. Zibo (100 mg / kg) monotreatment or its combination with Dapa (3 mg / kg) significantly increased plasma creatinine compared to vehicle (p<0.05), as shown in Table 12.

[0200] Table 12 Effects of Zibo, Dapagliflozin or their combination on plasma Na, K, glucose, urea and creatinine concentrations in Experiment 2

[0201]

[0202]

[0203] Effects of 30 mg / kg and 100 mg / kg zilpotentan with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and vehicle on plasma electrolytes, urea, glucose, and creatinine, N=8-12 / group. Data were analyzed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM.

[0204] In all dapagliflozin treatment groups, with or without co-administration of zilpotentan, 24-hour urine volume and urea excretion increased. No differences were observed in the zilpotentan monotherapy group compared to vehicle. No changes in Na, K, and creatinine were observed in any treatment group compared to vehicle, as shown in Table 13.

[0205] Table 13 Effects of Zibo, Dapa and combination on urine volume and Na, K, urea and creatinine in urine in Experiment 2

[0206]

[0207]

[0208] Effects of different doses of 30 mg / kg and 100 mg / kg zilpotentan with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and vehicle on urine volume, electrolytes, urea, glucose, and creatinine, N=8-12 / group. Data were analyzed using one-way ANOVA and Tukey's test for multiple comparisons. Data presented are mean ± SEM, *p<0.05 vs. vehicle, # p<0.05 versus the zilpotentan single-treatment group.

[0209] After seven days of treatment, kidney weights were increased in the Zibo 100 mg / kg + Dapa 3 mg / kg group compared to vehicle. Heart weights (whether normalized to tibia length or not) were increased in the Zibo 30 mg / kg group compared to vehicle alone, as shown in Table 14.

[0210] Table 14 Effects of Zibo and Dapa on kidney and heart weights in Experiment 2

[0211]

[0212] Effects of different doses of 30 mg / kg and 100 mg / kg zilpotentan with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and vehicle on kidney and heart weights. N=12 / group, *p<0.05 vs. vehicle. Data were analyzed using one-way ANOVA and Dunnett's test for multiple comparisons. Data presented are mean ± SEM.

[0213] The endothelial receptor A antagonist zilpotentan significantly reduced Hct concentrations in male Wistar rats fed a 4% salt diet. Co-administration of the SGLT-2 inhibitor dapagliflozin for 7 days significantly reduced the effect of zilpotentan on Hct concentrations. Dapagliflozin attenuated the zilpotentan-induced decrease in fluid retention (hematocrit) in rats.

[0214] Example 2

[0215] The effect of dapagliflozin on proteinuria was investigated in patients with CKD with or without type 2 diabetes. In this analysis of the phase 3 DAPACKD trial, the efficacy of dapagliflozin was evaluated to determine whether treatment with dapagliflozin could reduce proteinuria levels in patients with CKD.

[0216] In this trial, 4304 patients (estimated glomerular filtration rate 25-75 mL / min / 1 73 m 2, and urine UACR of 200-5000 mg / g) were randomized to receive dapagliflozin (10 mg) or placebo. Changes in proteinuria were assessed as the mean change in log-transformed UACR from baseline to the end of the study. UACR phase regression (defined as the transition from macroalbuminuria (≥300 mg / g) to micro or normoalbuminuria (<300 mg / g)) and UACR phase progression (defined as the transition from <3000 mg / g to ≥3000 mg / g) were additional endpoints.

[0217] In the entire population, the median (25th-75th percentile) UACR was 965 (472-1903) mg / g (compared with a median UACR of 934 (482-1868) for placebo). Dapagliflozin reduced UACR by 29.3% (95% confidence interval [CI], 25.2-33.1%; p<0.001) in patients with and without type 2 diabetes compared with placebo. Stratified by diabetes status, dapagliflozin reduced UACR by 35.1% (95% confidence interval [CI], 30.6-39.4%; p<0.001) in patients with type 2 diabetes and by 14.8% (95% confidence interval [CI], 5.9-22.9%; p<0.001) in patients without type 2 diabetes compared with placebo.

[0218] Among the 3860 patients with a baseline UACR ≥300 mg / g, dapagliflozin increased the likelihood of UACR regression (hazard ratio [HR] 1.81; 95% CI, 1.60-2.05). Among the 3820 patients with a baseline UACR <3000 mg / g, dapagliflozin reduced the risk of UACR progression (HR 0.41; 95% CI, 0.32-0.52). Regression and progression of proteinuria were further stratified by diabetes status and compared as follows: Fig. 9 and Fig.10 As demonstrated in this analysis, treatment with dapagliflozin reduced UACR in patients with CKD, regardless of diabetes status.

[0219] Example 3

[0220] A preclinical study of dapagliflozin in combination with zilpotentan is ongoing to show the effects of the single agents and the combination on certain biomarkers indicative of renal and cardiovascular function in male Dahl salt-sensitive (DSS) rats.

[0221] Example 3 The effects of zilpotentan alone and the combination of zilpotentan and dapagliflozin on body weight, blood pressure (mean arterial pressure-MAP, systolic blood pressure-SBP and diastolic blood pressure-DBP) and heart rate, urine protein albumin and creatinine, serum creatinine, hematology, echocardiography of heart and LV diameter and wall thickness, shortening fraction and ejection fraction, and kidney (left and right), heart and lung weights and tibia length in 7-9 week old DSS rats on a 4% salt diet will be evaluated in a 7 week study. Treatment will begin 12 days after starting the 4% salt diet.

[0222] Formulation for vehicle: 30% (w / w) PEG400, 2% (w / w) EtOH, 0.5% (w / w) HPMC (10000 cps), 0.1% (w / w) Tween 80 and 67.4% purified water.

[0223] Formulation for Zipotentan alone: ​​Suspension in 30% (w / w) PEG400, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000 cps, 0.1% (w / w) Tween 80 and 67.4% purified water.

[0224] Formulation for the combination of dapagliflozin and zilpotentan: suspension / solution in 30% (w / w) PEG400, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000 cps, 0.1% (w / w) Tween 80 and 67.4% purified water.

[0225] The effects of zilpotentan alone and dapagliflozin alone on body weight, blood pressure (MAP, SBP and DBP) and heart rate, urine protein albumin and creatinine, serum creatinine, hematology, echocardiography of heart and LV diameter and wall thickness, fractional shortening and ejection fraction, and kidney (left and right), heart and lung weights and tibia length will be determined according to the three groups summarized in Table 15 below:

[0226] Table 15 Study Groups of Example 3

[0227]

[0228]

[0229] Whole blood samples (20 μL) will be collected from Group B and Group C at Weeks 3 and 6 for PK assessment. An additional 200 μL whole blood sample will be collected at Week 1 for hematology Hct and MCV assessment (via Horiba ABX Micros ESV60). Urine will be collected at Weeks 1, 3, and 6 for clinical chemistry assessment (via RX ) to assess serum creatinine and urine protein albumin and creatinine. VisualSonics Vevo Echocardiographic images of rats lightly anesthetized with 1%-2% isoflurane were obtained using an ultrasound echocardiography system and an MX201 15MHZ microscanning transducer. M-mode (short axis) images of the left ventricle will be collected for LVEDD, LVESD, AWT, PWT, FS, and EF. The left kidney, lung, and heart will be harvested, decapsulated, and weighed. The length of the tibia will be measured and imaged by X-ray.

[0230] Clinical chemistry and hematology data, Echo data, and body and tissue weights (normalized to tibia length) will be analyzed using one-way ANOVA (GraphPad Prism version 7.0a). Statistically significant differences between groups will be tested using Dunnett's test. Blood pressure analysis will be evaluated as mean ± SEM for MAP, SBP, DBP, and heart rate in line graph form.

[0231] Example 4

[0232] A clinical trial of the combination of dapagliflozin and zilpotentan (ZENITH-CKD) is ongoing in a Phase 2b, multicenter, randomized, double-blind, placebo-controlled, parallel-group, dose-ranging study to evaluate the efficacy, safety and tolerability of zilpotentan and dapagliflozin in participants with CKD with an eGFR between 20 and 60 mL / min / 1.73 m2.

[0233] Study Design

[0234] The study will be conducted in 2 parts (Part A and Part B). Participants will be randomized for 12 weeks of treatment plus 2 weeks of follow-up in both study parts. All variables will be collected to verify inclusion criteria and other demographic data (such as race / ethnicity, serum creatinine, and height). All analyses (except for the interim analysis during Part A) will include data from both parts of the study.

[0235] Participants who meet the eligibility criteria will be randomized to receive the treatment described for Part A or Part B in addition to background topical SoC therapy. To maintain blinding, participants will receive active and placebo study interventions on each dosing day of Zibo / Dapa monotherapy. Participants who receive placebo only will take placebo for both study interventions.

[0236] In Part A, 132 eligible participants are planned to be enrolled and randomly assigned to one of four treatment groups, with 33 participants in each treatment group:

[0237] - Zipotentan 5 mg + dapagliflozin 10 mg once daily.

[0238] -Zilpotentan 5 mg once daily.

[0239] -Dapagliflozin 10 mg once daily.

[0240] - Placebo once daily.

[0241] An interim analysis of Part A data will be conducted when approximately 30 participants in each group (120 participants) have completed 6 weeks of treatment to assess changes in fluid-related measures (weight gain or BNP). If at least 5 participants in the zilpotentan 5 mg monotherapy group meet the specified criteria for the fluid-related measures, that group will be discontinued from the remainder of the study intervention period.

[0242] If the specified discontinuation criteria for the zilpotentan 5 mg monotherapy group are not met at the 6-week treatment interim analysis, a second interim analysis of available Part A data will be conducted after all randomized participants in Part A have completed 12 weeks of treatment to assess changes in fluid-related measures. If at least 5 participants in the zilpotentan 5 mg monotherapy group meet the specified criteria for fluid-related measures at the second interim analysis (at 12 weeks of treatment), that group will be discontinued from the remainder of the intervention period of the study.

[0243] In Part B, an additional 528 eligible participants are planned to be enrolled. Of these participants, 352 will be randomly assigned to the same 4 treatment groups as in Part A, with 88 participants in each treatment group. The remaining eligible participants will be randomly assigned to 2 additional treatment groups, with 88 participants in each treatment group:

[0244] - Zipotentan 0.25 mg + dapagliflozin 10 mg once daily.

[0245] - Zipotentan 1.5 mg + dapagliflozin 10 mg once daily.

[0246] - Zipotentan 5 mg + dapagliflozin 10 mg once daily.

[0247] -Zilpotentan 5 mg once daily.

[0248] -Dapagliflozin 10 mg once daily.

[0249] - Placebo once daily.

[0250] Participants who were randomized in Part A were not eligible for randomization in Part B.

[0251] Participants will be stratified by diabetes (DKD vs. non-DM CKD) and baseline eGFR (less than 45 mL / min / 1.73 m2 vs. greater than 45 mL / min / 1.73 m2) at randomization to ensure approximate balance between treatment groups within each subgroup. The number of randomized participants in each stratum will be monitored to ensure that the non-DM CKD subgroup is approximately a minimum of 30% and a maximum of 33% of the total number of randomized participants.

[0252] For each participant, the total duration of participation will be approximately 17 to 19 weeks. In each study part, the screening period may last up to 4 weeks before randomization. The first dose will be taken after randomization at the Day 1 Baseline Visit. In addition to the Baseline Visit, participants will visit the clinic 5 times during the next 12-week treatment period. Approximately 2 weeks after the last dose, participants will return to the clinic for a follow-up assessment.

[0253] Purpose and End Point

[0254] The primary endpoint will measure the effect of zilpotentan and dapagliflozin (combination and alone) on UACR relative to placebo. The change in UACR will be measured as log-transformed UACR from baseline to Week 12 (UACR (mg / g) = urine albumin (mg / dL) / urine creatinine (g / dL)). The secondary endpoints and objectives are summarized in Table 17 below.

[0255] Table 17 Secondary endpoints and other objectives of the ZENITH-CKD Ph2b trial

[0256]

[0257]

[0258]

Claims

1. Zipotentan, N-(3-methoxy-5-methylpyrazin-2-yl)-2-[4-(1,3,4-oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide, or a pharmaceutically acceptable salt thereof, in combination with dapagliflozin for the preparation of a medicament for reducing UACR and reducing the incidence of cardiovascular or renal death in human patients with chronic kidney disease.

2. The use according to claim 1, wherein zilpotentan or a pharmaceutically acceptable salt thereof and dapagliflozin are administered once daily in combination.

3. The use according to claim 2, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 5 mg, and dapagliflozin is administered at a dose of 10 mg.

4. The use according to claim 2, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 1.5 mg, and dapagliflozin is administered at a dose of 10 mg.

5. The use according to claim 2, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 0.25 mg, and dapagliflozin is administered at a dose of 10 mg.

6. The method according to claim 2, wherein the patient has an eGFR of 20-60 ml / min / 1.73 m 2 Patients with chronic kidney disease who are classified as stages 1-4.

7. The use according to claim 6, wherein the human patient is a chronic kidney disease patient classified as a stage 3-4 patient.

8. Zipotentan, N-(3-methoxy-5-methylpyrazin-2-yl)-2-[4-(1,3,4-oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide, or a pharmaceutically acceptable salt thereof, in combination with dapagliflozin for the preparation of a medicament for reducing UACR to <300 mg / g in human patients with chronic kidney disease.

9. The use according to claim 8, wherein zilpotentan or a pharmaceutically acceptable salt thereof and dapagliflozin are administered once daily in combination.

10. The use according to claim 9, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 5 mg, and dapagliflozin is administered at a dose of 10 mg.

11. The use according to claim 9, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 1.5 mg, and dapagliflozin is administered at a dose of 10 mg.

12. The use according to claim 9, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 0.25 mg, and dapagliflozin is administered at a dose of 10 mg.

13. The method according to claim 9, wherein the patient has an eGFR of 20-60 ml / min / 1.73 m 2 Patients with chronic kidney disease who are classified as stages 1-4.

14. The use according to claim 13, wherein the human patient is a chronic kidney disease patient classified as a stage 3-4 patient.

15. Zipotentan, N-(3-methoxy-5-methylpyrazin-2-yl)-2-[4-(1,3,4-oxadiazol-2-yl)phenyl]pyridine-3-sulfonamide, or a pharmaceutically acceptable salt thereof, in combination with dapagliflozin for the preparation of a medicament for reducing the risk of UACR progression to ≥3000 mg / g in human patients with chronic kidney disease.

16. The use according to claim 15, wherein zilpotentan or a pharmaceutically acceptable salt thereof and dapagliflozin are administered once daily in combination.

17. The use according to claim 16, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 5 mg, and dapagliflozin is administered at a dose of 10 mg.

18. The use according to claim 16, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 1.5 mg, and dapagliflozin is administered at a dose of 10 mg.

19. The use according to claim 16, wherein zilpotentan or a pharmaceutically acceptable salt thereof is administered at a dose of about 0.25 mg, and dapagliflozin is administered at a dose of 10 mg.

20. The use according to claim 16, wherein the patient has an eGFR of 20-60 ml / min / 1.73 m 2 Patients with chronic kidney disease who are classified as stages 1-4.

21. The use according to claim 20, wherein the human patient is a chronic kidney disease patient classified as a stage 3-4 patient.

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