Combination of zibotentan and dapagliflozin for the treatment of endothelin related diseases

Abstract

This disclosure relates to the combination of the endothelin receptor antagonist (ERA) zipotentan and the sodium-dependent glucose cocarrier protein 2 (SGLT-2) inhibitor dapagliflozin for use in the treatment of certain endothelin-related diseases.

Description

Technical Field

[0001] This disclosure relates to the use of the endothelin receptor antagonist (ERA) zibotentan in combination with the sodium-dependent glucose cocarrier protein 2 (SGLT-2) inhibitor dapagliflozin for the treatment of certain endothelin-related diseases. Prior Technology

[0002] Endothelin-1 (ET-1) is a potent vasoconstrictive peptide. ET-1 (regulated by endothelin A and B (ETA and ETB) receptors) is a highly potent systemic vasoconstrictor and a driver of kidney disease progression. In chronic kidney disease (CKD), ET-1 levels increase with the urinary albumin-to-creatinine ratio (UACR) and the severity of renal impairment (Grenda et al., Nephrol Dial Transplant. [Nephrology Dialysis and Transplantation] 2007; 22 (12): 3487–3494; Kohan Am J Kidney Dis. [American Journal of Kidney Disease] 1997; 29 (1): 2–26). The pathological effects of ET-1 (including proteinuria, vasoconstriction, and inflammation) are thought to be primarily driven by endothelial A (ETA) receptors (Goddard et al., Circulation. [Circulation] 2004; 109 (9): 1186–1193). ETA antagonists have demonstrated renal protective effects. In diabetic kidney disease (DKD), a 30% reduction in the urinary albumin-to-creatinine ratio (UACR) has been shown (Heerspink et al., Diabetes Obes Metab. 2018; 20(8): 1829–1835; Heerspink et al., Lancet 2019; 393(10184): 1937–1947). However, the clinical development of ETA receptor antagonists has been limited by problems such as fluid retention and hospitalization due to heart failure (Heerspink et al., Lancet 2019; 393(10184): 1937–1947). Zipotentan was an effective ETA receptor antagonist developed for the treatment of prostate cancer, but it was discontinued in 2011 due to insufficient efficacy in stage 3 and a 17% increase in the incidence of peripheral edema compared to placebo.

[0003] SGLT-2 inhibitors act as osmotic diuretics in glycosuria, thus increasing urine output and reducing volume overload largely independently of changes in systemic sodium load. SGLT-2 inhibitors block renal reabsorption of glucose, increasing glucose excretion and lowering 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 possess anti-inflammatory and anti-fibrotic properties similar to those of ERA (HJ Heerspink et al., Circulation (2016), 134(10): 752-772). SGLT2 inhibitors have been shown to be effective for DKD (Stephens et al., Diabetes Obes Metab. 2020; 22 Supplement 1: 32-45). The DAPA-CKD trial, which investigated the efficacy of dapagliflozin in CKD, was recently terminated early due to overwhelming efficacy. A posthoc analysis of the SONAR trial, presented at the American Society of Nephrology’s annual Kidney Week meeting in Washington, D.C. in 2019, showed that in a subset of DKD patients (n = 14) taking both the ETA antagonist atrasentan and the SGLT2 inhibitor, there was an increased decrease in UACR and a reduction in weight gain caused by atrasentan (an alternative to fluid retention) compared to atrasentan alone.

[0004] Zipotentan (N-(3-methoxy-5-methylpyridine-2-yl)-2-[4-(1,3,4-diazol-2-yl)phenyl]pyridine-3-sulfonamide) has the chemical structure of Formula I. .

[0005] Zipotentan (also known as ZD4054) has been disclosed as an endothelin receptor antagonist in WO 1996040681, along with detailed information on its chemical synthesis. The specific inhibition of endothelin A receptors by zipotentan has been reported by Morris et al., British Journal of Cancer (2005), 92, 2148-2152.

[0006] Dapagliflozin ((1S)-1,5-anhydride-1-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-glucanol) has the chemical structure of formula II. .

[0007] Dapagliflozin is a potent, highly selective, and orally active human renal sodium-dependent glucose transporter 2 (SGLT2) inhibitor (SGLT2i) approved for improving glycemic control in adults with type 2 diabetes (as an adjunct to diet and exercise) and reducing the risk of hospitalization for heart failure in adults with type 2 diabetes and established cardiovascular disease or multiple cardiovascular risk factors. Dapagliflozin's chemical synthesis details are disclosed in WO 2003099836.

[0008] When zipotentan, a specific inhibitor of endothelin A receptor, is combined with dapagliflozin, an SGLT-2 inhibitor, the diuretic effect of dapagliflozin can be mitigated by reducing the fluid retention side effects associated with the endothelin receptor antagonist zipotentan, thereby reducing the risks associated with each compound individually. Summary of the Invention

[0009] This specification demonstrates that dapagliflozin can reduce zipotentan-driven hemodilution, where hemodilution is defined as a decrease in hematocrit, and thus highlights the potential of the combination of zipotentan and dapagliflozin to demonstrate efficacy in the treatment of certain endothelin-related diseases.

[0010] Endothelin-1 (regulated by endothelin A and B (ETA and ETB) receptors) is a highly effective systemic vasoconstrictor and a driver of kidney disease progression.

[0011] ETA antagonist blockade can improve renal function, while ETB blockade is not optimal because ETB also clears circulating endothelin-1.

[0012] Because endothelin is present in many cardiorenal metabolic diseases, endothelin-related diseases are associated with increased vasoconstriction, proliferation, or inflammation. Examples of such endothelin-related diseases include hypertension, coronary artery disease, heart failure, renal and myocardial ischemia, and chronic kidney disease (CKD). In particular, CKD is associated with hypertension or diabetes (diabetic kidney disease, DKD). ERAs, through their acute (peripheral vasodilatory) and chronic (vasodilatory, vascular structure improvement, and sympathetic nerve activity modulation, antithrombotic, and anti-inflammatory) effects, may be beneficial in treating peripheral arterial occlusive diseases, including diabetic arterial disease.

[0013] SGLT-2 works by simultaneously inhibiting glucose and sodium uptake in the proximal tubules of the nephrons, which is thought to cause a resetting of the tubuloglomerular feedback, presumably leading to glomerular ultrafiltration. The efficacy of SGLT-2 inhibitors is believed to decrease with decreasing plasma glucose levels or glomerular filtration rate (GFR), thus SGLT-2 inhibitors have an inherently low risk of hypoglycemia. Therefore, the nature of SGLT-2 inhibitors may open a pathway 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).

[0014] Side effects associated with the pharmacological effects of SGLT-2 inhibitors include volume depletion / intravascular volume constriction, which may lead to dehydration, hypovolemia, orthostatic hypotension, or low blood pressure. Therefore, SGLT-2 inhibitors typically induce an increase in hematocrit (Hot, a marker of blood concentration) and increased blood viscosity (a presumed cause of vascular damage in the context of peripheral vascular disease).

[0015] Through the pharmacological action of SGLT-2 inhibitors, serum creatinine increases and eGFR decreases. Zipfortentan (an ERA that effectively blocks ETA receptors) is suitable for treating endothelin-related diseases when prescribed in combination with dapagliflozin. When an ERA is combined with an SGLT-2 inhibitor, the diuretic effect of this inhibitor and its potential pharmacological action in reducing the risk of heart failure may be suitable for mitigating the most prominent side effects commonly associated with ERAs, such as fluid retention and the potentially associated increased risk of congestive heart failure. This combination therapy can produce pharmacological effects on the identified endothelin-related diseases while maintaining a favorable side effect profile even at the optimal effective dose of zipfortentan, and possibly even at increased doses of zipfortentan compared to the maximum tolerated dose of zipfortentan alone. When zippotentan is used in combination with dapagliflozin, the increased dose of zippotentan available (e.g., due to reduced side effects) can allow for a broader effect on diseases caused by the harmful effects of the endothelin paracrine system, which is widely distributed throughout the body. This combination therapy improves the benefit / risk ratio. ERAs have been described as reducing hematocrit (Hot) through hemodilution. Therefore, zippotentan, 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 depleting effect. ERAs have been described as improving blood glucose levels through various mechanisms, such as increased blood flow and improved insulin signaling. Therefore, zippotentan, when used in combination with dapagliflozin, can have an additive or even synergistic effect on lowering blood glucose.

[0016] In a first aspect, ziprettan is provided for use in the treatment of human patients with chronic kidney disease (CKD), wherein ziprettan is administered in combination with dapagliflozin.

[0017] In another aspect, a method for treating chronic kidney disease (CKD) in patients requiring such treatment is provided, comprising administering to the patient a therapeutically effective dose of zipretem, wherein zipretem is administered in combination with a therapeutically effective dose of dapagliflozin.

[0018] In another aspect, the use of ziprettan in the preparation of a medicine for the treatment of human patients with chronic kidney disease (CKD) is provided, wherein ziprettan is administered in combination with dapagliflozin. Simple Explanation of the Diagram

[0019] [ [picture] [1] shows the blood concentration of zipretentate in individuals after oral administration of zipretentate in Experiment 1 of Example 1.

[0020] [ [picture] [2] shows the blood concentrations of zipretentate in individuals after oral administration of zipretentate alone or in combination with dapagliflozin in Experiment 2 of Example 1.

[0021] [ [picture] [3] shows the individual blood concentrations of dapagliflozin after oral administration of dapagliflozin alone or in combination with zipotentan in Experiment 2 of Example 1.

[0022] [ [picture] [4] shows the effect of zepotentan on Hct concentration in Experiment 1 of Example 1.

[0023] [ [picture] [5] shows the effect of zepotentan on body weight in Experiment 1 of Example 1.

[0024] [ [picture] [6] shows the effects of zipotentan, dapagliflozin and combination on Hct in Experiment 2 of Example 1.

[0025] [ [picture] [7] shows the effects of zipotentan, dapagliflozin and their combination on body weight in Experiment 2 of Example 1.

[0026] [ [picture] [8] shows the effects of dapagliflozin and zipotentan on diabetes, water and food intake in Experiment 2 of Example 1.

[0027] [ [picture] [9]] This study demonstrated the UACR-lowering effect of dapagliflozin in CKD patients (with or without type 2 diabetes) from the DAPA CKD Phase 3 trial.

[0028] [ [picture]

[10] shows the regression and progression of UACR in CKD patients (stratified by diabetes status) from the DAPA CKD Phase 3 trial. Implementation

[0029] In a first aspect, ziprettan is provided for use in the treatment of human patients with chronic kidney disease (CKD), wherein ziprettan is administered in combination with dapagliflozin.

[0030] In another aspect, a method for treating chronic kidney disease (CKD) in patients requiring such treatment is provided, comprising administering to the patient a therapeutically effective dose of zipretem, wherein zipretem is administered in combination with a therapeutically effective dose of dapagliflozin.

[0031] In another aspect, the use of ziprettan in the preparation of a medicine for the treatment of human patients with chronic kidney disease (CKD) is provided, wherein ziprettan is administered in combination with dapagliflozin.

[0032] In some implementations, chronic kidney disease (CKD) is defined as stage 1 to 4 CKD as defined by the Kidney Disease Improving Global Outcomes (KDIGO) guidelines.

[0033] In some implementations, CKD refers to stage 2-3 CKD.

[0034] In some implementations, CKD refers to stage 3-4 CKD.

[0035] In some implementations, CKD refers to Phase 4 CKD.

[0036] In some implementations, CKD refers to stage 3a or 3b CKD.

[0037] Zipotentan (N-(3-methoxy-5-methylpyridine-2-yl)-2-[4-(1,3,4-diazol-2-yl)phenyl]pyridine-3-sulfonamide) has the chemical structure of Formula I. .

[0038] Zipotentan is also known as ZD4054.

[0039] In this implementation, ziprentan or a pharmaceutically acceptable salt thereof is administered once daily.

[0040] In this implementation, the total daily dose of ziprettan is approximately 10 mg.

[0041] In this implementation, the total daily dose of ziprettan is approximately 5 mg.

[0042] In this implementation, the total daily dose of ziprettan is approximately 1.5 mg.

[0043] In this implementation, the total daily dose of ziprettan is approximately 0.5 mg.

[0044] In this implementation, the total daily dose of ziprettan is approximately 0.25 mg.

[0045] In this embodiment, ziprettan or its pharmaceutically acceptable salt-based tablet form is used.

[0046] In one embodiment, zipretane or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. In another embodiment, the composition comprises one or more pharmaceutical diluents, one or more pharmaceutical disintegrants, or one or more pharmaceutical lubricants.

[0047] Dapagliflozin ((1S)-1,5-anhydride-1-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-D-glucanol) has the chemical structure of formula II. .

[0048] In this implementation, dapagliflozin or a pharmaceutically acceptable salt thereof is administered once daily.

[0049] In at least one embodiment, dapagliflozin is in the form of a pharmaceutically acceptable solvate, mixed solvates, 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: .

[0050] In at least one embodiment, dapagliflozin is in the form of a pharmaceutically acceptable solvate, mixed solvates, 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.

[0051] In some respects described herein, the drug composition is administered orally to the patient. In some respects described herein, the drug composition is administered to the patient in tablet form.

[0052] In some aspects provided herein, pharmaceutical compositions containing dapagliflozin comprise a dose equivalent of about 2.5 mg / day to about 10 mg / day of dapagliflozin, administered to a patient. In some aspects provided herein, pharmaceutical compositions containing dapagliflozin comprise a dose equivalent of about 2.5 mg / day, about 5 mg / day, or about 10 mg / day of dapagliflozin, administered to a patient. In some aspects provided herein, pharmaceutical compositions containing dapagliflozin comprise a dose equivalent of about 5 mg / day of dapagliflozin, administered to a patient once daily. In some aspects provided herein, pharmaceutical compositions containing dapagliflozin comprise a dose equivalent of about 10 mg / day of dapagliflozin, administered to a patient once daily.

[0053] In one aspect, ziprettan is provided for use in treating CKD in a human patient, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3.

[0054] In one aspect, a method of treating CKD in a patient requiring such treatment is provided, the method comprising administering a therapeutically effective amount of zipretem to the patient, wherein the treatment comprises administering, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretem and ii) a therapeutically effective amount of dapagliflozin to the patient. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1-4. In another embodiment, the patient is a CKD patient classified as stage 3-4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2-3.

[0055] In one aspect, the use of ziprettan in the preparation of a medicament for treating CKD in a human patient is provided, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3.

[0056] In one aspect, ziprettan is provided for use in reducing UACR in a human patient, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0057] In one aspect, a method for reducing UACR in a patient requiring such treatment is provided, the method comprising administering a therapeutically effective amount of zipretem to the patient, wherein the treatment comprises administering, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretem and ii) a therapeutically effective amount of dapagliflozin to the patient. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1-4. In another embodiment, the patient is a CKD patient classified as stage 3-4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2-3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0058] In one aspect, the use of ziprettan in the preparation of a medicament for reducing UACR in a human patient is provided, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0059] In one aspect, ziprettan is provided for use in reducing the UACR of a human patient to < 300 mg / g, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0060] In one aspect, a method is provided for reducing the UACR to < 300 mg / g in a patient requiring such treatment, the method comprising administering to the patient a therapeutically effective amount of zipretentam, wherein the treatment comprises administering to the patient, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretentam and ii) a therapeutically effective amount of dapagliflozin. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1–4. In another embodiment, the patient is a CKD patient classified as stage 3–4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2–3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0061] In one aspect, the use of ziprettan in the preparation of a medicament for reducing the UACR of a human patient to < 300 mg / g is provided, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0062] In one aspect, ziprettan is provided for use in reducing the risk of progression in a human patient to a UACR ≥ 3000 mg / g, comprising administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0063] In one aspect, a method is provided for reducing the risk of progression to a UACR ≥ 3000 mg / g in a patient requiring such treatment, the method comprising administering to the patient a therapeutically effective amount of zipretem, wherein the treatment comprises administering to the patient, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretem and ii) a therapeutically effective amount of dapagliflozin. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1–4. In another embodiment, the patient is a CKD patient classified as stage 3–4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2–3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0064] In one aspect, the use of zipretem in the preparation of a medicament for reducing the risk of progression in a human patient to a UACR ≥ 3000 mg / g is provided, wherein the treatment comprises administering i) zipretem and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0065] In one aspect, zipotentan is provided for use in reducing the risk of eGFR decline in a human patient, comprising administering i) zipotentan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes. In some embodiments, this use reduces the risk of eGFR decline ≥ 30%. In some embodiments, this use reduces the incidence of eGFR decline ≥ 40%.

[0066] In one aspect, a method is provided for reducing the risk of eGFR decline in a patient requiring such treatment, the method comprising administering a therapeutically effective amount of zipretem to the patient, wherein the treatment comprises administering, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretem and ii) a therapeutically effective amount of dapagliflozin to the patient. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1–4. In another embodiment, the patient is a CKD patient classified as stage 3–4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2–3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes. In some embodiments, the method reduces the risk of eGFR decline ≥ 30%. In some implementations, this method reduces the incidence of an eGFR decrease of ≥ 40%.

[0067] In one aspect, the use of zipretem in the preparation of a medicament for reducing the risk of eGFR decline in a human patient is provided, wherein the treatment comprises administering i) zipretem and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes. In some embodiments, this use reduces the risk of eGFR decline ≥ 30%. In some implementations, this use reduces the incidence of eGFR decreases of ≥ 40%.

[0068] In one aspect, ziprettan is provided for use in reducing the risk of fluid retention (edema) in a human patient, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes. In some embodiments, the reduction in the risk of fluid retention (edema) can be measured by, for example, a decrease in Hct or weight gain.

[0069] In one aspect, a method is provided for reducing the risk of fluid retention (edema) in a patient requiring such treatment, the method comprising administering a therapeutically effective amount of ziprettan to the patient, wherein the treatment comprises administering, separately, sequentially, or simultaneously, i) a therapeutically effective amount of ziprettan and ii) a therapeutically effective amount of dapagliflozin to the patient. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1-4. In another embodiment, the patient is a CKD patient classified as stage 3-4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2-3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes. In some implementations, the reduction in the risk of fluid retention (edema) can be measured by, for example, a decrease in Hct or an increase in body weight.

[0070] In one aspect, the use of ziprettan in the preparation of a medicament for reducing the risk of fluid retention (edema) in a human patient is provided, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes. In some implementations, the reduction in the risk of fluid retention (edema) can be measured by, for example, a decrease in Hct or an increase in body weight.

[0071] In one aspect, ziprettan is provided for use in reducing total body water in a human patient, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0072] In one aspect, a method for reducing total body water in a patient requiring such treatment is provided, the method comprising administering to the patient a therapeutically effective amount of ziprettan, wherein the treatment comprises administering to the patient, separately, sequentially, or simultaneously, i) a therapeutically effective amount of ziprettan and ii) a therapeutically effective amount of dapagliflozin. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1-4. In another embodiment, the patient is a CKD patient classified as stage 3-4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2-3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0073] In one aspect, the use of ziprettan in the preparation of a medicament for reducing total body water in a human patient is provided, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0074] In one aspect, ziprettan is provided for use in lowering blood pressure in a human patient, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0075] In one aspect, a method for lowering blood pressure in a patient requiring such treatment is provided, the method comprising administering to the patient a therapeutically effective amount of ziprettan, wherein the treatment comprises administering to the patient, separately, sequentially, or simultaneously, i) a therapeutically effective amount of ziprettan and ii) a therapeutically effective amount of dapagliflozin. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1-4. In another embodiment, the patient is a CKD patient classified as stage 3-4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2-3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0076] In one aspect, the use of ziprettan in the preparation of a medicament for lowering blood pressure in a human patient is provided, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0077] In one aspect, ziprettan is provided for use in reducing the risk of elevated blood pressure in a human patient, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0078] In one aspect, a method is provided for reducing the risk of elevated blood pressure in a patient requiring such treatment, the method comprising administering to the patient a therapeutically effective amount of ziprettan, wherein the treatment comprises administering to the patient, separately, sequentially, or simultaneously, i) a therapeutically effective amount of ziprettan and ii) a therapeutically effective amount of dapagliflozin. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1-4. In another embodiment, the patient is a CKD patient classified as stage 3-4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2-3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0079] In one aspect, the use of zipretem in the preparation of a medicament for reducing the risk of elevated blood pressure in a human patient is provided, wherein the treatment comprises administering i) zipretem and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0080] In one aspect, ziprettan is provided for use in human patients to achieve a composite endpoint of eGFR reduction ≥ 40%, attainment of end-stage renal disease (ESKD), and incidence of cardiovascular or renal death, comprising administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0081] 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 patients requiring such treatment, the method comprising administering a therapeutically effective amount of zipretentam to the patient, wherein the treatment comprises administering, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretentam and ii) a therapeutically effective amount of dapagliflozin to the patient. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1–4. In another embodiment, the patient is a CKD patient classified as stage 3–4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2–3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient who does not have type 2 diabetes.

[0082] In one aspect, the use of ziprettan in the preparation of a medicine for reducing the composite endpoint of eGFR decline >40% in human patients, achieving end-stage renal disease (ESKD), and the incidence of cardiovascular or renal death is provided, wherein the treatment comprises administering i) ziprettan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0083] In one aspect, ziprettan is provided for use in reducing the incidence of end-stage renal disease (ESKD) in human patients, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1–4. In another embodiment, the human patient is a CKD patient classified as stage 3–4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2–3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0084] In one aspect, a method is provided for reducing the incidence of end-stage renal disease (ESKD) in patients requiring such treatment, the method comprising administering a therapeutically effective amount of zipretem to the patient, wherein the treatment comprises administering, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretem and ii) a therapeutically effective amount of dapagliflozin to the patient. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1–4. In another embodiment, the patient is a CKD patient classified as stage 3–4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2–3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0085] In one aspect, the use of zipretentan in the preparation of a medicament for reducing the incidence of end-stage renal disease (ESKD) in human patients is provided, wherein the treatment comprises administering i) zipretentan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0086] In one aspect, ziprettan is provided for use in reducing the incidence of cardiovascular or renal death in human patients, comprising administering i) ziprettan and ii) dapagliflozin separately, sequentially, or simultaneously to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0087] In one aspect, a method is provided for reducing the incidence of cardiovascular or renal death in patients requiring such treatment, the method comprising administering a therapeutically effective amount of zipretentam to the patient, wherein the treatment comprises administering, separately, sequentially, or simultaneously, i) a therapeutically effective amount of zipretentam and ii) a therapeutically effective amount of dapagliflozin to the patient. In another embodiment, the patient is a CKD patient. In another embodiment, the patient is a CKD patient classified as stage 1-4. In another embodiment, the patient is a CKD patient classified as stage 3-4. In another embodiment, the patient is a CKD patient classified as stage 4. In another embodiment, the patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the patient is a CKD patient classified as stage 2-3. In another embodiment, the patient is a CKD patient with type 2 diabetes. In other embodiments, the patient is a CKD patient without type 2 diabetes.

[0088] In one aspect, the use of zipretentan in the preparation of a medicament for reducing the incidence of cardiovascular or renal death in human patients is provided, wherein the treatment comprises administering i) zipretentan and ii) dapagliflozin, separately, sequentially, or simultaneously, to the human patient. In another embodiment, the human patient is a CKD patient. In another embodiment, the human patient is a CKD patient classified as stage 1-4. In another embodiment, the human patient is a CKD patient classified as stage 3-4. In another embodiment, the human patient is a CKD patient classified as stage 4. In another embodiment, the human patient is a CKD patient classified as stage 3a or 3b. In another embodiment, the human patient is a CKD patient classified as stage 2-3. In another embodiment, the human patient is a CKD patient with type 2 diabetes. In other embodiments, the human patient is a CKD patient without type 2 diabetes.

[0089] In any of the foregoing aspects, the methods and uses may also relate to patients who receive at least one SGLT2-I alone (e.g., dapagliflozin, empagliflozin, canagliflozin, etc.) or a combination thereof with at least one standard CKD care agent. In this regard, the standard CKD care agent may be an ACE-I (e.g., captopril, enalapril, and lisinopril) and / or an ARB (valsartan, losartan, and irbesartan).

[0090] In any of the foregoing aspects, the methods and uses may be relevant to the baseline patient. In some implementations, the methods and uses may be relevant to the baseline patient compared to a patient receiving at least one SGLT2-I alone or in combination with standard CKD care.

[0091] In one aspect, a kit is provided that contains: A first pharmaceutical composition containing ziprettan and a pharmaceutically acceptable carrier; and A second pharmaceutical composition containing dapagliflozin and a pharmaceutically acceptable carrier.

[0092] Terms such as "treating" or "treatment" or "alleviating" refer to therapeutic measures that cure, slow, alleviate, or stop the progression of a diagnosed pathological condition or disorder. Therefore, patients requiring treatment include those already diagnosed with or suspected of having the disorder. Patients or subjects requiring treatment may include those diagnosed with CKD.

[0093] "Therapeutic effective amount" or "effective amount" means the amount by which at least one compound of this disclosure or a pharmaceutical composition comprising at least one such compound effectively produces 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 usually 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 preventative benefit) are within the skill of a person skilled in the art. The optimal dose of a therapeutic agent may depend on the patient's constitution, weight, and / or blood volume. The effectiveness of treatment in patients can usually be monitored using assays suitable for the disease, disorder, and / or condition being treated or prevented, such assays being familiar to those skilled in the art and described herein. The level of the compound administered to a patient can be monitored by determining the level of the compound (or its metabolites) in biological fluids such as blood, blood fractions (e.g., serum), and / or urine and / or other biological samples from the patient. During the treatment regimen, the level of the compound can be measured using any method practiced in the art for detecting the compound or its metabolites. [ ]

[0094] Alternatively, the pharmacological and / or physiological effects may be prophylactic, i.e., the effect completely or partially prevents the disease or its symptoms. In this regard, the disclosed methods include administering a "prophylacticly effective amount" of the drug (e.g., zipretane 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). "Prophylacticly effective amount" refers to the amount that effectively achieves the desired preventative outcome (e.g., prevention of CKD or disease flare-ups) within the required dose and time period.

[0095] As used herein, the terms "subject" and "patient" are used interchangeably. In some respects, a subject refers to a person.

[0096] As used herein, the term "end-stage renal disease (EKSD)" refers to (i) a persistent eGFR < 15 mL / min / 1.73 m2, (ii) chronic dialysis, or (iii) a kidney transplant. In some embodiments, "persistent" means that a second eGFR test at a 3-month interval confirms a similar eGFR measurement.

[0097] As used herein, the terms "administer," "administering," and "administration" refer to methods that enable the delivery of drugs (e.g., zipretane or a pharmaceutically acceptable salt, solvate, mixed solvate, complex, or prodrug as described herein, and dapagliflozin or a pharmaceutically acceptable salt, solvate, mixed solvate, complex, or prodrug as described herein). Administering techniques that can be used with the drugs 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 respects, zipretane and dapagliflozin are administered orally.

[0098] The terms "drug formulation" and "drug composition" refer to preparations in a form that enables the bioactivity of one or more active ingredients to be effective and that do not contain any additional components that would have unacceptable toxicity to a subject to be administered the formulation. Such formulations may be sterile.

[0099] "Pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier that is commonly used in the art to be used with therapeutic agents and together constitute 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 other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0100] "Sterile" preparations are clean (aseptic) or substantially free of live microorganisms and their spores.

[0101] As used herein, the term "produce" refers to, for example, esters and carbonates that can be converted into zipretentam or dapagliflozin, for example, under physiological conditions or by solvent degradation. Therefore, the term "produce" includes pharmaceutically acceptable metabolic precursors of zipretentam or dapagliflozin. The term "produce" also includes covalently bound carriers that release zipretentam or dapagliflozin in vivo when such produce is administered to a patient. Non-limiting examples of produce include esters and carbonates.

[0102] Various forms of precursor drugs 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 Prodrugs”, 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 Sciences, 77, 285 (1988); and (5) N. Kakeya et al., Chem Pharm Bull, 32, 692 (1984).

[0103] It should be understood that, in all cases where the language "comprises" is used to describe an aspect herein, other similar aspects describing "consisting of" and / or "consisting essentially of" are also provided. In this disclosure, "comprises," "containing," and "having" can mean "includes," etc.; "consisting essentially of" is open-ended, allowing for aspects beyond what is described, provided that the essential or novel features described are not altered by aspects beyond what is described, but excluding prior art aspects.

[0104] Unless explicitly stated or obvious from the context, as used herein, the term "or" is to be understood as included.

[0105] As used herein, the terms “about” and “approximately” when used to modify numerical values ​​or ranges indicate that deviations of 10% above or below the value or range are still within the intended meaning of the value or range being described. It should be understood that, in all cases where the language “about” or “approximately” is used to describe numerical values ​​or ranges herein, other similar references to the specified numerical values ​​or ranges (without “about”) are also provided. Experimental Procedure [Example] [1]

[0106] The effects of dapagliflozin and zipotentan on hematocrit (Hct) concentration in male Wistar rats fed a 4% salt diet were investigated.

[0107] In Experiment 1, zippotentan was administered orally once daily at doses of 30, 100, or 300 mg / kg for 14 days. On day 7, all three doses of zippotentan resulted in a significant decrease in Hct concentration compared to the mediator (p < 0.05). On day 14, compared to the mediator, higher doses of zippotentan (100 mg / kg and 300 mg / kg) showed a significant decrease in Hct concentration (p < 0.05), but the lower dose of zippotentan (30 mg / kg) did not.

[0108] In Experiment 2, Hct concentrations were measured 7 days after administration of ziprettan (30 or 100 mg / kg) or dapagliflozin (3.0 mg / kg) or a combination thereof to male Weiss rats fed a 4% salt diet. Ziprettan (30 or 100 mg / kg) significantly reduced Hct concentrations. Co-administration of dapagliflozin (3.0 mg / kg) and ziprettan (30 mg / kg) resulted in Hct concentrations that were not significantly different from those in the drug-treated animals. As revealed by a one-way ANOVA and a post-hoc Tukey test, co-administration of dapagliflozin (3.0 mg / kg) and ziprettan (100 mg / kg) attenuated the effect of ziprettan on Hct concentrations (ziprettan -4.00 ± 0.87 vs. ziprettan + dapagliflozin -1.72 ± 0.68, #p < 0.05). Zipotentan does not affect the effect of dapagliflozin on urinary glucose excretion. Materials and methods

[0109] For formulations of zipotentan alone: ​​a suspension in 30% (w / w) TEG, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000 cps, 0.1% (w / w) Tween 80, and 67.4% purified water.

[0110] Preparations for dapagliflozin alone: ​​a solution in 30% (w / w) TEG, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000 cps, 0.1% (w / w) Tween 80, and 67.4% purified water.

[0111] Formulation of a combination of zipretentam and dapagliflozin: a suspension / solution in 30% (w / w) TEG, 2% (w / w) EtOH, 0.5% (w / w) HPMC 10000 cps, 0.1% (w / w) Tween 80, and 67.4% purified water.

[0112] The study consisted of two experiments.

[0113] In Experiment 1, the effect of zipretentan on hematocrit (Hct) concentration was determined after 14 days of oral (po) QD administration of zipretentan. Three days prior to the first administration of the study drug, animals were randomly assigned based on body weight (BW) to the following groups: 1) the carrier, 2) zipretentan (30 mg / kg), 3) zipretentan (100 mg / kg), and 4) zipretentan (300 mg / kg). Animals were weighed daily before administration throughout the experiment. Blood samples (20 µL) were collected via tail vein from conscious animals 4 and 24 hours after the first dose, and via the retroorbital route from anesthetized animals 24 hours after the last dose, for bioanalysis. Blood samples (100 µL) were collected via tail vein 1–2 hours after administration on days 7 and 14 for Hct concentration and hemoglobin (Hb) measurement. Twenty-four hours of urine were collected between doses nine and ten for the measurement of urinary glucose, urine volume, and electrolytes. Animals were terminated 24 hours after the last dose. On the day of termination, animals were anesthetized with 5% isoflurane, and blood was collected via the retroorbital route (in heparin-coated microvettes, Sarsted). The blood was centrifuged at 3,500 rpm for 10 minutes at 4°C, and plasma was collected and stored at -80°C for analysis. Animals were then euthanized by removing the heart. The heart and kidneys were removed and weighed. A 4 mm thick tissue section was 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.

[0114] In the second experiment (Experiment 2), the effects of zipretem and dapagliflozin (alone or in combination) on Hct concentration and Hb were determined. Two to three days prior to administration of the study drugs, seventy-two animals were randomly assigned to the following groups based on BW and Hct concentration: 1) the mediator, 2) zipretem (30 mg / kg), 3) zipretem (100 mg / kg), 4) dapagliflozin (3 mg / kg), 5) zipretem (30 mg / kg) + dapagliflozin (3 mg / kg), and 6) zipretem (100 mg / kg) + dapagliflozin (3 mg / kg). For practical reasons, the study was conducted in two consecutive tanks, each containing 36 animals (6 animals / group). The compounds were administered orally via a quick dose (QD) for seven days. Blood samples were collected for bioanalysis on days 3 and 7, 4 and 24 hours after the first dose, and 24 hours after the last dose, for Hct concentration and Hb analysis, as described in Experiment 1. Twenty-four-hour urine samples were collected between the third and fourth doses. Food and water intake was measured over 24 hours between days 2 and 3 and between days 6 and 7. Two animals were in each cage, and the food and water intake per animal in each cage was assessed by dividing the cage's food and water intake by two.

[0115] During the termination, blood sampling, and sample analysis, the experimenters were unaware of the animal handling and allocation.

[0116] The blood and urine concentrations of zipotentan and dapagliflozin were determined by a bioanalytical method using liquid chromatography-mass spectrometry (LC-MS / MS).

[0117] Whole blood samples used for bioanalysis of dapagliflozin and zipretem were processed using the same procedure. 20 µL of whole blood was precipitated with 150 µL of acetonitrile containing an internal standard and rapidly vortexed, followed by centrifugation at 3220 g for 20 min at 4°C. The supernatant was transferred to fresh deep-well plates and diluted 1:1 with water before LC-MS analysis. Matrix-matched calibration samples and blanks were processed in the same manner as the study samples. Zipretem 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.

[0118] Weighted linear regression (1 / X2) was applied using Waters TargetLynx software XS V4.2 SCN986 to determine sample concentrations based on matrix-spiked calibration standards within the ranges of 0.010 µM – 10.0 µM (for dapagliflozin) and 0.75 µM – 376 µM (for zipotentan). Calibration sample residuals showed a homoscedastic distribution. Analytical bias was <15% across the concentration ranges of both analytes.

[0119] Samples were analyzed using reverse-phase high-performance liquid chromatography (HPLC) with a rapid gradient elution. Compounds were detected using a Waters Xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA). Chromatographic separation was performed using an ACQUITY BEH C18 1.8 µM, 2.1 × 50 mm column at 40°C. Mobile phase A consisted of an aqueous solution of 2% acetonitrile and 0.2% formic acid (A), and mobile phase B consisted of an acetonitrile solution of 0.2% formic acid (B). Separation was achieved using the following elution gradient: 0 min to 1.5 min, 4% B to 95% B; 1.5 min to 2.3 min, held at 95% B; 2.3 min to 2.4 min, 95% B to 4% B and held at 4% B until 2.7 min. The flow rate was 0.7 mL / min. Zipotentan was eluted after 0.82 min and detected using multiple reaction transition monitoring (425.3 m / z > 139.25 m / z) in positive electrospray mode.

[0120] Samples were analyzed by reversed-phase high-performance liquid chromatography with rapid gradient elution. Compounds were detected using a Waters Xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation, Milford, Massachusetts, USA). Mobile phase A consisted of 1 M ammonium acetate / acetonitrile / water (0.1 / 2 / 97.5, v / v / v), and mobile phase B consisted of 1 M ammonium acetate / acetonitrile (0.1 / 99.5, v / v). Separation was performed using an ACQUITY BEH C18 1.8 µM, 2.1 × 50 mm column from Waters Corporation with the following gradients: 0 min to 2.5 min, 4% B to 60% B; 2.5 min to 2.9 min, 60% B to 95% B; 2.9 min to 3.4 min, maintained at 95% B; and from 3.4 min to 3.5 min, mobile phase B was restored to 4% B and maintained at 4% B until 4 min. The flow rate was 0.6 mL / min, and the column temperature was 40°C. Dapagliflozin was eluted after 2.19 min and detected in negative electrospray mode via a transition of 407.1 > 329.07 for dapagliflozin and 413.0 > 335.13 for the 13C6-dapagliflozin internal standard.

[0121] Bioanalysis of zipotentan in rat urine: 50 µL of PBS containing 5% BSA was added to 50 µL of urine sample, followed by 500 µL of acetonitrile containing nifedipine as an internal standard. The sample was vortexed for 1 min and centrifuged at 2400 g for 5 min at 4°C. 400 µL of the supernatant was transferred to a new plate and dried under heated nitrogen. The sample was reconstituted, vortexed, and centrifuged in 100 µL of deionized water / formic acid (100 / 0.2, v / v) before analysis. Matrix-matched calibration samples, control samples, and blanks were processed in the same manner as the study samples.

[0122] Samples were analyzed on an Acquity i-class UPLC system (Waters Corporation, Milford, Massachusetts) coupled with an API 4500 (Sciex LLC, Framingham, MA, USA). Mobile phase A consisted of acetonitrile containing 0.2% formic acid, and mobile phase B consisted of water containing 0.2% formic acid. Separations were performed using an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm particle size, from Waters Corporation) with the following gradients: 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. Qipotentan (425.1 m / z > 361.2 m / z) was analyzed in positive ionization mode with a source temperature of 700°C and an ion spray voltage of 5000 V.

[0123] In addition to the minimum QC criteria (with a CV% of 13.6% and a bias of 21.0%), the lower limit of quantitation (LLOQ) is 0.010 µM, and the analytical bias is < 15%.

[0124] Bioanalysis of dapagliflozin in rat urine: 50 µL of PBS containing 5% BSA was added to 50 µL of urine sample, followed by 500 µL of acetonitrile / formic acid (100 / 0.5, v / v) containing nifedipine as an internal standard. The sample was vortexed for 1 min and centrifuged at 2400 g for 5 min at 4°C. 450 µL of the supernatant was transferred to a new plate and dried under heated nitrogen. The sample was reconstituted and vortexed in 400 µL of 1 M ammonium acetate / water / formic acid (20 / 75 / 5, v / v / v). The sample was further purified using SOLA HRP (10 mg) solid-phase extraction (SPE) plates (ThermoFisher Scientific, Waltham, MA, USA) on a liquid handling robot. The plate was acclimatized with 450 µL of methanol, followed by 450 µL of 1 M ammonium acetate / water / formic acid (20 / 75 / 5, v / v / v). Samples were loaded onto SPE plates and washed twice with 450 µL of 1 M ammonium acetate / water / formic acid (20 / 75 / 5, v / v / v) and 450 µL of acetonitrile / water (95 / 5, v / v). Samples were eluted twice with 250 µL of acetonitrile / deionized water / acetic acid (80 / 20 / 1, v / v / v) and dried under a heated nitrogen stream. Samples were reconstituted in 100 µL of deionized water, vortexed, and centrifuged before analysis. Matrix-matched calibration samples, control samples, and blanks were processed in the same manner as the study samples.

[0125] Samples were analyzed on an Acquity i-class UPLC system (Waters Corporation, Milford, Massachusetts) coupled with an API 4500 (Sciex LLC, Framingham, Massachusetts, USA). Mobile phase A consisted of 1 M ammonium acetate / acetonitrile / water (0.5 / 95 / 4.5, v / v / v), and mobile phase B consisted of 1 M ammonium acetate / acetonitrile / water (0.5 / 25 / 74.5, v / v / v). Separations were performed using an Acquity UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 µm particle size, Waters Corporation) with the following gradients: 0 min to 3.0 min, 5% A to 50% A; 3.0 min to 3.5 min, 50% A to 100% A; 3.5 min to 4 min, 100% A to 5% A. The flow rate was 0.45 mL / min, and the 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 a 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% (10.3% CV at the lowest QC standard, 0.03 µM).

[0126] Plasma and urinary 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.

[0127] For Hct and Hb analysis, 100 µl of blood was collected from the tail vein of rats and measured using a CG8 column. The results were analyzed using an iSTAT instrument (Abbott Point of Care Inc., Abbot Park, IL, USA).

[0128] Check that the data for each measurement feature conforms to a normal distribution, and apply logarithmic transformation to non-normally distributed data using R where appropriate. Since the data are grouped according to univariate zipotentan dose or combination therapy, use one-way ANOVA to analyze traits measured at a single time point (GraphPad version 8 or the R statistics package emmeans) and use a linear mixed-effects model in R (nlme package) to analyze multiple measurements of body weight. Use the Dunnett test (R or the emmeans package in GraphPad v8) (when comparing treatment groups with mediator groups) or Tukey comparisons (R or the emmeans package in GraphPad v8) (for specific pairwise comparisons of treatment groups) to test for differences between groups. Use model diagnostic plots in R statistics to check all model assumptions. Results are expressed as mean ± standard error of the mean (SEM) for pharmacodynamic parameters and mean ± standard deviation (SD) for compound concentrations in blood and urine. Significance is set at p < 0.05. result

[0129] Confirmation of exposure to zipretem was achieved by measuring concentrations of zipretem above the LLOQ in all blood samples collected from animals that received zipretem at 4 or 24 hours after the first dose or 24 hours after the last dose. [surface] [1] and [surface] [2], [picture] [1] and [picture] [2]). [ [surface] [1] [experiment] [1] [The Qi Botengtan in the middle () [Zibo] [)blood concentration () [µmol / L] [)] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Zibo] [(] [300 mg / kg] [)] 4 hours after the first dose < LLOQ(10) 53.8 ± 17.40(10) 134 ± 43.8(10) 247 ± 46.0(10) 24 hours after the first dose < LLOQ(10) 4.68 ± 3.93(10) 39.1 ± 38.1(10) 176 ± 108(10) 24 hours after the last dose 0.272 ± 0.146(6) 7.39 ± 10.38(9) 39.0 ± 32.4(10) 249 ± 193(10) The data presented are mean ± SD (sample size). LLOQ lower limit of quantitation SD standard deviation [ [surface] [2] [experiment] [2] [The Qi Botengtan in the middle () [Zibo] [) and dapagliflozin () [Dapa] [ ] blood concentration [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [30 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] Dapagliflozin (µmol / L) 4 hours after the first dose - - - 1.94 ± 0.517(11) 1.90 ± 0.309(12) 2.07 ± 0.292(11) 24 hours after the first dose - - - 0.086 ± 0.052(11) 0.133 ± 0.043(11) 0.212 ± 0.0649(12) 24 hours after the last dose - - - 0.048 ± 0.019(12) 0.111 ± 0.042(12) 0.172 ± 0.050(12) Zipotentan (µmol / L) 4 hours after the first dose - 61.1 ± 8.67(12) 161 ± 31.0(10) - 59.9 ± 19.1(12) 150 ± 18.8(11) 24 hours after the first dose - 7.80 ± 6.19(12) 63.9 ± 46.9(12) - 4.59 ± 2.69(11) 44.3 ± 28.8(12) 24 hours after the last dose - 22.9 ± 27.2(12) 136 ± 123(12) - 5.40 ± 5.29(12) 42.4 ± 26.7(12) The data presented are mean ± SD (sample size). LLOQ lower limit of quantitation SD standard deviation - Not detected; all concentrations were below LLOQ.

[0130] Concentrations of dapagliflozin above the LLOQ were measured in all blood samples collected from animals that received dapagliflozin 4 or 24 hours after the first dose or 24 hours after the last dose to confirm exposure to dapagliflozin. [picture] [3], [surface] [2]). In Experiment 2, concentrations of zipretane above LLOQ were measured in all urine samples collected overnight from animals that received zipretane between Day 3 and Day 4. [surface] [3]), and the concentration of dapagliflozin above LLOQ was measured in all urine samples from animals that received dapagliflozin. [surface] [3]). [ [surface] [3] [experiment] [2] [Dapagliptin excreted in urine ( )] [Dapa] [) and Ziptentan () [Zibo] [ ] quantity [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [30 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] Dapagliflozin (µmol) - - - 0.198 ± 0.0637(12) 0.293 ± 0.0600(11) 0.343 ± 0.107(12) Zipotentan (µmol) 0.00127 ± 0.00180(5) 2.17 ± 1.88(12) 3.21 ± 1.20(11) - 1.11 ± 0.531(11) 5.24 ± 2.93(12) The data presented are mean ± SD (sample size). LLOQ lower limit of quantitation SD standard deviation - Not detected, all concentrations below LLOQ

[0131] In Experiment 1, measurable concentrations of zipretem were detected in six blood samples from control animals on day 14. The apparent concentrations of zipretem 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 concentration in the vehicle animal, compared to the mean of 7.39 µM in the 30 mg / kg group). In Experiment 2, no concentrations of zipretem or dapagliflozin above the LLOQ were found in blood samples from control animals, but concentrations of zipretem above the LLOQ were detected in all five urine samples from control animals. The amount of zipretem excreted in urine in these samples was at least 190-fold lower than the mean amount excreted in urine in the zipretem-treated group (showing the lowest amount) (30 mg / kg zipretem + 3 mg / kg dapagliflozin).

[0132] In Experiment 2, compared with the group receiving zippotentan alone, the blood concentration of zippotentan in the combination of zippotentan and dapagliflozin was 3 to 4 times lower 24 hours after the last dose.

[0133] Compared with the mediator, zipostentan significantly reduced Hct concentrations at all doses on day 7 (30 mg / kg, 100 mg / kg, and 300 mg / kg) and at doses of 100 mg / kg and 300 mg / kg on day 14 (p < 0.05). [picture] [4]).

[0134] For each dose of zipostemma (30 mg / kg, 100 mg / kg, and 300 mg / kg), body weight was measured daily and compared with that of the carcass. There were no differences in body weight change between the zipostemma treatment groups and the carcass treatment groups. [surface] [4], [picture] [5]). On day 10, the animals lost weight due to being placed in a metabolic cage. [ [surface] [4] [] [Qi Botengtan() [30 mg / kg] [、] [100 mg / kg] [and] [300 mg / kg] [Comparison of weight between [ ] and the medium] [ ] [Handling Method] [Comparison] [difference] [*] [Standard Error] [p] [value] Zibo 30 mg / kg vehicle 0.02 0.01 0.14 Zibo 100 mg / kg vehicle 0.02 0.01 0.12 Zibo 300 mg / kg vehicle 0.01 0.01 0.73 * Differences in means are expressed on a log scale and estimated using a linear mixed-effects model and Dunnett’s post-hoc test.

[0135] Compared with the mediator, zipostentan significantly reduced Hb at all three doses on day 7 (30 mg / kg, 100 mg / kg, and 300 mg / kg) and at two higher doses on day 14 (100 mg / kg and 300 mg / kg). [surface] [5]). [ [surface] [5] [experiment] [1] [Zhongqi Botengtan () [Zibo] [)right] [Hb] [The function] [parameter] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Zibo] [(] [300 mg / kg] [)] Hb (g / L) on day 7 157.4 ± 1.68 146.4 ± 2.51* 141.5 ± 2.02* 143.2 ± 1.79* Hb (g / L) on day 14 162.2 ± 1.77 156.8 ± 2.43 148.5 ± 2.49* 150.3 ± 3.13* Effects of zipotentan at concentrations of 30 mg / kg, 100 mg / kg, and 300 mg / kg on hemoglobin (Hb). N = 8–10 mice / group, *p < 0.05 relative to the medium. Data analysis was performed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM.

[0136] like [surface] [6] As shown, compared with the mediator, the levels of Na, K, glucose and urea in plasma did not change in response to different doses of ziprettan. [ [surface] [6] [experiment] [1] [Zhongqi Botengtan () [Zibo] [ ] on plasma [Na] [、] [K] The roles of glucose and urea [ ] [parameter] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Zibo] [(] [300 mg / kg] [)] Na (mmol / L) 136.4 ± 0.70 137.0 ± 0.96 137.2 ± 0.97 139.4 ± 0.86 K (mmol / L) 4.35 ± 0.11 4.50 ± 0.06 4.52 ± 0.06 4.76 ± 0.09 Glucose (mmol / L) 9.23 ± 0.29 9.76 ± 0.28 9.65 ± 0.30 9.34 ± 0.13 Urea (mmol / L) 4.60 ± 0.25 4.64 ± 0.20 4.77 ± 0.25 4.94 ± 0.39 Effects of different doses of zipretem on plasma Na, K, glucose, and urea levels. N = 8-10 animals / group. Data analysis was performed using one-way ANOVA and Dunnett's test for multiple comparisons. The data is presented as mean ± SEM.

[0137] As shown in Table 7, compared with the mediator, there were no changes in urine volume, Na, K, urea, creatinine and glucose levels in response to different doses of ziprettan. [ [surface] [7] [experiment] [1] [Zhongqi Botengtan () [Zibo] [Regarding urine volume and urine composition] [Na] [、] [K] The roles of urea, creatinine, and glucose [ ] [parameter] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Zibo] [(] [300 mg / kg] [)] Urine volume (ml) 24.79 ± 2.85 28.19 ± 2.99 23.88 ± 2.02 29.09 ± 2.74 Na (mmol) 13.42 ± 1.27 14.05 ± 0.95 13.01 ± 1.07 13.76 ± 0.76 K (mmol) 1.62 ± 0.07 1.57 ± 0.05 1.52 ± 0.06 1.44 ± 0.03 Urea (mmol) 4.73 ± 0.42 6.19 ± 1.71 4.04 ± 0.39 4.60 ± 0.19 Creatinine (µmol) 86.84 ± 3.13 88.8 ± 3.3 82.8 ± 2.5 82.9 ± 3.3 Glucose (mmol) 0.05 ± 0.0 0.01 ± 0.0 0.02 ± 0.0 0.18 ± 0.1 Effects of different doses of zipotentan on urine output, Na, K, glucose, creatinine, and urea. N = 8-10 animals / group. Data analysis was performed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM.

[0138] Following 14 days of zipretentan treatment, compared to the mediator, renal weight increased at a dose of 300 mg / kg, both after and without tibial length normalization, and cardiac weight increased at 100 mg / kg zipretentan, although this increase was not statistically significant after tibial length normalization. [surface] [8]). [ [surface] [8] [experiment] [1] [Zhongqi Botengtan () [Zibo] [The effect of [ ] on kidney and heart weight] [parameter] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Zibo] [(] [300 mg / kg] [)] Kidney (g) 2.06 ± 0.03 2.13 ± 0.04 2.16 ± 0.05 2.25 ± 2.74* Heart (g) 1.00 ± 0.01 1.00 ± 0.02 1.05 ± 0.01* 0.98 ± 0.00 Tibial length (mm) 39.35 ± 0.23 39.26 ± 0.26 39.00 ± 0.13 39.09 ± 0.21 Kidney / Tibia Length (mg / mm) 52.0 ± 0.89 54.00 ±0.80 55.0 ±1.65 57.0 ±1.39* Heart / Tibia Length (mg / mm) 25.00 ± 0.44 25.00 ± 0.47 27.00 ± 0.37 25.00 ± 0.24 Effects of zipotentan at doses of 30 mg / kg, 100 mg / kg, and 300 mg / kg on kidney and heart weight. N = 8–10 animals / group. Data analysis was performed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM, *p < 0.05 relative to the treatment.

[0139] Hct data were analyzed using a linear mixed-effects model to estimate the marginal means and standard deviations within the mediator and treatment groups at day 3, day 7, and throughout the study for post-hoc comparisons. The treatment group was compared to the mediator group (Dunnett's test) to determine whether it had an effect on Hct compared to the mediator-treated control group, and the zippotentan treatment group was compared to the zippotentan plus dapagliflozin group (Tukey's test) to determine whether the combination therapy reduced the effect of zippotentan on Hct.

[0140] There was no difference in the absolute value of Hct at the baseline among the groups. [picture] [6A]). On day 3, compared with the mediator, ziprettan (30 mg / kg and 100 mg / kg) administered with or without dapagliflozin (3 mg / kg) reduced Hct ( [picture] [6B], [surface] [9A]). On day 7, ziprettan (30 mg / kg and 100 mg / kg) reduced Hct compared to the mediator, and both doses resulted in similar Hct to the mediator when co-administered with dapagliflozin (3 mg / kg). [(picture] [6C], [surface] [9B]). Overall, ziprettan 100 mg / kg plus dapagliflozin had a higher Hct level than ziprettan 100 mg / kg alone ( [surface] [9C]). [ [surface] [9A] [In the process of] [3] [Days, between paired treatment groups and between treatment groups and normal controls] [Hct] [Level Comparison] [ ] [Group] [1] [Group] [2] [Estimated value] [Standard Error] [p] [value] Dapa (3.0 mg / kg) vehicle 0.03 0.02 0.42 Zibo(100 mg / kg)+Dapa(3.0 mg / kg) vehicle -0.05 0.02 0.02 Zibo (100 mg / kg) vehicle -0.08 0.02 0.0009 Zibo(30 mg / kg)+Dapa(3.0 mg / kg) vehicle -0.06 0.02 0.01 Zibo (30 mg / kg) vehicle -0.10 0.02 0.0001 Zibo(100 mg / kg)+Dapa(3.0 mg / kg) Zibo (100 mg / kg) 0.03 0.02 0.08 Zibo(30 mg / kg)+Dapa(3.0 mg / kg) Zibo (30 mg / kg) 0.04 0.02 0.05 * Differences in means are expressed on a logarithmic scale and estimated using a linear mixed-effects model and Dunnett’s post-hoc test (for comparison with the medium) and Tukey’s comparison (for pairwise comparisons).

[0141] On day 7, zipostem monotherapy with 30 mg / kg and 100 mg / kg induced a decrease in Hct, but this effect disappeared when zipostem was combined with dapagliflozin. [picture] [6C], [surface] [9B]). [ [surface] [9B] [In the process of] [7] [Days, between paired treatment groups and between treatment groups and normal controls] [Hct] [Level Comparison] [ ] [Group] [1] [Group] [2] [difference] [*] [Standard Error] [p] [value] Dapa (3.0 mg / kg) vehicle 0.01 0.02 0.92 Zibo (100 mg / kg) + Dapa (3 mg / kg) vehicle -0.03 0.02 0.52 Zibo (100 mg / kg) vehicle -0.08 0.02 0.0003 Zibo (30 mg / kg) + Dapa (3 mg / kg) vehicle -0.05 0.02 0.12 Zibo (30 mg / kg) vehicle -0.06 0.02 0.02 Zibo (100 mg / kg) + Dapa (3 mg / kg) Zibo (100 mg / kg) 0.06 0.02 0.009 Zibo (30 mg / kg) + Dapa (3 mg / kg) Zibo (30 mg / kg) 0.01 0.02 0.93 [ [surface] [9C] [Comparison of processing groups and media at all time points] [Hct] [Differences in averages] [ ] [Group] [1] [Group] [2] [difference] [Standard Error] [p] [value] Dapa (3 mg / kg) vehicle 0.01 0.012 0.85 Zibo(100 mg / kg)+Dapa(3 mg / kg) vehicle -0.03 0.012 0.10 Zibo (100 mg / kg) vehicle -0.06 0.012 < 0.0001 Zibo (30 mg / kg) + Dapa (3 mg / kg) vehicle -0.04 0.012 0.01 Zibo (30 mg / kg) vehicle -0.05 0.012 0.0003 Zibo(100 mg / kg)+Dapa(3 mg / kg) Zibo (100 mg / kg) -0.03 0.012 0.01 Zibo (30 mg / kg) + Dapa (3 mg / kg) Zibo (30 mg / kg) -0.01 0.012 0.46

[0142] Dapagliflozin is known to increase weight loss (Hansen et al., Endocr Pract. [Endocrinology Practice] 2014; 20(11): 1187-1197). To assess the effect of combination therapy on weight loss, body weight was measured daily and compared using a linear mixed-effects model. As expected, animals treated with the vector gained weight during administration, and dapagliflozin monotherapy prevented this increase in body weight compared to the vector (p < 0.05), while zipotentan alone or in combination with dapagliflozin did not prevent it (p < 0.05). [picture] [7] [,surface]

[10] ). [ [surface]

[10] ] [:Intergroup comparisons of weight changes across the treatment groups throughout the study.] [ ] [Group] [1] [Group] [2] [difference] [Standard Error] [P] [value] Zibo (30 mg / kg) vehicle 0.01 0.01 0.06 Zibo (100 mg / kg) vehicle 0.01 0.01 0.11 Dapa (3 mg / kg) vehicle -0.03 0.01 < 0.001 Zibo (30 mg / kg) + Dapa (3 mg / kg) vehicle 0.00 0.01 > 0.99 Zibo(100 mg / kg)+Dapa(3 mg / kg) vehicle -0.01 0.01 0.14 Zibo(100 mg / kg)+Dapa(3 mg / kg) Zibo (100 mg / kg) 0.04 0.01 < 0.001 Zibo (30 mg / kg) + Dapa (3 mg / kg) Zibo (30 mg / kg) 0.01 0.01 0.07 As expected, animals treated with the vector gained weight during the feeding period. Compared with the vector, dapagliflozin monotherapy prevented this increase in body weight (p < 0.05), while zipotentan alone or in combination with dapagliflozin did not prevent it. [picture] [7]).

[0143] Dapagliflozin significantly increased urinary glucose excretion, while zipostemma had no effect. [picture] [8A]). Co-administration of zipretane did not alter the dapagliflozin-mediated increase in urinary glucose ( [picture] [8A]). Zipotentan did not alter water and food intake compared to the medium. Dapagliflozin treatment increased water and food intake compared to the medium, such as [picture] [8B] and [picture] [8C] As shown. When compared with similar levels observed in the medium and in animals given dapagliflozin alone, animals given dapagliflozin and zipotentan showed significantly increased water and food intake (respectively, respectively). [picture] [8B] and [picture] [8C] [). ] [ [surface]

[11] [experiment] [2] [Zhongqi Botengtan () [Zibo] [) and dapagliflozin () [Dapa] [)right] [Hb] [The function] parameter vehicle Zibo (30 mg / kg) Zibo (100 mg / kg) Dapa (3 mg / kg) Zibo (30 mg / kg) + Dapa (3 mg / kg) Zibo(100 mg / kg)+Dapa(3 mg / kg) Baseline Hb (g / L) 159.4 ± 2.44 160.2 ± 1.62 158.5 ± 2.00 157.9 ± 2.34 159.3 ± 2.17 158.7 ± 1.04 Hb (g / L) on day 3 159.1 ± 2.20 144.4 ± 2.32* 147.5 ± 1.96* 164.4 ± 1.42* 151.3 ± 1.62# 152.4 ± 1.02# Hb (g / L) on day 7 157.5 ± 1.91 148.6 ± 1.86 144.8 ± 1.69* 159.1 ± 1.88* 149.7 ± 2.80 152.9 ± 1.99# Effects of 30 mg / kg and 100 mg / kg zippotentan with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and the mediator on hemoglobin (N = 8–12 animals / group), *p < 0.05 relative to the mediator, #p < 0.05 relative to the zippotentan monotherapy group. Data analysis was performed using one-way ANOVA and Tukey's test for multiple comparisons. Data are presented as mean ± SEM.

[0144] Compared to the mediator, plasma Na, K, glucose, and urea concentrations were not altered by treatment with ziprettan, dapagliflozin, or a combination thereof. [surface]

[12] As shown. Compared with the medium, Zibo (100 mg / kg) monotherapy or its combination with Dapa (3 mg / kg) significantly increased plasma creatinine (p < 0.05). [surface] As shown in

[12] . [ [surface]

[12] [experiment] [2] [Zhongqi Botengtan () [Zibo] [), Dapagliflozin () [Dapa] [) or combinations thereof on plasma [Na] [、] [K] The effects of glucose, urea, and creatinine concentrations [parameter] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [30 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] Na (mmol / L) 138.9 ± 0.90 139.1 ± 1.23 138.4 ± 0.76 137.6 ± 0.74 138.4 ± 0.96 139.2 ± 0.87 K (mmol / L) 3.99 ± 0.68 4.17 ± 0.07 4.13 ± 0.06 3.68 ± 0.10 3.96 ± 0.05 4.0 ± 0.08 Glucose (mmol / L) 8.64 ± 0.28 8.96 ± 0.19 8.73 ± 0.17 8.98 ± 0.28 8.49 ± 0.39 9.41 ± 0.21 Urea (mmol / L) 4.51 ± 0.30 4.47 ± 0.25 4.25 ± 0.18 4.34 ± 0.14 3.95 ± 0.18 3.91 ± 0.12 Creatinine (µM) 15.35 ± 0.75 17.58 ± 0.68 18.08 ± 0.62* 14.62 ± 0.44 16.64 ± 0.52 17.98 ± 0.92* Effects of 30 mg / kg and 100 mg / kg zipotentan with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and the mediator on plasma electrolytes, urea, glucose, and creatinine, N = 8–12 animals / group. Data analysis was performed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM.

[0145] In all dapagliflozin treatment groups, with or without co-administration of zipotentan, 24-hour urine volume and urea excretion increased. No difference was observed in zipotentan monotherapy groups compared to the mediator. No changes in Na, K, and creatinine were observed in any treatment group compared to the mediator. [surface] As shown in

[13] . [ [surface]

[13] [experiment] [2] [Zhongqi Botengtan () [Zibo] [), Dapagliflozin () [Dapa] [) and combinations affecting urine volume and urine composition. [Na] [、] [K] [The role of urea and creatinine] [parameter] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [30 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] Urine volume (ml) 22.59 ± 2.10 19.08 ± 2.98 19.08 ± 2.98 44.13 ± 5.85* 48.00 ± 5.71*,# 53.21 ± 4.82*,# Na (mmol) 11.58 ± 0.87 10.67 ± 0.74 10.03 ± 0.95 12.60 ± 1.54 13.18 ± 1.41 14.13 ± 1.19 K (mmol) 1.26 ± 0.07 1.27 ± 0.05 1.16 ± 0.06 1.40 ± 0.07 Note: There seems to be a possible error in the original text where the value in line is likely incorrect as it was written as "44.13" instead of "43.13" in the original Chinese. The translation above has been done as accurately as possible based on the provided text. 1.48 ± 0.07 1.31 ± 0.07 Urea (mmol) 5.07 ± 0.38 4.61 ± 0.36 4.11 ± 0.34 7.43 ± 0.44* 6.88 ± 0.41*,# 6.95 ± 0.39*,# Creatinine (µmol) 70.64 ± 3.02 71.33 ± 2.6 68.18 ± 3.6 76.42 ± 3.5 79.09 ± 2.9 77.00 ± 3.5 Effects of different doses of 30 mg / kg and 100 mg / kg zipotentan with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and the mediator on urine output, electrolytes, urea, glucose, and creatinine, N = 8–12 animals / group. Data analysis was performed using one-way ANOVA and Tukey's test for multiple comparisons. Data are presented as mean ± SEM, * p < 0.05 relative to the mediator, # p < 0.05 relative to zipotentan monotherapy.

[0146] After seven days of treatment, kidney weight increased in the Zibo 100 mg / kg + Dapa 3 mg / kg group compared to the mediator alone. Heart weight (whether normalized or unnormalized to tibial length) increased in the Zibo 30 mg / kg group compared to the mediator alone. [surface] As shown in

[14] . [ [surface]

[14] [experiment] [2] [Zhongqi Botengtan () [Zibo] [) and dapagliflozin () [Dapa] [The effect of [ ] on kidney and heart weight] [ ] [parameter] [vehicle] [Zibo] [(] [30 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [30 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] [Zibo] [(] [100 mg / kg] [)] [+ Dapa] [(] [3 mg / kg] [)] Kidney (g) 1.89 ± 0.04 2.05 ± 0.04 1.90 ±0.05 2.01 ± 0.05 2.07 ± 0.06 2.12 ± 0.03* Heart (g) 0.89 ± 0.01 0.96 ± 0.01* 0.93 ± 0.01 0.85 ± 0.02 0.91 ± 0.01 0.88 ± 0.01 Tibial length (mm) 39.28 ± 0.19 39.28 ± 0.22 39.42 ± 0.15 38.85 ± 0.23 39.88 ± 0.22 39.32 ± 0.17 Kidney / Tibia Length (mg / mm) 48.33 ± 1.31 52.49 ± 1.15 48.20 ± 1.35 51.96 ± 1.34 52.02 ± 1.62 53.99 ± 1.13* Heart / Tibia Length (mg / mm) 22.82 ± 0.45 24.75 ± 0.27* 23.82 ± 0.46 22.08 ± 0.46 23.00 ± 0.33 23.59 ± 0.36 Effects of different doses of 30 mg / kg and 100 mg / kg zipotentan, with or without 3 mg / kg dapagliflozin, 3 mg / kg dapagliflozin alone, and the vehicle on kidney and heart weight. N = 12 animals / group, *p < 0.05 relative to the vehicle. Data analysis was performed using one-way ANOVA and Dunnett's test for multiple comparisons. Data are presented as mean ± SEM.

[0147] The endothelial receptor A antagonist zipretem significantly reduced hematocrit (Hct) concentrations in male Weiss rats fed a 4% salt diet. Co-administration of the SGLT-2 inhibitor dapagliflozin for 7 days significantly reduced the effect of zipretem on Hct concentrations. Dapagliflozin attenuated zipretem-induced reduction in fluid retention (hematocrit) in rats. [Example] [2]

[0148] The effects of dapagliflozin on proteinuria were investigated in CKD patients with or without type 2 diabetes. This analysis of the phase 3 DAPA CKD trial evaluated the efficacy of dapagliflozin to determine whether treatment with dapagliflozin could reduce proteinuria levels in CKD patients.

[0149] In this trial, 4304 patients (estimated glomerular filtration rate of 25–75 mL / min / 1.73 m² and urinary UACR of 200–5000 mg / g) were randomized to receive dapagliflozin (10 mg) or placebo. Changes in proteinuria were assessed as the log-transformed mean change in UACR from baseline to the end of the study. UACR regression (defined as the transition from massive proteinuria (≥ 300 mg / g) to micro- or normoproteinuria (< 300 mg / g)) and UACR progression (defined as the transition from < 3000 mg / g to ≥ 3000 mg / g) were additional endpoints.

[0150] In the entire population, the median (25th–75th percentile) UACR was 965 (472–1903) mg / g (compared to a median UACR of 934 (482–1868) for placebo). Compared with placebo, dapagliflozin reduced UACR by 29.3% in patients with and without type 2 diabetes (95% confidence interval [CI], 25.2%–33.1%; p < 0.001). Stratified by diabetes status, compared with placebo, dapagliflozin reduced UACR by 35.1% in patients with type 2 diabetes (95% confidence interval [CI], 30.6%–39.4%; p < 0.001) and by 14.8% in patients without type 2 diabetes (95% confidence interval [CI], 5.9%–22.9%; p < 0.001).

[0151] In 3860 patients with baseline UACR ≥ 300 mg / g, dapagliflozin increased the likelihood of UACR regression (hazard ratio [HR] 1.81; 95% CI, 1.60–2.05). In 3820 patients with 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 according to diabetes status and as follows: [picture] [9] and [picture]

[10] As this analysis illustrates, treatment with dapagliflozin reduces UACR in CKD patients regardless of their diabetes status. [Example] [3]

[0152] A preclinical study of dapagliflozin in combination with zipretentam is underway to demonstrate the effects of the single agent and combination on certain biomarkers indicative of renal and cardiovascular function in male Dahl salt-sensitive (DSS) rats.

[0153] Example 3 will evaluate the effects of zipretem alone and the combination of zipretem and dapagliflozin on body weight, blood pressure (mean arterial pressure - MAP, systolic blood pressure - SBP, and diastolic blood pressure - DBP), heart rate, urinary protein, albumin, and creatinine, serum creatinine, hematology, cardiac ultrasound of heart and LV diameter and wall thickness, fractional shortening, and ejection fraction, as well as kidney (left and right), heart, and lung weight and tibia length in 7–9 week old DSS rats on a 4% salt diet during a 7-week study. Treatment will begin 12 days after initiation of the 4% salt diet.

[0154] The formulation for the medium consists of 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.

[0155] Formulations for zipotentan alone: ​​suspensions 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.

[0156] Formulations for the combination of dapagliflozin and zipotentan: suspensions / solutions 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.

[0157] The effects of zipotentan and dapagliflozin alone on body weight, blood pressure (MAP, SBP, and DBP) and heart rate, urinary protein, albumin and creatinine, serum creatinine, hematology, cardiac and LV diameter and wall thickness, fractional shortening and ejection fraction by echocardiography, and kidney (left and right), heart and lung weight and tibia length will be determined based on the three groups summarized in Table 15 below. [ [surface]

[15] [] [Example] [3] [Research Group] [ ] [Group] [diet] [Test Item] [Dosage] (mg / kg) [Nominal Dosage Concentration] (mg / mL)* [Dosage / volume] (mL / kg) [Frequency and Duration] [Sample size] A 4% NaCl in food vehicle N / A N / A 5 QD, Day 12 until completion of the study 14 B 4% NaCl in food Qi Botengtan 0.75 mg / kg 0.15 5 12 C 4% NaCl in food Dapagliflozin 3 mg / kg 0.6 5 12 Qi Botengtan 0.75 mg / kg 0.15

[0158] Whole blood samples (20 μL) from groups B and C will be collected at weeks 3 and 6 to assess PK. An additional 200 μL whole blood sample will be collected at week 1 to assess hematological Hct and MCV (via Horiba ABX Micros ESV60). Urine will be collected at weeks 1, 3, and 6 for clinical chemistry assessment (via RX Daytona®) to evaluate serum creatinine, urinary protein, albumin, and creatinine. Cardiac ultrasound images of rats lightly anesthetized with 1%–2% isoflurane will be obtained using a VisualSonics Vevo 3100® echocardiography system and an MX201 15 MHz 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. Tibial length will be measured and imaged by X-ray.

[0159] One-way ANOVA (GraphPad Prism version 7.0a) will be used to analyze clinical chemistry and hematology data, Echo data, and body weight and tissue weight (normalized to tibial length). The Dunnett test will be used to examine for statistical significance between groups. Blood pressure analysis will be assessed as mean ± SEM of MAP, SBP, DBP, and heart rate in linear plot form. [Example] [4]

[0160] A phase 2b, multicenter, randomized, double-blind, placebo-controlled, parallel-group dose-range clinical trial (ZENITH-CKD) is underway to evaluate the efficacy, safety, and tolerability of dapagliflozin and zippotentan in participants with CKD and eGFR between 20 and 60 mL / min / 1.73 m2. Research Design

[0161] The study will be conducted in two parts (Part A and Part B). In both study parts, participants will be randomly assigned to receive 12 weeks of treatment plus 2 weeks of follow-up visits. All variables will be collected to validate 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 study parts.

[0162] In addition to receiving background local SoC therapy, eligible participants will be randomly assigned to receive either the treatment described in Part A or Part B. To maintain blinding, participants will receive both the active agent and placebo study interventions on each dosing day of receiving Zibo / Dapa monotherapy. Participants receiving placebo only will receive placebo for both study interventions.

[0163] In Part A, the plan is to recruit 132 qualified participants and randomly assign them to 4 treatment groups, with 33 participants in each group: - Zipotentan 5 mg + Dapagliflozin 10 mg once daily. - Zipotentan 5 mg once daily. - Dapagliflozin 10 mg once daily. - Placebo once daily.

[0164] Interim analysis of partial A data will be performed in approximately 30 participants in each group (120 participants) after they have completed 6 weeks of treatment to assess changes in fluid-related measures (weight gain or BNP). A group will be discontinued from the remainder of the study intervention if at least 5 participants in the zipotentiamine 5 mg monotherapy group meet the specified criteria for fluid-related measures.

[0165] If the specified discontinuation criteria for the zipotentan 5 mg monotherapy group are not met at the interim analysis of 6 weeks of treatment, a second interim analysis of available Part A data will be performed after all randomized participants in Part A have completed 12 weeks of treatment to assess changes in humoral-related measures. If, at the second interim analysis (at 12 weeks of treatment), at least 5 participants in the zipotentan 5 mg monotherapy group have humoral-related measures that meet the specified criteria, that group will be discontinued from the remainder of the study intervention.

[0166] In Part B, the plan is to recruit an additional 528 qualified participants. Of these, 352 will be randomly assigned to the same four treatment groups as in Part A, with 88 participants in each group. The remaining qualified participants will be randomly assigned to two additional treatment groups, with 88 participants in each group. - Zipotentan 0.25 mg + Dapagliflozin 10 mg once daily. - Zipotentan 1.5 mg + Dapagliflozin 10 mg once daily. - Zipotentan 5 mg + Dapagliflozin 10 mg once daily. - Zipotentan 5 mg once daily. - Dapagliflozin 10 mg once daily. - Placebo once daily.

[0167] Participants who are randomly assigned in part A cannot be randomly assigned in part B.

[0168] Participants will be stratified at randomization by diabetes (DKD vs. non-DM CKD) and baseline eGFR (below 45 mL / min / 1.73 m2 vs. above 45 mL / min / 1.73 m2) 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 comprises approximately 30% to 33% of the total number of randomized participants.

[0169] For each participant, the total duration of participation will be approximately 17 to 19 weeks. Within each study segment, the screening period may last up to 4 weeks, followed by randomization. Participants will receive their first dose after randomization at their baseline visit on Day 1. In addition to the baseline visit, participants will visit the clinic 5 times over the next 12 weeks of treatment. Approximately 2 weeks after the final dose, participants will return to the clinic for a follow-up assessment. Purpose and End Point

[0170] The primary endpoint will measure the effect of zipretem and dapagliflozin (combination and alone) relative to placebo on UACR. Changes in UACR will be measured as log-transformed UACR from baseline to week 12 (UACR (mg / g) = urinary albumin (mg / dL) / urinary creatinine (g / dL)). Secondary endpoints and objectives are summarized in Table 17 below. [ [surface]

[17] [ZENITH-CKD Ph2b] [Secondary endpoints and other objectives of the experiment] [ ] [Purpose] [Destination Description] To determine the changes in UACR of several doses of ziprettan combined with 10 mg dapagliflozin relative to 10 mg dapagliflozin alone. Changes in UACR from baseline to week 12 with logarithmic transformation. To determine the changes in office systolic and diastolic blood pressure (BP) relative to placebo in several doses of zipretentan combined with 10 mg dapagliflozin, as well as zipretentan alone and 10 mg dapagliflozin alone. Changes in blood pressure from baseline (visit 2) to week 12. To characterize the dose-response relationship (the relationship between different doses of zipostem / fixed dose of dapagliflozin and the decrease in UACR). Changes in the least squares mean of UACR from the three Zibo / Dapa dose groups and the dapagliflozin monotherapy group at week 12 To determine the effects of different doses of zipostemma and dapagliflozin (in combination and alone) on eGFR. Changes in eGFR from baseline to week 1. Changes in eGFR from baseline to week 12. Changes in eGFR from baseline to week 14. Changes in eGFR from week 1 to week 12. To evaluate the safety and tolerability of different doses of ziprententan and dapagliflozin (combination and alone) relative to placebo. AE / SAE / DAE. Vital signs. Clinical laboratory testing. 12-lead ECG assessment. Events of particular interest ( Changes in body fluid-related quantities). To evaluate the pharmacokinetics of different doses of zipotentan and dapagliflozin in plasma. Plasma concentrations of zipotentan and dapagliflozin. Exploratory analysis of zipretane metabolites (partial B only). Plasma concentration of zipotentan metabolites. To evaluate placebo-corrected changes in weight in response to different doses of zipretentam and dapagliflozin (combination and alone). Weight changes throughout the intervention period. To explore the relationship between zipotentan dose / exposure and safety / PD variables Zipotentan dose / exposure relative to safety and PD variables. Safety / PD variables included blood assessments of NT-proBNP, BNP, creatinine, and cystatin C, as well as urine assessments of albumin and creatinine. To evaluate the effects of zipotentan and dapagliflozin (in combination and alone) relative to placebo on plasma / serum K+, Na+, uric acid, BUN, fasting plasma glucose, hematocrit, heme, ET-1, ELDR, CT-proET-1 and peptidin levels. Changes in plasma / serum concentrations of K+, Na+, uric acid, BUN, fasting plasma glucose, hematocrit, heme, ET-1, ELDR, CT-proET-1, and peptidin levels over time during the study period. To evaluate the effects of zipotentan and dapagliflozin (combination and alone) relative to placebo on cardiovascular biomarkers in the blood. Evaluation of changes in cardiovascular biomarkers in the blood over time during the study period. To evaluate the effects of zipotentan and dapagliflozin (combination and alone) relative to placebo on fluid volume and distribution. Evaluation of changes in body fluid volume and distribution over time during the study. Changes in total body water, extracellular water, and intracellular water. The results will be obtained using a bioimpedance spectroscopy instrument. To collect and store plasma, serum, and urine samples for potential future exploratory studies, this study aims to explore biomarkers involved in zipotentan and dapagliflozin (combination and alone) relative to placebo or in cardiorenal disease, PK, PD, safety, and tolerability. Evaluation of changes in blood and urine biomarkers related to cardiorenal mechanisms, inflammation, and fibrosis during the study.

[0171] none

[0172] none

Claims

1. Use of ziprettan (N-(3-methoxy-5-methylpyridine-2-yl)-2-[4-(1,3,4-diazol-2-yl)phenyl]pyridine-3-sulfonamide) or a pharmaceutically acceptable salt, ester or solvation thereof in combination with dapagliflozin or a pharmaceutically acceptable salt, ester or solvation thereof, ziprettan, dapagliflozin, which are used to prepare a medicine for reducing the urinary albumin-to-creatinine ratio (UACR) and reducing the incidence of cardiovascular or renal death in patients with chronic kidney disease.

2. As requested in item 1, wherein the drug is administered once daily.

3. As claimed in claim 2, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 5 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

4. As claimed in claim 2, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 1.5 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

5. As claimed in claim 2, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 0.25 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

6. For the purposes of claim 2, wherein the patient is a chronic kidney disease patient classified as stage 1-4 with an eGFR of 20-60 ml / min / 1.73 m2.

7. As requested in item 6, wherein the patient is a chronic kidney disease patient classified as stage 3-4.

8. Use of ziprettan (N-(3-methoxy-5-methylpyridine-2-yl)-2-[4-(1,3,4-diazol-2-yl)phenyl]pyridine-3-sulfonamide) or a pharmaceutically acceptable salt, ester or solvation thereof, in combination with dapagliflozin or a pharmaceutically acceptable salt, ester or solvation thereof, ziprettan, dapagliflozin, for the preparation of a drug for reducing the urinary albumin-to-creatinine ratio (UACR) to < 300 mg / g in patients with chronic kidney disease.

9. As requested in item 8, wherein the drug is administered once daily.

10. As claimed in paragraph 9, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 5 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

11. As claimed in paragraph 9, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 1.5 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

12. As claimed in paragraph 9, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 0.25 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

13. As requested in item 9, wherein the patient is a patient with chronic kidney disease classified as stage 1–4 with an eGFR of 20–60 ml / min / 1.73 m2.

14. As requested in item 13, wherein the patient is a patient with chronic kidney disease classified as stage 3-4.

15. Use of ziprettan (N-(3-methoxy-5-methylpyridine-2-yl)-2-[4-(1,3,4-diazol-2-yl)phenyl]pyridine-3-sulfonamide) or a pharmaceutically acceptable salt, ester or solvation thereof in combination with dapagliflozin or a pharmaceutically acceptable salt, ester or solvation thereof, ziprettan, dapagliflozin, which is used to prepare a medicine for reducing the risk of progression of the urinary albumin-to-creatinine ratio (UACR) to ≥ 3000 mg / g in patients with chronic kidney disease.

16. As requested in claim 15, wherein the drug is administered once daily.

17. As claimed in claim 16, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 5 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

18. As claimed in claim 16, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 1.5 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

19. As claimed in claim 16, wherein zipretane or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of about 0.25 mg and dapagliflozin or a pharmaceutically acceptable salt, ester or solvate thereof is administered at a dose of 10 mg.

20. For the purposes of claim 16, wherein the patient is a patient with chronic kidney disease classified as stage 1–4 with an eGFR of 20–60 ml / min / 1.73 m2.

21. As requested in item 20, wherein the patient is a patient with chronic kidney disease classified as stage 3-4.

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